Neuroanatomical Localization, Pharmacological Characterization and Functions of CGRP, Related Peptides and Their Receptors

NeuroscienceandBiobehavioralReviews,Vol.21,No. 5, pp. 649-678, 1997 CopyrightO 1997Publishedby ElsevierScienceLtd Printedin GreatBritain, All rightsreserved 0149-7634/97~32.C41 + .00

Pergamon @

PII: S0149-7634(96)00023-1

NeuroanatomicalLocalization,Pharmacological CharacterizationandFunctionsof CGRP, Related PeptidesandTheirReceptors DENISE VAN ROSSUMa’b, UWE-KARSTEN

HANISCH”C AND ~MI

QUIRION”*

aDepartments of Pharmacology

and Therapeutics, and Psychiatry, McGill UniversiV, Douglas Hospital Research Centre, Verdun, Qukbec, Canada H4H 1R3. bpresent a&iress: Max-Planck-Institut fi”r Himforschung, 60528 Frankfurt a, M., Germany Cpresent address: Max-Delbriick-Centrum @”rMolekrdare Medizin, 13122 Berlin, Germany

VAN ROSSUM, D. U.-K. HANISCH AND R. QUIRION. Neuroanatomical localization, pharmacological characterization and jimctions of CGRP, related peptides and their receptors. NEUROSC1 B1OBEHAV REV 21(5)649-678, 1997.—Cafcitonin generelated peptide (CGRP) is a neuropeptide discovered by a molecular approach over 10 years ago. More recently, islet amyloid polypeptide or amyfin, and adrenomedullin were isolated from human insulinoma and pheochromocytoma respectively, and reveafed between 25 and 50% sequence homology with CGRP. This review discusses findings on the anatomical distributions of CGRP mRNA, CGRP-like immunoreactivity and receptors in the central nervous system, as well as the potential physiological roles for CGRP. The anatomical distribution and biological activities of amylin and adrenomedrdlirrare also presented. Based upon the differentirdbiological activity of various CGRP analogs, the CGRP receptors have been classified in two major classes, namely the CGRP1 and CGRP2 subtypes. A third subtype haa also been proposed (e.g. in the nucleus accumbens) as it does not share the pharmacological properties of the other two classes. The anatomical dishibution and the pharmacological characteristics of amylin binding sites in the rat brain are different from those reported for CGRPbut share several similarities with the safmon calcitorrirrreceptora. The receptors identified thus far for CGRP and related peptides belong to the G protein-coupled receptor superfamily. Indeed, modulation of adenylate cyclase activity following receptor activation has been reported for CGRP, arnylin and adrenomedullin. Furthermore, the binding affinity of CGRP and related peptides is modulated by nucleotides such as GTP. The cloning of various cafcitonin and most recentfy of CGRP1 and adrenomedullin receptors was reported and reveafed structural similarities but afso significant differences to other members of the G protein-coupled receptors. They may thus form a new subfamily. The cloning of the arnylin receptor(s) as well as of the other putative CGRP receptor subtype(s) are still awaited. Finally, a broad variety of biologicrd activities has been described for CGRP-like peptides. ‘flrese incIude vasodilation, nociception, glucose uptake and the stimulation of glycolysis in skeletat muscles. These effects may thus suggest their potential role and therapeutic applications in migraine, subarachnoid hemorrhage, diabetes and pain-related mechanisms, among other disorders. O 1997 Elsevier Science Ltd. CGRP

amylin

cafcitonin

adrenomedullin

receptor subtype

1. INTRODUCTION

CALCITONIN gene-related peptide (CGRP) is a 37 amino acid peptide generated from the alternative splicing of the calcitonin gene. Two forms have thus far been isolated, namely CGIWa and CGRP6 (also called CGFW-I and CGRP-11). CGRP shares about 25% sequence homology with calcitonin and adrenomedullin and about 5090 with amylin. Since the discovery of CGFW, several groups have reported on the presence of CGRP mRNA as well as of CGRP-like immunoreactivity in both central and peripheral nervous systems. A wide variety of biological effects on various tissues including the brain, heart, smooth and skeletal muscles have been reported for CGRP. These

rat brain

effects are mediated by the activation of specific plasma membrane receptor sites. The pharmacological characterization of these binding sites revealed different affinities and potencies for CGRP, calcitortin, amylin, adrenomedullin and related artalogues and fragments, suggesting the existence of receptor subtypes for each peptide. The main goal of this review is thus to summarize recent findings with respect to this family of peptides, from their anatomical distribution in the central nervous system and that of their respective binding sites, to the biological effects and potential pathophysiological roles in brain as well as in selected peripheral tissues. The reader should also refer to previous reviews for complementary information (154,275,338).

*Mailing address: Dr. R&ni Quirion, Douglas Hospital Research Centre, 6875 boul. Laaafle, Verdun, Qu6bec, Canada H4H 1R3. Tel: 514-762-3048 Fax: 514-762-3034.

650

VAN ROSSUM ET AL.

