INTERN ATlON AL
REVIEW OF CYTOLOGY A SURVEY OF CELLBIOLOGY
ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN GARY G. BORISY PIET BORST BHARAT B. CHATTOO STANLEY COHEN RENE COUTEAUX MARIE A. DIBERARDINO CHARLES J. FLICKINGER OLUF GAMBORG M. NELLY GOLARZ DE BOURNE YUKIO HIRAMOTO YUKINORI HIROTA K. KUROSUMI GIUSEPPE MILLONIG ARNOLD MITTELMAN AUDREY MUGGLETON-HARRIS DONALD G. MURPHY
ROBERT G. E. MURRAY RICHARD NOVICK ANDREAS OKSCHE MURIEL J. ORD VLADIMIR R. PANTIC W. J. PEACOCK DARRYL C. REANNEY LIONEL I. REBHUN JEAN-PAUL REVEL L. EVANS ROTH JOAN SMITH-SONNEBORN WILFRED STEIN HEWSON SWIFT K. TANAKA DENNIS L. TAYLOR TADASHI UTAKOJI ROY WIDDUS ALEXANDER YUDIN
INTERNATIONAL
Review of Cytology A SURVEY OF CELLBIOLOGY
Editor-in-Chief
G. H. BOURNE St. George's University School of Medicine St. George's, Crenadri West Indies
Editors
K. W. JEON
M. FRIEDLANDER
Department of Zoology University of Tennessee Knoxville, Tennessee
Jules Stein Eye Institute U C L A School of Medicine Los Angeles, California
VOLUME106
I987
ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers
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Contents
Biochemical Transmitters Regulating the Arrest and Resumption of Meiosis in Oocytes EIMEISATOA N D S. S. KOIDE
I . Introduction ........................................................... I1 . Factors Sustaining Meiotic Arrest ....................................... I11 . Factors Inducing Resumption of Meiosis ................................. IV . Mechanism of Meiotic Resumption ...................................... References ............................................................
i
2 13 19 28
Morphology and Cytochemistry of the Endocrine Epithelial System in the Lung D . w . SCHEUERMANN 1. Introduction ........................................................... I1 . Light Microscopic Aspects ............................................. 111. Argentaffinity and Argyrophilia ......................................... IV . Cholinesterase Activity ................................................. V . Neuron-Specific Enolase ............................................... VI . Aspects of Induced Fluorescence ........................................ VI1 . Immunocytochemistry for Regulatory Peptides ............................ VIII . Electron Microscopic Aspects ........................................... I X . Location .............................................................. X . Innervation ........................................................... XI . Concluding Remarks . . . . . . . . ....................................... References ............................................................
35 39 43 45 46 48 53 55 71 73 79 80
Intrinsic Nerve Plexus of Mammalian Heart: Morphological Basis of Cardiac Rhythmical Activity? JOSEF MORAVECA N D
MlRElLLs MORAVEC
I . Introduction ........................................................... I1 . Autonomic Innervation of the Heart ..................................... V
89 91
vi
CONTENTS
111. Intracardiac Ganglionic Cells ............................................ IV . Terminal Nerve Plexus ................................................. V . New Developments in Studies of the Autonomic Nervous System .......... V1 . Morphological Basis of the Rhythmical Activity of the Heart: A Working Hypothesis ............................................................ VII . Conclusion ............................................................ References ............................................................
96 119 132 135 139 139
Structural and Functional Evolution of Gonadotropin-Releasing Hormone ROBERT P. MILLARAND JUDYA . KING I. I1 . 111. IV .
Introduction ........................................................... Structure and Distribution of GnRH and Related Molecular Forms .......... Biological Activity of GnRH ............................................ Conclusions ........................................................... References ............................................................
149 150 163 174 171
Excitons and Solitons in Molecular Systems
.
A . S DAVYDOV 1. Introduction ........................................................... I1 . The New Concept of Energy Transport along Protein Molecules ............ 111. History of Observation of Solitary Waves ................................ IV . Nonlinear Phenomena in Biology ........................................ V . Solitons in Real a-Helical Protein Molecules .............................. VI . Solitons in Discrete Models: Numerical Calculations ...................... VII . Solitons and the Molecular Mechanism of Muscle Contraction .............. VIII . Intracellular Dynamics and Solitons ..................................... IX . The Laser Raman Scattering by Metabolically Active Cells ................. X . Possible Mechanism for Anesthesia ...................................... XI . Electron Transfer along Protein Molecules ............................... XI1 . Electrosolitons Pairing in Soft Molecular Chains .......................... References ............................................................
138 187 189 192 199 201 204 207 213 214 216 221 223
The Centrosome and Its Role in the Organization of Microtubules I . A . VOROBJEV AND E . S . NADEZHDINA I . Introduction ........................................................... 11. Ultrastructure of Centrioles and Basal Bodies .............................
I11 . The Ontogenesis of Basal Bodies and Centrioles .......................... IV The Organization of the Centrosome and Its Behavior in a Cell Cycle ....... V . The Biochemistry of Centrioles and Basal Bodies .........................
.
227 229 239 244 249
CONTENTS
vii
V1 . Assembly of Microtubules on Microtubule-OrganizingCenters (MTOCs) in Virro
.................................................................
VII . Assembly of Microtubules on Microtubule-OrganizingCenters in Vivo ....... VIII . The Centrosome and the Cell ........................................... IX . Localization and Orientation of Centrioles in Cells ........................ X . Conclusion ............................................................ References ............................................................
INDEX
......................................................................
257 265 272 276 280 284 295
This Page Intentionally Left Blank
INTERNATIONAL REVIEW OF CYTOLOGY. VOI.. IIK
Biochemical Transmitters Regulating the Arrest and Resumption of Meiosis in Oocytes' EIMEISATO* A N D
s. s. KOIDEt
'Depurtment of' Animul Sciences, Fuculty c d Agricultrrre, Kyoto University, Kyoto 606, Jupun, and 'Center for Biomedicrrl Reseurch, The Poprrlution Coimcil. N e w York, New York 10021
I. Introduction
Germ cells migrate to the genital ridge from the yolk sac region during early embryonic development. In the genital ridge, the female germ cells start to divide and differentiate into oogonia. They enter meiosis and become primary oocytes. Nuclear division progresses to the diplotene stage of the first meiotic prophase and is arrested. The chromosomes decondense and are distributed diffusely throughout the oocyte nucleus. Progression of meiosis to the diplotene stage occurs before or shortly after birth. The oocytes may remain arrested at the dictyate stage for a prolonged period. Subsequently, a follicle develops enclosing the oocyte which contains a large clear nucleus designated as the germinal vesicle. It is generally accepted that the surge of luteinizing hormone (LH) during each ovarian cycle triggers the resumption of meiosis of the mature oocyte enclosed within Graafian follicles (Channing ef al., 1980, 1982a,b; Tsafriri, 1978b,c). The resumption of meiosis follows a sequence of programmed events while the oocytes are situated within the preovulatory follicles. The process is designated as oocyte maturation and is characterized by a series of biochemical, morphological, and functional changes that take place within the nucleus, highlighted by the following events: ( I ) dissolution of the nuclear membranes manifested as germinal vesicle breakdown (GVBD), (2) chromatin condensation and the formation of distinct chromosomes, (3) formation of the first meiotic spindle, (4)translocation of the spindle to the peripheral region, (5) formation and extrusion of t h e first polar body, (6) formation and positioning of the second meiotic division, (7) rearrest at the second metaphase. To better understand the biochemical mechanisms involved in oocyte maturation, in v i t r o culture systems have been developed. Pincus and Enzmann (1935) were the first to demonstrate that rabbit oocytes removed 'The authors dedicate this paper to Dr. Haruo Kanatani, pathfinder of starfish oocyte maturation and discoverer of I-methyladenine.
I Copyright B ' IYX7 hy Ac;idrmic h e \ \ . Inc. All right.. ol' repriiductiun in tiny limn rewrved.
2
EIMEI S A T 0 A N D S. S. KOIDE
from follicles resume meiosis spontaneously and mature under in vitro culture without the addition of hormones. This phenomenon of spontaneous maturation has been observed in all mammalian species examined (Biggers, 1973). Oocytes with adhering cumulus cell complexes or denuded from cumulus cells are widely used as models to study oocyte maturation. Studies with preovulatory follicles cultured in vitro demonstrate the interdependency of the various cells and fluid of the follicles and yield pertinent information on the resumption of meiosis triggered by LH added to the culture medium. The contrasting results obtained using denuded oocytes and follicle-enclosed oocytes suggest that maturation of mammalian oocytes is prevented by the follicular cells or factors in follicular fluid. By removing these factor(s) resumption of meiosis proceeds spontaneously. The involvement of the follicular cell-oocyte complex in the regulation of meiotic arrest was further investigated by coculturing isolated oocytes with follicular cells. Several review articles are available on various aspects of mammalian oocyte maturation. General and historical accounts have been covered by Donahue (1972), Tsafriri (1978b,c, 1984), and Masui and Clarke (1979). Technical problems relating to the in vitro culture of oocytes were discussed by Biggers (1973) and McGaughey (1978). Biochemical events involved in mammalian oocyte maturation have been presented by Mangia and Canipari (1977) and Wassarman et al. (1978). Morphological and ultrastructural changes were described by Albertini (1984). Informative review articles on the hormonal control and factors regulating oocyte maturation have been published (Lindner et ul., 1974, 1977, 1983; Schuetz, 1974; Channing and Tsafriri, 1977; Baker, 1979; Thibault, 1977; Channing et al., 1978, 1980, 1981, 1982a,b; Tsafriri and Bar-Ami, 1982; Tsafriri et al., 1982a; McGaughey, 1983; Eppig, 1980a). Cell-to-cell communication of cumulus-oocyte complexes has been discussed by Schuetz (19781, Moor (1983), and Dekel (1984). In the present article factors sustaining meiotic arrest and regulating resumption of meiosis in mammalian oocytes are discussed. A hypothesis of the sequence of events during oocyte maturation is proposed, based on our recent results. 11. Factors Sustaining Meiotic Arrest
Oogonia undergo the initial stages of the first meiotic division to reach the dictyate stage of prophase. The oocyte may remain in meiotic arrest for a prolonged period until activated shortly before ovulation or may undergo atretic degeneration. This suspended metabolic state of oocytes is an unusual phenomenon and has attracted the attention of many in-
ARREST A N D RESUMPTION OF MEIOSIS IN OOCYTES
3
vestigators. To identify the factors that sustain meiotic arrest, studies have been conducted with fully grown oocytes obtained from untreated follicles. Although the in vitro results may not fully reflect the physiological state, the findings are relevant and significant. Isolated oocytes will resume meiosis spontaneously when placed in hormone-free media (Pincus and Enzmann, 1937; Edwards, 1965), while follicle-enclosed oocytes remain in the dictyate stage (Tsafriri, 1978b; Lindner rt al., 1983). These findings suggest that the follicular microenvironment plays a dominant role in the physiological stability of oocytes at the dictyate stage. To clarify the role of various structural elements sustaining meiotic arrest, it has been found that oocytes in contact with granulosa cells remain arrested in the dictyate stage (Foote and Thibault, 1969; Sat0 rt al., 1982). Also follicular fluid and extracts of granulosa cells suppressed the occurrence of spontaneous maturation (Tsafriri and Channing, 1975a,b; Tsafriri et al., 1976, 1977; Tsafriri, 1978a; Hillensjo et al., 1978; Stone et al., 1978; Channing et ul., 1983; Eppig and Downs, 1984; Downs r t ul., 1985). These preliminary studies indicate that granulosa cells produce factors that sustain meiotic arrest. The meiotic-arresting factors to be discussed are cyclic nucleotides, cyclic nucleotide-potentiating factor, and maturation inhibitory peptides or meiosis-arresting peptides. A. CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE (CAMP) There are substantial number of reports supporting the hypothesis that cAMP maintains meiotic arrest in oocytes. This contention is based on the finding that the derivatized CAMP,dibutyryl cAMP (db CAMP),blocks the spontaneous resumption of meiosis of isolated cumulus-enclosed and cumulus-free oocytes cultured in vitro (Cho et al., 1974; Magnusson and Hillensjo, 1977; Dekel and Beers, 1978, 1980; Nekola and Smith, 1975; Ahren et al., 1978). Also activators of adenylate cyclase and inhibitors of phosphodiesterase elevate intraoocyte cAMP and prevent GVBD (Nekola and Smith, 1975; Hillensjo, 1977; Hillensjo et al., 1978; Dekel and Beers, 1978; Ekholm rt al., 1984; Hubbard and Terranova, 1982; Dekel rt ul., 1984; Powers and Paleos, 1982; Olsiewski and Beers, 1983; Sat0 and Koide, 1984a). Unmodified cAMP added to the suspending medium of oocytes did not influence the occurrence of GVBD. The finding that unmodified cAMP does not influence GVBD while the derivatized cAMP is an effective inhibitor is attributed to the low uptake of the unmodified cAMP by the oocytes, its low plasma membrane permeability, instability, and rapid metabolism (Hillensjo et al., 1978). To render credence to the hypothesis that cAMP is the regulator of
4
ElMEl SAT0 A N D S. S. KOIDE
meiotic arrest, the level of this nucleotide in oocytes during the resting stage and following resumption of meiosis was determined. The cAMP content of resting oocytes was estimated to be 6.3 5 0.7 fmoVoocyte (Moor and Heslop, 1981). Treatment with gonadotropin to induce maturation did not affect the cAMP level of oocytes. The addition of 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase activity, blocked the occurrence of spontaneous GVBD and induced a rise in the intracellular cAMP level of cumulus-free oocytes (Vivarelli et al., 1983). In further support of this hypothesis, Schultz et al. (1983a,b) found that the level of oocyte cAMP decreased significantly during the period when the oocyte resumed meiosis. This fall in cAMP can be inhibited with IBMX at the same time preventing the occurrence of GVBD. This decrease in the oocyte’s cAMP level precedes GVBD and occurs concomitantly with a paradoxical rise in cAMP of the follicular fluid and cumulus cells. These findings suggest that the resumption of meiosis in mammalian oocytes is triggered by a fall in oocyte cAMP level, similar to that observed with amphibian oocytes (Masui and Clarke, 1979). In the Xenopus oocyte progesterone probably acts by inhibiting adenylate cyclase (Sadler and Maller, 1985). Cholera toxin, an activator of adenylate cyclase, inhibited spontaneous GVBD of cumulus-enclosed oocytes but not of cumulus-free oocytes (Dekel and Beers, 1980). The inability of cholera toxin to block GVBD of denuded oocytes is not clear in view of the fact that the oocytes are able to synthesize cAMP and that zona-free oocytes possess adenylate cyclase activity (Schultz et al., 1983a.b; Urner et al., 1983; Sat0 and Koide, 1984a; Bornslaeger and Schultz, 1985). The lack of response of denuded oocytes may be due to the lag period between the time of toxin exposure and the increase in cAMP level (Moss and Vaughan, 1979). The occurrence of a lag period before the resumption of meiosis is further supported by the observation that the fall in cAMP takes place earlier with denuded oocytes compared to cumulus-enclosed oocytes (Dekel and Beers, 1980). We have demonstrated that forskolin, an activator of adenylate cyclase, blocked GVBD of cumulus-free oocytes (Sato and Koide, 1984a). These findings using cholera toxin and forskolin suggest that oocytes do possess adenylate cyclase that lacks the stimulatory GTP-binding regulatory subunit. In this case the enzyme will not be affected by cholera toxin since adenosine diphosphoribosylation of the regulatory subunit will not take place (Gill, 1982). During the initial period of oocyte maturation, protein synthesis takes place leading to GVBD. Richter and McGaughey (1981) reported that the oocytes synthesized stage-specific polypeptides during meiotic maturation and that db cAMP blocked the synthesis of some of these polypeptides.
5
A R R E S T A N D RESUMPTION OF MEIOSIS IN OOCYTES
The relationship of cAMP to protein synthesis is not clear. There are reports indicating that db cAMP did not alter the rate of protein synthesis nor the spectrum of proteins synthesized (Stern and Wassarman, 1974). Nonetheless the clearest evidence indicates that cAMP is involved in meiotic arrest. Determination of the level of cAMP in oocyte at the dictyate stage is a critical factor to test the validity of this hypothesis.
B. CAMP-POTENTIATING FACTORS lntraoocyte level of cAMP may be the physiological factor sustaining meiotic arrest. In addition follicular fluid contains a CAMP-potentiating factor(s) (Eppig et d . , 1983; Eppig and Downs, 1984; Freter and Schultz, 1984; Downs et al., 1985; Racowsky, 1983; Sat0 et ul., 1985). A factor was identified in porcine follicular fluid that blocks mouse oocyte maturation in vitro when combined with cAMP (Eppig and Downs, 1984). The substance was identified to be hypoxanthine (Fig. I ) (Downs et d . , 1985). Based on these findings it was proposed that the active factor is produced by a CAMP-dependent process in the granulosa-cumulus cells. The factor is transported to the oocyte through cytoplasmic channels that couple the cumulus cells to the oocyte. Alternatively, an inactive factor is taken up directly by the oocyte and activated by a CAMP-dependent process. The biochemical steps involved in the activation of the factor and the mech-
Hypoxant hine
Cyclic adenosine 3: 5cpyrophosphate ( C A P P I
0
II
HO-
p- 0- CHz
I 0
I HO-
P
H ’
H d
-0
H OH
II
0
FIG.I .
Diagram of follicular substances that sustain meiotic arrest in mammalian oocytes.
6
ElMEI SAT0 AND S. S. KOIDE
anism of its action are not clear. The presumptive control of oocyte maturation is that there is a decrease in the levels of both the follicular fluid factor and CAMP. A reduction of these factors may result from a fall in their production by the cumulus cells or alternatively a block in their transport from the cumulus cells to the oocyte. These events will trigger the resumption of meiosis. We have recently purified a factor from bovine follicular fluid that inhibits mouse oocyte maturation in combination with cAMP (Sato et ul., 1985). The factor was purified by extraction with 70% ethanol, chromatography on a Dowex 1-X8 column, and reversed-phase high-performance liquid chromatography. The physicochemical properties of the follicular fluid substance are similar to that of cyclic adenosine 3’3’-pyrophosphate (cAPP) (Fig. 1). Both the follicular fluid factor and cAPP in combination with db cAMP blocked mouse oocyte maturation in combination with cAMP and inhibited protein kinase activity. 17P-Estradiol inhibits maturation of denuded porcine oocytes (McGaughey, 1977). This steroid is effective when used in a chemically defined medium containing bovine serum albumin (BSA) or dextran (Richter and McGaughey, 1979), but not in a BSA-free medium (Racowsky and McGaughey, 1982). The inhibition is reversible (Richter and McGaughey, 1979). Testosterone can potentiate the maturation-arresting activity of db cAMP (Richter and McGaughey, 1981; Racowsky, 1983). Androgens, however, may modulate CAMP-induced meiotic arrest in vitro by being converted enzymatically to 17P-estradiol via the aromatase system (Racowsky, 1983). This conversion to 17P-estradiol as the mediator of the meiotic arrest is supported by the observations that follicle-stimulating hormone (FSH) and cAMP stimulate aromatase activity (Lacroix et ul., 1974; Moon et al., 1975; Armstrong et al., 1979; Lindsey and Channing, 1979; Anderson et al., 1979).
C. OOCYTEMATURATION INHIBITOR (OMI) Inhibition of spontaneous maturation of isolated rabbit oocytes by follicular fluid was first described by Chang (1955). Tsafriri and Channing (1975a,b) demonstrated a similar factor in porcine follicular fluid designated as oocyte maturation inhibitor (OMI). Other investigators claim that follicular fluid does not influence oocyte maturation (Liebfried and First, 1980a,b; Racowsky and McGaughey, 1982; Fleming et al., 1983). This controversy has not been resolved. An explanation was offered by Channing et al. (1982a) for the apparent contradictory results. They suggested
ARREST AND RESUMPTION OF MEIOSIS IN OOCYTES
7
that follicular fluid contains an inhibitory factor and a maturation-inducing factor, and varying content of these two factors can account for the different results reported. The inhibitor and inducer were separated by chromatography on CM-Sephadex column (Channing et al., 1982a). The inducer has not been characterized. Several factors with maturation inhibitory activity are present in the follicular fluid. OM1 is a peptide with an estimated molecular weight of 2000 (Tsafriri et al., 1976; Stone et al., 1978). A similar factor was extracted from granulosa cells (Centola et al., 198I). The granulosa cell factor when added to the culture medium prevented oocyte maturation (Tsafriri, 1978b). suggesting that granulosa cells produced OMI. Also it was found that an extract prepared from granulosa cells of small follicles was more potent than those obtained from large follicles, indicating that its content decreases as the follicles mature, paralleling the physiological state of the follicles (Channing et al., 1982a; Tsafriri et al., 1982a,b; Tsafriri and BarAmi, 1982). Another oocyte maturation inhibitory factor was discovered in follicular fluid. Its properties differ from that of OM1 (Chari et al., 1983). It apparently potentiates the inhibitory potency of CAMP (Eppig and Downs, 1984) and is identified as hypoxanthine (Downs et al., 1985). A third inhibitory factor is an immunoreactive prolactin-like substance that cross-reacts with anti-prolactin antiserum (Baker and Hunter, 1978; Channing et al., 1982a). When anti-prolactin antiserum is added to the medium containing follicle-enclosed porcine oocytes, maturation is accelerated (Baker and Hunter, 1978), suggesting that prolactin might induce oocyte maturation. It has been further suggested that this hormone may act indirectly on the oocyte by stimulating the granulosa cells to synthesize OM1 (Channing et al., 1982a,b). The production of OM1 is blocked by testosterone and dihydrotestosterone (Channing et al., 1982a). Since androgens or estrogens fail to influence oocyte maturation directly, follicular androgens probably act by decreasing OM1 production by the granulosa cells (Channing et al., 1982a). OM1 acts on cumulus cells instead of directly on the oocytes since denuded oocytes will undergo spontaneous maturation in the presence of OM1 (Hillensjo et al., 1979). There are multiple effects attributed to OM1 on the cumulus cells. It inhibits the spontaneous maturation of cumulus-enclosed porcine oocytes, prevents morphological differentiation of cumulus cells, and blocks progesterone secretion by cumulus cells (Schaerf et d., 1982). These data suggest that cumulus cells take up OM1 and transport it to the oocyte where it can sustain meiotic arrest. An alternative possibility is that OM1 promotes the production of yet another inhibitor, e.g., CAMP. It may also act by preventing the formation of an oocyte maturation inducer.
8
EIMEI S A T 0 AND S. S. KOIDE
D. GRANULOSA CELLFACTOR(GCF) When isolated porcine oocytes are in contact with porcine granulosa cells, maturation is inhibited (Foote and Thibault, 1969; Sat0 et nl., 1977). Isolated oocytes will remain in the dictyate stage when juxtaposed to a layer of granulosa cells. The oocytes will undergo maturation when detached from these cells. Granulosa cells obtained from small follicles were more potent in preventing the spontaneous maturation of isolated oocytes than cells from Graafian follicles (Tsafri and Channing, 1975a), suggesting that GCF is the active agent within the follicles. Other investigators claim that granulosa cells did not affect the occurrence of spontaneous maturation of isolated oocytes (Liebfried and First, 1980a,b). Nonetheless they found that segments of follicular wall attached to the oocytes can prevent the occurrence of GVBD. The addition of LH to hemisectioned follicles induced resumption of meiosis of the oocytes. The inhibitory potency of granulosa cells can be demonstrated providing the cells are in contact with each other (Sato et al., 1977, 1980, 1982, 1984b, 1986). The mere coculturing of oocytes with a granulosa cell layer (about lo7 cells) obtained from medium-sized (2-5 mm) follicles did not prevent the occurrence of spontaneous GVBD. Inhibition was observed only when the oocytes were in direct contact with the granulosa cells. Maturation block can be induced with cumulus-enclosed oocytes by having a portion of the granulosa cell layer in contact with the cumulus cells, whereas to induce maturation block of denuded oocytes the entire surface has to be enclosed by the granulosa cells (Sato et al., 1982), indicating that meiotic arrest is dependent upon cell-to-cell communication between the cumulus-oocyte complex and the granulosa cells. These findings further suggest that the inhibitory factor is located on the surface of the granulosa cells or may be a component of the extracellular matrix of the granulosa cell layer. The inhibitory factor can be extracted from the surface of granulosa cells with a buffer containing I M urea and 5 mM ethylenediaminetetraacetate (EDTA) (Sato and Koide, 1984b; Sat0 et al., 1986). This buffer was used to dissociate sea urchin embryo (Kondo and Sakai, 1971) to extract surface components from cultured fibroblasts (Igarashi and Yaoi, 1975) and sperm-aggregating factor from Spisula oocytes (Sato et al., 1983). Cells treated with the urea-EDTA solution recover without any deleterious effect. The maturation inhibitory factor was extracted from bovine granulosa cells with a buffer containing 1 M urea and 5 mM EDTA and purified by gel filtration on Sephadex G-25. Two protein peaks were obtained (Fig. 2). The material in the second (minor) peak at a concentration of 400 pg (dry weight)/ml of culture medium completely prevented spontaneous maturation of isolated mouse oocytes. At a lower concentration (50 pg/ml),
ARREST AND RESUMPTION OF MEIOSIS IN OOCYTES
9
O.81
Fraction no.
FIG.2. Gel filtration of peptides extracted from bovine granulosa cells on Sephadex (3-25 column. Column size: 1.5 x 75 cm. Arrows indicate position of reference markers: insulin. 6 kDa: bradykinin, 1.2 kDa. Fraction I (tube numbers 24-32) and fraction 2 (tube numbers 33-49) were pooled. Fraction 2 possessed oocyte maturation-preventing activity. Effective concentration was 400 pg/ml.
it blocked GVBD by 58% (Fig. 3). Peak I (major) possessed slight inhibitory activity. Inhibition was 58 and 6% at concentrations of 4000 and 500 pg/ml, respectively. The factor in peak 2, designated as granulosa cell factor (GCF), was further purified by affinity chromatography on Con ASepharose 4B column. The unabsorbed fraction contained the maturationpreventing activity showing that the factor is probably devoid of sugar moieties. The inhibitory effect of GCF was found to be reversible at lower concentrations. At a high concentration (400 pg/ml), the oocytes remain in meiotic arrest even after washing and transfer to the control medium. At a concentration of 200 pg/ml, approximately 10% of the oocytes have undergone GVBD. The remainder of the oocytes resumes meiosis after washing. GCF at a concentration of 50 pg/ml permits GVBD in 18% of the oocytes after 2 hours of incubation which increased gradually to 35% by the end of 6 hours. During this period, 86% of control oocytes have undergone GVBD. When oocytes are cultured in medium containing GCF for 3 hours and transferred to control medium, 76% of oocytes undergo GVBD compared to 35% of unwashed oocytes. The possibility of contaminating EDTA or urea to account for the inhibitory effect was excluded by experimental design (Sato et ul., 1984b, 1986). GCF is a peptide since it is destroyed by Pronase but not by DNase, RNase, or glycosidase. Its estimated molecular weight has been determined to be less than 6000 by gel filtration on Sephadex G-25. Thus, GCF and OM1 are related compounds possessing common properties.
10
EIMEl SAT0 A N D S. S. KOIDE 100,
’0°1
B
,
100
50 (3
1 2 3 4 5 6 Incubation time (hrs)
FIG.3. Effect of bovine granulosa cell factor (GCF) on the time course of spontaneous GVBD of isolated mouse oocytes. Fraction purified by gel filtration on Sephadex (3-25 was Control medium; -0, GCF in the medium throughout the experiment: used. -, 0-0, GCF in medium for 3 hours, oocytes washed three times, and resuspended in control medium. Concentrations tested were 400 p,g of GCF/ml of medium (A), 200 pg/ml (B). 50 p,g/ml (C). Values are mean 2 SD ( n = 5 ) .
E. CALCIUM Plasma membranes of many cells are usually impermeable to Ca’+. When the cells are stimulated or activated they become sensitive to exogenous Ca” . These findings suggest that the mechanism of activation of mammalian oocytes and other cells may involve influx of exogenous calcium. External Ca2+is essential in maintaining mouse oocytes viable in the culture medium (Paleos and Powers, 1981; De Felici and Siracusa, 1982). Small meiotically incompetent oocytes and early embryos do not require exogenous Ca” in the medium for survival, indicating that the
AKKESI‘ AND RESUMPTION OF MEIOSIS IN OOCYTES
II
Ca” requirement is restricted to specific stages in the growth and development of oocytes (De Felici and Siracusa, 1982). Various hypotheses have been proposed to account for the Ca” requirement of the dictyate oocytes. One possibility is that there is an activation of the membrane calcium pumps and internal calcium buffering systems in the oocytes upon release from the ovary. In the absence of extracellular calcium, the intraoocyte calcium level will fall below that required to sustain metabolic activities. Another reason is that calcium may be necessary for the repair of membrane injury sustained during the mechanical release of the oocytes from the follicles (Okamoto et al., 1977). The effect of calcium was studied by using the calcium ionophore A21 387. Although the ionophore does not influence the spontaneous maturation of isolated rat oocytes, it can induce GVBD in follicle-enclosed rat oocytes (Tsafriri and Bar-Ami, 1978), suggesting that calcium may trigger the resumption of meiosis. This thesis is supported by the report that the total calcium concentration of cumulus-enclosed rat oocytes increases paralleling the serum LH level (Batta and Knudsen, 1980). The rise in oocyte calcium and serum LH levels occurs during the time maturation is initiated. Unfortunately the oocyte calcium level was not determined with the onset of GVBD. Although lowering calcium or magnesium content of the medium appears not to influence the resumption of meiosis of isolated bovine oocytes with adherent cumulus cells, oocyte maturation was blocked when cultured in calcium- and magnesium-free medium (Liebfried and First, 1979). We have demonstrated that there is a dramatic decrease in the incidence of GVBD of oocytes incubated in a Ca”- and Mg”-free medium (Sato er al., 1980). This finding supports the thesis that calcium and magnesium ions are essential ingredients for the occurrence of GVBD. It is interesting that db CAMP-induced meiotic arrest in mouse oocytes can be overcomed by elevating the extracellular calcium level: although at a higher concentration of db cAMP (0.2 mM), the block cannot be reversed with calcium (Paleos and Powers, 1981). Moreover, the ionophore is able to induced GVBD in oocytes treated with db cAMP (0.1 mM) (Powers and Paleos, 1982).These findings suggest that calcium and cAMP may regulate oocyte maturation by influencing a common mechanism. The proposed mechanism is based on the premise that the intracellular reservoir of calcium is sufficient to promote spontaneous GVBD in virro. The addition of db cAMP to the medium may create a need for exogenous calcium (Powers and Paleos, 1982). It is postulated that db cAMP reduces cytoplasmic Ca’+ level by stimulating the calcium pumps of the membrane (Berridge, 1975) as found in other cell systems. To elucidate the role of intracellular Ca2+in the resumption of meiosis of oocytes, further technical
12
ElMEI SAT0 AND S . S . KOlDE
advancement in the method of measuring intracellular Ca” translocation needs to be developed. It is clear that the metabolism and action of calcium ions in eukaryotic cells are regulated by calmodulin. This protein and Caz+are involved in cyclic nucleotide metabolism, protein phosphorylation, microtubule assembly, and calcium flux. To demonstrate the participation of calmodulin in the resumption of meiosis in mouse oocytes, the effect of calmodulin antagonists on GVBD in isolated mouse oocytes was examined. W7 [ N ( 16-aminohexyl)-5-chloroI -naphthalene-sulfonamidehydrochloride] at concentration of 5 x lo-’ M or greater inhibited GVBD (E. Sato and S. S. Koide, unpublished data). The block exerted by W7 is partially reversible. W5 [N-(6-aminohexyl)-1 -naphthalene-sulfonamide hydrochloride], a calmodulin antagonist with less specificity than W7, did not inhibit maturation of isolated oocytes at a concentration of I x M. The meiotic block in cumulus-free and cumulus-enclosed oocytes induced with W7 was not reversed by estrogen, progesterone, or a combination of estrogen and progesterone. These findings suggest that calmodulin may be involved in the resumption of meiosis. Sperm, ethanol, and phorbol ester activate cellular processes associated with a sustained oscillation of [Caz’Ii in mouse oocytes (Cuthbertson and Cobbold, 1985). Although the [Ca”li responses are significantly different with each inducer, activation of the mouse eggs resulted. We hypothesized that oocyte membranes contain a meiotic-arresting component (Sato et al., 1984a). When oocytes are recovered from the ovaries of Spisula, they are arrested in the dictyate stage and possess a large germinal vesicle. Oocyte maturation is signaled by the dissolution of the germinal vesicle which can be induced in Spisula oocytes by sperm, KCI, or serotonin (Allen, 1953; Hirai et al., 1984). Our results show that trypsin induces GVBD of Spisula oocytes only in the presence of Ca” . The maturation-promoting activity of this protease can be blocked by a membrane component(s) (Sato et al., 1984a). These observations suggest that the oocyte membrane component may sustain meiotic arrest and that the hydrolysis of this component may be an early step in the induction of oocyte maturation in Spisula. Since the induction of GVBD with trypsin is dependent upon Ca”, the membrane component may sustain meiotic arrest by preventing Ca” influx. This point needs to be clarified. Proteolytic enzymes may mediate oocyte maturation induced by sperm and may account for the subsequent events associated with fertilization. The proposed hypothesis is as follows: upon sperm-egg interaction, proteolytic enzymes are liberated, hydrolyzing the meiotic-arresting component of the membrane, promoting calcium influx, and triggering the resumption
A R R E S T A N D R E S U M P T I O N OF M E I O S I S I N O O C Y T E S
13
of meiosis. The existence of the meiotic-arresting factor in mammalian oocyte membranes has to be verified.
111. Factors Inducing Resumption of Meiosis
A. GONADOTROPINS LH is considered to be the physiological agent that induces maturation of mammalian oocytes. The hormone acts on follicular cells and not directly on the oocyte. This thesis is based o n the finding that oocytes undergo GVBD when whole ovaries or isolated follicles in organ culture are treated with LH or HCG (human chorionic gonadotropin) (Baker and Neal, 1972; Lindner et a/., 1974; Tsafriri er al., 1972). However, FSH is equally effective and its action is not due to contaminating LH (Lindner et a / . , 1974; Neal and Baker, 1975). The minimal effective dose of FSH required to induce resumption of meiosis in follicle-enclosed oocyte is lower than that of LH (Neal and Baker, 1975). Furthermore, anti-LH antiserum raised against the p-subunit of LH abolishes LH action on the follicular cells, but not that of FSH (Lindner er d.,1974). The capability of LH and FSH to induce oocyte maturation varies with the species. With isolated follicles obtained from swine (Baker et al., 1975) or women (Baker and Neal, 1974),neither LH nor FSH was effective, while both hormones induce maturation of sheep follicle-enclosed oocytes in organ culture. The resulting eggs can be fertilized and develop into viable young embryos when transplanted into suitable recipients (Moor and Trounson. 1977; Staigmiller and Moor, 1984). Cultures of follicle-enclosed oocytes of rat (Lindner rt a / . , 1974), mouse (Baker and Neal, 1972), rabbit (Thibault et d.,1975). and sheep (Hay and Moor, 1975) were used to elucidate the mechanism of LH induction. An early action of LH on follicles is the stimulation of adenylate cyclase activity. Stimulation of the cyclase will increase intracellular cAMP level (Tsafriri et a / . , 1972; Marsh et al., 1973; Nilsson el a / . , 1974) which undoubtedly plays an important role in the resumption of meiosis (Marsh, 1976; Tsafriri et al., 1972). cAMP and its derivatives may promote meiosis under certain conditions. For example, elevating follicular cAMP levels by microinjection of this nucleotide into the follicle (Tsafnn et a / . , 1972) or preincubation of follicles with db cAMP (Hillensjo et al., 1978) promotes resumption of meiosis. The involvement of cAMP in oocyte maturation is further supported by the findings that cAMP levels in isolated follicles are elevated within 5 minutes after exposure to forskolin. reaching a plateau at 15 minutes (Dekel
14
EIMEI SAT0 AND S. S. KOIDE
and Scherizly, 1983). Forskolin mimics LH by stimulating cAMP production in rat ovarian follicles and inducing GVBD in follicle-enclosed oocytes. Derivatized cAMP or cyclic nucleotide phosphodiesterase inhibitors blocks LH-induced maturation of follicle-enclosed oocytes (Hillensjo et al., 1979; Dekel et al., 1981). LH failed to induce GVBD in follicleenclosed rat oocytes exposed to db cAMP (Tsafriri et al., 1972; Hillensjo et al., 1978). Moreover, db cAMP and related compounds in vitro prevent the occurrence of spontaneous GVBD of isolated oocytes. Thus elevating cAMP level in follicular cells will promote oocyte maturation, while cAMP acting directly on the oocyte prevents GVBD. To explain this paradoxical action of cAMP on oocyte maturation, it is hypothesized that, at the time when cumulus-oocyte complexes are isolated from follicles, cAMP level is sufficient to sustain meiotic arrest. Because oocytes contain an active phosphodiesterase, the intraoocyte cAMP level falls rapidly, triggering oocyte maturation. It is proposed that the oocyte phosphodiesterase is activated following the isolation procedure whereby the oocyte is removed from the action of an inhibitor of the enzyme present in the follicular fluid. LH-induced oocyte maturation is dependent on the stimulation of adenylate cyclase by the hormone and to the subsequent rise in cAMP levels within the follicular cells. The apparent contrasting findings to reconcile is the high cAMP level in granulosa cells accompanied by a fall of cAMP level in the oocyte (Channing and Tsafriri, 1977). A postulated mechanism is that follicular cells are interlinked by gap junctions. As a consequence of the rise in the cAMP level in the granulosa cells, the cell-to-cell communication system is disrupted and the transport of metabolites from the cumulus cells to the oocyte is blocked. It has been proposed that LH in some indeterminate manner terminates cell-to-cell communication in the cumulus-oocyte complex and that nucleotide transport to the oocyte ceases promoting the resumption of meiosis (Dekel and Kracier, 1978; Dekel ef al., 1981, 1984). This hypothesis that the uncoupling of the oocyte and cumulus cells is a prerequisite for the resumption of meiosis has been questioned by the findings that oocytes and cumulus cells remain interlinked to one another even after the resumption of meiosis (Moor et ul., 1980; Eppig, 1982) and that transport of CAMP from the cumulus cells to the oocyte may not take place under physiological condition. An alternative hypothesis is that LH may prevent the action of a maturation-arresting factor or a CAMP-potentiating factor produced by the granulosa cells on the oocyte. This hypothesis is supported by the findings that gonadotropins stimulate the production of the extracellular matrix and promote the accumulation of glycosaminoglycans in the interstitial spaces between the cumulus cells. The glycosaminoglycan components can interact with the granulosa cell factor and neutralize its meiotic-
A R R E S T A N D RESUMPTION OF MEIOSIS IN OOCYTES
15
arresting activity, thereby promoting resumption of meiosis (Sato et ul., 1984b, 1986). Steroids have been implicated as regulators of oocyte maturation. After the LH surge, the concentrations of estradiol in follicular fluid decline, initially followed by a marked rise in the synthesis of progesterone (Thibault, 1977; Eiler and Nalbandov, 1977; Dorrington and Armstrong, 1980). The relative concentrations of progesterone to estradiol in the follicular fluid is elevated at the onset of oocyte maturation (Thibault, 1977; Gerard et a / . , 1979). Numerous steroids were added to the culture medium of isolated oocytes to determine their ability to influence oocyte maturation. Conflicting results were obtained (McGaughey, 1983). Bae and Foote (1975) demonstrated that progesterone added to the culture medium stimulated maturation of bovine and rabbit oocytes. Other investigators were unable to demonstrate any effect of progesterone on the maturation of isolated porcine oocytes with or without adherant cumulus cells in vitro (Richter and McGaughey, 1979). The LH induction of oocyte maturation in cultured Graafian follicles obtained from rat ovaries was not impaired when hormone-induced steroidogenesis was completely suppressed with 17P-ol-3-one)or cyanoketone (2a-cyano-4,4,17a-trirnethylandrost-5-enaminoglutethimide (Tsafriri et al., 1972). Also GVBD was not induced in cultured rat follicles treated with progesterone, 20a-dihydroprogesterone, or 17P-estradiol. None of the steroids tested inhibited LH-induced GVBD. Thus they concluded that the resumption of meiosis induced by LH is not dependent upon its ability to influence the rate and pattern of follicular steroidogenesis (Lieberman et al., 1976). B. GONADOTROPIN-RELEASING HORMONE (GnRH) The principal function of GnRH is to promote LH and FSH release from the pituitary gland. An additional response attributed to GnRH agonist is to mimic LH in hypophysectomized rats by inducing resumption of meiosis and dispersion and mucification of cumulus cells (Ekholm et a / . , 19811. GnRH and its agonists stimulate maturation of follicle-enclosed oocytes in an in vitro culture system in a dose-dependent manner (Hillensjo and LeMaire, 1980). However, these hormones did not influence the spontaneous GVBD of isolated oocytes (Anderson and Hillensjo, 1982). Also the GnRH antagonist effectively abolished the stimulating effect of a GnRH agonist to induce GVBD of oocytes in isolated preovulatory rat follicles and yet did not influence the inducing action of LH. These findings suggest that GnRH acts via the follicular cells and does not directly affect the oocytes, and its action appears to be independent of LH effect on meiosis.
16
EIMEl SAT0 A N D S. S. KOlDE
c. REGULATORSOF MEIOTICCOMPETENCE As oocytes develop they mature and acquire the ability to resume meiosis beyond the dictyate stage. This property is acquired during specific stages of development. Oocytes recovered from mice younger than 15 days postpartum are unable to undergo GVBD in vitro (Szybek, 1972). Similarly the proportion of oocytes recovered from prepubertal rabbits (70-90 days of age) that had matured to the second metaphase in vitro was lower than adult rabbits (Thibault, 1977). We have found that about 50% of the oocytes recovered from prepubertal swine undergoes spontaneous GVBD (Sato et al., 1977), indicating that the capacity to resume meiosis is acquired during the late stages of oocyte growth and development. Although the exact developmental age of the animal when oocyte competence is acquired has not been determined, it probably corresponds to the time period when oocytes attain full growth. One of the criteria used is the size of the oocytes. Incompetent oocytes recovered from small follicles were significantly smaller than competent oocytes recovered from larger antral follicles, indicating that there is a correlation between maturation competence and fertilizability (Sorensen and Wassarman, 1976). However, the average diameter of competent rat oocytes explanted on day 20 was 61.8 & 1.2 pm compared to 76.5 ? 0.8 pm for incompetent oocytes (Bar-Ami and Tsafriri, 1981), suggesting a lack of correlation between oocyte size and competence. The acquisition of competence occurs at a defined period of growth and development when some essential components are being produced. Immaturity may result from a deficiency of specific components required for maturation, for example, in the assembly of spindle proteins as found in immature amphibian oocytes (Brachet, 1977). One of the essential components may be RNA (Iwamatsu and Yanagimachi, 1975). RNA synthesis increases gradually during growth and development in mouse oocytes (Moore et d.,1974) and decreases when they reach their maximal size at the Graafian follicle stage shortly before ovulation. Investigations to determine the precise period when the developing oocytes attain competence to resume meiosis have been carried out. In the mouse, oocytes acquire the capacity to resume meiosis shortly before the appearance of the antrum in the primary follicle (Pincus and Enzmann, 1937). This belief is based on the findings that oocytes attain their maximal size at this time, and it is the earliest stage when meiotic maturation figures can be identified in oocytes of atretic follicles (Engle, 1927).The strongest support of this thesis is the finding that 83-91% of the oocytes removed from antral follicles (300-600 pm in diameter) progressed to the first or
ARREST AND RESUMPTION OF MEIOSIS IN OOCYTES
17
second metaphase, whereas 98% of those isolated from preantral follicles failed to undergo spontaneous maturation (Erickson and Sorensen, 1974). There is species variability in the potential to undergo maturation. In swine and cattle, 90-100% of the oocytes isolated from Graafian follicles (1015-mm diameter) undergoes GVBD, while 50% of the oocytes removed from small follicles (2-5 mm in diameter) resumes meiosis. This indicates that the potential to undergo maturation is acquired during the maturation of follicles (Sato et al.. 1977). The acquisition of meiotic competence, i.e., the ability to resume meiosis, may be influenced by hormones that regulate follicular development. This thesis is based on the findings that oocytes from hypophysectomized rats, performed on 15 days postpartum, fail to undergo maturation (Bar-Ami rt ul., 1983). The ability of the oocytes to resume meiosis can be reversed on administering FSH to the hypophysectomized rats, but not with LH. Furthermore, 17P-estradiol administered as an implant to hypophysectomized rats partially restored meiotic competence to the oocytes within 24 hours, while progesterone and androstenedione were not effective. Also coadministration of inhibitors of steroidogenesis with FSH to the hypophysectomized rats did not restore meiotic competence to the oocytes. These findings suggest that FSH is involved in the induction of meiotic competence and that its action is partially mediated by estrogens produced within the follicles. D.
MATURATION PROMOTORS in
Vitro VERSUS in v i v o
The observed spontaneous maturation of isolated oocytes in vitro may or may not reflect the natural occurrence of maturation under in vivo conditions (Motlik and Fulka, 1976).The changes associated with maturation appear earlier in isolated oocytes cultured in vifro compared to those induced to mature within the follicles after HCG injection (Thibault, 1977). The observed time lag in vivo corresponds to the duration when HCG released from the injection site reaches a critical level to initiate its action. In the swine, this time lag is quite prolonged (Motlik and Fulka, 1976; Sat0 et al.. 1978a,b). Nonetheless, the rate of in vitro maturation is affected by the culture conditions and hormones added to the media (Sato et d., 1978a,b). For example, the pH of the culture medium influences the maturation time of porcine oocytes. At pH 6.8-7.0, the time required to progress to the second metaphase was delayed by several hours, compared to pH 7.2-7.4. The delay occurs at the early stage of the process leading to GVBD, while the time from the first to the second metaphase was not appreciably changed.
18
ElMEl S A T 0 A N D S. S. KOIDE
Denuded and cumulus-enclosed oocytes resume meiosis spontaneously at the same rate when cultured in vitro. FSH added to the medium caused a delay in the occurrence of meiotic resumption of cumulus-enclosed oocytes, but not with denuded oocytes. The temporal sequence of events with cumulus-enclosed oocytes paralleled the process of oocyte maturation in vivo (Salustri and Siracusa, 1983). LH added to the culture medium of rat oocytes accelerated the progress of GVBD (Lopata et al., 1977; Kaplan et al., 1978). The period of dissolution of the nuclear membrane ranged from 105 to 130 minutes after isolation determined by cinemicrography. This period is shortened to 45-80 minutes after the addition of LH to the medium (Lopata et al., 1977).
E. MATURATION-PROMOTING FACTOR(MPF) The formation of MPF during maturation of mammalian, amphibian, and invertebrate oocytes has been well documented (Masui and Clarke, 1979). Hybrid cells formed by fusing dictyate-arrested oocytes obtained from sexually immature mice and maturing oocytes with Sendai virus will undergo germinal vesicle dissolution and proceed to the first metaphase (Balakier, 1978). Using a similar method, fused porcine and rabbit oocytes were prepared. The presence of MPF in the maturing oocytes was demonstrated (Fulka, 1983). It should be pointed out that the capability to produce MPF is acquired on reaching maturity because small oocytes from sexually immature mice do not undergo GVBD in vitro (Szybek, 1972; Sorensen and Wassarman, 1976). The direct demonstration of MPF production in mouse oocytes undergoing spontaneous maturation was carried out by Kishimoto et al. (1984), who showed that the cytoplasm from maturing mouse oocytes induced GVBD when microinjected into starfish oocytes. MPF in the Xenopus oocyte cell-free system induces phosphorylation of lamins A and C followed by a gradual depolymerization of the nuclear lamina and finally to the dissolution of the nuclear envelope (Miake-Lye and Kirschner, 1985). It should be pointed out that MPF from oocytes (meiosis) and somatic cells (mitosis) is interchangeable and is active over the broad evolutionary range of species (Sunkara et al., 1979; Nelkin et al., 1980; Kishimoto et al., 1982, 1984; Miake-Lye and Kirschner, 1985). The oncogene product, ras protein, microinjected into Xenopus oocyte, induces GVBD (Birchmeier et a/., 1985), simulating MPF action, while the protooncogene product of ras was less effective. It is noteworthy that the intraoocyte CAMP level did not change and yet cholera toxin blocked ras action. To clarify how MPF and ras protein induce GVBD is a fertile field of study.
ARREST A N D RESUMPTION OF MEIOSIS IN OOCYTES
19
IV. Mechanism of Meiotic Resumption A. PERMEABILITY OF CUMULUS-OOCYTE COMPLEXES The major cellular elements within the follicles are the granulosa cells. The granulosa cells are heterogeneous, showing structural variation and differences in hormone binding. They are found in association with a variety of tissue structures and cellular components, i.e., juxtaposed with the follicular wall, interconnected to other granulosa cells, and immersed in antral fluid (Amsterdam et al., 1976; Gilula et al., 1978). The innermost layer of the cumulus, the corona radiata, is composed of granulosa cells surrounding the oocyte. These cells possess cytoplasmic processes that pierce through the intervening zona pellucida and are in contact with the oolemma (Bjorkman, 1962; Odor, 1960; Zamboni, 1974). There are gap junctions in the regions of contact between the cumulus cells and the oocyte (Amsterdam et al., 1976; Lindner ef al., 1977; Anderson and Albertini, 1976).Gap junctions have been implicated as structural pathways for cellto-cell communication (Gilula et al., 1972). Through these junctions, the exchange of nutrients between the cumulus cells and the oocyte takes place during follicular development. These specialized membrane structures contain channels that permit intercellular exchange of metabolites with molecular weights less than 1000 (Flagg-Newton et al., 1979; Anderson and Albertini, 1976). The cumulus cells may serve an important function in transporting essential substances from granulosa cells to the surface of the oocytes. The entire cumulus layer is connected by gap junctions (Gilula et al., 1978). The cumulus-oocyte complex acts as a single unit in the transport of small essential nutrients. Whether or not the entire cellular components of the follicle are interconnected by gap junctions has not been verified. It is unlikely that they are interconnected, since there is a marked regional variation in the response of the granulosa cell population to gonadotropins. The involvement of the cumulus cells on oocyte maturation and in the resumption of meiosis has not been clarified. Removal of cumulus cells from hamster and rat oocytes accelerated the process of spontaneous maturation (Gwatkin and Andersen, 1976; Dekel and Beers, 1980). The maturation rate was slower in cumulus-deprived mouse oocytes (Cross and Brinster, 1970). Differences in the maturation rate was not detected by other investigators who studied cumulus-enclosed and cumulus-free mammalian oocytes (Cross, 1973; Binor and Wolf, 1979). The morphological features of the cumulus cells suggest that these cells may influence the permeability of the oocyte or the transport of essential
20
ElMEl S A T 0 AND S . S. KOlDE
nutrients to the oocyte (Heller et a / . , 1981). That is to say cumulus cells act cooperatively in the transfer of metabolites from granulosa cells or follicular fluid to the oocytes. This thesis is supported by demonstrating significantly higher uptake of radiolabeled leucine, uridine, and ribonucleosides by cumulus-enclosed oocytes than by denuded oocytes (Cross and Brinster, 1974; Wassarman and Letourneau, 1976; Heller and Schultz, 1980). Cumulus cells can facilitate the entry of tritium-labeled choline, uridine, and inositol into oocytes via the gap junctions (Moor et ul., 1980). Active transport of metabolites by the cumulus cells may take place by the following processes: the metabolities may ( I ) undergo structural changes that would facilitate their passage through the membranes, (2) increase the permeability capacity of the oolemma, (3) promote formation of direct communicatingjunctions between cumulus cells and the oocyte. The most probable cause is due to an increase in membrane permeability, since direct oocyte-cumulus cells contact is a prerequisite for the facilitated transfer of metabolites (Moor et al., 1980). FSH effectively decreases the flow of small molecules from the cumulus cells to the oocytes (Moor et a/., 1980). Steroids may be involved as mediators of gonadotropin suppression and in the maintenance of junctional competence between cumulus cells and oocytes. Several studies revealed that the degree of metabolic dependency between cumulus cells and oocytes decreased during meiotic maturation (Cross and Brinster, 1974; Moor et ul., 1980; Heller and Schultz, 19801, suggesting that the permeability of metabolites from the cumulus cells to the oocyte decreased with the resumption of meiosis. In antral follicles prior to the preovulatory stage, the oocyte is surrounded by tightly packed cumulus cells. The cumulus cells in contact with the oocyte become elongated and send fine processes radiating toward the oocyte to form the corona radiata (Eppig, 1982; Dekel and Phillips, 1979; Gilula et ul., 1978). Following the preovulatory LH surge, the follicles mature, accompanied by the disintegration of the cumulus structure, resulting from the accumulation of glycosaminoglycans in the intercellular spaces (Dekel et ul., 1979) and a decrease in the permeability of the cumulus-oocyte complexes. In the follicles with fully expanded cumulus cell masses isolated during the late pre- and postovulatory periods, the oocytes have undergone nuclear membrane dissolution. These findings indicate that the following three events are linked, i.e., alterations in the cumulus cell mass, decreased permeability of cumulus-oocyte complexes, and resumption of meiosis. It should be pointed out that the granulosa cell layer from large Graafian follicles containing the expanded cumulusoocyte complex possesses meiotic-arresting activity. This effect was
ARREST A N D RESUMPTION OF MEIOSIS IN OOCYTES
21
demonstrated by attaching oocytes to the granulosa cell layer from medium-sized follicles (Sato e t ul., 1977). This finding supports the thesis that the expanded cumulus contains substances that prevent the action of the meiotic-arresting factor or that the oocyte is committed to undergo maturation. Various components of the extracellular matrix were tested for their ability to block the action of meiosis-arresting factor. Hyaluronic acid, chondroitin sulfate, heparin, heparan sulfate, and dextran sulfate at concentrations of 500 pg/ml or less did not influence the spontaneous maturation of isolated mouse oocytes in vitro. Heparin and heparan sulfate at concentrations exceeding 100 & n l . however, blocked the inhibitory action of purified GCF (Sato et al., 1984b, 1986). GCF was purified by gel filtration on Sephadex (3-25 and by reversed-phase high-performance liquid chromatography. Heparin did not influence the maturation inhibitory activity of db CAMP, forskolin, calmodulin antagonist (W7), or IBMX (Sato ct al., 1984b, 1986). When purified GCF was applied to a heparinagartose column, the factor was eluted in the adsorbed fraction, suggesting that GCF interacts with heparin directly. This direct interaction of GCF and heparin may account for the loss of meiotic-arresting activity after incubation with heparin (Sato et al., 1984b. 1986). It should be pointed out that the predominant glycosaminoglycan in the cumulus-expanded intracellular matrix is hyaluronic acid. The physiological relevance of heparin-GCF interaction and the high content of hyaluronic acid in cumulusexpanded follicles need to be clarified. The present results indicate that the cumulus cells may play a key role in meiotic arrest and in the resumption of meiosis. During the expansion of the cumulus mass, the glycosaminoglycans of the extracellular matrix may nullify the activity of the meiotic-arresting factors produced by the granulosa cells and/or present in the follicular fluid, triggering the resumption of meiosis.
B. CUMULUS DIFFERENTIATION At the time of cumulus expansion, meiotic maturation of oocyte takes place within the follicle (Dekel et al., 1979; Schuetz and Swartz, 1979). Other studies suggest that the dissociation of the cumulus-oocyte junctions may follow rather than precede GVBD in the rat (Hillensjo et ul., 1979; Dekel and Kraicer, 1978) and rabbit (Szollosi et al., 1978). The administration of pregnant mare serum gonadotropins (PMSG) may initiate GVBD without inducing changes in the cumulus masses (Vermeiden and Zeilmaker, 1974). In mice, 3 hours after the injection of HCG, more than 90% of the oocytes isolated from large Graafian follicles had undergone
22
ElMEI SAT0 AND S. S. KOIDE
GVBD, although cumulus expansion and changes in intercellular communication were not observed (Eppig, 1982). In growing follicles, gap junctional contacts are detected in the cumulusoocyte complex; while in the Graffian follicles, the numbers are reduced and the cumulus layer appears to undergo disorganization. At the time of ovulation, cell-to-cell communication is disrupted, and the remaining cumulus cells become loosely arranged with bullous cytoplasmic extensions (Moor et al., 1980), suggesting that the reduction in intercellular communication may be a consequence of cumulus expansion which continues after oocyte maturation. Dekel et al. (1979) pointed out that cumulus expansion is a gradual process in that the accumulation of glycosaminoglycans takes place initially at the periphery and gradually involves the central portion of the cumulus cell mass. The final stage is the disruption of the cumulus attachment to the oocyte (Dekel et al., 1979), while the disintegration of cell-to-cell communication between cumulus-granulosa cells may occur at the beginning of cumulus dispersion. The end result is that the oocyte escapes from the maturation inhibitory effect of the granulosa cells due to the binding of the meiotic-arresting factor and from the reduced flow of cAMP to the oocyte (Sato et ul., 1984b, 1986). Cumulus expansion of isolated rat follicles cultured in a chemically defined medium is stimulated by gonadotropins (Hillensjo ef al., 1976). Both LH and FSH can induce cumulus expansion in isolated cumulus-oocyte complexes. However, other investigators reported that FSH and not LH induced cumulus expansion in isolated mouse cumulus-oocyte complexes (Dekel and Kraicer, 1978; Dekel et al., 1979; Eppig, 1979a,b, 1980a,b). Although there is a general consensus that LH is the only gonadotropin capable of stimulating follicular events associated with ovulation, FSH appears to be responsible for cumulus expansion (Eppig, 1979a,b). Also follicular fluid obtained from PMSG-primed mice was very active in stimulating cumulus expansion of isolated cumulus-oocyte complexes, demonstrating that follicular fluid contains a FSH-like activity capable of stimulating extracellular matrix synthesis. The mediating factor may be cAMP because db CAMP,phosphodiesterase inhibitors, and cholera toxin stimulate cumulus expansion in vitro (Hillensjo, 1977; Dekel and Beers, 1978; Eppig, 1979a,b). The granulosa and cumulus cells differ in their capacities to produce glycosaminoglycans in response to gonadotropins. The cumulus and granulosa cells originate from a single layer of follicular cells surrounding the primordial oocyte (Franchi et al., 1962). Gonadotropins stimulate mucification of the cumulus and not of the granulosa cells (Dekel er al., 1979). It is noteworthy that the ability of FSH to induce cumulus expansion in vitro is dependent upon the addition of fetal bovine serum (FBS) to the
ARREST A N D RESUMPTION OF MEIOSIS IN OOCYTES
23
cultured medium (Eppig, 1980a,b). FSH stimulates the synthesis of glycosaminoglycans in oocyte-cumulus cell complexes in the presence or absence of FBS. In the presence of FBS the glycosaminoglycans are retained within the complexes, while in the absence of FBS they are released into the culture medium. Containment of glycosaminoglycans within the complex corresponds to cumulus expansion or mucification. The active factor in FBS, promoting cumulus expansion by inducing retention of the glycosaminoglycans within the complex (Eppig, 1979a,b, 1980a,b), has been found to be a protein with a molecular weight exceeding 10,000. Follicular fluid from preovulatory follicles contain components similar to that of FBS (Hadek, 1963; Edwards, 1974) and potentiate the action of FSH in promoting cumulus mucification. Based on these findings, it was hypothesized that the active factor(s) in follicular fluid is a core component of the extracellular matrix of cumulus cells which binds the glycosaminoglycans produced to form the mucified matrix. Since follicular fluid contains an FSH-like factor that stimulates cumulus expansion, why is it that cumulus expansion does not occur spontaneously and is dependent upon a LH surge or on HCG administration? It is postulated that there may be components in Graafian follicles that block the activity of the FSH-like factor (Eppig, 1980a,b, 1981a,b; Eppig and WardBailey, 1984). Sulfated glycosaminoglycans may be involved in suppressing the activity of the FSH-like substance, since these compounds block the incorporation of radiolabeled glucosamine into the extracellular matrix components of cultured cumulus-oocyte complexes from mice (Eppig, 1981a,b). The order of potency of sulfated glycosaminoglycans to inhibit FSH-induced cumulus expansion correlates with the degree of sulfation: heparin > heparan sulfate > chondroitin sulfate B > chondroitin sulfate C > chondroitin sulfate A (Eppig, 1981a). The sulfated glycosaminoglycans may inhibit the synthesis of the extracellular matrix components in response to FSH. Eppig and Ward-Bailey (1984) showed that sulfated glycosaminoglycans suppress the synthesis of glycosaminoglycans after the development of partial cumulus expansion. We proposed that heparin and heparan sulfate accumulate in the extracellular spaces between cumulus cells at the beginning of the expansion and interact with the granulosa cell factor with maturation-preventing activity (Sato ef af., 1984b, 1986). C. FOLLICULAR GLYCOSAMINOGLYCANS
The contents of glycosaminoglycans in follicular fluid is about 0.2-0.3% ( w h ) (Jensen and Zachariae, 1958; Yanagishita et al., 1979). The glycosaminoglycans of follicular fluid are composed of a core protein with an estimated molecular weight of 400,000 with an average of 20 dermatan
24
EIMEI SAT0 AND S . S. KOlDE
sulfate chains and 350 sialic acid-containing oligosaccharides (Yanagishita et ul., 1979). The glycosaminoglycans of rat and porcine granulosa cells are chemically similar (Yanagishita and Hascall, 1979; Yanagishita et al., 1979). The follicular fluid glycosaminoglycans are produced by the granulosa cells. It is known that rat granulosa cells cultured in vitro synthesize and secrete glycosaminoglycans into the medium at a linear rate (Yanagishita et al., 1979). The mechanism of how the glycosaminoglycans accumulate in the intercellular spaces is not clear. Yanagishita and Hascall ( 1979) reported that 90% of the glycosaminoglycans secreted by rat granulosa cells are susceptible to hydrolysis by chondroitinase A, B, and C, indicating that the major constituent produced is chondroitin sulfate. Heparan sulfate was not detected in porcine follicular fluid (Yanagishita and Hascall, 1979). Heparin-like substances, chondroitin sulfate, and dermatan sulfate were identified in rat ovarian tissue and follicles (Gebauer et al., 1978; Ax and Ryan, 1979a,b), indicating that these substances were synthesized in the follicles and may block the maturation inhibiting activity of GCF. (Sato et ul., 1984b, 1986). It was also observed that the production of glycosaminoglycans varied with the size of the follicles. Granulosa cells from large porcine follicles produced slightly less glycosaminoglycans compared to the cells from small follicles (Schweitzer et al., 1981). As the follicles mature, the content of glycosaminoglycans decreased markedly in porcine and bovine follicular fluids (Ax and Ryan, 1979a,b; Grimek and Ax, 1982). The production of glycosaminoglycans was studied by determining the rate of incorporation of radiolabeled precursors. FSH stimulated the incorporation of precursors into rat and porcine follicular glycosaminoglycans (Mueller et ul., 1978; Ax et al., 1978; Ax and Ryan, 1979a,b). In contrast, LH decreased glycosaminoglycans production in rabbit follicles in vitro (Zachariae, 1957) and in rat ovarian slices in vitro (Gebauer et al., 1978). As described in an earlier section, db CAMP can mimic FSH stimulation of glycosaminoglycans production by porcine and rat granulosa cells (Ax and Ryan, 1979a; Schweitzer et al., 1981). Progesterone, in contrast, decreased glycosaminoglycans synthesis by rat ovarian slices (Gebauer et al., 1978) and porcine granulosa cells (Schweitzer et al., 1981). During the occurrence of cumulus expansion, glycosaminoglycans accumulate in the intercellular spaces between the cumulus cells. The major component produced by the mouse cumulus-oocyte complexes in response to gonadotropin is hyaluronic acid (Eppig, 1979a,b). Also, Ball et al. (1982) reported that, during the expansion of bovine cumulus-oocyte complexes induced with FSH or CAMP, massive amounts of glycosaminoglycans accumulated in the matrix enveloping the cumulus cells. The intercellular matrix material was isolated and subjected to electrophoretic analysis.
ARREST A N D RESUMPTION OF MEIOSIS IN OOCYTES
25
The radiolabeled glycosaminoglycans comigrated with reference hyaluronic acid. The substance was resistant to chondroitinase A, B, and C and nitrous acid degradation and hydrolyzed by hyaluronidase. It was concluded that the glycosaminoglycans of the intercellular matrix produced by bovine cumulus-oocyte complexes are rich in hyaluronic acid (Ball et al., 1982). A portion of the glycosaminoglycans of the extracellular matrix of the cumulus should contain heparin-like substance, since ovarian tissues are known to produce this substance (Gebauer et al., 1978; Ax and Ryan, 1979a).
D. A HYPOTHETICAL
S C H E M E OF O O C Y T E
MATURATION
The pertinent facts relating to oocyte maturation can be summarized as follows: isolated follicle-enclosed oocytes remain arrested in the dictyate stage (Tsafriri, 1978a; Lindner et al., 1983). When released from the follicles, the oocytes undergo spontaneous maturation (Pincus and Enzmann, 1935; Biggers, 1973), indicating that meiotic-arresting factors within the follicles maintain the oocyte in the dictyate stage. Several maturationpreventing factors have been identified in follicular fluid and granulosa cells. Resumption of meiosis of cumulus-enclosed oocytes can be prevented by being in contact with the granulosa cell layer (Sato et al., 1982, 1984b).The meiotic-arresting activity of the granulosa cells is demonstrable only when they are adherent to the cumulus-oocyte complex (Sato et a l . , 1982). Based on these studies, a substance with meiotic-arresting activity from the intercellular matrix and the external surfaces of bovine granulosa cells has been isolated (Sato et al., 1984b, 1986) and designated as the granulosa cell factor (GCF). Other maturation inhibitory factors found in follicular fluid are OM1 and hypoxanthine. The latter substance acts only in combination with cAMP (Tsafriri, 1984; Downs e / al., 1985). Follicular fluid may contain another nucleotide with meiotic-arresting activity in combination with cAMP (Sato e/ ul., 1985). Among these arresting factors, GCF is the most potent agent. GCF and OM1 possess similar properties and may possibly be identical substances. Nonetheless, at least two types of factors appear to sustain meiotic arrest: a peptide and a base or nucleotide. During cumulus expansion, cells in the periphery of the cumulus separate and begin to disperse, while the oocyte undergoes maturation (Dekel et al., 1979). The resumption of meiosis in oocytes surrounded by dispersed cumulus is not inhibited even when the cumulus-oocyte complexes are in contact with a layer of adherent granulosa cells (Sato et d., 1982).Thus the dispersion of the cumulus cells, resulting from the accumulation of glycosaminoglycans in the intercellular matrix, will promote maturation.
26
ElMEl S A T 0 AND S. S. KOIDE RESTING OOCYTE c o o c y t e cytoplasm
Follicular flLid inhibitors
FIG.4. Hypothetical scheme of follicular factors inducing meiotic arrest and promoting maturation of mammalian oocytes. A factor, GCF, located on the surfaces and in the intercellular spaces of granulosa cells sustains meiotic arrest by acting on the cumulus-oocyte complex by direct contact, increases CAMP level within the cumulus-oocyte complex, and inhibits calcium transport. Additional associated meiotic-arresting substances are the oocyte
ARREST A N D RESUMPTION OF MEIOSIS IN OOCYTES
27
LH stimulates the preovulatory follicle to produce glycosaminoglycans which accumulate on the surface and in the intercellular spaces of the cumulus cells (Eppig, 1979a,b). Following cumulus expansion, the oocytes undergo maturation. We have demonstrated that among the glycosaminoglycan, heparin and heparan sulfate interact with GCF and nullify its maturation inhibitory activity (Sato et al., 1984b, 1986). A consequence of cumulus expansion is a decrease in the permeability of the oolemma and the transport of essential nutrients from cumulus cells to the oocyte (Heller and Schultz, 1980; Moor et af., 1980). Based on the above findings, the following hypothesis is proposed to explain the mechanism of meiotic arrest and the resumption of meiosis (Fig. 4). GCF is located on the external surface and in the intercellular spaces of the granulosa cells. By direct contact between the granulosa cells and the cumulus-oocyte complex, GCF can sustain meiotic arrest. GCF acts on the cumulus cells and stimulates an increase in the intraoocyte level of CAMPand suppresses calcium transport. Gonadotropins stimulate the production and accumulation of glycosaminoglycans in the intercellular spaces of the cumulus cells. The net result is a disruption of the gap junctions between cumulus cells and between the cumulus cells and oocytes, impeding the transport of metabolites within the cumulus-oocytes complexes. The glycosaminoglycans can prevent the action of GCF on the oocyte by binding the factor. Cumulus expansion results in preventing the action of the maturation inhibitors on the oocyte by binding these factors, reducing the flow of essential nutrients to the oocyte, and disrupting the cell-to-cell communication system within the follicles. In this manner, the oocyte is protected from the arresting influence of the maturation inhibitory factors and resumes meiosis. Protein phosphorylation and dephosphorylation may be involved in meiotic arrest and the resumption of meiosis. In mouse oocytes, phorbol esters prevent spontaneous GVBD (Urner and Schoderet-Slatkine, 1984). Stith and Maller (1985)reported that phorbol esters can induce maturation of Xenopus oocytes, providing they are primed initially with inositol I ,4,5trisphosphate (IP,) which facilitates Ca” release (Berridge, 1984). Microinjection of IP, alone into Xenopus oocytes induced Ca2+release without GVBD (Busa et al., 1985). Tumor-promoting phorbol ester, phospholipase C. and diacylglycerol, l-oleoyl-2-acetylglycerol,induced GVBD maturation inhibitor and CAMP-potentiating factors including hypoxanthine and a novel nucleotide. Luteinizing hormone stimulates the production and accumulation of glycosaminoglycans. specifically of heparin-like substances, that surround the cumulus4ocyte complex. The intercellular matrices prevent the action of CGF on the oocyte and interfere with the flow of metabolites to the oocyte. promoting the resumption of meiosis.
