Calcitonin Gene-related Peptide (CGRP) Andrew W. Taylor* Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114, USA * corresponding author tel: (617) 912-7452, fax: (617) 912-0113, e-mail:
[email protected] DOI: 10.1006/rwcy.2002.1306.
SUMMARY Calcitonin gene-related peptide (CGRP) is a 37 amino acid long neuropeptide with one intrachain disulfide link. It exists as two isoforms encoded by two separate genes with no discernible distinction in their activities. CGRP and its isoforms are conserved evolutionarily and can be interchanged in functional experiments. CGRP, a product of sensory nerve endings in CNS and periphery, mediates vasodilation and may have an important role in mediating inflammatory edema. It inhibits dendritic cell activation of TH1 cells and the inflammation associated with autoimmune disease and endotoxin-activated macrophages. CGRP may also have a role in regulating the development and activation of T lymphocytes.
BACKGROUND
Discovery CGRP was discovered initially as an mRNA splice variant of calcitonin (CT) (Rosenfeld et al., 1993). Antibodies to the C-terminus of the amino acid sequence predicted by the splice variant revealed the presence of CGRP protein in neural fibers. The second isoform was discovered when chromosomal fragments were probed and two hybridization products were detected (Steenbergh et al., 1985). The second CGRP gene lacks CT introns and thus produces CGRP directly. Antibodies against the predicted amino acid sequences of CGRP I and II
Cytokine Reference
protein, which differ from each other in humans by three amino acids, were used to quantify their presence in tissues of the central nervous system, pituitary, and thyroid (Petermann et al., 1987).
Alternative names CGRP I is also known as CGRP and CT/CGRP; CGRP II is also known as CGRP.
Structure Most is understood about the protein sequences of CGRP of humans and rats. The primary structure of CGRP is known to have one disulfide chain that links the Cys2 to Cys7 (Tippins et al., 1986). This is evolutionarily conserved between the isoforms and is also found in the sequences for CGRP in rats and chickens. The amino acid sequences differ by three amino acids between isoforms and between species (Tippins et al., 1986; Petermann et al., 1987). These amino acid substitutions do not affect the affinity or the biological activity of CGRP.
Main activities and pathophysiological roles The most commonly described pathophysiologic function of CGRP is its vasodilatory activity (Brain et al., 1985; Beglinger et al., 1991). This regulation of blood flow in tissues may be important in early stages
Copyright # 2002 Published by Elsevier Science Ltd
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of inflammation. Another pathophysiologic function of CGRP is its suppression of macrophage and dendritic cell activation (Nong et al., 1989; Hosoi et al., 1993; Taylor et al., 1998; Liu et al., 2000). This activity of CGRP prevents macrophages and dendritic cells from inducing and responding to adaptive immunity (Nong et al., 1989; Hosoi et al., 1993; Asahina et al., 1995a; Niizeki et al., 1997; Kitazawa et al., 2000). This immunosuppressive activity of CGRP promotes antigen-specific tolerance (Kitazawa et al., 2000). In addition, CGRP regulates the development of T cells (Boudard and Bastide, 1991; Bulloch et al., 1998). These activities indicate that the main function of CGRP is to regulate dilation of blood vessels and suppress immunity in peripheral tissues.
GENE AND GENE REGULATION
Accession numbers Human CGRP I: XM_030255 Human CGRP II: XM_030255 Rat CGRP I: M11597 Rat CGRP II: M11596 Mouse CGRP I: AF330212 Mouse CGRP II: NM_054084
Chromosome location Human chromosome 11 for both isoforms.
Relevant linkages On the same arm of chromosome 11 in humans are insulin, insulin-like growth factor II, c-ras-1, and the parathyroid hormone genes. Although close in association, linkages between CT/CGRP and other genes have yet to be identified.
ribonucleoprotein (sRNP) complexes that bind to an intron enhancer element found between the fourth and fifth exon of the primary CT/CGRP I mRNA transcript. It is considered that the sRNP causes recognition of the fourth exon and enhances the poly(A) site at the end of the exon. There is then cleavage of the fifth±sixth exon of the mRNA and calcitonin mRNA is generated. The CGRP I mRNA is the default transcript of the CT/CGRP I mRNA in the brain where the fourth exon is cleaved out of the primary transcript.
Cells and tissues that express the gene The mRNA transcripts of CGRP are found in the trigeminal ganglion, neural fibers of the midbrain, hypothalamus, lateral medulla, amygdala, and pituitary (Rosenfeld et al., 1983; Jonas et al., 1985). CGRP mRNA is detected in most cranial nuclei, in the ventral horn of the cervical and lumbar regions of the spinal cord, and the dorsal root ganglion (Rethelyi et al., 1989).
PROTEIN
Accession numbers Human CGRP I: XP_030255 Human CGRP II: XP_030255 Rat CGRP I: AAA40847 Rat CGRP II: AAA40850 Mouse CGRP I: AAK06841 Mouse CGRP II: NP_473425
Sequence See Figure 1 for comparison between human and rat CGRP I and II sequences.
Regulatory sites and corresponding transcription factors
Description of protein
There is no understanding of the regulation of CT and CGRP gene expression; however, since the expression of CGRP mRNA is limited to neurons it can be speculated that the regulation is through tissue-specific promoters. There is a clear understanding that the rearrangement of the CT/CGRP I mRNA (Lou and Gagel, 1998) is regulated by small
There is in all CGRP molecules a disulfide bond in the N-terminus between C2 and C7. This disulfide chain appears to be important for CGRP functionality (Tippins et al., 1986; Juaneda et al., 2000). Loss of this sequence of amino acids produces an antagonist of CGRP bioactivity, even though the disulfide loop is not the receptor recognition site.
Calcitonin Gene-related Peptide (CGRP) 3 Figure 1 Amino acid comparison of human (hu) and rat (r) CGRP isoforms. Differences in the amino acid sequences are indicated relative to the amino acid sequence of human CGRP I. Conserved sequences are indicated with a hyphen (-). There is a conserved disulfate bond between Cys2 and Cys7.
hu CGRP I hu CGRP II r CGRP I r CGRP II
1 2 3 4 Ala Cys Asp Thr Asn Ser Asn Ser Asn -
5 6 Ala Thr -
7 8 9 Cys Val Thr -
10 11 His Arg -
hu CGRP I hu CGRP II r CGRP I r CGRP II
14 15 Gly Leu -
20 21 22 Gly Gly Val Met -
23 24 25 26 Val Lys Asn Asn Ser Asp Asp -
27 hu CGRP I Phe hu CGRP II r CGRP I r CGRP II -
16 Leu -
17 Ser -
18 Arg -
28 29 Val Pro -
30 Thr -
31 32 33 34 Asn Val Gly Ser -
Important homologies Only the precursor proteins of calcitonin and CGRP contain domains of identical sequences. Since the mRNA processing excludes the exons for CGRP in the calcitonin mRNA transcript and excises out the calcitonin exon to create CGRP transcripts, there is little protein sequence homology between the bioactive proteins. CGRP is extremely homogeneous between species and between the two isoforms (Jonas et al., 1985).
19 Ser -
12 Leu -
13 Ala -
35 36 37 Lys Ala Phe Glu -
CELLULAR SOURCES AND TISSUE EXPRESSION
Cellular sources that produce CGRP is produced by sensory fibers of the central nervous system and in sensory termini throughout the body (Franco-Cereceda et al., 1987; Petermann et al., 1987; Dalsgaard et al., 1989; Kurz et al., 1995). Nonneural sources of CGRP have not been found, with the exception of a medullary thyroid carcinoma described as a source of CGRP II (Petermann et al., 1987).