28
EIMEI S A T 0 AND S. S. KOIDE
in follicle-enclosed rat oocytes (Aberdam and Dekel, 1985). These findings suggest that Ca" release in combination with diacyglycerol promotes GVBD. Ca" and diacylglycerol are known to activate protein kinase C (Nishizuka, 1984), which mediates protein phosphorylation. Since diacylglycerol and IP, are hydrolytic products of phospholipase C action on phosphatidylinositol (Berridge, 1984), progesterone may stimulate this membrane enzyme on interaction with its receptor.
ACKNOWLEDGMENTS This study was supported in part by grant numbers HD 13184 from NICHD. NIH. GA PS 8506 ( S . S . K.), and 8429 (E. S . ) from the Rockefeller Foundation and grant numbers 60304036 and 60760207 from the Ministry of Education, Science and Culture. Japan ( E . S.).
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INTEKNATIONAL REVIEW OF CYTOLOGY, VOL. lob
Morphology and Cytochemistry of the Endocrine Epithelial System in the Lung D.W.SCHEUEKMANN Institrrtc>of Histology and Micwscwpic Anritomy, University of’Antwerp, 2020 Antwerp, Belgium
I. Introduction In his light microscopic study of 1938, Feyrter first described what he called “Helle Zellen” (clear cells), due to their almost transparent cytoplasm in hematoxylin- and eosin-stained sections, lying dispersed throughout the epithelial tissue of various organs. He assumed them to be endocrine-like cells belonging to a widespread endocrine epithelial system with paracrine, neurocrine, and hemocrine functions, located in various organs including the respiratory system. Later, in an extensive study, Frohlich (1949) provided a precise description of these clear cells in the epithelium of the tracheobronchial tract of several mammals (rabbit, cat, guinea pig, dog, wether, and monkey) including man (executed persons) by means of different conventional staining methods. Strikingly, in mammals, these clear cells-particularly those in man-were found to occur not only as solitary elements; indeed, Frohlich also outlined and illustrated the existence of distinctive groups of clear cells forming round or oval corpuscular structures. According to the same author, these clear cells are situated in the epithelial tissue, close to the basement membrane, the apical cytoplasm contacting the airway lumen only occasionally. He attributed to these cells a neurosensory function and realized a relationship with the dispersed endocrine epithelial system originally outlined by Feyrter ( 1938). Moreover, Frohlich provided the first demonstration of nerve endings in close contact with the basal cytoplasm of both solitary and groups of pulmonary clear cells, an observation later confirmed by several authors (e.g., Glorieux, 1963; Shul’ga, 1965; Lauweryns ef al., 1972, 1974; Hung et al., 1973; Lauweryns and Cokelaere, 1973b; Hung and Loosli, 1974; Lauweryns and Goddeeris, 1975; Wasano, 1977; Hung, 1980; Scheuermann et al., 1983a,b; Scheuermann, 1984; Stahlman and Gray, 1984). The early investigators demonstrated the reactivity of pulmonary clear cells to argentaffin and/or argyrophilic silver techniques and argued that they might possibly have a chemoreceptive and neurosecretory function, acting primarily at the pulmonary level (Frohlich, 1949; Feyrter, 1953, 35 Copyright l a 19x7 by Academic Pre% Inc. All rightr of reproduclion in any Iorm r r w v e d .
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1954, 1958). Since these first histological observations, the presence of endocrine-like clear cells among seemingly darker epithelial cells has been revealed by both light and electron microscopy in the extra- and intrapulmonary airways of several mammals, including man (Glorieux, 1963; Bensch et al., 1965; Lauweryns and Peuskens, 1969; Lauweryns et al., 1970, 1972; Cutz and Conen, 1972; Ericson et al., 1972; Hage, 1972, 1973a,b, 1974, 1980; Hung et a l . , 1973, 1979; Cutz et al., 1974, 1975, 1978a,b; Hung and Loosli, 1974; Jeffery and Reid, 1975; Lauweryns and Goddeeris, 1975; Hung, 1976, 1980; Hage et al., 1977; Hernandez-Vasquez et al., 1977, 1978a,b; Wasano, 1977; Sorokin and Hoyt, 1978; Edmondson and Lewis, 1980; Palisano and Kleinerman, 1980; Dey et al., 1981, 1983; Keith et al., 1981, 1982; Wasano and Yamamoto, 1981; Carabba et al., 1982; Sarikas et al., 1982; Pearsall et al., 1985), birds (Cook and King, 1969; Walsh and McLelland, 1974; Wasano and Yamamoto, 1979), amphibians (Rogers and Haller, 1978, 1980; Wasano and Yamamoto, 1978; Goniakowska-Witalinska, 1980a,b, 1981), and a reptile (Scheuermann et al., 1983a,b). These endocrine-like cells occur isolated or in distinctive groups of two or three cells, as well as in large clusters of more than 100 cells (Hoyt er al., 1982a,b) within the epithelium at every level of the bronchoalveolar tract. Combined histochemical, fluorescence microscopic, and ultrastructural investigations have shown the clear cells of the pulmonary epithelium to contain intracytoplasmic chemical mediators, such as 5-hydroxytryptamin (5-HT) (Lauweryns et al., 1974, 1982; Rogers and Haller, 1978; Keith et al., 1982; Scheuermann et al., 1983a) and neuropeptide hormones (bombesin, Wharton et al., 1978; Dayer et a l . , 1985; gastrin-releasing peptide; Iwanaga, 1983; Tsutsumi et al., 1983a,b; calcitonin, Becker er al., 1980; leu-enkephalin, Cutz et al., 1981), thereby displaying in many aspects a similarity to the elements of the APUD (amine precursor uptake and decarboxylation) endocrine system conceived by Pearse (1969, 1977). The structural resemblance of these cells to some known receptor cells (Cook and King, 1969; Lauweryns et al., 1972; Lauweryns and Peuskens, 1972; Hung et d., 1973; Wasano, 1977; Cutz et al., 1978a,b; Scheuermann et al., 1983a) has led to their classification in the paraneuronic system of Fujita (1977), indicating their functional relation to neurons. In the course of recent years, a plethora of names has been coined on the basis of morphological and presumed functional features to designate these cells. For instance, they have been referred to as Feyrter cells (Moosavi et al., 1973; Hernandez-Vasquez et al., 1977, 1978a,b; Taylor, 1982) after the pathologist who first described them. But they were also called enterochromaffin-like cells (Ericson et al., 1972) and Kultschitzkylike cells (or K cells) (Bensch et al., 1965; Cutz et al., 1974, 1975). since
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they display many features similar to those found in their counterparts in the gastrointestinal tract (Bensch et al., 1965; Cutz and Conen, 1970, 1972; Lauweryns et al.. 1970; Terzakis et al., 1972; Jeffery and Reid, 1975; Breeze and Wheeldon, 1977; Capella et al., 1978; Sorokin et ul., 1983). Another term suggested was argyrophil cells (Lauweryns and Peuskens, 1969; Taylor, 1977) or even AFG k e . , the initial letters of argyrophilic, fluorescent, and granulated) cells (Lauweryns et al., 1970). Other terms which have been proposed are chromafln-type cells (Basset et al., 1971), endocrine cells (Hage, 1972, 1973a,c, 1974), endocrine-like cells (Hage, 1976; Cutz and Conen, 1972; Ewen et al., 1972; Hage et al., 1977), neurosecretory cells (Becci et al., 1978) or neurosecretory-appearing cells (Terzakis et al., 1972). small granulated cells (McDowell et al., 1976), small granule endocrine cells (Sorokin and Hoyt, 1978), biogenic aminecontaining cells (Eaton and Fedde, 1978), or neuroendocrine cells (Keith et al., 1981). Their amino acid uptake characteristics have inspired some authors to call them APUD cells (Hage, 1973a; Sidhu, 1979), since they fulfill the principal criterium of this system (Pearse, 1969, 1977). However, except for the Feyrter and Kultschitzky terms, none of these names specify whether they are situated intraepithelially or in the pulmonary connective tissue. Since it was demonstrated that both solitary and groups of neuroendocrine cells containing biogenic amines may belong to intrapulmonary ganglia (McLean and Burnstock, 1967a,b; Bliimcke, 1968; Bock, 1970; Mann, 1971; Jacobowitz et al., 1973; Knight, 1980; Scheuermann and De Groodt-Lasseel, 1983; Scheuermann et al., 1983b, 1984a,b,c, 198% two populations of endocrine cells of the respiratory system should be included in the APUD series. Accordingly, it seems necessary to maintain a terminology which refers to the characteristic location of the cells in the epithelial tissue. This shortcoming applies equally to the term dense-core granulated cells introduced by Jeffery and Corrin (1984) in a recent study on the structural analysis of the respiratory tract. Indeed, the intraepithelial, granule-containing cells and the small intensely fluorescent cells occurring in the ganglia of the pulmonary interstitium are characterized by an abundance of dense-cored vesicles and are both assumed to have a chemoreceptor and endocrine or paracrine function (Bock, 1970; Knight, 1980; Scheuermann et al., 1983b, 1984a,b,c). Since the present work deals with neuroendocrine epithelial cells-and not with endocrine cells of the intrapulmonary ganglia-it seems justified to use the term neuroepithelial endocrine (NEE) cells. The well-demarcated epithelial organoid structures, composed of aggregated NEE cells-for the first time extensively described by Glorieux (1963), who called them “corpuscule kpithelial” (i.e., epithelial corpuscle)-will be referred to in the course of this review as neuroepithelial
38
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SCHEUERMANN
bodies (NEBs), a term proposed by Lauweryns et al., (1972) because of their prominent nerve supply. The endocrine clear cells grouped in NEBs have been compared to the solitary endocrine cells, lying dispersed in the epithelium of the respiratory system and described in human fetuses (Cutz and Conen, 1970, 1972; Hage, 1973a,b; Cutz et al., 1973, in children (Lauweryns and Peuskens, 1969; Cutz and Conen, 1970; Lauweryns et al., 1970; Rosan and Lauweryns, 1971; Cutz et al., 1975; Lauweryns and Goddeeris, 1975), in the adult human lung (Cutz and Conen, 1970; Terzakis et al., 1972; Lauweryns and Goddeeris, 1975; Hage et al., 1977) as well as in various mammalian species (Jeffery and Reid, 1973, 1975; Cutz et al., 1974; King et al., 1974; Hernandez-Vasquez et al., 1977; Sorokin and Hoyt, 1978; Edmondson and Lewis, 1980; Palisano and Kleinerman, 1980; Lauweryns et al., 1985), in birds (Cook and King, 1969), and in amphibians (Wasano and Yarnamoto, 1978; Goniakowska-Witalinska, 1980a). The corpuscular appearance of groups of NEE cells in NEBs and their conspicuous innervation are considered by some authors to be a separate neuroendocrine cell system, distinct from solitary NEE cells (Lauweryns and Cokelaere, 1973a; Lauweryns et d., 1974, 1978, 1985; Lauweryns and Goddeeris, 1975; Lauweryns and Liebens, 1977; Loosli and Hung, 1977; Hung et d., 1979; Sonstegard et al., 1979; Foliguet and Cordonnier, 1981). However, in developing rabbit lungs, it was demonstrated by electron microscopy (Sorokin et al., 1982) that scattered solitary clear cells appear very early during gestation as the first population in the pulmonary epithelium undergoing differentiation into NEE cells and that mature NEBs are derived from them. According to these authors, embryonal undifferentiated precursor cells, situated in the primary pulmonary epithelium, are observed in various stages of transformation to NEE cells, which subsequently appear in groups of two to three cells that will finally mature, at least partly, to NEBs. Similarly, Stahlman and Gray (l984), investigating electron microscopic preparations of the fetal human lung, describe putative neuroendocrine cells which, during development, differentiate into either singly occurring neuroendocrine cells or into NEBs. In a combined immunohistochemical and ultrastructural investigation of the development of NEE cells in the human lung from the early fetal to the perinatal period, Cutz et al. (1984) demonstrated the presence, in the canalicular stage, of NEE cells occurring either solitarily or packed in NEBs. As observed by these authors, the NEE cells grouped in NEBs share identical ultrastructural features with the isolated NEE cells. Both light and electron microscopic investigations seem to indicate that single NEE cells and NEBs represent stages of differentiation of one and the same embryonal precursor cell. In agreement with these findings, studies have shown that in the res-
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39
piratory system both the diffuse endocrine cells and the NEBs, which may be involved in the production of amines and/or polypeptide hormones, share similar histochemical properties (Cutz et al., 1984). Moreover, the innervation of NEE cells is not restricted to NEBs. Nerve terminals with synaptic contact have been described at the base of single NEE cells in the infant bronchial epithelium (Lauweryns et al., 1970). Unmyelinated axons were also found in close association with individual NEE cells of the pulmonary system in the rabbit (Hung, 1980), hamster (Edmondson and Lewis, 1980), rat (Jeffery and Reid, 1973), and guinea pig (DiAugustine et al., 1984). Since functions of these endocrine cells remain still unknown, the pulmonary single NEE cells and those arranged in NEBs will be treated as a single neuroepithelial endocrine system. 11. Light Microscopic Aspects
Epithelial cells with transparent cytoplasm are observed in routine light microscopic preparations of the entire respiratory tract, in particular, when using, after fixation with formaldehyde, hematoxylin-eosin, the trichrome method of Masson, or a modification of the Goldner-Masson staining method (Frohlich, 1949; Feyrter, 1958). However, since the translucent appearance is not a specific morphological feature, the solitary NEE cells remain in this way relatively inconspicuous. Conversely, NEBs can be readily detected by conventional staining techniques, forming clearly demarcated epithelial corpuscles. In some species, they protrude slightly into the airway lumen (Hung et al., 1973, 1979; Cutz et al., 1978b), but in others they are enveloped in the epithelium, indenting the underlying connective tissue (Pearsall et al., 1985). They can also be observed in a pitlike depression of the pulmonary epithelium. In most species, the more centrally located cells of the NEBs are characterized by a well-ordered appearance consisting of nonciliated cells, joined side-by-side, with their longitudinal axes more or less at right angles to the basal lamina, albeit slightly inclined to the center of the luminal surface. The shape of these corpuscular cells is almost columnar with a more or less oval nucleus. Sometimes, at the branching region of the airways, the NEE cells appear stratified or form ajumble of cells, often with a pyramidal form, the apex of which is directed to the airway lumen and the broad face situated against the basement membrane. Some profiles of NEBs do not contact the lumen of the airways, but seem isolated from the surface by dark nonciliated cells stretched over the luminal and lateral cytoplasm as it expands (Fig. 1). They are assumed to be modified Clara cells (Cutz et ul., 1978b; Hung et al., 1979; Pearsall e f al., 1985). From serially cut sections, it is apparent that, in most animal species,
FIG. I . In some sections, the NEB of the red-eared turtle appears elongated, with granulecontaining cells in a palissade-like row lying on the basal lamina. Most of the apical surfaces are covered with flattened Clara-like cells. The NEB is separated from capillaries by a narrow subendothelial space containing collagen fibers. To the left, Clara-like cells abut on ciliated epithelial cells. Silver method applied to semithin section of Epon-embedded material according to Lopez e/ d.(1983). Light microscopy. x 1100. FIG.2. NEB in the epithelial lining of the bronchiolus of a neonatal rabbit composed of yellow fluorescent, elongated cells, as revealed by formaldehyde-induced fluorescence. The contours at the base of the individual cells are hardly visible, because of their close apposition and very intense fluorescence. The emission and excitation spectra of the fluorophore of this NEB is rendered in Fig. 5 . Fluorescence microscopy. x 800. FIG.3. The same NEB of the neonatal rabbit as in Fig. 2, revealed by the argyrophilic method according to Grimelius. Light microscopy. x 800. FIG. 4. Whole-mount stretch preparation of the red-eared turtle lung treated for formaldehyde-induced fluorescence. An extensive group of intensely yellow-fluorescent neuroendocrine epithelial cells, with, in their neighborhood, a few solitary and grouped neuroendocrine epithelial cells. Green-fluorescent nerve fibers running to the yellow-fluorescent NEB. Fluorescence microscopy. x 90.
THE ENDOCRINE EPITHELIAL SYSTEM IN THE LUNG
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a small portion of the apical cytoplasm often reaches the airway lumen (Fig. I). In most species, there is a striking similarity in NEB architecture and morphology; their size is highly variable (for review, see Foliguet and Cordonnier, 1981). However, in the toad lung, a NEB appears covered by a dark apical cell, provided with a single cilium protruding into the airway lumen (Rogers and Haller, 1978, 1980). The basement membrane rests on an usually thin lamina propria, which envelops one or more capillaries close to the NEE cells (e.g., Lauweryns and Goddeeris, 1975; Scheuermann er al., 1983a; Hung, 1984; Pearsall et ul., 1985). Fascicles of smooth muscle may closely approach the base of the NEE cells (Pearsall er al., 1985). In order to detect the NEE cells, besides conventional light microscopy, various convenient staining methods have been used, including the use of masked metachromasia (Solcia et ul., 1968) and such methods as lead hematoxylin (Solcia et ul., 1969) or periodic acid-Schiff and lead hematoxylin (Sorokin and Hoyt, 1978). Acid hydrolysis which precedes staining enhances the metachromasia to basic dyes of secretory granules in endocrine cells attributed to sidechain carboxyl or carboxamide groups of granule proteins (Pearse, 1969), whereas the diffuse basophilia, due to RNA, DNA, and acid polysaccharides, is not realized by extraction of these acid substances (Solcia et ul., 1968). A consecutive treatment with basic dyes after HCI hydrolysis stained the endocrine cells in pancreatic islets, in thyroid and parathyroid endocrine cells, in the gastroenteric endocrine cells, and in the adenohypophysis (Solcia et ul., 1968). It was believed that probably all cells from the APUD series contain metachromatic substances in their secretory granules in a “masked” form which can be unmasked by HCI hydrolysis (Bussolati et d.,1969; Fujita and Kobayashi, 1974). This technique was therefore applied to demonstrate the presence of endocrine cells in the lung. According to Hage ( 1972, 1976, 1980), HCI-toluidine blue-positive cells are distributed throughout the pulmonary epithelium of human fetuses, whereas in the human adult lung, these have not been found (Hage et d.,1977; Hage, 1980), nor, for that matter, in the rabbit, guinea pig, mouse (Hage, 19741, and rat (Cutz et al., 1974). The lead hematoxylin method is frequently used in light microscopy for staining secretory granules in endocrine cells known to produce polypeptide hormones (Solcia et al.. 1969). Some authors report a positive lead hematoxylin reaction in NEE cells of the lung of human fetuses (Hage, 1980) and in carcinoid lung tumors (Hage, 1976), but not in the normal adult human lung (Hage er al.. 1977). Lauweryns and Cokelaere (1973a) demonstrated weak lead hematoxylin-positive NEBS in the lung of neonatal animals (rabbit and mouse). However, Sorokin and Hoyt (1978) furnished
42
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D. W. SCHEUERMANN
evidence that, in lungs from young mice, rats, hamsters, kittens, as well as in late fetal and neonatal rabbits, lead hematoxylin alone produces little if any staining of the NEE cells. Conversely, a staining method which combines the periodic acid-Schiff (PAS) method to lead hematoxylin and which is applied to plastic-embedded sections seems particularly useful for granule-containing cell populations in the lung (Sorokin and Hoyt, 1978). The cells are recognizable by the magenta coloration of the cytoplasm, frequently heavier toward the cell base. In plastic-embedded material of control lungs and S h y droxytryptophan pretreated animals examined for the formaldehyde-induced fluorescence (FIF) technique and consecutively stained with the PAS-lead hematoxylin method, the NEE cells revealed a magenta staining corresponding precisely to sites of 5-HT fluorescence (Sorokin and Hoyt, 1978). In a systematic study on the infracardiac lobe of the hamster lung, five different types of NEE cells have been identified by the PAS-lead hematoxylin method (Hoyt et al., 1982a). Types I, 11, and V bear granules of about 0.2 pm in diameter, whereas types 111 and IV contain larger granules. Types I and I1 can be readily segregated, since only the granules of the first type stain reddish-pink without affinity for lead hematoxylin. Of the coarse-grained PAS-positive types 111 and IV, only the latter show affinity for lead hematoxylin. Type V cells appear reddish-purple with PAS and the small granules of these cells are stained by lead hematoxylin. Whereas types I, 11, 111, and 1V encompass columnar cells whose granules tend to accumulate at the basal pole of the cell, type V cells are apparent by their spheroidal shape. Types I, 11,111, and V may occur both solitarily and in organized clusters, whereas it was demonstrated that the PASpositive and lead hematoxylin-positive coarse-grained type IV cells, which never occur as single cells, are usually present in large NEBS. These structural differences of NEE cell subtypes are signifcant in view of the preferential relationship which each of them displays with nonendocrine cells and tissues, whether occurring solitarily or in organized clusters. For instance, NEE cell types I and I11 can be found in relation to the capillary network of the pulmonary artery; types 11, IV, and V may be linked with the capillaries of the pulmonary circuit; types I1 and IV can be related predominantly to smooth muscle cells. From these data, it might be concluded that, at least in the hamster lung, the NEE cell subtypes may have different functions, independent of their single or clustered appearance. The neuronal-like cytochemical features of NEE cells, which allow their detection in light microscopy, include silver techniques, cholinesteraseand neuron-specific enolase cytochemistry, FIF, and immunocytochemical detection of 5-HT and peptide hormones.
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111. Argentaffinity and Argyrophilia
The argentaffin method to reveal silver-reducing cells was first developed by Masson (l914), demonstrating that after formalin fixation the Kultschitzky cells contain an endogenous substance which reduces alkaline solutions of silver salts, resulting in these cells being impregnated by metallic silver; ever since, the term argentaffinity points to impregnation of the cells by silver after reduction of an alkaline silver solution without additional treatment with an extraneous reducing agent. After it was demonstrated that Kultschitzky cells contain 5-HT (Erspamer and Asero, 1952), Barter and Pearse (1953, 1955) showed that Kultschitzky cells in formalin-fixed tissue and synthetic 5-HT models reacted in a similar way with the usual argentaffin method, demonstrating that the silver-reducing power of these cells after formalin fixation is due to 5-HT. It was shown that, after subsequent treatment with alkaline or neutral solutions of silver salts and a weak extraneous reducing agent, not only were the argentaffin cells stained but also a wide range of other cells and tissue components (Hamperl, 1932). This other way to reveal tissue structures with silver salts is usually called the argyrophilic method, which is most frequently used for the demonstration of bronchopulmonary NEE cells; however, the NEE cells of the respiratory tract, being the pulmonary counterparts of the Kultschitzky cells, is the case in which it seems more obvious to apply the argentaffin reaction. Cytoplasmic argyrophilia was revealed in some clearly isolated NEE cells and in NEBS of paraffin-processed lung tissues from human fetuses and children (Hamperl, 1952; Feyrter, 1954, 1958; Hage, 1972, 1973b, 1976; Lauweryns and Peuskens, 1972; Cutz et a/., 1975; Lauweryns and Goddeeris, 1975), of adult human lungs (Tateishi, 1973; Lauweryns and Goddeeris, 1975; Hage et al., 1977; Hage, 1980), of rabbits, rats, sheep fetuses, and neonatal rats (Lauweryns et al., 1972, 1973, 1974; Moosavi et al., 1973; Hage. 1974, 1976; Cutz et al., 1975; Hung, 1980; Sorokin et al., 1982), of adult rabbits, dogs, and cats (Frohlich, 1949; Lauweryns et al., 1973; Taylor, 1977), and of a young armadillo (Cutz et al., 1975). This reaction yields fine, dark-brown argyrophilic granules either diffusely scattered throughout the cell or concentrated in its basal portion (Fig. 3). In this way, the solitary, pulmonary NEE cells of most species usually appear fusiform, pyramidal, or flask shaped, resting on the basement membrane, with a dark-stained, narrow, cytoplasmic, apical process pointed toward the airway lumen. In adult man, most NEE cells, observed only by Tateishi (1973) as extending to the airway lumen, lack luminal contacts. Some cells revealed dark, long, basolateral cytoplasmic processes extending along the basement membrane or between other epithelial
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cells. The NEBs exhibiting a strong and massive argyrophilia can be readily identified at the light microscopic level throughout the entire respiratory system. The argentaffin reaction on paraffin sections of formalin-fixed material in Bouin’s solution and immersed in Fontana’s ammoniacal silver nitrate solution (often with omission of gold toning and counterstaining) revealed, in fetal and neonatal rabbit NEE cells (Lauweryns et al., 1972, 1973; Sonstegard et al., 1982) and in NEBs of the turtle lung (Scheuerrnann et al., 1983a), dark-brown deposits in the cytoplasm, preferentially in the basal portion of the cell (Fig. 8). Consequently, these NEE cells possess silverreducing properties after formalin fixation. Nevertheless, several investigators observed little if any argentaffin reaction in NEE cells within the wall of the intrapulmonary airways of man and different animals (Lauweryns and Peuskens, 1969; Hage, 1972, 1974, 1976, 1980, 1984; Cutz et al., 1975). In the lung of Polypterus, NEE cells appeared to reduce the ammoniacal silver salt, some of them strongly, while in others this was only barely visible; hence, they may be assumed to be argentaffin. As shown by consecutive serial sections, the argyrophilic technique applied to the same cells impregnated them with silver as well (D. W. Scheuerrnann and M. H. A. De Groodt-Lasseel, unpublished work). Since the argyrophilic reaction seems more likely to occur than the argentaffin reaction, the former will also be positive whenever argentaffin cells are demonstrated. It therefore appears that argentaffin NEE cells are argyrophilic, whereas some argyrophilic cells do not seem to be argentah. This finding is consistent with reports by Tateishi (1973) and Hage (1980), who demonstrated several argyrophilic NEE cells in the human adult lung to be nonargentaffin. Hage (1980, 1984) attributed a negative argentaffin reaction to the very low concentration of the biogenic amine, as shown by the FIF technique. In this interpretation, it is not necessary to consider argentaffin and argyrophilic cells as distinct cell types. In fact, they have been thought of as representing different stages in the secretory cycle of a single cell (Hamperl, 1952), a hypothesis supported by electron microscopic investigations (Ratzenhofer and Leb, 1965; Ratzenhofer, 1966a.b) and by cytochemical studies on 5-HT fluorescence and argyrophilia (Penttila, 1966, 1967). It is known that the argentaffin silver technique requires a critical level of silver reduction, i.e., a minimal cytoplasm-reducing ability to effectively demonstrate amine-containing cells by light microscopy. As we have seen, the argentaffinity reflects the endogenous capability of the formalin-fixed cytoplasm to reduce silver salts; therefore, this reaction has a histochemical value. Although the argyrophilic reaction is more likely to occur than the argentah reaction, it will obviously become
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positive whenever the argentaffin reaction is demonstrated. With the argyrophilic technique, however, the silver ions, which are mainly caused by addition of an extra reducer, deposit metallic silver on different tissue constituents. This staining characteristic mainly reflects the physical properties and not a chemical composition; it is therefore histochemically unspecific. As a result, the argyrophilic cells are by far more numerous than the argentaffin cells, but they are also much less precisely defined. In summary, a positive argentaffin reaction is indicative of the presence of reducing substances, whereas a negative reaction could imply that the reducing capacity is minimal. The situation is quite different for the argyrophilic reaction caused by treatment with an extraneous reducing agent. Although it is believed that here too the reducing substance of the cytoplasm first partly reduces the silver salt, whereafter additional silver is deposited on top, the latter deposit also occurs on a range of other tissue constituents that seem unrelated to the argentaffin components: it is not yet clear how and why this process occurs. It might be explained by the fact that 5-HT is closely linked to other nonamine components, which may interfere with its reactivity. Hence, as far as the NEE cells of the respiratory system are concerned, it is not surprising that different authors argue that the argyrophilic reaction is not entirely reliable (Cutz et af.. 1974; Sorokin et uf., 1982), probably due to species differences or to the age of the animals, as well as the development and maturation of the endocrine cells. Certainly these possibilities and restrictions should be taken into account when dealing with a comparative distribution of argentaffin and argyrophilic cells. Both silver methods can be applied to semithin sections of Aralditeembedded material according to the method described by Lopez ut af. (1983). Hence, the solitary NEE cells as well as the NEBs are readily identified in the lung of the adult monkey, pig, and red-eared turtle throughout the length of the airways by their low cytoplasmic density. The silver-impregnated granules are most numerous in the cytoplasm facing the basal lamina (Fig. 1). IV. Cholinesterase Activity
Cholinesterase activity was observed by some authors throughout the cytoplasm of NEE cells in the fetal lung of the rabbit (Lauweryns and Cokelaere, 1973a; Hung, 1980. 1984; Sonstegard et af., 1982)and rat (Morikawa rt d.,197th). Moreover, it has been demonstrated that NEBs of the embryonic rat lung, differentiating in virro and segregated from the central nervous system, revealed acetylcholinesterase-containing granules
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(Morikawa et al., 1978b). These observations are in agreement with the original view of Pearse (1969) that high levels of cholinesterase may characterize the cells of the APUD series. Indeed, monoamine-containing cells have been reported to contain cholinesterase activity, such as the chief cells of the carotid body (Koelle, 1950, 1951; Ballard and Jones, 1971; Korkala and Waris, I977), the cardiac (aorticopulmonary) glomus bodies (Papka, 1975, 1980), the catecholamine-containing cells in the pulmonary ganglia of the calf, goat, and fetal sheep (Mann, 1971), in the pelvic paraganglia (Thompson and Gosling, 1976), in adrenomedullary cells (Palkama, 1967), and in some adrenergic neurons (Jacobowitz and Koelle, 1965). As yet, the functional significance of this enzyme in monoaminecontaining cells is not clear. Since most of these cells receive a synaptic input from cholinergic nerve terminals, they are sensitive to acetylcholine. Acetylcholinesterase hydrolyzes and thereby inactivates the neurotransmitter acetylcholine. Hence, this cholinesterase, synthetized in NEE cells, transported on the outer surface of the cellular envelope, and released from this site, is likely to participate in modifying the microenvironment of the NEE cells, regulating the responsiveness to acetylcholine after its release from presynaptic axon terminals. It might also be that the presence of acetylcholinestemse is related to that of active acetylcholine metabolism in NEE cells, since, in some biogenic amine-containing cells, choline acetyltransferase (Ballard and Jones, 1972) as well as the uptake of [3H]choline was demonstrated (Fidone et al., 1976). Indeed, acetylcholine probably plays a role in modulating chemosensory discharge (Eyzaguirre and Zapata, 1968). Moreover, it was demonstrated that cultures of cells from pheochromocytoma may synthetize, store, and release acetylcholine (Greene and Rein, 1977). In adrenergic tissue (Burn and Rand 1959, 1965)and in parathyroid C cells (Welsch and Pearse, 1969), the release of catecholamines is mediated or facilitated by acetylcholine, so that cholinesterase activity may be correlated with physiological states of these cells.
V. Neuron-Specific Enolase
Neuron-specific enolase is a protein which, in light microscopic immunocytochemistry, was found distributed in neuronal perikarya, dendrites, and axons (Pickel et al., 1976; Schmechel et ul., 1978a). It was shown to be a major neuronal protein correlated with neuronal development and differentiation; observations have apparently established it to be essential to the neuronal function (Marangos et al., 1978; Marangos
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and Schmechel, 1980). Since immunocytochemical investigations have revealed neuron-specific enolase to be present in endocrine cells of the APUD series (Schmechel et a/., 1978b) and in the larger cell group called paraneurons (Fujita et al., 1983), this enzyme seems to be a functional marker for the diffuse neuroendocrine system (Marangos et al., 1981; Tapia et a / . , 1981). Consistent with this finding, immunostaining using antibodies to neuron-specific enolase was found in both single NEE cells and in NEBS of the respiratory tract (Cole et a / . , 1980; Tapia et al., 1981; Wharton et al., 1981; Polak and Bloom, 1982, 1984; Sheppard et al., 1984). Although in the adult human lung distinctly immunostained NEE cells can be found, a considerably larger number is present in lungs from human neonates and perinates (Polak and Bloom, 1984). In serial sections of the human fetal lung, at least three different types of NEE cells on the basis of their immunoreactive content were identified containing ( 1) neuron-specific enolase, 5-HT, and bombesin; (2) neuronspecific enolase and 5-HT; and (3) neuron-specific enolase only (Wharton et a/., 1981). This enzyme was for the first time observed in lungs of 16week-old human fetuses; they assumed it to be a useful marker for the immunocytochemical detection of NEE cells in the lungs at any age and regarded its presence as indicative of the starting functional activity of these cells, since, in neuronal tissue, its appearance coincides with the initiation of synaptic contacts (Marangos et al., 1979). Some authors reported the first immunoreactive cells for neuron-specific enolase in the human fetal lung at about 8 weeks gestation, i.e., before neuropeptide could be detected (Sheppard et al., 1984), an observation strengthened by immunostaining for 5-HT and the electron microscopic findings of cytoplasmic secretory granules (Cutz et a / . , 1984). In contrast herewith, is a report by Takahashi and Yui (1983), who detected the first immunoreactive cells for 5-HT in the human fetal lung at 12 weeks. In rats chronically exposed to asbestos fibers, an increased number of large and irregular clusters of NEE cells may be demonstrated in the bronchopulmonary tree, using antibodies to neuron-specific enolase (Cole et a / . , 1982; Sheppard et a/., 1982; Polak and Bloom, 1984). According to these and other authors, using this antibody, neuroendocrine tumors can be identified in the lung (Tapia et a/., 1981; Polak, 1983). Although immunocytochemistry has detected neuron-specific enolase mostly in neurons and paraneurons (Marangos et a / . , 1981), it appeared that immunostaining for this neuronal protein in tissues and cells includes the reaction with a hybrid form of enolase (Marangos et al., 1980; Kato et al., 1982; Haimoto et al., 19851, warning that this way to detect NEE cells must be used with care.
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VI. Aspects of Induced Fluorescence
Recent microscopic studies with the FIF method (Falck and Owman, 1965) have made it clear that many NEE cells and NEBs in the wall of the respiratory system contain biogenic amines (Lauweryns and Peuskens, 1969, 1972; Lauweryns et al., 1970, 1973; Hage, 1972, 1976; Cutz et al., 1974; Cutz, 1982; Scheuermann et al., 1983a, 1984a; Scheuermann, 1984). After treatment with formaldehyde vapor, these cells exhibit a fluorescence of variable intensity (Fig. 4). The ring-closing condensation reaction, occumng between formaldehyde and indolylethylamines or catecholamines, yields heterocyclic compounds. The subsequent dehydrogenation, in the presence of proteins, produces strongly fluorescent dihydro-P-carbolines (Corrodi and Jonsson, 1965) and dihydroisoquinolines(Corrodi and Hillarp, 1964), respectively. There are authors who make use of a recently developed glyoxylic acidinduced fluorescent method for the demonstration of biogenic amines in NEE cells (Rogers and Haller, 1978; Hung, 1980). Because of the low endogenous amine content in APUD cells of some animals, it is difficult to reveal NEE cells directly by fluorescence microscopy without treatment with an amine precursor. Others, without treatment, are apparently devoid of a cellular store of biogenic amines. They can be revealed by in vivo or in vitro treatment of the specimens with an amine precursor, (3,4-dihydroxypheny1)-L-alanine or ~-5-hydroxytryptophan,which demonstrates their amine-handling properties (Ericson er al., 1972; Hage, 1973a, 1974, 1980,1984;Cutz etal., 1974,1975;Walsh and McLelland, 1974;Sonstegard et al., 1976; Hage et al., 1977; Lauweryns et al., 1977; Palisano and Kleinerman, 1980; Dey et al., 1981). The fluorescence appears predominantly in the basal, paranuclear cytoplasm; the nucleus is free of fluorescence (Figs. 2 and 7). After treatment with sodium borohydride solution, the fluorescence is nullified and then reestablished by further exposure to formaldehyde vapor, demonstrating the specificity of the histochemical reaction for monoamines (Corrodi et al., 1964). As determined in intrapulmonary NEBs of the rabbit (Lauweryns et al., 1973, 1974, 1977) and in single NEE cells of the same animal (Dey et al., 1981), as well as in human NEE cells (Keith er al., 1981), where the maximum intensity of the fluorescence emission is situated between 520 and 530 nm, it seems likely that the amine involved is 5-HT. Special caution, however, is required by the fact that the maximal emission intensity of catecholamines, usually about 480 nm, may under certain conditions be situated in the wavelength range from 500 to 540 nm, thus appearing yellowish. This spectral shift of catecholamines may occur when high concentrations are present, provided the catecholamine-protein ratio
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is very high (Caspersson et d . ,1966; Corrodi and Jonsson, 1967;Jonsson, 1967a. 1971a,b; Bjorklund et d . , 1968; Bjorklund and Falck, 1973; Laszlo, 1975). An exact differentiation between the fluorophores of indolylethylamine derivatives and catecholamines can only be achieved by microspectrofluorometric readings of excitation spectra. Indeed, at neutral pH, the maximal excitation intensity for catecholamine fluorophores is usually higher (i.e., 410 nm) as compared with that of fluorophores from indolylethylamine compounds (around 385 nm). Furthermore, upon acidification. the excitation maxima of catecholamine fluorophores change from 410 to 370 nm, with additional excitation peaks at 320 nm (Bjorklund c’t al.. 1972a.b) and, in an extended excitation range from 240 to 450 nm. a peak at about 260 nm (Reinhold and Hartwig, 1982; Scheuermann et d . , 1984b). On the other hand, it was found that, in some tissues, 5-HT and its metabolic precursor, 5-hydroxytryptophan, may be present simultaneously (Hartwig and Reinhold, 1981). In addition to the excitation peak at 385 nm, excitation recordings conducted in an extended wavelength range yield a distinct clear excitation peak at 310 nm. Moreover, the relative height of the peak at 385 nm, as compared to the excitation peak at 310 nm, appears much higher for 5-HT than for 5-hydroxytryptophan (Reinhold and Hartwig. 1982). Thus, microspectrofluorometrically in an extended excitation range, it is possible to distinguish clearly the fluorophores of 5-HT from those of 5-hydroxytryptophan. Additionally, the differentiation between fluorophores of indolylethylamine derivatives and catecholamines can be performed by studying the rate of fading of the fluorescence: the decrease in fluorescence intensity of the 5-HT fluorophore upon irradiation with the most effective wavelength is more rapid than that of the catecholamine fluorophores (Jonsson, 1967b). Moreover, the final fading rate of the 5-hydroxytryptophan fluorophore is much less pronounced than that of the 5-HT fluorophore (Reinhold and Hartwig, 1982). After formaldehyde condensation, the microspectrofluorometric measurements revealed, in NEBS of several vertebrates (rabbit, pig, and turtle), a maximal emission from 520 to 530 nm and an excitation maximum at 385 nm (Fig. 5). Furthermore, the characteristic spectral shift of catecholamine fluorophores after acid treatment do not materialize. These results allow a classification of the monoamine content in the NEE cells of the lung as an indolylethylamine derivative. Also, the excitation spectra revealed a much higher ratio of the 385-310 peak as compared to S h y droxytryptophan (Fig. 6). After irradiation at the most effective wavelength, the photodecomposition is very rapid, with a loss of 50% of the original fluorescence intensity during the first minute, followed by a much
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Qamar 1
0.5
c.
200
300
400
500 nm
FIG.5 . Excitation (left) and emission (right) spectra recorded by formaldehyde-induced fluorescent, neuroendocrine epithelial cells of the neonatal rabbit. FIG. 6. Microspectrofluorometric excitation spectrum from formaldehyde-induced fluorescent, neuroendocrine epithelial cells of the neonatal rabbit. The extended excitation range from 240 to 460 nm with the characteristic height of the peaks at 385 and 310 nm makes a differentiation possible between 5-hydroxytryptamine and 5-hydroxytryptophan.
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slower fading rate and ending in a final intensity decrease of about 70% after 5 minutes (Fig. 9). Hence, these microspectrofluorometric recordings point to the presence of 5-HT. Upon staining the same section with the argyrophilic reaction, the shape, size, location, and distribution of fluorescent cells correspond to that of the argyrophilic cells (Figs. 2 and 3) (Cutz et al., 1975; Dey et ul., 1981), although the argyrophilic reaction is assumed to be less sensitive than the FIF (Palisano and Kleinerman, 1980). When staining was camed out using the argentaffin reaction, those cells, which show a basal yellow fluorescence with spectral characteristics of 5-HT, revealed dark-brown deposits in the basal cytoplasm, indicating that the yellow fluorescent cells are identical to the argentaffin cells (Dey et al., 1981; Scheuermann et al., 19834. On the basis of their fluorescence color, following formaldehyde condensation, it has been suggested (Eaton and Fedde, 1977) that the NEBs of the mouse lung may occur in two different groups, one comprising yellow, quickly fading fluorescent cells, claimed to produce and store an indolylethylamine compound, whereas the other consists of blue-green fluorescent cells producing catecholamines. Consistent with this finding, in whole mounts of the lizard lung, yellow fluorescent cells and cells which fluoresce a faint green have been observed (McLean and Burnstock, 1967b). As mentioned before, the formaldehyde-induced, typically yellowish fluorescence, is in itself, i.e., without objective microspectrofluorometric measurements, inconclusive for the critical identification of a biogenic amine. As we have reported in the turtle lung, the yellow fluorescent cells show the microspectrofluorometric characteristics of 5-HT (Scheuermann et al., 1983a; Scheuermann, 1984). They are situated in the epithelial tissue and belong to the NEBs. The blue-green fluorescent cell groups, which appear as small intensely fluorescent cells, belong to pulmonary ganglia, located in the intraparenchymal connective tissue (Scheuermann et ul., 1984b,c). After formaldehyde condensation and microspectrofluorometry, the latter cells show excitation and emission maxima at about 415 and 480 nm, respectively, corresponding to the occurrence of catecholamines (Bjorklund et al., 1975). Moreover, the fading reaction follows the typical course for catecholamines. After acidification and analysis of the relative intensities of the excitation maxima, the identification of the catecholamine at the cellular level was performed (Scheuermann et al., 1984b). In the lungs of the monkey, pig, rat, and rabbit, these two types of intensely fluorescent cells, i.e., 5-HT-containing NEBs and intraganglionic, catecholamine-containing, small intensely fluorescent cells with the characteristic microspectrofluorometric recordings, can be demonstrated as
9
0
1
2
3
4
5 min
FIGS.7 A N D 8. 5-HT fluorescence and argentaffinity demonstrated consecutively in the same tissue section of the basal portion of a NEB in the lung of the red-eared turtle. (Fig. 7) Cluster of yellow-fluorescent cells occurring in the trabecular epithelium. Note the nerve fibers. located in juxtaposition to the base of the yellow-fluorescent cells and emitting a blue-green fluorescence (arrow). x 1050. (Fig. 8) The same section as in Fig. 7 after subsequent exposure to the Masson-Hamper1 silver technique, showing argentaffinity of granular material in the basal portion of the cells (arrow). Cells displaying a basal formaldehyde-induced fluorescence are identical to those which reveal argentaffin granules in the basal portion of the cell. x 1050.
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well (D. W. Scheuermann. unpublished observations). Immunocytochemistry carried out at the light microscopic level, using a specific antiserum against 5-HT, confirmed the presence of 5-HT in both NEBS and solitary NEE cells (Cutz et a/., 1982; Lauweryns e l a / . , 1982; Sonstegard et al., 1982; Memoli el al., 1983). It should be made clear that the bluegreen fluorescent cells described in the mouse lung (Eaton and Fedde, 1977) contain catecholamines and do not belong to the NEBs, being intraganglionic, small intensely fluorescent cells located in the lung interstitium. It can be assumed that both populations of APUD cells, i.e., the NEE cells as well as the small intensely fluorescent cells of the intrapulmonary ganglia, might each serve as an origin for lung tumors belonging as they do to elements of the diffuse peripheral endocrine system (Scheuermann et a / . , 1983b).
VII. Immunocytochemistry for Regulatory Peptides Since NEE cells and NEBs of the respiratory system have many features in common with cells from the APUD series initially described by Pearse (1966, 1968, 1969), they may be expected to produce polypeptide hormones. Indeed, as revealed by recent immunohistochemical studies in paraffin sections, pulmonary NEE cells may contain a variety of regulatory peptides. Bombesin-like immunoreactivity was detected in NEE cells of the bronchial and bronchiolar epithelium of the fetal and newborn human lung, both in single cells and in groups of cells (Wharton et a/., 1978; Johnson et a / . , 1982; Stahlman e l al., 1982, 1985); it was shown to be particularly abundant in the second trimester to term (Track and Cutz, 1982). Furthermore, bombesin-like immunoreactivity was detected in NEE cells at all levels of the tracheobronchiolar tract of the adult human lung (Cutz et d.,1981, 1984; Polak and Bloom, 1984) and in NEE cells of the airways of adult rats (Marchevsky and Kleinerman, 1982). Calcitonin-like immunoreactivity was revealed in single cells as well as in some NEBs of human fetuses and neonates (Becker et al., 1980; Stahlman et d.,1982, 1985), in fetal and adult human lungs (Cutz et a / . . 1981), FIG.9. Two fading curves from NEB cells and adjacent varicose nerve fibers. demonstrating the different photodecomposition rate of both fluorophores. The lower curve shows a rapid decomposition of the fluorescence in NEB cells after irradiation with the most effective wavelength. which is especially pronounced in the first minute and finally results in a total fluorescence decrease of about 70% after 5 minutes, typical of formaldehyde-induced 5-HT fluorophore. The upper curve renders the slow and gradual decrease (-20%) in fluorescence intensity of the nerve fibers under identical conditions. i.e., typical of formaldehyde-induced catecholamine fluorophores. [Fig. 7-9 modified from Scheuermann c’t ul. ( 1983a). Reprinted with permission of publisher.]
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D. W. SCHEUERMANN
and in NEBs of adult rats (Marchevsky and Kleinerman. 1982). However, calcitonin-positive NEE cells appeared most numerous in neonates compared with fetal and adult lungs. Leu-enkephalin antiserum revealed immunostaining in the peripheral airways of the fetal, neonatal, and adult human lung, but only in a few single NEE cells (Cutz et al., 1981). This was also true, to the same extent, during hyperplasia of pulmonary NEE cells in patients with bronchiectasis and bronchial epithelial neoplasms (Memoli et al., 1983). Immunoreactivity to gastrin-releasing peptide, the mammalian counterpart of amphibian bombesin (McDonald et al., 1979; Iwanaga, 1983; Tsutsumi et al., 1983b), was found in the fetal and adult human lung, both in single NEE cells and in NEBs (Takahashi and Yui, 1983; Tsutsumi et al., 1983a,b). However, bombesin immunoreactivity should be attributed to gastrin-releasing peptidelike molecules, which are present in the human lung (Price et al., 1983; Yoshizaki et al., 1984). Consecutive sections of the fetal human lung, alternatively immunostained for 5-HT and gastrinreleasing peptide, revealed the coexistence, within the same NEE cell, of a biogenic amine and a peptide (Takahashi and Yui, 1983). Chemical and immunocytochemical studies confirmed the simultaneous occurrence of neuropeptides and amines in NEE cells and lead to the concept that the coexistence of different bioactive substances in neuroendocrine cells is the rule rather than the exception (for reviews, see Owman et al., 1973; Fujita and Kobayashi, 1974; Pearse, 1976; Hokfelt et al., 1980; Sundler et al., 1980; Fujita, 1983): Using serial sections, immunoreactivity for 5-HT, bombesin, and somatostatin have been detected in the same NEBs of the fetal monkey lung (Dayer et al., 1985). According to their immunoreactivity, four groups of NEBS can be distinguished, i.e., NEBs containing ( I ) 5-HT, bombesin, as well as somatostatin; (2) 5-HT and somatostatin; (3) 5-HT and bombesin; (4) 5-HT only. Will et al. (1985) showed that, in the monkey, NEBs may also yield cholecystokinin immunoreactivity. Whether these peptides are present in different or in the same cells remains to be clarified. Although the simultaneous localization of more than one peptidergic antigen in a single cell has not frequently been reported, the coexistence of gastrin-releasing peptide and calcitonin was demonstrated within a subpopulation of NEE cells of the human bronchial tree using a serial section technique (Tsutsumi et al., 1983b). Whether these different peptides coexist in the same secretory granules of the NEE cells is unknown, although a coexistence in the same granules was recently described for met-enkephalin and oxytocin within nerve terminals of the posterior pituitary gland (Adachi et al., 1985). Species differences exist in relation to the immunoreactivity to regulatory peptides. For instance, in cats, bombesin
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is located in .scattered NEE cells of the upper respiratory tract and only occasionally in the bronchiolar epithelium, whereas immunoreactivity to bombesin could not be confirmed in rats and guinea pigs (Ghatei et al., 1982).Apparently, the NEE cell neoplasms of the respiratory system may express a larger spectrum of neuropeptides than has been found so far in normal NEE cells, e.g., they may demonstrate ectopic ACTH immunoreactivity (Gould et al., 1983a,b; Tsutsumi et al., 1983a; Polak and Bloom, 1984; Said, 1984). Since the above-mentioned peptides revealed in the NEE cells of the respiratory tract are known to be widely distributed in the central and peripheral nervous system (Becker et al., 1979; Yanaihara et al., 1981; Yui et al., 1981), it may be that they discharge peptides either into intercellular spaces, acting at least locally as neurotransmitters and/or neuromodulators, or as circulating hormones into adjacent blood capillaries. VIII. Electron Microscopic Aspects
The NEE cells, whether occurring solitarily or clustered, may have various aspects and can be clearly recognized as intraepithelial elements (Fig. 10). The shape of single NEE cells is variable; but triangular, flask-shaped, or pear-shaped cells were regularly encountered, extending from the basement membrane to the airway surface, where they may terminate in a narrow tuft of microvilli (Fig. 18). Others, resting with a broad, basal pole on the basement membrane, prolong their narrow apical portion toward the luminal surface, without actually reaching the airway lumen; it is not unlikely that the latter transforms into the former. Grouped cells appear mostly in a palisade-like arrangement spanning the height of the epithelium between the airway lumen and the underlying connective tissue. Sometimes, the NEE cells revealed a stratification. The basal cells are oval or polygonal, resting on the basement membrane as well as interdigitating with the overlying rather pear-shaped cells with a major portion of the cytoplasm at their base. Some cells of the superficial layer contact the airway lumen by means of a narrow process (Fig. 11) (Ericson et d., 1972; Lauweryns et al., 1972; Terzakis ef al., 1972; Hung et al., 1973, 1979; Moosavi et al., 1973; Tateishi, 1973; Cutz et d., 1974; Hage, 1974; Walsh and McLelland, 1974; Lauweryns and Goddeeris, 1975; McDowell et ul., 1976; Taira and Shibasaki, 1978; Edmondson and Lewis, 1980; Johnson et al., 1980; Goniakowska-Witalinska, 1981; Scheuermann et al., 1983a,b, 1984a). As a result of the irregular course of the apical portion of these cells, it is not always possible in the same section to observe both the cell body and the apical
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process contacting the airway lumen. However, microvillous projections can be found arising from the apical surfaces. In some species, NEE cells of a NEB arising from a basal body may extend a modified or primary solitary cilium into the intercellular space or into the airway lumen (Rogers and Hailer, 1980; Scheuermann rt ul., 1983a). In transverse sections of the proximal portion of the cilium, the axonemal pattern appears as 9 + 0, representing only nine peripheral doublets without a central pair. Somewhat more distally, one of the peripheral paired tubules may be observed to be displaced gradually toward the center of the cilium, while eight pairs are arranged evenly around the periphery, resulting in an 8 + 1 axonemal configuration. An associated centriole connected to striated rootlets can be observed as a diplosomal basal structure. A similar kind of cilium has been reported in some neurons and sensory cells with a well-known receptor function (Barnes, 1961; Meyer and Bencosme, 1965; Dubois and Girod, 1970; Munger, 1971; Vigh and Vigh-Teichmann, 1973; Afzelius, 1975; Vigh-Teichmann et ul., 1976a,b. 1980; Kataoka, 1974). Although it should be borne in mind that the function of the single cilium remains unknown, the occurrence of such a modified cilium in some sensory receptor cells is suggestive of the detection of environmental conditions, such as chemical, osmotic, or local mechanical changes. Moreover, receptors on the microvillous apical cell membrane might recognize stimuli from the airway lumen and transduce them by an intracellular mechanism which triggers off or arrests the release of secretory granules. The morphological features of the apical portion of the NEE cells of the respiratory system are similar to those of the gastroenteric endocrine cells; hence, the hypothetical term of “taste cells,” proposed by Fujita and Kobayashi (1974) in their study on the gut, might be borrowed to designate similar cells in the lung. In lower vertebrates, as reported in the toad lung, when discussing light microscopic aspects, the apical surface of the NEE cells of a NEB can be observed in electron micrographs to be completely covered by an apical cell, the luminal pole of which is comparable with that of well-known receptor cells (Rogers and Hailer, 1980). This apical cell, provided with microvilli and a primary cilium, also contains microtubules, bundles of microfilaments, rough endoplasmic reticulum, and dense-cored granules.
FIG. 10. NEB in the trabecular epithelium of the red-eared turtle. The neuroendocrine epithelial cells form an organoid structure between the basement membrane and the flattened Clara-like cells (arrow) covering most of the apical surface. Some neuroendocrine epithelial cells ( * I extend from the basement membrane to the airway lumen. The capillary lumen (C) is surrounded by a thin-walled endothelium and a narrow, subendothelial space. At lower left, the pulmonary ciliated epithelium. x 5600.
FIG.I I . Superficial neuroendocrine epithelial cell of a NEB of the red-eared turtle covered by cytoplasm from Clara-like cells. The remainder opens onto the air space and bears microvilli. Intercellular junctions (arrow). Note the concentration of dense-cored vesicles in the basal portion of the cell. x 8500.
FIG. 12. Putative neuroepithelial endocrine cell in the lung of a developing red-eared turtle, observed in mitotic division. The cytoplasm contains a moderate number of characteristic dense-cored vesicles. x 10,500.
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It was proposed that the apical cell functions as a receptor-transducer cell and that the underlying NEE cells serve as an additional source of peptides-biogenic amines to be released on stimulation of the apical cell. The combination of NEE cells buried in the respiratory epithelium with a specialized epithelial cell that could serve as a chemoreceptor stimulating the secretion by contacting NEE cells was also observed in the fetal human lung (Stahlman and Gray, 1984). In the adult human tracheobronchial tract, NEE cells occur singly (Bensch r t d . , 1965; Gmelich et a / . , 1967; Basset el ul., 1971; Terzakis c’t ul., 1972; Tateishi, 1973; Bensch et ul., 1968), adjacent to the basement membrane, rarely extending to the airway surface (McDowell et ul., 1976). Conversely, in the fetal and neonatal human lung, numerous NEE cells extend from the basement membrane to the luminal surface, terminating in a microvillous border (Hage, 1973b; Stahlman and Gray, 1984). In scanning electron microscopy, the luminal surface of NEBs of the fetal rabbit (Cutz et id., 1978b) and rat (Carabba et d . , 1985) appears partially covered with nonciliated cells showing dome-shaped protrusions, which make them similar in appearance to Clara cells (Fig. 14) (Kuhn et a / . , 1974; Kuhn, 1976). Correlated scanning and transmission electron microscopy revealed that, in neonatal mouse lungs, the Clara-like cells failed to display both the large amounts of endoplasmic reticulum and the secretory granules described as characteristic for mature Clara cells (Hung et r i l . , 1979). However, numerous large mitochondria and accumulations of particulate glycogen did occur (Sonstegard et ul., 1982). The differences in ultrastructural features between Clara cells of neonatal and adult mammals may reflect the immaturity of the former (Smith et al., 1974). Yet, in the red-eared turtle, the boundary of NEBs was outlined by flattened nonciliated cells containing numerous mitochondria as well as membranebound secretory granules and conspicuous cisterns of smooth and rough endoplasmic reticulum, which are all cytoplasmic features of Clara cells (Scheuermann et ul., 1983a). Interdigitating basolateral cytoplasmic processes of NEE cells may extend between other epithelial cells (Bensch et d . , 1965; Lauweryns and Peuskens, 1969; Tateishi, 1973; Dey et ul., 1981). Along the lateral interfaces next to the lumen of adjacent epithelial cells, tight junctions have been observed in the lung of the adult rat, the adult hamster (Edmondson and Lewis, 1980), and the adult mouse (Hung et ul., 1973; Hung and Loosli, 1974). In the adult human lung, the lateral cell membranes of NEE cells are linked to adjoining cells by desmosomal structures (Hage et ul., 1977). Junctional complexes, composed of zonulae occludens, zonulae. and maculae adherens, linking NEE cells to adjacent nonendocrine cells, are reported in the toad lung (Rogers and Haller, 1978). In the red-eared turtle,
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NEE cells are interconnected and linked to adjacent Clara-like cells by small desmosomes (Scheuermann er a / . , 1983a). As yet, there is no physiological evidence that would indicate a possible coupling between NEE cells. In large NEE cell clusters of the rat and hamster lung, narrow interstitial spaces reveal expansions, forming channels in which microvilli are seen to extend (Moosavi et al., 1973; Edmondson and Lewis, 1980). These intercellular canaliculi-like spaces resemble the channels between other endocrine cells, such as the hypophysis (Rennels, 1964) and the adrenomedullary cells (Wetzstein, 1957; Coupland, 1965; Elfvin, 1965; Coupland and Weakley, 1970; Grynszpan-Winograd, 1975). Hematogenous and interstitial substances may be transported through the channels in order to reach the receptor site of the basolaterdl cell membrane of the NEE cells, to which these may respond by triggering off or arresting a hormone release, in analogy to the D-glucose activation process described in the islets of Langerhans of the rat (Niki P r ul., 1974). The nucleus, usually spherical or ovoid with some small indentations, is located in the bottle-shaped cells at the entrance to the narrowed portion as well as in the polygonal cells almost at the center. This nucleus contains patches of dense chromatin, situated along the nuclear membrane and surrounding a prominent nucleolus. The nuclear envelope shows numerous pores. In the human fetal lung (Stahlman and Gray, 1984) and in the lung of the developing red-eared turtle, a putative NEE cell was observed in mitotic division (Fig. 12). A well-developed Golgi complex is often composed of greatly distended sacculi and different kinds of vesicles, partly filled with an electron-dense content (Fig. 16). It is usually present in a supranuclear position, although sometimes a lateral Golgi complex can be observed as well. This pattern, more evident in NEE cells abutting on the luminal endings, suggests some double functional polarity. There are investigations providing morphological indications for the involvement of the Golgi complex in the formation of dense granules (Gmelich et ul., 1967; Lauweryns and Cokelaere, 1973a; Hage, 1974; Taira and Shibasaki, 1978; Scheuermann et al., 1983a). FIG.13. Long strands of rough endoplasmic reticulum with attached and free polyribosomes visualized in the lateral region of a granule-containing cell in the red-eared turtle. x 38,000. FIG.14. NEB in the bronchiolus of a neonatal rabbit is largely covered by protruding nonciliated Clara-like cells. The uncovered part of neuroepithelial endocrine cells can be recognized in a craterlike depression between the Clara-like cells, where the stubby microvillous projections of the narrow tips of these cells reach the airway lumen. Arrow indicates the exposed surface of the NEE cells. Adjacent bronchiolar epithelium is composed of ciliated cells. Scanning electron microscopy. x 3200.
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In most species, the cytoplasm contains rather small mitochondria (Ericson et a/., 1972; Cutz et a/., 1974, 1975). Multivesicular bodies, lysosomes, pinocytotic vesicles, microtubules, and a few glycogen particles are present in variable numbers. The granular endoplasmic reticulum is usually dispersed in the cytoplasm, but sometimes a configuration with attached and free polyribosomes may resemble, to some degree, the Nissl substance of neurons (Fig. 13). Bundles of branching microfilaments, frequently packed in sheaves (Fig. 15) (Cook and King, 1969; Cutz and Conen, 1972; Ericson e f a / . , 1972; Lauweryns et a / . , 1972; Terzakis rf d . , 1972; Hung et ul., 1973; Hung and Loosli, 1974; Cutz et a / . , 1975; Taira and Shibasaki, 1978; Johnson et a / . , 1980; Scheuermann et a / . , 1983a; Hung, 1984; Pack and Widdicombe, 1984; Stahlman and Gray, 1984; Pearsall ef a / . , 1985), are considered by many authors as a major distinguishing feature of these cells. Although the role of microfilaments is as yet unknown, their presence is of special importance in the pathology-oriented classification of carcinoids of the bronchopulmonary tumors (Gould et a / ., 1983b). In electron micrographs, the most striking feature of the NEE cells is the presence of numerous vesicles with granular cores, referred to as dense-cored vesicles (DCV), which vary in number, sometimes scattered throughout the cytoplasm, but frequently tending to accumulate in the broad basal portion of the cell (Figs. 17 and 19). The shape of the cells, with their deep broad pole, the location of the nucleus, and the basal position of the secretory granules indicate that the pole of discharge is directed toward the connective tissue, in which capillaries are closely adjacent to the basis of the NEE cells. These features, in addition to the fact that the capillary endothelium sometimes appears fenestrated (Lauweryns ef d . , 1974; Scheuermann et d . , 1983a), strongly suggest that the NEE cells may function as endocrine glands. The DCV are usually spherical, but can also be irregular in shape. A limiting smooth-surfaced membrane encloses the osmiophilic content entirely. Usually there is a clear halo between the dense core and the membrane, ranging in width from a few nanometers to 20 nm. In larger vesicles, the dense core may be at the center of the vesicle or adhering eccentrically to the limiting membrane. The external diameter usually ranges from 60 to 200 nm in diameter (Bensch et a / . , 1965; Cook and King, 1969; Ericson et a / . , 1972; Lauweryns et a / . , 1972; Terzakis et d . , 1972; Hung ef a / . , 1973; Moosavi et a/., 1973; Cutz et a / . , 1974; Hung and Loosli. 1974; Walsh and McLelland, 1974; Cutz and Orange, 1977; Hage et u/., 1977; Becci et a / . , 1978; Taira and Shibasaki, 1978; Johnson et d . , 1980; Hung, 1984; DiAugustine and Sonstegard, 1984; Stahlman and Gray, 1984). The size of the DCV is sometimes described as specific for each animal species.
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F a . 15. Sheaves of microtilaments can be observed in the cytoplasm between the nucleus ( N ) and the Golgi complex ( G ) of a neuroepithelial endocrine cell of the red-eared turtle. x 40,000.
FIG. 16. Golgi complexes of a neuroendocrine epithelial cell of the red-eared turtle with electron-dense materials in some Golgi vesicles, indicated the involvement of this organelle in the formation of the specific granules. x 2 6 . 0 0 .
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FIG. 17. Base of a NEB, showing numerous secretory granules concentrated near the basement membrane. Some granules (arrow) contact the plasma membrane. In the connective tissue, numerous nonmyelinated nerve fibers occur immediately below the NEB. x 14,000.
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e.g., mouse, 107 nm, and rabbit, 142 nm (Hage, 1974). However, both the size and electron density of the DCV vary greatly not only from cell to cell, but even within the same cell (Lauweryns et ul., 1972, 1974; Taira and Shibasaki, 1978; Goniakowska-Witalinska, 1981). When the core is less dense, it may show a faintly granular substructure. In some DCV, a central constriction is seen, which appears to divide the granule into two portions, containing two dense cores as in a hourglass. They possess the same fine structure as the neurosecretory granules found as a normal component in the adrenomedullary cells. Different fixation and staining procedures may affect the ultrastructural appearance of the membrane-bound granules (Chen et a l . , 1969; Matthiessen et al., 1973; Schafer et ul., 1973). The dense core of the DCV in adult human bronchial NEE cells, stained intensely with phosphotungstic acid at low pH. suggests the presence of a glycoprotein (McDowell et d., 1976). Since glycosylation of proteins in order to form glycoproteins appears to be one of the functions of the Golgi complex (Dauwalder et ul., 1972), the close relationship between maturating DCV and the Golgi complex does not seem surprising. The material contained in the halo of the vesicle may be variously extracted by the fixation fluids or by other substances used for dehydration and embedding, thus accounting for the different electron density of the granule halos (Schafer et ul., 1973). but it is hard to accept that the procedures used for tissue processing can produce different effects in the same sample on identical cell components. Ultrastructural studies have revealed as many as three types of NEE cells in fetal human lungs (Cutz and Conen, 1972; Hage, 1972, 1973b, 1980; Capella et ul., 19781, mainly on the basis of the fine-structural morphology of their cytoplasmic secretory granules. The first and most frequent cell type, called P, cells, bears small secretory granules with a mean diameter of about I10 nm. Two varieties of granules may be present: ( I ) membrane-bound, spherical granules displaying a thin clear halo interposed between the central dense core and the membrane; (2) spherical to ovoid vesicles containing a small, eccentrically situated core of variable electron density. These P, cells are provided with an extensive rough endoplasmic reticulum, an expanded Golgi complex, scattered small vesicles, and microtubules. P, cells are located in all parts of the bronchial tree of the developing lung and give a positive argyrophilic reaction, whereas only
FIG. 18. Enlargement of the luminal pole of a N E B , showing the apical portion of a neuroepithelial endocrine cell bearing a tuft of rnicrovilli. Junctional complexes (arrow).The Golgi complexes are supranuclear. The apical cytoplasm contains a moderate number of characteristic dense-cored vesicles and lysosomes. X 17,000.
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the cells with vesiculated granules are argentaffin. They can be observed forming clusters similar to the corpuscular arrangement of NEBS. The second type, called P2 cells, contains slightly larger, spherical granules with a mean diameter of about 140 nm and provided with a moderately electron-dense core surrounded by a thin transparent rim. Likewise, PI cells are found in all parts of the bronchial tree of the human fetus. The third type, P, endocrine cells, possess large, spherical, membranebound granules with a mean diameter of 190 nm, displaying a homogeneous electron-dense content. In the normal fetal human lung, these P, cells are restricted to the larger bronchial tubes, where they are present in small numbers. According to Hage (1973b), P, and P2 cells may be merely different functional stages of the same cell. In the normal adult human lung, she observed only a single type of endocrine cell, which she called Pa cells; they are provided with spherical, dense-cored. secretory granules characterized by their uniform size and homogeneous appearance. The diameter of the vesicle ranges from 110 to 140 nm (Hage et ul., 1977). There is a narrow clear rim between the core and the surrounding membrane, similar to the secretory granules of the PI cells in the fetal human lung. The P, cells of the fetal human lung and the P, cells from the adult human lung resemble certain endocrine cells in the stomach and pancreas (Solcia e t d.,1975; Capella et ul., 1978). A similar ultrastructural identification of NEE cells in the fetal human lung, which contain distinctive secretory granules, was described by Stahlman and Gray (1984). Some authors correlate these distinct types of NEE cells, defined by the features of vesicle appearance, with the three types of NEE cells distinguished according to their immunoreactivity to neuron-specific enolase, whether or not combined to 5-HT and bombesin (Polak and Bloom, 1982). However, it should be pointed out that biochemical data (Scrutton and Utter, 1968) as well as ultrastructural immunocytochemical observations on neuroendocrine cells (Zabel and Schafer, 1985) indicate that neuron-specific enolase apparently is not associated with secretory granules. Nevertheless, in electron micrographs of the fetal human lung, gastrin-releasing peptide immunoreactivity was found in cytoplasmic granules (Iwanaga, 1983) similar to the DCV of the cell type classified as P, (Hage, 1973b). FIG.19. Enlargement ofthe basal part o f a NEB. showing secretory granules of various shapes and electron density. At the upper right, a capillary separated from the neuroendocrine epithelial cell by a subendothelial space containing collagen fibers. x 28,000. FIG. 20. Part of ii NEB of the red-eared turtle stained with the Masson-Hamper1 argentaftin reaction on ii grid. Intense deposition of silver grains is shown in the granules. A slight background precipitation of silver is seen over the cytoplasm and nucleus. x 15.000.
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Some authors feel inclined to assume that every ultrastructural type of NEE cell produces one specific peptide, i.e., that each peptide is located in a different type of NEE cell (Cutz et al., 1981). However, this finding is difficult to reconcile with the observation, in serial sections of immunostained material, that the same NEE cell may contain different peptides (Tsutsurni et al., 1983b; Zabel, 1984). These peptides may even be present in the same secretory granule, as was demonstrated for met-enkephalin and oxytocin within nerve terminals of the neurohypophysis (Adachi et al., 1985). Conclusive correlation of the ultrastructurally defined types of NEE cells with the presence of specific peptides requires the application of a double immunocytochemical staining technique for the simultaneous demonstration of coexistent neuropeptides at the electron microscopic level. In fetal and adult human trachea as well as in the trachea of adult rabbits, Cutz et al. (1975) found NEE cells with only one type of spherical granules, measuring about 100 nm in diameter and displaying a homogeneous dense core surrounded by a clear space of 16-18 nm. DiAugustine et al. (1984) also described one kind of NEE cells in the trachea of the guinea pig. Conversely, in the tracheal mucosa of lamb and armadillo, Cutz et ul. (1975) demonstrated two distinct types of NEE cells whose DCV differ not only in their mean diameter, i.e., 168 versus 112 nm for the lamb and 175 versus 125 nm for the armadillo, but also in their configuration and electron density. It seems likely that NEE cells in the lung of man and various animals have ultrastructural differences particularly with respect to their DCV. This diversity could reflect species variation (Hage, 1974). but the differences might just as well be due to variations in the physiological and/or pathological state. Ultrastructural studies have revealed that a number of human bronchial carcinoid tumors and oat cell carcinomas of the lung (e.g., Bensch et ul., 1965, 1968; Toker, 1966; Gmelich et al., 1967; Hachmeister and Okorie. 1971; Hattori et al., 1972; Gould et a l , , 1983a,b, 1984) is composed of DCV-containing cells. Compared to the normal adult human bronchial epithelium, the secretory granules in these cells may display a wider range in size and shape, as well as a greater variation in both electron density and ultrastructural configuration (for reviews, see Hage et al., 1977; Taira and Shibasaki, 1978; Gould et ul., 1983a,b, 1984; Hage, 1984). This heterogeneity of the granules points to a similarity with some enterochrornafin cells of the human gastric mucosa (Pearse et al., 1970; Hage, 1973d; Solcia at ul., 1975),which reflects the common entodermal origin of the gut wall and the bronchoalveolar tree. It has been reported that morphometrical analysis, after glutaraldehyde fixation, of fetal rabbit NEBS might provide evidence for the existence
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of two types of DCV within the same cell (Lauweryns et al., 1972, 1974; Sonstegard et al., 1979). Type I DCV are wedge or ovoid, with a diameter of about I34 nm, containing an electron-dense amorphous core, usually with a narrow clear halo subjacent to the limiting membrane. In this halo, acetylcholinesterase was detected (Lauweryns and Cokelaere, 1973a). Near the center, the electron-opaque core may contain a compact deposit surrounded by a more grayish periphery extending up to the limiting membrane. Type I1 DCV have a more spherical shape with a diameter of about 112 nm and a less electron-dense core surrounded by a distinct, large, perigranular, clear halo of about 15-20 nm. Formalin pretreatment blocks the active sites of catecholamines but does not prevent the indolamines from reacting with glutaraldehyde in order to form a SchifT monobase. Since this is necessary for the subsequent reaction with potassium dichromate in order to obtain electron-opaque deposits, the formalin-glutaraldehyde-dichromate method is considered to be specific for indolamines (Wood, 1967; Jaim Etcheverry and Zieher, 1968). The latter method was applied to NEE cells, demonstrating that only type 1 DCV yield dense reactive granules, in contrast to type 11 DCV, which can be seen after uranyl acetate staining only (Lauweryns et d., 1972, 1977). This observation therefore suggests that only type I DCV contain 5-HT. In addition. the latter authors consider the possibility of a direct conversion of DCV I1 into DCV I, which may represent their mature form (Lauweryns et a / . , 1977). Thus, the differences in size, shape, and characteristics of the DCV within the same NEE cell are assumed to represent stages of granular genesis (Lauweryns et a / . , 1977; Sonstegard et al., 1979). These results might parallel the different maturation stages during the development of some granular vesicles in the adrenergic neuron (Machado, 1971). Their formation in adrenergic fibers seems to be initiated by an agranular vesicle in which the development takes the place of an eccentric small core attached to the vesicle membrane. The size of the core increases after further accumulation of dense or semidense material, finally resulting in a vesicle with a large dense core, apparently forming a mature DCV. This comparison is supported by the fact that secretory granules of NEE cells display transitional stages with regard to the density of their content. Thus, besides vesicles which are almost empty, there are granular vesicles whose dense core area is extremely small, i.e., with a large clear halo up to the limiting membrane, as well as others with a large dense core filling the vesicle nearly completely, i.e., up to the membrane. In controlled studies of hypoxia on neonatal rats, DCV reveal ultrastructural changes indicating almost the reverse phenomenon of what was suggested in connection with the development (Moosavi et al., 1973). A
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widening of the clear halo between the osmiophilic core and the limiting membrane goes hand-in-hand with a decrease in size and electron density of the core, which often becomes not only minute but eccentric, lying attached to the limiting membrane. Moreover, many empty vesicles are formed. Hence, one gains the impression that type 1 and type 11 DCV are the extremes of a continuous series of different degrees of amine and/or polypeptide filling. Thus, the type 11 DCV would contain little if any biogenic amines, which are hardly detectable by the poorly sensitive aldehyde-osmium tetroxide method. The morphological variations of DCV observed in chronic hypoxia might be considered as an enhancement of variations observed in DCV of normal controls. Quantitative fluctuations between these different types of granular vesicles occurring under normal conditions may reflect physiologic changes in the oxygen content of the inhaled air. Using Fontana’s ammoniacal silver technique at the fine-structural level (Hgkanson et al., 1971), the membrane-bound granules of the bronchopulmonary NEE cells, at least those of the frog (Rogers and Haller, 1978) and the turtle (Scheuermann et ul., 1983a, 1984a), stained selectively by silver deposits over the dense core of the vesicles, demonstrating the argentaffin reaction of the DCV (Fig. 20). This is in agreement with electron microscopic studies of Ericson and co-workers (l972), who have shown and 5-hydroxytryptophan are that tritiated 3,4-dihydroxyphenyl-~-alanine taken up and incorporated into DCV of NEE cells of the mouse lung (trachea). This indicates the presence in the granules of a synthetized amine from exogenous precursors. Moreover, electron microscopic immunocytochemical studies on NEE cells of the respiratory system of human fetuses revealed that gastrin-releasing peptide immunoreactivity is located in DCV (Iwanaga, 1983). Thus, the results obtained so far by light and electron microscopy seem to indicate that, as has also been assumed for endocrine cells in other organs, the DCV of the NEE cells belonging to the respiratory system may be considered as a storage site of biogenic amines and polypeptide hormones (Owman et ul., 1973). There is evidence suggesting that a biogenic amine, whether in combination with coexisting substances, is liberated from the NEE cells by vesicular exocytosis, i.e., direct extrusion of the entire vesicular content to the extracellular space after fusion of the vesicular limiting membrane and the basolateral plasma membrane. Invaginations of the plasma membrane, sometimes containing an amorphous material, similar in size to the DCV content, are indicative of this phenomenon (Lauweryns and Cokelaere, 1973a; Taira and Shibasaki, 1978; Scheuermann et al., 1983a). This suggests that the secretion from the NEE cells could be directed to struc-
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tures below the basement membrane, such as capillaries, smooth muscle cells, and mucosal glands. Granular release at the luminal surface has never been observed. In bronchopulmonary NEE cells of the rabbit, during hypoxia or after intake of certain drugs, such as nicotin or reserpin, the DCV are clearly shifted to the basal pole of the cell. Eventually their bounding membranes are in contact with the basal cytoplasmic membrane, a feature which is rather exceptional in normal animals (Lauweryns et d . , 1977). The content of the granular material appears to empty into the intercellular space at an increased rate by emiocytosis. This phenomenon is correlated with a decrease of 5-HT, as shown by FIF (Lauweryns et d . , 1977). IX. Location
The presence of NEE cells in the epithelium of the respiratory system of man and every vertebrate species examined is well established, although they are not evenly distributed. As early as 1949, Frohlich reported argyrophilic cells to occur mainly at the bifurcations of large and small bronchi as well as at the sites of transition from the bronchioli terminalis to the bronchioli respiratorii. This was later confirmed by several investigators (e.g., Lauweryns et d.,1972; Lauweryns and Goddeeris, 1975; Hage, 1976; Hung et al., 1979; Foliguet and Cordonnier, 1981; Cutz et (11.. 1984; Sarikas et ul., 1985a.b). Single NEE cells were found distributed over almost the entire respiratory system [e.g., larynx (Ewen et a / . , 1972; Kirkeby and Rgmert, 19771, trachea (Ericson et al., 1972; Cutz et al., 1975; Dey et al., 19x1, 19831, and bronchi and bronchioli terminalis (Lauweryns and Peuskens, 1969; Terzakis et d.,1972; Moosavi et d.,1973; Hernandez-Vasquez et al., 1977; Stahlman and Gray, 1984; Stahlman et d . , 1985)l. whereas NEBS seemed to be restricted to the intrapulmonary airways (Cutz et d.,1975). Frohlich (1949) and various later investigators reported that the number of these NEE cells increases in a distal direction up to the smallest bronchi and that the distance between NEE cells increases from the bifurcations of the bronchioli respiratorii onward. As for the upper airways, the ventral mucosa of the trachea revealed more NEE cells than the dorsal mucosa. predominantly in the cranial segment (Dey et d . , 1981),a finding confirmed in the guinea pig (Kirkeby and Rgmert. 1977; DiAugustine et a/., 1984) and rat (Kleinerman et al., 1981). In the lungs of fetal and newborn rabbits (Lauweryns e t al., 1972) and mice (Hung et al., 1979), the number of NEE cells appears high. Hemandez-Vasquez and co-workers ( 1977, 1978a) have shown the number of identifiable NEE cells in fetal rabbit to decrease from 26 to 29 days,
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followed by an increase between 29 days of gestation and I day of extrauterine life, and finally by an initial decrease after birth. The decrease observed in the rat pulmonary NEE cells after the fourth postnatal day is consistent with this pattern (Moosavi et al., 1973). In the adult rat, these cells occur predominantly in the trachea, gradually decreasing in the smaller airway branchings (Kleinerman et al., 1981). Postnatally, the NEB density and average diameter in rabbits were found to decrease in conjunction with the increase in lung volume (Redick and Hung, 1984). Systematic studies have revealed large numbers of NEE cells in fetal human lungs in the early canalicular period (Cutz and Orange, 1977). These were more numerous in proximally differentiated bronchial tubes than in terminal buds. Thereafter, a gradual decrease in the number of NEE cells was observed, although the number per airway remained unchanged (Cutz and Orange, 1977). In the lungs of infants and adult man, NEE cells were found to be more numerous in small bronchi and proximal bronchioli, as compared with major bronchi and bronchioli terminalis (Lauweryns and Peuskens, 1969; Tateishi, 1973). The number of NEE cells is generally reported to decrease with age (for review, see Cutz, 1982; Keith and Will, 1982; DiAugustine and Sonstegard, 1984; Hung, 1984; Pack and Widdicombe, 1984). In some mammalian species, after an initial increase in NEE cells demonstrable in the fetal lung, an apparent decrease close to term was reported, followed by a considerable increase at birth. Since the apparent decrease close to term may be the result of a depletion of cytoplasmic secretory material, some authors ascribed an important role in the respiratory adaptation at birth to the activity of these cells (Lauweryns et al., 1982; Hernandez-Vasquez et al., 1978a; Cutz et al., 1984; Redick and Hung, 1984). However, in contrast to other mammalian species studied, maturation of NEE cells in hamsters does not appear to have been completed at birth (Sarikas et al., 1985a). The significance of these findings is strengthened by quantitative studies in this animal, demonstrating that, I day before birth, most peripheral bronchioles were devoid of NEE cells (Sarikas et al., 1985b). Obviously, it is not inconceivable that the apparently higher frequency of NEE cells in developing lungs of some animal species might be due to their early differentiation, or possibly, also to the smaller dimensions of the fetal lung, i.e., it could be argued that, with peri- and postnatal development of alveoli and growth of the airways, the NEE cells are distributed over an enlarged surface, as a result of which they cannot readily be detected. It appears that the number, as well as the presence, of NEE cells in the different parts of the airways varies not only according to the age of an animal, but also with respect to the species involved. Therefore, in the
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author's opinion, it is not yet possible to draw a final conclusion with regard to the actual significance of NEE cells at birth.
X. Innervation Using a modified silver impregnation technique for the staining of nervous tissue, Frohlich (1949) revealed, in the bronchial epithelium of rabbits and cats, fine nerve terminals to the very surface of the NEE cells and entering NEBs. He therefore suggested that these cells constitute an afferent' chemosensitive system comparable to the specific cells in the carotid and aortic bodies. Following Frohlich, a number of investigators has observed both light and electron microscopically a distinct innervation of the single and grouped NEE cells in the pulmonary tree of various animal species and in man (Cook and King, 1969; Lauweryns et al., 1970; Hung et al., 1973; Jeffery and Reid, 1975; Hung, 1976, 1980, 1984; Taira and Shibasaki, 1978; Goniakowska-Witalinska, 1980a, I98 I ; Al-Ugaily et a / . , 1983; Stahlman and Gray, 1984). Some authors described nerve endings on NEBs only (Lauweryns et al., 1972, 1974, 1985; Lauweryns and Cokelaere. 1973a; Cutz et al., 1974; Hung and Loosli, 1974; Rogers and Haller, 1978, 1980; Scheuermann et al., 1983a); others have not observed them on solitary NEE cells (Bensch et al., 1965; Terzakis et al., 1972; Cutz et al., 1974, 1975; Hage, 1974; Hage et al., 1977; Hage, 1980), which argues in favor of a subclassification in multicellular NEBs and solitary NEE cells. Different methods have been used to differentiate the nerves associated with NEE cells in the respiratory system. In silver-impregnated sections, unmyelinated nerve endings are described near the basement membrane and surrounding the epithelial cells of NEBs in newborn infants (Lauweryns and Peuskens, 1972) and various vertebrate species (Lauweryns et ul., 1972. 1973, 1974; Hage, 1976; Hung, 1980; Scheuermann et a / . , 1983a). They apparently originate from bundles of nerve processes running in the subepithelial connective tissue, where they are ensheathed by Schwann cells. Although some nerve endings may be traced in the immediate vicinity of argyrophil NEE cells, the pronounced argyrophilia of the latter often impedes a clear recognition of nerve terminals. Some authors reported a dense network of acetylcholinesterase-positive fibers in apposition to NEE cells [lamb (Cutz and Orange, 19771, neonatal rabbit and mouse (Lauweryns and Cokelaere, 1973a), fetal rabbit (Hung, 'The author uses the terms afferent and efferent for terminals on NEE cells of nerve fibers that conduct to and from the central nervous system, respectively.
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1980), fetal rat (Morikawa et al., 1978a,b), and fetal human lungs (Taylor and Smith, 1971)l. In some species, a blue-green fluorescent nerve plexus with microspectrofluorometric characteristics of catecholamines was demonstrated by the FIF method in the subepithelial connective tissue, with presumed nerve terminals contacting the yellow fluorescent NEBS (Lauweryns et al., 1972; Hung, 1980; Scheuermann et al., 1983a; Redick and Hung, 1984). The recorded peak of maximum emission is situated at 480 nm, with an excitation maximum at 4 10 nm, characteristic for catecholamines (Bjorklund r t al., 1975). After irradiation at the most effective wavelength, the photodecomposition showed a slow, almost linear decrease in fluorescence intensity, with a loss of less than 20% of the original intensity (Fig. 9). This fading characteristic is in accordance with the results of excitation and emission maxima, arguing for a catecholamine-dependent FIF (Ritzen, 1966; Bjorklund rt al., 1972a,b), and is in contrast with those results obtained for 5-HT-containing cells. In electron microscopic studies, isolated or small groups of nerve fibers and presumed nerve terminals are reported invaginating between NEE cells of a NEB (Hung et al., 1973; Lauweryns and Cokelaere, 1973a; Hung and Loosli, 1974; Lauweryns et al., 1974; Rogers and Haller, 1978, 1980; Goniakowska-Witalinska, 1981; Scheuermann et al., 1983a; Hung, 1984; Stahlman and Gray, 1984) or in close apposition to the basolateral plasma membrane of a single NEE cell (Lauweryns ef al., 1970; Hung et ul., 1973; Jeffery and Reid, 1973; Hung, 1976; Goniakowska-Witalinska, 1980a; Stahlman and Gray, 1984). From the examination of serial sections, it appears that the same nerve fiber may innervate, after branching, several NEE cells by means of bulbous and basket endings or fusiform “en passant” dilatations (Bensch et al., 1965). As a result, the course of one nerve terminal may display a range of appearances. The axoplasm is characterized by the presence of neurotubules. neurofilaments, and small mitochondria (e.g., Hung, 1984). Sometimes, glycogen particles are condensed in larger amounts. Many nerve endings feature an accumulation of various types of vesicles. Clusters of densely packed agranular vesicles of about 60 nm in diameter are almost always present in the approximately oval nerve endings (Figs. 21 and 23) (Cutz et al., 1974; Rogers and Haller, 1978; GoniakowskaWitalinska, 1980a; Scheuermann et al., 1983a). Between the clear vesicles, a few large granular vesicles can usually be observed, ranging in diameter from 90 to 110 nm. These nerve terminals and NEE cells are separated by an extracellular space, about 20 nm wide. Some authors reported nerve processes forming junctions with NEE cells, quite specific for synapses. Features characteristic of these synapses are the presence of cytoplasmic
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densities attached asymmetrically to apposed plasma membranes of both the nerve fiber and the NEE cell, as well as numerous small clear vesicles forming clusters close to the junctional material (Lauweryns ef u / . , 1970, 1972. 1974; Lauweryns and Cokelaere. 1973a; Rogers and Haller, 1978; Scheuermann et ( I / . , 1983a; Hung, 1984; Stahlman and Gray, 1984).These structures display the characteristics of efferent cholinergic nerve endings. Furthermore, electron microscopic observations have shown that some adrenergic nerve fibers appear related not only to blood vessels, but also to NEE cells, forming distinct synaptic contacts (Rogers and Haller, 1978). In addition to clear vesicles, these are characterized by small granular vesicles of about 60 nm in diameter, typical of adrenergic nerve varicosities (Rogers and Haller. 1978; Scheuermann et uI., 1983a; Stahlman and Gray, 1984).They can be correlated with the nerve endings observed using the FIF method, since this kind of vesicle is shown to store noradrenaline (Bisby and Fillenz. 1971). Rogers and Haller (1978) argue that the function of the adrenergic nerve fibers might be efferent, since there is no indication of transmission from the NEE cells to the nerve varicosities. In certain vertebrates. two kinds of synaptic regions may be recognized on the same afferent nerve terminal, arranged side-by-side. One region is postsynaptic' and the other presynaptic' to the NEE cell. In the former, an accumulation of small, dense-cored, and clear vesicles occurs along the surface of the presynaptic membrane thickenings in the cytoplasm of the NEE cell. In the latter synaptic component, clusters of small, agranular vesicles (25-50 nm in diameter) are aggregated near dense presynaptic projections on the surface membrane of the NEE cell, without accumulation of dense-cored vesicles in the NEE cell (Rogers and Haller, 1978). These complex paired synaptic contacts were described for the first time as reciproccd synupses in the central nervous system (Reese and Shepherd, 1972). In the peripheral nervous system, they were encountered in the carotid body (King et a / . , 1975; McDonald and Mitchell, 1975; Osborne and Butler, 1975) and cardiac ganglia (Yamauchi el d.,I975a,b). It has been suggested that the regions of reciprocal synaptic junctions, where the nerve terminal is postsynaptic to the NEE cell, are involved in a synaptic mechanism from NEE cell to nerve terminal, while the adjacent synaptic component interacts in the reverse direction. Nerve profiles containing another type of filled vesicle with a wider range in size (80-225 nm in diameter) and a moderately electron-dense content appear in apposition to single NEE cells and to NEBS (Stahlman and Gray, 1984), without yielding synaptic structures. These large granular 'The author uses the tcrms presynaptic and postsynaptic to designate the direction of synaptic transmission.
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vesicles may be compared with similar granules observed in peptidergic neurosecretory systems (Rinne, 1966; Bargmann et af., 1967; Krisch, 1974; Gibbins and Haller, 1979; Helen and Hervonen, 1981), which are presumed to belong to nonadrenergic, noncholinergic pathways (Baumgarten et a l . , 1970; Burnstock, 1982). Other enlarged nerve endings on NEE cells are crowded with slender mitochondria (Fig. 22). These terminals have been observed close (6-20 nm) to the plasma membrane of NEE cells of the respiratory system in different mammalian species, e.g., in the mouse (Hung et af., 1973) and rabbit (Lauweryns and Cokelaere, 1973a), and in nonmammalian species, e.g., in birds (Cook and King, 1969; King et al., 1974) and reptiles (Scheuermann et ul., 1983a). Some authors considered these nerve endings as characteristic for sensory (afferent) nerve fibers (Cook and King, 1969; Lauweryns and Cokelaere, 1973a; King et al., 1974; Rogers and Haller, 1978; Hung, 1980; Stahlman and Gray, 1984), with mitochondria serving as a source of energy for the transformation of the stimulus into a nerve impulse. As revealed by serial sections of the turtle lung, the number of mitochondria appeared to vary greatly from one region of a given nerve ending to another (Scheuermann et al., 1983a), confirming observations on nerve terminals in the carotid body (Verna, 1973). In comparison with the innervation of other tissues, the nerve terminals, tightly packed with mitochondria, are mostly considered as sensory (Rees, 1967; Bock et al., 1970; Chiba and Yamauchi, 1970; Kobayashi, 1971; Kondo, 1971; Munger. 1971; Chiba, 1972; Verna, 1973; King et al., 1974). Other authors attributed the accumulation of mitochondria to a degenerative change associated with a process of aging (Seitelberger, 1971; Leonhardt, 1976). However, in the latter case, mitochondria display various stages of disintegration and transitional forms between mitochondria
FIG.21. Cross section through a bundle of subendothelial nerve fibers in the lung of a red-eared turtle near neuroendocrine epithelial cells. Some nerve fibers, partially encased by Schwann cell cytoplasm, contain ( I ) peptidergic granules (PI,(2) cholinergic granules (arrow), (3) adrenergic nerve varicosities (arrowhead). x 19,000. FIG.22. A nerve ending associated with a neuroendocrine epithelial cell in the red-eared turtle lung. No synapse is present. but the nerve ending is densely packed with numerous slender mitochondria. x 47,000. FIG.23. Nerve terminal on the perikaryonal region of a neuroendocrine epithelial cell in the red-eared turtle lung filled with small clear vesicles. Part of a synapse is visible between the arrows. The basal lamina of the neuroendocrine epithelial cell runs at the outer side of the nerve terminal. x 56.000.
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and dense bodies. Nevertheless, in the lateral vestibular nucleus of the rat, dendritic growth cones packed with slender mitochondria are suggestive of regeneration (Sotelo and Palay, 1968). Alternating degenerative and regenerative processes might be considered to occur in nerve fibers abutting the NEE cells as suggested for nerve terminals in the carotid sinus (Knoche and Addicks, 1976). Although no strictly morphological criteria exist for the establishment of the afferent or efferent nature of the NEE cell innervation, selective nerve degeneration experiments are of considerable importance. It was shown that, after unilateral cervical infranodose vagotomy, degenerating intraepithelial axons appeared in the trachea of both the rat (Hoyes and Barber, 1981) and the cat, as well as in the bronchi (Das et a / . , 1979). This is in agreement with the concept that there exist intraepithelial afferent nerve fibers in the tracheobronchial tree. Recently it was shown that most axon endings in the NEBS rapidly degenerate after unilateral sectioning of the homolateral vagus nerve below the nodose ganglion, a process which does not take place after homolateral supranodose vagotomy. Hence, it appears that the cell bodies of these nerve endings are located in the nodose ganglion (Lauweryns and Van Lommel, 1983; Lauweryns et a / . , 1985). Selective labeling with tritiated amino acids of the nodose ganglia proved that the wall of the respiratory system possesses an afferent innervation, at least in the adult hen (Bower et al., 1978), where labeled fibers are observed to enter groups of NEE cells, suggesting a role as receptor. Although great differences in the innervation of the lung may exist among animal species (Richardson, 1979), it appears that the NEE cells are provided with a cholinergic, adrenergic, and nonadrenergic, noncholinergic innervation. The cholinergic and adrenergic nerve terminals are generally considered to be stimulatory, the nonadrenergic, noncholinergic nerve terminals playing an inhibitory role. Although the unequivocal identification of afferent nerve terminals remains difficult, the presumed function of NEE cells, i.e., subserving as chemoreceptors of the airways, leads to assume the existence of an afferent innervation. Finally, it should be mentioned that the morphology and histochemistry of the pulmonary NEE cells were compared to the structure of type 1 cells of the carotid body. The dual innervation with afferent and efferent pathways as well as the morphological features that are produced after hypoxic conditions have both cell types in common (e.g., Keith and Will, 1982; Gould et d.,1983b; Becker, 1984). Nonetheless, the NEE cells differ from principal cells of the carotid body in several respects. For instance, the former contain 5-HT, while type I cells of the carotid body store catecholamines (for review, see Verna, 1979). Moreover, the latter are situated
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in the carotid body among ganglionic cells, which were never observed between NEE cells. In addition, NEE cells seem to react directly to hypoxia of inhaled air, whereas, in contrast to cells of the carotid body, these do not respond to hypoxemic conditions (Lauweryns et al., 1977; Cutz et a / . , 1982). In consequence, NEE cells do not appear to be vascular chemoreceptors. Conversely, granule-containing cells of the intrapulmonary ganglia have many structural features in common with both type I cells of the carotid body and glomus cells of the aorticopulmonary bodies (Verna, 1979; Papka, 1980; Bock, 1982; Scheuermann et a / . , 1984b). The latter three types of cells contain catecholamines in their secretory granules and, being associated with ganglionic cells, are thought to have a receptor-secretory function. It should be given thought whether the reaction of the lung to hypoxemic conditions may be effectuated through stimulation by vascular chemoreceptors, including the granule-containing cells of the pulmonary ganglia and their release of catecholamines, since the latter substances may produce vasoconstriction in the lung (Bergofsky, 1980; Becker, 1984). The functional relations between the NEE cells, sensing oxygen levels in the pulmonary airways, and the vascular chemoreceptors to oxygen levels in the blood, e.g., the carotid body, the aortic bodies, and perhaps the granule-containing cells of the pulmonary ganglia, are not yet fully understood. A further investigation of the efferent and afferent innervation pattern of the receptors located near gas and blood in the lung is thus required. XI. Concluding Remarks
To date, the typical histological, histochemical, and ultrastructural characteristics as well as the bioactive substances of NEE cells of the lung are fairly well-known. The function of these cells, however, is at present far from elucidation and therefore remains subject to speculative thought. The shape of the NEE cells, whether solitary or grouped into clusters, with a narrow apical portion bearing villous projections into the airway lumen, is indicative of a receptor function. The basal portion is found adjacent to capillaries and may be synaptically connected with varicosities of subepithelial nerve fibers. Ultrastructurally, the nervous connections are suggestive of both afferent and efferent innervation. Furthermore, most of these cells are located in strategic positions at the bifurcations of the bronchial tree. It seems likely that these structures perceive changes in
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the intraluminal environment of the lung, upon which they respond by releasing their secretory products. This assumption is supported by the fact that the NEE cells are degranulated by hypoxia releasing 5-HT. Possibly 5-HT, presumably released in association with polypeptides, could then influence their specific target cells via synaptic structures, local action, or in a vascular way. The central nervous system may modulate, by means of efferent cholinergic nerve fibers, the release from NEE cells of 5-HT and/or polypeptides in response to intraluminal stimuli, e.g., changes in the airway gases. This assumption is supported by the fact that synaptic contact between nerve terminals and NEE cells has been demonstrated with certainty. Thus, the secretory products released from NEE cells may activate afferent nerve terminals, evoking local or systemic reflex changes. The release and diffusion of neurally active substances into nonsynaptic intercellular spaces may provide a morphological basis for a paracrine function, e.g., a local response of the bronchial and vascular smooth muscle and perhaps of the intrapulmonary neuronal plexuses. Moreover, the release of 5-HT and/or polypeptides may be transported by the blood stream, either systemic or specific, from the NEE cells to remote targets. The regularity with which capillaries, at times fenestrated, are observed in proximity to the enlarged basal foot of NEE cells provides a strong indication that bioactive substances secreted by the NEE cells diffuse into the blood circulation, making them widely accessible. Much work remains to be done in the field of lung endocrinology. An improved knowledge of the secretory activities of the NEE cells and a clarification of the functional relationships between these cells and the nervous system represent challenges demanding further research. REFERENCES Adachi. T., Hisano, S.. and Daikoku, S. (1985). J . Hisrochem. Cytochem. 33, 891-899. Afzelius, B. A. (1975). In "Handbook of Molecular Cytology" (A. Lima-de-Faria. ed.), pp. 1219-1242. North-Holland Publ., Amsterdam. Al-Ugaily, L. H., Pack, R. J . . and Widdicombe, J . G . (1983). J . Physiol. (London) 340,54P. Ballard, K. J . , and Jones. J . V. (1971). J . Physiol. (London) 219, 747-753. Ballard, K . J . . and Jones, J . V. (1972). J . Physiol. (London) 227, 87-94. Bargmann, W., Lindner, E., and Andres, K. H . (1967). Z . ZelUorsch. 77, 282-298. Barnes, B. G . (1961). J . Ulrrustruct. Res. 5, 453-467. Barter, R . , and Pearse. A. G. E. (1953). Nutitre (London) 172, 810. Barter, R . , and Pearse, A. G . E. (1955). J . Purhol. Bucreriol. 69, 25-31. Basset, F., Poirier, J . , Le Crom, M., and Turiaf, J . (1971). Z . Zellforsch. 116, 425-442. Baumgarten, H. G . , Holstein, A.-F., and Owman, C. (1970). Z . Zellforsch. 106, 376-397. Becci, P. J . , McDowell, E. M., and Trump, B. F. (1978). J . Null. Cuncer Inst. U.S.61, 551-561.
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INTEKNATIONAL KI;.VIEW 0 1 : CYTOLOGY. VOI. IM
Intrinsic Nerve Plexus of Mammalian Heart: Morphological Basis of Cardiac Rhythmical Activity?' JOSEF MORAVECA N D MIREILLEMORAVEC Utiitk dc~Patliologie Ctrrtliovtrsc.rrluirt~tie I'ltistitrct Nationcil de In Stititc; ot tte Itr RechivchtJ MPdicwle ( I N S E R M ) , Hfipittrl LPon Bertiurd, 94456 LirneilBrPvmnes CPdex, Frcinw
I. Introduction
The purpose of the rhythmical beats of the heart remained unexplained until the early seventeenth century, when Harvey (1628) made his important discovery that the heart beats in order to circulate the blood (cf. Noble, 1979). However, even after this decisive step, the mechanism of the initiation of cardiac beats remained obscure. For several centuries, the inherent rhythmicalactivityof the heart has beenconsidered as amajor example of vital forces. At the same time, different mechanistic conceptions have been postulated. Each investigator tried to use the knowledge of physics and chemistry available to him in order to assess the process of heartbeating. At present, the heartbeat is considered an electromechanical process initiated by the nodal tissue (Noble, 1979) and distributed through the ventricular myocardium via the conduction system. The microanatomy of the latter has been progressively elaborated since the original descriptions of Purkyne (1843, Aschoff (1910). Tawara (1906). and Keith and Flack (1907). More recently, various components of the intracardiac conduction tissue have been thoroughly examined by modern electrophysiological methods (Weidman, 1967; Noble and Tsien, 1968; Rougier ~t d., 1969; Winegrad. 1979; Coraboeuf, 1982). This allowed the identification of different transmembrane currents, which are now considered as responsible for the repetitive depolarizations of pacemaker cells and for the normal intracardiac conduction. The fact that some of the specialized cells can continue to fire even upon their isolation (Cranefield, 1978; Noble, 1979) led to the contention that most of their rhythmical activity depends on kinetic properties of different ionic channels, which were shown to coexist in their cell membranes (Noble and Tsien, 1968; Rougier et d., 1969; Coraboeuf, 1982). 'This work i\ a tribute t o J. E. Purkyne on the occasion of the forthcoming bicentennial of his hirth (December 17. 1787).
89 C'opyrlght (11 19x7 hy Av;idmuc Pru\\. Inc. All right\ of reprodoclion in any form rewrved.
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JOSEF MORAVEC AND MlRElLLE MORAVEC
However, as Cranefield (1983) pointed out, it is not certain that the electrical behavior of disaggregated cells, which are used for the voltageclamp and voltage-patch studies, is wholly comparable to those that can be observed in situ. The cells of the conduction system of the heart were shown to constitute complicated networks in which operate not only several types of specialized cells (James, 1973; Cranefield, 1983), but also connective elements (Thornell et al., 1976), nerve fibers (Bojsen-Mgiller and Tranum-Jensen, 1972), and ganglionic cells (Moravec et af., 1985; Moravec and Moravec, 1984). The existence of multiple intercellular interactions can thus be expected at different levels of the conduction system. This would explain why the resting potential, source impedance, and intercellular coupling vary from one part of the heart to the other (Cranefield, 1983) and why some of the electrical properties of one single cell can change upon its isolation from the neighboring structures (Mendez et al., 1969). It became evident that one part of the above pluricellular network can impose a decisive load on its other parts. The nature of the latter might be very variable. Apart from direct electrical stimulation via the electrical synapses or through local membrane currents, the electrical properties of specialized cells can be modulated by mechanical stretch (Brooks and Lu, 1972; Irisawa, 1978) and by neurotransmitter release from intracardiac nerves (Sarnoff and Mitchell, 1962; Jacobowitz, 1967; Randall, 1976) as well as from nonneuronal storage sites (Lignon and Le Douarin, 1978; Pollack, 1978). The appropriate interaction between different components of the conduction system and a permanent feedback from the surrounding structures seems to be necessary for the initiation and propagation of the excitation wave to proceed optimally (Cranefield, 1983). The contribution of the nervous system to the control of cardiac pacemaker is believed to vary from one animal species to another. The crustacean hearts seem to be entirely neurogenic (Irisawa, 1978). In this case, the triggering signal is provided by a cell-driven oscillator of the cardiac ganglion (Selverston and Moulins, 1985). A similar situation was also shown to occur in the leech, where rhythmical depolarizations of the heart are under the control of an oscillatory network composed of a group of ganglionic cells interconnected by reciprocal inhibitory synapses (Stent et al., 1979). In contrast, the generation of rhythmical heartbeats in mammals has been considered essentially myogenic (Irisawa, 1978), resulting from slow diastolic depolarizations of pacemaker cells (Noble and Tsien, 1968) or from subthreshold oscillations of their membrane potentials (Irisawa, 1978; Cranefield, 1983; Goto, 1986). The role of the nervous system is often restricted to an external modulation of the inherent muscular pacemaker
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91
(Randall, 1976; Levy and Martin, 1979). This situation results from the fact that current knowledge of cardiac innervation of the mammalian heart has been derived from classical, physiological, and microanatomical observations (Dogiel, 1882; Gaskell, 1886; Langley and Orbelli, 1910; Nonidez, 1939; Kuntz, 1953; Randall, 1976). According to these studies, the pacemaker area of the heart receives abundant cholinergic and adrenergic postganglionics originating from the extracardiac ganglia (Randall, 1976; Levy and Martin, 1979; Armour and Hopkins, 1984). However, apart from this extrinsic component, abundant intramural ganglionic cells can be identified in light and electronic microscopy, mainly at the level of the interatrial septum and all along the sulcus terminalis (Kuntz, 1953; Jacobowitz et al., 1967; Abraham, 1969; Ellison and Hibbs, 1976; Papka, 1976; Rossi, 1978). In rat and in other rodents, some of these ganglionic cells seem to be structurally associated with different portions of the intracardiac conduction system (Nielsen and Owman, 1968; Weihe et al., 1984; Moravec and Moravec, 1984). This finding may suggest that, also in mammals, some of the electrophysiological properties of the intrdcardiac conductive tissue may result from the intimate cooperation between specialized cells and the intrinsic nervous components. In other words, the conduction system of the heart should no longer be regarded as a specialized muscular tissue, but rather as a highly differentiated neuromuscular organ (Oppenheimer and Oppenheimer, 1912; Wensing, 1965; Bojsen-MZller and Tranum-Jensen, 1972; Irisawa, 1978; Moravec and Moravec, 1984).
11. Autonomic Innervation of the Heart
A. GENERAL ORGANIZATION, ANATOMICAL, A N D PHYSIOLOGICAL EVIDENCES FOR DUALINNERVATION According to classical physiological (Sarnoff and Mitchell, 1962; Randall, 1976; Armour and Hopkins, 1984) and histochemical studies (Jacobowitz et a / . , 1967; Ehinger et al., 1968; Abraham, 19691, the autonomic nerve supply to the heart of different vertebrates is still considered in terms of Dale’s principle, i.e., in terms of a dual (adrenergic and cholinergic) innervation (Dale, 1953; Yamauchi, 1973; Levy and Martin, 1979; Armour and Hopkins, 1984). This separation of the autonomic nervous system into its two major divisions is based on the original observations of Gaskell and Langley (Gaskell, 1886; Langley. 1921) concerning the anatomy and pharmacology of the autonomic nervous system. However, the possibility of the existence of a third category of the autonomic system has been
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suggested as early as in 1898, when Langley introduced the term “intrinsic nerve system.” He also suggested that peripheral nerves act on different tissue “receptors” (cf. Brooks, 1981). Today this field has been reopened; new transmitters and new receptors have been identified (Burnstock, 1969; Gershon, 1977; Lundberg et al., 1984), and the role of the intramural plexuses is now being reexamined (Burnstock and Bell, 1974; Brooks, 1978; Leranth and Unguary, 1980; Nozdrachev and Vataev, 1981; Wood, 1981; Weihe et al., 1984). It seems to be accepted that sympathetic and parasympathetic fibers feed into the intramural plexuses of peripheral organs which, per se, can mediate reflexlike reactions and exert a local control (Hillarp, 1959; Yamauchi, 1%9; Selverston et al., 1976; Brooks, 1981; Wood, 1981). Some authors do not hesitate to extend these new concepts also to the field of regulatory mechanisms of cardiovascular function (Brooks and Lange, 1977; Priola et al., 1977; Brooks, 1981; Drake-Holland et al., 1982; Weihe et al., 1984). B.
SYMPATHETIC
INNERVATION
OF THE
MAMMALIAN HEART
I . Efferent Sympathetic Pathways to the Heart The cell bodies of the preganglionic sympathetic neurons are located in the intermediolateral columns of the upper eight thoracic segments of the spinal cord (Henri and Calaresu, 1972). The preganglionic fibers emerge from the spinal cord through the white rami communicates of the first six thoracic segments (Gaskell, 1886; Randall et al., 1957; Seagdrd et al., 1978; Levy and Martin, 1979) and enter the paravertebral chain of sympathetic ganglia. In some animals, the preganglionic sympathetic fibers can also arise from the spinal cord via the ventral roots of the last two cervical segments (Levy and Martin, 1979). Most of these sympathetic preganglionics transit throughout the ipsilateral stellate ganglion and pass through the ventral or dorsal limbs of the ansa subclavia to the inferior cervical ganglion. The synapses between the preganglionic and postganglionic neurons are believed to take place in the cervical and upper thoracic ganglia including the stellate ganglion (Kuntz, 1953; Wacksman el al., 1969; Wechsler et al., 1969; Tollack et al., 1971; Levy and Martin, 1979). Only few sympathetic spinal fibers reach the heart directly without interruption (Kunz, 1953; Brown, 1967). The presence of these sporadic preganglionic fibers may have a serious impact on the control of cardiac function: they can be expected to innervate a distinct population of the intracardiac ganglion cells. A bulk of these noninterrupted presynaptic fibers (large myelinated fibers of ACYcategory) has been identified in the ventrolateral cardiac nerve which supplies selectively the atrioventricular
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junction of the dog heart (Seagard et ul., 1978). It is not known whether a similar situation also prevails in other species, but it should be noted that a recent electron microscopic analysis of serial sections of the interatrial septum of the rat heart (Moravec and Moravec, 1984; Moravec et ul., unpublished data) has revealed the presence of adrenergic neurons in this area. The traditional affirmations concerning the absence of sympathetic ganglia in the terminal nerve plexus of the mammalian heart (Hirsch et al., 1963; Jacobowitz, 1967; Yamauchi, 1973; Randall, 1976; Levy and Martin, 1979; Armour and Hopkins, 1984) will possibly need a detailed verification. 2 . Afferent Symputhetic Fibers from the Heart In addition to the above efferent nerve fibers, the sympathetic nerves of the heart also contain afferent nerve fibers which, like the corresponding preganglionics, are the components of the first to the sixth thoracic nerves (Kuntz, 1953). At least two types of afferent fibers can be distinguished by means of studies of conduction velocities and according to fiber diameters (Armour rt al., 1975; Seagard et al., 1978): myelinated A6 fibers (2-5 k m in diameter) and unmyelinated C fibers (below 1 km). Reflexes carried by these fibers may play a role in the fine regulation of cardiac performance (Uchida, 1979; Malliani, 1979) and in the control of cardiac rhythm (Shepherd, 1985). The possible involvement of these fibers in the transmission of anginal pain has been suggested by curative effects of the sympathectomy or stellate ganglionectomy (Uchida et al., 1971). The majority of the sympathetic reflexes is centrally coordinated. However, the existence of short reflex loops, mediated by either mediastinal (Armour and Hopkins, 1984) or stellate ganglia (Bosnjak et al., 1982), is not excluded. In addition, the existence of the intramural axonal reflexes should also be considered (Leranth and Unguary, 1980; Brooks, 1981).
c. PARASYMPATHETIC I N N E R V A T I O N OF THE MAMMALIANHEART I. Efferent Purusymputhetic Pathways to the Heart The parasympathetic efferent innervation of vertebrate heart involves two cholinergic synapses arranged in series. The presynaptic nerve fibers are provided by the right and left vagi (Randall, 1976). These fibers originate from the ipsilateral regions of the brain stem, e.g., from nucleus ambiguus and dorsal motor nucleus of the vagus (Weiss and Priola, 1972; Levy and Martin, 1979). Some cardioinhibitory neurons can also be detected in the intermediate zone of the dog and some other species so far examined (Cabot and Cohen, 1980; Armour and Hopkins, 1984). They
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JOSEF MORAVEC A N D MIREILLE MORAVEC
synapse on intracardiac ganglionic cells that are mostly associated with the subepicardial layer of the posterior aspect of the atria (Kuntz, 1953; Loffelholz, 1982; Moravec and Moravec, 1984). The cardiac postganglionic nerve fibers differ from motor nerves supplying skeletal muscles in that they terminate by extensive varicose end fibers, ramifying in the effector organ, from which acetylcholine is released by action potentials invading en passant one varicosity after the other (Loffelholz, 1982). As to the neuromuscular cholinergic synapses, they have been considered as quite rudimentary. Some authors (Boeke, 1936; Abraham, 1969) have even concluded that they are absent. Only recently, small cholinergic synapses similar to those of skeletal muscle spindles could be demonstrated in the electron microscope material, at least at the level of the atrioventricular junction (Moravec-Mochet et al., 1977). In some cases, the terminal cholinergic fibers run parallel with the preterminal adrenergic fibers of the autonomic ground plexus (Hillarp, 1959; Ehinger et ul., 19701, and, occasionally, cholinergic and adrenergic fibers can share the same Schwann cell sheath. This led to the contention that some of the vagosympathetic interactions (Levy and Martin, 1979) may result from this particular structural configuration (Napolitano et al., 1965; Ehinger et al., 1970; Brooks, 1981). However, direct proof that the two fibers are really functionally interacting is lacking. 2 . Afierent Parasympathetic Fibers from the Heart The presence of afferent fibers in nearly all vagal branches to the heart was suggested by the electrophysiological studies (Nettleship, 1936; Sleight and Widdicombe, 1965; Oberg and Thoren, 1972; Coleridge et NI., 1973). They could be subdivided into two categories: medullated (myelinated) fibers and unmedullated (unmyelinated) C fibers. The receptors associated with the myelinated fibers predominate in the atria (Shepherd, 1985). They have been also described in the right and left ventricles and in coronary vessels (Sleight, 1979). However, they are rather sparse and their reflex effects unknown. The majority of ventricular receptors with unmyelinated C fibers is confined to the left ventricle (Coleridge et ul., 1964). They are involved in depressor reflexes such as the pronounced bradycardia elicited by left ventricular distension (Thoren, 1979) and in body fluid volume regulation (Oberg, 1979). It has been also suggested that these receptors are essential for the circulatory response to acute exercise and to other stressful conditions. Their stimulation may prevent an excessive tachycardia normally occurring at high levels of circulating catecholamines. In contrast to the profusion of data concerning the physiological role of vagal cardiac mechanoreceptors, relatively little is known about their structure. Their identification in the light microscope (methylene blue or
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95
silver staining) proved not to be easy. Most of the structures described (i.e., compact encapsulated endings and diffuse unencapsulated endings) are derived from myelinated axons (Nettleship, 1936; Nonidez, 1939; Khabarova, 1961; Abraham, 1969; Floyd, 1979). The contribution, if any, from unmyelinated fibers is not known. The latter are believed to subserve the diffusely distributed end-nets (Floyd, 1979; Shepherd, 1985).
D. FINESTRUCTURE OF INTKACARDIAC
MECHANORECEPTORS
Our knowledge of the fine structure of cardiac receptors supplied by either sympathetic or parasympathetic fibers is also rather sparse (TranumJensen, 1975; Yamauchi, 1979; Moravec and Moravec, 1982). As pointed out by Tranum-Jensen (l979), the uncertainties and limitations relating to the ultrastructural identification of cardiac receptors are determined by the arbitrary choice of tissue samples, selected on the basis of the preliminary light microscopic examination (Floyd, 1979). The detailed description of the intracardiac sensory corpuscles and, mainly, the understanding of their relations to the surrounding structures are not possible without the systematic use of serial semithin and thin sections (Thaemert, 1970; Moravec-Mochet et al., 1977; Moravec and Moravec, 1982). Until present, the existence of two types of intracardiac mechanoreceptors has been suggested by electron microscopy studies. ( I ) Unencapsulated end organs (baroreceptors), associated with 5- and 10-pm-thick myelinated fibers, have been described in the subendocardium of the right and left atria of the pig (Tranum-Jensen, 1979) and rat hearts (Moravec and Moravec, 1982). These terminals were covered by a basement membrane and partly devoid of Schwann cell sheath. Their cytoplasm contained small filiform mitochondria (0.2 x 1.0 pm), bundles of microfilaments, and numerous vesicles of the endoplasmic reticulum associated with abundant glycogen granules. Similar structures were also described in the rat aorta (Yamauchi, 1979) and in human atria (Chiba and Yamauchi, 1970). (2) Muscular spindlelike structures, previously suggested by Lawrentjew and Gurritsch-Lasowskaja (1930), have been described in the rat atrioventricular junction (Moravec-Mochet et al., 1977; Moravec and Moravec, 1982). In this case, agglomerates of nodal cells, surrounded by a common basal lamina and elaborated connective sheath, were innervated by efferent cholinergic fibers terminated by small en grappe synapses. Other types of spiral fibers wrapped the same nodal cell corpuscles and interpenetrated into deep invaginations of muscular cell membranes, in a manner which was similar to the afferent fibers of skeletal muscle spindles (Landon, 1972; Ovalle, 1972). The absence of any basal lamina in the intermembrane space (less than 20 nm) as well as the characteristic cytoplasmic content
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JOSEF MORAVEC AND MIREILLE MORAVEC
of these relatively large nerve endings (numerous small mitochondria, abundant glycogen granules, and few small vesicles), strongly suggested that the above spiral fibers were sensory in nature. Similar coiled endings were formerly described using optical microscopy with samples of right and left ventricular myocardium (King, 1939; Plechkova, 1948; Khabarova, 1961).According to a more recent study of silverstained serial sections of the entire rat heart (Moravec et ul., 1985). these spindlelike structures are selectively located in the upper portion of the interventricular septum and all along the specialized tissue of the atrioventricular junction, namely, in the reticular portion of the atrioventricular node and in proximal portions of the right and left branches (Moravec et a / . , 1985).This association of the sensory innervation with the specialized tissue may have a functional significance (Brooks and Lu. 1972; Pollack, 1974), which we shall discuss in the following sections.
111. Intracardiac Ganglionic Cells
As the vagus nerves enter the chest, they pass in the vicinity of the cervical sympathetic ganglia. The postganglionic adrenergic and preganglionic cholinergic fibers then course together to the heart, sharing some of their satellite cells (Nonidez, 1939; Kuntz, 1953; Levy and Martin, 1979). They ramify in the pericardiac nerve plexus located between the aortic arch and pulmonary veins and supply both the heart and great vessels (Nonidez, 1939; Kuntz, 1953; Randall et al., 1972; Goldberg and Randall, 1973; Levy and Martin, 1979). The microanatomical description of these mixed cardiac nerves was considerably improved by the recent physiological studies of Randall and colleagues (Hageman et al., 1975; Hondenghem et al., 1975; Goldberg and Randall, 1973; Randall, 19761, who studied the respective contributions of sympathetic and parasympathetic fibers to different cardiac nerves of the dog heart. They also established the cartography of peripheral projections of the individual cardiac branches to the epicardial layers of the left and right cardiac cavities. According to these studies, the proportions of sympathetic and parasympathetic efferents are variable in different left and right branches. So are the respective contributions of the left- and right-side nerves to the innervation of different cardiac structures, such as the sinoatrial node and the atrioventricular junction. It has been demonstrated that, for embryological reasons (Taylor, 19771, the pacemaker area receives the branches from the right vagosympathetic trunk, while the atrioventricular junction is innervated quasi-entirely from the left side (West and Toda, 1967; lrisawa et al., 1971; Hondenghem et al., 1975). However, species differences in the overlap and in the specificity of the projection areas of cardiac nerves
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(Burnstock, 1969; Yamauchi, 1969; Cabot and Cohen, 1980; O’Shea and Evans, 1985) are not negligible. A. EVIDENCE FOR INTRINSIC GANGLIONIC CELLS One of the most significant contributions of the above physiological studies was the confirmation of the earlier morphological observations (Lawrentjew and Gurwitsch-Lasowskaja, 1930; Vitali, 1937; Field, I95 I ; Hirsch. 1962; Abraham, 1969) concerning the selective innervation of the intracardiac conductive tissue and the adjacent portions of the right atrium. In contrast to the ventricular myocardium which is innervated by sparse individualized nerve fibers, often directed to the coronary vessels (Abraham, 1969; Somlyo, 19731, the sinus node and, mainly, the structures of the atrioventricular junction are literally enmeshed by abundant terminal fibers of the intracardiac nerve plexus (Akkeringa, 1949; Wensing, 1965; Bojsen-MGller and Tranum-Jensen, 1972; Moravec and Moravec, 1984). In addition to the extrinsic nerves, intracardiac ganglionic cells provide the bulk of terminal nerve fibers to both the conduction system and the ventricles. The existence of the latter was recognized during the early decades of the last century (Purkynb, 1845; Ludwig, 1848). Later on, they have been reported in different parts of the heart in both lower species 1963; Mc Mahan and Kuffler, 1970; Taxi, 1976) (Dogiel, 1882; Falck ef d., and mammals, including man (Kuntz, 1953; Rossi, 1955; Khabarova, 1961; Hirsch, 1963, 1970; Abraham, 1969; Papka, 1976). A majority of these ganglionic cells is concentrated in the subepicardium of the base of the heart, around the ostia of great vessels and in the coronary sulcus. where they form more or less delimitated subepicardial ganglia. Isolated ganglionic cells can also be found scattered in the meshes of the terminal nerve plexus located in the wall of the right atrium and in the interatrial septum all along the sulcus terminalis (Nielsen and Owman, 1968; Ehinger et a/., 1968; Ellison and Hibbs, 1976; Papka, 1976). The presence of ganglionic cells in the ventricular myocardium. except for the upper segment of the interventricular septum, has been reported less frequently (Smith, 1971; Bolton, 1976; Loffelholz and Pappano, 1985). According to denervation studies, some of the intracardiac ganglionic cells can survive a long time after cardiac denervation. This can be obtained by surgical interventions, such as mediastinal neural ablation (Jacobowitz r t al., 1967) and cardiac autotransplantation (Napolitano ef al., 1965; Potter et u / . , 1965) or immunologically (Zaimis et a / . , 1970; Gabella, 1976). The selective destruction of the sympathetic component can be also obtained by chronical administrations of guanethidine (Burnstock et [ I / . , 1971) or by a single injection of 6-OH-dopamine (Tranzer and Thoenen, 1967). After most of these interventions, functionally active intrinsic nerve components
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JOSEF MORAVEC A N D MIREILLE MORAVEC
could be demonstrated both at the level of the atrioventricular junction (Ljima et al., 1974) and in the ventricular myocardium of different species (Priola et al., 1977; Drake-Holland et al., 1982). The persistence of more than one-half of cholinergic and adrenergic postganglionic fibers in the atrial and ventricular walls was also confirmed morphologically (Napolitano et al., 1965; Potter et al., 1965). These data suggest that the majority of postganglionic fibers is derived from the intracardiac ganglion cells, which constitute an intrinsic nervous component similar to the intramural nervous system of Langley (1921). It is now widely accepted that this special division of the autonomic nervous system takes part in the innervation of most of peripheral tissues supplied by the autonomic nerves. According to recent studies, the intramural plexus is composed of a network of terminal nerve fibers and abundant ganglionic cells with surprisingly high synaptic plasticity (Brooks. 1981; Gershon and Erde, 1981; Wood, 1981). Apart from the two classical neurotransmitters (i.e., acetylcholine and noradrenaline). a number of neuromodulators (substance P, vasoactive intestinal peptide, neurotensin, etc.) as well as a series of putative transmitters (ATP, serotonin, etc.) were shown to contribute to its function (Burnstock, 1969; Hokfelt, 1979; Gershon et al., 1981). Some of the latter compounds were recently identified in the ganglion cells of the intrinsic nerve plexus of the guinea pig and rabbit hearts (Crowe and Burnstock, 1982; Weihe et al., 1984). This confirms the sporadic suggestions concerning the functional autonomy of the intracardiac nerve plexus. Experimental evidences in favor of such a hypothesis will be presented in the forthcoming sections. B. REGIONALSPECIALIZATIONS OF THE INTRINSIC NERVEPLEXUS OF THE INTERATRIAL SEPTUM At the first sight, the distribution of the atrial ganglionic cells might seem rather arbitrary. However, after having examined series of silverstained serial sections from a large number of animals, Moravec et al. (1984, 1986) suggested that, at least in the rat, the distribution of the intracardiac ganglia respects a reproducible pattern. In agreement with other authors (Abraham, 1%9; Bojsen-Mgller and Tranum-Jensen, 1972; Papka, FIG. I . (A) Light microscopic view of a small epicardial ganglion adjacent to the insertions of the ascending aorta and superior vena cava (arrow). An agglomerate of chromaftin cells surrounding a capillary can also be seen on the same picture (arrowhead). (From the Am. J . Anal.. 1984, 171, 307-320.) (8) Electron microscopic view of a small unipolar neuron of the epicardial ganglion. Note abundant membrane-bound ribosomes in its cytoplasma as well as the axonal profiles surrounding the cell body. (Arrow) Axonal hillock.
INTRINSIC NERVE PLEXUS OF MAMMALIAN HEART
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FIG.2. The epicardial ganglion as seen on a silver-stained paraffin section of a formaldehyde-fixed heart. Note presence of both unipolar and multipolar neurons (arrows).
1976), they regularly found a large epicardial ganglion between the root of the aorta and superior vena cava, i.e., close to the sinoatrial node. Apart from the unipolar neurons, which might be the second ganglion cells of cholinergic paths to the heart (Papka, 1976; Ellison and Hibbs, 1976), sporadic multipolar neurons could also be demonstrated (Figs. 1 and 2). In electron microscopy, most of the neuronal bodies presented abundant Nissl bodies (Fig. I B). Occasionally, neurosecretory-like profiles could also be seen on the same sections. Abundant ganglion cell bodies were also present all along the posterior branch of the sinoatrial ring bundle (SARB) of Bojsen-MZller and Tranum-Jensen ( I 972), which courses parallel to the sulcus terminalis and interconnects the sinus node area with the coronary sinus and posterior part of the atrioventricular node. Some of these nerve cells constitute a discontinuous intraseptal ganglionic lamina (Fig. 3), which sends several nerve branches in a forward direction to the structures of the atrioventricular junction, i.e., the atrioventricular node and bundle of His. Some of the peripheral extensions of these nerve fibers join the nerve plexus surrounding the right and left branches (Fig. 4). These nerve fibers follow a similar course as the previously described middle internodal pathway of James (1963).
FIG.3. ( A ) The section through the ganglionic lamina, which runs parallel to the sulcus terminalis in the interatrial septum. Note paucity of the periganglionic capsule. (From the Am. J. A n u f . . 1984, 171, 307-320.) (B) A ganglion cell found in the vicinity of a large nerve trunk running from the intraseptal ganglionic lamina in a forward direction to the specialized structures of the atrioventricular junction. ( C )An isolated unipolar ganglion cell as revealed by the silver staining in the interventricular septum. (From the “Advances in Myocardiology.” Vol. 6. pp. 13-23. Plenum, 1985.)
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FIG.4. Schema illustrating the relationships between the atrioventricular (AV) specialized tissue and different components of the intrinsic nerve plexus of the interatrial septum; reconstruction from serial semithin sections. IAS, Interatrial septum; SVC, superior vena cava; HB, bundle of His; MV, mitral valve; CS,coronary sinus; TV, tricuspid valve; IVS. muscular interventricular septum; AV, aortic valve; MS, membraneous septum; LB. left branch; RB. right branch; AVN. atrioventricular node; ---,retroaortic ganglion; @) , ganglionic lamina; A, chromaffm cells; @) , glomeruli; 0 , neurosecretory cells: ‘)rr , coiled endings.
In contrast, the anterior branch of the SARB, as previously identified by cholinesterase reaction (Bojsen-Mdller and Tranum-Jensen, 1971, 1972), does not contain any ganglionic cells (Moravec et al., 1985). It is composed of a strand of small muscle cells surrounded by a fine plexus of terminal nerve fibers in a manner which is similar to that encountered in the sinus node and in the accessory atrioventricular node (Anderson, 1972). It would seem that the anterior branch of the SARB, located in the upper portion of the interatrial septum, represents a neuromuscular link between these two structures. From the topographical point of view, the posterior and anterior branches of the SARB follow the courses of the third and first “specialized pathways” for the intraatrial conduction (James, 1963; Viragh and Challice, 1973; Hiraoka and Sano, 1976; Anderson et a / . , 1978), which were found to have their electrophysiological counterparts in, respectively, the posterior and anterior inputs to the atrioventricular node (Paes de Carvalho et al., 1959; Emberson and Challice, 1970; Spach et a / . , 1971; Janse et al., 1978). All of the above interatrial nerve pathways converge to the specialized
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FIG.5 . Small ganglion cells as found in the nodal interstitium. Note abundant microtubules, microvesicles, and small mitochondria in their cytoplasma. The glycogen granules are sometimes encircled by a single membrane (arrow). The cell bodies are surrounded by a discontinuous Schwann cell sheath (Sw), which they share with numerous neurites (Ne). N, Nucleus; arrowhead, membrane-bound ribosomes; Nc, nodal cell.
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tissue of the atrioventricularjunction, where they ramify and feed in the terminal nerve plexus constituted by the intrinsic ganglionic cells (Fig. 5 ) . These small ganglion cells were shown to invest the atrioventricularjunction of different species (Rossi, 1955; Abraham, 1969). At several occasions, it was suggested that presence of these cells might be of particular importance for the function of the intracardiac conductive tissue (Wensing, 1965; Bojsen-Mpller and Tranum-Jensen, 1972; Moravec and Moravec, 1984).
c. CHOLINERCIC OR ADRENERGIC CHOICE: PRESENT STATEOF THE QUESTION The ganglionic cells of mammalian hearts have been generally described as unipolar cholinergic neurons responsible for the second ganglionic relay of parasympathetic fibers to the heart (Jacobowitz, 1%7; Yamauchi, 1973; Pdpka, 1976; Ellison and Hibbs, 1976). The existence of intracardiac adrenergic neurons has been considered excluded for embryological reasons; the cardiac mesenchyme of birds and mammals is believed to induce invariably the expression of cholinergic phenotype in all ganglionic precursors migrating to the heart (Yamauchi, 1973; Viragh and Challice, 1977; Cabot and Cohen, 1980; Le Douarin ef al., 1981; Smith, 1983). The fact that most of the intracardiac neurons are intensely AChE-positive and, at the same time, devoid of specific fluorescence induced by formaldehyde vapors (Falck ef al., 1963; Jacobowitz, 1967; Ehinger ef al., 1968; Yamauchi, 1973) was often considered a confirmation of this rule. However, it should be noted that a small fraction of intracardiac ganglionic cells may be derived from the aortic plexus which is an adrenergic structure, similar to sympathetic ganglia and adrenal medulla (Le Douarin and Cochard, 1983; Le Douarin and Smith, 1983). It is not excluded that, during the folding of the primitive cardiac tube (Anderson ef al., 1978; Viragh and Challice, 1977), some of these adrenergic cells invest the ostia of neoformed great vessels and the intracardiac septum. The fact that they exhibit only moderate specific fluorescence should be considered in the light of the following data. Neurons devoid of specific fluorescence are present in the sympathetic ganglia of many species (Norberg, 1967; Gabella, 1976). The injection of nialamide [an inhibitor of the monoarnine oxidase (MAO)] produces a general increase in their fluorescence (Hamberger and Norberg, 1963). A similar observation was also reported by Jacobowitz (1967). who, after the administration of another inhibitor of the MA0 (i.e., tranylcypromine), could reveal fluorescent neurons similar to those of sympathetic ganglia in rat and guinea pig cardiac nerve plexus. Their yellow-green fluorescence
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was further enhanced by a consecutive administration of catecholamine precursors, such as DL- or L-dopa (Jacobowitz, 1967). All of these observations may illustrate the heterogeneous distribution of the neurotransmitter between different compartments of adrenergic neurons (Gabella, 1976). It has been estimated that each neuronal body of the superior cervical ganglion of the cat contains on average 0.4 pg of norepinephrine, while the terminal varicosities of the same neuron totalize up to 150 pg of norepinephrine (Dahlstrom and Haggendal, 1966). It would seem that some of the sympathetic ganglionic cells may operate with low transmitter concentrations, except for their peripheral ramifications (Ehinger et ul., 1970). A similar situation also occurs in the intramural nerve plexus of the gut (Read and Burnstock, l%9). Also in this case, drastic interventions, such as the use of MA0 inhibitors and simultaneous administration of catecholamine precursors, are necessary in order to reveal the presence of adrenergic neurons (Burnstock and Bell, 1974). The autonomic neurons devoid of specific fluorescence have often an intense acetylcholine esterase activity (Hamberger et al., 1965), which was frequently considered as a histochemical manifestation of their cholinergic or, more exactly, parasympathetic nature (Jacobowitz, 1967; Yamauchi and Lever, 1971; Yamauchi, 1973). However, it has been shown that acetylcholinesteraseactivity can sometimes be associated with a weak, moderate, or strong fluorescence (Eranko, 1966). In the hedgehog, all nerve cells of sympathetic ganglia exhibit regularly an intense acetylcholinesterase reaction (Cauna et ul., 1961). This acetylcholinesterase of sympathetic neurons has been separated into external and internal pools. The former is believed to be involved in cholinergic ganglionic transmission (Mc Isaac and Koelle, 1959; Gabella, I976), the latter is possibly implicated in catecholamine synthesis, since it is associated with the Nissl bodies (Koelle r t ul., 1974; Black ef ul., 1979; Smith, 1983). I n vitro, adrenergic neurons synthetize both acetylcholine and norepinephrine, and it was shown that acetylcholine secreted by a sympathetic neuron triggers the release of norepinephrine from the same cell via its own nicotinic receptors (Burn. 1975). For all these reasons, the acetylcholinesterase activity cannot be any more regarded as the best marker of cholinergic (parasympathetic) neurons (Johnson et al., 1981; Potter et ul., 1981). Therefore, some of the nerve cells of mammalian hearts could, in fact, be sympathetic ganglionic cells, despite of the fact that they are frequently acetylcholinesterase-positive. A similar situation was previously described in the frog heart (Falck ct d., 1963; Taxi, 1976). In this latter species, an adrenergic component was described in the intramural nerve plexus of the atrioventricular junction. A neurosecretory component was also described in Bidder's ganglia, which
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are located in the interatrial septum close to the atrioventricular valves (Bidder, 1868) and which do not seem to receive any vagal fibers (Marceau, 1946; Taxi, 1976).
D. EVIDENCE FOR INTRINSIC ADRENERGIC NEURONSIN THE NERVEPLEXUS OF THE ATRIOVENTRICULAR JUNCTION TERMINAL The existence of sympathetic preganglionic fibers in the heart of higher vertebrates still remains rather controversial. Most of the acetylcholinesterase-containing fibers are considered as vagal preganglionics and/or postganglionics (Jacobowitz, 1%7; Randall, 1976; Levy and Martin, 1979). However, it has been suggested that some of the sympathetic preganglionic fibers can transit through the stellate ganglia without interruption (Gabella, 1976). This has been confirmed, at least for the dog heart, by the denervation studies of Seagard et al. (1978). These authors have performed dorsal root ganglionectomy on left thoracic segments T,-T4. The peripheral cardiac nerves were examined 3 weeks later. At that time, totality of thin myelinated afferent fibers (up to 15% of total fibers) was degenerated in most of the left cardiac branches. The only exception was the ventrolateral cardiac nerve, which contained the least amount of degeneration (less than 5% of total fibers). In this nerve, intact large myelinated fibers (Aa category) persisted for a long time after the surgery. The authors of the above study concluded that these thick A a fibers are sympathetic preganglionics. The presence of myelin sheath and absence of any electrical activity during the stimulation of reflectogenic areas of the heart (Armour et al., 1975) would argue in favor of that possibility. Another significant point is the selectivity of peripheral projections of the ventrolateral cardiac nerve; this nerve was found to innervate selectively the atrioventricular junction where, also in the dog (Abraham, 1%9), abundant ganglionic cells are present. In this connection, it should be noted that large neurosecretory cells, closely associated with the specialized structures of the atrioventricular junction of the rat heart, have been revealed by a recent electron microscopic study (Moravec and Moravec, 1984; Moravec et al.;1986). In that FIG.6. An electron microscopic view of the perihissian ganglion of the rate pretreated by a single injection of 5-OH-dopamine.Note the presence of a large neurosecretory neuron with an eccentric position of its nucleus (N), separated from the rest of the cell body by a profound infolding of the neurolemrna (arrow). The central element is accompanied by several cellular processes which have the same cytoplasmic content, i.e.,abundant small mitochondria and two kinds of electron-dense microvesicles (35 and 60 nm in diameter) (insert). The size of these vesicles, as well as their disposition in clusters, reminds one of the cytological descriptions of adrenergic neurons. Nc, Nodal cells: Co, collagen: arrowheads, emergence process; asterisks, vacuoles.