Posttranscriptional modifications A CGRP precursor peptide is proteolytically processed before CGRP is stored in neural vesicles (Rosenfeld et al., 1983). The precursor peptide is an 81 amino acid N-terminal peptide of CGRP that is identical to the N-terminal peptide of calcitonin. In addition, there is a C-terminal pentapeptide. The mature CGRP peptide is freed of the N-terminal and C-terminal peptides. There is no evidence that CGRP is glycosolated. The molecular weight of bioactive CGRP I is 3786.91 Da and CGRP II is 3790.89 Da.
Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Any sensory stimulation will release CGRP from sensory nerve endings. CGRP is released by UV irradiation, neurogenic stimulation, and capsaicin treatment of skin (Franco-Cereceda et al., 1987; Niizeki et al., 1997; Sauerstein et al., 2000). There is a constitutive level of CGRP in mouse eyes which
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reflects a tonic release of CGRP from neural endings of the iris and ciliary body (Uusitalo et al., 1989; Taylor et al., 1998). Capsaicin treatment of tissues results in up to 70% depletion of CGRP from sensory nerve endings (Franco-Cereceda et al., 1987).
RECEPTOR UTILIZATION There are four putative G protein-coupled receptors for CGRP (CGRPr) (Juaneda et al., 2000). CGRPr1 has been cloned. It is a receptor complex of the CGRP-binding protein associate with RAMP1 (receptor-associated membrane protein 1) or with RCP (receptor component protein). This receptor has equal affinity for CGRP I and II. A second CGRP receptor, CGRPr2, is reported based on its sensitivity to the agonistic activity of a linearized CGRP analog; CGRPr1 is insensitive to the agonistic analog. CGRPr2 also has equal affinity for CGRP I and CGRP II. The two receptors are expressed on different tissues. CGRPr1 is expressed by atrium, pulmonary, spleen, and neuroblastoma cells. CGRPr2 is expressed by vas deferens, urinary bladder, liver, and colonic epithelium-derived cells. The other receptors are the amylin and adrenomedullin receptors. Each of these have higher affinity for their respective ligands but also bind CGRP I and II at a lower affinity than CGRPr1 and CGRPr2. The association of CGRP bioactivities to the expression of specific CGRP receptors has yet to be determined.
IN VITRO ACTIVITIES
In vitro findings In vitro assays demonstrate an immunosuppressive and anti-inflammatory role for CGRP. CGRP induces apoptosis in Con A-stimulated thymocytes (Bulloch et al., 1998). In addition, CGRP suppresses Con A-stimulated proliferation of splenocyte T cells without inducing apoptosis or suppressing IL-2 production (Boudard and Bastide, 1991; Bulloch et al., 1998). Moreover, there is no evidence that CGRP affects the activation of primed or armed T cells (Taylor et al., 1998). Neurons of the thymus and spleen contain immunoreactive CGRP, suggesting that CGRP could be involved in a potential neuroregulatory mechanism of T cell development. CGRP suppresses endotoxin-induced generation of reactive oxide intermediates and nitric oxide by macrophages (Taylor et al., 1998). CGRP induces
IL-10 synthesis while suppressing IL-12 synthesis by macrophages and dendritic cells (Kitazawa and Streilein, 2000; Liu et al., 2000). Dendritic cells treated with CGRP prior to their adoptive transfer in vivo induce antigen-specific tolerance to both delayed-type hypersensitivity and contact hypersensitivity reactions (Hosoi et al., 1993; Asahina et al., 1995a). Thus, in vitro assays demonstrate CGRP suppression of antimicrobial and proinflammatory activity of macrophages and dendritic cells. Therefore, CGRP interferes with antigen presentation to T cells, possibly leading to immune tolerance. In vitro, CGRP is an immunosuppressive and antiinflammatory neuropeptide.
Regulatory molecules: Inhibitors and enhancers A potent antagonist of CGRP binding to its receptors is the amino acid sequence of CGRP 8±34 (Juaneda et al., 2000; Kitazawa and Streilein, 2000). It antagonizes CGRP binding to CGRPr1 and CGRPr2. A linearized analog of CGRP is a potent agonist of only CGRPr2 (Juaneda et al., 2000).