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work, the adrenergic components of cardiac innervation were prelabeled by a false precursor of catecholamines, i.e., 5-OH-dopamine(Tranzer and Thoenen, 1967; Chiba, 1973). The size of these neurosecretory neurons (up to 35 km in diameter) and the presence of two kinds of electron-dense vesicles (sporadic large ones and clusters of small vesicles with the respective diameters of 60 and 30 nm) (Fig. 6) resembled those of other sympathetic neurons (Matthews, 1974; Gabella, 1976; Taxi, 1976). However, the true nature of the transmitter, which could be released under physiological conditions [i.e., noradrenaline, adrenaline, or dopamine (Gabella, 1976; Drake-Holland et al., 1982; Geffen and Jarrott, 1977)], was not elucidated. These cells had one or two thick peripheral extensions, containing the same granular material as their cell bodies (Fig. 7). which resembled the dendritic collaterals of the adrenergic ganglion cells (Taxi and Droz, 1969; Jacobowitz, 1974) and, in particular, the 5-OH-dopaminecumulating dendritic specializationsof the guinea pig atrium (Chiba, 1973). One of the most interesting features of these perinodal ganglion cells is apparent in their immunohistochemical properties (Fig. 8a, b). According to our recent study (Moravec et al., unpublished data), cell bodies and peripheral extensions of these cells contain both neuropeptide Y (NPY) and C-terminal-flankingpeptide of neuropeptide Y (C-PON) (Fig. 8d, e). They also react with the anti-TH sera (Fig. 8c). This seems to suggest that catecholamines of the electron-dense vesicles, as observed in electron microscopy, can be synthetized in situ together with other prospective cotransmitters of adrenergic neurons (Lundberg et al., 1985; Van Noorden et al., 1985). In contrast, the perinodal ganglion cells do not react with anti-SP nor with anti-VIP sera which, in turn, gave positive results in other portions of the intracardiac nerve plexus, i.e., in the epicardial ganglia and in the intraseptal ganglionic lamina (Moravec et al., unpublished data). Most of these intramural, “short adrenergic neurons” (Sjostrand, 1%5), were surrounded by several thin layers of Schwann cells, which constituted a discontinuous sheath of loose myelin. Except for their dendrites, which frequently shared their Schwann cell sheaths with small cholinergic neurites, they were only sparsely innervated. Their nuclei were often eccentric, having been separated from the rest of the cell body by profound infoldings of the cell membranes (Fig. 6). This may explain the absence of nuclear profiles on some of the thin sections examined. FIG.7. (A) A dendritic-like adrenergic varicosity as seen in the nodal interstitium of the rat pretreated by a single injection of 5-OH-dopamine. Note two sizes of electron-dense vesicles and the presence of a continuous Schwann cell sheath (Sw). (From the Am. J . Anut.. 1984, 171, 307-320.) (B) A dendritic-like varicosity contrasted by 5-OH-dopamine, this time uncompletely coated only by the neighboring satellite cell (Sc). Note two enlarged axons synapting on the surface of this neurosecretory element of the perinodal interstitium (arrows). (From the Am. J . Anut.. 1984. 171, 307-320.)
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The distinction between axons and thick dendritic collaterals was not easy without the use of 5-OH-dopamine (Moravec et al., 1985). In hearts of rats pretreated with this false precursor of catecholamines, the axons could be distinguished from the rest of neuronal body by their clear appearance. They contained microtubules and microfilaments, but only a few mitochondria and sparse vesicles (Fig. 9). Glycogen granules were sometimes surrounded by a single membrane (Fig. 9) in a manner which was similar to that described in the postganglionic fibers of sympathetic neurons of Rana esculenta by Taxi (1976) and in the bull frog by Berthold (1966). The initial segment of the axon was not myelinated. This fits well with the contention that this part of the nerve cell should be considered as its functional axon hillock (Palay et al., 1968; Kuffler and Nichols, 1976; Taxi, 1976). On some sections, even the Schwann cell sheath seemed to be discontinuous and, at these locations, several whorls of axonal profiles approached the ganglion cell body. However, the peripheral portion of the axon rapidly acquired a sheath of compact myelin. It was remarkable that this myelin developed in the external layers of Schwann cells, having been separated from the axon by several other layers of Schwann cell cytoplasm (Fig. 9). These sleeved fibers (Pick, 1962) resembled those described as typical for the postganglionic emergence processes of frog sympathetic neurons (Taxi, 1976). Another noteworthy feature of these large adrenergic neurons was their fragility face-to-face the isotonic solutions used for the perfusion fixation (2.3% glutaraldehyde in 45 mM sodium cacodylate) and for washing of samples (I50 mM sodium cacodylate buffer). Large exploding vacuoles frequently found in the perikarya of these cells would suggest that their intracellular milieu was strongly hyperosmolar. A similar situation was
FIG.8. Serial cryostat sections through the atrioventricular junction of the rat heart counterstained with pontamine sky blue. The nerve elements of the specialized tissue and the interventricular septum were visualized by indirect immunocytochemistry with fluorescein-conjugated secondary antibody. (a) A dense protein gene product (PGP)-positive nerve network interpenetrating His' bundle (HB) and the arising right branch (RB). An agglomerate of PGP-immunoreactive ganglion cells (arrow) supplying nerve bundles (arrowhead) into the interventricular septum (IVS). x 120. (b) A detail of the PGP-immunoreactive nerve cells (arrows)with their thick dendrites (arrowheads) interpenetrating the interventricular septum (IVS). x 300. (c) Tyrosine hydroxylase-immunoreactive nerve cell (arrow) at the junction of interventricular septum (IVS) and His' bundle (HB). ~ 4 0 0 (d) . Neuropeptide tyrosine (NPY)-immunoreactive nerve cell (arrow) of the atrioventricular junction with its thick dendritic projection interpenetrating the interventricular septum (IVS). HB, His' bundle. x 400. (e) C-PON-immunoreactive nerve cell (arrow) in the connective tissue separating His' bundle (HB) from the interventricular septum (IVS). Note a thin (axonic) projection of this cell (arrowhead). ~ 4 0 0 The . authors would like to thank Professor J. Polak who supplied the samples of sera used in this work and Professor J. Taxi who allowed us to use his fluorescence microscope.
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previously described at the level of terminal nerve fibers innervating the specialized cells of the atrioventricular node of the rat (Moravec-Mochet et al., 1977).
E.
CYTOLOGICAL ANALOGIES BETWEEN SENSORY NEURONS A N D THE NEUROSECRETOKY CELLS OF THE ATRIOVENTRICULAR JUNCTION
The irregular shape of the above cells, as well as the presence of an irregular satellite cell sheath, giving rise to several lamellae of loose or compact myelin, remind one of the cytological descriptions of autonomic ganglia (Forssmann, 1964; Hess, 1965; Gabella, 1976; Taxi, 1976) and those of sensory neurons (Hess et al., 1969; Peters et al., 1976; Pannese, 1981). The myelinated nerve cell bodies are numerous in the acoustic and vestibular ganglia in all classes of vertebrates, from elasmobranchs to man. They occur occasionally in other sensory ganglia, especially those of fish, amphibians, and reptiles. Most of these myelinated nerve cells are bipolar, some pseudounipolar. The myelin sheaths surrounding the nerve cell bodies extend along the initial portions of their axons up to the first nodes of Ranvier. Under the polarized light, the perikaryal myelin presents the same characteristics as those surrounding the nerve fibers (Scharf, 1958; Pannese, 1981). According to electron microscopic studies (Rosenbluth and Palay, 1961; Rosenbluth, 1967; Merck et al., 1975; Perre et al., 1977; Pannese. 1981). several types of perykaryal myelin can be distinguished. In some cases, the myelin sheath is built of a varying number of lamellae, each consisting of a layer of satellite cell cytoplasm bounded by the plasma membranes of satellite cells and separated from the nearby ones by a narrow space (loose myelin). In other cases, the perikaryal myelin displays a highly regular pattern. The ctyoplasm between the plasma membranes has disappeared, and the space between the lamellae is obliterated. In such cases, the perikaryal myelin is very comparable to the compact myelin surrounding the nerve fibers (Pannese, 1981). Between these two extremes of loose and compact myelin, a wide range of perikaryal myelin sheaths, such as single satellite cell layers, multiple layers of satellite cells, pseudomyelin FIG.9. (A) A large nerve process as seen in the perihissian glomerule, which might be either the emerging axon of an intrinsic neuron acquiring progressively its myelin sheath or a peripheral myelinated fiber loosing it myelin. Note the glycogen inclusions in the cytoplasma (arrowheads). SW. Schwann cells. (B) Section through a perinodal glomerule presenting a peripheral nerve fiber surrounded by an irregular layer of Schwann cell cytoplasm developing an eccentric sheath of compact myelin. Similar sleeved fibers characterize the postganglionic emergence processes of sympathetic ganglia. (Arrows) Loose myelin, (arrowheads) compact myelin. (asterisk)glycogen.
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and, finally, compact myelin, can be found in sensory (Rosenbluth, 1967; Merck et al., 1975; Stensaas and Fidone, 1977; Pannese, 1981) and autonomic (Hess, 1965; Taxi, 1965; Takahashi and Hama, 1965) ganglia. The irregularity of their cell shape is another common feature of some sensory and autonomic neurons (Gabella, 1976; Peters et u l . , 1976; Pannese, 1981). Their cell bodies are often folded, which results in the eccentric position of their nuclei (Takahashi and Hama, 1965; Cantino and Mugnani, 1975). The axon is first directed proximally then, very close to the preganglionic fibers, it bends and courses distally where it becomes myelinated (Pannese, 1981). Several thick and short intracapsular dendrites (unipolar or pseudounipolar neurons) arise from these cells (Gabella, 1976). The cells of this type were described in dorsal root ganglia (Pannese, 1981) and in two types of peripheral sensory ganglia, i.e., the acoustic and vestibular ganglia (Peters et al., 1976). Some authors also pointed out the presence of irregular myelinated nerve fibers in autonomic ganglia, such as the paravertebral ganglia of frog (Taxi, 1976) and the ciliary ganglion of birds (Hess, 1965; Hess et al., 1%9). In this latter case, it was suggested that the time at which the myelin lamellae occur (before hatching in the chick and during the second posthatching period in the pigeon) correlates well with the appearance of electrical coupling and the bidirectional conduction through the developing ciliary ganglion (Hess et a!., 1%9; Manvitt ef al., 1971; Gabella, 1976). According to these data, the myelin sheath of the above ganglionic cells might be essential for the perception and transmission of local electrical phenomena. The presence of ganglionic cells of similar cytology at the level of the atrioventricularjunction might therefore be of considerable significance for the integration of local electrical phenomena and for the feedback regulation of cardiac activity (James, 1973; Pollack, 1974).
F. EVIDENCE FOR SENSORY NEURONSIN THE TERMINAL NERVE PLEXUS OF THE ATRIOVENTRICULAR JUNCTION Some of the ganglionic cells of the terminal nerve plexus of the atrioventricular junction could also be involved in the perception of mechanical phenomena related to ventricular systoles (Brooks and Lu, 1972; Irisawa, 1978). The existence, at the level of the intracardiac specialized tissue, of a nonidentified self-governed device involved in the control of the spontaneous activity of the heart has been suggested by several authors (James, 1973; Irisawa, 1978; Pollack, 1978; Brooks, 1981). According to our data (Moravec-Mochetet al., 1977; Moravec et al., 198% the density of sensory coiled endings in and around the atrioventricular junction is surprisingly high. Recently, we studied the atrioventricular junctional area of the rat heart, using thick serial silver-stained sections (Moravec, 1985).
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The results of that study were quite significant. The specialized tissue of the atrioventricular junction was embedded in a connective tissue of the membraneous septum, which separates the left ventricular cavity from the right atrium, i.e., the arterial portion of the systemic circulation from its venous return (Anderson el a/., 1978; Moravec and Moravec, 1982). All along the course of the atrioventricular node, the bundle of His and the upper portions of right and left branches, agglomerates of large granular ganglion cells, could be identified (Fig. 10). Some of these cells had thick dendritic projections, which interpenetrated the upper portion of the interventricular septum and the reticular portion of the atrioventricular node (Moravec et a/., 1986). They terminated by large coiled endings enrolled around the respective muscle cells (Fig. I I), which, in electron microscopy, could be identified as sensory in nature (Moravec-Mochet ef al., 1977; Moravec and Moravec, 1982). The size of these ganglion cells (up to 35 pm), as well as the presence of a granular material in their cytoplasm, strongly suggested that these sensory neurons were identical to the abovedescribed adrenergic cells of the atrioventricular junction. Their axons encountered the enlarged terminal nerve fibers of the intraatrial neuromuscular pathways described in one of the preceding sections. At this level, abundant axodendritic and dendrodendritic glomeruli (De Castro, 1932; Gabella, 1976) could be identified by means of a combined phasecontrast and electron microscopic examination of the alternate thin and semithin sections (Moravec and Moravec, 1984). Further work, i.e., the use of serial-thin sections, will be necessary in order to follow the course of axons of these cells and to study the ultrastructure of their active zones as well as their relationships to the surrounding myelin. The association of an adrenergic component with the function of different sensory receptors is not new. Already Bernard (1851) had suggested that sympathetic nerves modulate the sensory input to the brain (cf. Gabella, 1976), and Bechterev (1896) presented the sympathetic ganglia as an offshoot of the central nervous system, which conducts impulses both centripetally and centrifugally from one functional unit to another (cf. Khabarova, 1961). However, direct proof for this integrative function of the sympathetic system has been lacking until recently. At present, the possibility that the sympathetic pathways may be involved in sensory functions has been open anew (Gabella, 1976; Leranth and Unguary, 1980; Brooks, 1981). For a while it seemed forgotten that, in many organs, autonomic nerves terminate in intramural plexuses (Meissner, 1857; Auerbach, 1864; Langley, 19211, which possess a high level of intrinsic activity and responsiveness (Langley, 1921; Brooks, 1981; Gershon and Erde, 1981; Wood, 1981). These latters may affect, or even elicit, the function of surrounding structures. Today, there are multiple examples of the role of an intrinsic adrenergic component in the local control of different myogenic
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FIG. 10. A large sensory neuron of the perihissian ganglion as revealed by silver impregnation. Note several short dendrites terminated by large coiled endings surrounding a strand of muscle cells (arrowheads) and several large whorls of the axonal process (arrow). In contrast to the unipolar neurons of cardiac ganglia. this cell has a dark and granular cytoplasm similar to the above neurosecretory neurons. found in the electron micrograph at the same location. VS, Interventricular septum.
systems (Pollack, 1978; Leranth and Unguary, 1980; Cranefield, 1983; Weihe et al., 1984). As concerns the function of sensory organs, catecholaminergic components were already found in different types of mechanoreceptors, such as skeletal muscle spindles (Paintal, 1973; Barker, 1974), acoustic receptors (Spoendlin and Lichtensteiger, 1966), baroreceptors (Aars, 197 1 ; Belrnonte et al., 1972; Chiba, 1973), and in cardiac chemoreceptors (Knoche et al., 1970; Eyzaguirre et al., 1977). The association of large neurosecretory elements with myelinated nerve fibers was also described in frog hearts, namely, at the level of Bidder's ganglia (Bidder, 1868; Taxi, 19761.' 'Quite recently it has been suggested that the primary sensory neurons of cranial nerves, as well as some of the sensory neurons of dorsal root ganglia of adult rat, may express an adrenergic phenotype (Jonakait e/ a / . , 1984; Katz ct d..1983). This later disappears when they were disconnected from their respective projection areas which, in most cases, belong to the cardiovascular system (Katz and Black, 1986). The centripetal transport of trophic factors from the target tissues seems to be necessary for the catecholaminergic traits of cranial sensory neurons to be maintained (Katz and Black, 1986).
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FIG.I I . ( A ) A detail of the coiled ending (arrow) surrounding a strand of specialized cells of the reticular segment of the AVN (atrioventricular node) as revealed by the silver impregnation technique. (B) An electron micrograph view of a section through the presumptive sensory corpuscle. Several profiles of the same nerve fiber (arrows) can be found in the vicinity and within an agglomerate of nodal cells. Note the predominance of very small mitochondria and small empty vesicles (30 nm in diameter). The nerve elements are accompanied by a discontinuous satellite cell sheath (Sw), which is lost at the site of the terminal nerve-muscle interactions (arrows). (From the "Advances in Myocardiology." Vol. 6, pp. 13-23. Plenum, 1985.)
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Another finding, which seems to be of particular significance for our discussion, is the existence of peripheral mechanosensory neurons, inserting their dendrites into the fine strands of skeletal muscle fibers that were found operating in crayfish and other crustaceans (Alexandrowicz, 1951; Eyzaguirre and Kuffler, 1955; Bullock et al., 1977). They were shown to provide a powerful proprioceptive feedback to the neural networks involved in the generation of motor patterns responsible for rhythmic swimmeret movements (KuMer and Nichols, 1976). The fact that, in the heart, the perception of electrical and mechanical phenomena, affecting the atrioventricular junction during each contraction cycle, seems to be in the hands of the intrinsic adrenergic neurons (Figs. 6, I I and 12) (Moravec and Moravec, 1984) might thus be of considerable significance. The cyclic catecholamine release from the above neurosecretory cells can be expected to affect both specialized tissue and cholinergic components of the cardiac nerve plexus (Jacobowitz, 1967; Yamauchi et al., 1973; Pollack, 1978). In this way, the catecholamine (and neuropeptide) recycling may contribute to the intrinsic autoregulation of the pacemaker activity (Pollack, 1977; Cranefield, 1983) and intracardiac conduction (Pollack, 1974; Irisawa, 1978). This can also explain some still badly understood aspects of vagosympathetic (Levy and Martin, 1979) and vagus-cardiac pacemaker (Spear et al., 1979; Michaels et al., 1983) interactions. IV. Terminal Nerve Plexus According to light microscopic studies, the intramural cardiac ganglia contain unipolar, bipolar, and multipolar neurons (Davies et al., 1952; Abraham, 1969; Ellison and Hibbs, 1976). They also contain many nerve fibers in transit, including a number of sensory fibers and fibers which originate from, or are directed toward, other ganglia (Gabella, 1976). Most of the preganglionic fibers were believed to be preganglionic inhibitory branches from the vagus nerves. However, with fluorescence microscopy, numerous adrenergic fibers running through the cardiac plexus could also be identified (Ehinger et al., 1968; Nielsen and Owman, 1968; Forsgren, 1985). Most of these adrenergic fibers originated from the inferior cervical FIG.12. (A) A section through a preterminal nerve fiber of the autonomic ground plexus of the atrioventricular junction in a 5-OH-dopamine-pretreated rat. Note the coexistence of an adrenergic (ADR) and a cholinergic (ACH) nerve fibers sharing the same Schwann cell (Sw) sheath. Nc, Nodal cell. (B) A free adrenergic varicosity (arrow) as seen in the A V nodal interstitium in a 5-OH-dopamine-treated rat. (C) An adrenergic neuromuscular junction encountered in the compact zone of the A-V node of a rat pretreated with 5-OH-dopamine. Note the absence of basement membrane in the intermembrane space, the densification of synaptic membranes, and an equivocal postsynaptic apparatus (arrows).
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ganglion (Wacksman et al., 1%9) or from the thoracic paravertebral ganglia (Gabella, 1976). Only some of them persisted after surgical sympathetic denervation (Potter et al., 1965). The contribution of an intrinsic adrenergic component to cardiac innervation is therefore not excluded. The persistence of the tyrosine hydroxylase activity as well as that of dopaminebinding sites in chronically denervated dog hearts (Drake-Holland et al., 1982) would argue in this sense. Terminal varicosities of these adrenergic fibers impinge on ganglion cells forming very small terminals whose detection in both optical and electron microscopy requires optimal conditions (Nielsen and Owman, 1%8; Ehinger et al., 1970). Some of these adrenergic varicosities can form axosomatic synapses with the intramural ganglion cells and, sometimes, they can approach cholinergic postganglionic axons (Jacobowitz, 1967; Ehinger et al., 1970; Yamauchi, 1973). The existence of adrenergic neuromuscular junctions was also suggested. In our work, we found them in the rat atrium (Fig. 12).
A. AUTONOMIC GROUNDPLEXUS From the morphological point of view, this terminal portion of cardiac innervation resembles Hillarp’s description of the autonomic “ground plexus” (Hillarp, 1946, 19591, which has been identified in most of the peripheral organs supplied by autonomic nerves (Burnstock and Bell, 1974; Gabella, 1976; Taxi, 1976; Brooks, 1981; Gershon and Erde, 1981). According to this concept, the terminal autonomic axons invest a finely meshed network of Schwann cells. Each Schwann cell sheath contains several axons derived from different neurons (Yamauchi, 1973), enabling a convergence of nerve fibers to the same effector cell. The terminal nerve fibers course within the plexus for longer distances and innervate en passant several effector cells. According to some authors, the adrenergic and cholinergic axons can also form reciprocal synapses (Ehinger et ul., 1970) (Fig. 12A). However, there is no proof that the appositions of adrenergic and cholinergic fibers do have a functional significance. At the electron microscopic level, the autonomic ground plexus has a certain number of morphological features, which are common to all organs presenting an intrinsic innervation (Taxi, 1965; Rogers and Burnstock, 1966; Yamauchi, 1973; Taxi, 1976). It consists of varicose branching fibers enveloped by Schwann cells which interpenetrate the interstitial spaces. Close to the neuroeffector junctional area, the Schwann cell sheaths become discontinuous and, through these exposed foci, the transmitter substance can be released (Fig. 12B). In some cases, rudimentary neuromuscular synapses could also be identified (Richardson, 1964; Yamauchi, 1973; Moravec-Mochet et d . , 1977).
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I . Sympathetic Postganglionic Fibers The density of adrenergic nerves is higher in the atria than in the ventricles in almost all mammalian species, except for the cat (Nielsen and Owman, 1968; Yamauchi, 1973; Levy and Martin, 1979) and the bat heart (O'Shea and Evans, 1985). In the case of this hibernating animal, the density of adrenergic innervation is quite uniform in both the atria and the ventricles. In other species, the adrenergic axons are the most numerous in the auricular appendages and around the structures of the conductive tissue, namely, at the level of the sinus node (Ehinger et al., 1968; Yamauchi. 1973). The atrioventricular node, mainly its reticular portion, also receives the adrenergic innervation. Its compact zone, on the other hand, is supplied essentially by abundant cholinergic axons (Thaemert, 1970; Bojsen-Mgller and Tranum-Jensen, 197 I ; Moravec-Mochet et ul., 1977). There seems to be a controversy as to the adrenergic innervation of the ventricular segments of the conductive tissue, i.e., that of the bundle of His, Purkyne cells, and moderator bands (Yamauchi, 1973). These structures were shown not to have any adrenergic terminals at all, at least in the dog and pig hearts (Dahlstrom et al.. 1965; Bojsen-Mgller and TranumJensen, 1971). However, using the glyoxylic acid technique (Axelsson e t d . , 1973), which gives better results than the classical Falck and Hillarp method, Forsgren (1985) succeeded to demonstrate numerous adrenergic varicosities in the ventricular conduction system of bovine and human hearts. He suggested that catecholamine release from these intraventricular adrenergic endings might be responsible for the development of postischemic or postinfarction ventricular tachyarrhythmias. As to the adrenergic innervation of both ventricles, there seems to be a general consensus that it is less extensive than that of atria. Although adrenergic axons can be found distributed within the ventricular myocardium (Jacobowitz et al., 1967; Winckler, 1969; Yamauchi, 1973), the adrenergic terminals are almost exclusively concentrated to the perivascular spaces (Nielsen and Owman, 1968). Adrenergic neuromuscular junctions were found only occasionally (Ehinger et al., 1970) (Fig. 12C). There is now experimental evidence that some of the intracardiac adrenergic fibers do not degenerate after chronical cardiac denervation (Jacobowitz, 1967; Potter et al., 1965). This may suggest that they are, at least some of them, derived from the intrinsic adrenergic cells. The existence of the latter was suggested at many occasions by fluorescence and electron microscopic studies (Truex, 1950; Jacobowitz e t al., 1967; Yamauchi, 1973; Ellison and Hibbs, 1976; Moravec and Moravec, 1984). According to the biochemical analysis (Gabella, 1976), up to 50% of norepinephrine of the pacemaker area may be provided by the intracardiac chromafin cells and by the above-mentioned intramural adrenergic neu-
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rons, which can be supposed rather resistant to the effects of both surgical and chemical denervation (Potter et al., 1965; Jacobowitz, 1967; Tranzer and Thoenen, 1967; Burnstock and Bell, 1974; Gabella, 1976; Drake-Holland et al., 1982).
2. PARASYMPATHETIC POSTGANGLIONIC FIBERS In comparison with the efferent synapses of skeletal muscles, the autonomic nerve endings on smooth and cardiac muscle cells are small, and their ultrastructure resembles that of embryonic skeletal muscle end plates and that of “en grappe” endings of the intrafusal fibers (Yamauchi, 1973; Uehara et al., 1976). The pre- and postsynaptic specializations, typical for neuromuscular synapses (Couteaux, 1978), are often rudimentary or missing (Couteaux, 1961 ; Yamauchi, 1973; Moravec-Mochet et al., 1977). Only occasionally, close membrane-to-membrane appositions of presumptive cholinergic terminals and specialized muscle cells have been observed in the cardiac nerve plexus of different species (Couteaux and Laurent, 1958; Viragh and Porte, 1961; Thaemert, 1970; Taxi, 1976). However, the interpretation of these structures was questioned by Yamauchi (1969). Some authors (Moravec-Mochet et al., 1977) found these endings, leaving a gap less than 20 nm and devoid of any basal lamina, associated with terminal arborizations of large coiled fibers, which were similar to sensory innervation of skeletal muscle spindles (Uehara et al., 1976). Some of these sensory specializations seemed to be supplied by large intramural sensory neurons (see above), at least at the level of the atrioventricularjunction. As concerns the parasympathetic postganglionics, characterized by empty vesicles (30-50 nm in diameter), even after the administration of 5-OH-dopamine (Chiba, 1973; Yamauchi, 1973; Moravec and Moravec, 1984), they were suggested to be derived from cholinergic neurons of the parasympathetic cardiac ganglia (Hirsch et al., 1963; Yamauchi, 1973; Ellison and Hibbs, 1976). In optical and electron microscopy, these fibers were strongly AChE-positive and so were the plasma membranes of the adjacent muscle cells (Hirano and Ogawa, 1967; Jacobowitz et al., 1967; Yamauchi, 1973). Up to 70% of these cholinergic axons remain intact after the cardiac autotransplantation (Napolitano et al., 1%5; Potter et al., 1965) and mediastinal neural ablation (Jacobowitz et al., 1967). This proves that FIG.13. ( A ) A small en grappe efferent ending with empty vesicles (50 nm in diameter) presumably cholinergic. bl, Basal lamina; arrow, agglomerate of synaptic vesicles: Nc, nodal cell. (From the J . Ultruslrucr. Res.. 1977,58, 196-209.) (B) A small presumptive cholinergic ending with scarce vesicles and small mitochondria. Note the persistence of the basal lamina in the synaptic cleft and rudimentary postsynaptic folds (arrows). Nc, Nodal cell. (From the J . Ulrrustrucr. Res., 1982, 81, 47-65.
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a majority of cholinergic postganglionic fibers is derived from the intrinsic ganglion cell of the cardiac nerve plexus (Jacobowitz, 1967; Loffelholz and Pappano, 1985). The distribution of the cholinergic fibers and, mainly, the density of cholinergic neuromuscular synapses vary throughout different cardiac compartments. Only in hibernating animals, a diffuse distribution of vagal postganglionic fibers was recently reported (O’Shea and Evans, 1985). Apart from this exception, both atria were comparatively more innervated than the ventricles in most mammalian species examined (Jacobowitz et al., 1967; Yamauchi, 1973; Levy and Martin, 1979). After surgical denervation, the number of cholinergic nerves in the left atrium was considerably reduced, while only minor reduction was observed in right atria and in the ventricles (Jacobowitz et al., 1967). As concerns the conduction system of the heart, an accumulation of cholinergic synapses (small en grappe endings with empty vesicles and minor pre- and postsynaptic specializations)was mentioned in the compact zone of the atrioventricular node (Yamauchi, 1973) and in the bundle of His (Moravec-Mochet et a / . , 1977). More elaborated synapses (very small en plaque terminals), similar to those of polar regions of skeletal muscle spindles (Uehara et al., 1976), predominated in the superficial, reticular layers of the atrioventricular node of the rat (Moravec and Moravec, 1982) (Fig. 13A and B). According to Thaemert (1970), who studied the tail of the mouse atrioventricular node on serial sections, every nodal cell in that portion of the mouse heart receives at least one cholinergic terminal. In addition to these efferent synapses, the presence of afferent nerve varicosities was also described in this part of the atrioventricular junction (Fig. 14) (Moravec-Mochet et al., 1977),as well as at the level of the mole sinoatrial node (Kikuchi, 1976). This double (efferent and afferent) innervation of different segments of the intracardiac conduction system of mammals may suggest that it should be considered as an intrinsic spindlelike organ, similar to the in situ mechanoreceptors of lower species (Kuffler and Nichols, 1976). B. CHROMAFFIN CELLS The first description of clusters of small granular cells within, or close to cardiac ganglion of the dog, is that of Truex (1950). In 1961, Viragh FIG. 14. A muscular spindlelike structure of the atrioventricular node of the rat. Note double [sensory (arrow) and motor (arrowheads)] innervation of a group of nodal cells surrounded by a common basal lamina. (Inset) Detail of the sensory ending. Note the small mitochondria with concentric cristae, accumulation of glycogen, and scarce empty vesicles. The intermembrane space (less than 20 nm) is devoid of basal lamina. Abundant pinocytic vesicles surround the nerve ending. (From the J . Ulrrustrucr. Res., 1977, 58, 1%-209.)
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and Porte (1961) published their ultrastructural observations concerning the “cellules particulihres,” which they found in rat cardiac ganglia. There is no doubt that these cells are identical with the chromaffin cells, which have been consequently identified by histochemical (Jacobowitz, 1967; Nielsen and Owman, 1968; Winckler, 1969) and electron microscopic studies (Ellison and Hibbs, 1974; Yamauchi et al., 1975; Moravec and Moravec, 1984). These cells occur grouped in small clusters surrounding a capillary (Fig. 15). They are either isolated in the wall of the interatrial septum or associated with the intracardiac ganglia. In this case, they can share their satellite cell sheath with the adjacent nerve structures. They contain large secretory granules (75-120 nm in diameter), each with an eccentric dense core surrounded by an electron-translucent zone. The density of their secretory granules can be enhanced by the preliminary administration of 5-OH-dopamine (Moravec and Moravec, 1984). On the other hand, in contrast to the terminal adrenergic varicosities, the chromaffin cells are less sensitive to chemical and immunological denervation, as well as to the effects of reserpine (Iversen er al., 1966; Burnstock and Bell, 1974). Some authors also reported their persistence in surgically denervated hearts (Jacobowitz, 1967). The chromaffin cells were found associated with cardiac ganglion of the turtle heart (Yamauchi, 1973), in which they formed reciprocal inhibitory synapses with cholinergic neurons. A similar situation also occurs in rat and guinea pig hearts (Ellison and Hibbs, 1974; Yamauchi et al., 1975). These observations strengthened the view of Jacobowitz (1967), according to which, the chromaffin cells of the heart should be considered adrenergic interneurons modulating ganglionic transmissions in the terminal nerve plexus. In fact, some authors suggested that the chromaffin cells, as seen in electron microscopy, are morphologically similar to small intensely fluorescent (SIF) cells of Norberg et al. (1966), which were found associated with sympathetic ganglia. According to Ellison and Hibbs (1974), these two types of catecholaminecontaining cells are one and the same. Phylogenetically, they might be derived from specific granule-containing cells, which are evenly distributed in the heart of the hagfish and concentrated to the atrioventricularjunction in lampreys and higher vertebrates (Lignon and Le Douarin, 1978). These cells probably recycle catecholamines during each cardiac cycle (Pollack, 1977) and are thought to take part in adrenergic control of the heart via their dual action on cardiac muscle cells and intracardiac cholinergic neurons. A different point of view was formulated by Burnstock and Bell (1974). These authors considered the SIF cells as an intermediate cytological class between short adrenergic neurons of Sjostrand (1%5) and chromaffin cells of the adrenal medulla. As to their function, Burnstock and Bell were in
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FIG.15. Cluster of small chromafin cells (5 kum in diameter) found in the interstitial space of the atrioventricular conductive tissue. Note the irregularity of the Schwann cell sheath (Sw), the presence of several neurites (arrows), and large vesicles with eccentric electron-dense cores. G, Golgi apparatus. (From the Am. J . Anal.. 1984. 171, 307-319.)
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favor of the possibility that the SIF cells, which are most abundant in the autonomic ganglia, can play the role of the interneurons. On the other hand, the chromaffin cells, which are often associated with blood vessels, may function as chemoreceptors involved in the control of the systemic and coronary circulation. The same opinion was also shared by James et al. (1972) and by Ellison and Hibbs (1974). However, apart from SIF and chromafin cells, another adrenergic component of the cardiac plexus (i.e., the above-described large sensory neurons of the atrioventricularjunction) could take an active part in the intrinsic parasympathetic-sympathetic interactions (Levy and Martin, 1979). This would considerably enhance the number of synaptic patterns available and improve the function of the intracardiac oscillatory networks, providing both a focal neuromodulation and a phasic sensory input into the system. These two mechanisms are believed to be essential for the control of oscillatory discharges produced by different neuromuscular motor pattern generators (Selverston and Moulins, 1985). It has been also suggested that the fluorescent cells of the atria may contain, not only catecholamines, but also serotonin and other related substances (Angelakos et al., 1969; Nee1 and Parsons, 1986). In this connection, it should be noted that serotonin might act as a neurotransmitter, at least at the level of the myenteric plexus (Gershon, 1977). Several other studies have indicated that adrenergic axons are responsible neither for the synthesis nor for the high affinity uptake of this compound; according to embryological studies (Gershon and Thompson, 1973), the development of serotoninergic components of the myenteric plexus precedes the ingrowth of adrenergic axons. This would indicate that the uptake of serotonin must be accounted for by intramural neurons which can survive in organotypic cultures. A similar situation may also occur in the cardiac nerve plexus, since serotoninergic neurons have been demonstrated in the hamster (Sole et al., 1979) and in the rat (Votavova et a / . , 1971) hearts. Another prospective source of serotonin and of other vasoactive substances was identified at the level of the interatrial septum of the rat. In this species, a richly innervated lacunar body containing numerous mast cells was invariably present in the proximity of the reticular portion of the atrioventricular node (Fig. 16). A similar structure was also described close to the accessory atrioventricular node (Anderson, 1972), and it was suggested that these secretory cells might be involved in different cardiogenic reflexes (James et al., 1972). A local action of the 5-OH-tryptamine and histamine on the neural structures of the atrioventricularjunction is also not excluded, if we take into account the recent demonstrations of serotoninergic receptors encountered in other divisions of the autonomic and cerebrospinal nervous system (Gershon, 1977). One of the structures which might be particularly sensitive to 5-HT action is the sensory network
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FIG. 16. Detail of the paranodal lacunar body showing the presence of mast cells with large secretory granules of variable density. These cells are supposed to synthetize and store the 5-OH-tryptamine (serotonin).
composed of substance P (SP)-immunoreactive nerve fibers and SP-containing nerve cells, which was recently described in the rat and guinea pig atria (Weihe et al., 1986; Moravec et al., unpublished data).
C. INTERSTITIALCELLS Several species of supporting cells can be distinguished in the intracardiac nerve plexus of different vertebrates. Typical Schwann cells and small satellite cells with fibrous cytoplasm and few organelles tightly surround the large nerve profiles and accompany small terminal neurites into the intracardiac interstitium. This perineural sheath is lost as the nerve fibers join the terminal nerve plexus, for example, at the level of the atrioventricular node. Here the Schwann cells are substituted by large irregular cells which seem to be interconnected by their peripheral processes (Yamauchi et d.,1973; Moravec and Moravec, 1984). The cytoplasms of these cells contain abundant ribosomes arranged in rosettes and associated with the endoplasmic reticulum. Their principal process contains few microtubules, and, often, a large collagen bundle seems to be inserted into its base. The cell membrane of these cells is devoid of basal
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lamina and presents particular specializations,such as microvilli and cilia with their basal bodies (Fig. 17). Their principal extensions are closely associated with the muscle cell membranes in which attachment plaques could be identified (Yamauchi et al., 1973). Sometimes they also receive adrenergic endings. This association resembles axosomatic synapses, at least in the fish heart (Yamauchi, 1973) and in the atrioventricularjunction of the rat (Moravec and Moravec, 1984). In this case, the above intercalary cells were found in nodal interstitium and in the interstitial spaces of the adjacent interventricular septum close to the sensory specializations of the terminal nerve plexus. Some of these glial cells can undergo cell divisions, since typical mitotic figures can be found in the optical and electronic microscopes (Moravec and Moravec, 1984). The above cytological description resembles that of the autonomic interstitial cells of Cajal (1894), which are structurally and functionally interposed between the postganglionic axons and the effector cells of the intramural plexus of the gut and other tissues receiving autonomic innervation (Thuneberg, 1982). According to histological studies, the interstitial cells can share some of their staining properties with the autonomic neurons themselves. However, an exhaustive embryological study will be necessary, for it is possible to state whether they have diverged from the same precursors as ganglion cells or whether the ingrowing autonomic nerve fibers have attracted the mesenchymal elements from the target tissues (Taxi, 1965; Rogers and Burnstock, 1966; Yamauchi et al., 1973). Most of the authors interested in the physiological role of the interstitial cells came to the conclusion that these cells represent a special type of glial cells characteristic for the intramural division of the autonomic nervous system. Their presence in the heart confirms that this intrinsic nervous component is also present in hearts of different vertebrates. However, an interesting hypothesis was recently postulated by Thuneberg (1982), who, after an exhaustive morphological and physiological study, considered the interstitial cells of the gut as the “intestinal pacemaker” cells. He found them structurally associated with the electrophysiologically identified foci, which initiated peristaltic sequences of isolated intestinal segments under study. Whether a similar situation also holds in the heart is not known. However, the presence of interstitial cells in the sinus venosus of the fish heart (Yamauchi et al., 1973) as well as their association with the atrioventricular junction of the rat heart (Moravec and Moravec, 1984) are quite significant in the light of these data. One may conclude, together with Yamauchi et al. (1973), that the interstitial cells of the nodal tissue represent a specialized cell population playing a role in the modulation of the autonomic nerve input directed to the effector myocardial cells.
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FIG. 17. Interstitial cell (Ic) found in the upper portion of the interventricular septum of the rat heart. Note its irregular shape and its close relationships with the adjacent myocytes. The cytoplasm contains rough endoplasmic reticulum and abundant free ribosomes. Co. Collagen fiber; arrows, microvilli. (From the Am. J . Anat., 1984, 171, 307-319.)
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V. New Developments in Studies of the Autonomic Nervous System
In the above sections, the nerve supply to the heart is still considered in terms of the dual (adrenergicand cholinergic) innervation (Dale, 1953). According to this classical principle, each nerve cell makes and releases only one transmitter. However, morphological and neurochemical studies performed is recent years strongly suggest that single neurons may contain and release more than one active compound (Furshpan et al., 1976; Burnstock, 1978; Potter et al., 1981; Lundberg et al., 1983; Smith, 1983). This concept of cotransmission holds for both sympathetic and parasympathetic innervation of the heart (Forssmann et al., 1982; Reinecke et al., 1982; Gu et al., 1983; Lundberg et al., 1983) as well as for the intrinsic cardiac neurons (Gu et al., 1984; Hassall and Burnstock, 1984; Weihe et al., 1984). Generally speaking, the coexistence of the classical neurotransmitters, i.e., norepinephrine or acetylcholine, with a series of putative neurotransmitters (ATP and serotonin) and neuromodulators [substance P, neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), neurotensin, etc.)] could be demonstrated in all divisions of the autonomic nervous system, i.e., in the autonomic ganglia, postganglionic nerve fibers, and intramural neurons (Hokfelt et al., 1977; Leranth and Feher, 1983; Cummings et al., 1984; Lundberg et al., 1984; Elfvin, 1983). In some cases, the two traditionally antagonistic transmitters, i.e., norepinephrine and acetylcholine, were shown to coexist in a single neuron (Patterson, 1978; Burnstock, 1978). It would seem that each neuron, being a cell body supplied with a complete set of genes, possesses the potential ability to synthetize the entire set of enzymes for all transmitter substances. Its actual phenotypic expression is determined by the environmentalfactors and the type of its interactions with the target tissue (Potter et al., 1974; Furshpan et al., 1976; Patterson, 1978; Smith, 1983). It has been suggested that, during the organogenesis, the migrating precursors of nerve cells, having been exposed to different microenvironmental conditions, develop distinct phenotypes leading to different transmitter choices (Patterson, 1978; Le Douarin, 1980, 1984). According to Gershon (Gershon et al., 1981), the microenvironmental signals provided by the target tissues at critical points of their development can explain the sequential changes occurring in the phenotypic expression of peripheral nerve cells during their ontogeny (Le Douarin, 1984) and in conditioned cell cultures (Patterson, 1978). This plasticity of nerve cell precursors can account for the multiplication of the neuronal types and for the diversity of neurotransmitters encountered in some portions of the autonomic nervous system, such as the enteric nervous plexus (Gershon and Erde, 1981; Cummings et al., 1984; Wood, 1984) and the terminal nervous plexus of the heart (Forssmann et a / . ,
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1982; Gu e t a / . , 1983; Dalsgaard et a/. 1985; Hassall and Burnstock, 1984; Moravec and Moravec, 1984; Weihe et a/., 1984). Although it is not easy to reconcile all available data, it would seem that VIP-like immunoreactivity predominates in the perivascular nerves of mainly parasympathetic origin. VIP and PHI (peptide histidine isoleucine) (Lundberg and Tatemoto, 1982) are considered as strong candidates for the atropin-resistant vasodilatation seen upon the parasympathetic stimulation. In the heart, VIP has been shown to be responsible for coronary vasodilatation and positive inotropic effects, which have been reported in dog and cat heart preparations (Said et al., 1972). VIP-immunoreactive cell bodies were demonstrated in the epicardial ganglia, and VIP-immunoreactive fibers predominated in the conduction system. Some of these VIP-containing nerve fibers were in close contact with the cells of the atrioventricular node (Weihe et al., 1984). On the other hand, the NPY and the C-terminal-flankingpeptide of neuropeptide Y (C-PON) have been shown to coexist with norepinephrine in sympathetic nerves around blood vessels. NPY itself induces local vasoconstriction and hypertension upon the intravenous administration (Lundberg and Tatemoto, 1982). The vasoconstrictor response to NPY is long lasting as compared to norepinephrine. The response to NPY is not associated with any postinfusion hyperemia which is seen after the administration of norepinephrine. Finally, the effect is resistant to a-adrenoreceptor-blocking agents and persists after the sympathectomy (Lundberg et a/., 1984). In the heart, the NPY-immunoreactive fibers were detected in the atria of different species including man (Gu et a / . , 1983). Lundberg et a / . (1983) suggested that NPY coexists with both dopamine (3-hydroxylase and norepinephrine. NPY-immunoreactive cell bodies with weak tyrosine hydroxylase-irnmunoreactivity were also found in the rat and mouse hearts (Gu et d.,1984; Moravec et al., unpublished data). According to Hassall and Burnstock (19841, NPY is not exclusively associated with the extrinsic nerves to the heart; it can be synthetized by cultures of the intrinsic cardiac neurons obtained from newborn guinea pigs, the hearts of which are not yet innervated by the extrinsic nerves. In this case, NPY does not coexist with norepinephrine, but it is not excluded that this would be the case in sitir where these intrinsic cardiac neurons still contain dopamine P-hydroxylase and tyrosine hydroxylase (Gu et al., 1984). Another peptide which has been found associated with the intrinsic ganglion cells, at least in the gut (Costa et al., 1982), is substance P. This compound has been identified as an atropine-resistant excitatory neuromodulator secreted by the network of multipolar intestinal interneurons (Costa rt a/., 1981; Cummings et a/., 1984; Wood, 1984). The activation of the entire network is triggered by serotonin release from neurons with
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Dogie1 I morphology (unipolar neurons); the activity within the network is terminated by an inhibitory action of the optoid peptides. These are known to hyperpolarize and inhibit the intramural neurons by increasing their GK (cf. Wood, 1984). Another substance P-mediated action is the antidromic stimulation of afferent C fibers involved in local vasodilatation (Hokfelt et al., 1975; Lundberg et al., 1984). The association of substance P with sensory nerve fibers investing various peripheral tissues, including the heart, is now rather well established (Lundberg et al., 1984; Weihe et al., 1984; Nee1 and Parson, 1986). In this respect, it should be noted that a small proportion of substance P containing ganglion cells could be identified in sympathetic ganglia (Hokfelt et al., 1975; Leeman, 1980). In particular, the superior and the middle plus inferior cervical ganglia as well as the upper thoracic (T3-T,) ganglia of the rat contain the substance P-immunoreactive primary nerve fibers sensitive to capsaicin treatment (Tsunoo et al., 1982; Papka et al., 1981). According to Forssmann et al. (Weihe et al., 1984), various receptor structures of the heart (coronary chemoreceptors, baroreceptors, etc.) could be interconnected, via axonal collaterals of substance P-positive afferents, with the efferent sympathetic and parasympathetic pathways far below the level of their respective central integration centers. Abundant substance P-immunoreactive nerve fibers were found branching on the intracardiac cholinergic and VIP-ergic nerve bodies. These authors suggested that the cardiac substance P-containing afferents may modulate the actual postganglionic nervous input to the heart. They can be also involved in short, intrinsic, substance P- and VIP-mediated feedback loops necessary for the fast beat-to-beat autoregulation of cardiac electrical activity (Covell et al., 1981; Weihe et al., 1984).
The above distinction between the VIP, NPY, and substance P-ergic nerve fibers should be considered as rather didactic; it is not excluded that some of the neuropeptides coexist in a single nerve cell (White et al., 1985). It has been demonstrated that different neuropeptides can be derived from the same precursor synthetized by a unique gene. The choice of a given amino acid sequence, that is a given phenotypic expression, takes place at the transcriptional level, different mRNA being used for the synthesis of different neuropeptides (White et al., 1985). This situation may contribute to the multiplicity of neuronal phenotypes as well as to the diversity of interneuronal connections encountered in the terminal nerve plexuses. This, in turn, seems to argue against the idea that the intramural plexuses should be considered as a diffuse parasympathetic ganglia in which direct synaptic connections between preganglionic fibers and postganglionic cholinergic neurons are the unique basis of organ function (Wood, 1984).
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Some of the ancestral conceptions concerning the exclusivity of both adrenergic and cholinergic centrifugal transmission in the autonomic nervous system (Dale, 1953) do not hold any longer. At present, the peripheral extensions of the autonomic nervous system are more and more considered in terms of pluricellular networks with a high degree of histotopographical specialization providing a nonnegligible functional autonomy (Leranth and Unguary, 1980; Brooks, 1981; Weihe et al., 1984; Wood, 1984). This evolution is nothing else but the rehabilitation of Langley’s concept of the functionally independent intrinsic nervous system of peripheral organs (Langley. 1921). At the same time, the recent progress in the electron microscopic and immunohistochemical techniques gave rise to the contention that this special category of the autonomic nervous system is present, not only in the intestinal wall (Selverston et a / . , 1976; Gershon and Erde, 1981; Wood, 1984), but also in other organs receiving the autonomic innervation including the heart (Crowe and Burnstock, 1982; Reinecke et a / . , 1982; Lundberg e t a / . , 1983; Gu et al., 1984; Hassall and Burnstock, 1984; Moravec and Moravec, 1984; Weihe et al., 1984). According to the electrophysiological and pharmacological studies (Hirst e t a / . , 1973; Erde at a / . , 1980; Nozdrachev and Vataev, 1981; Wood, 1981; North, 1982), the enteric nervous system is considered as the best example of an independent integrative system composed of subsets of interneuronal circuits that control the respective intestinal segments and ensure the appropriate function of the whole organ (Wood, 1984). Each segment is composed of a network of interneurons that processes the sensory information and controls the activity of the respective motor neurons. The stereotyped reflex behavior is mediated by the integrative circuits which also generate the motor patterns of the individual intestinal segments. The synaptic connections between the adjacent neuronal subsets ensure their reciprocal coordination that is necessary for peristaltic movements. The role of the central nervous system is restricted to the modulation of these preprogrammed circuits. The intestinal tract can thus be considered as a self-governed fluid-propelling system possessing its own little brain (Weems, 1981). VI. Morphological Basis of the Rhythmical Activity of the Heart: A Working Hypothesis
A similar situation may also prevail in the mammalian heart, the complex structure of which is also derived from a primitive cardiac tube (Anderson et d.,1978). The development of the intracardiac septum and the separation of the outflow trunk into the ascending aorta and pulmonary artery
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are initiated by the migration of the ectomesenchymal elements derived from the cranial neural crest (Kirby, 1985). The presence of ganglion cells within the interatrial septum also depends on the migration of the ectodermic elements (Kirby and Stewart, 1984). It has been suggested that neural crest cells from both cranial (S,-S,) and thoracic segments 6,"Szo)may contribute to the embryogenesis of the intracardiac conduction system (Taylor, 1977; Stewart and Kirby, 1985). In postnatal life, they may constitute a heterogeneous nerve network interconnecting the upper segments of the left ventricle with the specialized structures of the right atrium (Wensing, 1965; Bojsen-M#ller and Tranum-Jensen, 1972; Weihe et al., 1984; Forsgren, 1985). Such a neuromuscular network may provide for the anatomical substratum of the intracardiac servomechanism involved in the local control of the electrical activation of the heart and the propulsion of the blood through the cardiac cavities (Fig. 18). The strategic position of richly innervated structures of the atrioventricular junction between the venous return and arterial portion of the systemic circulation is, in this respect, quite significant. In other words, the intracardiac conduction system of higher vertebrates resembles a neuromuscular oscillatory system similar to the oscillatory networks responsible for rhythmical movements of some of lower species (Fields and Kennedy, 1965; Bullock et al., 1977; Connor, 1985). The following observations seem to argue in the sense of such a possibility. ( I ) Presence of sensory specializations (mechanoreceptors)which is necessary for beat-to-beat modulation of these oscillatory systems (Weihe et a/., 1984; Selverston and Moulins, 1985). In some cases (Altman, 1982), a phasic sensory input is responsible for the generation of the oscillatory patterns to be observed. As concerns the rhythmical depolarizations of the nodal tissue, it has been experimentally demonstrated that they are under the control of mechanical stretch. It has been shown that a phasic sensory input can both entrain the pacemaker as well as reset it to a new rhythm (Brooks and Lu, 1972; Pollack, 1977; Irisawa, 1978). (2) Existence of the intracardiac neurosecretory (adrenergic and peptidergic) neurons which, together with the highly specific histotopography of the intrinsic multitarget nerves, seems to suggest that multiple and complex transmitter interactions can occur at the level of the interatrial septum. This local neuromodulation may explain some of the physiological properties of the cardiac pacemakers (existence of true pacemaker cells and follower cells) and intracardiac conduction tissue [dual atrioventricular node (AVN) entry, slow and fast intranodal pathways, AVN-NH conduction delay, etc.] (Janse et al., 1978). In particular, it has been suggested that a sudden catecholamine release from tissue-binding sites may trigger the sinus node (SN) depolarizations (Cranefield, 1978; Pollack, 1978). Catecholamine re-
I37
IN'IKINSIC NERVE PLEXUS OF MAMMALIAN HEART CNS
'
I
' ''
\ \
\
\
\ \
MECHANO-
VENTRICLES
RECEPTOR
SA N O D E
I
Fic;. 18. Model of the functional arrangement of the neuromuscular network of the interatrial septum responsible for the intrinsic generation of rhythmical coordinated contractions of cardiac cavities. The sinus node can be considered a motor pattern generator. The rate of its triggered depolarizations as well as the intracardiac conduction are supposed under the control of the intrinsic nerve plexus. which constitutes a local integrative component. The latter can process the efferent stimuli from the C N S as well as the sensory feedback from intracardiac mechanoreceptors necessary for a fast beat-to-beat modulation of cardiac excitability and contraction. SA. Sinoatrial node.
cycling between the intracardiac adrenergic cells and tissue receptors could thus be responsible for the sustained rhythmical activity of different cardiac preparations (Pollack, 1977; Cranefield, 1978; Zipes, 1981). According to our data, large sensory neurons of the atrioventricular junction may contribute to catecholamine traffic at the level of the interatrial septum (Fig. 19). The existence of a positive sensory feedback between the upper segment of the interventricular septum and the S N area is therefore not excluded. The adrenergic nature of this sensory feedback could be particularly adapted for the control of the heart rate: the same neurotransmitter (norepinephrine or dopamine?) may stimulate directly the nodal cells (Cranefield, 1978; Irisawa, 1978). having at the same time, an inhibitory effect on cholinergic nerves supplying the pacemaker area (Levy and Martin, 1979; Sole clt d.,1982; Loffelholz and Pappano, 1985). This multiplicity of norepinephrine action may considerably amplify its overall effect on
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JOSEF MORAVEC AND MIREILLE MORAVEC
HlSSlAN
FIG. 19. Sinus node and atrioventricular junction as relaxation oscillators. Schema of the extrinsic innervation and of the local adrenergic feedback between the interventricular septum and cardiac pacemaker. Phasic stimulations of the sensory neurons of the AV junction may be coresponsible for cyclic catecholamine release and reuptake resulting in triggering and synchronization of the sinus node. It can be also involved in the modulation of vagal input to the heart, which would amplify indirectly the effects of sympathetic stimulation. PG. Epicardial cholinergic ganglion; ISG, intraseptal ganglion; SG, stellate ganglion; C,-T,, cervical and upper thoracic sympathetic ganglia; SN, sinus node; AVN. atrioventricular node; RB, right branch; LB, left branch.
the electrical properties of the pacemaker cells. The above catecholamine release is believed to be phasic, since it is under the control of mechanical events accompanying cardiac contractions (Brooks and Lu, 1972; Pollack, 1977; Weihe et al., 1984). This may explain the dynamic nature of vagusSN interactions (Jalife et al., 1983; Michaels et a/., 1983) and some badly understood aspects of sympathetic-parasympathetic interactions (Levy and Martin, 1979; Levy, 1984). These latter observations suggest that different segments of the intracardiac conduction system should not be considered functionally isolated. It would seem that specialized cells together with the respective nervous components constitute a self-governed nonlinear dynamic system with variable transit functions (Yates, 1983; Van Rossum et al., 1984). Such a highly organized network would not only react to input stimuli, but it should be able to process the input informations and control its own environment (Aplevich, 1968). These are the obvious prerequisites for the efficient and well-integrated cardiac function (Wurster, 1984).
INTRINSIC NERVE PLEXUS OF MAMMALIAN HEART
I39
VII. Conclusion
The above hypothesis will need a thorough verification. The morphological data presented in this review have to be completed by new electrophysical and pharmacological evidence. However, the upward axonal projections of the adrenergic neurons of the atrioventricular junction may provide for the retrograde limb of the connecting loop transforming the two cardiac pacemakers into a system of coupled relaxation oscillators (Van der Pol, 1940). In such a system, the atrioventricularjunction should not be any more considered as functionally subordinated to the sinus node and simply transmitting the excitation values from the right atrium to the left and right ventricles. The electromechanical events that accompany any heartbeat and which are perceived at this level may, after local processing influence the timing of the forthcoming sinus node depolarization and modulate the execution of the next mechanical systole (Covell et d., 19811. The need for such an arrangement has been predicted by James in his original "Brief Review" (James, 1973). At that time, the internodal communications were not completely understood, but according to James, the closing limb of the internodal loop should combine both a neural (reflex) factor and mechanical (hemodynamic)events. According to our data, both of these parameters might be interacting at the level of a single cell of the terminal nerve plexus of the rat atrioventricular junction (Moravec and Moravec, 1984; Moravec et a / . , 1986).
ACKNOWLEWMENTS
Professor R. Couteaux and Professor J. Taxi are thanked for their encouragement and for their critical advice. We also would like to thank Mrs. M.-T. Dronne for her patience in typing our article and Mrs. A. Courtalon for her skillful technical assistance.
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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. I W
Structural and Functional Evolution of Gonadotropin-Releasing Hormone ROBERTP. MILLARA N D JUDY A. KING Medical Reseurch Council Regulatory Peptides Research Unit, Department of’ Chemical Pathology, University of Cape Town Medical School and Groote Schuur Hospital, Observatory 7925, Cape Town, South Africa
I. Introduction Gonadotropin-releasing hormone (GnRH)I is a hypothalamic decapeptide (Fig. I ) which regulates reproduction by stimulating the release of pituitary gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), which in turn stimulate gonadal activity (Schally, 1978). Following the structural elucidation of GnRH from porcine and ovine hypothalamus, it became generally accepted that the decapeptide was a unique molecular form. This belief arose from the following: 1. Studies purported to have shown nonribosomal biosynthesis of GnRH and thereby excluded the possibility of ribosomally biosynthesized prohormonal forms. 2. It was assumed that GnRH was confined to the central nervous system and, thus, was unlikely to be present in other tissues in a modified form. 3. Immunological and low-resolution chromatographic studies demonstrated that GnRH in the hypothalamus of nonmammalian vertebrates was identical to the mammalian peptide. This notion of a lack of interspecific differences in GnRH structure in vertebrates was supported by the demonstration that mammalian GnRH was biologically active in a wide range of mammalian species and in nonmammalian vertebrates (Schally, 1978).
A number of factors argued against the view of a nonribosomal synthesis of GnRH and a universal conservation of the GnRH structure.
( I ) The GnRH structure, comprising exclusively L-amino acids, a-amino peptide linkages, and a cyclized NH,-terminal Glu and COOH-terminal amide, is characteristic of ribosomally synthesized peptides. In contrast, ‘Abbreviations: GnRH, Gonadotropin-releasing hormone; mGnRH, mammalian GnRH: cGnRH 1. chicken GnRH I (Gln‘--GnRH); cGnRH 11. chicken GnRH I I (His’, Trp’. TyPGnRH): sGnRH. salmon GnRH (Trp’, Leun-GnRH); LH. luteinizing hormone; FSH, folliclestimulating hormone; HPLC. high-performance liquid chromatography. I49 Copyright Q 1987 by Academic Press. Inc. All riphls of reproduction in any form reserved.
I50 Pig/Sheep
ROBERT P. MILLAR A N D JUDY A. KING pGlu'
-
His2
-
T r p 3 - Serb - T y r 5
-
Gly6 - Leu7 - Arg8 - Pro9 - Glyl0.NH2
Chicken I
Gln
Chicken I 1
His
Trp Trp
Salmon FIG.
I.
- Tyr - Leu
Structures of vertebrate GnRHs.
the presence of D-amino acids (e.g., in prokaryote antibiotic peptides) and unusual peptide linkages (y-glutamyl in glutathione and p-alanyl in carnosine in eukaryotes) characterizes peptides synthesized by nonribosomal mechanisms. (2) The related neurohypophyseal peptides, vasopressin and oxytocin, had been shown convincingly to be processed by proteolytic cleavages from ribosomally synthesized precursors. Moreover, the neurohypophyseal peptides exhibit structural heterogeneity in vertebrates, which is consistent with conservative single nucleotide base changes in the triplet codons (Acher, 1983). (3) Accepting that GnRH is synthesized ribosomally, it appeared likely that different GnRH molecular forms would have arisen in vertebrates during 400 million years of evolution, as is the case with the neurohypophyseal hormones (Acher, 1983). Research over the past 7 years has now established that there is considerable diversity in the structure of GnRH and related molecular forms. Higher molecular-weight prohormonal forms have been demonstrated, while structural variations in vertebrate hypothalamic GnRHs have been shown in several species from the major vertebrate classes. These structural differences have been confirmed by the isolation and structural analysis of GnRHs from a single species of bird, amphibian, and teleost (Fig. I). GnRH-like peptides, which may differ structurally from the mammalian hypothalamic peptide, have also been found in mammalian tissues, such as the extrahypothalamic brain, testis, and ovary. In this report, we review current knowledge on the molecular heterogeneity of GnRH, the biological actions of GnRH and related molecules, structure-activity relations, and the evolution of the hormone. 11. Structure and Distribution of GnRH and Related Molecular Forms
A. PROHORMONAL GnRH The existence of higher molecular-weight immunoreactive forms of GnRH in hypothalamic extracts suggested that these might constitute prohormonal species. In both rat and sheep hypothalami, immunoreactive
EVOLUTION OF GONADOTROPIN-RELEASING HORMONE
I5 I
GnRH peptides of molecular weights of >5K, 3K, and 2K were detected in addition to decapeptide GnRH (Millar et al., 1977, 1978, 1981a). Since the 5K peptide eluted in the void volume of a Sephadex G-25 column, it was possible that it had a molecular weight of greater than 5K. Recent studies in our laboratory using Sephadex G-50 and high-performance liquid chromatography (HPLC) gel permeation chromatography under denaturing conditions indicate that the precursor is approximately 8K-1OK in size. Extensive studies provided evidence for the prohormonal nature of the largest peptide. The peptide was specifically retained on immunoaffinity columns using an appropriate antiserum directed toward the central sequence of GnRH; it was not dissociated by rigorous treatment with 8 M urea and 6 M guanidinium hydrochloride and was not an immunoassay artifact, as it did not bind or degrade '251-labeledGnRH. Physiological manipulations, which altered hypothalamic GnRH content, also changed the occurrence of the putative prohormonal forms, and in tissues lacking GnRH, no immunoreactive higher molecular-weight material was detected (Millar et al., 1978, 1981a). Specific chemical modifications of each of the amino acids comprising the GnRH sequence resulted in a similar loss of immunoreactivity of both GnRH and the 25K prohormonal form when quantitating with appropriate antisera requiring the particular residues for binding (Millar et al., 1978, 1981a). These studies also demonstrated that proteolytic cleavage of GnRH and the prohormones with a range of enzymes (except trypsin) yielded appropriate losses of immunoreactivity with the different antisera. The presence of relatively higher proportions of the prohormonal forms in hypothalamic regions containing GnRH cell bodies compared with a paucity of prohormonal forms in the stalk median eminence, supported the classical concept of neuronal precursor-peptide processing for GnRH biosynthesis (Millar et al., 1978, 1981a). These observations have been more thoroughly demonstrated by immunocytochemistry (King and Anthony, 1983, 1984). The prohormonal forms were also more prevalent in the microsomal fraction than in purified synaptosomes (nerve endings) which contained exclusively fully processed decapeptide GnRH (Millar et al., 1981a). HPLC separation of the synaptosomal extract confirmed that only decapeptide GnRH is present in contrast to the presence of both somatostatin-28 and somatostatin-14 in synaptosomes (Kewley er al., 1981). Other studies addressed the question as to the localization of the GnRH sequence within the prohormonal peptide. The interaction of the 35K putative prohormonal GnRH species with seven region- and/or conformation-specific GnRH antisera indicated that the molecule is modified at both the NH2- and COOH-termini (Millar et al., 1978, 1981a). This indication of both NH2- and COOH-terminal extensions to the GnRH sequence was supported by the demonstration that aminopeptidase and carboxy-
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peptidase digestions led to increases in immunoreactivity (Millar er a/., 1978, 1981a). The 5K GnRH species was partially converted by hypothalamic peptidases into an immunoreactive peptide eluting in the position of GnRH. Progressive trypsin digestion released a 2K- to 3K- immunoreactive species followed by complete conversion to a single form migrating in the position of GnRH on Sephadex (3-25 (Millar et al., 1977, 1978, 1981a). It was suggested, therefore, that there are both NH,- and COOHterminal modifications or extensions to GnRH in the putative prohormonal GnRH and that trypsin-sensitive cleavage sites (basic amino acids) are interposed between the GnRH sequence and peptide extensions. This arrangement is common to many prohormones which are processed by trypsin-like cleavages at pairs of basic amino acids, followed rapidly by removal of the exposed basic residues by carboxypeptidase-B-like activity (reviewed by Douglass et al., 1984). It was also proposed that prohormonal GnRH is characterized by Gln in position one of the GnRH sequence, which is spontaneously (or enzymatically) cyclized after cleavage of the NH,-terminal extension to give rise to pGlu' (Millar er a/., 1981a). In addition, the amide (GIY'~*NH,) was presumed to arise from the amino group of an additional Gly preceding the basic residues. This now seems a likely possibility, as structural analysis of prohormonal forms of 1 I propeptides with COOH-terminal amides has revealed that all have this additional Gly, and recent studies on a pituitary amidating enzyme using synthetic peptide substrates have demonstrated an absolute requirement for Gly in this oxidative transamidation (Bradbury et d . , 1982). Subsequent studies demonstrated 26K and I .8K higher molecular-weight immunoreactive forms of GnRH in extracts of rat hypothalamus, cortex, and placenta (Gautron et al., 1981). The 26K peptide was recognized almost exclusively by NH,-terminus-directed antiserum, suggesting it is COOH-terminally extended. Unfortunately, the authenticity of the 26K species is uncertain, as rigorous dissociating conditions were not used. It is also uncertain as to whether both NH,- and COOH-extensions were present, as a middle-directed antiserum which would recognize such forms was not employed. The primary immunoprecipitable GnRH translation product of mouse and human hypothalamic mRNA in reticulocyte lysate was recently shown to be a 28K peptide (Curtis and Fink, 1983; Curtis et a / . , 1983). Allowing for the cleavage of a signalAeader sequence, this is comparable to the 26K peptide reported above. Although the above studies provided a persuasive argument for the existence of prohormonal GnRH, final proof lies in the isolation and sequence analysis of the putative prohormone and demonstration of its conversion to GnRH by the tissues concerned. The demonstration of incorporation of [3H]tyrosine into GnRH by human placental trophoblast (Tan and
I53
EVOLUTION OF GONADOTROPIN-RELEASING HORMONE
Rousseau, 1982) indicates the potential of this tissue for these biosynthetic and processing studies. An alternative approach to establishing the structure of prohormonal GnRH is by recombinant DNA technology. Several laboratories initiated programs aimed at the difficult goal of elucidating the sequence of GnRH mRNA. Our laboratory was unable to convincingly demonstrate GnRH clones in hypothalamic cDNA libraries using labeled synthetic oligomers (17-mers) coding for both NH,- and COOH-terminal sequences of GnRH. However, several clones which hybridized with these oligomers were identified in a cDNA library of a human buccal tumor cell line which produced GnRH. Recently, a major breakthrough occurred in establishing the sequence of a human GnRH gene and human placental mRNA encoding a precursor form of GnRH (Seeburg and Adelman, 1984). The cDNA sequence codes for a protein of 92 amino acids in which the GnRH decapeptide is preceded by a signal peptide of 23 amino acids and followed by a Gly-Lys-Arg sequence, as expected for enzymatic cleavage of the decapeptide from its precursor (Gubler et al., 1983) and arnidation (Bradbury et ul . , 1982) of the COOH-terminus of GnRH (Fig. 2). The next apparent cleavage site is Ly~'"-Lys''~,which suggests that a 53 amino acid peptide is also processed from the precursor. Single basic amino acid residues (Fig. 2) might, however, also be cleavage sites as in several other prohormones. Since hormone precursors can generate several biologically active peptides, we recently synthesized the sequence 14-26 and tested the effects of this peptide on cultured human pituitary cells. The peptide did not affect thyrotropin or prolactin release but, surprisingly, stimulated both LH and
PRE -23
GnRH -1 1
CS 10
11
Gh H s TP Ser Tyr oly Leu Arg Pro oly Gly lys Arg
14
CARBOXYL-TERMINAL EXTENSION 27 37 46 50 53 66 LYS
Arg
Arg ArgLys
PCS 69
lys lys le
FIG.2. Schematic diagram of the structure of the human placental GnRH precursor as determined by nucleic acid sequencing of the corresponding cDNA (Seeburg and Adelman, 1984). The precursor consists of a signal sequence (PRE) of 23 amino acids followed immediately by the GnRH decapeptide sequence. Cleavage of the signal peptide reveals a NHZ-terminusGin which cyclizes (enzymatically or spontaneously) to pyro-Glu. The GnRH sequence is followed by a Gly. which is the donor for the COOH-terminal amide of GnRH, and Lys-Arg, which is a conventional dibasic amino acid cleavage site (CS). This is followed by a 53 amino acid peptide before a second potential cleavage site, suggesting that this peptide is released together with GnRH. The positions of single basic amino acid residues are also shown, as these may be cleavage sites as in the precursors of vasopressin, enkephalins, somatostatin. cholecystokinin, growth hormone-releasing factor, epidermal growth factor, and nerve growth factor.
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ROBERT P. MILLAR A N D JUDY
A.
KING
FSH release (R. P. Millar, P. J. Wormald, and R. C. deL. Milton, unpublished). GnRH antagonists did not affect the stimulation, suggesting that the effects are mediated by a separate receptor. The physiological significance of the presence of a second gonadotropin-releasing peptide in the GnRH precursor requires clarification.
B. GnRH IN MAMMALIAN TISSUES 1. Hypothalamic GnRH GnRH was originally isolated from porcine hypothalami by virtue of its ability to stimulate the release of pituitary gonadotropic hormones (Matsuo et al., 1971; Scha4ly et al., 1971). Subsequently, the same structure was established for ovine hypothalamic GnRH (Amoss et al., 1971). Hypothalamic GnRH in several other mammalian species has identical chromatographic and immunological properties to porcine-ovine GnRH (designated mGnRH) (Fig. 1). 2 . Extrahypothalamic Brain GnRH The presence of immunoreactive GnRH in extrahypothalamic brain regions is well documented (Endroczi and Hilliard, 1965; Silverman and Krey, 1976; Ibata et al., 1983), but few studies have investigated the molecular nature of the peptide(s). The sheep pineal gland has a GnRH molecular form which is identical to the hypothalamic peptide in its interaction with different region-specific antisera and which cannot be distinguished using gel filtration chromatography, cation-exchange chromatography, or higher resolution HPLC (King and Millar, 1981a; Millar and Tobler, 1981; Millar et al., 1981b). These studies demonstrated a second form of GnRH which was structurally distinct from the hypothalamic hormone. This GnRH species was of similar size to GnRH as it comigrated with the decapeptide on Sephadex G-25, brlt was less positively charged and eluted earlier on cation-exchange chromatography. The peptide had similar properties to a chicken hypothalamic GnRH (Gln8-GnRH) (see below) in coeluting on cation-exchange chromatography and reversed-phase HPLC. Interactions with NH2- and COOH-terminal-directed antisera and resistance to degradation by aminopeptidase and carboxypeptidase A supported the studies with antisera, which indicated the presence of pGlu' and Gly" NH, in the molecule. The pineal gland form of GnRH had intrinsic LH-releasing activity, but decreased the LH response to GnRH, suggesting that it may be a weak agonist. The presence of Gln8-GnRH in the sheep pineal gland has recently been confirmed using high-resolution HPLC systems, which were specifically designed to separate GnRH an-
EVOLUTION OF GONADOTROPIN-RELEASING HORMONE
I55
alogs (J. A. King and R. P. Millar, unpublished). The occurrence of a GnRH species in the pineal gland, which is similar to hypothalamic GnRH of a lower vertebrate (Gln'-GnRH in chicken hypothalamus), is reminiscent of reports on the presence of vasotocin in the mammalian pineal gland (Pavel, 1979). The finding of Gin'-GnRH in the pineal gland poses the possibility that this molecular species is present in other areas of the nervous system and serves a neurotransmitter or neuromodulator role. 3. Gonadal GnRH
GnRH has direct effects on the gonads of laboratory animals (Hsueh and Erickson, 1979; Clayton et al., 1981; Sharpe and Cooper, 1982), and high-affinity binding sites for GnRH analogs have been demonstrated in the Leydig cells of the testis (Labrie et al., 1978; Bourne et al., 1980; Perrin et al., 1980; Sharpe and Fraser, 1980; Fraser et al., 1982; Millar ef al., 1982) and in the granulosa cells of the ovary (Harwood et al., 1980; Pieper el al., 1981; Hazum and Nimrod, 1982; Marian and Conn, 1983). Testicular extracts were reported to displace '2'I-labeled GnRH in radioreceptor assays (Sharpe et al., 1981). Immunoreactive GnRH species from acetic acid-extracted and immunoaffinity-purified rat testicular material (Dutlow and Millar, 1981) have been characterized by region-specific antisera and by gel filtration and HPLC. Molecular species of -IOOK, 32K, 5K, and IK were all found to interact strongly with a COOH-terminaldirected antiserum and poorly with middle- and NH,-terminal-directed antisera, suggesting they contain the COOH-terminal sequence of hypothalamic GnRH. Only the lower molecular-weight forms displaced "'Ilabeled GnRH binding to rat pituitary GnRH receptors. A recent report of a similar study on rat testis extracts also suggests that the testicular material shares COOH-terminal sequences in common with GnRH and displaces "'I-labeled GnRH in the radioreceptor assay (Bhasin el al., 1983). In contrast, another study on rat testicular GnRH showed that the high-molecular-weight form could be dissociated to molecules of the same size as GnRH, which reacted more poorly with COOHterminal-directed antisera than with an antiserum specific for the NH2and COOH-terminus and one recognizing the middle region of the molecule (Paul1 et al., 1981 ;Turkelson et al., 1983). In porcine follicular fluid, three immunoreactive GnRH peptides in the molecular range 30K-50K have been detected. The immunoreactivity of these peptides was found to increase after trypsin digestion (M. S. Hendricks and R. P. Millar, unpublished). Since our recent studies with hypothalamic prohormonal GnRH have shown that rigorous conditions are necessary to dissociate high-molecular-weight forms of GnRH, much of the work on gonadal GnRH should be regarded with caution. This is emphasized by the studies of Turkelson
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ROBERT P. MILLAR AND JUDY A. KING
et al. (1983). Since the gonadal GnRH examined may be bound to other peptides, the antisera studies defining sequences in common with hypothalamic GnRH must also, therefore, be regarded with caution, as certain sequences may be obscured by the binding peptide.
4. GnRH in Other Tissues Immunoreactive GnRH detected in the placenta has immunological, chromatographic, and biological properties identical to the hypothalamic decapeptide (DePalatis et al., 1980; Khodr and Siler-Khodr, 1980; Lee el al., 1981;Tan and Rousseau, 1982). Higher molecular-weight forms (26K) have also been detected in this tissue (Gautron et al., 1981). Elucidation of the sequence of human placental GnRH cDNA (Seeburg and Adelman, 1984) has now confirmed the existence of a GnRH in the placenta with the identical structure of the hypothalamic peptide. The cDNA sequence codes for a 69 amino acid precursor (-8K). GnRH-like immunoreactive material has been demonstrated by radioimmunoassay or immunohistochemical techniques in the mammalian pancreas (Seppala and Wahlstrom, 1980a), submandibular gland (C. Dutlow and R. P. Millar, unpublished), olfactory system (Phillips et al., 1980; Dluzen and Ramirez, 1983; Witkin and Silverman, 1983), milk (Baram et al., 1977; Sarda and Nair, 1981; Amarant et al., 1982; Hazum, 1983), and in certain mammary tumors (Seppala and Wahlstrom, 1980b). Specific binding sites for GnRH have been demonstrated in the placenta (Currie et al., 1981; Belisle et al., 1984) and adrenal gland (Bernard0 et a / . , 1978; Pieper et al., 1981; Eidne et al., 1985a). A GnRH-like peptide has been described in the amphibian adrenal gland (see below), but there are no reports of GnRH in the mammalian adrenal gland. C. INTERSPECIFIC HETEROGENEITY IN GnRH
I . Birds In the early studies on bird hypothalamic GnRH, one study suggested an identity (Jeffcoate et al., 1974) of bird GnRH with mammalian GnRH (mGnRH), but others indicated that there were structural differences (Jackson, 1971; King and Millar, 1979a, 1980; Hattori et al., 1980). Chicken (Gallus domesticus) and pigeon (Colurnba livia) hypothalamic GnRHs had a similar molecular size to the mammalian peptide, but were less positively charged and differed immunologically (King and Millar, 1979a, 1980). Antisera directed toward the middle region of mGnRH and those recognizing the COOH-terminal three amino acids gave lower quantitation and nonparallel radioimmunoassay displacement curves. Antisera requiring the
EVOLUTION OF GONADOTROPIN-RELEASING HORMONE
I57
extreme NH,- and COOH-termini and tolerant of certain amino acid substitutions in the middle region of the molecule gave high quantitation and parallel displacement curves (King and Millar, 1979a, 1980). Examination of overlapping sequence requirements of these antisera indicated that the alteration in this form of chicken hypothalamic GnRH, designated chicken GnRH I (cGnRH I), resided in the position of Arg' (Fig. I ) . The difference was investigated in more detail by noting the interaction with the different antisera after selectively modifying the molecule at constituent amino acid residues of GnRH in turn by specific chemical and enzymatic treatment (King and Millar, 1982a). These data showed that cGnRH I differed at Arg'. The difference in isoelectric points of cGnRH I and mGnRH was compatible with a neutral amino acid substitution for Arg' of mGnRH. On the basis of evolutionary probability of amino acid interchange for Arg, Gln was a likely candidate. The putative cGnRH I (Gld-GnRH) (Fig. I ) was synthesized and shown to have identical immunological, chromatographic, and biological properties to natural cGnRH I (King and Millar, 1982a). Other GnRH analogs with substitutes of Ser, Trp, Leu, Met, Ile, Phe, His, Asn, Glu, Cit, Orn, and Lys in position eight had properties different from that of natural cGnRH I. The use of a specific antiserum raised against synthetic Gln'-GnRH, which has an absolute requirement for glutamine in position eight (King et al., 1983), confirmed the assignment of glutamine to position eight. Concurrent studies culminated in the purification of 17 pg of cGnRH I from 250,000 chicken hypothalami using a combination of affinity chromatography, cation-exchange HPLC, and reversed-phase HPLC. Amino acid analysis of an acid hydrolysate showed an absence of Arg and the presence of an additional Glu, compatible with the proposed structure (King and Millar, 1982b,c). Sequence analysis was consistent with the location of Gln as a replacement for Arg in the eighth position (King and Millar, 1982b; J. Spiess, J. A. King, and R. P. Millar, unpublished). Another group has confirmed the structure of this cGnRH I as Gln8-GnRH (Miyamoto et al., 1983). Subsequently, a second form of GnRH, His',Trp',Tyr'-GnRH [designated chicken GnRH I1 (cGnRH 11) (Fig. I I], was isolated from chicken hypothalamus (Miyamoto et al., 1984). Both forms of GnRH stimulate gonadotropin secretion, but it remains to be determined whether only one or both forms are released into the hypothalamopituitary portal system and reach the pituitary gland. GnRH has not been isolated from any other species of birds. Recent studies using chromatographic, immunological, and bioassay assessment have established the presence of cGnRH I and cGnRH I1 in ostrich (Struthio cumelus) hypothalamus (R. C. Powell, R. P. Millar, and J. A. King, unpublished) (Table I).
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ROBERT P. MILLAR A N D JUDY A. KING
hYLOGENETIC
TABLE I DISTRIBUTION OF THE FOURKNOWN VERTEBRATE BRAINGnRHs GnRH structure
Group Mammal Bird Reptile
Amphibian Teleost
Elasmobranch
mGnRH cGnRH 1 cGnRH I1 sGnRH
Species
.
Pig" Sheep" Chicken" Ostrich (Strurhio camelus)b Alligator (Alligator mississippiensis)b Lizard (Podarcis sicula sicuIa)b Lizard (Cordylis nigra)b Skink (Calcides ocellatus)b Frog (Rana cafesbeiuna)" Toad (Xenopus l a e W b Salmon (Oncorhynchus ketay Codfish (Gadus morhua morhua)" Hake (Merluccius capensis)b Tilapia (Tilapia sparrmanii)b Coris julis" Mullet (Mugil cepha/us)b Milkfish (Chanos chanos)b Trout (Salmo gairdneri)b Dogfish (Poroderma africanum)b
+ +
+ + + +
+ + + + + +
+ + + +
+
+ + + + + + + + + + +
"Determined by amino acid composition andor sequence analysis. "Determined on the basis of immunological and chromatographic studies and (in most cases) assessment of LH-releasing activity in a chicken pituitary cell bioassay.
GnRH immunoreactivity is also present in the extrahypothalamic brain of birds (King and Millar, 1980; Jozsa and Mess, 1982; Sterling and Sharp, 1982; Knight et al., 1983). In ostrich extrahypothalamic brain, the presence of immunoreactive and bioactive cGnRH I and cGnRH 11 has recently been demonstrated (H. Jach, R. C. Powell, R. P. Millar, and J. A. King, unpublished). 2. Reptiles Immunoreactive GnRH material in extracts of lizard (Mabuya capensis) and tortoise (Chersine angulata) hypothalami exhibited immunological and charge (cation-exchange chromatography) properties different from those of mGnRH and similar to those of chicken and teleost GnRHs (King and
EVOLUTION OF GONADOTROPIN-RELEASING HORMONE
159
Millar, 1979a, 1980).The data pointed to alterations in the vicinity of Leu7 of mGnRH. Since GnRH in reptile species is less positively charged than mGnRH, as in cGnRH I (King and Millar, 1979a, 1980), it appears that Arg' is also not present in the GnRHs of these reptiles. More extensive immunological and chromatographic studies have demonstrated that the major form of lizard (Cordylis nigra) brain GnRH is identical to salmon brain GnRH, Trp7, Leu'-GnRH (sGnRH) (Table I) (Fig. 1). The peptide coeluted with sGnRH in a cation-exchange and three reversed-phase HPLC systems, which were specifically designed to separate the four known natural vertebrate GnRHs and a range of GnRH analogs (Powell et al., 1985). The interaction of this immunoreactive peptide with antisera directed against different regions of mGnRH, cGnRH I, and sGnRH was similar to the relative interaction of sGnRH with these antisera. The peptide also had LH-releasing activity similar to that of sGnRH in a chicken pituitary cell bioassay (Powell et al., 1985). In addition, three structurally related GnRH molecular forms were detected. One of these had HPLC properties and chicken LH-releasing activity identical to that of cGnRH 11. In lizard (Podarcis sicula sicula) brain, three GnRH forms have been demonstrated. One of these had immunological and HPLC properties, and LH-releasing activity in a chicken pituitary cell bioassay, identical to sGnRH. The other two are novel GnRH forms, which exhibit some cross-reaction with several GnRH antisera, but cannot be identified as any of the known GnRHs. These peptides also had chicken LH-releasing activity (J. A. King, R. C. Powell, G. Ciarcia, and R. P. Millar, unpublished) (Table 1). The presence of cGnRH I1 has been demonstrated in skink (Calcides ocellatus tiligugu) brain, using chromatographic, immunological, and biological techniques (R. C. Powell, G. Ciarcia, R. P. Millar, and J. A. King, unpublished) (Table I). Alligator (Alligator mississippiensis) hypothalamic GnRH was thought to differ from mGnRH in position eight on the basis of immunological data (Lance, 1985). Recent immunological and high-resolution HPLC chromatography studies on alligator brain extracts have indicated the presence of two GnRH molecular forms, these being identical to cGnRH I and cGnRH I1 (R. C. Powell, V. Lance, R. P. Millar, and J. A. King, unpublished) (Table I). The alligator brain GnRHs also had LH-releasing activities identical to those of cGnRH I and cGnRH I1 in a chicken pituitary cell bioassay. GnRH is also present in extrahypothalamic areas of the reptile brain (King and Millar, 1980; reviewed by Peter, 1983). The regional distribution of the different GnRH types within the reptile brain has not been investigated, but it is likely that specific GnRHs are confined to specific localities within the brain.
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ROBERT P. MILLAR A N D JUDY A. KING
3. Amphibians Hypothalamic GnRH in frogs (Rana pipiens and Rana catesbeianu) (Alpert et al., 1976; Jan et al., 1979; Branton et al., 1982; Eiden et al., 1982)and Xenopus laevis (Deery, 1974; King and Millar, 1979a. 1980)and in the toad (Bufogariepensis) (King and Millar, 1980) was shown to have identical physicochemical properties to that of mGnRH. Purification of frog (R. catesbeiana) brain GnRH revealed a single species with an amino acid composition identical to that of mGnRH (Rivier et al., 1981) (Table I). In X . laevis brain, two forms of GnRH, which have identical chromatographic and immunological properties to mGnRH and cGnRH 11, have been identified (J. A. King and R. P. Millar, unpublished) (Table I). These peptides also had LH-releasing activities similar to those of mGnRH and cGnRH I1 in a chicken pituitary cell bioassay. Hypothalamic immunoreactive GnRH content in X . laevis has been shown to vary in relation to season and reproductive physiological state (King and Millar, 1979b), with high concentrations in reproductively active frogs collected in the breeding season and with low levels in sexually quiescent frogs collected in the nonbreeding season. The role of the two forms of GnRH found in the frog brain in amphibian reproduction has not been determined. Immunological and HPLC studies have revealed that frog (R. catesbeiana) retinal extracts have the mammalian type of peptide in addition to a more hydrophobic species, which has HPLC properties similar to sGnRH and which is thought to differ from mGnRH at Arg' (Eiden et ul., 1982). This additional species of GnRH is the major form found in the frog sympathetic ganglion (Jan et al., 1979; Eiden and Eskay, 1980; Eiden et al., 1982) and adrenal gland (Eiden et al., 1982). Branton et al. (1982) have reported that the GnRH form found in ganglion predominated in brain extracts of metamorphic frog (R. catesbeiana) tadpoles, while mGnRH predominated in the brains of postmetamorphic frogs and adults. In X . laevis tadpoles, the radioimmunoassayable GnRH (middle-directed antiserum 1076) had properties similar to mGnRH (King and Millar, 1981b). The GnRH form detected in the ganglion might also have been present, but was not detected by the antiserum used. Brain immunoreactive GnRH content has been shown to increase steadily during metamorphosis, with a rapid rise at postclimax (King and Millar, 1981b). The functions of the two forms of GnRH during metamorphosis remain to be determined. GnRH-like immunoreactivity has been demonstrated in the brain, retina, and sympathetic ganglion of numerous other amphibians (reviewed by Crim and Vigna, 1983; Peter, 1983), but the nature of the GnRH has not been identified.
EVOLUTION OF GONADOTROPIN-RELEASING HORMONE
161
4. Fish Teleost fish (tilapia, Sarotherodon mossambicus) brain GnRH was found to differ from mGnRH in the vicinity of Leu’ (King and Millar, 1979a, 1980). The teleost peptide was less positively charged, suggesting the absence of Arg’ (King and Millar, 1980). Similarly, codfish (Gadus morhuu morhua) brain GnRH (Barnett et al., 1982; Jackson and Pan, 1983) and winter flounder (Pseudopleuroncctes americanus) brain GnRH (Idler and Crim, 1985) were shown to be immunologically different from mGnRH. Purification and sequence analysis of salmon (Oncorhynchus keta) brain GnRH (designated sGnRH) revealed the structure Trp’, Leu8-GnRH (Sherwood et al., 1983) (Fig. I). The salmon brain form of GnRH appears to be widespread in teleost fish. In all the species which have thus far been investigated using immunological and chromatographic techniques, the major GnRH molecular form has identical properties to Trp’, Leu*-GnRH, as in salmon brain (Table I). These species include hake (Merluccius capensis) pituitary gland (King and Millar, 1983, tilapia (Tilapia sparrmanii) brain (King and Millar, 1985), Cork julis brain (R. C. Powell, G . Ciarcia, R. P. Millar, and J. A. King, unpublished), codfish brain (Jackson and Pan, 19831, and mullet (Mugil cephalus), milkfish (Chanos chanos), and trout (Salmo gairdneri) brain (Sherwood et a / . , 1984). Multiple forms of GnRH have been described previously in various species of teleost fish (Barnett et al., 1982; Jackson and Pan, 1983; Sherwood er a / . , 1983. 1984; King et a / . , 1984a; Idler and Crim, 1989, although the nature of these molecular variants of GnRH has not been identified. In recent immunological and chromatographic studies, we have demonstrated that, in addition to sGnRH, hake pituitary gland and tilapia brain also contain cGnRH I (King and Millar. 1985) (Table 1). Higher molecular-weightforms of GnRH have been reported in codfish brain (Barnett at al., 1982; Jackson and Pan, 1983) and in winter flounder brain (Idler and Crim, 1985). Amongst elasmobranch fish, dogfish (Poroderma ufricanum) hypothalamic GnRH was shown to differ from mGnRH in the vicinity of Leu’ (King and Millar, 1980). Recently, two forms of GnRH were identified in dogfish ( P . ufricanum) brain, which have identical chromatographic and immunological properties to sGnRH and cGnRH I1 (R. C. Powell, R. P. Millar, and J. A. King, unpublished) (Table I). These GnRHs also had LH-releasing activities identical to those of sGnRH and cGnRH 11 in a chicken pituitary cell bioassay. Immunoreactive GnRHs have also been detected in another dogfish (Squalus acanthias) (Jackson, 1980; Sherwood and Sower, 1985) and in ratfish (Hydrolasus colliei) (Jackson, 1980). In Scyliorhinus canicula brain, immunoreactive sGnRH is reported to be absent (Breton et ul., 1984).
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ROBERT P. MILLAR AND JUDY A. KING
Considering cyclostomes, immunoreactive GnRH has been demonstrated in hagfish (Heptatretus hexatrema) brain (King and Millar, 1980), Pacific hagfish (Eptatretus stouti) brain (Jackson, 1980), Pacific lamprey (Entosphenus tridentata) brain (Crim et al., 1979a), Western brook lamprey (Lampetra richardsoni) brain (Crim el al., 1979b), and landlocked sea lamprey (Petromyzon marinus) brain (Sherwood and Sower, 1985). The molecular nature of GnRHs in these primitive fish has not been identified. GnRH immunoreactivity is present in the extrahypothalamic brain of fish (King and Millar, 1980; reviewed by Crim and Vigna, 1983; Peter, 1983). However, detailed analysis of the nature of GnRH in fish brain (excluding the hypothalamus) has not been undertaken, and it remains uncertain as to whether there is a differential distribution within the brain of the multiple forms of fish GnRH. 5 . Invertebrates and Lower Organisms Immunoreactive and biologically active GnRH has been demonstrated in the hepatopancreas of the grass shrimp (Penaeus monodon) (Wan et al., 1984). The structure of this GnRH-like material has not been ascertained. A substance with GnRH-like biological activity has been reported in extracts of oak (Avena sativa) leaves (Fukushima et al., 1976).
D. GnRH-RELATED MOLECULES In addition to the various forms of GnRH which have been identified in the different vertebrate species, there are several peptides which have a degree of sequence homology with GnRH (Fig. 3). The sequence homGnRH
mGnRH Prolactin
cGnRH 1 cGR P
pGlu' --Pro147-
His
Trp
His
-
Pro
-
-
Ser
y.
Tyr
Gly
Ser .. Gly
-
Leu
Trp - Ser - Tyr
-
Gly
Val .. Trp
pGlu' H-Alal
.
Leu - Gln
-
Pro - Gly
-
1
1
Pro -%r
- Leu - Gln156--
Leu
-
1
Leu .. Arg
-
Pro
Gly1'*NH2
Gln - Pro - Gly1'*NH2]
Glya J S e r -
Pro
-
Alal'--
FIG. 3. Structural homologies of yeast a-mating factor and mammalian prolactin with mGnRH. and chicken gastrin-releasing peptide (cGRP) with cGnRH 1. "Denoted as being homologous, since the amide of mGnRH (and presumably cGnRH I) is derived from an additional Gly at the COOH-terminus of the mGnRH precursor (see Fig. 2).
EVOLUTION OF GONADOTROPIN-RELEASING HORMONE
I63
mology of yeast a-mating factor with mGnRH is sufficient to allow specific binding to rat pituitary GnRH receptors and the stimulation of LH release from cultured gonadotrophs, albeit at relatively high concentrations (Loumaye et d . , 1982).
111. Biological Activity of GnRH
A. INTERSPECIFIC GONADOTROPIN-RELEASING ACTIVITIESOF VERTEBRATEGnRHs
Synthetic ovine-porcine GnRH is biologically active at low doses in a wide variety of domestic and laboratory mammals and also in feral mammalian species, including rock hyrax, impala, blesbok, Soay sheep, and hyena (Millar and Aehnelt, 1977; Lincoln, 1979; Illius et al., 1983), suggesting that the GnRH structure and the GnRH receptor have been conserved throughout the class Mammalia. cGnRH I has low gonadotropin-releasing activity using sheep pituitary cells in v i m (Millar and King, 1983a) and in the rat in vivo (Sandow e? al., 1978) (Table 11) (Fig. 4). sGnRH is slightly more active using rat pituitary cells (Sherwood er al., 1983) and sheep pituitary cells (Millar e? al., 1986) in vitro, while cGnRH I1 exhibits a further enhancement in LHreleasing activity in sheep pituitary cells in vitro (MiUar er al., 1986) (Table 11) (Fig. 4). In birds, cGnRH I and mGnRH are equipotent in releasing LH from chicken pituitary cells in vitro, with low ED,,s of 3 x lo-" M (Millar and King, 1983a) (Table 11) (Fig. 4). They are also equipotent in vivo in TABLE II INTERSPECIFIC GONADOTROPIN-RELEASING ACTIVITIES OF VERTEBRATE GnRHs" GnRH type Mammalian Chicken I Chicken II Salmon
Vertebrate class Mammalb
Bird'
100
100 100
Oor 0
600 250
+d
2 8 5
Reptile'
?
+
Amphibianc
Fish
100 100
100 100 100 100
7
100
"Activity of the peptides is shown as a percentage of the activity of mGnRH, except for the reptile studies where 0 indicates no activity and + indicates activity. "ln vitro pituitary cell LH response. 'In vivo bioassay. "o-Arg6-cGnRH I I .
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ROBERT P. MILLAR A N D JUDY A. KING
I
I
lo-'' 10"
I
I
I
I O - ~ lo-' PEPTIDE (M)
I
I
lo5
1
10"
1
1
I
loi11 0 "
,
(
lo7
PEPTIDE (MI
FIG.4. LH-releasing activity of mammalian GnRH (-), chicken GnRH I (-.-.-.-). chicken GnRH I1 (----), and salmon GnRH (............) in sheep pituitary cells ( A ) and in chicken pituitary cells (B). Data were adapted from Millar and King (l983a) and from unpublished results.
chickens (Johnson et al., 1984; Sterling and Sharp, 1984) and in quail (Chan et a/., 1983). The two peptides have identical affinities for chicken pituitary GnRH receptors (Millar and King, 1983a). cGnRH I1 is 5.6 times more potent than cGnRH I in releasing LH from chicken pituitary cells (Fig. 4) and 13.5 times more potent in releasing FSH (Millar et al., 1986). sGnRH is 2.5 times more potent than cGnRH I in releasing L H (Fig. 4) and 1.8 times more potent in releasing FSH (Millar et a / . , 1986). In turtles and snakes, mGnRH and cGnRH I are apparently inactive (Licht et al., 1984). In contrast, other studies reported some ability of mGnRH to stimulate the reproductive system in turtles (Callard and Lance, 1977; Licht, 1980). In male alligators, mGnRH was shown t o stimulate plasma testosterone (Lance et al., 1986). Administration of DArg'-cGnRH I1 to female iguanas stimulated plasma estradiol, which ellicited reproductive behavior in males (Phillips et al., 1985) (Table 11). Among amphibians, mGnRH (i.e., one of the frog GnRH forms) stimulates gonadotropic hormone secretion, steroidogenesis, and spawning in anurans (McCreery et al., 1982; Licht et al., 1984). mGnRH has been reported to stimulate spermatogenesis and ovulation in newts (Mazzi et al., 1974; Vellano et al., 1974). cGnRH I is equipotent with mGnRH in stimulating gonadotropin secretion in frogs in vivo (Licht et a / ., 1984) (Table 11). The doses required to achieve these effects are considerably higher than those required to induce gonadotropin release in rats, despite the fact that one of the natural hypothalamic hormones in frogs has the structure of mGnRH. A lower affinity of mGnRH for frog pituitary receptors and/or more rapid degradation of mGnRH may account for this relatively
EVOLUTION OF GONADOTROPIN-RELEASING HORMONE
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poor activity. We have observed very rapid degradation of mGnRH by X . laevis plasma (R. P. Millar, unpublished). The sensitivity of frog pituitaries to GnRH in vitro suggests that the receptor-binding affinity is high (P. Licht, personal communication). mGnRH and its analogs have relatively poor gonadotropin-releasing activity in fish when compared with doses required in mammals (Ball, 1981; Crim et al,, 1981; Donaldson et al., 1981; King and Millar, 1981~;Peter, 1983; L. W. Crim, 1984). The structural characterization of salmon brain GnRH as Trp’, Leu*-GnRH (sGnRH) (Sherwood et al., 1983) suggested that this might be due to interclass specificity of GnRH. However, mGnRH and sGnRH are equally effective in stimulating plasma testosterone and 17-P-estradiol in tilapia in vivo (King et al., 1984b), and the peptides are equipotent in stimulating gonadotropin release from superfused goldfish pituitary cells (MacKenzie et al., 1984). All three peptides (mGnRH, cGnRH I , and sGnRH) are equipotent in stimulating gonadotropin secretion in goldfish (Peter et al., 1985) and salmon (L. W. Crim, personal communication) in vivo (Table 11). These observations of interspecific differences in gonadotropin-releasing activities of vertebrate GnRHs (Table 11) emphasize the high specificity of the mammalian GnRH receptor and a relative nonspecificity (“promiscuity”) of the pituitary GnRH receptor in the nonmammalian vertebrates. This question has been addressed in more detail by testing gonadotropin-releasing activity of synthetic GnRH analogs with substitutions in positions five, seven, and eight, which vary among natural vertebrate GnRHs (see below). Aside from GnRH action in regulating pituitary gonadotropin secretion, the peptide(s) appear to act as a neurotransmitter and directly affect reproductive behavior in rats (Moss and McCann, 1973; Pfaff, 1973; Moss et d.,1979). The specificity of these actions has not been thoroughly investigated, although Ac-GnRH (5-10) enhances lordotic behavior in female rats while GnRH (I-6)-NH2 is ineffective (Dudley et d.,1983). Evidence that another form of GnRH exists in mammalian brain (as found in the pineal gland) may indicate that these neurotransmitter effects are not mediated by mGnRH, but by a different form of the peptide. The presence of GnRH receptors in extrapituitary tissues in mammals, such as the testis, ovary, placenta, and adrenal gland (see references in Section II.B,3, and in human mammary carcinoma tissue (Miller et al., 1985; Eidne et al., 1985b)and the biological effects of GnRH and analogs on these tissues point to local sources of GnRH-related peptides which affect these tissues. In view of the evidence for a local production of these GnRH-like peptides, a paracrine or autocrine regulatory system appears to pertain. The specificity and molecular size of gonadal GnRH receptors
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ROBERT P. MILLAR A N D JUDY A. KING
suggests that they are identical to the pituitary receptor (Reeves et al., 1980), although other studies have found some differences in binding specificity (Perrin et al., 1980; Millar et al., 1982). Evidence has been presented which indicates that testicular GnRH differs structurally from mGnRH (Dutlow and Millar, 1981; Paul1 et al., 1981). However, cGnRH I has a low relative activity in inhibiting gonadal steroidogenesis, similar to its activity in the pituitary (R. P. Millar and A. J. Hsueh, unpublished). Sequence analysis of human placental GnRH mRNA has confirmed an identity with the hypothalamic peptide (Seeburg and Adelman, 1984). mGnRH stimulates human p-chorionic gonadotropin secretion by human placenta in vitro (Belisle et af., 1984), but comparative studies of the effects of other vertebrate GnRHs have not been undertaken. Studies on extrapituitary biological actions of GnRHs in nonmammalian vertebrates are even more limited. In birds, studies have demonstrated a direct effect of mGnRH on gonadal steroidogenesis (Hertelendy et al., 1982) and behavior (Cheng, 1977). mGnRH and sGnRH stimulate nerve firing in bullfrog spinal ganglia (Jan et al., 1980; Jan and Jan, 1983). A GnRH with properties similar to sGnRH has been extracted from this tissue (Eiden et a / . , 1982) and is thought to resemble an amphibian sympathetic neurotransmitter (Jones et al., 1984). mGnRH was 10-fold more potent than sGnRH in pressor activity in the toad (Wilson, 1985). The amating factor in yeast, which has structural homology with mGnRH (Fig. 3), stimulates fusion of gametes, indicating an early evolution of GnRHrelated peptides in regulating reproduction. B. STRUCTURE-ACTIVITY RELATIONSOF GnRH FOR GONADOTROPIN RELEASE
I . Structural Significance of Position Eight Amino Acid Arg in position eight of mGnRH appears to be essential for gonadotropinreleasing activity in mammals. A conservative substitution with Lys reduces biological activity from 10 to 25% (Sandow et al., 1978; Milton et al., 1983). Substitution of Arg' with Gln, Leu, Om, His, and Cit results in low LH-releasing activity in the rat in vivo (Sandow et a f . , 1978), as well as from sheep pituitary cells in culture (R.P. Millar, J. A. King, and R. W. Roeske, unpublished). Of the neutral amino acids, Phe, Trp, Cit, Met, and Leu retain the most LH-releasing activity (5-9%), while Ser, Ile, and Asn have 0.2-2% relative activity (R. P. Millar, J. A. King, and R. W. Roeske, unpublished). Acidic amino acid substitution (e.g., Glu) results in the lowest activity (0.04%). These findings are in accordance with the concept that His', Tyr', and Arg' form a combined unit of hy-
EVOLUTION OF GONADOTROPIN-RELEASING HORMONE
I67
drogen bonding important for stabilizing the molecule for biological activity (Shinitzky and Fridkin, 1976). We have proposed that this interaction limits the potential number of GnRH conformers and favors the occurrence of a "preferred" conformer which interacts with the mammalian GnRH receptor (Milton et al., 1983). This relative limitation on conformers is indicated by the narrow titration range (< 1.74 pH units) of His', as monitored in Trp3 fluorescence in the native mGnRH, and also in Lys'GnRH, which retains substantial biological activity. Neutral amino acid substitution results in a titration range of > 1.74 pH units, reflecting a heterogeneous population of His residues in the poorly active analogs, and presumably a greater heterogeneity of GnRH conformers (Milton et al., 1983). cGnRH I falls into this group and has a relative potency of 1-5% in mammalian systems (Sandow et al., 1978; Millar and King, 1983a; Milton et al., 1983). In the bird, these structural requirements of the receptor clearly do not pertain, as cGnRH I is equipotent with mGnRH in chickens (Johnson et al., 1984; Sterling and Sharp, 1984) and quail (Chan et al., 1983) in vivo and in chicken pituitary cells in vitro (Millar and King, 1983a; Milton et al., 1983). Using chicken pituitary membranes, these biological effects were shown to be directly related to receptor-binding affinity (Millar and King, 1983a). We have now defined the requirements of the chicken pituitary GnRH receptor in more detail by comparing the ability of a range of position eight-substituted GnRH analogs to release LH from dispersed chicken pituitary cells. Relative to cGnRH I, Arg' and Phe8 analogs have full LH-releasing activity. Met', His', and Leu' analogs exhibit about 30% activity. Ser', Trp', Cit', and Ile' analogs have 1&20% activity; and only the acidic residue Glu' had low LH-releasing activity (R. P. Millar, J. A. King, and R. W. Roeske, unpublished). Thus, accepting the validity of the structural conformer stabilization model for the mammal, one must conclude that the avian receptor is promiscuous and binds a number of GnRH conformers present in the unstabilized analogs which have substitutions for Arg'. However, this proposal does not exclude the possibility that the importance of a basic amino acid in position eight of GnRH for biological activity is simply related to a charge interaction with a negative charge at the binding site of the mammalian GnRH receptor. Studies on the influence of the position eight amino acid on gonadotropin-releasing activity in reptiles, amphibians, and fish are much less extensive. As indicated above, cGnRH I is active in amphibians and fish (Licht et al., 1984; Peter et al., 1985), cGnRH I1 in certain reptiles (Phillips et al., 1983, and sGnRH is active in fish (King et al., 1984b; MacKenzie et al., 1984; Peter et al., 1985) (Table 11). Although these findings are not sufficiently extensive to draw definite
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ROBERT P. MILLAR AND JUDY A . KING
conclusions regarding the requirements for the amino acid in position eight in these nonmammalian vertebrates, they do indicate that the pituitary GnRH receptor in these classes is similar to that of the bird in tolerating considerable amino acid variation in this position.
2. Effects of Conformational Constraint on GnRH Bioactivity In support of the postulate that Args plays a role in stabilizing GnRH conformation and that this is important for receptor binding and biological activity in mammals, Freidinger et al. (1980) reported that a GnRH analog containing a y-lactam bridge (between the C of Gly6 and N of Leu7) which stabilizes the (3-turn of residues five to eight of GnRH, was 9 times more potent than native GnRH in stimulating LH release from dispersed rat pituitary cells. This analog is also 9-10 times more active in sheep pituitary cells (R. P. Millar, J. A. King, R. W. Roeske, unpublished). On the basis of the hypothesis that the avian receptor does not require Arg' for conformational stabilization and will bind a number of GnRH conformers, it follows that the y-lactam conformational restraint would not enhance bioactivity in the chicken bioassay. In a recent study, we observed that the stimulation of LH release from dispersed chicken pituitary cells by native mGnRH and the y-lactam analog over the dose range of 10-11-10-6M was indistinguishable (R. P. Millar, J. A. King, and R. W. Roeske, unpublished). These findings, therefore, support the concept that conformational stabilization is less important for GnRH interaction with its receptor in the chicken and quail, and probably in birds in general. The effect of this conformational constraint on GnRH activity in other nonmammalian vertebrates has not been established. 3. Influence of Positions Five and Seven on Biological Activity Studies in mammalian bioassays established that some modifications such as N-methyl-Leu7 actually enhance activity (Ling and Vale, 1979, presumably through stabilizing the (3-turn of GnRH (Chandrasekaren et al., 1973). Ile7-, Nle7-, Ser7-, and (Boc)Lys7-substituted mGnRH analogs retain significant activity in mammalian systems (Sandow et al., 1978), suggesting a degree of nonspecificity of the mammalian receptor for the amino acid in this position. Trp7-mGnRH has high activity when compared with the parent compound (R. P. Millar, J. A. King, and R. C. deL. Milton, unpublished) and the presence of Trp7 in cGnRH I1 and sGnRH appears to enhance binding to the mammalian receptor when compared with cGnRH 1. The mammalian receptor is, however, not totally indiscriminate in binding position seven-substituted analogs, as Gly7, Ala7, Val7, Lys7,
EVOLUTION OF GONADOTROPIN-RELEASING H O R M O N E
169
Arg7, D-L~u'.and Pro7 substitution leads to a marked decline in activity (Sandow et al., 1978). The mammalian GnRH receptor appears to differ from the chicken GnRH receptor in its requirements for the amino acid in position five. His5enhances biological activity in the chicken and mammalian bioassays when incorporated in cGnRH 11. This substitution in mGnRH results in an increase in activity in the mammal, while a marked decrease in activity ensues in the chicken (R. P. Millar, J. A. King, and R. C. deL. Milton, unpublished). Ala' and Pro' substitution results in reduced gonadotropinreleasing activity in the mammal, while Phe'-mGnRH retains substantial activity (Sandow et ul., 1978). Our observation that sGnRH (Trp7,L e d mGnRH) is 2.5-fold more active than cGnRH I (Gln8-mGnRH) in stimulating LH release from chicken pituitary cells indicates that the avian receptor is also tolerant of alterations in position seven of GnRH. However, it is clear that the combination of substitutions in positions seven and eight can be important as Gln7, Leu8-mGnRH has only 4% relative potency in the chicken pituitary cell system (King et al., 1983). As LeunmGnRH has 30% relative activity, it appears that Gln in position seven is largely contributory to the decline in activity. cGnRH 11 (His', Trp7, Tyr8-mGnRH) is more active than cGnRH I and mGnRH in stimulating gonadotropin release from dispersed chicken pituitary cells (Millar et al., 1986). Since sGnRH, which shares Trp7 in common with cGnRH 11, was also more active than cGnRH I , this residue appears to be responsible for the enhancement in activity. Indeed, Trp7mGnRH is 2.8 times more active than mGnRH in the chicken system (R. P. Millar, J. A. King, and R. C. deL. Milton, unpublished). It also appears that His substitution for Tyr' is acceptable to the chicken receptor and may even contribute to the enhanced activity of cGnRH 11. However, the nature of other residues in positions seven and eight is clearly important since His'-mGnRH was actually much less active than any of the natural GnRHs, and His'. Trp7-mGnRH was less active than cGnRH 11 (R. P. Millar, J . A. King, and R. C. deL. Milton, unpublished). Comprehensive data on the influence of amino acids five and seven for GnRH activity in other nonmammalian vertebrates are lacking. The high biological activity of all the naturally occurring vertebrate GnRHs suggests a considerable tolerance of amino acid substitutions in these positions.
4. Structural Requirements for Superactive GnRH Agonists It is now well established that substitution of D-amino acids for Gly", N-methyl-Leu for Leu7, and N-ethylamide for Gly"' - NH2 in GnRH en1978) hances gonadotropin-releasing activity of the peptide (Sandow et d.,
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ROBERT P. MILLAR A N D JUDY A. KING
(Fig. 5). In view of the less basic nature and different hydrophobicity of both forms of chicken GnRH and sGnRH and the different requirements of the nonmammalian vertebrates’ pituitary GnRH receptors, the same principles for producing agonists with enhanced activity need not necessarily apply. a. Analogs of Mammalian GnRH. D-Trp6-mGnRH exhibits an enhancement of 26-fold in stimulating L H release from chicken pituitary cells, which is similar to the 36-fold increased activity in sheep pituitary cells (Millar and King, 1983b). o-Ser6(Bu‘),Prog-NHEt-mGnRH,which has enhanced activity in mammals, exhibits increased activity in chickens in vivo (Sterling and Sharp, 1984). On the other hand, ~-His~(Bzl),Pro’-NHEtmGnRH, which has a relative potency of about 50 in the mammal, exhibits only a 4-fold enhancement in the chicken system (R. P. Millar and J. A. King, unpublished). Similarly D-Leu6-mGnRH, which is 27 times more active than mGnRH in releasing L H from rat pituitary cells, has no enhanced activity in chicken pituitary cells (Hasegawa et d . , 1984). A new (J. E. Rivier and H. GnRH agonist, ~-Glu~(anisole),Pro~-NHEt-mGnRH Anderson, unpublished), also displayed no enhancement in activity in chicken pituitary cells in contrast to a 4-fold increase in activity in the sheep pituitary cell bioassay. Similarly D - ~ A ~ ~ ~ ( E ~ , ) , P I - o ’ - N H E ~ - ~ G ~ R H , which is a very active analog in the rat, is only twice a s active as mGnRH in the chicken pituitary cell bioassay (J. J. Nestor and R. P. Millar, unpublished). It is clear, therefore, that the enhanced activity of mGnRH agonists in mammalian systems is Frequently not paralleled by their activity in the chicken. This is likely to be due to the differences in GnRH receptors described earlier and may be related to the fact that conformational constraint of GnRH does not affect biological activity in the chicken system to the same extent as in the mammal. Fewer data on comparative potencies of mGnRH agonists in other nonmammalian vertebrates are available. ~-His~(Im-Bzl),Pro’-NHEt-rnGnRH is 45 times more potent than mGnRH in the bullfrog (McCreery et a f . ,
AGONIST
pGlul
ANTAGONIST
.\“:“i 1
D-AMINO ACID (ESPECIALLY AROMATICS)
-
His2
-
Trp3
1 1
D-AMINO ACID (ESPECIALLY BULKY AROMATICS)
-
Ser4
-
Tyr5
-
Gly
-
Leu7
ETHYLAMIDE
- Arg8 -
D-AMINO ACID (ESPECIALLY BASICS)
Fa. 5. Structure of GnRH agonists and antagonists.
Pro9
-
1
Glylo*NH2
.c
D-Ala
EVOLUTION OF GONADOTROPIN-RELEASING HORMONE
171
1982) in accordance with mammalian data. This analog is approximately 4 times more active than mGnRH in the goldfish (Peter et al., 1985), which is similar to our observations in the chicken (R. P. Millar and J. A. King, unpublished). In turtles, this analog does not increase plasma gonadotropins or steroid levels (Licht et al., 1982). In the frog, D-Ser"(Bu'),Pro9NHET-mGnRH has been shown to stimulate testicular steroidogenesis both in vivo and in vitro (Pierantoni et a l . , 1984). ~-Ala",pro'-NHEtmGnRH, which is 14 times more active than mGnRH in the rat, is active in the African catfish (de Leeuw et al., 1985),but does not exhibit enhanced activity in the goldfish (Peter et al., 1985). Other information on the actions of GnRH analogs in lower vertebrates has been reviewed (L. W. Crim, 1984). b. Analogs ofChicken GnRH I . D-T~~"-cG~RH I is 26 times more potent than the parent peptide in the chicken pituitary cell bioassay (Millar and King, 1983b), which is identical to the increased activity when ~ - T r p "is incorporated in mGnRH. This analog is 100 times more potent than cGnRH I in the sheep pituitary cell bioassay (R. P. Millar and J. A. King, unpublished). In goldfish, D-Trp"-cGnRH I exhibited only a 5-fold increase in activity (Peter et al., 1985), suggesting a difference in the GnRH receptor in birds and fish. However, the analog was approximately 20 times more active than the parent peptide in trout (L. W. Crim, personal communication). D-hArg"(Et,)-cGnRH I exhibited a 5-fold increase in activity in the chicken pituitary cell bioassay (J. J. Nestor and R. P. Millar, unpublished). c. Analogs of Chicken GnRH ZZ. D-Arg" incorporation in cGnRH I1 led to a 4-fold increase in activity in the chicken pituitary cell bioassay (Millar rt al., 1986). D-hArg"(Et,) resulted in no enhancement in the chicken system, but increased the activity 46-fold in the sheep pituitary system when compared with the poor activity of the parent peptide (J. J. Nestor and R. P. Millar, unpublished). The D-Arg" analog appears to exhibit good activity in the iguana (Phillips et al., 1985). d. Analogs of Salmon GnRH. D-Arg", Pro9-NHEt-sGnRH had an 8fold increase in activity in chicken pituitary cells (R. P. Millar, J. E. Rivier, and J. A. King, unpublished) and a 12-fold increase in the goldfish (Peter rt ul., 1985). D-His" (Im-Bzl), Pro9-NHEt-sGnRH was also 8 times more active in the chicken system, but exhibited no significant increase in activity in the goldfish as did a D-Ala" analog (Peter et al., 1985). In summary, it is apparent that the majority of GnRH analogs which are superactive agonists in mammals exhibits relatively little or no increase in activity in the chicken and probably also the other nonmammalian vertebrates. This emphasizes the differences in structural requirements of the respective receptors. Differences in metabolic clearance may con-
I72
ROBERT P. MILLAR A N D JUDY A. KING
tribute in the in vivo studies, but are unlikely to be of much significance in the in vitro bioassays. Although there is a much closer parallelism of activity in birds and fish, there may also be differences in these receptors, as certain analogs (e.g., DHis6 (Im-Bzl), Pro9-NHEt-sGnRH)have marked differences in activity in the chicken and goldfish bioassays. The studies on GnRH analog activity in the chicken pituitary cell bioassay provide the most comprehensive data and demonstrate that, in general, substitution of a particular D-amino acid for Gly6 results in a similar increase in activity when substituted in any of the naturally occurring vertebrate GnRHs. An exception to this is D-hArg6(Et,) substitution in sGnRH, which is considerably more active than the corresponding mGnRH, cGnRH I, and cGnRH 11 analogs. With the limited information available, it is not possible to formulate definite rules concerning the types of GnRH analogs likely to exhibit enhanced activity in nonmammalian vertebrates, as has emerged for analogs in mammals. However, the sequence of activity of the D-hArg6(Et,)analogs (sGnRH-A > cGnRH 11-A > cGnRH I-A > mGnRH-A) relates to the relative hydrophobicity of these analogs, as has been demonstrated for GnRH analogs in mammals (Nestor et al., 1984).
5 . Structural Requirements for GnRH Antagonists GnRH analogs which have antagonist activity in mammals (Fig. 5 ) were tested for their ability to inhibit the LH responses of sheep and chicken pituitary cells to cGnRH (J. A. King and R. P. Millar, unpublished). The analogs were also tested alone for their intrinsic LH-releasing activity. The most potent antagonist in the chicken pituitary bioassay was Ac~-Phe',~-pCl-Phe~,D-Trp-'.~,D-Ala'~-mGnRH with an IC,, of 5 x lo-' M and no intrinsic LH-releasing activity. In the sheep pituitary cell bioassay, this analog was intermediate in activity with an IC,, of 2.5 x M. ~-pGlu',~-Phe',~-Trp'.~-mGnRH had slightly lower antagonist activity in the chicken system while, it was a very weak antagonist in the sheep bioassay. Ac-~-pCl-Phe'~~,~-Trp~~~,~-Ala'~-mGnRH had intermediate anhad h e similar ~ tagonist activity in both bioassays, and ~ - P h e * , ~ - T r p ' , ~ - P activity in the chicken bioassay, but was a weak antagonist in the sheep. The most active antagonist in the sheep bioassay was Ac-D-pCI-Phe'.',DTrp',~-Phe~,~-Ala''-mGnRH (IC,, lo-'' M ) . This antagonist had the M ) and weakest antagonist activity in the chicken bioassay (IC,,, displayed agonistic activity when administered alone, with an ED,,, of M. There are, therefore, marked differences in the antagonistic and agonistic activities of these analogs in the chicken and sheep bioassays, which fur-
EVOLUTION OF GONADOTROPIN-RELEASING HORMONE
I73
ther indicates differences in the receptors with respect to their GnRH structural requirements for interaction with the NH2-terminal part of the molecule. Differences in this regard are perhaps unexpected, as the sequence of the NH,-terminal region of GnRH (amino acids 1-4) has been completely conserved in vertebrate GnRHs, and a similar conservation of the receptor in its interaction with this region might have been anticipated. An antagonist incorporating the natural Trp7of cGnRH I1 and sGnRH rather than the Leu7 of rnGnRH has been shown to have increased potency in rats (Folkers et al., 1984). Among lower vertebrates, a mGnRH antagonist (Ac-dehydro-Pro',pCI~-Phe',~-Trp',~,NaMeLeu~-mGnRH) has been shown to block mGnRHinduced gonadotropin release in the bullfrog (McCreery et al., 1982). DPhe2,Phe3,D-Pheb-mGnRHinhibits mGnRH-stimulated gonadotropin release in trout (Crim et al., 1981). 6. Pituitary Desensitization Since pituitary desensitization to prolonged and/or high doses of GnRH is a receptor-mediated event in the rat (Smith and Conn, 1984), it was of interest to determine the influence of the differences in the nonrnammalian receptor on expression of desensitization. When dispersed chicken pituitary cells suspended in a Biogel column were stimulated with 2-minute M cGnRH I every 30 minutes for 3% hours, a LH response pulses of was associated with every pulse. In contrast, a definite desensitization of the pituitary cells occurred when they were continuously perifused with M cGnRH I or 10-7M D-Trp6-cGnRHI (Millar and King, 1984). After 100 minutes of perifusion, LH release declined to basal levels. In view of our difficulty in satisfactorily quantitating GnRH receptors in chicken pituitary cells or membranes (Millar and King, 1983a),we have been unable to determine whether the receptor "down-regulation" plays a major part in pituitary desensitization, as in the rat (Zilberstein et al., 1983). In chickens in vivo, daily injections of D - S ~ ~ ~ ( B U ' ) , P ~ ~ ~ - N H E ~ - ~ G ~ did not reduce pituitary responsiveness to the analog (Sterling and Sharp, 1984). Prolonged doses of D - S ~ ~ ~ ( B U ' ) , P ~ O ~ - N H E have ~ - ~been ~ G ~reRH ported to induce desensitization in lizards in vivo (Ciarcia et al., 1983). while in turtles this desensitization phenomenon has not been observed (Licht et al., 1982). The perifused bullfrog pituitary continues responding to sustained high doses of GnRH and lacks the phenomenon of desensitization (McCreery and Licht, 1983). The goldfish pituitary is reported to exhibit desensitization when exposed to superactive GnRH analogs (Peter, 1980).
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ROBERT P. MILLAR AND JUDY A. KING
IV. Conclusions
It is evident that heterogeneous molecular forms of GnRH are present within vertebrates. GnRH structure also varies in different tissues within the same vertebrate species and even within the same tissue, as occurs in the brains of several nonmammalian vertebrate species. At present, four forms of GnRH have been structurally characterized: the original GnRH from porcine and ovine hypothalamus; Gln8-GnRH and His’,Trp7,Tyr8-GnRHin chicken hypothalamus; and Trp7,Leu8-GnRHin salmon brain. Immunological, chromatographic, and biological properties indicate the presence of other forms of GnRH in mammalian and nonmammalian tissues which await characterization. All of the GnRHs exhibit gonadotropin-releasing activity, especially in nonmammalian vertebrates in which the pituitary receptor is relatively nondiscriminatory in binding GnRHs with substitutions in positions five, seven, and eight. However, it is conceivable that the GnRHs have other biological activities, such as is demonstrated by the stimulation of growth hormone secretion in fish, actions on the placenta, ovary, testis, and adrenal gland in certain mammals, and effects in the central and peripheral nervous systems. It appears, therefore, that the basic GnRH structure has been recruited through evolutionary selective processes to serve diverse functions. The specificity of these actions is achieved in a variety of ways: (1) through the evolution of different molecular forms of GnRH, which interact with tissue-specific receptors; (2) through paracrine regulation by anatomically closely localized cells, which does not allow the peptide to enter the general circulation in concentrations sufficient to bind other GnRH receptors. In this system, which appears to pertain in the gonads, the GnRH and receptors in different tissues can be identical; (3) through the existence of a “private” conducting system, as is found in the hypothalamohypophyseal portal system; and (4) through intimate contact between the secretory and target cells, as occurs in neuronal communication. In addition to the heterogeneity of GnRHs within vertebrates and in tissues of the same species, it is apparent that at least two forms of the peptide are generally present in the hypothalamus (or brain) of nonmammalian vertebrates. In mammals, only a single molecular form has been isolated, and only a single GnRH sequence has been detected in the mammalian genome (Seeburg and Adelman, 1984). However, the presence of a different form of GnRH in the mammalian pineal gland suggests that two or more forms are present in the mammalian brain (and possibly hypothalamus). Conceivably, the detection of only one GnRH-coding sequence in the mammalian genome may be due to significant structural
EVOLUTION OF GONADOTROPIN-RELEASING HORMONE
I75
differences in other form(s) which do not hybridize well with the mGnRH probe. The functional significance of two hypothalamic GnRHs in vertebrates has not been established. In the chicken, both forms of hypothalamic GnRH stimulate pituitary secretion of LH and FSH at concentrations appropriate for physiological regulation (
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ROBERT P. MILLAR AND JUDY A. KING
erance of amino acid substitutions in position eight, it is evident that they are not identical in this regard and display distinct differences. The mammalian pituitary GnRH receptor shows some similarity to the nonmammalian receptors in its tolerance of substitutions in positions five and seven. Distinct differences do exist, however, as is evident in the acceptance of His’ substitution for activity in the mammal, while this change causes a decline in activity in the chicken. The conservation of the NH,-terminal sequence of GnRH during evolution suggests that this part of the molecule plays a vital role in interacting with the receptor. Appropriate amino acid substitutions in this region produce molecules which still bind the receptor, but do not activate gonadotropin release (antagonists) (Sandow, 1982). Cross-linking these antagonists by antibodies reestablishes gonadotropin release (Conn et al., 1982; Gregory et al., 1982), suggesting that the structural information in the NH,-terminus of GnRH is to do with receptor aggregation required for initiating intracellular biochemical events which mediate gonadotropin release. There is some indication that the avian receptor domain, which interacts with the NHI-terminal region of GnRH, may also differ from that of the mammal in that a series of GnRH antagonists exhibited different properties in mammalian and chicken bioassay systems. The mammalian and avian GnRH receptors also differ in molecular weight. Using a ligandimmunoblotting technique (Eidne et al., 1985a), we recently demonstrated a binding protein for GnRH in chicken pituitary membranes which was -7,000 Da larger than the well-established 60,000-Da mammalian pituitary GnRH receptor or receptor-binding subunit. The presence of two forms of GnRH in representative species of the major vertebrate classes suggests that gene duplication occurred early in vertebrate evolution or even preceded the earliest vertebrates. Consideration of the nucleotide-coding sequences for the known vertebrate GnRHs reveals that cGnRH I1 is the most different from the other GnRHs. as a minimum of three nucleotide changes are required to account for the amino acid changes. Since cGnRH I1 is also the most commonly represented form in vertebrates, it may have arisen early after duplication of the gene and been highly conserved during evolution in a similar way to vasotocin (Acher, 1983). A single base change is sufficient to account for an interchange between cGnRH I and mGnRH and a double base change for cGnRH I cf sGnRH and mGnRH cf sGnRH. It is possible, therefore, that these forms represent the other evolutionary arm of the duplicated gene in which there has been greater diversity of structures akin to that of the oxytocin-like peptides, which comprise some six known structures in vertebrates (Acher, 1983).The distinctly different activities of oxytocin and vasopressin reemphasize the possibility that some of the “GnRHs”
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may have functions unrelated to the stimulation of gonadotropin release. Nevertheless, GnRH may have a very ancient evolutionary origin as a regulator of reproduction in view of the structural similarity between mGnRH and the yeast a-mating factor. The multiplicity of actions of GnRHs in reproduction, where they act in the central nervous system to stimulate reproductive behavior, in the pituitary to stimulate gonadotropic hormones, in the gonad to affect steroidogenesis, in mammary carcinoma cells to affect growth, and in the placenta to affect human chorionic gonadotropin secretion, appears to be a remarkable conservation of function within a major physiological system. However, it is apparent that GnRHlike sequences are present in other peptides with nonreproductive functions. A sequence in chicken gastrin-releasing peptide has close homology with cGnRH I, and sequences having homology with mGnRH occur in prolactin (Fig. 3). There appears to be considerable plasticity in the cooption of certain peptide sequences for diverse functions during the course of evolution.
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OF CYTOLOGY.VOL. 106
Excitons and Solitons in Molecular Systems A. S . DAVYDOV Institirte jbr Theoretical Physics, Academy of Sciences of the Ukrainian SSR, Kiev 252130, USSR
I. Introduction The chemical and physical research methods used on a wide scale have made it possible to study biological phenomena at the molecular level. The laws which determine the properties of atoms and molecules making up objects of inanimate matter are also operative in complex molecular systems forming living organisms. The overwhelming majority of scientists agree that all the various manifestations of life can eventually be explained on the basis of the same physical and chemical laws which govern nonliving systems. Certainly, it should be kept in mind that biological systems possess a number of highly specific properties which distinguish them from inanimate matter. The most important of these are reproduction and adaptation to a changing environment, extremely fine control, and self-consistency of all the biological processes occurring in living organisms that ensure their life activity. The extreme variety of biological organisms does not imply a multiformity of chemical units from which they are built. Such a diversity is determined by a large number of combinations of identical compounds and atomic groups. There exist many millions of different protein molecules in living organisms. However, all the proteins are constructed only from 20 different amino acid residues, and the basic molecules of heredity (DNA) are formed from only four types of nucleotides. It is well known that a quantitative complication of atomic systems in inanimate matter also leads to the appearance of new qualitative properties. The concepts of temperature, entropy, sound waves, and other fundamental collective excitations are applicable to a system of atoms and molecules but are not applicable to the individual atom. There can be no doubt that all the special characteristics of living organisms that distinguish them from inanimate matter are the result of the specific organization of complex molecular systems. Let us give some fundamental characteristics distinguishing the structures of living organisms and inanimate matter. I . All living organisms are essentially nonhomogeneous systems. When
I83 Copyrighf 0 1987 hy Academic Press. Inc. All righfs of reproduction in any form reserved.
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A. S. D A V Y D O V
studying inanimate nature, one can consider some systems of the same composition, density, and other characteristics. 2. All biological entities are open systems. Their life function is only made possible by a constant exchange of energy and matter with the surrounding medium. In studying inanimate matter, one can examine the properties of almost isolated systems. 3. Living systems are always far from thermodynamic equilibrium. They evolve only in one direction. The aging process cannot be directed backward. Any biological system comes into being, develops, and dies. When a complete thermodynamic equilibrium is established, all proteins disintegrate into separate amino acids. 4. Finally, it should be noted that living organisms have the unique ability to reproduce a rigorous order of their molecules, an ability inanimate matter does not possess. Inhomogeneity of biological systems and their states far from thermodynamic equilibrium do not allow us, without making sufficient stipulations, to use, in biology, the well-established concepts of entropy, free energy, and other thermodynamic functions characterizing the states of systems in thermodynamic equilibrium. In order to describe an inhomogeneous macroscopic system in a state far from thermodynamic equilibrium, it is necessary to have numerous data on its local densities, temperatures, composition, and other local characteristics. All these data cannot be included in one concept (the entropy or the quantity of information) or in two concepts-the quantity and the quality of information. These average characteristics carry little information. Attempts to determine the properties of solids consisting of a large number of atoms for describing the state of motion of individual atoms have long been abandoned. Many properties of such solids proved to be determined by a comparatively small number of collective, simultaneous, synchronous displacements of atoms, ions, and electrons. So, for example, in describing the sound propagation in a solid or a gas, the data on very complex movements of individual atoms are unnecessary. A sound wave represents a wavelike propagation of vibrations of mean-density particles. A successful theoretical description of biological phenomena at the molecular level is also possible only with a considerable simplification of the process. With such a simplification, we consider only primary degrees of freedom (usually collective) determining the phenomenon and neglect secondary ones. The proper choice of both the model and the methods for its description determine the advance of the theory. In this review, some problems of modern bioenergetics are discussed at the molecular level, and an important role of nonlinear processes is
ISXC'ITONS A N D SOLII'ONS I N MOLECULAR SYSTEMS
I85
shown. The process of the vibrational energy and electron transfer along protein molecules is studied on the basis of nonlinear equations. A new model of the molecular mechanism of muscular contraction in animals is presented using the concept of solitons. The possible role of solitons in other biological processes is investigated.
"CRISIS"I N BIOENERGETICS The most active part in cell bioenergetics is played by protein molecules. They are closely connected with the basic manifestations of life. All chemical processes in the cell take place with the participation of proteins-enzymes. Proteins transform chemical energy into mechanical energy and are responsible for cellular and intracellular movement. Proteins, in complexes with lipids in cellular and intracellular membranes, ensure the transport of substances and ions into and out of the cell. They participate in the process of respiration, ensuring the oxidation of food, with the purpose of providing energy for all the needs of the living organism. All proteins are polymeric molecules with a very high molecular weight. They are formed due to polymerization of various amino acids. An amino acid is a chemical compound in which an amino group (NH,), a carboxyl group (COOH), a hydrogen atom, and a group of atoms [ ( R ) , which distinguishes amino acids from one another] are joined to a carbon atom. There is a large number of amino acids in nature; however, the proteins of living organisms are made up only of 20 different amino acids. The polymerization of two amino acids occurs in the presence of enzymes and with the expenditure of energy (about 3.23-4.84 kcal/mol). The polymerization is accompanied by the formation of the water molecule from the separation of ;I hydrogen atom from the amino group of one amino acid and the hydroxyl group (OH) from the carboxyl group of another (Fig. I ) . At the same time, the nitrogen and the carbon of amino acid residues join together to form a peptide bond. Given the appropriate enzymes and energy, this process of polymerization is repeated, producing long polypeptide chains (proteins) with repeated groups of four atoms, H , N , C, and 0, called peptide groups (PGs). A segment of protein chain is presented in Fig. 2. A special fcaturc of PGs is their ability to form hydrogen bonds with one another. with water molecules, and with other molecules containing electronegativc atoms (0,N , etc.). Proteins (polypeptide chains) can take different spatial configurations (secondary and tertiary structures) determined by interactions between elements of the peptide chain and the aqueous environment. One of the most important and interesting spatial protein structures is an a-helical
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FIG. I . The polymerization of two amino acids.
structure first established by Pauling and Corey (1951) (see Pauling et al., 1954). The protein molecule coils into a helix due to three chains of hydrogen bonds between peptide groups: 1, 4, 7, etc., in the first chain; 2, 5, 8, etc., in the second; and 3, 6, 9, etc., in the third. As a result, the right helix, with a step of 5.6 A and diameter of 6 A, is formed (Fig. 3). It has been accepted that the universal unit of energy exploited by protein molecules for various kinds of “work” (movement, ion transfer, etc.) is the energy released by hydrolysis of adenosine triphosphate (ATP) molecules synthesized in specific organelles of the cell (mitochondria) during oxidation of food. The ATP molecule hydrolysis is the universal intermediate step of an enormous number of energy transformations in the cells of all living organisms, from the simplest bacteria to humans. The hydrolysis takes place under the influence of enzymes and coenzymes (ions of metals) in some sites of protein molecules incorporated into ionic pumps, muscular fibers, etc. As a rule, the released energy is used in some other parts of these molecules. In this connection, there arises an important problem concerning the mechanism of energy transfer of the hydrolysis of ATP molecules along large protein molecules. The 1973 meeting of the New York Academy of Sciences was devoted to the elucidation of the mechanism of energy transport in biological systems. Three problems were discussed. (1) Is there a crisis in bioenergetics? (2) What is the nature of the crisis? (3) How can the crisis be resolved? The free energy released under the hydrolysis of ATP molecules under
FIG.2. A segment of the protein chain consisting of two peptide groups (shaded).
EXCITONS A N D SOLITONS IN MOLECULAR SYSTEMS
I87
I FIG.3. The arrangement of peptide groups in the a-helix protein molecule. Peptide groups are denoted by ellipses and hydrogen bonds by lines connecting these groups.
-
normal physiological conditions is comparatively small, 12 kcal/mol (-0.52 eV). It is only 20 times greater than the average energy of heat motion. Consequently, this energy cannot be transferred by electron excitations of molecules requiring 2-5 eV. Taking into account small energy released under hydrolysis, some participants of the conference (see Green, 1973) maintained the view that the vibrational energy of atoms C and 0 incorporated into peptide groups of all proteins is transferred along protein molecules in biological systems. These vibrations are called amide I. They have an energy of about 0.21 eV and a comparatively large electric dipole moment (about 0.3 D) directed along the helix axis, ensuring a large resonance interaction between peptide groups. The vibrations of amide I are the strongest characteristic vibrations detectable in the spectra of infrared radiation absorption (in the region of frequencies 1650-1660 cm-') by all proteins. The energy transport of amide I vibrations has been disputed by the other participants of the conference. They asserted that, due to the short lifetime (of the order of seconds) of amide 1 vibrations of individual peptide groups in a condensed medium, these vibrations cannot take part in the excitation transfer at distances much larger than the dimensions of PGs. The supporters of the assertion that the energy is transferred by amide I vibrations failed to confirm its validity. The view on the necessity of elaborating special (empiric) laws governing the energy transformations in living organisms has not also been given an approval. The problem of crisis in bioenergetics was thus unsolved.
11. The New Concept of Energy Transport along Protein Molecules
Theoretical investigations carried out at the Institute for Theoretical Physics of the Academy of Sciences of the Ukrainian SSR since 1973 by Davydov and co-workers (Davydov and Kislukha, 1973, 1976a, b; Davy-
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dov, 1977a, b, 1979a; 1982; 1983a, b) have shown that the energy of ATP molecule hydrolysis can be transferred at large distances (in atomic scales) along a-helical protein molecules as special collective excited statessolitons. As will be shown below, solitons are the excitations described by nonlinear equations. For many years, linear equations, taking into account a linear response of the system to external effects, were used for theoretical interpretation of different phenomena in physics, chemistry, biology, and engineering. In this case, the increase in the input by N times results in the same increase in the response of the system. The classical linear equations in mechanics, electrodynamics, and quantum theory were based on the superposition principle, which allows one to represent any physical quantity as elementary noninteracting components. Using such equations, it has become possible to explain many properties of the systems made up of a great number of interacting particles by introducing the concept of collective excitations characterizing a simukaneous consistent motion of a very large number of particles. The concepts of elementary excitations of the type of phonons, excitons, magnons, etc.. have been introduced. All the elementary excitations are described by simple harmonic waves of definite length and frequency. Such waves have an infinite spatial extension. Therefore, they cannot transfer energy and information. Such excitations, localized and moving in space (wave packets), are a superposition of a large number of waves distinguished by the wavelength. In many media, the phase velocity of simple harmonic waves (displacement of their constant phase value) depends on the wavelength. Such media are called dispersive. When a wave packet moves, its separate monochromatic constituents move at different velocities; therefore, the spatial extension of a wave packet increases. The wave packet is said to “smear” over the course of time. This is one of the essential shortcomings of the energy transfer by the wave packet-type excitations. The second shortcoming is that, while traveling, the wave packets lose energy by transferring their energy to the atom vibrations, i.e.. heating the medium. It has recently been found that in nonlinear systems with dispersion, i.e., in the media where the phase velocity of simple waves depends on the length and amplitude of the wave, the ideal way for the energy transfer is its transport in the form of solitary waves, which are called solitons. Unlike the usual waves, which are periodic repetitions of elevations and cavities on the surface of water, or densities and rarefactions or deviations of the mean value of the other physical quantities, solitons are solitary elevations transferring as a unit with a constant velocity. It was shown by Davydov (1977a, b, 1982, 1983a, b) that, in a-helical protein molecules,
EXCI’I’ONS A N D SOLII’ONS IN MOLECULAR SYSTEMS
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solitons corresponding to intrapeptide vibrations amide 1 can appear which transport energy along the molecule without losses. 111. History of Observation of Solitary Waves
The first qualitative description of solitary waves on the water in a narrow channel connecting Edinburgh with Glasgow in Scotland was first made by Scott Russell (1844), a naval engineer who studied the design of channel barges, particularly the problem of finding the best shape of a ship’s hull, the shape which would offer the least resistance to the water. He noted in 1834 that, when a boat drawn along the channel by a pair of horses suddenly stopped, a part of water was separated. I t accumulated round the prow of the vessel in a state of violent agitation, then suddenly leaving it behind. rolled forward with great velocity. assuming the form of a large solitary elevation. a rounded. smooth and well defined heap of water, which continued its course along the channel apparently without change of form or diminuation of speed.