Bioassays used The most widely used bioassay for CGRP is to measure cAMP production in any cell that is receptive to CGRP (Boudard and Bastide, 1991; Krootila et al., 1991; Asahina et al., 1995b; Juaneda et al., 2000; Liu et al., 2000). For immune cells, defined cultures of macrophages and dendritic cells are treated with CGRP and endotoxin (Nong et al., 1989; Taylor et al., 1998; Kitazawa et al., 2000; Liu et al., 2000). The effects of CGRP are detected by measuring any changes in reactive oxygen intermediates, nitric oxide, or cytokine production. Further assessment of CGRP activity is assayed using the CGRP 8±34 antagonist and anti-CGRP antibodies.
IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS
Normal physiological roles Nerve termini in many tissues contain CGRP. Upon stimulation, UVB exposure, pain sensor activation, or
Calcitonin Gene-related Peptide (CGRP) 5 capsaicin treatment, CGRP is released and significant vasodilation follows (Franco-Cereceda et al., 1987; Niizeki et al., 1997; Franco-Cereceda and Liska, 2000; Sauerstein et al., 2000). In some tissues, such as the eye, the stimulated release of CGRP from neurons or the injection of CGRP into the ocular microenvironment breaks the blood barrier (Unger et al., 1985; Oksala and Stjernschantz, 1988; Krootila et al., 1991). Such findings of vasoactivity have suggested that CGRP is a mediator of inflammation. However, the vasodilation can only be achieved by injecting CGRP in excess of physiological concentrations. In addition, stimulating the nerves to release CGRP also releases other vasoactive neuropeptides and nitric oxide that enhance the vasoactivity in the tissues (Franco-Cereceda et al., 1987; Dalsgaard et al., 1989; Wang et al., 1996; Sauerstein et al., 2000). Other experiments have suggested that the normal physiological role of CGRP is to suppress immunity. The injection of a CGRP antagonist blocked antigenspecific tolerance mediated by CGRP in UVBexposed skin (Kitazawa and Streilein, 2000). Transgenic expression of CGRP by pancreatic betacells in NOD mice suppressed in vivo the production of inflammatory lymphokines by autoreactive T cells and suppressed the incidence of diabetes (Khachatryan et al., 1997). Therefore, the normal physiological role of CGRP is to suppress immunemediated inflammation by promoting tolerance to peripheral tissue antigens.
Species difference None ± the CGRP family of ligands and receptors are evolutionarily conserved in mammals.
Transgenic overexpression There is only one report of transgenic overexpression of CGRP (Khachatryan et al., 1997). The CGRP gene was placed under the insulin promoter to target the beta-cells of insulitis-prone NOD mice. The incidence of diabetes in the transgenic mice was suppressed by 68% in females and 100% in males. The composition of infiltrating immune cells in the prediabetic pancreas was not affected by the localized expression of CGRP by the beta-cells. This suggested that the locally elevated levels of CGRP in the pancreas prevented T cell production of lymphokines that would lead to destructive inflammation.
Pharmacological effects Intradermal injection of CGRP results in a localized reduction in the density of Langerhans cells in the skin (Niizeki et al., 1997). This reduction of antigenpresenting cells results in a suppression of contact hypersensitivity at the CGRP injection site. There is also a report that injections of CGRP suppress gastric acid production (Beglinger et al., 1991). Neurological studies on rats have demonstrated that the effects of CGRP on CNS processing of information are analogous to the actions of dopamine (Van Rossum et al., 1997).
Interactions with cytokine network UVB radiation-induced immune tolerance is mediated by a cytokine network initiated by CGRP (Niizeki et al., 1997). The UVB radiation release of CGRP from cutaneous nerve endings induces mast cell release of TNF that in turn mediates the loss of Langerhans cells from the skin and this suppresses contact hypersensitivity.
PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY
Normal levels and effects Normally, the end process of sensory neurons in all tissues contain CGRP protein as detected by immunohistochemical staining. CNS tissues constitutively express CGRP from 0.03 to 34 pmol/g of tissue, with the highest levels of CGRP in the spinal cord (Petermann et al., 1987). The pituitary and thymus contain CGRP I (1.6 and 1.51 pmol/g respectively) and CGRP II (76 and 0.2 pmol/g). The levels of CGRP I and II are not significantly different, except in the pituitary. Skin has a basal release level of about 5 pM of CGRP, which doubles with neural stimulation (Sauerstein et al., 2000). Serum levels of CGRP are approximately 50 pM (Ashina et al., 2000).
Role in experiments of nature and disease There is an elevation in the concentration of CGRP in cerebral spinal fluid of humans with chronic
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headaches (Sarchielli et al., 2001). This is in contrast to individuals with chronic peripheral pain resulting from osteoporosis, herniated lumbar disks, or hip fractures where the CSF levels of CGRP are lower than average (Lindh et al., 1999). There is no change in peripheral blood CGRP levels in either group. Vitreoretinopathy elevates CGRP levels in ocular aqueous humor (Troger et al., 2000). Infarction of the heart releases CGRP from the C-fibers of the coronary arteries (Franco-Cereceda and Liska, 2000). The released CGRP appears to attenuate the myocardial infarction through CGRP-mediated vasodilation. There are four identified polymorphisms of the human calcitonin/alpha-CGRP gene (Buervenich et al., 2001). One of the polymorphisms is an intronic 16 base pair microdeletion that cosegregates with unipolar and bipolar depressive disorders in one family.
References Asahina, A., Hosoi, J., Biessert, S., Stratigos, A. and Granstein, R. D. (1995a). Inhibition of the induction of delayed-type and contact hypersensitivity by calcitonin gene-related peptide J. Immunol. 154, 3056±3061. Asahina, A., Moro, O., Hosoi, J., Lerner, E. A., Xu, S., Takashima, A., and Granstein, R. D. (1995b). Specific induction of cAMP in langerhans cells by calcitonin gene-related peptide: relevance to functional effects Proc. Natl Acad. Sci. USA 92, 8323±8327. Ashina, M., Bendtsen, L., Jensen, R., Schifter, S., Jansen-Olesen, I., and Olesen, J. (2000). Plasma levels of calcitonin gene-related peptide in chronic tension-type headache Neurology 55, 1335± 1340. Beglinger, C., Born, W., MuÈnch, R., Kurtz, A., Gutzwiller, J. P., JaÈger, K., and Fisher, J. A. (1991). Distinct hemodynamic and gastric effects of human CGRP I and II in man Peptides 12, 1347±1351. Boudard, F., and Bastide, M. (1991). Inhibition of mouse T-cell proliferation by CGRP and VIP: effects of these neuropeptides on IL-2 production and cAMP synthesis J. Neurosci. Res. 29, 29±41. Brain, S. D., Williams, T. J., Tippins, J. R., Morris, H. R., and MacIntyre, I. (1985). Calcitonin gene-related peptide (CGRP) is a potent vasodilator Nature 313, 54±56. Buervenich, S., Xiang, F., Sydow, O., Jonsson, E. G., Sedvall, G. C., Anvret, M., and Olson, L. (2001). Identification of four novel polymorphisms in the calcitonin/alpha-CGRP (CALCA) gene and an investigation of their possible associations with Parkinson disease, schizophrenia, and manic depression Human Mutat. 17, 435±436. Bulloch, K., McEwen, B. S., Nordberg, J., Diwa, A., and Baird, S. (1998). Selective regulation of T-cell development and function by calcitonin gene-related peptide in thymus and spleen Ann. N.Y. Acad. Sci. 840, 551±562. Dalsgaard, C.-J., Jernbeck, J., Stains, W., Kjartansson, J., Hñgerstrand, A., Hokfelt, T., Brodin, E., Cuello, A. C., and Brown, J. C. (1989). Calcitonin gene-related peptide-like