In such a way, this singular phenomenon was described by Scott Russell. It is interesting that the exceptional stability and self-organization of solitary waves was noted only by him. For a long time, solitary waves have not been a matter of interest for researchers. Only in the fifties, after the death of Scott-Russell (1844). two Dutch scientists, Korteweg and de Vries (1895), proposed a mathematical description of solitary waves on the surface of water in a shallow channel using a nonlinear differential equation, which is now briefly called KdV equation. For a one-dimensional system in the continuum approximation it is written as
The simplest solution of this equation has a form of a bell-like excitation M(X,
I ) = 3v sech’ [ f i ( x - vt)/2]
(2)
traveling with velocity v. This form of the traveling excitation, Eq. (2). is determined by a mutual compensation of the nonlinearity effect due to the second term, Eq. ( l ) , and the dispersion effect due to the third term. Dispersion leads to the spatial spreading and changes the form of the excitation consisting of many simple harmonic waves. In linear systems, this spreading is not compensated due to the independence of waves. In nonlinear systems, an intensive interaction of monochromatic constituents takes place, causing the redistribution of energies between them. If this
190
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redistribution is such that the energy of more quickly moving waves of the wave packet is taken and transmitted to retarded waves, then a stable excitation is formed which propagates as a unit without spreading. It is the soliton’s great stability that has recently stimulated numerous attempts to use them for the explanation of many phenomena in different fields of nonlinear optics, physics of condensed state, field theory, gravitation theory, and other sciences, in particular, in biology. To illustrate the applications of this phenomenon in biology, we note a recent attempt of Rowlands (1982)to use a concept of solitons for describing propagation of a blood impulse in arteries of animals. Toward the end of 1950s much more interest was generated in solitons in view of investigations of physical processes in plasma aimed at solving the problem of thermonuclear synthesis. In 1958, the Soviet physicist Sagdeev showed that, in plasma, solitary waves may propagate in ways similar to those on the fluid surface. Consideration of nonlinear effects in real systems is connected with great mathematical difficulties. Earlier, the influence of small nonlinearity was usually studied by perturbation theory methods. So, for example, in the linear (harmonic) theory of atom vibrations relative to the equilibrium positions in the lattice of a solid, the excited states were described by a system of noninteracting phonons (plane waves). With allowance for small nonlinearity between phonons, there appeared an interaction leading to the energy exchange between them and to establishing thermodynamic equilibrium, heat transfer, thermal expansion of a body, etc. However, in strongly nonlinear systems-ferroelectrics in states close to phase transitions accompanied by displacements of equilibrium positions of atoms, shock waves, and turbulence in condensed media, the interaction of a powerful laser radiation with a substance, etc.-the allowance for nonlinearity by the methods of the perturbation theory is inadmissible. Although the mathematical analysis of nonlinear media had been started by Riemann as early as 1860 (in his classical work on nonlinear wave propagation; see Riemann, 1892) remarkable progress in studying nonlinear systems was not achieved until a hundred years later, progress due to investigationscamed out by numerical methods using powerful computers. Creation of a computer with the capacity of billions of real number operations per second has made it possible to extend tremendously the practical usage of the methods for describing different nonlinear phenomena. The combination of analytical and numerical methods of investigation resulted in the creation of so-called synergetic approximation (Zabusky, 1967). Any scientific theoretical investigation is connected with the idealization
EXCITONS A N D SOLITONS IN MOLECULAR SYSTEMS
191
and simplification of the phenomenon under study which results in the creation of a mathematical model. Analytical and numerical solutions of such a model allow one to understand certain properties of a real phenomenon. The proper choice of a model determines the success in explaining the phenomenon. Powerful computers make it possible to create progressively more complicated models and obtain results that contribute to a more complete understanding of the phenomenon and, in some cases, predict new phenomena. Excitations in the form of solitary waves (solitons) together with ordinary extended waves are inherent in many nonlinear and dispersive dynamic systems. However, analytical and numerical solutions on a computer are well developed only for one-dimensional and quasi-one-dimensional systems. Numerical calculations are especially effective in studying nonlinear phenomena. Fermi, Pasta, and Ulam in Los Alamos (Fermi ef uf., 1955) were apparently the first to demonstrate the high efficiency of a computer used to analyze nonlinear phenomena. They intended to describe quantitatively the thermalization of a primary long-wave excitation of a small number of the degrees of freedom in a one-dimensional chain of identical particles coupled by nonlinear springs. According to a generally accepted view presented by Debye as early as 1914, it has been expected that, due to the nonlinear interaction between particles, the energy transferred to the system should uniformly be distributed in all the degrees of freedom of the chain and, in particular, in short-wave ones, i.e., a thermal equilibrium will be established. To the authors’ surprise, it turned out that there was no thermalization. Over some time, the chain returned to the primary excitation state. Thus, the Fermi-Pasta-Ulam problem that changed the traditional concepts of scientists arose. Fairly interesting properties of solitary waves described by the KdV equation were also obtained from numerical calculations by Zabusky and Kruskal (1965). According to Eq. (2), the velocity of a solitary wave is proportional to the wave amplitude (v). The solitary waves with greater amplitude leave behind those with less amplitude. Studying collision, in which one wave passes through another, they discovered that under small phase variation the waves do not change their shape and velocity. Therefore, to emphasize such a particle-like behavior of solitary waves, Zabusky and Kruskal have called them solitons. The ending “on” is used in Greek to denote a particle. A great contribution in the development of mathematical theory of solitons has been made by the works of Zakharov and Faddeev (1971), Zakharov and Shabat (1974), Cologero and Degasperis (1976), Bullough and Caudrey (1980), and Zakharov et uf. (1980).
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IV. Nonlinear Phenomena in Biology Physiological activity of protein and other molecules in living organisms is due to weak interactions: Van der Waals’ forces, ionic, hydrophilic and hydrophobic interactions, and hydrogen bonds. A hydrogen bond, in particular, determines all unusual properties of water which are important for the existence of living systems. Determining the secondary and higher order structures of huge polymeric molecules, internal and external membranes, without which there would be no individual cells and a great number of biological processes of vital significance, the weak interactions. in their turn, are also dependent on these structures. Therefore, all biological systems are essentially nonlinear. Bioenergetics dealing with the mechanism of the energy release and transfer by a cell are initially connected with the elucidation of the role of proteins which catalyze and maintain living processes. The primary and spatial structures of many proteins have been found using chemical, crystallographic, spectral, and other methods. But still, information about their dynamic properties is incomplete. The equilibrium therniodynamics and mechanics based on linear equations cannot explain the mechanisms of the energy transformation and transfer in biological systems. Among diversified protein molecules, a keen study has been made of a-helical molecules with the quasi-periodic arrangement of idential elements, i.e., peptide groups. A large number of protein molecules, i.e., myosin, tropornyosin, and troponin, have a basically linear a-helical structure. The others, even globular ones, contain considerable linear ahelical sections. So, for example, a hemoglobin molecule, which is a constituent of blood erythrocytes, has 32 a-helical sections; a bacteriorhodopsin molecule incorporated into a proton pump of membranes in halobacteria (microorganisms living in salt lakes) has seven a-helical sections. Many problems in bioenergetics are due to the nonlinear character of excited states in a-helical proteins. A study of the excited states of cihelical protein molecules was first made by Davydov and Kislukha using nonlinear theory (1973, 1976a, b). It was shown that, in such molecules, very stable excitations, solitons, can be excited by means of a chemical reaction. In Davydov’s papers (1977a, b, 1979a, 1983a, b), this theory was developed and used to substantiate the transfer of vibrational excitation amide I along a-helical proteins at large distances. Initially, a simplified model of a molecule (one chain of peptide groups formed by hydrogen bonds between them) was studied. The excitations corresponding to the intrapeptide vibrations amide I characterized by a dipole moment of the transition d (-0.35 D) parallel to the axis chain were
EXCI‘I’ONS A N D SOLITONS IN MOLECULAR SYSTEMS
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investigated. The resonance dipole-dipole interaction J = d’/d tends to distribute this excitation along the whole chain. In a nonlinear approach the intrapeptide vibrations. amide 1 is connected in a self-consistent way with displacements of equilibrium positions of PGs. These displacements cause a local chain deformation. This local deformation crcates a potential well which prevents the excitation smearing caused by a jumplike transition of the intrapeptide excitation from one peptide group to the neighboring one due to the resonance dipole-dipole interact ion. The self-consistcnt interaction between intrapeptide vibrations and the local deformation results in a compound excitation propagating along the chain without energy loss and change in the shape and linear dimensions. During analytical calculations. a discrete chain with peptide groups situatcd in the lattice points ~ ( r i= 0, 2 I , . . .) was changed by a continuous chain, i.e., the continuum approximation ( n r r +-x) was used. In the works of Davydov and co-workers (a general review of these papers is given in Davydov’s monograph, 1984), it was shown that the distribution of intrapeptide groups of amide I vibrations propagating along the chain of PGs with a constant velocity is characterized by the square of thc modulus of the function’ 18
$(x, t ) = N u , t ) exp[i(k.r - Er/h)]
( 31 in which E is the total excitation energy involving the chain deformation energy propagating together with the intrapeptide excitation. The value of the wave number k is connected with the velocity I J and the energy J of the resonance interaction of amide I vibrations of the neighboring groups by the equality
k
=
hv/2ti2J
(4)
The amplitude real function @ ( x , t ) in the system of coordinates
5
= x
- X,) -
I’t
(5)
moving together with the excitation satisfies the nonlinear Schrodinger equation
‘The complex function e x p ( i a ) = cos a
+
i sin a .
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A. S. DAVYDOV
The real function p(s) characterizing the decrease in distance between PGs [a + a - p(s)] in the deformation region is determined by the expression’ p([) = 2XQ2(()/K(l
-
S
S’),
= V/Vo
(7)
in which the coupling constant x determines the interaction of amide 1 vibrations with the displacements of equilibrium positions of PGs in the deformation region. K is the chain longitudinal elasticity coefficient connected with the longitudinal sound velocity in the chain and the peptide group mass M by the relation vo
(8)
=
The nonlinearity parameter G in the nonlinear Schrodinger equation is determined by the formula G
=
4 ~ * / ~ (-1 s’),
s # 1
(9)
The chain deformation accompanying the propagation of the intrapeptide excitation has the energy W
=
Mv,’,(2a3)-’( I
+ s’)
I
p’(s)d(
(10)
The total energy of the propagating excitation involving the deformation energy Eq. (10) is determined by the expression E =
so - 2J
-
A
+W
( 1 1)
where ( 0 is the energy of an isolated amide I vibration and is the spectral parameter of Eq. (6). The above expressions characterize two possible types of excitations of the chain of peptide groups: excitons and solitons, which will be given special consideration.
A. EXCITONS
If the velocity of the collective excitation exceeds that of the longitudinal sound (s* > I),the chain deformation has not enough time to follow the intrapeptide excitation. In this case, the nonlinearity parameter (9) is negative and Eq. (6) in an infinite chain has no stationary solutions normalized by the condition (6a). In a finite chain of sufficiently large length (Nu >> a), such solutions have the form ’Note that in the recent monograph of Davydov (1984) the value x is used which is twice of that utilized in (7) and in his pioneer works on solitons.
EXCITONS A N D SOLITONS IN MOLECULAR SYSTEMS
@(c) = N - ” : ,
0
6
I95
N >> 1
uN,
In this case, the excitation is described by the function in Eq. (3) taking the form of a plane wave +(x,
r)
=
N-”2 exp[i(kx
-
Er/h)l
(12)
Hence, according to Eq. (7), all interpeptide distances u change by the same value p,,([)
=
-2X/NK(S’
- 1)
(13)
which tends to zero at N + x. In the same approximation, A = W = 0. The excitation described by the functions in Eqs. (12) and (13) is called an exciton. The excitons transport only the intrapeptide excitation energy. In this case at small velocities, this energy is determined by the expression E,,(v) = E,,(o)
+ +mv2 = EJO) + h2F/2m
(14)
where
E,,(O) =
t,, - h’/mu2 = to - 25
(15)
The energy levels corresponding to different values form the exciton band. The E,,(O) characterizes the position of the exciton band “bottom,” m is the effective exciton mass. At ? < I , the plane waves of Eq. (12) are also the solutions of Eq. (6). However, the slow excitons described by the above solutions are metastable, since in this case there are more stable localized excitationssolitons which are considered below. According to Eq. (12), the exciton states with the definite value of k (energy) are distributed uniformly along the whole length of the chain [ I +cx(.x,t) 1 = constant]. Therefore, they transfer no energy and information. Usually, in the chain, the states described by a superposition of plane waves in Eq. (12)-a wave packet-are excited. Since the exciton states in Eq. (12) are dispersive (the phase velocity of these waves hk/2m depends on the wavelength), wave packets, made up of them, smear. The exciton states, corresponding to the excitation of the amide I vibrations in real protein molecules characterized by a more complex distribution of peptide groups, are also described by plane waves. They are excited when the proteins absorb infrared radiation. The absorption spectra of infrared absorption by proteins were studied in detail by Chirgadze and Nevskaya (1976). They have shown that, using the exciton theory developed by Davydov (1951, 1968) for the interpretation of such spectra, one can obtain very important information about the secondary structure of protein molecules. In particular, studying the infrared absorption spec-
196
A. S. DAVYDOV
tra, they determined the magnitude of the dipole moment of the amide I vibrations (0.35 D) and the energies of the resonance interactions between peptide groups which occupy different positions in proteins with the configurations: a-helix. (3-sheets, and globular conformation. The exciton excitation by radiation of frequency w takes place on condition that the conservation law of energy Aw = E(k) and quasi-momentum hk = hw/c is fulfilled and if the projection of electric field intensity of the wave in the direction of the transition dipole moment (d)is different from zero. In this case, the wave packet, whose spatial extension is about the radiation wavelength, i.e., exceeds by thousands of times the distance between PGs, is excited. Instability of excitons formed is due to the wave packet smearing, the energy loss for the phonon radiation, and the possible transition to a more stable state-the soliton of lower energy. The intensive energy loss by excitons for the phonon radiation is caused by the great value of the coupling coefficient ( x ) of amide 1 vibrations with displacements of equilibrium positions of PGs. Owing to this strong coupling, the exciton, when moving, excites significantly the vibrations of PGs relative to their equilibrium positions. €4.
SOLITONS
At s’
W.3
=
(16)
sech(Q0
at Q
=
muGI2h’
=
2mux’/~h’(1 - s2)
A
=
h’Q2/2m
(17)
According to Eq. (71, the local chain deformation in the excitation region is characterized by the function
The excitations described by the functions in Eqs. (16) and (18) are called solitons. They are distributed along the chain of the dimensions A t = 2 d Q (Fig. 4). When solitons move with constant velocity v , neither the excitation region nor the form of the excitation distribution along the chain undergoes changes. The function p(C;) characterizes the decrease in distances between PGs in the excitation region. This decrease is due to the displacements of equi-
EXC‘ITONS AND SOLITONS IN MOLECULAK SYSTEMS
I97
FIG.4. The distribution of the probabilities of intermolecular excitations (vertical lines) and the distances between peptide groups in the region of the soliton excitation.
librium positions nu, which are occupied by PGs in the absence of excitation. Such displacements are determined by the function
pcs,
= K(
I
2x ,[ I
-
tanh(Q[)]
p([) = - a-dp
- .S-)
4
(19)
All the above cxpressions are valid only when the equality3 SZ
<< 1
(20)
is satisfied. The energy of the chain deformation is W
=
mci’(I
+ 4s’)/3 x’ri’
(21)
and the total energy, involving the deformation energy of an exciton moving with velocity v , is determined by the expression E,‘,l(LJ)
=
E,,m +
;Mod
(22)
where EJO) is the energy of a soliton at rest
E,JO)
=
E,,(O)
-
mu’X4/3K’h’
(23)
Here E,,(O) is the energy of an exciton at rest determined by Eq. (15). Thus, the energy of a soliton at rest is less than that of an exciton at rest by the value (Fig. 5) AE E,,(O) - E,JO) = m ~ ’ ~ ~ / 3 ~ ’ h ’ (24) Hence due to the connection between intrapeptide excitation and the lattice deformation their total energy decreases. The multiplier M,,,in Eq. (22) can be called the effective mass of a soliton ’The region where the theory can be applied is violated at lim s’ = 1 by using a harmonic approximation to describe the displacements of PGs from their equilibrium positions. In the works of Davydov and Zolotariuk (1983a.b.c). it was shown that, with allowance for anhermonicity. all the quantities characterizing solitons have also finite values in the lim .Y’ = I.
I98
A. S. D A V Y D O V
FIG.5. The dependence of the soliton and the exciton energies on their velocities,
11:
(1) the soliton energy, (2) the exciton energies, (3) the energy of metastable excitons. and vu is the longitudinal sound velocity.
M,,,
=
( + $;)
m I
-
The effective soliton mass in soft chains (small K) exceeds significantly that of an exciton m ,since the soliton motion is accompanied by the local chain deformation. The soliton with a large mass can transfer great kinetic energy at small velocities. The presence of the energy gap in the spectrum of excited states of the chain between exciton and soliton states [Eq. (24)] proves to be one of the reasons for the very high stability of a soliton. In order to destroy a soliton, i.e., to split it into a free exciton and a deformation which then relaxes into a thermal motion, it is necessary to expend a considerable amount of energy. According to Eq. (24), such energy is proportional to the fourth degree of the coupling parameter (x) and inversely proportional to the square of the elasticity coefficient (K) of the chain. Therefore, the soliton stability is especially great in soft chains, where the interaction between PGs is determined by weak hydrogen bonds. It is interesting to note that the stability of soliton is great at large values of the coupling parameter x. On the contrary, as was noted in Section IV,A, large values of x shorten the lifetime of excitons, due to the intensive formation of vibrations of PGs relative to their equilibrium positions, i.e., due to the production of phonons. Since solitons always move with velocity less than that of longitudinal sound in the chain, they do not emit phonons. In other words, their kinetic energy is not transformed into the energy of thermal motion. This is the second important characteristic that ensures the high stability of solitons in soft molecular chains of the type of a-helical protein molecules.
EXCLTONS A N D S O L I T O N S IN M O L E C U L A R S Y S T E M S
199
Finally, the third causative factor in the high stability of solitons is their topological stability. When a soliton moves along the chain, the displacement of equilibrium positions of PGs takes place [Eq. (19)l. To the right of a soliton (E>O), all PGs are in undisplaced positions nu and to the left (&O) they are displaced equally
In order to destroy a soliton, it is necessary that all the left-hand peptide groups of the chain be returned to their initial position. As is known, at the moment of light absorption by molecular systems, the coordinates of heavy particles cannot be displaced (the Franck-Condon principle). Since the formation of a soliton is connected with the displacement of equilibrium positions of heavy PGs, the probability for soliton excitations by light is very small. For the same reason, the probability of light radiation is considerably small. The theory of this problem has been developed in the work of Davydov and Eremko (1977). Solitons may be excited by local effects. One such effect is a chemical reaction. The probability of the excitation is the highest when such a local effect takes place at the end of a molecular chain. This statement is based on the consideration of topological stability of solitons.
V. Solitons in Real a-Helical Protein Molecules As was mentioned above, three chains of PGs appear in a protein molecule when the a-helical structure is being formed. Therefore, in a more detailed theoretical description of excited states of the molecule, it is necessary to take into consideration the resonance interaction between PGscomponents of different chains. Apart from possible PG displacements along each chain, one must take into account the change in distance between the chains, i.e., the change in the helix diameter. Such complications of a mathematical model were fulfilled in the works of Davydov et ul. (1978), Eremko and Sergienko (1980, 1982), and Scott (1981, 1982a, b, c , 1983).
From the data on the spectra of the infrared radiation absorption by proteins, Chirgadze and Nevskaya (1976) obtained values of the resonance interactions between neighboring PGs in a single chain ( J = 7.8 cm-’ = 1.55 x I0-I’ J ) and between PGs in neighboring chains ( L = 12.4 cm-‘ = 2.46 x lo-” J). The solutions of coupled differential equations, taking into account both the interaction J and L, the change in the pitch, and the diameter of the helix, characterize the simultaneous propagation of the excitation along
200
A . S. DAVYDOV
A
FIG.6 . T w o types of solitons can form in the a-helix: ( A ) ii symmetric soliton in which amide 1 vibrational energy is shared equally among the three spines. and ( B ) an ahymmetric soliton in which amide I energy is shared with an insignificant shift in phases.
three chains. In this case, one intrapeptide vibration amide I corresponds to three types of stationary solitons:
I . symmetric solitons (excitations propagate along the thrcc chains in a phase) and 2. two kinds of asymmetric solitons.
In the case of symmetric solitons in the region of excitation, the pitch of the helix decreases and the diameter increases. In the case of asymmetric solitons, the excitations propagate along the three chains with insignificant shift in phases. The diameter of the helix, then, increases and a local bend of the molecule occurs (see Fig. 6). The energy of asymmetric solitons is less than that of symmetric ones. According to the numerical calculations performed by Scott ( 198?c), this difference (expressed in units of cm- ') is equal to AE,
=
15.4 cm-'
If, in the molecule, a nonstationary state is excited, which corresponds to the superposition of symmetric and asymmetric solitons, then pulses appear which correspond to the energy AEl. These pulses correspond to a jump of the excitation from one chain of PGs to the neighboring one. The excitation jump between peptide chains is due to the resonance interaction L between PGs of the neighboring chains. A rough estimate of the jump frequency is determined by the equality v = ~12.rrh= 3.7 x 10'' sec-l
which corresponds to the period -2.7 x IO-I'sec.
EXCITONS AND SOLITONS IN MOLECULAR SYSTEMS
20 I
VI. Solitons in Discrete Models: Numerical Calculations
In order to obtain analytical results, models of infinite chains are usually used in the continuum approximation. The limited discrete chains can be investigated only by means of modern computers. The powerful digital computers exploited in calculations have made it possible to propose and settle new problems in the region of nonlinear phenomena, the solution of which earlier seemed impossible. Numerical calculations of the soliton excitations of protein molecules have greatly contributed to a more complete understanding of these states. The first numerical integration of the discrete differential equations proposed in the work of Davydov e t d.(1978) to describe the excited states of the three chains of PGs in an a-helical protein molecule was performed by Hyman rrl. (1979) with the help of the computer at the Los Alamos Scientific Laboratory. They studied symmetric excited states in the three spines of PGs. Each spine contained 200 peptide groups. The protein molecule was characterized by the following quantities: 6 = 0.205 eV = 0.328 x lo-’‘ J ; sound speed v,, = I . I5 x lo4 m/sec, M = 70 x mass of proton, J = 7.8 cm-’ = 1.55 x lo-” J , L = 12.4 cm-’ = 2.46 x I O - ” J ; u = 5.4 A. The initial conditions at t = 0 were taken as I,
for
17
= 1
(26)
and PI,<$= 0, for all 11, where the subscript a = I , 2, 3 denotes the number of a spine; ti = I , 2, . . ., 200 specifies a particular unit cell along a spine. The calculations were performed for different values of the nonlinear coupling coefficient of intrapeptide amide I vibrations with displacements of equilibrium positions x of PGs. It was shown that for the initial conditions in Eq. (261, distinct solitons are formed and propagate in the molecule with x = 3 x l o - ” N. Solitons with close to the critical value propagate along the molecule with velocity v = I .26 x 10’ m/sec. Therefore, the distance 1000 A could be traversed by a soliton within 80 psec. Based on the above calculations. Hyman rt ul. (1979) arrived at the conclusion that “numerical studies of Davydov‘s nonlinear dynamic model of a-helical proteins confirm his prediction on the formation of solitons.” This conclusion is very important, for the authors have used the values of the model parameters corresponding to real protein molecules. Their work is also interesting in the respect that the process of formation of solitons from the detinite initial state is investigated, and the role of the chain discreteness is clarified. Kuprievich and Kudritskaya (1982) have conducted an independent
202
A . S. DAVYDOV
theoretical investigation of the value x on the basis of an initial quantumchemical calculation of the electronic structure of a formamide dimer. This molecule consists of two peptide groups coupled by a hydrogen bond, the value of which was determined by taking into account the change in the C = 0 spring constant due to the change in the hydrogen bond length. Using the data on the two values of the hydrogen bond lengths, Kuprievich and Kudritskaya estimated the position of x between 0.3 x IO-I'and 0.5 x IO-'ON. Careri (1973) has estimated x empirically by comparing amide 1 energies and hydrogen bond lengths of various polypeptide crystals. He found x to be about 0.62 x lo-"' N . Lomdahl et ul. (1984) noted that "these estimates for x indicate that the level of nonlinearity in real systems is sufficient to allow self-focusing (soliton-like) excitation to be formed." Eilbeck (l979), in Heriot-Watt University (Edinburg, Scotland), made a 16-mm computer film that demonstrates the propagation of an internal vibrational excitation amide I from an edge peptide group along a PG chain. This film vividly shows that, for an above threshold value of the coupling parameter, the excitation propagates along the protein molecule in the form of a soliton, i.e., in the form of a distinct local pulse with the width of several PGs. Its shape and width remain constant during the motion. Eilbeck's film (1979) and numerical calculations of Hyman et cil. (1979) show that the soliton is formed at the very beginning of the chain of the peptide group. Therefore, solitons can arise within comparatively short sections of the a-helical protein molecule. Discreteness of the real protein molecule leads to a small periodic modulation of its motion. The modulation period equals to the time at which a soliton traverses the distance between the neighboring PGs. According to Scott's calculations (1981, 1982a. b), this period corresponds to the energy (Fig. 7)
AE2 = 125.7 cm-' Since the solitons in various proteins excited in different ways have different velocities, the values AE, are expected to undergo significant changes. The numerical calculations performed by Lomdahl ez (11. (1984) confirmed the particle-like properties of solitons. When two peptide groups are excited at the opposite ends of the polypeptide chain, there appear two solitons moving one toward the other with the velocity constituting three-eights of that of a longitudinal sound. It turned out that, in colliding,
EXCI’I‘ONS A N D SOLITONS IN MOLECULAR SYSTEMS
4
203
I
0 50 100 150 200n FIG.7. Plots of the distribution of probabilities of intermolecular excitations along peptide groups ( n ) of the chain for several values of the nonlinearity parameter x ( x 10 “I N ) [Scott, A. C.. 1982a. Phys. R c v . A26 ( I ) . S78-595.1
they pass through each other, retaining their velocity and shape. Thus, the collision of solitons resembles the elastic collision of two particles. Most studies have been done on the Davydov soliton theory at zero temperature. Sometime ago, Davydov (1980) (see also Davydov, 1985) had extended the theory to account for thermal effects. He had obtained a nonlinear Schrodinger equation with a temperature-dependent coefficient for a nonlinear term. Recently, Lomdahl and Kerr (1985) have used the classical fluctuationdissipation relationship by adding to the equation for the displacement of momentum operators by some damping force and noise force. They obtained an essential result: at biologically relevant temperature ( T = 300 K), the random noise forces prevent Davydov self-trapping from occurring. I think they obtained this result by introducing an unjustified strong interaction of solitons with a noise force. The Eilbeck film (1979) demonstrates clearly the stability of soliton relative to their interaction with acoustic waves. The wave packet of acoustic waves was excited simultaneously with the soliton. Transferring more rapidly than the soliton, the acoustic wave packet is reflected a few times by the chain ends and passes through the soliton, leaving it unchanged. No interaction was observed after the acoustic wave packet was converted into a noise. Lomdahl and Kerr (1985) noted (p. 1236) that, using parameters for the a-helix, they had calculated from the theory extended by Davydov (1980) to account for the thermal effects that “solitons shorrld exist rip t o T = 370 K . ” I t is very important that the nonlinear parameter G,, of the Schrodinger
204
A. S.
DAVYDOV
equation increases with increasing soliton velocity VC,)+ G,J(1 - v ’ h : ) , where v0 is the velocity of sound. Lomdahl and Kerr considered only resting soliton at the contour of the chains. The disagreement between a quantum theoretical analysis by Davydov and classical numerical studies of Davydov solitons by Lomdahl and Kerr was investigated by Alwyn Scott from the University of Arizona. H e has shown in the paper “On Davydov solitons at 310 K” that the decay of soliton obtained in the numerical calculations i s forbidden by the FranckCondon principle which was not been taken into account in numerical studies by Lomdahl and Kerr. Alwyn Scott has indicated that biological Davydov solitons can be formed and propagate at physiological temperature 3 10 K. The transition from soliton state to exciton state i s forbidden because of the “topological stability” o f the biological solitons (see p. 28).
VII. Solitons and the Molecular Mechanism of Muscle Contraction
One of the most interesting problems in bioenergetics is to explain on the molecular level the question of how the chemical energy of hydrolysis of ATP molecules is transformed to mechanical energy in different intracellular and intercellular movement processes in living organisms. Of all such movements, the best studied turn to be the movements caused by transversely striated muscles (Figs. 8 and 9). Modern phenomenological theories of muscle contraction (Huxley, 1957; Murray and Weber, 1974)are based on the concept that the sliding of thin protein filaments relative to thick ones in sarcomeres of contractile muscles, which is observed experimentally, is due to an active movement of the heads of myosin molecules incorporated into thick protein filaments (Fig. 10).
FIG.8. Diagram of a sarcomere in striated muscle: Z. transverse membrane called the Z line: (1. the length of a sarcomere in a relaxed muscle; I , thin filaments, which contain an F-actin polymeric chain of globular actin molecules, globular tropomyosin molecules. and a-helix tropomyosin molecules: 2. thick filaments, which contain about 200-400 myosin molecules; 3. heads of myosin molecules.
FK;.9. I)iagr;im of the contraction of the length ofthe sarcomere according to the sliding filament model. I . A relaxed muscle; 2. a displacement of the thin filament relative t o the thick one.
I"" FIG. 10. Phenomenological model for the contraction of striated muscles [Murray. J. A . Sci. A m . . 1974, 230 (2). 59-71]: a, thick filament: b. head of the myosin molecule; c . thin filament; d. Z line; I, position of the head of the myosin molecule in a relaxed muscle; 2 elongation of the head of the myosin molecule and formation of a bond with the actin molecule of the thin filament: 3, rotation of the head accompanied by a displacement of the thin filament; 4, separation of the head of a myosin molecule from the thin filament and its return to the initial state.
M..and Weber.
206
A. S. D A V Y D O V
It is supposed that, under the ATP hydrolysis, the head of the myosin molecule elongates, forming a link (“cross-bridge”) with an active molecule of the thin fiber, then turns, displacing the thin fiber relative to the thick one toward the sarcomere center, and, finally, detaches from the thin fiber, regaining the previous size and the position in a thick filament. Having joined the new ATP molecule in the presence of Ca” ions, it can repeat this cycle. The idea of the bridges, which are joined between thick and thin filaments, that pull the fibers, causing their displacement and breakage does not explain the molecular nature of this phenomenon. The following questions remain unsolved. How is the ATP hydrolysis energy used in the elongation, the cross-bridge formation, the pull force, and the cross-bridge breaking? What is the molecular mechanism of change in the myosin molecule head that leads to these phenomena? And, finally, why does only the head of the enormous myosin (molecular weight 500,000), having, besides a head, a long “tail” formed by an a-helical protein, take an active part in the contraction? Academician Frank (1982) wrote: “The transfer of the basic kinetic mechanism only on the bridges comprising 7% of muscular substrate seemed unconvincing for me.” Using theoretical investigations of solitons in helical protein molecules, Davydov (1973, 1974a, b, 1975, 1976, 1979a) proposed a new hypothesis to elucidate the mechanism of the shortening of sarcomere lengths. According to this hypothesis, the calcium ions reaching the first series of the myosin molecule heads at the ends of thick filaments initiate the hydrolysis of the ATP molecules attached to them. The energy released generates solitons in long helical sections of myosin molecules. These move from the heads of the myosin molecules to their ends, placed in the region of the sarcomere center. As was shown in Section V, the motion of a soliton along a protein molecule is accompanied by a local bending and increasing in the diameter of a molecule. Therefore, in the region of the soliton motion, the thick filament formed by a bundle of myosin molecules is swollen (Fig. 11). The motion of this swollen region of a thick filament from its end to the center causes the displacement of thin (actin) protein filaments attached to Z plates (limiting the sarcomere size) to the sarcomere center. Analogous phenomena take place at another end of the thick filament. The motion of thin filaments from opposite ends causes the shortening in the length of each sarcomere and, consequently, the contraction of the length of the muscle fiber. According to this model, the heads of myosin molecules, located on the surface of thick fiber, attach themselves to thin filaments and also detach from them (as in the model of “bridge” formation and breaking).
EXCITONS AND SOLITONS IN MOLECULAR SYSTEMS
-1
-2
3
3
207
z
w
FIG. I I . A section of the right-hand half of the sarcomere. The movement of solitons. originating in the heads of myosin molecules along the tails inside the thick filament. lead5 to its “swelling” and the movement of the “swollen part” to the middle of the sarcomere. The middle-sized arrows ( I and 2) show the direction of the movement of the myosin heads, pushing the thin filaments to the middle of the sarcomere. Smaller arrows (3 and 4) are moving away from t h e thin filaments.
This movement, however, is not due to elongation, turn, and contraction of the heads themselves, but to the motion of solitons inside the thick fiber along a-helical parts of myosin filaments, causing the swelling of a thick fiber. The motion of solitons is accompanied by the motion of “swollen” regions of thick filaments. In this case, the kinetic energy of solitons is converted into the contraction energy or the energy of tension, if a load is applied to the muscle. In this model, all the parts of the myosin molecule, not only its head, are active contractile elements. Expending their kinetic energy on the work necessary to contract the muscle fiber, the solitons are slowed down and, stopping near the centers of the thick filaments (H-band), are annihilated, giving up the rest of their energy to thermal motion. Thus, only the kinetic energy of solitons is used in the contraction of the muscle fibers. As a good example of the mechanism of displacement of thin filaments, due to the motion of the swollen region of thick filaments in which solitons are moving, there may serve the vibromotors of Ragulskis and co-workers (1976). In these motors, elastic oscillations, excited in piezoelectric films in the form of pulses, move along their surface and, under the action of friction caused by the sliding, give rise to the rotation or to a consecutive movement of the bodies, which are in contact with them.
VIII. Intracellular Dynamics and Solitons
For a comparatively long time, the concept of a living cell as a certain quantity of structureless liquid (cytoplasma) that fills its shell has been shared by many scientists. However, the studies of cell with the help of electron microscopes revealed that a cell contains many incorporationsorganelles surrounded, in turn, by the membranes. Such organelles are mitochondria that synthesize ATP molecules, ribosomes and their com-
208
A. S. DAVYDOV
plexes, polysomes involved in the synthesis of proteins necessary for the cell, lysosomes and baglike formations containing the enzymes digesting foreign molecules, etc. All these incorporations do not float freely in cytoplasma, but occupy certain positions in the cell. Late in the 1960s, our understanding of the intracellular structure was essentially advanced by using high-voltage electron microscopy with the energy of the electron beam of 10 MeV, 10 times higher than the energy of standard electron microscope (Porter and Tucker, 1981). This, on the one hand, sharply increased the resolving power of the microscope and, on the other hand, allowed us to investigate the three-dimensional structure of cells up to several micrometers-thick layers rather than in the finest slices of the cell. The first investigations of the spatial structure of cells by means of the high-voltage electron microscopes have already shown that the whole cellular interior is spanned by a network of protein microfilaments and tubules which retain the organelles of the cell in definite sites, determine the form of the cell, its change, and all the movements inside the cell. Such a network of microfilaments and microtubules has been called the cytoskeleton. The first information of the presence of the cytoskeleton in each cell was met with distrust. It has been supported by the fact that the cytoskeleton structure proved to be unstable, dependent on external environment and the stage of functioning and development of the cell. Microfilaments and tubules of the cytoskeleton are made up of bundles of protein molecules about 300-1500 A long. These molecules tend to form longer aggregates by means of polymerization: head-to-tail and tailto-tail configurations. The protein molecules incorporated into the cytoskeleton determine all the internal movements in the cell, the energy transport, and the internal bond. All these actions are performed due to the energy released under hydrolysis of ATP molecules. The process of hydrolysis is, apparently, controlled by the change in the concentration of calcium ions in the muscle fibers. At present, the concept that all types of movements, both of muscle fibers and all intercellular and intracellular movements in nonmuscle cells, are determined by a small number of contractile proteins formed by actin, myosin, tropomyosin, and troponin protein molecules finds more and more support (Lazarides and Revel, 1979). In muscle fibers, these proteins fill sarcomeres, forming a network of parallel-running thin and thick filaments. In nonmuscle fibers, the contractile proteins form a less-ordered thin structure, which is a constituent of the cytoskeleton. Actin fibers have been separated from nonmuscle cells only in 1966. It has been established that these fibers have physical, chemical, and bio-
EXCI’I‘ONS A N D SOLITONS I N MOLECULAK SYSTEMS
209
logical properties identical with the properties of muscle actin molecules. A detailed picture of the arrangement of actin molecules in the cell was obtained by using the method of immunofluorescence. In the method of immunofluorescence, a molecule of fluorescent dye is attached to specially prepared antibodies linked selectively with a definite type of protein molecules. In the cell, an antibody with a fluorescent molecule dye is attached to the corresponding protein. When the cell is illuminated by ultraviolet radiation, the fluorescent dye radiates the light, which allows one to determine the arrangement of the protein molecules inside the cell. By means of immunofluorescence,it was discovered that actin molecules form long filaments (threads) (with the size close to that of the cell size across) with a thickness of about 60 A. One end of such fiber is attached directly to the outer (cytoplasmic) membrane. It was especially difficult to detect myosin filaments in nonmuscular cells. The myosin molecules in muscle fibers are arranged in thick bundles 150-170 A in diameter and 1.5 nm long. The myosin filaments in nonmuscular cells are shorter and thinner. They are also formed by myosin molecules which are arranged tail-to-tail (as in muscular fibers) so that their “heads” are placed at the ends of the filament. However, the diameter of the filament itself differs only slightly from the diameter of actin filament; therefore, using electron microscopes, one cannot distinguish them from actin filaments. Due to the low concentration of myosin molecules in the cytoplasm, they cannot be separated from cells. Thus, the idea that there are no myosin molecules in nonmuscular cells has been enhanced. Only the use of immunofluorescence has made it possible to establish the availability of myosin and tropomyosin in nonmuscular cells. The presence of contractile actin, myosin, and tropomyosin molecules in nonmuscular cells confirms the hypothesis of a general way of converting the chemical energy of ATP molecule hydrolysis into a mechanical motion. Such a process should be realized by a sliding mechanism, which differs in some details from that in muscular fibers (see Section VII). In the muscular fibers, thick filaments lie parallel to one another in the center of the sarcomere, forming a hexagonal structure at the distance of about 450 A from one another. One end of thin actin filaments (length of about I nm) is attached to Z plates, separating one sarcomere from another, and the second end enters the space between thick filaments in such a way that each thick filament is surrounded by six thin ones. In nonmuscular fibers, myosin filaments have no fixed position-they “tloat” in the cytoplasm, attaching to their ends (where the heads of myosin molecules are placed) the ATP molecules. If such a comparatively
210
A . S. DAVYDOV
short myosin filament turns to be near the actin filament then, in the presence of calcium ions, the ATP molecule hydrolysis occurs. The energy released is converted into soliton excitations, propagating from the filament ends to its center. The soliton motion is accompanied by the motion of swollen regions of thick filaments, which involve actin filaments attached to them. If near the myosin filament there are opposite ends of actin filaments attached to the outer membrane by the other ends, the soliton motion in a myosin filament results in the cell deformation (Fig. 12). Since helical sections constitute a considerable part of the protein structure of the cell cytoskeleton, they can transfer the energy and information from one part of the cell to another with the help of propagating soliton excitations. Transmembrane glycoproteins play an important role in the life activity of the cell. Glycoproteins represent a covalent combination of protein molecules with a ramified chain of carbohydrate residues-polysaccharides. The long protein fraction is in the a-helical conformation. It penetrates the entire thickness of the outer membrane of the cell to form the membrane channel. A ramified polysaccharide portion is on the cell surface. Some glycoproteins are closely connected with microfilaments and tubules of the cytoskeleton of the cell. Thus glycoproteins connect the inside and the outside of the cell (Fig. 13). The glycoproteins are crucial to the livelihood of the cell. They are implicated in cellular identity, cellular adhesion, intercellular communication, and transmembrane signaling. They can convey the signals that originate on the cellular exterior through the binding of hormones, neurons, immunoglobulins, and other molecules. Thus, nonlinear dynamics of solitons may provide a key for understanding the mechanism of transmission of information from the outside to the inside of the cell. A characteristic feature of this effect is its resonance nature. It is observed only in a narrow
FIG.12. Cytoskeletal filament system. Bipolar (double-headed) myosin molecules ( I ) bind to actin filaments ( 2 ) . having opposite polarity (small arrows). In the presence of ATP and calcium ions, the chemical energy of ATP can convert into contractive movement by creation of solitons in myosin molecules. Movement of the swollen actin filament anchors the filament to the inside of the cell membrane or some other organelle. The sliding of myosin and actin filaments past one another could then move two attachment sites closer together.
EXCITONS A N D SOLITONS IN MOLECULAR SYSTEMS
*///
21 I
cytoplasm inside the cell
FIG.13. Schematic diagram of the plasma membrane: I , hydrophobic segment of the ahelix protein: 2, hydrophilic segment of the a-helix protein: 3 . globular protein: 4, phospholipids: 5 . cholesterol: 6. oligosaccharide side chains; 7. glycolipid.
range of frequencies, from 0.05% to a few percentages of the medium frequency. Up to now, there are no sufficiently elaborated concepts on the nature of such an effect. Academician Devjatkov (1973) has noted “From the scientific viewpoint the explanation of the mechanism of resonance effect of radiation and also of the other radiation effects represents specific interest. But we still have no rigorous scientific foundation throwing light on the action of millimetre electromagnetic waves.” In a number of studies, Frohlich (1980) suggested that the sections of cellular membranes with incorporated protein molecules can be affected by radiation. According to Frohlich, in a membrane of about 0.01 mm thick, there can appear vibrations with the frequency of about 0.5 x 10” Hz. Since the membranes in the living cell are polarized, such vibrations can be excited by electromagnetic radiation. It is still an open question how such vibrations can influence the life activity of the cell. One of possible mechanisms of the resonance effect of electromagnetic radiation on living organisms was proposed by Eremko (1983, 1984) on the basis of the concept of an active participation of solitons in the vital activity of the cell. Solitons are ideal carriers of the ATP molecules hydrolysis energy along protein molecules. Under the influence of electromagnetic radiation, solitons disintegrate into rapidly relaxating excitons and a local deformation of the molecule. As a result of this process, the energy transfer efficiency decreases. Due to the Franck-Condon principle, during the optical transition accompanied by the soliton disintegration into an exciton and the local molecule deformation, the positions of heavy PGs do not change (Fig. 14). A vertical transition into the exciton state takes place with the wave num-
212
A . S . DAVYDOV
2q--&* 0
-A 0
Po
p
14. Soliton photodissociation. The dependence of the sum of exciton energy and local deformation ( I ) and soliton (2) on relative decrease (p) in interpeptide distance. Fici.
ber k = 0 under the fixed position of PG in the initial state characterized by the function in Eq. (18). In this case, the energy expenditure exceeds the soliton deformation binding energy of Eq. (24)
6E
=
x4/3u2a4J
by the value of the chain deformation energy W of photodissociation is determined by ho
=
36E
=
=
26E. Thus, the energy
x4/~?a4J
Using the parameters J = 7.8 cm-I, K = 19 Nlm, and x = (3.2 - 4) x lo-’’ N , we find that the resonance frequency corresponds to the radiation wavelength X (4.6-8.8 mm). This magnitude coincides qualitatively with the region of the wavelengths of electromagnetic radiation interacting actively with living organisms. Since in real protein molecules the energy transfer of the ATP molecule hydrolysis can be realized by three types of solitons, one symmetric and two asymmetric ones (see Section V), three resonance frequencies can be observed in the absorption spectrum. Two of them (corresponding to asymmetric solitons) slightly differ in their energy. Thus, the soliton representations can explain the qualitatively “enigmatic” nature of the resonance radiation effect on living organisms. Besides the electromagnetic radiation absorption, the process of photodissociation can manifest itself under the shift of laser-scattering frequencies. Phonons, expending a part of their energy to provide the soliton photodissociation, shift their frequency into the red region (Stokes scattering). Probably, it was just this process that had taken place in the work of Webb and Stoneham (1977) when they studied the laser Raman-scattering spectra by living bacteria. The authors observed the discrete shifted lines only when the bacteria were in the active metabolic state. These
EXCITONS AND SOLITONS IN MOLECULAR SYSTEMS
213
lines disappeared when the bacteria were in the inactive state. In the spectrum of Bucilliis megaterium bacteria, three shifted frequencies have been discovered: 5.7 x 10". 3.75 x lo", and 4.3 x 10" Hz. In the spectra of Escherichia coli in the range of 6 X 10 to 6.6 x 10" Hz, five frequencies have been observed. The differential cross sections of the Stokes component of the Raman scattering corresponding to the soliton photodissociation have been calculated using a simple model of one-dimensional molecular chain (Vakhnenko et ul., 1984).
IX. The Laser Raman Scattering by Metabolically Active Cells At the beginning of the 1980s, an attempt was made to study the laser Raman scattering by bacterial cells active metabolically (Webb, 1980; Bannikov and Rozhkov, 1980; Drissler and Santo, 1983). In Webb's paper ( 1980), nine resonant frequencies in anti-Stokes laser scattering by bacteria at 300 K have been observed only in the metabolically active state of bacteria. The ratio of the intensity of Stokes lines to anti-Stokes lines denoted that the Raman active state reflects some vibrational movements accompanying the activity of bacteria, rather than it is caused by thermal excitation. Under the radiation scattering, the energy of these vibrations is carried out by photons increasing, thus, their frequency. Independent of these experimental works, Scott (1981, 1982a, b) has calculated theoretically the soliton vibrations. He showed that the propagation of solitons along a-helical proteins is accompanied by the excitation transfer from one chain of PGs to another with the period of TI = 2 x lo-'' sec, and there also appears the modulation of motion with the period TI = 8/3 x 10- l 3 sec, which is caused by the discreteness of arrangement of PGs in the molecule. These periods correspond to the energies in c m - ' units [Ei = (cJTi)-'cm-'1 equal to E l = 17 cm-' and E2 = 125 cm-'. When comparing his own calculations with experimental data, Scott (1982a,b) found out that the observed anti-Stokes band frequencies are in a good agreement with the sums and differences of El and E2. The analysis of theoretical and experimental data led theorists and experimentators (Lomdahl et al., 1982)to the conclusion that the agreement between the calculated and measured frequencies of vibrations confirms the idea that solitons determine a possible mechanism of transport and storage of biological energy in protein molecules. It is important that these experimental and theoretical investigations have been performed independently and were compared only after they have been published. Al-
214
A . S. DAVYDOV
though the date reported by Webb (1980) had been confirmed by Drissler and Santo (1983),4 there was the suggestion that the observed spectral lines arose from the scanning of unstable preparation [See M. S. Cooper and N. M. (1983), Am. Phys. Lett. A 98, 138-140; L. Puria and 0. P. Gandhi (1984), Phys. Lett. A 102, 380-382; S. Kimoshita, H. Kumiko, and T. Kushida (1980), J . Phys. Jpn 43,3 14-321 .] Resolution of this question was considered to be so important that the Los Alamos scientists mounted a careful experiment using an optical multichannel analyzer to record the spectral data. Use of the optical multichannel analyzer eliminated the artifacts suggested by the above authors. The results of this experiment were negative; no lines of that sort, reported by Webb, were observed. Instead, it appeared that at least some of the lines reported by Webb could be assigned to transient fluorescence that occurs during cell division (Layne et a / . , 1985). The remarkable agreement between the spectral lines reported experimentally by Webb and those calculated numerically by Scott remains unexplained. X. Possible Mechanism for Anesthesia Molecules of a wide class of intravenous anesthetics form hydrogen bonds with protein molecules placed inside cytoplasmic membrane and outside of it. Such an attachment of anesthetic molecules to proteins inhibits the normal functioning of the cell. One possible inhibiting mechanism under the attachment of anesthetic molecules to proteins was proposed by Layne (1984) on the basis of the soliton model. The molecules of anesthetics contain the groups of atoms H, N , C, and 0 and very similar to the peptide groups involved in proteins. So, for example, the barbiturates molecule contains four such groups in its ring; the hydantoins molecule contains three and the glutethimides and succinimides molecules contain two. When a barbiturates molecule is attached to a protein molecule, the hydrogen bond between neighboring PGs in a protein molecule is weakened or even broken, since new hydrogen bonds of 0 and H atoms of a peptide group with the atoms of the HNCO group of barbiturates are formed (Fig. 15). When the hydrogen bond is ruptured (or weakened), the distance between the neighboring PGs in the protein molecule increases, which leads to the fact that a soliton propagating along the PG chain stops or 4Authors have noted that a highly efficient method of synchronization is required.
EXCITONS A N D SOLITONS IN MOLECULAR SYSTEMS
215
: 1
FIG. 15. The binding of a barbiturate changes the localized structure within the a-helix: normal spine; (2) spine distorted by barbituric acid-the hydrogen bond is weakened and its bond length is increased by the distance AR = 0.6 A; (3) barbiturate.
(I)
decelerates, and, consequently, the energy and information transfer along a protein molecule is violated. To compute the effect of the barbiturates molecule attachment to the &-helical protein molecule, Layne suggested that the distance between PGs in the region of attachment increases by 0.8 A, which corresponds to the decrease in the energy of hydrogen bond by 55%. In this case, due to the increasing in the distance between PGs, the energy of their resonance interaction (4 decreases by 40%. It has further been assumed that the elasticity coefficient (K) and the coupling parameter (x) decrease proportionally to the decrease in the energy of the hydrogen bond. In numerical calculations, the PG chain perturbation in the region of three PGs was taken into account. The numerical calculations have revealed the soliton to be well shaped before entering the perturbation region; however, having traversed through the perturbation band, it significantly degrades emitting phonons. These phonons move at the sound velocity exceeding the soliton velocity. Thus, Layne's numerical calculations show that the attachment of an anesthetic molecule to proteins decreases their ability to transfer the energy and information. Layne suggests that the decrease in efficiency of the energy and information transfer by solitons when barbiturates are attached to proteins would be important in two cellular structures: 1. in helical proteins of the inner mitochondria1 membrane, which participate in the ATP synthesis and electron transport, and 2. in the membrane proteins of neurons, which are responsible for
216
A. S. D A V Y D O V
chemical reception and signal transduction. Namely, the predominant parts of the protein molecules constitute the a-helix and are capable to bind barbiturates, which violate their normal functioning in the transport of information. energy, and electrons by solitons. Barbiturates exhibit their activity when the concentration is between 100 and 200 mM and when they are associated with about I% typical membrane proteins.
XI. Electron Transfer along Protein Molecules Many biological phenomena (photosynthesis, cell respiration, enzyme activity, etc.) are associated with the electron transfer from donor to acceptor molecules through molecular structures, which are usually called chains of electron transport. Such an electron transfer has commonly been implied to be caused by quantum-mechanical tunneling. The idea has first been proposed by De Vault et al. (1967). The most complete and detailed theoretical investigation of possible tunneling under the electron transfer between the components of the chain of the electron transport in chloroplasts and chromatophores was made by Grigorov and Chernavskii (1972) and by Blumenfeld and Chernavskii (1973). The tunneling mechanism for the electron transfer can be realized only at the distances not exceeding 10-12 A. However, in a number of works, the electron transfer at the distances of about 30-70 A was discovered. In this case, the centers between which an electron is transferred are separated by a large number of atoms and atomic groups. The electron transfer at such large distances is unlikely to be realized by simple tunneling. One of the possible explanations of the electron transfer at large distances is connected with the assumption that this process is facilitated by the participation of protein molecules situated between donor and acceptor electrons. The problem concerning the electron transfer along protein molecules has long been discussed in the literature. So, for example, Szent-Gyorgyi (1941) suggested that protein structures can possess semiconductor properties and have conduction bands through which electrons would migrate. This viewpoint has not been confirmed. From the data on the spectra of light absorption by protein molecules, it follows that the width of the forbidden band for the proper electrons of the molecule is not less than 4.8 eV. This rules out completely the possibility of the electron conduction of protein in dark reactions. Proteins are dielectrics rather than semiconductors.
EXCI’IONS A N D SOLITONS IN MOLECULAR SYSTEMS
217
Petrov o r r i l . (1978) emphasized the fact that, when the electron is transferred from donor to acceptor, it is necessary to consider not the proper electrons of a protein molecule, but the extra one, having been got in a protein molecule from a donor. Such an electron can get into the conduction band of the protein molecule without overcoming the forbidden energy band. The authors considered the electron motion along peptide bonds of the basic polypeptide chain of a protein molecule. The subbarrier electron transfer along a quasi-one-dimensional chain of monomers was also studied by Davydov (1972) without regard for displacements of the equilibrium positions of monomers. It was shown that, when a donor molecule is attached to the chain, the wave function of its outer electron is distributed along the chain decreasing exponentially. Consequently, there increases the probability for the electron transfer to an acceptor molecule attached to another end of the chain. The calculations performed arc valid only for rigid structures in which monomers are coupled by chemical bonds. The next step in the development of the model of donor-acceptor electron transfer through proteins was made by Ukrainskii and Mironov (1979), who have studied the electron transfer along the chains of peptide groups coupled by weak hydrogen forces in a-helical protein molecules. They considered that peptide groups, arranged in an a-helical protein molecule due to large constant dipole electric moments caused by asymmetric charge didribution in a group (Fig. 161, form, for an outer (excess) electron, their own conduction band. which lies below the conduction band of the moleciile electrons. However, the electron motion in this band was investigated in an adiabatic approximation taking into account only slight displacements of peptide groups from fixed equilibrium positions. The interaction of an electron with such displacements was considered, using linear equations by the perturbation method. This interaction leads to a significant deceleration of electrons.
Fic;. 16. The distrihution of electric charges on atoms of the peptide group.
218
A. S. D A V Y D O V
Under the strong coupling of an electron with monomer (PG) displacements, which is realized in the problems of motion of an excess electron along comparatively soft chains of PGs in protein molecules, the perturbation theory cannot be applied. The investigations performed by Davydov (l979a) have shown that the processes of an excess electron transfer along a-helical protein molecules are determined by their specific properties. Peptide groups due to asymmetric distribution of electric charges have a comparatively large constant electric dipole moment-about 4 D. Therefore, they form potential wells capable in retaining outer electrons entering the molecule from the donors. The energy of the bound state &, and the wave functions of an electron in the field of electric dipole were calculated in the work of Turner and Anderson (1968). The overlap of wave functions of an excess electron in the chain with periodically arranged dipole electric moments (Fig. 17) leads to the exchange interaction D,and the collectivization of electron states. The energy level totransforms into the energy band of the states for a quasi-particle with the effective mass m
=
h2/2a2D,
(27)
The state of the quasi-particle motion in the energy band called the conduction band (without consideration of displacements of PGs from their equilibrium positions) is described by a plane wave with the definite value of the wave number k = 2 d X . In the long-wave approximation, this state corresponds to the energy E&k) = EJO)
+ h2kk'/2m
(28)
The phase velocity in this state v = hk/2m depends on the wavelength A; consequently, there occurs dispersion, which is sensitive to the effective mass m. The energy of the conduction band bottom EJO) is less than that of the lowest level &, in an isolated well, due to the exchange interaction between the neighboring PGs EJO) =
to -
h2/ma2 =
to - 2 0 ,
(29)
FIG. 17. The arrangement of permanent electrical dipole moments in the peptide group of the a-helix protein molecule.
EXCITONS A N D SOLITONS IN MOLECULAR SYSTEMS
219
A quasi-particle in the energy band of Eq. (28) possesses an electron charge; however, its effective mass m can essentially differ from the mass of a free electron. In spite of this, the quasi-particle usually preserves its name-the electron. In general, the concept of rigid PG fixation in an a-helical protein molecule turns to be inconsistent due to a weak hydrogen bond between PGs. The strong quasi-particle (electron) interaction with the PG chain described by the deformation potential u leads to the change in intrapeptide distances in the region where a quasi-particle is located. Therefore, the energy states of a quasi-particle in the chain are of complicated character due to the interaction between the quasi-particle and the nonlocal deformation. It is impossible to describe these states by means of the perturbation theory. Theory without perturbation has been used by Davydov (1979~).It resulted in the fact that the electron movements were described by the system of nonlinear differential equations. It has been shown that such a system of nonlinear equations for the quasi-particle (extra electron) admits two types of states divided by the energy gap. 1. Unstable state of motion in the conduction band with the velocity exceeding that of a longitudinal sound in the chain. Such states are described by plane waves. Stipulated by the fast quasi-particle motion, the chain deformation fails to follow it; therefore, the PG equilibrium positions do not change. However, due to strong coupling with displacements, the electron generates sound waves and decelerates. 2. The soliton-like stable state with the energy disposed under the conduction band bottom of the quasi-particle. In this state, the quasi-particle is connected with the local chain deformation and moves with it is a whole with the velocity less than the sound velocity in the chain. The state stability is greater, the larger the parameter of the deformation potential u.
The energy gap between the states I and 2 is proportional to the fourth power of u. The larger u, the higher is the deceleration of the quasi-particle motion described by the plane wave in state 1. However, the larger u, the higher is the stability of the soliton state 2 with the energy under the conduction band bottom of a quasi-particle. The system of nonlinear equations characterizing stationary states of an extra electron in the PG chain with account of its interaction with the displacements of PGs equilibrium positions describes a relative decrease in distances between the neighboring peptide groups and the distribution of amide 1 vibrations along the chain. In the continuum, approximation i n a system of coordinates,
220
A. S. DAVYDOV
6 = i ’ ( x - x,, - v t ) moving with constant velocity v , the wave function of an extra electron in the PG chain has the form +(x, t ) =
@(t)exp[i(kx - Et/h)],
k
=
mv/h
(30)
Then the decrease in distances between the neighboring PGs (in units is characterized by the function
~ ( 6 )=
uQ2(6)/Mvi(l
-
N)
(30a)
s’)
proportional to the square of the electron overlapping wave function (30), satisfying the Schrodinger nonlinear equation
and the normalization condition
I
LIZ‘J
- LIZ0
W[)& =
I,
L >> a
(3 1 a)
where L is the chain length. In this equation, the nonlinearity coefficient of the energy degree is determined by the expression
G = U2/KU2(1
-
S’),
S =
V/V0
(32)
Here u is the parameter characterizing the electron binding energy with deformation, M is the peptide group mass, K is the parameter that characterizes the elastic properties of the chain. v,, = M < a is the longitudinal sound velocity in the chain. At the velocity v less than that of longitudinal sound v,,, the equation describes the electron motion accompanied by the local deformation. In this case @&)
=
a sech(q6)
pSol(~)=U@’([)/KU’( 1 -
(33) S’)
(34)
where q = ma’/2u?iZ(1 - s’)
(35)
The electron in the state described by the functions of Eqs. (33) and (34) will be called the electrosoliton. At v << v, the electrosoliton transfers along the chain, the electron electric charge, and the energy including that of the chain deformation
where E,(O) is the energy of the conduction band bottom of Eq. (29)
EXCITONS AND SOLITONS IN MOLECULAR SYSTEMS
22 I
A = (r4/48~'u4D,
(37)
The value A is determined by the energy released under the coupling of the electron and the local chain deformation. M,,, is the electrosoliton effective mass
M,,,
=
m[l
+ Mu4/6x2h'(l
- s2)]
It exceeds the mass m of a quasi-particle (electron). When moving, the electrosoliton loses no energy. The stability of electrosolitons as well as that of solitons corresponding to intrapeptide amide 1 vibrations is due to the intercompensation inherent in nonlinearity and dispersion effects. According to Eq. (371, the electron binding energy with the chain deformation is proportional to the fourth power of the deformation potential ((r) and is inversely proportional to the elastic coefficient (K) square. At the same time, the large values u stipulate the electrons deceleration in the conduction band of a quasi-particle due to the intensive phonon radiation. These dependences are analogous to the phenomenon of metalic superconduction. Metals in which electrons are weakly coupled with lattice deformation of ions (silver and copper) have good conductivity, but are not transformed into the superconducting state at low temperatures. Metals with strong coupling of electrons with lattice deformation (lead, etc.) are bad conductors in the normal state, but are transformed into the superconducting state at low temperatures. The exceptional electrosolitons stability makes a-helical sections of protein molecules the ideal guides for transporting electrons from donor to acceptor molecules. To the electron motion in the conduction band of a quasi-particle there corresponds the value s2 > I ; in this case, the nonlinearity coefficient of Eq. (32) has the negative value, and Eq. (31) has the solution @(t)= and the function of Eq. (30) is transformed into the plane wave
a,
+(x, t ) =
v'Xexp[i(kx
-
~t/h)]
In this state of motion, the probability for electron distribution along the chain is independent of coordinates, since I +(x, t ) I ' = d L . As has already been mentioned, such excitations are unstable.
XII. Electrosolitons Pairing in Soft Molecular Chains
I
If a pair of excess electrons gets into a section of a-helical protein molecule, then, as it has been demonstrated by Brizhik and Davydov (1984), electrons couple in the singlet spin state in the local deformation region
222
A . S. D A V Y D O V
made up by these electrons. The local deformation of the chain gives birth to attractive interaction between two electrons. Due to the nonlinearity effect, the potential well formed by one electron attracts another one with the opposite spin which, in turn, deepens the well. Suppose the dimensionless value g = mu’G/R characterizes the autolocalization of one electron so that the probability of its spacial distribution in the system of coordinates is determined by the square of an amplitude function
=
iG sech(igS)
(39)
Then, without allowance for the electrons coulomb repulsion, the amplitude function characterizing the spacial distribution of each electron in the pair is defined by @&)
=
sech(gS)
(40)
Comparing Eqs. (39) and (40), we see that in pairing the effective nonlinearity parameter is doubled, and the localization of each electron in the pair ( 2 d g ) decreases by a factor of two. The energy gain in pairing is determined by the expression E ~ ” ~ (=V ma4(I ) - Ss2)/4~*1i3u’(I-
2)
(41)
from which there follows that pairing is advantageous energetically only at small velocities. The paired solitons moving with constant velocity v << vo have the energy-involving that of the chain E,,(V) =
m[V’U4/3KZU’h2]-k
-$bundV’
Their effective mass Mbnd
=
2m(1
-k 2MU4/3K3h2U’)
exceeds more than twice the mass of an isolated electrosoliton of Eq. (38). Consequently, when velocity increases, the kinetic energy of the pair increases faster than the sum of energy of each soliton, which moves with the same velocity. Thus, electrosolitons pairing is stipulated by the electrons nonlinear coupling with the inertial local chain deformation. The solution to a Schrodinger nonlinear equation describing inertialess field with the selfaction contains no energetically bound states. The effect of electrons pairing in the singlet spin state manifests itself in some biological phenomena connected with the electron movements. So in redox reactions, the pairs of electrons are transferred from one mol-
EXCITONS AND SOLII‘ONS IN MOLECULAR SYSTEMS
223
ecule to another. In internal membranes of mitochondria and chloroplasts, under the ATP molecule synthesis, not a single electron is transferred, but the pairs of electrons are transferred (Hinkle and McCarty, 1978).
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Pauling, L.. and Corey. R. B. (1951). Proc,. Nrtrl. A c d . Sci. U . S . A . 37, 729-740. Pauling. L.. Corey. R. B.. and Hayward. R. (1954). Sci. A m . 191, 51. Petrov, E. G.. Ukrainskii. I. I..and Kharkyanen. V. N . (1978). J . 7%por. Biol. 73. 29-50. Porter. K. R.. and Tucker, J. B. (19x1). Sci. A m . 244(3), 40-51. Ragulskis. K. M., Bansevichus. R. J.. DidLgalvis. K. B . , and Ragulskiene, V. 1. (1976). “Theory o f Machines and Mechanisms.” Nauka. Moscow. Riemann, B. ( 1x92). “Uber die Firtpflanzung ebener Luftwellen von endlicher Schwingungsweite,” Gottingen Abhandlungen, Bd. VIII, p. 43. Werke. 2nd Edition. p. 157. Leipzig. Rowlands. S . (1982). J . B i d . Phys. 10, 199-200. Sagdeev. R. Z. (1958). / / I “Problems in Plasma Theory and Problem of Controlled Thermonuclear Reactions.” Actrd. Sci. USSR 4, 384 (in Russian). Scott. A. C. (1981). Pliys. Lett. A 86(1). 60-62. . A 26( I), 578-595. Scott. A. C. ( 19831).P / I ~ sRc\,. Scott. A. C. ( 1982b). P/iy.s. Scr. 25, 6S 1-658. Scott. A . C. (1982~).I r i “Modern Problems o f Solid and Biophysics.” pp. 176-179. Naukova Dumka. Kiev. Scott. A . C. ( 1983). / ! I “Structure and Dynamics. Nucleic Acid and Protein” (R.Clementy and K . Sarma, eds.). pp. 389-404. Adenine. New York. Scott-Russell. J. (1844). Report on Waves. / / I “Rep. 14th Meeting o f the British Assoc. for Advancement of Science.” London: John Murray. Scott-Russell. J. ( 1844). Report on Waves. Proc.. R. Soc.. Edinhitr~li.319-320. Szent-Gyiirgyi, A. (1941). N t r / r t r ~(London) 148, 157; Scieriw 93, 609. Turner. I. E.. and Anderson, V . E. (1968). Phvs. Re\,. 174, 81-89, Ukrainskii. 1. I..and Mironov. S. L. (1979). Prcpr. ITPAcud. Sci. Ukr. SSR. ITP-78-6lK3 Kiev. . ULr. Vakhnenko. A. A,, Gaididei. Yu. B., and Eremko, A. A. (1984). Prepr. I 7 P A c ~ r dSci. SSR. 1TP-84-13E. Kiev. Webb, S . J. (1980). P h y s . Rep. 60(4). 201-204. Webb. S. J.. and Stoneham, M . R. (1977). Pliys. Lett. A 60(3).267-268. Zabusky. N . J. (1967). Irr “Nonlinear Partial Differential Equations” (W. F. Ames. ed.). No. 4. pp. 222-258. Academic Press. New York. Zabusky. N . J . . and Kruskal, M. D. (1965). Pliys. RN. Lett. 15, 240-243. Zakharov. V. E., and Faddeev. L. D. (1971). Fitnct. A n d . I/.$ Appl. 3 4 ) . 18. Zakharov. V. E., and Shabat, A. B. (1974). Fr/rict. Anti/. I t s Appl. 8(3). 43. Zakharov. V. E.. Manakov. S. V.. Novikov. S. P.. and Pitaevskii, L. P. (1980). “Theory of Solitons. Inverse Problem Method.” Nauka. Moscow.
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INTERNATIONAL REVIEW OF C Y I O L O G Y . VOL.. loh
The Centrosome and Its Role in the Organization of Microtubules I . A.
VOROBJEV A N D
E.
s. NADEZHDINA
A. N . Bidoziwky Ltrhortitoty oj’Mokiwrlmr Biology Litid Bioorganic ChiJniistry, Moscow State University, M o s c m i 119899, USSR
I. Introduction The present review deals with the centriole and associated structures. We shall consider its tine structure and function in the cell. By the function of the centrosome, we imply the organization of microtubules (MTs), for no other functions have been detected as yet (even though they are presumed). A number of reviews have already been published on the subject (de Harven, 1968; Brown et al., 1983; Fulton, 1971; Mclntosh, 1983; Peterson and Berns. 1980; Pitelka, 1974; Stubblefield and Brinkley, 1967; Tucker, 1983; Wolfe, 1972) and even a monograph has appeared (Wheatley, 1982), along with a large number of reviews and mini reviews on contiguous problems, such as the role of MTs in cells, assembly of MTs and its regulation, etc. However, the past few years have yielded many important experimental data directly related to the topic indicated in the title. These data prompt us to query some of the earlier notions. We have tried to emphasize these new data by comparing them both with the well-known facts and with those which have received less attention, though they might have been obtained long ago. At present, various investigators use different terms to denote the same structures. So to avoid confusion, we shall define our terminology first. Pickett-Heaps (Pickett-Heaps, 1969) formulated the concept of a microtubule-organizing center (MTOC). According to this concept, MTOCs are the structures from which or on which MTs start their assembly. Subsequent studies have revealed that the centrioles and the structures surrounding them (the ccntrosome) operate as MTOCs (we shall not consider the role of kinetochores in the organization of MTs). Electron microscopy has shown that MTs entering the cell center may be attached there to electron-dense structures-the “microcenters.” The size of the microcenters correlates with that of the MTs. Therefore, Borisy and Could (1978) designated these microcenters as microtubule-nucleating centers (MTNCs). For clarity, we shall henceforth use these two terms: MTOC-a center, mainly observed in a light microscope by using the immunofluorescence 227 Copyright 0 1987 by Academic Press, Inc. All righla of reproduction in any form reberved.