immunoreactivity in nerve fibers in the human skin: relation to fibers containing substance P-, somatostatin-, vasoactive intestinal polypeptide-like immunoreactivity Histochemistry 91, 35±38. Franco-Cereceda, A., and Liska, J. (2000). Potential of calcitonin gene-related peptide in coronary heart disease Pharmacology 60, 1±8. Franco-Cereceda, A., Henke, H., Lundberg, J. M., Petermann, J. B., HoÈkfelt, T., and Ficher, J. A. (1987). Calcitonin generelated peptide (CGRP) in capsaicin-sensitive substance P-immunoreactive sensory neurons in animals and man: distribution and release by capsaicin Peptides 8, 399±410. Hosoi, J., Murphy, G. F., Egan, C. L., Lerner, E. A., Grabbe, S., Asahina, A., and Granstein, R. D. (1993). Regulation of Langerhans cell function by nerves containing calcitonin generelated peptide Nature 363, 159±163. Jonas, V., Lin, C. R., Kawashima, E., Semon, D., Swanson, L. W., Mermod, J. J., Evans, R. M., and Rosenfeld, M. G. (1985). Alternative RNA processing events in human calcitonin/calcitonin gene-related peptide gene expression Proc. Natl Acad. Sci. USA 82, 1994±1998. Juaneda, C., Dumont, Y., and Quirion, R. (2000). The molecular pharmacology of CGRP and related peptide receptor subtypes Trends Pharmacol. Sci. 21, 432±438. Khachatryan, A., Guerder, S., Palluault, F., Cote, G., Solimena, M., Valentijn, K., Millet, I., Flavell, R. A., and Vignery, A. (1997). Targeted expression of the neuropeptide calcitonin gene-related peptide to cells prevents diabetes in NOD mice J. Immunol. 158, 1409±1416. Kitazawa, T., and Streilein, J. W. (2000). Hapten-specific tolerance promoted by calcitonin gene-related peptide J. Invest. Dermatol. 115, 942±948. Krootila, K., Uusitalo, H., and Palkama, A. (1991). Intraocular and cardiovascular effects of calcitonin gene-related peptide (CGRP)-I and -II in the rabbit Invest. Ophthalmol. Vis. Sci. 32, 3084±3090. Kurz, B., VonGaudecker, B., Kranz, A., Krisch, B., and Mentlein, R. (1995). Calcitonin gene-related peptide and its receptor in the thymus Peptides 16, 1497±1503. Lindh, C., Liu, Z., Welin, M., Ordeberg, G., and Nyberg, F. (1999). Low calcitonin gene-related, peptide-like immunoreactivity in cerebrospinal fluid from chronic pain patients Neuropeptides 33, 517±521. Liu, J., Chen, M., and Wang, X. (2000). Calcitonin gene related peptide inhibits lipopolysaccaride-induced interleukin-12 release from mouse peritoneal macrophages, mediated by the cAMP pathway Immunology 101, 61±67. Lou, H., and Gagel, R. F. (1998). Alternative RNA processing ± its role in regulating expression of calcitonin/calcitonin generelated peptide J. Endocrinol. 156, 401±405. Niizeki, H., Alard, P., and Streilein, J. W. (1997). Calcitonin gene-related peptide is necessary for ultraviolet B-impaired induction of contact hypersensitivity J. Immunol. 159, 5183± 5186. Nong, Y. H., Titus, R. G., Ribeiro, J. M. C., and Remold, H. G. (1989). Peptides encoded by the calcitonin gene inhibit macrophage function J. Immunol. 143, 45±49. Oksala, O., and Stjernschantz, J. (1988). Effects of calcitonin generelated peptide in the eye Invest. Ophthalmol. Vis. Sci. 29, 1006± 1011. Petermann, J. B., Born, W., Chang, J. Y., and Fisher, J. A. (1987). Identification in the human central nervous system, pituitary, and thyroid of a novel calcitonin gene-related peptide and partial amino acid sequence in the spinal cord J. Biol. Chem. 262, 542±545.
Calcitonin Gene-related Peptide (CGRP) 7 Rethelyi, M., Metz, C. B., and Lund, P. K. (1989). Distribution of neurons expressing calcitonin gene-related peptide mRNAs in the brain stem, spinal cord, and dorsal root ganglia of rat and guinea-pig Neuroscience 29, 225±239. Rosenfeld, M. G., Mermod, J. J., Amara, S. G., Swanson, L. W., Sawchenko, P. E., Rivier, J., Vale, W. W., and Evans, R. M. (1983). Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing Nature 304, 129±135. Rosenfeld, M. G., Mermod, J. J., Amara, S. G., Swanson, L. W., Sawchenko, P. E., Rivier, J., Vale, W. W., and Evans, R. M. (1993). Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing Nature 304, 129±135. Sarchielli, P., Alberti, A., Floridi, A., and Gallai, V. (2001). Levels of nerve growth factor in cerebrospinal fluid of chronic daily headache patients Neurology 57, 132±134. Sauerstein, K., Klede, M., Hilliges, M., and Schmelz, M. (2000). Electrically evoked neuropeptide release and neurogenic inflamamtion differ between rat and human skin J. Physiol. 529, 803±810. Steenbergh, P. H., , HoÈppener, J. W. M., Zandberg, J., Lips, C. J., and Jansz, H. S. (1985). A second human clacitonin/CGRP gene FEBS Lett. 183, 403±407. Taylor, A. W., Yee, D. G., and Streilein, J. W. (1998). Suppression of nitric oxide generated by inflammatory macrophages by calcitonin gene-related peptide in aqueous humor Invest. Ophthalmol. Vis. Sci. 39, 1372±1378. Tippins, J. R., DiMarzo, V., Panico, M., Morris, H. R., and MacIntyre, I. (1986). Investigation of the structure/activity relationship of human calcitonin gene-related peptide (CGRP) Biochem. Biophys. Res. Commun. 134, 1306±1311. Troger, J., Kremser, B., Stockl, T., Kralinger, M., Schmid, E., Kunze, C., and Kieselbach, G. F. (2000). Elevated levels of
calcitonin gene-related peptide in aqueous humor of patients with proliferative vitreoretinopathy Graefes Arch. Clin. Exp. Ophthalmol. 238, 237±242. Unger, W. G., Terenghi, G., Ghatei, M. A., Ennis, K. W., Butler, J. M., Zhang, S. Q., Too, H. P., Polak, J. M., and Bloom, S. R. (1985). Calcitonin gene-related polypeptide as a mediator of the neurogenic ocular injury response J. Ocul. Pharmacol. 1, 189±199. Uusitalo, H., Krootila, K., and Palkama, A. (1989). Calcitonin gene-related peptide (CGRP) immunoreactive sensory nerves in the human and guinea pig uvea and cornea Exp. Eye Res. 48, 467±475. Van Rossum, D., Hanisch, U. K., and Quirion, R. (1997). Neuroanatomical localization, pharmacological characterization and functions of CGRP, related peptides and their receptors Neurosci. Biobehav. Rev. 21, 649±678. Wang, Z. Y., Alm, P. and HaÊkanson, R. (1996). The contribution of nitric oxide to endotoxin-induced ocular inflammation: interaction with sensory nerve fibers Br. J. Pharmacol. 118, 1537±1543.
LICENSED PRODUCTS Bioactive peptides and antagonists of CGRP I and II can be obtained through Penninsula Laboratories (Bachem AG), www.penlabs.com U.S. Patents #4,549,986 and #4,530,838 (Salk Institute).