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technique with antitubulin antibodies. It is the area of cytoplasm from which MTs grow. MTNC-an electron-dense body (usually too small to be visible in a light microscope) to which MTs are directly attached. We shall apply the term “centrosome” to centrioles and specific structures surrounding them (MTNC in particular) located in a definite region of the cytoplasm-devoid of ribosomes and membranes. The definition of “centriole” does not appear to cause controversy. The centriole is a cylindrical structure in the cytoplasm whose wall consists of MTs and interconnecting structures. A similar structure, lying near the plasma membrane and forming a kinocilium, is called a basal body. The main part of the centriole and basal body is the centriolar cylinder, the radially symmetric structure consisting of nine interconnected triplets of MTs. But it must be stressed that the centriolar cylinder is not an equivalent of the centriole or the basal body, because the latter two organelles may contain some other important structures (sattelites, connectives, etc.) We shall use the term “pericentriolar satellite” the way Bessis (1964) understood it; he was the first to describe satellites as dense “massules” attached to the centriole. It would be premature to define other centriolerelated structures; their description will be given later. The centrosome can organize MTs in four different patterns (Fulton. 1971):as a mitotic spindle, as an interphase network, as axonemes (ciliary or flagellar), or as a new centriole (basal body). We shall concentrate below on the role of the centrosome for assembly of MTs in mitosis and in interphase, since it is in this field that the greatest progress has been made in the last few years. Other completely different functions are attributed to centrioles: perception of external signals (Albrecht-Buehler, 1981), regulation of cell metabolism (as a “pacemaker”) (Bornens, 1979), etc. These speculations may contain some rational grains, but, at the present time, their discussion appears premature to us due to the lack of experimental data for or against them. Yet it becomes clear that the role of centrosomes in the cell is not limited to a mere formation of MTs. Organizing the MT network, the centrosome is involved in creating a spatially organized asymmetric form of the cell (Solomon, 1980; Tucker, 1983; Mitchison and Kirschner, 1984a). Centrioles perform other functions (to be considered in the final section) that can hardly be related to MT organization. It is not accidental that the centriole has been named as the central enigma of cell biology (Wheatley, 1982). We have tried our best to adhere to facts only with a minimum of speculations. Our review considers a variety of subjects: the ultrastructure of centrioles and MTNCs, the genesis of centrioles and basal bodies in cells,
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the biochemistry of centrioles, its role in the assembly of MTs, and its properties as an particularly independent organelle. 11. Ultrastructure of Centrioles and Basal Bodies
The fine structure of centrioles and basal bodies was described in the 1950s, at the height of electron microscopic investigations of cell organelles (de Harven and Bernhard, 1956; Burgos and Fawcett, 1956; Bessis and Breton-Gorius, 1957; Yamasa, 1956; Amano, 1957; Fawcett and Porter, 1954; Manton et al., 1956). As it became clear from the very first works, the ultrastructure of these organelles is quite similar: this fact confirmed the Henneguy and Lenhossek hypothesis on their homology. New circumstantial and substantive descriptions appeared in the 1960s (reviews: de Harven, 1968; Fulton, 1971; Wolfe, 19721, after the introduction of aldehyde fixation (Sabatini et al., 1963). Centrioles proved to be relatively constant in form and size: they are cylindrical structures, about 0.2 pm in diameter and 0.2-0.5 pm in length. Basal bodies have the same diameter as centrioles, but they are somewhat longer. In some cases (spermatids of insects), a basal body may be as long as 8 pm (Friedlander and Wahrman, 1970). Most of the information on the fine structure of both centrioles and basal bodies may be obtained from the ultrathin sections perpendicular to the centriolar cylinder axis (Fig. 1). In this case, the structure itself turns to be radially symmetric (it has a symmetry axis of the ninth order), which makes it possible to obtain rotation images according to the method of Markham and co-workers (Markham et d.,1963). These images show the radial-symmetrical structures in greater contrast against the nonsymmetrical details of the negative. The first three-dimensional centriole model based on rotation images was suggested in 1967 (Stubblefield and Brinkley, 1967).The reconstruction of a basal body on the basis of a complete series of cross-sections was accomplished in 1972 (Anderson, 1972) (Fig. 21, and a similar reconstruction of the centriole was accomplished in 1980 (Vorobjev and Chentsov, 1980) (Fig. 3). The ultrastructure of centrioles and basal bodies abounds in minute details; unfortunately, these details have received no comprehensive and systematic description. Here we shall not enumerate all the observations bearing on the subtle structural features of centrioles and basal bodies, for this in the main has been done in previous reviews (Stubbletield and Brinkley. 1967; Fulton, 1971; Wolfe, 19721, but rather we will consider an overall centriolar structural model and structural distinctions of centrioles and basal bodies.
FIG. I . (A-C) Serial sections of the centriole in an interphase pig embryo (PE) cell. Sections 1, 5 , and 6 (first, proximal; sixth, distal end). ap. Appendages; con, connectives (A-C links); h, hub; rl, ring of links (A-A links);tb, triplet bases; st. pericentriolar satellite. Bar, 50 nm.
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FIG.2. Model of the basal body. (Reproduced from The Jorrrnd c$Ce// Eicdogy, 1972. 54, 260 by copyright permission of the Rockefeller University Press and courtesy o f R. G . W. Anderson.)
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A FIG.3. Model of the centriole from a mammalian cell. (From Vorobjev and C h e n t w v . 1980.) ( A ) Three-dimentional reconstruction. (B-D) Cross sections of proxinial. middle. and
distal parts of the centriole, respectively.
Already Bernhard and de Harven (1960) demonstrated that the centriole is a polar structure, i.e., it has nonequivalent ends. A daughter centriole is formed at one end, while a ciliary axoneme grows from the other. Since in basal bodies the end from which the cilium grows is oriented to the outer surface of the cell, it has been named distal. In centrioles, the distal end is one from which a stereocilium or a primary cilium grows. Correspondingly, the end where the founding of a daughter centriole takes place is named proximal. The plus ends of centriolar microtubules are at the distal end of the centriolar cylinder (Snell r t d., 1974). A three-dimensional reconstruction of centrioles and basal bodies revealed differences in their fine structure along the cylinder; these differences followed a regular pattern from one end to the other (Stubblefield and Brinkley. 1967: Anderson, 1972; Vorobjev and Chentsov. 1980). A general description of the centriolar
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ultrastructure in the cells of vertebrates proceeding mainly from the data of Vorobjev and Chentsov (1980) follows. The list of other references is given at the end of the description. The most conspicuous component of the centriole is nine MT triplets stretching along the full length of the centriolar cylinder. The slope of the triplets to the radius decreases from 70-80" to 50-55" from the distal on to the proximal end; the diameter of the internal lumen of the centriolar cylinder does not change, and its external diameter at the proximal end increases somewhat. The middle and outer microtubules of the triplets may have an incomplete wall at the distal end, and the outer MTs may not be present at all. The data in the literature on centrioles with double MT are usually based on photographs obtained from sections of the centriolar cylinder's distal part. The triplets of MTs are interlinked by different kinds of connectives: in the proximal part, these are the connectives between the inner and outer MTs of neighboring triplets, and in the distal part, they form a continuous ring linking the inner MTs of all the triplets. The photographic images of the system of connectives may create an illusion of a fourth MT adjacent to the triplet, which is not the case. In the lumen of the centriolar cylinder closely to its distal end, is found an amorphous hub. This hub is attached by its short protrusions to the ring of connectives linking the triplets. The protrusions are not organized in a strictly symmetric pattern. At the distal end ofthe centriolar cylinder, the hub is replaced by accumulations of electron-dense material. This material may be girdled by a 5-nm-thick fiber in the form of an incomplete circumference. Sometimes, the hub also occupies the distal part of the centrioles. In the proximal part, the lumen of the centriolar cylinder is usually free of the electron-dense material. The proximal part of the cylinder has triplet bases located along the triplets, inside the cylinder; in the middle part and at the distal end, there remain only columns of electron-dense material at the junctions of the inner and middle MTs of the neighboring triplets. Along the full length of the centriole, the triplets are surrounded from the outside by a "rim" of amorphous material. the matrix. The matrix is most clearly distinct in the proximal part of the centriole. The matrix is a constant centriolar Component persisting during the whole cell cycle. It differs from the mitotic halo by higher electron density and smaller diameter. There exists yet another structure, which may be located in the luman of the centriolar cylinder, in its proximal part. This is the pinwheel (for a detailed description, see Section 111). In contrast to the amorphous hub. the pinwheel is a strictly radial-symmetric structure, and it has adorned
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many rotation photographs. Yet in vertebrate cell centrioles, the pinwheel occurs only at their initial formative stages and disappears later. It may remain in mature organelles in the fungi and mosses, and in some invertebrates, insects in particular. The distal end of the centriole has appendages attached to the triplets from the outside. The appendages are not an obligatory centriolar component (thus they may be absent in some of Chinese hamster cell lines), but they are common for most cell types. Their position does not follow a completely regular pattern with respect to the axis of symmetry, and therefore, appendages are weakly contrasted on rotation images. Should the centriole form a stereocilium or a primary cilium, its appendages are practically symmetric, the way they are in basal bodies (see below). Immature daughter centrioles do not have appendages (see Sections 111 and IV), and so only one of the two centrioles in an interphase cell carries them, while the other does not. [Triplet MTs have been described by Ringo (1967). Ross (1968), Brinkley and Stubblefield (1970), Heath and Greenwood (1970), Kiefer (1970), Wolfe (19701, and McNitt (1974). The hub, triplet bases, and triplet interconnectives have been described by Dahl(1963), Murray et al. (1965), Dingle and Fulton (1966), Stubblefield and Brinkley (l967), de Harven (l968), Perkins (l970), Konishi et al. (l973), and Rattner and Phillips (1973). Appendages have been described by Cachet and Thiery (1964), Murray et af. (196% Doolin and Birge (1966), and Stubblefield and Brinkley (1967).] Besides the structures listed above, one may come across others, i.e., nonobligatory ones, in the lumen of the centriolar cylinder; these are the membrane-bound vesicle or dense granule (Sorokin, 1962; Doolin and Birge, 1966; Vorobjev and Chentsov, 1977). In these cases, the contents of even two centrioles in a cell are different. Unfortunately, the fine structure of centrioles has been studied only for a small number of plant and animal species and more often than not. the data relate to spermatogenic cells alone, where the ultrastructure may exhibit considerable aberrations (see below). Yet, even n from the available scant evidence, it is clear that the centriolar structure may vary for different species. The known distinctions in the centriolar structure may pertain to the structure (the length of the amorphous hub, the length of the unclosed part of triplets of microtubules, to certain details in the organization of the system of connectives, the presence or absence of appendages) of the centriolar cylinder’s distal part. The structure of the cylinder’s proximal part is more conserved. There exist two variants only, with an empty lumen or with a pinwheel connected by the radial spokes to the triplets. It should be emphasized once again that the basic centriolar structure, a cylinder composed of 9 MT triplets is the most universal one, and ab-
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errations are exceptionally rare. Centrioles, composed of 60-80 single MTs, were described for spermatogenic cells of the fly Sciuru (Phillips, 1967) and so were the centrioles and basal bodies of the coccids Eirnerci intrstitiu/i.s and Eirneru mugnu, consisting of nine single MTs (Chesin. 1967). Nevertheless, somatic cells of many plants, fungi, and of some animals may contain MTOCs other than centrioles. Usually these are balls, disks. or plates of electron-dense material (Friedlander and Wahrman. 1970; Zickler. 1970: Aist and Williams, 1972; Franke and Reau, 1973; McDonald t>t d . , 1977; Gifford and Larson, 1980; Dave and Godward, 1982). Zoospores or spermatozoa with flagella in such species may have centrioles (basal bodies) of usual structure (Renaud and Shift, 1964; Reichle, 1969; Dingle and Fulton, 1966). If flagella or cilia are never formed, e.g., in angiosperms, diatoms, or higher fungi, then the centrioles are not observed. Pickett-Heaps (1969) suggested that the centriolar cylinder is only a template for casting the axonemes of cilia and flagella, i.e., it is a potential basal body incorporated within MTOC for equivalent distribution among daughter cells in mitosis. In a way, the Pickett-Heaps’ hypothesis develops the Henneguy and Lenhossek suggestion on the homology of centriole and basal bodies (Henneguy, 1898; Lenhossek, 1898). Indeed, centrioles in vertebrate cells may form a primary cilia. Such cilia are partially or completely immersed in the cytoplasm; they may be several micrometers long or quite short. Only one centriole of the pair (the mother centriole) is capable to form a cilium (Bernhard and de Harven. 1960; Sorokin, 1962; Albrecht-Buehler and Bushnell, 1979; Rieder and Borisy, 1982). A cross section of primary cilia may show a random disposition of MTs, even though they may form something like type 9 + 0 axonemes (Jensen et al., 1979; Albrecht-Buehler and Bushnell, 1979; Vorobjev and Chentsov, 1982). At first, these cilia were considered a curiosity belonging to certain cell types (Barnes, 1961; Sorokin, 1962; Dahl, 1963; Doolin and Birge, 1966; Allenspach and Roth, 1967; Dingemans. 1969; Rash et d., 1969; Wheatley, 1969; Fonte et u/., 1971; Chung and Keefer, 1976). Yet it is clear now that this phenomenon is a rule rather than an exception especially for cultured cells. The primary cilia disappear from cultured cells before mitosis or at its beginning (Fonte et a / . , 1971; Archer and Wheatley. 1971; Rieder el d.,1979; Tucker et ( I / . , 1979a). The disappearance of primary cilia may occur immediately after the cell receives a proliferation signal (Tucker rt d . , 1979b; Lockwood and Pendergast, 1980). Probably, the primary cilia are most typical of cells in the G,,period. Centrioles are also responsible for the formation of single stereocilia of sensory cells (von Narnack, 1963; Flock and Duvall, 1965; Schmidt, 1969; Wolff, 1969; Tilney Pt a / . , 1980). In stereocilia as in primary cilia, axonemes may be significantly modified or may be absent altogether (Tilney et d . ,
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1980; Matera and Davis, 1982), but they may be of a regular structure as well (Wolff, 1969). As a stereocilium is formed, the pair of centrioles migrates to the cell surface; one (the mother centriole) attaches to the plasmalemma and forms an axoneme, while the other (the daughter centriole) lies nearby and has no axoneme (Bernhard and de Harven, 1960). In either case, the centrioles contact the membranes (the plasmalemma if a stereocilium is formed, and the intracellular vesicle if it is a primary cilium). Their appendages become more sturdy and symmetrical and are attached to the membrane (Vorobjev and Chentsov, 1982). Centrioles lying at the base of stereocilia are traditionally regarded as basal bodies, and this is a clear example for tracing the homology of these two organelles. Sometimes (for example in sea urchin blastomeres) the kinocilia are formed in the same way as stereocilia: their basal bodies simultaneously act as a pair of centrioles, which are the only MTOCs of these cells (Tilney and Goddard, 1970). Both centrioles and basal bodies are not the obligatory cell organelles. Although they are widespread in eukaryotes, there are organisms whose cells are devoid of them (angiosperms). In some cases, the cells may have only basal bodies and no centrioles (for example, Flagellata and Infusoria). Others, however, have only centrioles and no basal bodies (some fungi and mosses). Only vertebrates, insects, and echinoderms reliably have both these organelles. With animals, centrioles are present in most of the cells. Basal bodies are present only in special cases: in multiciliate cells of ciliated epithelia, in single-cilium sensory cells, and in spermatozoa. The centrosome restructuring during spermatozoon flagellum formation is an independent area of research to which a large number of workers have been devoted (reviews: Phillips, 1970; Danylova, 1982). Spermatozoa are the example of the cell’s uttermost specialization which applies to all the intracellular structures without exception. Changes of the centrioles in spermatozoa and the structure of the basal bodies thus obtained are highly variable and species specific. Frequently, the spermatozoon basal body is replaced by oddly packed electron-dense material (Phillips, 1970). The question is to what extent the changes in the basal bodies of spermatozoa are reversible and what happens to them after fertilization (in the zygote); we shall consider this in Section VIII. On the whole, however, we shall try not to refer to works on spermatogenesis and spermatozoa because of the highly specific nature of the subject. So, the homology and structural similarity of centrioles and basal bodies may be of practical interest only in the case of ciliated epithelia of different animals (vertebrates and invertebrates). Basal body triplets of MTs, unlike centriolar ones, are hardly detectable
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on cross-sections simultaneously. To explain this phenomenon, it was suggested that the triplets are coiled axially in the centriolar cylinder (Gibbons, 1961; Fawcett. 1966; Phillips, 1970; Anderson, 1972). As in the centriole, the lumen of the basal body cylinder may have an amorphous hub at the distal end (Reese, 1965; Anderson, 1972). or a pinwheel at the proximal end (Friedlander and Wahrman, 1970), but it does not have membranebound vesicles and a fine fiber in the distal part, which are often described in centrioles. The system of triplet connectives has not been studied in basal bodies in such detail as in centrioles. The total length of the basal body exceeds, as a rule, that of the centriole (Wolfe, 1972). To some extent, this is due to a special transition zone, the necklace, which, in basal bodies, is located at the distal end after the appendages (Thornhill, 1967; Gilula and Satir, 1972). Only doublets remain in the transition zone instead of the triplets of MTs (the triplet's outer MT is absent); these doublets are arranged strictly in a circle. Each one is attached to the plasmalemma via a system of connectives. A basal plate is found in kinocilia, above the transition zone, to separate the basal body from the axonema (Perkins and Amon, 1969; Wolfe, 1972; Rubin and Cunningham, 1973). The basal body appendages (often designated in the literature as transition fibers) diverge from the outer surface of triplets and are attached to the membrane (Szollosi, 1964; Reese, 1965; Anderson, 1972). They are arranged strictly symmetrically around the centriolar cylinder of the basal body and are similar in size to the appendages of centrioles (the basal body appendages may be somewhat larger). In the proximal part, striated rootlets diverge from the basal body (one or several in different cell types). Each rootlet is a bundle of thin (about 5 nm) fibers with transverse periodic striations. In multicellular animal cells, the striation period of the rootlets is equal to 60-70 nrn (Dahl, 1963; von Narnack, 1963; Doolin and Birge, 1966; Matsusaka. 1967; Schmidt, 1969; Olson and Rattner, 1975; Stephens, 1973, and in Protozoa, the striation period of the rootlets is equal to 12-78 nm (Dingle and Fulton, 1966; Dingle and Larson, 1981; Simpson and Dingle, 1971; Brown et d., 1976; Amos et a / . , 1979; Larson and Dingle, 1981; Salisbury, 1982):Striated rootlets around centrioles have been described as well (Sakaguchi, 1965; Loweryns and Boussaw, 1973; Wachmann and Hennig, 1974; Vorobjev and Chentsov, 1977). They are much thinner than those of basal bodies; as a rule, they are not directly linked to the centriolar cylinder. On the contrary, the basal bodies are immured into a rootlet by their proximal end (Anderson, 1972). The number of pericentriolar rootlets varies; there may be as many as 10 and more per centriole (Loweryns and Boussaw, 1973).
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In many cells, the centrioles are surrounded by dense masses attached to them, i.e., the pericentriolar satellites (Bessis et a l . , 1958; Bernhard and de Harven, 1960; David-Ferreira, 1962; Bessis, 1964; Vorobjev and Chentsov, 1977). Satellite counts on serial cross sections of centrioles in axolotl hemopoietic tissue cells point to the variable number of satellites (from two to thirteen, and more often, from four to seven); they may be located along the full length of the centriolar cylinder (Vorobjev and Chentsov, 1977). Tissue culture cells contain fewer satellites (one to five per centriole), but their number varies as well (Vorobjev and Chentsov, 1982). The satellites are composed of a head which serves as a MT-nucleating center (MTNC), and of a foot, fixing the head to the centriolar cylinder (Bessis et al., 1958; Dalcq, 1964; de The, 1964; Szollosi, 1964; Vorobjev and Chentsov, 1977). The satellite foot is conical in shape, and it often exhibits transverse striation. The satellite is attached to two or three triplets of the centriolar MTs (Szollosi, 1964; Vorobjev and Chentsov, 1977).
Satellites are found in basal bodies as well. Commonly designated as basal feet, they have the same structure as the centriolar satellites. The basal bodies and the centrioles differ in the number of satellites. Most of the investigated basal bodies in ciliated epithelia contain only a single satellite (Fawcett and Porter, 1954; Gibbons, 1961; Szollosi. 1964; Anderson, 1972; Afzelius, 1980). The basal body satellites are positioned in a definite manner-in the cilium effective stroke plane (Gibbons, 1961; Afzelius, 1980). Thus, all the satellites are oriented in the same direction within a cell (Gibbons. 1961; Afzelius, 1980). Under diseases resulting in immotile cilia syndrome, the satellites of ciliated epithelium cells show random disposition (Afzelius, 1980). So, unlike the centrioles, the basal bodies have a bilateral symmetry, sometimes, a functionally conditioned one. Unfortunately, no comparative studies have been made on the structure of centrioles and basal bodies taken from the same object. Nor is it known how the structure of a centriole forming a single cilium changes (besides the changes in the position of appendages). The available data on the ultrastructure of centrioles and basal bodies enable us to make the following important conclusion: in the cells of multicellular animals, the basal body is an organelle with a more narrow specialization than that of the centriole. The centriole may have all the basal body structures, but the variability of the centriolar structure within one and the same organism is higher than that of the basal bodies. So, we conclude that the centriole in the cells of multicellular animals is a primary structure capable of different functional restructurings, while the basal body is just the result of one of them. This presumption is largely at var-
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iance with the Pickett-Heaps’ hypothesis about centriolar “passivity.” (Pickett-Heaps. 1969). It should be noted that in Protozoa, too, the organization of basal bodies is rigidly determined to a much greater extent than the organization of the centriolar apparatus of multicellular organisms. Possibly, the rigid organization of basal bodies is due to the necessity of spatial orientation of kinocilial beating. The ultrastructure of centrioles and basal bodies may reveal mutant deviations. Thus, in certain lines of Chinese hamster cells, the centrioles lack some of the triplets (McGill el al., 1976; Vorobjev, 1979); triplets of MTs, different in length, are found in M I 0 green monkey cells (1. A. Vorobjev, personal communication), etc. Yet, no centriolar structure in mutant cell lines have been obtained so far. Chlamydomonas mutants may exhibit motility disturbances and changes in the connectives linking the basal bodies, but they reveal no changes in the basal bodies themselves (Wright et al., 1983). So, mutations in the centriolar structure are either lethal or affect its functions and thus cannot be analyzed.
Ill. The Ontogenesis of Basal Bodies and Centrioles The principal stages of the formation of centrioles and basal bodies are much similar for different groups of eukaryotes. New centriolar cylinders are formed in four modes: I . Next to the preexisting centrioles, - end-to-end and in direct contact with them (Heath and Greenwood, 1970; Moser and Kreitner, 1970; Heath, 1974); 2. Near and perpendicular to the lateral surface of the mother centriole or the basal body (Bernhard and de Harven, 1960; Gall, 1961; Murray et d.,1965; Sorokin, 1968; Allen, 1969; Millecchia and Rudzinska, 1970, etc.). Although this process is traditionally named replication, it has nothing in common with DNA replication; 3. Around the aggregations of fibrogranular material, the deuterosomes (Stockinger and Cireli, 1965; Dirksen and Crocker, 1966; Steinman. 1968; Sorokin. 1968; Dirksen, 1971) or blepharoplasts (Mizukami and Gall, 1%6). Both, in the second and in the third case, the daughter structures (procentrioles) seems not to be directly linked either to the mother centrioles or to the deuterosomes; 4. Finally, centrioles may be formed de novo, when the nascent centriolar cylinders are not associated with any special intracellular structures.
Dr now formation of centrioles has been described for sea urchin eggs both for parthenogenesis and for artificially activated eggs (Dirksen, 1961;
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Koichi and Masao, 1971 ; Kato and Sugiyama, 1971; Miki-Noumurd, 1977; Kallenbach and Mazia, 1982), and it has also been described for dividing mouse blastomeres (to a stage of 8-16 blastomeres) (Szollosi rt d.,1972). Probably, it takes place under karyoplast regeneration (Zorn rt (11.. 1979). An attempt at a more in-depth analysis into the finding of centrioles during their de n o w formation yielded no definite result (Kallenbach, 19831, i.e.. it was not possible to identify procentrioles at their early formative stages. The second mode applies to centriole and basal body formation in the cells of both multicellular animals and Protozoa; in some cases, the basal bodies may be formed by the third mode (in multiciliate cells as a rule). Ciliogenesis in multiciliate cells enables one to trace the formation of a multitude of basal bodies. In each cell, there are many centriolar cylinders at different formative stages; therefore, this process is easily and sufficiently well investigated (Reese, 1965; Steinman, 1968; Sorokin, 1968; Kalnins and Porter, 1969; Anderson and Brenner, 1971; Jirsova et d., 1974) (Fig. 4). At the earliest discernible stage, the basal bodies are shaped like short cylinders, 65-120 nm long and about 160 nm in diameter. The walls of the cylinders are composed of amorphous substance, and the inner lumen of the cylinder is equal to 80-85 nm. A cartwheel, about 30 nm in diameter with nine symmetric pins, appears in the lumen. Longitudinal sections show that the pins are located in rows, one over another. Then MTs appear at the pin ends (the inner or the A tubules of future triplets), parallel to the central axis. As the number of the MTs reaches nine (according to the number of pins), middle (B) and outer (C) tubules of the triplets are added in succession from the outside. Connectives are formed between the A and the C tubules of neighboring triplets. At this stage, the pin ends are interconnected by a solid electron-dense ring. The triplets in the juvenile basal body are oriented practically radially (Fig. 5). Later they turn around to form an angle of about 50 with the radius. Then the basal body cylinder is elongated, mainly at the expense of the growth of the triplet microtubules. The cartwheel grows to reach half of the length of a normal basal body (or even less). N o cartwheel is present, as a rule, in the mature basal bodies of multiciliate cells (see Wolfe, 1972, for review), but it may occur fairly often in basal bodies of the Protozoa (Allen, 1969; Tamm, 1972; Wright et al., 1979). The ultrastructure of replicating centrioles has been investigated in one work only (Vorobjev and Chentsov, 1982) (Fig. 5). On the whole, the centriolar foundation and growth resemble that of the basal bodies with the exception that the incipient stages, i.e., the amorphous cylinder and the cartwheel without MTs, are not detectable. The minimal centriolar structure includes both the cartwheel and the single or double MTs of future triplets attached to the pin ends. Such a procentriole is about 100
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24 I
FIG.4. A diagrammatic interpretation of the development of the basal body. (a) Earliest stage of formation. Neither the annulus, nor the cartwheel is completed. (b) The cartwheel is complete, and the first A tubule is completely initiated. (c)The triplet tubules are growing. The tubules tend to be progressively less complete from the base to the apex. (Reproduced from 7 7 1 Jorrrncrl ~ c~f’CdlBio/ogy, 1971. 50, 18, 22, 26 by copyright permission of the Rockefeller University Press and courtesy of R. G. W. Anderson.)
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FIG.5. Cross section of the procentriole. a, Axis; sp. spokes. Note incomplete triplets of MTs attached to the ends of the spokes (arrowheads). Bar, 50 nm.
nm in length and about 150 nm in diameter. The MTs are not quite symmetric about the central axis. Subsequently, complete MT triplets are formed, grow, and thereby cause centriolar cylinder elongation. The cartwheel grows to reach only half of the centriolar length at most. During the initial stages, the triplets are located almost on a radius, but then they turn through at an angle of 55-70'. The cartwheel disappears in a mature daughter centriole. The MT triplets become interconnected immediately upon their formation. Yet, the final system of triplet connectives takes shape only as the cartwheel has disappeared. The formation of mature basal bodies is completed upon their departure from the mother centriole (basal body) or from the deuterosome (Reese, 1965; Sorokin, 1968; Anderson and Brenner, 1971). Likewise, complete maturation of the centrioles occurs as the daughter centriole departs from the mother centriole (after the disruption of the diplosome) in a cell cycle subsequent to its foundation (Vorobjev and Chentsov, 1982; Rieder and Borisy, 1982). Thus, centriole and basal body formation is composed of three stages: foundation of the axial structure and MT triplets on it; elongation of the
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triplets and formation of a centriolar cylinder; and final restructuring of the centriolar cylinder, corresponding to the functional maturation of the organelle. Centriolar maturation consists of some structural changes. First, the cartwheel in the proximal part of the cylinder disappears and the amorphous hub appears in the distal part. The absence of a ring of connectives and the presence of a cartwheel are certain indications that the centriole is immature (in vertebrate cells). Yet in invertebrate cells, it may persist even in a mature centriole (Lutz and Huebner, 1980). Second, connectives between the inner MTs of the triplets are formed. Third, appendages appear at the distal end of the centriole. Prior to that, the daughter centrioles have only outer projections, ribs, along the triplets in the distal part. The appendages appear during the centriole’s second mitosis (Vorobjev and Chentsov. 1982). And finally, as we have noted above, the mature centriole is surrounded by MTNCs, i.e., pericentriolar satellites, mitotic halo, and free microtubular convergence foci. The immature centriole has few, if any, such structures around it. Centriole formation by the end-to-end replication mode has been described for some of the fungi and mosses (Heath and Greenwood, 1970; Moser and Kreitner, 1970; Heath, 1974). At the initial stage and in this case, the cartwheel outgrows the mother centriole. Then, at the pin ends, triplets of MTs of the daughter centriole are formed. However, the triplets of the mother and daughter centrioles are coiled in different directions, so the entire diplosome shows central symmetry. The triplets become elongated and form a centriolar cylinder. The cartwheel does not grow. As a result, the daughter centriole thus formed is connected with the mother via a common axis. The two centrioles diverge only as the cell prepares for subsequent division. In all the cases described, the radial symmetry of the centriolar cylinder is determined at its earliest formative stage: the cartwheel has a symmetry of the ninth order. The MTs formed at the pin ends may not be quite symmetric at first. The triplets become linked not only with the pins but with each other as well, and the structure of the centriolar cylinder attains to strict symmetry. It is suggested that the cartwheel symmetry is determined, in turn, by some finer structure within the wheel (Satir and Satir, 1964); but there is no evidence in favor of this suggestion. The morphological similarity of nascent centrioles and basal bodies does not mean, however, that the processes of their formation are set off and controlled by some common mechanisms. Quite the contrary, the regulation of centriolar replication is cardinally different from that of ciliogenesis. It has been proved that the duplication (replication) of centrioles sets
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is in the S period, though not necessarily at its beginning (Robbins et al., 1968; Wheatley, 1968; Erlandson and de Harven, 1971; Rattner and Phillips, 1973; Vorobjev and Chentsov, 1982). Replication of the two centrioles in a cell is strictly simultaneous, and so it may be touched off by a signal from a common source. Of major significance in this connection would be data on centriolar duplication in cells with multiple centrioles, but such data are poorly represented in the literature: we have found only two descriptions of possible simultaneous centriolar replication in young megakaryocytes and in regenerating liver cells (Wheatley, 1968; MoskvinTarkhanov and Onischenko, 1978). Elongation of the daughter centrioles is completed in mitosis, and their divergence takes place at the beginning of interphase. Complete maturation includes the whole subsequent cell cycle (Vorobjev and Chentsov, 1982; Rieder and Borisy, 1982). In cells retiring from the cell cycle in the G, period, no maturation of the daughter centriole occurs, i.e., it does not form appendages and can form no cilia (Vorobjev and Chentsov, 1982). Ciliogenesis in the monkey and rabbit oviduct is hormone induced (Anderson and Brenner, 1971; Anderson, 1974) and thus does not directly depend on the cell cycle. Replication of basal bodies in Protozoa is not coupled to DNA synthesis also (Younger et al., 1972). The basal body formation process is asynchronous within a single cell, i.e., one can frequently see basal bodies at different formative stages (Reese, 1965; Sorokin, 1968; Kalnins and Porter, 1%9; Anderson and Brenner, 1971). Thus centriolar replication is closely related to the cell mitotic cycle, while basal body formation is only related to cell differentiation. Unfortunately, we do not know whether the typical centrioles and basal bodies can coexist in one cell. Mammalian ciliated epithelium in which the cell multiplication zone is separated from the ciliary formation zone in space or in time, e.g., in the trachea or the oviduct, may not have such cells; earthworm olfactory epithelium has no centrioles in the ciliated cells (Rhein et al., 1981). Yet, it has been proved that ciliated cells of the epithelia of certain invertebrates can enter the mitotic cycle, i.e., they incorporate [3H]thymidine, and mitotic cells are found among them (Kaganovskaya, 1976; Punin, 1981; Zavarsin et al., 1984). Centrioles and basal bodies ought to coexist in such cells, and replication of both is of undeniable interest for study. IV. The Organization of the Centrosome and Its Behavior in a Cell Cycle
We shall consider centrosome dynamics in the cell cycle by beginning with metaphase, i.e., the key moment when one morphologically integral unit divides into two separate cells. In mitosis, the centrosome forms mi-
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totic spindle poles. In metaphase, each pole has a pair of mutually perpendicular centrioles, i.e., the diplosome (Robbins and Gonatas, 1964; Krishan and Buck, 1965; Murray et al., 1965; Roos, 1973). The mother and the daughter centrioles are clearly distinct in the diplosome (Murray et al., 1965; Robbins et al., 1968). The mother centriole has both ends free and is surrounded laterally by fine fibrillar material, the halo, which becomes particularly distinct upon detergent treatment of the cells. The halo is 150-200 nm wide, and the diameter of its fibers fluctuates between 3 and 8 nm. Most of the fibers are about 5 nm thick (Vorobjev and Chentsov, 1982). The second daughter centriole has its proximal end immersed in the halo. This centriole is at the proximal end of the mother centriole, perpendicular to its axis. The halo is a convergence focus of spindle MTs. In some cases, the MTs, radiating from the halo, may get into the lumen of the daughter centriole (Vorobjev and Chentsov, 1982). In metaphase, the daughter centriole is equal in length to the mother centriole or may be slightly shorter (Robbins and Gonatas, 1964; Krishan and Buck, 1965; Roos, 1973); the fine structure of the two centrioles is different (Vorobjev and Chentsov, 1982; see Section 111). In some cell lines (most frequently, in Chinese hamster cells), spherical electron-dense bodies, otherwise known as pericentriolar satellites, may surround the diplosome (Starosvetskaya, 1969; Gould and Borisy, 1977; the Peterson and Berns review, 1980; Rieder and Borisy, 1982). But, since these structures are not attached to the centrioles and do not make contact with the MTs, it would be wrong, we believe, to designate them as satellites. The chemical nature of the bodies is obscure. It is presumed they may be viral particles in some cases (Wheatley, 1974). In metaphase, the location of the mother centrioles in pig embryo (PE) cells and in Chinese hamster cells, as well as in PE cells in anaphase, follows a regular pattern, i.e., the centrioles are predominantly perpendicular to the spindle axis and this orientation is statistically reliable (Vorobjev and Chentsov, 1982). The daughter centrioles are scattered at random with respect to the spindle. The mother and the daughter centrioles retain their mutually perpendicular position throughout mitosis until mid telophase (Robbins and Gonatas, 1964; Krishan and Buck, 1965; Murray et al., 1965; Allenspach and Roth, 1967; Robbins and Jentzsch, 1969; Roos, 1973; Ates and Sentein, 1977; Vorobjev and Chentsov, 1982). According to some data, the fibrillar halo may be lost in anaphase (Robbins and Gonatas, 1964; Robbins and Jentzsch, 1969), or it may persist to telophase (Roos, 1973; Vorobjev and Chentsov, 1982). In all the cases, the number of MTs radiating from the mother centrioles successively decreases in the second half of mitosis, and only few MTs remain at the pole in telophase (Robbins and Gonatas,
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VOROBJEV A N D E. S. NADEZHDINA
1964; Robbins and Jentzsch, 1969; Ates and Sentein, 1977; Vorobjev and Chentsov, 1982). The centrosome undergoes a profound restructuring during cell transition into interphase. First, the mitotic halo disappears, and the two centrioles retain only a thin rim of electron-dense material (the centriolar matrix), which surrounds them during the entire cell cycle and must be a structural component of the centrioles. Second, the mother and the daughter centrioles lose their mutually perpendicular orientation and the diplosome disintegrates. This may also occur late in the telophase (Rattner and Phillips, 1973). Moreover, the two centrioles may depart from each other as far as 2-3 p m (Rattner and Phillips, 1973; Vorobjev and Chentsov, 1982). After the initial departure of the centrioles early in the G, period, they come back together and, prior to mitosis, do not lie farther than I pm from each other. Thus, a single centrosome, comprising the two centrioles and the surrounding structures, is formed in most interphase cells. Yet the mutual disposition of the two centrioles in interphase has nothing to do with their disposition in mitosis, when a diplosome exists. In interphase cells, the two centrioles are not perpendicular to each other as a rule (Vorobjev and Chentsov, 1977; Gudima et al., 1983a,b; de The, 1964; Schaffer, 1969; Albrecht-Buehler and Bushnell, 1980; Rieder and Borisy, 1982) (Fig. 6). Pericentriolar satellites appear on the mother centriole. The heads of the satellites function as MTNCs. In addition to the satellites in interphase, many cell lines (3T3, CHO, PtK2, and L), as well as hemopoietic tissue cells and blood cells, contain dense convergence foci of microtubules not attached to the centrioles; these are so-called free MTNCs (Rattner and Phillips, 1973; Vorobjev and Chentsov, 1977; the McIntosh review, 1983). Both the satellites and the free MTNCs persist during most of the interphase and disappear as the cells prepare for mitosis (Erlandson and de Harven, 1971; Vorobjev and Chentsov, 1982). The other centriole usually has no satellites or other MTNCs in interphase; the mother centriole has several times more microtubules around it. Consequently, only one of the two centrioles is active in interphase in the same way as it is in mitosis. Dalcq (1964) was the first to point out the difference between the two centrioles in an interphase cell. One had ignored this distinction for a long time until a close study of the centriolar maturation process during the cell cycle made it possible to explain the phenomenon: the centrioles differ in interphase because one of them (the daughter centriole) has no appendages at this time. Its structure becomes completely identical only in the next mitosis (in proliferating cells).' The absence of appendages may ac'The differences between the two centrioles may or may not persist in nonproliferating cells (like neurons). This question needs special study.
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FIG. 6. Scheme of the centrosome in interphase mammalian cell. m, Mother (active) centriole with pericentriolar satellites; d. daughter (inactive) centriole; b. electron-dense bodies; s, striated rootlet. MTs radiate in the main from satellites and free MT convergence foci. Some M’I’ may be attached to the centriolar matrix.
count for the fact that the daughter centriole does not form a cilium (Vorobjev and Chentsov, 1982). The doubling (replication) of centrioles takes place in the S period. This fact, independently verified in a number of mammalian cell cultures, has in effect been proved (Robbins ef al., 1968; Erlandson and de Harven. 1971 ; Rattner and Phillips, 1973; Vorobjev and Chentsov, 1982). However, a more delicate question has yet to be answered: at what moment of the S period does centriolar replication set in? Probably, this event takes place at different times in different cell types. The finding of procentrioles is synchronous at both centrioles. They are found near the proximal end of the mother centrioles in direct contact with their matrix. These two, now mother, centrioles preserve their structural and functional differences in the S period and early in the G, period (Vorobjev and Chentsov, 1982; Rieder and Borisy, 1982): only one of them carries appendages and is capable of forming a cilium. This centriole also has pericentriolar satellites (though this does not apply to all the cell types). As the cell prepares for division, centrosome restructuring takes place (in the G, period). The pericentriolar satellites disappear and so do most of the MTs, radiating from the cell center. This happens in the middle of the G, period (Vorobjev and Chentsov, 1982). At the end of the G, period, numerous small membrane vesicles appear around the centrioles, and their number reaches a maximum in the late prophase. The vesicles disappear rapidly in prophase-prometaphase transition (Robbins et al., 1968; Vorobjev and Chentsov, 1982). Late in the G , period, MTs radiate from both mother centrioles and asters appear. The MTs are attached directly to the
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centriolar matrix. The matrix rim becomes larger, and it transforms into the mitotic halo, surrounding the mother centrioles. The well-defined mitotic halo is observed in late prophase. The daughter centrioles elongates during the G, period, and they may attain up to Y4 of the length of the mother centrioles toward the end of this period. The elongation of the daughter centrioles is completed in mid or late mitosis (Krishan and Buck, 1965; Stubblefield and Brinkley, 1967; Vorobjev and Chentsov, 1982). As shown recently, the structural differences between the centrioles of a dividing cell may persist as late as anaphase, i.e., only one centriole has appendages, while the other has no appendages (Vorobjev and Chentsov, 1982). So, the cell always has only one completely developed (mature) centriole. The centriolar formation process, beginning from the foundation, thus takes a 1 '/z cell cycle (Vorobjev and Chentsov, 1982) (Fig. 7). Consequently, in typical mammalian cells, MTOCs consist of a pair of centrioles surrounded by MTNC structures. In mitosis, there is one such structure, the mitotic halo. The pattern is different in interphase. In some cells, the centriolar surfaces, the matrix, act as interphase MTNCs; in others, the matrix is supplemented with pericentriolar satellites. Furthermore, MTOCs may include MTNCs without structural links to the centrioles. These MTNCs resemble dense granules or small balls, 30-70 nm in diameter (Stubblefield and Brinkley, 1967; Starosvetskaya and Kazanjev, 1977; Peterson and Berns, 1980). In other cells, the balls are interlinked by connectives of less dense substances (de The, 1964; Vorobjev and Chentsov, 1977, 1983). In either case, the MTs approach the balls only; and in the case involving satellites, the microtubules approach the satellite head. Finally, clouds of electron-dense fibrillar material with radiating MTs may be found in the centriolar region (Gould and Borisy, 1977; Schliwa et al., 1978, 1979). The latter case is most typical of fish melanophores in which as many as loo0 MTs may radiate from one MTOC (Schliwa et al., 1978). Cells without centrioles but with basal bodies may exhibit a highly diversified MTNC structure. The basal body satellite heads act as MTNCs (Tilney and Goddard, 1970) and so do the connectives between the basal bodies and their rootlets (Bouck and Brown, 1973; Hyams and Chasey, 1974; Wright et al., 1979; Roobol et al., 1982). In Protozoa, a pair of basal bodies may carry several rootlet-type MTNCs, with a definite number of microtubules radiating from each (Bouck and Brown, 1973; Brown et ul., 1976; Wright et al., 1979). In cells without basal bodies, various plates, disks, or electron-dense balls operate as MTOCs (reviews. Raykov, 1978; Heath, 1980). In all the cases, MTOCs undergo corresponding restructuring in the mitotic cycle.
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FIG. 7. Generd scheme of the centriole cycle in PE cells ( A ) Metatelophase, ( B ) the beginning of interphase. (C) G , period. (D)second half of the S period to the first half of the G , period. and ( E ) the end of the G, period to prophase,. (From The, Jorrrirtrl of’ Cell B i d ~ g . 1982, ~ , 93, 949 by copyright permission of the Rockefeller University Press.)
V. The Biochemistry of Centrioles and Basal Bodies
The present knowledge of the centriolar structure at the molecular level is strikingly poor, especially compared to what has been achieved with respect to such cell components as ribosomes or mitochondria. We do not even know the rough composition of centriolar proteins and MTNC material, let alone their molecular structure. We know very little about the chemistry of basal bodies, though the first steps in this direction have already been made. The reason for this lag lies to some extent in the insufficient attention given to the problem. Yet researchers confront formidable objective difficulties as well. The critical stage in the study of the biochemistry of any cell organelle is its isolation as an individual fraction. This stage has not yet been reached for centrioles, and only a few research teams have succeeded in obtaining basal body preparations suitable for biochemical in-
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vestigations (Snell et al., 1974; Gould, 1975; Anderson, 1977; Stearns and Brown, 1979; Anderson and Floyd, 1980). To isolate some component as a fraction, one begins by selecting an adequate model object, i.e., one readily available for experimental use and containing the required component in a sufficiently high concentration. Such objects are the heart muscle (for mitochondria) or the brain (for microtubules). After the isolation procedures have been practiced on the model object, it becomes possible to obtain the desired fraction from other sources as well. Yet this technique is not good for centrioles. The main obstacle for centriole isolation is that normal cells have only two centrioles. They make up about 0.01% of the average cell volume. So even if the centriolar yield is loo%, large quantities of the starting material are needed for obtaining a sufficient amount of centriolar fraction. No model objects have been found for centrioles as yet. Neuroblastoma-a cell culture with multiple centrioles-appears to hold promise in this respect (Sharp et al., 1981) and recently a centriolar preparation has been obtained from it (Ring et al., 1980; Mitchison and Kirschner, 1984a). To test fractions of isolated cell components, one determines the activity of marker enzymes or specific biological activity; one may also perform direct analyses to detect specific proteins or nucleic acids, etc. None of these procedures was suitable for detecting centrioles. Electron microscopy remained for a long time the only method for assaying the centriolar content in preparations. It is a labor-consuming procedure, which requires comparatively large amounts of material. This drawback made it difficult to apply various linear gradients or chromatographies for isolating centrioles. Certain headway has been made by using specific autoimmune antibodies against centrioles, i.e., in this case, it becomes possible to detect centrioles in fractions by immunocytochemical procedures (Maro and Bornens, 1980; Ring et al., 1980; Mitchison and Kirschner, 1984a). There is also some progress in evolving the assay for biological activity of centriole assembly of MTs in an asterlike pattern (see Section VI). Only a few communications on enriched centriolar preparations have been published (Blackburn et al., 1978; Nadezhdina et al., 1979; Mar0 and Bornens, 1980; Mitchison and Kirschner, 1984a); yet no pure preparation is available. All we have said about centriolar isolation pertains in a way to basal bodies. Yet, there is a good object for isolating basal bodies, i.e., the unicellular flagellated algae. Basal bodies make up a significant part of their small cells. Pure preparations of basal bodies were isolated from Chlamydomonas (Snell et al., 1974; Gould, 1975) and from Polytomella (Stearns et al., 1976). Spermatozoa seem to be likewise appropriate for basal body isolation, but preparations obtained from them are not free of
THE CENTROSOME AND MICROTUBULE ORGANIZATION
25 1
contaminations (Maller er al., 1976; Ishikawa er al., 1979; Esponda and A h a , 1983). A major difficulty in isolating both centrioles and basal bodies is to separate the required organelles from the surrounding fibrillar structures. In the case of centrioles, these are mainly the intermediate filaments; in the case of basal bodies, these are mainly a complex of striated rootlets, linking fibers, and special connectives. It is only after several years of purposeful work that Anderson and Floyd succeeded in isolating a pure basal body fraction from ciliated epitchelium of rabbit oviduct (Anderson, 1974, 1977; Anderson and Floyd, 1980). No pure basal bodies have been isolated from Infusoria, though some crude preparations of cortex were obtained (Hufnagel, 1969; Gavin. 1980, etc.). The tight connections of centrioles and basal bodies with filaments may be useful at the initial stages of isolation. In softly homogenized cells, the centrioles remain bound to the nuclei (via the fibrillar structures, see also Section VIII) and may be isolated in a complex (Bornens, 1977; Nadezhdina et al., 1978; Mar0 and Bornens, 1980). The centriolenucleus complex can be used as an intermediate stage in centriolar purification, since in effect, all other cytoplasmic structures can be removed during its isolation. Furthermore, the centrioles can be separated from the nuclei upon additional homogenization and centrifugation (Nadezhdina er d.,1978, 1979; Maro and Bornens, 1980). In a similar way, cortex preparations of multiciliate cells may be an intermediate step in isolating basal bodies (Anderson, 1977). On the other hand, in order to avoid mechanical homogenization leading to disruption of MTNC substance, Ring et al. (1980) and Mitchison and Kirschner (1984a) proposed to lyse the cells in a very lowionic-strength solution. Such lysis releases the centrioles into the solution. Thus, concerning centriole and basal body isolation, we may conclude that 15-min centrifugation at 20,000 g is necessary to precipitate these organoids. Their buoyant density is fairly high and equals about 1.30 g/ cm3, which excludes the possibility of equilibrium density centrifugation in sucrose solution for their purification. Yet, purification is possible by means of short-term centrifugation ( I hour at 70,000-100,000 g ) in a 55% layer of sucrose gradient. Centrioles and basal bodies are not destroyed by EDTA and Triton X-100, and so these substances can be used to remove chromatin and cell membrane components. Centrioles may be highly sensitive to proteases (Snyder, 1980; Kuriyama, 1984), and hence it is recommended that they be isolated in the presence of protease inhibitors, phenylmethylsulfonyl fluoride (PMSF) in particular. A homogenization regime should likewise be carefully selected. Thus, isolation procedures for centrioles and basal bodies involve centrifugation in various sucrose gradients.
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Since the MTNC fibrogranular material is labile to mechanical destruction and is readily detached from the centrioles in cell homogenization (even if it is gentle homogenization), it has not been possible to obtain its preparation so far, and the prospects appear to be bleak. It seems that Mitchison and Kirschner (1984a) alone have succeeded in obtaining MTNC-bound centriolar fractions by dispensing with all mechanical homogenization procedures. Proceeding from direct assays of basal body preparations and from the data of histochemistry, immunocytochemistry, autoradiography , as well as cell inhibitor effects, it has been established that the centrosome consists predominantly of protein; the presence of nucleic acids (RNAs) is not excluded either. Yet in speaking of the biochemical composition of centrioles, it should be kept in mind that the ultrastructure of these organelles is an extremely complex one (see Sections I1 and III), and different structural elements may perform completely different functions. So it is essential to find out not only what kind of protein is in the whole composition of the centrioles, but also where this protein is localized. It has been shown that in salt solutions (NaCI and KCl) over 0.2 M and at an acid or alkaline pH in vitro, selective destruction of centriolar microtubules and destruction of basal bodies takes place (Nadezhdina et d . , 1980; Gavin, 1977, 1980). The matrix layer and certain cylinder lumen structures forming a characteristic centriolar “rim” remain intact (Nadezhdina et al., 1980). This indicates that the MTs and the matrix are comparatively independent elements of centrioles and basal bodies, which is in good agreement with the different functions of these elements: the MTs of centrioles and basal bodies organize a ciliary axoneme, while the cytoplasmic microtubules may radiate from the matrix. It has been found recently that the cartwheel, an obligatory structure of juvenile centrioles, is capable of self-assembly in vitro from a homogenate of Tetrahymena basal bodies (Gavin, 1984). Tetrahymena basal bodies were dissolved in 1 M KCI. On dialyzing the solution against a diluted KCI solution, cartwheel-type structures were reconstructed in it. Possibly, the cartwheel is yet another independent structure of centrioles and basal bodies required for their assembly only. A direct analysis of basal body and centriole preparations by SDSPAGE has shown them to contain dozens of polypeptides (Wolfe, 1972; Gould, 1975; Anderson and Floyd, 1980; Cavin, 1984). Tubulin is found in significant amounts among basal body proteins: it has the same electrophoretic motility and divides into a- and p-subunits as the tubulin of cytoplasmic MTs or cilia (Wolfe, 1972; Gould, 1975; Anderson and Floyd, 1980). This tubulin is capable of assembling into MTs at about the same conditions as other types of tubulin. It can bind to colchicine in such a
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way that M colchicine completely blocks the formation of basal bodies during mass ciliogenesis in frog embryo (Steinman, 1970).Colchicine and other MT poisons have no effect on the basal bodies, an added proof that their MTs, like those of axonemes, are not in equilibrium with the dissolved tubulin pool. Basal body MTs can be disassembled in concentrated salt solutions (Nadezhdina et al., 1980; Gavin, 1980, 1984) or by changing the pH of solutions. In this case, the basal MTs are less stable in high-ionicstrength solutions than the axoneme or the cortex MTs (Gavin, 1984, 1980). A detailed investigation of basal body tubulins has revealed that their peptide maps differ from those of ciliary tubulins, even though they have the same isoelectric point (Anderson and Floyd, 1980). The presence of tubulin in centrioles and basal bodies has also been demonstrated by immunofluorescent and immunoelectron staining by tubulin antibodies (Gordon et al., 1977; Mitchison and Kirschner, 1984a). Yet, these antibodies bind worse to centrioles and basal bodies than to other MT structures. Some explanation is that the centriolar cylinder MTs are covered with a matrix layer which may impede the access of the antibodies. It is more probable, however, that a set of antigenic determinants of tubulin of centrioles and basal bodies differs from that of other varieties of tubulin; this is quite consistent with their distinctions in peptide maps. It is obvious that centriolar MTs and basal bodies consist of tubulin. According to some authors, tubulin may be contained in the oligomeric form in MTNC material, notably, in the mitotic halo (Pepper and Brinkley, 1977, 1979; Dustin, 1983). Indeed, the halo can bind antitubulin antibodies. Under the reconstitution of a net of cell MTs destroyed by cold or colcemid, the immunofluorescent staining by antitubulin antibodies shows MTOC as a fluorescent ring (Bershadsky et al., 1979b), i.e., the antibodies bind to the MTNC material surrounding the centrioles rather than to the centrioles themselves. It is presumed that tubulin oligomers may serve as specific primers for the assembly of MTs on MTNC, and if so, this is a cytoplasmic, and not a centriolar, tubulin. On the other hand, even though it is presumed that MTNC should contain many MAPs (microtubular-associated proteins), antibodies against MAPs do not bind to MTOC as a rule (Kuznetsov et al., 1980; Thompson et al., 1983; Vallee and Bloom, 1983; Bloom et al., 1984), with the exception of the only sample of antibodies against protein MAP-I, which Sherline and Mascardo use for immunofluorescent staining of centrioles (Sherline and Mascardo, 1982a). No cilium ATPase (dynein) has been found in basal bodies (Anderson and Floyd, 1980). Yet an ATP cytochemical reaction reveals significant ATPase activity in centrioles and basal bodies (Matsusaka, 1967; Abel et al., 1972; Nayak, 1972; Dentler, 1977; Anderson, 1977; Ohta and Ishikawa,
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1979; Burnasheva and Solovjeva, 1980). At the electron microscopic level, the reaction product is localized in the centriolar cylinder matrix, in the appendages, in the striated rootlets, and around the centriolar microtubules (Matsusaka, 1967; Anderson, 1977; Burnasheva and Solovjeva, 1980). Anderson (1977) described this enzyme (ATPase) in basal body preparations isolated from rabbit oviducts. ATPase of basal bodies proved to be Ca2+-and Mg“-dependent, and the optimal Ca” concentration is 2 mM; 6 mM Ca2+inhibits the enzyme effect. The optimum pH for basal body ATPase is about 8.5. Dynein has somewhat different characteristics, i.e., it is not inhibited by Ca” and its optimum pH is about 10. The molecular weight and functions of centriolar and basal body ATPase are not yet known. Cytochemical investigations, as well as those involving monospecific antibodies, allowed the detection of purine phosphorylase in centrioles, i.e., an enzyme catalyzing the reversible reaction: purine nucleoside (guanosine, inosine, xanthosine) @I purine base + ribose-I-P(EC 2.4.2.1). In human leukocytes and fibroblasts, this enzyme is localized in small concentrations in the nucleus and throughout the cytoplasm with the exception of mitochondria. Its concentration is very significant in centrioles, both in interphase and in mitosis, though it is not clear how the centrioles are related to purine exchange (Oliver et al., 1981). Monospecifc (Welsh et al., 1978; Gordon et al., 1982) and some samples of monoclonal (Pardue et al., 1983; Deery et al., 1984) antibodies against calmodulin likewise bind to basal bodies and to centrioles, especially to the mitotic spindle poles. It was demonstrated that hamster trachea basal bodies contain calmodulin on the exterior surface of the triplets, i.e., in the matrix (Gordon et al., 1982). There can be no doubt that calmodulin may play a significant role in MTOC functioning: it is involved in Ca”dependent regulation of assembly and disassembly of MTs, so its presence in MTOC is no cause for surprise. Work is now in progress to obtain monoclonal antibodies against mitotic apparatus proteins. Clevenger and Epstein (1984) obtained antibodies against 100-kDa protein, localized in mitotic poles and in active chromatin of the interphase nucleus. Several other samples of monoclonal antibodies were obtained by Vandre and co-workers (1984a,b). In most cases, their antigens appeared to be phosphoproteins associated with spindle poles. These proteins are specifically phosphorylated in mitosis and probably participate in spindle MTs nucleation. Centrioles and basal bodies also contain a specific antigen, which no other cell components (notably, cytoplasmic MTs and cilia) have. In mammals, autoimmune antibodies are often generated against this antigen (Connoly and Kalnins, 1978; Maunory, 1979). The titer of such antibodies
+
-
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is particularly high in the blood of patients suffering from autoimmune diseases (Brenner and Brinkley, 1982; Tuffanelli et al., 1983; Hyams, 1984). The antigen is localized in the MTNC material (Maro and Bornens, 1980; Calarco-Gillam et al., 1983; Sauron et al., 1984) and must be either the 50- or the 75-kDa protein (Turksen et al., 1982; Sauron et al., 1984). This is an evolutionary conserved antigen, since antibodies against it are bound to centrioles and basal bodies from many objects. The antigen disappears from MTOC upon destruction of the pericentriolar material caused by superheating of the cells (Malawista et al., 1983). The basal bodies do not contain collagen, desmin, myosin, and actin (Anderson and Floyd, 1980). It is not so difficult to isolate striated rootlets bound to the basal bodies, i.e., they have a significant mass and are resistant to salt extractions (Stephens, 1975) and thus have been studied sufficiently well. In each of the investigated objects, the striated rootlets are composed of a protein specific to a given species. In mollusk gill epithelium, this protein (anchorin) has a molecular weight of 230-25OK (Stephens, 1979, about 250K in Tefruhymena (Williams et al., 19791, 170K in Naegleria gruberi (Larson and Dingle, 1981), 90K in Trichomonas (Amos et al., 1979), etc. So far, we have scant data on centrosome proteins. Even less is known about other possible components of the centrosome. Many investigators are certain that the centrosome contains nucleic acids, for formation of new centrioles (see Section 111) is much similar to template-guided replication, a process which must involve nucleic acids. In the course of the first investigations to isolate basal bodies (largely from Infusoria pellicles), significant amounts of DNA were detected by direct assay (Child and Mazia, 1956), by autoradiography (Smith-Sonneborn and Plaut, 1%9), and by acridine orange staining (Smith-Sonneborn and Plaut, 1967; Randall and Disbrey, 1965). Yet it was natural that less DNA was detected as the purity of the preparations increased. Finally, a thorough investigation revealed that the DNA of Infusoria pellicles belongs to the nuclear, mitochondrial, and bacterial fractions (the latter is from the bacterial feed for Infusoria) and has no fractions which could be basal body DNA proper (Hufnagel, 1969; Flavell and Jones, 1971). Inhibitors of DNA synthesis, such as fluorodesoxyuridine, arabinosylcytosine, and amethopterin, have no effect on the foundation of new centrioles in tissue-culture cells (Rattner and Phillips, 1973; De Foor and Stubblefield, 1974), while ethidium bromide, which suppresses DNA synthesis in Stentor, does not inhibit formation of new basal bodies (Younger et al., 1972). Even a prolonged effect of DNase causes no changes in the ultrastructure of basal bodies from Paramecium (Dippell, 1976). DNase treatment of lysed cells has no effect on the assembly of MTs from exogenous tubulin on the mitotic halo (Pepper and Brinkley, 1980).
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Thus, there is no evidence to support DNA presence within centrioles, basal bodies, or in the mitotic halo. Yet there are quite a few facts in favor of RNA there, though it is too early to draw final conclusions. RNA is always found in basal body preparations obtained from various objects, and its amount tends to increase as thorough purification procedures are applied (Hoffman, 1965; Hartman et al., 1974; Heidemann et al., 1977). Unfortunately, no published data are available on RNA in purest basal body preparations, isolated in recent investigations (Anderson and Floyd, 1980). Heidemann et al. (1977) determined 2-8 x g of RNA per basal body in a sufficiently pure preparation of basal bodies from Chlamydomonas. Tetrahymena pellicles, isolated in the presence of a detergent, were found to contain RNA which, hybridized with the cell DNA, is 35% noncompetitive with the ribosomal RNA, does not contain 4 S RNA, and has a sedimentation constant of 17 to 25 S (Hartman et al., 1974). An inhibitor of RNA synthesis, actinomycin D, blocked the finding of centrioles in tissue culture cells at 4 pg/ml (De Foor and Stubblefield, 1974) and the formation of new mitotic centers in dividing sea urchin zygotes (Went, 1977). Upon RNase treatment, Chlamydomonas basal bodies lost the ability to induce cytasters in nonfertilized Xenopus eggs (Heidemann et al., 1977), while basal bodies of mouse spermatozoa have a diminished capacity to induce assembly of MTs in vitro (Esponda and A h a , 1983). Considerable data have been accumulated on the presence of RNA in the mitotic halo. In newt tissue-culture cells, mitotic halo staining by the method of Bernhard, i.e., by uranyl acetate-EDTA on EM preparations, indicated RNA (Rieder, 1979). In order to obtain a truly positive staining, it was necessary to make use of the “thick,” instead of the conventional ultrathin, sections with subsequent observing in a I-MV EM. This positive staining (according to the Bernhard method) was removed by pretreatment with RNase (Rieder, 1979). In other investigations, the mitotic halo was shown to be reduced upon RNase treatment (Pepper and Brinkley, 1980), with the assembly of exogenous tubulin on it being strongly inhibited (Snyder, 1980). To verify the latter case, special assays were performed to exclude the possible protease effect on the halo, and various RNases were tested. It should be noticed, however, that Kuriyama (1984) denies that RNase has an inhibitory effect upon MT assembly on the mitotic halo. Berns and co-workers obtained interesting data on RNA role in the mitotic halo in experiments involving laser microbeam study of living cells (Peterson and Berns, 1978; Berns et al., 1977). If, prior to the microbeam study, the cells were treated with a dye which specifically binds to RNA,
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i.e., psoralen (4-aminomethyl-4,5,8-trimethylpsoralen), and the prophasic or prometaphasic spindle pole was irradiated by a beam with the wavelength corresponding to the dye absorption maximum, destructive changes would take place in the halo. The migration of the chromosomes to the irradiated pole in anaphase would be inhibited, and near the irradiated pole, there would be few, if any, MTs (Peterson and Berns, 1978).If another psoralen, binding to DNA but not to RNA, is used or if the pole is exposed to radiation in the same dose without prior staining of the cell, no changes in the halo would occur, and mitosis would proceed normally. Thereby some damages of the centriolar cylinder may be observed; but they exert no effect on mitosis (Peterson and Berns, 1978). Similar evidence was reported by Berns et al. (1976,1977)and Berns and Richardson (1977) in earlier studies when cells were treated (sensitized) by acridine orange instead of psoralen. The role of the centriolar RNA is quite obscure. Its amount is sufficient for coding several dozens of polypeptides. However, should we suppose that centriolar RNA is a centriolar genome, we should also suppose that RNA-dependent DNA polymerase or RNA replicase is present in the centriole. Another possibility is that the centriolar RNA may serve as a structural matrix, be it only at the early stages of centriole foundation, the way it is in the ribosome. The same role could be attributed to RNA in the mitotic halo as well, i.e., it may operate as a matrix in organizing the proteins which subsequently participate in the assembly or anchoring of MTs. Yet, the cell RNA inhibits MT polymerization in vitro (Zackroff et al., 1976; Meza et al., 1975). The mechanism of MT assembly on the centers must be a highly complex one and demands careful study. VI. Assembly of Microtubules on Microtubule-Organizing Centers (MTOCs) in Vitn,
Organization of a MT system is perhaps the most accessible function of the centrosome for recent methods of research. An asterlike growth of MTs from the centriolar region may be observed in living cells in mitosis or after the removal of MT poisons. We shall consider this question in Section VII. An “asterlike” assembly of MTs on the cell centers can be reproduced in vitro with the exogenous MT proteins and centrioles of detergent-permeabilized cells (McGill and Brinkley, 1975;Snyder and McIntosh, 1975; Gould and Borisy, 1977,etc.) or with preparations of isolated basal bodies and centrioles (Snell et al., 1974;Stearns and Brown, 1979;Telzer and Rosenbaum, 1979;Mitchison and Kirschner, 1984a;Kuriyama, 1984).Be-
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fore describing these experiments, it would be advisable to give a general view on cytoplasmic MTs and their properties, specifically, those which make an asterlike assembly on the centers possible. Tubulin, an essential protein for microtubular wall construction (Herzog and Weber, 1977; Rodionov et al., 1978; Stephens, 19821, constitutes 80% of the MT composition (Mohri, 1968). The other 20% is under MT-associated proteins (MAPS),a highly heterogeneous group of proteins with a molecular weight from 20 to 310 (Dentler et al., 1975; Murphy and Borisy, 1975; Cleveland et al., 1977; Berkowitz et al., 1977; Runge et al., 1979; Black and Kurdyla, 1983). A remarkable property of cytoplasmic MTs is their lability exhibited both in cells and in isolated preparations: the MTs may disassemble into subunits and then repolymerize from them. Axonemal and centriolar MTs are stable; they polymerize once and for all. Investigations in several laboratories on MTs from mammalian brain (a traditional object of biochemical research) revealed that, in the presence of MAPS, the optimal conditions for polymerization are as follows: the ionic strength should not exceed 100 mM, the pH should range from 6.7 to 6.9, the Mg2+concentration should range from 0.01 to 1 mM. the Ca2+ concentration should not exceed not above 0.01 mM, and the presence of nucleosidetriphosphates is also required (preferably GTP). The temperature interval of the reaction should be between 25 and 42°C. Under these conditions, the minimal protein concentration for polymerization should be lo-' mg/ml; it shows variations with different authors. The reaction of MT assembly proceeds in two stages: at first, high molecular primers, the tubulin oligomers, are formed, followed by elongation of the MTs from these primers. Formation of the primers is the slowest, rate-limiting step of the assembly (Bordas et al., 1983). The elongation proceeds until the MTs and the dissolved tubulin are in equilibrium, i.e., steady-state conditions. If the MAP fraction is removed from the MT preparation by ion-exchange chromatography, tubulin may still be polymerized into MTs, though the assembly requirements become more rigorous. For instance, it is possible to achieve polymerization of purified tubulin by increasing its concentration in the solution to 10 mg/ml (Sloboda and Rosenbaum, 1978) or by adding MT-stabilizing substances, such as sucrose, glycerol, polyethyleneglycol, or dimethylsulfoxide (Himes et al., 1977; Herzog and Weber, 1977, 1978), to the incubation mixture or by returning some of the MAP fractions (Cleveland et al., 1977; Kuznetsov et al., 1978). Purified tubulin may also be polymerized on exogenous stabile primers, for instance, on axonemal fragments (Allen and Borisy, 1974; Bergen and Borisy, 1980) or on prepolymerized MTs (Carlier et al., 1984; Mitchison and Kirschner, 1984b).
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Investigations of MT assembly on exogenous stable or labile MTs showed that MTs are polar structures: assembly of tubulin at one end of the MT proceeds faster than at the other end (Allen and Borisy, 1974; Dentler et al., 1974; Olmsted et al., 1974; Binder et al., 1975). In addition, there are such concentrations of tubulin when its polymerization takes places only at one end of the stable primer (Olmsted et al., 1974; Binder et al., 1975; Bergen et al., 1980). A thorough investigation of polymerization and depolymerization kinetics likewise confirmed the fact that the two MT ends are different: in labile MTs, the tubulin molecule association constant is higher at the plus end than at the minus end (Margolis and Wilson, 1978; Kirschner, 1980) (Fig. 8). Blockage of either end may cause drastic changes in the MT properties in the assembly systems (Bergen and Borisy, 1980; Mitchison and Kirschner, 1984b). The process of depolymerization of MTs, as shown by recent studies (Carlier et al., 1984; Hill and Chen, 1984; Mitchison and Kirschner, 1984b), is more complex. After dilution suspension of MTs in the steady state, the number of MTs decreases, though the median length of the remaining
B
I i
l-
a
NH
wn
I:
1; 0 0
c
O
PROTEIN CONCENTRATION
W
5U
FIG . Plot of :.I rate of tubulin polymerization versus monomer concentration. The shaded area denotes the concentration range in which -end-capped MTs remains stable and free MTs depolymerize. Cr , Critical concentration for +end; C;, critical concentration for -end; 6,critical concentration for free MTs. (A) Plot according to the data obtained by Bergen and Borisy (1980) for tubulin with MAPS. (8)The plot according to the data obtained by Mitchison and Kirschner (1984a) and Hill and Chen (1984) for the phosphocellulose, purified tubulin.
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ones may increase initially. The same appears to hold for the steady state as well, i.e., some MTs are depolymerized, but their disassembly is compensated by elongation of the remaining ones (Mitchison and Kirschner, 1984b). To study MTOC polymerization activity, almost all the authors used tubulin preparations that either were uncapable of spontaneous polymerization or that were able to polymerize sufficiently slow. If a suspension of isolated basal bodies is added to purified tubulin, and then the mixture is heated to 37°C in the presence of GTP, bundles of MTs grow from the ends of the basal bodies, i.e., the way they would have grown from the ends of axonemal fragments. Obviously, the MTs of basal bodies can operate as exogenous primers for tubulin assembly (Snell et al., 1974; Kuriyama and Kanatani, 1981; Esponda and A h a , 1983). Microtubules may grow also from the MTNCs associated with basal bodies; they may grow from the striated rootlets (Stearns and Brown, 1979) or from the connective between the two basal bodies in the pair (Roobol et al., 1982; Stearns and Brown, 1981; Esponda and Aliva, 1983). Stearns and Brown (1979) showed that incubation of Polytomella basal bodies in 1 mM Tris, pH 8.0, and 0.1 M EDTA deprives their ability to induce assembly of MTs from the rootlets. In the process of this extraction, four proteins with a molecular mass from 190 to 210 kDa are washed from the basal bodies into the solution. In solution, these proteins also can induce assembly of the purified tubulin into MTs and are incorporated into them, i.e., the proteins act as specific MAPS within MTNC. The centrioles of cultured cells are usually surrounded by MTNC fibrogranular substance. If, in the process of centriole isolation or construction of permeable cell models, this substance is washed away from the centrioles, exogenous tubulin MTs may grow from the ends of the centriolar cylinders, the way they do from the basal body ends (Gould and Borisy, 1977; Schliwa et al., 1979), and may grow separately, i.e., on MTNC substance fragment (Gould and Borisy, 1977). Yet if the centriole remains surrounded by MTNC substance, the MTs grow only from MTNC and form asterlike structures (McGill and Brinkley, 1975; Snyder and McIntosh, 1975; Telzer and Rosenbaum, 1979; Pepper and Brinkley, 1979; Schliwa et al., 1979; Bergen et al., 1980, etc.). Several experimental models were employed to study the polymerization capacity of centrioles. The simplest one involved detergent-permeabilized cells in which their MTs were destroyed by colcemid, with the centrioles being assessable to exogenous tubulin. Such a model ensures the optimal intactness of the centrioles and the surrounding structures. However, the samples thus obtained can be studied only on ultrathin sections or by the immunofluorescent microscopy (Snyder and McIntosh, 1975; McGill and
THE CENTROSOME AND MICROTUBULE ORGANIZATION
26 1
Brinkley, 1975; Brinkley et al., 1981), and this causes difficulties for obtaining quantitative data on the number and length of polymerizing MTs. The next step toward a noncellular system for studying MTOC functioning was made by Gould and Borisy (1977), who developed procedures in accordance with which a suspension of detergent-lysed and softly homogenized cells was sedimented on grids for EM. This preparation was composed mainly of nuclear-centriolar complexes (Gould and Borisy , 1977; Kuriyama and Borisy, 1981a), and it was possible to perform polymerization of MTs on the centrioles with subsequent EM after negative staining (Gould and Borisy, 1977; Kuriyama and Borisy, 1981b). However, in this case the nuclei interfered and made it difficult to observe the MTs, and so this method is suitable for mitotic cells (Telzer and Rosenbaum, 1979; Kuriyama, 1984). The culminating step for the present level of investigations was made by Mitchison and Kirschner (1984a). They isolated centrioles from interphase CHO cells and neuroblastoma in the enriched fraction and then polymerized tubulin on the centrioles in suspension. They then sedimented the preparations thus obtained on grids for EM. Special investigations showed that the number of MTs polymerized on each MTOC is proportional to the concentration of tubulin solution: a stronger concentration gave a larger number of polymerized MTs. Some degree of saturation was always achieved if, with the increase in the protein concentration, the number of microtubules on MTOC remained invariable or even slightly decreased (Brinkley et al., 1981; Kuriyama, 1984; Mitchison and Kirschner, 1984a). If a suspension with MTs already assembled on the centrioles was diluted, the number of MTs on each MTOC significantly decreased, though their length might increase (Mitchison and Kirschner, 1984a). On the other hand, the number of MTs assembling on each MTOC at optimal conditions also varies depending on MTOC properties. Several times as many MTs are polymerized on mitotic centrioles as on interphase centrioles (Snyder and McIntosh, 1975; Telzer and Rosenbaum, 1979; Kuriyama and Borisy, 1981b). This may be directly related to the presence of the mitotic halo. In general, the number of MTs formed on MTOC in vitro correlates with that of the MTs radiating from the same MTOC in vivo. Thus, Brinkley et al. (1981) demonstrated that, in detergent-permeabilized 3T3 and SV-3T3 cells, the purified tubulin is polymerized on one to two centers in each cell, with an average of twenty-four MTs radiating from each MTOC in 3T3 cells, and about nine MTs radiating from each MTOC in SV-3T3 cells. The same number of MTs was formed on MTOC in these cells in vivo, after the cells had been washed free of colcemid.
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It was shown (Schliwa et al., 1979) that, in fish melanophores, the number of MTs, polymerized from exogenous tubulin on one MTOC, depends on the amount of MTNC substance contained in it. Schweitzer and Brown (1984) also demonstrated that the number of MTs radiating from the centrosome in Con A-stimulated lymphocytes is 2-3 times higher than that of unstimulated cells. The same correlation was obtained by these authors after polymerization of tubulin on the centrosomes of lysed lymphocytes in vitro. A MTOC of the dispersed melanophore pigment granules contained much MTNC substance and as many as 388 f 70 MTs radiated from it in vivo. Yet in the case of pigment aggregation, its MTOC had much less MTNC substance and fewer MTs radiating from MTOC (60 k 7). After destruction of the MTs by colcemid and detergent lysis, the amount of MTNC substance in melanophore MTOC did not change. On these MTOCs were assembled about the same number of MTs from exogenous tubulin as in vivo for a given disposition of pigment granules (348 +: 65 in the dispersed and 28 f 8 in the aggregated state, respectively). Schweitzer and Brown (1984) also demonstrated that the number of MTs radiating from the centrosome in Con A-stimulated lymphocytes is 2-3 times higher than that of unstimulated cells. The same correlation was obtained by these authors after in vitro polymerization of tubulin on the centrosomes of lysed lymphocytes. Mitotic centers from CHO cells, containing different numbers of centrioles (one to four), had the number of polymerizing MTs directly proportional to the number of centrioles (Kuriyama, 1984).It should be noted that, in the preparation used, as a result of colcemid treatment of the cells, the mitotic halo must have surrounded each centriole and not only the mother centrioles. Consequently, the number of MTs correlated with the halo volume (the amount of MTNC material) in a given MTOC. A critical tubulin concentration at which it is polymerized into MTs on MTOC is very low, much less than a critical concentration for the assembly of MTs from purified tubulin (Kuriyama, 1984; Hill and Chen, 1984; Mitchison and Kirschner, 1984a). This concentration, however, corresponds to the one needed for tubulin assembly on stable fragments of axonemal MTs (Bergen et al., 1980; Mitchison and Kirschner, 1984a). At low tubulin concentrations, the growth of axonemes proceeds only from the plus end, and under the same conditions the MTs grow from MTOC (Bergen et al., 1980) (Fig. 9). No one has ever directly determined the polarity of MTs assembled on MTOC in vitro; however, by decoration of native MTs in the mitotic spindle with dynein or tubulin “hooks,” it was demonstrated that, in vivo, the plus end of the MTs is a distal one with respect to MTOCs (centrioles) (Haimo et al., 1979; Heidemann and McIntosh, 1980).
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FIG.9. Comparison of centrosome and flagellar seed-initiated MTs. C, Centrosome. Note that the polymerized tubules at the plus end ( + ) of the flagellar seed (arrows) are approximately of the same length as the centrosomal MTs. Bar, 2 pm. (From The Journal of Cell Biology, 1980, 84, I55 by copyright permission of the Rockefeller University Press and courtesy of Bergen el a / . )
It may thus be suggested that MTOC is a set of stable primers (MTNCs) for the growth of MTs, i.e., in the simplest case, of very short fragments of MTs or tubulin oligomers. Here the minus end of a MT is blocked in MTNC substance. Indeed, after short-term incubations of cells in the presence of colcemid or nocodazol, an aster of radiating MTs persists around MTOC, even though free MTs become completely destroyed (We-
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ber and Osborn, 1981). In the case of assembly of MTs on isolated yeast MTOCs in vitro, which are disks of electron-dense material, a regular number of microtubules is likewise formed, and the ends of the MTs are immersed in the MTOC substance and closed (Hyams and Borisy, 1978; Byers et al., 1978). The fact that MTNC primers are in existence is also proved by the blockage of assembly of exogenous MTs on mitotic centrioles by tubulin antibodies (Pepper and Brinkley, 1977, 1979). But biochemical analysis of MTOC is needed for the final conclusion about the presence of MT assembly primers and about the mechanisms of assembly on MTNC. Some data on the biochemical properties of MTOCs have already been obtained. Their polymerization activity is inhibited by 0.2-0.5 M KCI and KI, at pH of the solution over 7.5, after ultrasound treatment, and in heparin. All these cases involved MTOC treatment prior to the addition of exogenous tubulin (Kuriyama and Borisy, 1981b; Kuriyama, 1984; Mitchison and Kirschner, 1984a). Mitotic MTOCs exhibited high-endogenous protease activity, which completely suppressed MTOC polymerization activity in 3 minutes at 35°C or in 2 hour at 5°C (Snyder, 1980; Kuriyama, 1984). This protease was inhibited only at high concentrations of PMSF (1 mg/ml) or in 50% glycerol (Kuriyama, 1984). These observations are at variance with the results of the previous investigations in which assembly of MTs on the mitotic halo was inhibited by RNase (Pepper and Brinkley, 1980). At any rate, Kuriyama (1984) denies the inhibitory effect of RNase on MT assembly on the halo, but Snyder (1980) confirms it. Such high sensitivity to endogenous protease has not been shown for interphasic MTOCs. Probably, endogenous protease is involved in mitotic halo disassembly, which takes place in late mitosis. Despite significant achievements in purifying the system for polymerization of MTs on MTOC in vitro, one should remember that the reaction of polymerization of MTs is a very complex one and can be regulated by many independent factors in such a way that departure of some of the conditions from the optimal ones could be redressed by the others. Assembly of brain tubulin on MTOC at conditions from the optimal ones could be redressed by the others. Assembly of brain tubulin on MTOC at conditions selected for spontaneous assembly of MTs, most of which are not attached to MTOC in vivo either (Chalfie and Thomson, 1979; Tsukita and Ishikawa, 1980), is possibly not a sufficiently adequate model for studying MTOC performance, even though it was the only available one until recently. Entirely different observations were reported by Deery and Brinkley (1982, 1983) for the assembly of endogenous tubulin in 3T3 and SV-3T3 cells. As established by these authors, upon permeabilization of the cells by Brij 58, the tubulin is not washed away and can be assembled
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and disassembled at controlable ionic conditions. If, prior to detergent treatment, the MTs were destroyed by preincubation of the cells in the presence of colcemid, then at “normal” polymerization conditions, i.e., at 3 7 T , pH 6.9, and with the addition of 1 mM GTP, randomly scattered microtubules, about 5 pm long, were formed (though the length of the native MTs was 34 pm). An essential condition for assembly of normal length MTs, and only from MTOCs, was pH 7.6, and addition of GDP and ATP, as well as of 8-bromo-CAMP. If an aster of the remaining short MTs persisted in the permeabilized cells, “normal” conditions of assembly and even the presence of GDP instead of GTP in the system were sufficient for their elongation. The results of this investigation show that optimal conditions for initiating assembly of MTs on MTOCs may differ significantly from those selected for self-assembly of isolated MTs in vitro. The present data on MTOC polymerization activity in vitro enable us to draw the following conclusions: 1. MTOC is capable of inducing assembly of MTs under conditions excluding their spontaneous polymerization; 2. MTOC must contain MT growth primers. The number of primers is limited, depending on the physiological state of the cell; 3. The biochemical composition of the primers is not clear. RNA participation is believed probable but not proved unambiguously. They do not contain DNA. The primers may differ from MT fragments in some properties.
VII. Assembly of Microtubules on Microtubule-Organizing Centers in Vivo
Microtubules were discovered by EM. Yet it was difficult to study a MT pattern in cells on ultrathin sections because of their large extension. After specific antibodies against tubulin had been obtained (Fuller et al., 1975; Weber et al., 1975), it became possible to trace MT localization by light microscopy (by indirect immunofluorescence). It was immediately found that, in interphase cells, MTs form a fairly dense network; so the genesis of this network attracted attention next. It should be noted that, even before the immunofluorescent studies, Tihey and Goddard ( 1970) demonstrated that, in sea urchin blastula after colcemid has been washed away, MTs grow from MTNC surrounding the basal bodies in each blastula cell. To study the origin of MTs, a simple procedure of their preliminary destruction by colcemid or cold in tissue-culture cells was employed.
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Brinkley et al. (1976) and Osborn and Weber (1976)first showed that after 3T3 cells had been washed free of colcemid, MTs began to grow in an asterlike fashion from one (sometimes from two) centers. Then the number of MTs sharply increased and individual MTs in the central part of the cells became invisible. Gradually the system of MTs spread to the cell’s periphery and lost its distinct center, similar to the way it occurs in intact cells. Frankel (1976) obtained similar results by studying L cells and macrophages, though the distinct center persists in macrophages even after a complete reconstitution of the MT network. Background fluorescence of colcemid-depolymerized tubulin interfered with observations. Pretreatment of the cells with the detergent, Triton X100, in a solution protecting MTs before furation gave more distinct images (Osborn and Weber, 1977; Bershadsky et al., 1978b). They proved to be similar to the ones described above, yet short MTs, not bound to the center, became visible in most cases at the beginning of reconstitution (Spiegelman et al., 1979a; Bershadsky and Gelfand, 1981; de Brabander et al., 1982). These free MTs disappeared subsequently and all of the cytoplasm became filled with a network of long MTs. Moreover, coldresistant MTs, not bound to the center, were detected (Bershadsky et al., 1979b). But as a rule, most of the MTs during their reconstitution in the cells are bound to the common center (Fig. 10). A supposition was expressed already in the early studies that the centriolar region is the center from which MTs begin their growth, for it is there that a cilium emerged (Brinkely et al., 1975; Bershadsky et al., 1978a). Correspondence of MTOCs to centrioles was particularly proved convincingly in neuroblastoma cells in which multiple MT growth centers were detected (Spiegelman et al., 1979b); it was then demonstrated that the number of these centers correlated with the number of centrioles (Sharp et al., 1981). A comparison of immunofluorescent and EM images of the same cell also indicated the location of the MT growth center, the centnolar region (Sharp et al., 1981). Karsenti and coauthors furnished the most cogent evidence of the role of MTOC (Karsenti et al., 1984a). They succeeded in performing enucleation of the cells and also in removing centrioles from some of them. Reconstitution of the MT system in the cytoplasts with centrioles proceeded normally, and as in normal cells, a network of MTs was formed; but only few MTs were restored at the periphery of the cytoplasts without centrioles. Cold treatment ( +2-O°C, for 1.5-3 hour) and mild doses of colcemid (0.4-1.0 pg/ml, for 8-40 hour), destroying a system of cytoplasmic MTs, caused blobs of electron-dense material to appear around the centrioles; these blobs resembled MTNC material (Vorobjev and Chentsov, 1983,
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FIG. 10. MTs radiating from the centrosome. Cells were cooled (0°C for 2 hours) and then warmed to 37°C for 7 minutes (A) and 8 minutes ( B ) . Indirect immunofluorescence with antitubulin antibodies.
1985).As the cells were transferred to 37°C or after colcemid was washed away, MTs began their growth not only from the surface of the centrioles or the heads of the pericentriolar satellites, but also from the blobs. The cell center at that time showed many short, randomly scattered MTs (Vorobjev and Chentsov, 1983). Then the electron-dense blobs vanished, and the number of MTs, radiating from the center, decreased correspondingly back to the “normal” level; short MTs disappeared (Vorobjev and Chentsov, 1983). A similar picture showing the appearance of short disorderly MTs around the cell center was observed under a light microscope (de Brabander el al., 1982). Thus, colcemid and cold treatments induce a restructuring of the cell center: extra MTNCs are formed (possibly, under the effect of free tubulin). In mitotic cells, kinetochores also serve as a MT polymerization center after colcemid is washed away or after the cold-treated cells are heated (Witt and Borisy, 1980; Rieder and Borisy, 1981). The question of how MTOC works in the living cell is rather difficult and involves several aspects: first, the dynamics of MT growth (temporal order); second, how MTs can grow preferably in one direction (spatial
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order); and the last problem is why MTs start their polymerization only from MTOC and do not polymerize throughout the cytoplasm. The main part of experimental data and speculations available so far concerns the latter aspect. Experiments showed that the preferential growth of MTs from MTOC in vitro takes place when tubulin concentration is sufficient for MT elongation from the plus end, but too small for spontaneous polymerization. We do not know whether such a situation occurs in the living cell. But it was found that MTs are in dynamic equilibrium with tubulin monomer in vivo (Salmon et al., 1984; Saxton et al., 1984) and their turnover is rather quick (especially during mitosis) (Salmon et al., 1984; Saxton et al., 1984). Though the amount of MTs and the whole content of tubulin per cell protein may vary (Waterhouse et al., 1983), a definite concentration of the tubulin monomer is sustained (Ben-ZeCv et al., 1979; Cleveland et al., 1981, 1983). The simplest suggestion about the mechanism of MTOC operation boils down to the following: there exist MT growth primers within MTOC, and assembly conditions in the cell are such that MTs can grow only on primers of MTNC. If so, MTOC must have a definite number of such primers, each giving rise to one MT only (Borisy and Gould, 1978). In that case, the number of MTs and the direction of their growth depend only on MTOC. This idea agrees well with the results of MT assembly on the centers in vitro, when a definite number of MTs is formed, depending on the state of the cell. Yet at the beginning of MT reconstitution, free MTs also appear in the cell (Spiegelman et al., 1979a; Bershadsky and Gelfand, 1981; de Brabander et al., 1982). A possible explanation is that the system may not be in equilibrium during reconstitution of MTs, i.e., spontaneous polymerization of MTs may take place. A more complex suggestion implies that the minus ends of the MTs are blocked on MTOC (Kirschner, 1980; Tucker, 1984). As we have said in Section VII, if the free tubulin pool is limited, the MTs with the blocked minus end prove to be more competetive than the MTs with both ends free: after all, the former MTs (with blocked minus ends) grow, while the latter MTs (with both ends free) disassemble. Really, the free MTs in the cell, which are formed after colcemid or nocodazole has been washed out, do gradually disassemble (de Brabander et al., 1982). MTs with the free ends also disassemble in fragments excised from fibroblasts (Gelfand et al., 1984) and after laser-caused destruction of the spindle pole (Berns et al., 1977). But they do not disassemble in excised fragments of melanophores, where a new MTOC is formed (McNiven et al., 1984). The hypothesis advanced by de Brabander et al. (1980, 1982an-) alternative to the one on assembly in MTOC on primers-presumes that,
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in MTOC, a selective anchoring of the minus ends of the growing MTs takes place. According to this hypothesis, special condition MTOCs facilitate nucleation of MTs, around MTOCs. Then some of these MTs are anchored and acquire advantages for growth, while those not anchored disassemble, sustaining thereby the required tubulin concentration for the growing MTs (de Brabander, 1982; de Brabander et al., 1982). The action of taxol-a preparation that lowers the critical concentration for assembly-causes MTs of the cell to lose their links with the center and form a chaotic network (Schiff and Horwitz, 1980), since the anchored MTs are deprived thereby of their advantage for growth. As nocodazole has been removed from the cells with a diminished ATP level (under the effect of respiration inhibitors) in which the disassembly, but not the assembly, of MTs is slowed down (Bershadsky and Gelfand, 1981), the MTs likewise form a random network, for the MTs, not linked to the center, are not disassembled (de Brabander et al., 1982). It appears that special ionic conditions are obtained in the MTOC region which favor the assembly of MTs; thus Caz+concentration may decrease due to the operation of membrane transport systems (Silver et a / . , 1980; Aguas and Nickerson, 1981; Kierhart, 1981). How does the MTOC in a cell with assembled MTs maintain their spatial and temporal order? Under normal conditions, a system of cellular MTs is formed “from zero” only twice in a cell cycle: immediately before mitosis and after it (review, Brinkley et d . , 1980). Otherwise, the cell center only sustains the MT system and participates in its renewal. A thorough analysis of reconstitution of a MT system in interphase cells after cooling shows that the number of MTs bound to the centrosome in the beginning of reconstitution is much above the normal level. This observation led Vorobjev and Chentsov (1983) to suggest an idea that two systems of MTs exist in a cell. One forms a network in the cytoplasm and has no definite convergence foci. The other radiates from the centrioles. It is presumed that the radiating MTs persist bound to the cell center only for a definite time interval during their growth. After their growth is over, the MTs disengage and form a cytoplasmic network slowly departing from the center. New MTs may start growing on MTNCs, which the full-grown MTs have left. Consequently, the entire cell center operates as a conveyer feeding MTs into the cytoplasm (Fig. 11). The existence of the two MT systems in cultured cells has been demonstrated (Karsenti et al., 1984b). It was found that the peripheral network of MTs is less resistant to depolymerization by nocodazole than are the center-bound MTs. So the authors concluded that their observations corroborated the idea that, in the cell center, the MTs are anchored by one of their ends, while the MTs not bound to the center have both ends free.
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FIG. I t . The scheme of a conveyor assembly of MTs on the centrosome. MT starts growing from MTNC with its plus end. Then, after a certain period of time, it is detached from MTNC and may be depolymerized. for its minus end becomes free. The next MT starts growing from the same MTNC.
On the contrary, according to some other observations, in nocodazoletreated abnormally large Chinese hamster cells, MTs associated with MTOC are disassembled first and then only the peripheral ones disappear (Raes er al., 1984). This is attributed to the decreased functional activity of the centrioles in such cells. If the suggestion on the attachment of the MT minus end in the cell center is valid, then, in the steady-state condition of the cell, the MTs with both ends free should disassemble, and those with one free plus end should grow or remain in the same condition (Kirschner, 1980). The centrosome apparently exerts some effect on the number of MTs in the cellthe critical concentration of tubulin for the MTs bound to it is lower than for free ones. In the absence of the centrosome, as shown by Karsenti et al. (1984a) on centriole-free cytoplasts, the number of MTs is smaller. It is most probable that the number of MTs in centriole-free cytoplasts, is directly lined to the effective concentration of tubulin in them. The dependence is more complicated in cells and cytoplasts with centrioles. Since MTs with both ends free should disassemble, it is obvious that the time of their existence should be inversely proportional to the depolymerization rate. A more or less dense network of MTs may thus be formed only in cells where the time of growth of MTs radiating from the center is significantly less than the time of disassembly of the free MTs, or if the minus-end is protected, against disassembly. One may point to two extreme variants: neutrophils, where no free MTs are practically detected (Schliwa er al., 1982;Anderson et al., 1982),and fibroblasts, where
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27 I
cold-resistant MTs, not bound to the center, are found (Bershadsky et al., 1979a). In the latter case, an excised cell fragment with no center retains MTs for several hours (Gelfand et al., 1984). Moreover, reconstitution of a MT system in the axon of the neuron in Caenorhabditis elegans showed it to have relatively short MTs not bound to any common center (Chalfie and Thomson, 1979).Axons of other animals possibly contain such systems of MTs as well (Tsukita and Ishikawa, 1980). Regulation of the depolymerization rate of free MTs is a subject beyond the topic of the present work, and so we shall point to most probable options only. First, the rate may be dependent on MAPS (Jameson and Caplow, 1981); second, disassembly may be inhibited by modification of the MT ends (Mitchison and Kirschner, 1984b; Chalfie, 1982). In our opinion, the conveyer hypothesis of MT assembly explains the cause of stark distinctions in the disposition of MTs in different cells: in motile blood cells and in melanophores, the system is radial (Schliwa, 1975; Schliwa et al., 1978; Anderson et al., 1982; Schliwa et al., 1982); slowly moving cells (fibroblasts) form a network often without a clearly distinct center (Osborn and Weber, 1976);and in practically immotile epithelial cells, a MT convergence focus may not be detectable at all (Bershadsky et al., 1978a). According to the conveyer hypothesis, cells with a developing network of MTs would have an ever-increasing number of them farther away from the center. An increase in the number of MTs at some distance from the centrioles is indeed observed during radial spreading of fibroblasts on glass (Gudima et al., 1983a). The number of MTs radiating from the center should increase during a transition of the cell from immotility to motility, and this has also been observed in cultured hepatocytes and bibroblasts (de Brabander et al., 1978; Gudima et al., 1983b). Irrespective of a concrete mechanism for centrioles to operate as MTOCs, we should note that their role in the organization of cell MTs may change depending on different physiological conditions of the cells, and such changes have been demonstrated at least for two cases: for transition from interphase to mitosis and vice versa and for the aging of the cultured cells. As we have said above, during transition to mitosis, the interphasic forms of MTNC give way to the mitotic halo, and MTs are assembled into a spindle upon disassembly of the preceding interphase network. Obviously, other MTNCs-the chromosomal kinetochores specific for mitosis only-have a significant part to play in spindle formation. The injection of a fraction of interphasic centrioles isolated from neuroblastoma cells into Xenopus eggs revealed the ability of these centrioles to induce formation of cytasters only in interphase-activated eggs, while exogenous nuclei or chromosomes are needed for that in mitotic cells
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(with artifical delay of activation) (Karsenti et al., 1984a). According to the authors, conditions for assembly of MTs in mitotic cells are such that MTs cannot assemble even on the centrioles (and this explains disassembly of MTs at the end of the G , period); they reassemble only with the aid of additional chromatin-related factors. Two subpopulations have been detected in cultured Chinese hamster fibroblasts: normal fibroblasts and abnormally large ones, the polygonal cells. The latter are accumulated with the aging of the culture (in late passages). If, in the normal cells, nocodazole-induced depolymerization of MTs proceeded from the periphery to the center (the way it should be if a MT has its minus end anchored in the center), the pattern was reversed for the large cells: the MTs in the center were disassembled first, and thereupon, only those at the periphery (Raes et al., 1984).The mechanism of the latter process has received no satisfactory explanation as yet, but it is certainly connected with functional changes in MTOC. We may conclude that the role of the cell center in organizing a system of MTs needs further investigation and that the actual mechanism for its action may turn out to be more complex than hitherto believed. VIII. The Centrosome and the Cell
Microtubules are involved in sustaining a “spatially organized asymmetric form of the cell” (Solomon, 1980). Unlike actin filaments, which generate local forces bringing the cells or their parts into motion and which must have multiple organizing centers (Lazarides, 1976; Schliwa, 1982), the MTs organize the cell as a whole and usually have a single organizing center. As said above, the centrioles and the structures surrounding them operate as MTOCs in animal cells. The number of centrioles in the cells varies and so does the amount of MTNC substance. Nevertheless, experiments on MT growth initiation described in Sections VI and VII indicate that the number of MTOCs is as a rule one to two per cell (Brinkley et al., 1975; Osborn and Weber, 1976; Frankel, 1976; Watt and Harris, I 980). How does the cell regulate the number of MTOCs? First, by regulating the distribution of the MTNC substance. Usually it is present only in the centriolar region, and the number of MTOCs corresponds to the number of active centrioles. A diploid cell has two centrioles, of which only one is active (Dalcq, 1964; Schaffer, 1%9; Vorobjev and Chentsov, 1977, 1982). Additional MTOCs without centrioles are a rare exception (Schliwa et al., 1982, 1983; Sato et al., 1983). Such MTOCs consist of one or few aggregates of the MTNC substance. An increase in the number of such
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centriole-free MTOCs after 1000-rad irradiation is accompanied by a loss of their MT-polymerizing activity and the death of cells (Sato et al., 1983). Fewer MTs come to the centriole-free (containing only a halo fragment) pole of the pathological multipolar mitotic spindle than to the poles with centrioles (regardless of the number of these centrioles), and a contractile ring near the centriole-free pole takes a longer time to form (Keryer et al., 1984). Concentration of MTNC substance around the centrioles is still a great enigma. Since only the centrioles persists in the cell center, and MTNCs appear and disappear [for instance, in the cell cycle (Erlandson and de Harven, 1971; Vorobjev and Chentsov, 1982; Rieder and Borisy, 1982)], it seems probable that the centrioles act as a matrix for assembly or as a frame to which MTNC substance is attached in animal cells. The amount of MTNC depends on the physiological condition of the cells and may increase or decrease as, for instance, under dispersion and aggregation of granules in melanophores (Schliwa et al., 1979). Under polarization and motion of fibroblasts (Gudima et al., 1983a, b) or after lymphocyte stimulation (Schweitzer and Brown, 1984), the amount of MTNC substance increases. There is no ground for an assumption that the centrioles are directly involved in MTNC substance generation, for it would contradict the available data on the pathways of biosynthesis of proteins. Probably, a process similar to self-assembly of MTNCs and their accumulation in the cell center takes place, for instance, when centrioles are injected into eggs. Mitotic halo substance is accumulated on the centrioles, with cytasters being formed (Kuriyama and Kanatani, 1981; Hamaguchi and Kuriyama, 1982). Second, the number of centrioles and their distribution in the cell is regulated. As is known, diploid cells have only two centrioles. Since the centrioles double in tandem with DNA synthesis, the number of centrioles in polyploid cells should correlate with ploidy. Thus, tetraploid hepatocytes have four centrioles, and octaploid ones have eight (Onischenko, 1978). The same dependence of the number of centrioles on ploidy has also been observed for Allomyces (Borkhardt and Olson, 1979). At the same time, polyploid cells at the terminal stages of differentiation (megakaryocytes) and also cells (syncytia) formed via the fusion of dipolid cells (muscle fibers and giant cells appearing in inflammation processes) have fewer centrioles compared with their ploidy (Przybylski, 1971; Konishi et d., 1973; Sapp, 1976; Moskvin-Tarkhanov and Onischenko, 1978). One possible suggestion is that centriolar replication is inhibited or that resorption of some of the centrioles occurs (Mahovald et al., 1979). The reasons for the imbalance between ploidy and the number of centrioles deserves special investigation. It should be mentioned that only a deficit of centrioles
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is observed, but most probably, there exist no superfluous centrioles in the cells. Whether the cell can reconstitute the lost centrioles is still an open question. There is evidence that regenerating diploid karyoplasts from centrioles again, namely, two centrioles per cell (Zorn et al., 1979); but there are also opposing data that, during enucleation, some karyoplasts may retain centrioles, and only these karyoplasts are reconstituted into normal cells (Brown et al., 1980). Whatever the number of centrioles in a cell, they and the structures surrounding them are usually aggregated into a single cell center, and it is rarely that they are spaced far apart (Spiegelman et al., 1979b). Centrioles are found in a compact group even in giant syncytia, the homosynkaryons (Heidenhain, 1907; Matthews et al., 1967; Wang et al., 1979). Multiple centrioles are grouped at the spindle poles in heterokaryons, the neuroblastoma cells, and hepatocytes (Pera, 1975; Onischenko, 1978; Peterson and Berns, 1979; Ring e? al., 1982). Extra centrioles outside the mitotic spindle are rather an exception to the rule (Peterson and Berns, 1979; Brenner et al., 1977). As the cell changes its functional activity, they (the centrioles) may split. Centrioiar “splitting” takes place in neutrophils under the effect of chemoattractant or tumor promoter (Schliwa et al., 1982, 1983), and in cultured cells, under the effect of the growth factor and other mitogenetic agents (Sherline and Mascardo, 1982a, b). As a result of “splitting,” each centriole either active or inactive forms a MTOC of its own. Divergence of diplosomal centriolar pairs at the prophasic spindle poles is a process similar to “splitting.” This divergence is not synchronized with the condensation of chromosomes in the nucleus (Mole-Bajeret al., 1975; Aubin et al., 1980) and apparently does not depend on MTs (Snyder and McIntosh, 1975; Anderson et al., 1981; Cabral et al., 1983). Under prolonged incubation of cells in colcemid or in p-mercaptoethanol, the centrioles move apart and each may form its own MTOC (Watt and Harris, 1980; Watt et al., 1980), including a mitotic spindle pole (Went, 1977; Onischenko et al., 1979). Early embryogenesis, i.e., egg fertilization and division, may be a good model for studying cell MTOC regulation. Mature unfertilized eggs contain significant amounts of tubulin and MAPS, but very few MTs (Schatten et al., 1983; Balczon et al., 1983). Formation of MTs (asters and spindles) begins only after a spermatozoon penetrates the egg. It was believed earlier that the role of the spermatozoon is to bring the centrioles, absent in the egg, to initiate aster formation and division (Wilson, 1925). Indeed, after the spermatozoon enters the egg, its basal body loses its connection to the flagellum and becomes surrounded by osmiophilic material; MTs start growing from it, and a spermal aster is formed (Longo
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and Anderson, 1968). This picture was described with the aid of electron and light microscopy. Yet staining egg cells with tubulin antibodies revealed that no direct relationship exists between the sperm aster and the mitotic figure, and there is a period when, in the zygote, no MTs, emerging from any centers, are detected (Harris et al., 1980). It was also learned that some eggs may have a centriole (Zamboni, 1971; van Assel and Brachet, 1968). On the other hand, basal bodies (centrioles) are absent from many spermatozoa where they, in the course of spermiogenesis, are replaced by electron-dense material, which resembles a centriole by its contour only (Woolley and Fawcett, 1973). Finally, in mouse and rabbit fertilized eggs, though we see a classical combination of an egg without centrioles and a spermatozoon with basal bodies (Szollosi et al., 1972), no centrioles are present at the spindle poles during first divisions until a stage of 16 blastomeres. Instead, electrondense material (MTNC), organized into a single complex, is found (Burkholden e? al., 1972; Calarco-Gillam et al., 1983; Szollosi et al., 1972; Longo, 1974, 1976). Various treatments (hypertonic solution heating, microneedle prick, ammonia, heavy water, etc.) may induce parthenogenetic development of eggs in many animal species. Long-term effect of some of these agents results in the appearance of multiple asters, the cytasters, in the ooplasm. As shown in sea urchin and frog egg cells, such cytasters contain centrioles in the center (Koichi and Masao, 1971; Miki-Noumura, 1977; Dirksen, l%l). In sea urchin ooplasm, centrioles, close to a nuclear envelope or annulate lamellae, are frequently formed de novo (Kallenbach, 1982; Kallenbach and Mazia, 1982). Possibly, the latent centriolar precursors, found in significant amounts in the egg, are activated (Kallenbach, 1983). It is far more difficult to resolve the problem of the origin of multiple centrioles appearing upon injection of basal bodies or centrioles isolated from various objects. It was taken for granted that the basal bodies and centrioles thus injected underwent certain functional alterations and started functioning in the ooplasm as cytasters. In other words, they became surrounded by a halo from which MTs started growing (Heidemann and Kirschner, 1975, 1978; Maller et al., 1976; Heidemann et al., 1977; Kuriyama and Kanatani, 1981; Hamaguchi and Kuriyama, 1982; Karsenti et a l . , 1984a). Yet a thorough EM study or basal body labeling is needed, otherwise, it may not be excluded that the injected basal bodies operate only as promoters for the appearance of functioning centrioles in the ooplasm. It is known that only basal bodies or centrioles are capable of inducing cytaster formation (Hirano and Ishikawa, 1979; Heidemann and Kirschner, 1975). Their inducing activity is sensitive to proteases and RNase (Heidemann e? al., 1977).
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Egg cell cytasters, irrespective of their origin, divide synchronously with mitotic asters (Wilson, 1925). A mitotic aster can divide upon removal of the zygote nucleus, while the zygote is capable of regenerating the mitotic aster removed from the egg (Lorch, 1952). All these facts prove that egg centrioles, albeit not a fully autonomous structure, are sufficiently independent of the nucleus. It appears that an egg has a pool of centriolar and mitotic halo precursors, which are mobilized, due to various activating effects setting off cell division (Weisenberg and Rosenfeld, 1975). In the course of cell division, however, the extra cytasters are lost, and each cell of the embryo gains two centrioles (Wilson, 1925). Also, if the division sets in without centrioles at the mitotic poles, then centrioles appear at the morula stage two per cell (Szollosi et al., 1972). There is only one indication that father centrioles persist in the embryo, i.e., a giant centriole of the spermium Chrysopa carnea is detectable in one of the blastula cells (Friedlander, 1980).Thus, animal cells can “take count” of their centrioles and MTOCs. They lose this capacity during oocyte maturation and early division when the centrioles gain relative autonomy. It is desirable to look into a mechanisms of control which the cell establishes over the centrioles in the course of the organism’s ontogenesis.
IX. Localization and Orientation of Centrioles in Cells Centriolar location in cells was the object of many investigations in the past (reviews: Heidenhain, 1907; Wilson, 1925). The last few years have seen a renaissance in related studies, but at a higher level, with the aid of EM and immunofluorescent methods, and also, using cultured cells as an object. Since an EM shows the centriole as a differentiated structure and it has proved to be a polar structure (Vorobjev and Chentsov, 1980), one can now study not only the location of centrioles, but also their orientation in cells. It is hard to perceive some causal relationship between the facts of nonrandom disposition of centrioles described below and other intracellular processes. The presence of one MTOC in most animal cells accounts for cell disymmetry-the greater, the closer is the MTOC to some surface of the cell; conversely, a cell with a centrally located MTOC retains a spheric symmetry. As shown near the end of the last and early in this century, the centrioles in some cells may move away from the nucleus onto the surface intestine or trachea epithelial cells which are a case in point. In most instances, however, centrioles are located in the central region and are pushed aside
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by the nucleus only (Heidenhain, 1907). Two groups of cells may be singled out as the extreme types: those with perinuclear and those with periplasmalemmal disposition of centrioles. The first type is represented by motile cells (leukocytes and fibroblasts) and epithelial cells, still capable of division. The centrioles are located near the nucleus, often in its recess. They are frequently surrounded by Golgi complexes (Kupfer et al., 1982; Nemere et al., 1985). The centrioles of epithelial cells and fibroblasts may produce a primary cilium (Sorokin, 1962, etc.) (see Section 11; Allenspach and Roth, 1967), but they do not form specialized stereo- or kinocilia. The second type is represented by some differentiated immotile cells. Their centrioles lie far from the nucleus and close to the free surface, usually in the apical part (Heidenhain, 1907; Tucker, 1984). They may form a stereocilium [for instance, in sensory organs (Dahl, 1963; von Narnack, 1963; Tilney et al., 1980)] or serve as a matrix for basal bodies (Sorokin, 1968; Steinman, 1968; Dirksen, 1971). The centrioles moving toward the cell edge can participate in the formation of a specialized system of MTs, for example, in the growth of axon neurotubules (Spiegelman et al., 1979b), when they lie in the axon hill, or in forming an ulterior bundle of MTs in invertebrate nuclear erythrocytes (Cohen and Nemhauser, 1980). The centriolar shift to the erythrocyte periphery is genetically determined and controlled by one gene (Searle and Bloom, 1979). In secretory cells, the centrioles may be connected with secretory vesicle clusters (Zeligs, 1979). Tissue culture cells occupy an intermediate position between the two cell types described above: the distance between the nucleus and the plasmalemma is very small, and the centrioles, lying not far from the nucleus, may form an external cilium (Albrecht-Buehlerand Bushnell, 1979; Jensen et al., 1979; Rieder and Borisy, 1982). The nuclei and centrioles from cells of the first type and from cultured cells may be isolated together (Dales et al., 1973; Gould and Borisy, 1977), which suggests their mechanical cohesion (Bornens, 1977; Nadezhdina et al., 1979; Mar0 and Bornens, 1980; Nelson and Traub, 1982; Kuriyama and Borisy, 1981b). This cohesion is insensitive to detergents (Nadezhdina et al., 1979), i.e., the centrioles are attached not to the nuclear membrane, but to some submembrane structures of the nucleus. No concrete structures, responsible for the centriolenucleus association have been detected so far. Intermediate filaments are the putative candidates, even though centriole-nucleus complexes may be isolated in cells having no intermediate filaments (Nelson and Traub, 1982). The centriolar connection to the nucleus is weakened by cytochalasin B (Maro and Bornens, 1980), and therefore, when cells are enucleated with cytochalasin, the centriole remains in the cytoplast (Goldman et al., 1975). There is contradictory evidence concerning the effect of colcemid or nocodazole on the centriole-
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nucleus association. Aronson was the first to demonstrate a colcemiddependent centriolar connection to the prophasic nucleus in dividing sea urchin eggs (Aronson, 1971). But centrioles were also found to form MTindependent links to chromosomes (Bahr and Sugler, 1977). In tissue-culture cells, under the effect of colcemid or nocodazole, the centrioles may retreat to the cell periphery (de Brabander and Borgens, 1975), and it is more difficult to isolate them together with the nuclei (Maro and Bornens, 1980). During cell enucleation in the presence of nocodazole or cytochalasin B, some of the centrioles transfer into karyoplasts (Karsenti et al., 1984b),as the authors believe, because the MTs anchor the centrioles inside the cells. Centrifuged cells are an exciting example of centriole-nucleus association (Fais et al., 1984). If the cultured cells are centrifuged at 20,00040,OOO g, in such a way that the centrifugal force is parallel to the substrate plane, the nucleus is shifted in the centrifugal direction and so does the centriole in its wake: they push back other heavier organelles. In the presence of cytochalasin B, the centriole does not follows the nucleus, and the distance between them significantly increases. A single cell center causes cell asymmetry. It may be that there is some mechanism regulating the location of cell center in polarized cells. Motile cells, observed in v i m , are the most convenient model for studying this problem. It was shown that, in fibroblasts and endothelial cells polarized at the monolayer edge, the centrioles move to the front that becomes the leading edge of the cell (Gotlieb et al., 1981; Gudima et ul., 1983b) (Fig. 12). The same holds true for polarization of neutrophils in a chemoattractant gradient (Malech et al., 1977). The data on moving cells are more contradictory. Thus Gudima and coauthors write that, in moving fibroblasts polarized after attachment on the glass surface, the centrioles are always (in 100% of the cells) located between the nucleus and the leading edge of the cell (Gudima et al., 1983a,b). Albrecht-Buehler and Bushnell, contrary to what they had described in a summary of their work, observed lateral location of the centrioles (in 60% of the cells) in the direction of the fibroblast movement (Albrecht-Buehler and Bushnell, 1979). It may be that the authors traced the direction of cell movement by the phagokinetic track and not by the leading edge. In moving neutrophils, according to some data, the centrioles migrate toward the leading edge (Bessis and Breton-Gorius, 1967; Schliwa et al., 1982), and according to others, they remain in the center of the cell (Anderson et al., 1982; Gudima et al., 1984). No cogent explanation of these different results has been offered. In moving lymphocytes (T killers), the centrioles are located in the uropod (Gudima et al., 1984), i.e., in the part of the T killer by which it attaches to the target cell (Geiger et al., 1982).
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FIG.12. Centrioles in a moving fibroblast. A, Side view; B, top view: m. mother centriole: d. daughter centriole; c, cilia; N . nucleus: le, leading edge of the cell.
How are the centrioles located with respect to each other? During replication in animal cells, the daughter centriole is formed near the proximal end of the mother centriole and at 90”. In most (if not in all) cells investigated to date, the two centrioles after mitosis are not perpendicular to each other (Bessis, 1964; Murray et al., 1965; Robbins et al., 1968; Erlandson and de Harven, 1971; Schaffer, 1969; Fawcett, 1966; Vorobjev and Chentsov, 1977). Unfortunately, this fact is seldom mentioned in reviews and manuals. The mother and daughter centrioles lose their mutually perpendicular orientation at the end of mitosis or at the beginning of the interphase that follows it (Robbins et al., 1968; Erlandson and de Harven, 1971; Vorobjev and Chentsov, 1982). Thereby the centrioles can move far apart, to a distance of several micrometers (Vorobjev and Chentsov, 1982), and come together subsequently. A similar divergence of the centriolar pair was also described for centriolar replications in PtK, cells (Rieder and Borisy, 1982). Activation of neutrophils causes the centrioles to split rapidly as far as 1 Fm and more (up to 10 pm), whereas they are quite near to one another in the control cells (Schliwa et ul., 1982; Sherline and Mascardo, 1982a). It is believed that “centriolar splitting,” as the author calls it, is related to the work of actin microfilaments (Schliwa et al., 1982; Sherline
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and Mascardo, 1982b). The same explanation applies to centriolar splitting in the course of the transition from mitosis to interphase: as the cells spread, actin filaments are restructured in them. Since the centriole is a cylinder-like structure and a polar one at that, one may assume, proceeding from only geometrical concepts, that the centriolar cylinder can take a definite position in the cell-for instance, perpendicular or parallel to the substrate on which the cell lives, and in mitosis, a definite position with respect to the spindle axis is possible, etc. Nonrandom (predominantly perpendicular) disposition of the centrioles with respect to the substrate plane was described for PE cells early in interphase (Vorobjev and Chentsov, 1982), for normal mouse fibroblasts at the stage of radial spreading and during polarization (Gudima et al., 1983a),and for 3T3 fibroblasts polarizing at the monolayer edge (Gudima et al., 1983b). In spreading fibroblasts, centriolar orientation depends on the presence of MTs and does not change upon destruction of microfilaments (Gudima et al., 1983a). In mitosis the active centrioles are located mainly perpendicularly to the spindle axis at the stage of metaphase and anaphase [see Tables 1 and 111 in Vorobjev and Chentsov (1982)l. One should also point to the phenomenon of nonrandom disposition of centrioles in tissues, when they are found at one and the same level in a layer of epithelial cells (Heidenhain, 1907; Wilson, 1925; Tucker, 1984), in a malignant tumor strand (Schaffer, 1969), or are shifted within the cells in the course of embryogenesis (Foe and Alberts, 1983; Tucker, 1984).
X. Conclusion Since the publication of Fulton’s review (197I), we have learned much about the centrioles. Yet as it often happens, the new data have produced far more questions than answers. At present, we have a thorough knowledge of the three-dimensional centriolar structure (in mammals, at any rate). A large number of EM observations enable us to conceive an overall image of both centrioles and basal bodies. These organelles comprise, besides nine MT triplets, a large number of fine substructures. A detailed study into the genesis of centriolar cylinders and experiments on dissociation in vitro of isolated centrioles bring us to the conclusion that the most conspicuous centriolar cylinder component, the triplets, is not a structural basis of this cylinder; this role appears to be reserved for the centriolar matrix, a rim of amorphous material, surrounding the triplets and the axial hublike structures (the cartwheel and the pinwheel).
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Bernhard and de Harven (1960) showed that the centriole is a polar structure. Today we know that the polarity of the centriolar cylinder manifests itself, on the one hand, in the orderly organization of its substructures; and on the other, centriolar cylinder polarity is a sequel to the polarity of the MTs within the centrioles and basal bodies. The past few years, owing to the appearance of immunofluroescent methods for detection of cytoskeletal structure, have provided some fresh insights into the role of centrioles in organizing cell MTs. Although not all the MTs in interphase cells are connected to the centrosome, it has a virtual monopoly on forming new MTs. The MTs grow from the center toward the periphery; they have their minus ends in the cell center and the plus end at the periphery. It is the plus-ends that are elongated. Consequently, the cell center seems to regulate only the finding of MTs; other factors must account for their growth. The number of MTs growing from the cell center varies. It may vary depending on the cell cycle, the functional changes of the cell, and during its morphogenetic reactions. The number of MTs radiating from the cell center apparently does not depend how dense the MT network is in a cell, but on the renewal rate of that network. As indicated by immunofluorescent investigations, the MT system undergoes two drastic alterations in the cell cycle: as the cell enters mitosis, the cytoplasmic MTs disassemble and form a spindle; upon completion of mitosis, the spindle is destroyed and reconstitution of the cytoplasmic network takes place. Formation of MTs in interphase and in mitosis is related to different structures of the cell center, and so a regular succession of these structures (pericentriolar satellites in interphase and the halo in mitosis) in the cell cycle precedes a restructuring of the MT system and, most probable of all, is the cause of this restructuring. Back in the early 1960s, Dalcq (1964) pointed to the nonequivalence of the two centrioles in a cell. Detailed studies of centrioles in a cell cycle have made it possible to explain their nonequivalence, as well as the fact that at least two centrioles are present in a cell (if they are there at all!). The point is that the centriolar formation process, beginning in the S period of the cell cycle, embraces a cell cycle and a half and thus only one mature centriole, capable of performing all of its functions, gets into the cell after division, and one immature centriole, capable of replication only, gets into the cell after division. In the 1970s, centrioles were detected in fractions of isolated nuclei. Subsequent investigations have shown the centrioles to be structurally connected to the cell nucleus, and this association does really exist in a living cell.
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The nonrandom disposition of centrioles in a cell was described long ago at the light microscopic level (review: Wilson, 1925), and the latest EM observations enable us to speak about the nonrandom location of the centriolar cylinders in vitro. In certain events in the life of cultured cells (spreading and motion), the centriolar cylinders may be perpendicular to the substrate plane; and during cell division (in meta- and anaphase), the mother centrioles may be perpendicular to the spindle axis. The least results appear to have been achieved in the field of biochemical studies of centrioles and basal bodies. The only relevant fact is that the cell center and the basal bodies contain no DNA, but they contain RNA. Of late there has been a gradual resurgence of the concept of centrioles as the central structures in the life of an animal cell. Two independent approaches are applied: one focuses on the unique geometrical properties of the centrioles, their orderly structure with the ninth order central symmetry. The centrioles are attributed the role of an intracell gyroscope (Bornens, 1979); it is also presumed that they ensure a higher probability of the cell turning at 40” and not at any other angle (Albrecht-Buehler, 1981).
The other approach, in the footsteps of Pickett-Heaps (1974), includes MTOCs on the evolutionary standpoint. The centrioles are regarded as derived from the basal bodies of ancient Flagellata. According to this approach, the centrioles are no more than a structure for template-guided axonemal polymerization; they are put by in a cell for emergence, if the need arises to construct a cilium or flagellum. Furthermore, it is supposed (Onischenko, 1982) that, in the process of evolution in animal cells, a structural unification of centrioles and mitotic and cyloplasmic MTOCs into a single polyfunction system, the cell center, took place. Besides, having originated as a motility organ, the flagella and cilia, in the case of multicellular animals, acquired a function of reception. It is suggested therefore that centrioles, as derivatives from basal bodies, have retained the former receptory functions of the cilia and evolved into “intracellular receptors” (Chentsov, 1984). For all the attraction of the above-sketched suggestions, the only waterproof concept of centriolar functions is as follows: orderly initiation of the growth of the MT network. Concerning further trends in centriolar studies, we would like to stress these points: 1. Hitherto centriolar investigations have not involved mutant cell lines or mutant species, except for a few studies on Chlumydomonus basal bodies. We have found only one reference in the literature to the possibility
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of hereditary changes in the behavior of human centrioles (Afzelius, 1980). No such observations have been registered with respect to animals. So, the cytogenetic trend in centriolar apparatus studies is still an open area of research. 2. The centriole possesses considerable difficulties for biochemical analysis. Most of the functions ascribed to it are not accessible to such analysis at present and require preliminary phenomenological investigation. On the other hand, until recently, centriolar behavior could be described only with the aid of EM on serial sections. This is the most reliable, even though the most labor-consuming, and thus, inefficient method. Lately, autoimmune antibodies against centrioles have been found (Connoly and Kalnins, 1978; Maunory, 1979), which should certainly speed up investigations. However, obtaining autoimmune antibodies, unlike the conventional procedure for polyclonal antibodies, is more difficult and this may be the reason why antibodies against centrioles are not as widespread as antibodies against tubulin. Therefore isolation of a centriolar (or basal body) antigens is an urgent problem. 3. No good experimental models have been found as yet for studying centriolar functions (apart from the direct induction of MT polymerization). Only initial steps have been taken toward this end. Thus it has been shown that centriolar splitting occurs under nonspecific activation of granulocytes (Schliwa et al., 1982)and under stimulated proliferation of certain cultural cells (Sherline and Mascardo, 1982a, b); nonrandom orientation of the centrioles to the substrate in morphogenetic reactions of cells in vitro has been discovered (Gudima et al., 1983a, b). In all cases, the effect applied to a portion of the cells is from 15 to 50%. As the next step, it is essential to find some methods for comprehensive effects (i.e., in 100% of the cells) on the location and transition of centrioles, or on their activity in MT polymerization. 4. The most straightforward approach would be to obtain cells without centrioles. Such eosinophils have been obtained recently by microirradiation (Koonce et al., 1984), as well as cytoplasts, by enucleation in the presence of nocodazole and cytochalasin B (Karsenti et al., 1984b),and by a line of centriole-free cells (Debec et al., 1982). Comparing the life of the centriole-free cells, we may gain a direct understanding of what the centriole is needed for.
ACKNOWLEDGMENTS We thank Professor Yu. S. Chentsov for critical remarks and fruitful discussion. We also thank Dr. R. G. W. Anderson and Dr. 0.G . Borisy for their permission to reproduce their pictures and Dr. L. G . Bergen for a copy of his figure.
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Index
A A 21387, oocyte maturation and. I I Acetylcholine, neuroendocrine cells and, 46 Acet ylcholinesterase in intracardiac neurons, 104, 105 in neuroepithelial cells, 69, 73 in parasympathetic postganglionic fibers, I22 Actin fibers. within cells, 208-209 Actinomycin D. centriole formation and. 256 Adenosine triphosphatase. in basal bodies and centrioles, 253-254 Adenosine triphosphate bioenergetics and, 186-187, 188 muscle contraction and, 204, 206 Adrenergic neurons, in terminal nerve plexus of, 119-120 AV junction, 105-1 I I Agonists superactive, of GnRH, 169- 170, I7 I- I72 D-Trph and, 170-171 D-Arg* and, 171 Amide I vibrations, energy transport and. 187. 189. 192-194 Amphibians. GnRH in, 160. 164-165 Anaphase, centrioles and, 248 Anesthesia, possible mechanisms for, 214216 Antagonists. of GnRH, structural requirements for, 172-173 Antibodies, to proteins of mitotic spindle, 254
Antigen, specific to centrioles and basal bodies, 254-255 Appendages of basal bodies, 237 of centrioles, 234, 246, 248 Argentaffinity, of neuroendocrine cells of lung, 43, 44-45, 51, 70 Arginine residue, importance for GnRH activity, 166-168 Argyrophilia, of neuroendocrine cells of lung, 43-44, 51 Atrioventricular junction neurosecretory cells, cytological analogies with sensory neurons, 113-1 14 terminal nerve plexus, adrenergic neurons in. 105-1 I I sensory neurons in. 114-1 19 Autoimmune diseases. antigen of centrioles and basal bodies and, 254255 Autonomic ground plexus. of terminal nerve plexus of heart parasympathetic postganglionic fibers, 122-125 sympathetic postganglionic fibers, 120121 Autonomic nervous system, new developments in study of, 132-135
B Barbiturates, mechanism of action, 214216 295
296
INDEX
Basal bodies, 228 biochemistry of, 249-257 microtubule assembly and, 260, 265 occurrence of, 236 reorganization of, 252 ontogenesis of, 239-244 triplet microtubules of, 236-237 ultrastructure of, 229-239 Bioenergetics, crisis in, 185-187 Biology, nonlinear phenomena in, 192-194 excitons, 194-196 solitons, 196-199 Birds, GnRH in, 156-158. 163-164 Bombesin, in neuroepithelial cells. 36, 47, 53. 54-55, 67
Brain. extrahypothalamic GnRH in, 154155
C
Calcitonin. neuroepithelial cells and, 53-54 Calcium meiotic arrest and, 10-12. 27-28 muscle contraction and, 206 Calmodulin in centrioles, 254 meiotic arrest and. 12 Catecholamines chromafin cells and, 126, 128 induced fluorescence of neuroepithelial cells and. 48-49. 51, 74 sensory neurons of A V junction and. I19 CellW centrosome and, 272-276 metabolically active, laser Raman scattering, 64, 213-214 organization and behavior of centrosome in. 244-249 Centriolar cylinder, 228 function of, 235 Centrioles biochemistry of, 249-257 definition of. 228 duplication of, 247 functions attributed to, 228 functions of, 235-236 localization and orientation in cells. 276280
microtubule assembly and, 260-262, 266-267
occurrence of, 236 ontogenesis of, 237-244 ploidy and, 273-274 ultrastructure of, 229-239 Centrosome cell and, 272-276 definition of, 228 microtubule concentration control and. 270
organization and behavior in cell cycle, 244-249
Chemical mediators, in pulmonary clear cells, 36 Cholecystokinin. in lungs. 54 Cholera toxin, 18 germinal vesicle breakdown and, 4 Cholinesterase activity, in neuroendocrine cells of lung, 45-46 Chromafin cells, of autonomic ground plexus of heart, 125-129 Cilia basal bodies and, 232 on neuroepithelial cells, 57 primary, centriole and, 235 Conformational restraint, GnRH bioactivity and, 168 Connectives of basal bodies, 237 of centriolar microtubules, 233, 243 Cumulus differentiation, oocyte maturation and, 21-23
OM1 and, 7 Cumulus-oocyte complexes, permeability of. meiotic resumption and, 19-21 Cyclic adenosine monophosphate meiotic arrest and, 3-5, 6, 11, 25 potentiating factors, 5-6 oocyte maturation and, 13-14 Cytasters, formation of, 275-276 Cytoplasm, microtubular network in. 269 Cytoskeleton. intracellular movement and, 208
D
Denervation, of intracardiac ganglion cells, 97. 106
Dense-cored vesicles, in neuroepithelial cells, 62-65, 67, 68-71
297
INDEX Deoxyribonucleic acid, in basal bodies, 255-256, 257 Depolymerization, of microtubules, 259260. 268 Diacylglycerol. meiosis and, 27-28 Dibutyryl CAMP germinal vesicle breakdown and, 3, 4, 6, II
oacyte maturation and, 13, 14
E Electron microscopy, of neuroepithelial cells in lung, 55-71 Electron transfer, along protein molecules, 216221 Electrosolitons, pairing in soft molecular chains, 22 1-223 Energy transport. along protein molecules. new concept of, 187-189 Enolase, neuron-specific, of neuroendocrine cells. 46-47, 67 Estradiol oocyte maturation and, 15, 17 meiotic arrest and. 6 Excitons, nonlinear phenomena in biology and. 194-196
F Fertilization, microtubule assembly and, 274-275 Fetal bovine serum. oocyte maturation and. 22-23 Fish, GnRH in, 161-162, 165 Fluorescence chromaffin cells and. 126, 128 induced, in neuroepithelial cells, 48-53, 74 in sympathetic ganglia. 104 Follicle stimulating hormone, oocyle maturation and, 13. IS, 17, 18. 20. 22-23. 24 Follicular fluid. 23 meiotic arrest and, 5 , 6-7 Forskolin germinal vesicle breakdown and, 4 oocyte maturation and, 13-14
G
Ganglionic cells intracardiac adrenergic, 104- I05 cholinergic or adrenergic choice, 104I05 cytological analogies between sensory neurons and neurosecretory cells of atrioventricular junction, 113I I4 evidence for intrinsic adrenergic neurons in terminal nerve plexus of AV junction, 105-1 I I evidence for intrinsic ganglionic cells. 96-98 evidence for sensory neurons in terminal nerve plexus of AV junction. 114-1 19 regional specialization of intrinsic nerve plexus of interatrial septum, 98- I03 in sinoatrial ring bundle, 98, 100-101 Gap junction, oocyte maturation and, 1920 Gastrin-releasing peptide GnRH and, 162 in lungs, 54, 67 Glucosaminogl ycans follicular, oocyte maturation and, 23-25 oocyte maturation and. 14. 21, 22-23 Glutamine, in chicken GnRH, 157 Glycoproteins, cell functions and. 210 Golgi complex, of neuroepithelial cells, 60, 65
Gonad(s). GnRH in. 155-156 Gonadotropin(s). resumption of meiosis and. 13-15 Gonadotropin-releasing hormone biological activity of structure-activity relationships, 166I73 vertebrate interspecific activities, 163I66 extrapituitary sources, 165-166 historical background, 149-150 oocyte maturation and, I5 related molecules 162-163 structure. distribution and related molecular forms interspecific heterogeneity. 156-163
298
INDEX
in mammalian tissues, 154-156 prohormonal forms, 150-154 Granules of chromaffin cells, 126 in neuroepithelial cells, 42, 62, 6 5 4 7 , 68 Granulosa cell(s), oocyte maturation inhibitor and, 7 Granulosa cell factor meiotic arrest and, 8-10, 25, 27 properties of, 8-10
H Heart autonomic innervation of general organization, 91-92 parasympathetic, 93-95 sympathetic. 92-93 morphological basis of rhythmical activity of, working hypothesis, 135-137 terminal nerve plexus of, 119-120 autonomic ground plexus, 120-125 chromaffin cells, 125-129 interstitial cells, 129-131 Heartbeat, mechanism of, historical background, 89-91 a-Helix, excited states in, 192 Heparin, oocyte maturation and, 21, 23 Hub, of centriole, 233. 234, 243 Human chorionic gonadotropin, oocyte maturation and, 13, 17, 21 Hyaluronic acid, in follicular cells 2425
I Irnmunocytochemistry, for regulatory peptides, in neuroepithelial cells, 53-55 Immunoreactivity, of prohormonal GnRH, 151
Innervation of heart general organization, 91-92 parasympathetic, 93-95 sympathetic, 92-93 of neuroepithelial cells 73-79 Inositol I , 4, 5-triphosphate, meiosis and, 27, 28 Interatrial septum, intrinsic nerve plexus, regional specializations of, 98-103 Interphase. centrosome and, 246 Interstitial cells, of autonomic ground plexus, 129-131 Intracellular dynamics, solitons and, 207213 Intrinsic nerve plexus, of interatrial septum, regional specializations of, 98I03 Invertebrates, GnRH in, 162 3-lsobutyl-l-methylxanthine,germinal vesicle breakdown and, 4 Isolation, of centrioles and basal bodies 250-252
K Kinetochores, microtubule assembly and, 267. 271-272
5-H ydroxydopamine
chromaffin cells and, 126 neurons of atrioventricular junctions and, 106-1 1 I postganglionic parasympathetic fibers of autonomic ground plexus of heart and, 122 5-Hydroxytryptamine, in neuroepithelial cells. 36, 43, 44, 47, 48, 49-53, 54, 67. 69, 70 Hypothalamus, GnRH in, 154 Hypothetical scheme, of oocyte maturation, 25-28 Hypoxanthine, meiotic arrest and, 5, 7, 25 Hypoxia, neuroepithelial cells and, 6970
L Laser Raman scattering, by metabolically active cells, 213-214 Leucine residue, GnRH bioactivity and, 168-169 Light microscopy, of neuroepithelial cells, 39-42 Living organisms, fundamental characteristics of. 183-184 Lung, location of neuroepithelial cells, in 71-73 Luteinizing hormone meiotic arrest and, 8, I 1 resumption of meiosis and, 13-15. 20, 22. 27
INDEX
M
299
definition of, 227-228 microtubule assembly and, 262-265 properties of, 264 regulation of, 272-274 Mitochondria, nerve endings on neuroepithelial cells, 77-78 Mitosis centriolar localization and, 279-280 microtubule organization and, 271-272 Mitotic halo, RNA in, 256-257 Molecular chains, soft, electrosoliton pairing in, 221-223 Motility, cellular, centriolar localization and, 278 Muscle contraction, molecular mechanism. solitons and, 2 W 2 0 7 Myosin filaments, within cells, 209-210
Mammalian tissues, gonadotropin-releasing hormone in extrahypothalamic in brain, 154-155 gonadal, 155-156 hypothalamic, 154 other tissues, 156 Mammals, interspecific activity of GnRH in, 163 Matrix, of centriole, 233 Maturation promoters, in v i m versus in V ~ \ W ,17-18 Maturation-promoting factor, production by oocytes, 18 Meiosis, factors promoting resumption of gonadotropins, 13-15 gonadotropin-related hormones, 15 maturation-promoters in vitro versus in i d w , 17-18 N maturation-promoting factor, 18 regulation of meiotic competence, 16-17 Neuroepithelial cells (bodies) Meiotic arrest. factors sustaining, 2-3 argentafinity and argyrophilia of, 4 3 4 5 CAMP, 3-5 aspects of induced fluorescence in, 48CAMP potentiating factors, 5-6 53 calcium, 10-12 cholinesterase activity of, 45-46 granuloma cell factor, 8-10 electron microscopy of, 55-71 oocyte maturation inhibitor, 6-7 immunocytochemistry for regulatory Meiotic competence, regulation, 16-17 peptides in, 53-55 Meiotic resumption, mechanism of innervation of, 73-79 cumulus differentiation, 21-23 light microscopic aspects, 39-42 follicular glucosaminoglycans. 23-25 location of. 71-73 hypothetical scheme of, 25-28 neuron-specific enolase of, 46-47 permeability of cumulus-oocyte comNeuropeptide y plexes, 19-21 action on heart, 133 Metaphase, centrosome in, 244-245 neurons of atrioventricular junction and, Microtubule(s) 108 assembly Neurosecretory cells, of atrioventricular in viiro. 257-265 junction, in v i w , 265-272 cytological analogies with sensory neubasal body formation and, 240 rons. 113-1 14 centriole and, 233 Neurotransmitters, cardiac function and, centriole formation and. 240, 242. 243 98 disposition, cell type and, 271 Nonlinear phenomena, in biology, 193Microtubule-nucleatingcenter, 227 I94 definition of, 228 excitons. 194-196 in interphase, 246, 248 solitons, 1%-199 microtubule assembly and, 260 Norepinephrine Microtubule-organizingcenters in autonomic ground plexus of heart, 121 assembly of microtubules on in superior cervical ganglion. 104 in tifro. 257-265 Nucleus, centriolar localization and, 277in vivio, 265-272 278
300
INDEX
0
Oncogene product, oocyte maturation and, 18
Oocyte maturation factor, 25 follicular fluid and, 7 Organelles, in neuroepithelial cells, 62
P Parasympathetic innervation, of mammalian heart afferent pathways, 94-95 efferent pathways, 93-94 Parasympathetic postganglionic fibers, of autonomic ground plexus of heart, 122- I25 Peptidases, prohormonal GnRH and, 152 Peptides, regulatory, immunocytochemistry of, 53-55 Pericentriolar satellite, 228 components of, 238 mitosis and, 245 Pineal gland, GnRH in, 154-155 Pinwheel, of centriolar cylinder, 233-234 Pituitary, desensitization to GnRH, 173 Placenta, GnRH in, 156 Plasma membrane, meiotic arrest and, 1213 Preganglionic fibers, uninterrupted, to heart. 92-93 Primers, in microtubule-organizing centers, 268 Progesterone, oocyte maturation and, 15 Prohormonal forms, of gonadotropin-releasing hormone, 150-154 Prolactin, GnRH and, 162 Prolactin-like substance, oocyte maturation and, 7 Prophase, centrioles and, 248 Protein(s) bioenergetics and, 187 a-helical, solitons in. 199-200 infrared spectra, 199 exciton states and. 195-196 Protein molecules electron transfer along, 216-221 energy transport along, new concept of, 187- I89 Protein synthesis, oocyte maturation and, 4-5
Pulmonary clear cells historical background, 35-39 nomenclature and, 36-38 Purine phosphorylase, in centrioles. 254
R Radiation, electromagnetic, cell activity and, 211-212 Receptors cardiac, 94-95 for GnRH. 165-166, 167 Reptiles, GnRH in, 158-159, 164 Ribonucleic acid, in basal bodies, 256-257 Ribonucleic acid synthesis, in oocytes, 16 Rootlets, of basal bodies, 237
S Satellites, of basal bodies, 238 Sensory neurons cytological analogies with neurosecretory cells of AV junction, 113-1 14 in terminal nerve plexus of AV junction, 114-1 I9
Serotonin, in atrial cells, 128 Sinoatrial ring bundle, ganglion cells in. 98, 100-101 Solitary waves history of observation of, 189-191 properties of, 191 Soliton definition of, 188 in discrete models: numerical calculations, 201-204 intracellular dynamics and, 207-213 molecular mechanism of muscle COntrdCtion and, 204-207 nonlinear phenomena in biology and, 196-199 in real a-helical protein molecules, 199200 stability of, 198-199 Somatostatin, in lungs, 54 Spermatozoa, flagellum formation in, 236 Staining, of neuroepithelial cells, 41-42 Sterocilia. formation of, 235-236 Steroids, oocyte maturation and. 15, 20 Striated rootlets microtubule assembly and, 260 proteins of, 255
301
INDEX
Struct ure-acti vit y relations of GnRH, for gonadotropin release effects of conformational restraints. I68
pituitary desensitization, 173 postion eight amino acid, 166-168 positions five and seven, 168-169 structural requirements for antagonists, 172-173 structural requirements for superactive agonists. 169-172 Substance P, action of on heart. 133-134 Sympathetic innervation, of mammalian heart afferent fibers. 93 efferent pathways, 92-93 Sympathetic postganglionic fibers, of autonomic ground plexus of heart, 120-121
Terminal nerve plexus of atrioventricular junction, sensory neurons and, 114-119 of heart, 119-120 autonomic ground plexus, 120-125 chromaffin cells. 125-129 interstitial cells, 129-131 Tubulin in basal bodies and centrioles. 252-253 conditions for polymerization of, 258259
Tyrosine residue, GnRH bioactivity and, I69
V Vagotomy, innervation of neuroepithelial cells and, 7, 8 Vasoactive intestinal peptide autonomic nervous system and, 132 in heart. 133
T
Telophase, centrioles and, 245-246 Testosterone, oocyfe maturation and. 6. 7
Y Yeast. a-mating factor, GnRH and, 162
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