1.1. Calcitonin

gene-related

peptide

(CGRP)

splicing process: CGRPCYvs. calcitonin The calcitonin/CGRP gene is expressed in specific cell types of both the endocrine and nervous systems. The gene is alternatively spliced to yield MRNA encoding calcitonin in thyroid C-cells or the neuropeptide CGRP in a subset of central and peripheral neurons (7,244,293,294). The rat as well as the human calcitonidCGRP gene consists of six exons (Fig. 1). The calcitonin MRNA contains exons 1 to 4 with a poly(A) tail at exon 4 whereas CGRW includes exons 1,2,3,5 and 6 with a poly(A) tail at exon 6. Structural comparison of human calcitonin and human or rat CGRP peptides revealed -25% homology. Interestingly, srdmon calcitonin exhibits a greater structural similarity to human CGRP than to human calcitonin (Fig. 2). The high homology between these peptides suggests that calcitonin and 1.1.1. Alternative

Exonl

Exon2 Exon3

CGRP exons are derived from a common ancestral gene and that the calcitonin/CGRP gene arose from duplication and sequence divergence events (212). The high degree of homology (86–89%) between chicken and human CGRP suggests that CGRP is a phylogenetically well conserved peptide. The expression of the calcitonin/CGRP gene is regulated by a number of hormones and second messengers including glucocorticoids (351) and cAMP (384). Leff et al. (204) have proposed that the specific calcitonin splicing is the “default” pathway and that CGRP-specific splicing requires a dominant neuronal trans-acting factor. A good candidate for this factor is a protein component of the spliceosome, SMN, as this protein is expressed in only a limited range of tissues and cell types, such as the brain and the heart (198,204). The cell types that express SMN are essentially those that are capable of correctly splicing the calcitonird CGRP transcript to produce CGRP MRNA. However, the

Exon4

Exon5

Cakitonin

CGRP

Exon6

5’

3’

Tranecriptkxr Cleavage Pdyadenytatkn mRNA aptlcing

a$!!l&

‘A”’AAA

Cakltonln mRNA 5’

CGRPmRNA 3’

5’

3’

POlyA

PotyA

Translation

Caicitoninprecurrjor

poattransiationel Procaaein9

CGRP precuraor

N-terminal and C-terminai peptidae P

32aa-Caicitonin

A

1

37aa-CGRP 1

FIG. 1. Alternative rnRNA spIicing of the catcitorsirrgene generating calcitonin and CGRP in a tissue-spexific manner.

RECEPTORS

OF CGRP AND RELATED PEPTIDES

651

human CGRPa

AC D T A T C V T H R L A G L L S R S GG V V K N N F V P T N V G S K A F-NH2

human CGRPB

‘ - N-----

rat CGRPa

S- N--

rat CGRPB

S: N- --

t

t

rat amyiin

K- N--

aalmon cakitanht

cw--

human calcitanin

cw-

1 –------------;

M--S

----------------: ----

------D- ---

-‘-

-------

‘ --

------NH2 -‘-‘-

D- -----

E‘ ---

‘-NH2

‘--NH2

I

--

A- Q---

N F- V H- SN N FG A I L S S----S0 E- HK LQ T*

●

* * * Y P R--

M LG T Y TO D F NK F H T*

●

* *

LGK-

G: R FG- : T VQ K--

●

N T Y-NH2 T--

G T P-NH2

- PQ- A I - VG - P-NH2

HQ i YQ FTD K D- D- VA-

R S K i - PQG Y-NH2

human ADM Y RQ SM NN FQG L R S F FIG. 2. Amino acid sequence of CGRP and related peptides. Amino acids in common to human CGRP are indicated by dash (-). Spaces inserted to allow for sequence comparison tie indicated by a star (A).

inclusion of exon 4 is also a complex event which involves calcitonin-specific uridine branch acceptor and the poly(A) tail of exon 4 as well as a regulatory element within exon 4 (2,33,58,83). The full identification and/or combination of the factors involved for each route thus remains to be determined in order to clearly establish the specificity of CGRP/calcitonin RNA splicing. 1.1.2. CGRP@ An mRNA product of a related gene was identified in rat brain and thyroid, encoding the protein precursor of a peptide differing from CGRPa by only one or three amino acids in rat and human, respectively. The mRNA encoding this peptide was referred to as CGIW3 (8). The second gene encoding this second CGRP peptide was isolated from a cosmid library and was shown not to be subject to the mechanism of alternative gene expression as no second calcitonin peptide was produced. Consequently, only the CGRP peptide could be derived from this second gene (324). Overall, the distribution of the hybridization signal for either CGRPcI or CGRP/3 was similar in the central and peripheral nervous systems (8). However, some evidence suggests that these two CGRP-related peptides and their respective receptors can be differentially distributed in certain tissues such as primary sensory neurons (248), intrinsic enteric neurons (326) and the human hypothalamus (136). This differential expression pattern would result from a selective regulation of the CGRP genes. For example, dibutyryl cAMP was reported to predominantly stimulate the expression of the CGIW3 gene in thyroid carcinoma cell line (384). Moreover, the levels of mRNA for CGRPCYwere increased whereas that of the CGIW3 mRNA was decreased or unaltered in rat spinal motoneurons that were subjected to either axotomy, crushing or axonal flow blockade (175,270,299). Therefore, these reports suggest that these two peptides maybe differentially regulated. It has also been shown that CGRIYI can have distinct biological activities such as its ability to suppress gastric

acid secretion (22,23). Moreover, Jansen (160) has shown that the relaxation induced by CGRPa, but not that of CGRFY3,in the guinea pig basilar artery was antagonized by the CGRP antagonist, CGRp&qT. These findings suggest that the a and 6 forms of CGRP may act through different CGRP receptor subtypes although further studies will be necessary to confirm this interesting possibility. 1.1.3. CGRP structure All species variants of CGRP have 37 residues, a phenylalanylamide C-terminal and a disulfide bridge between positions 2 and 7 (Fig. 2). Circular dichroism and nuclear magnetic resonance studies have indicated that CGRP has a solvent-dependent tendency to adopt an a-helical secondary structure. Indeed, a significant increase in the a-helical content of rat CGRP, varying from 20% to 60%, in purely aqueous medium over buffer containing 5090 trifluoroethanol (TFE) respectively, has been observed (36,217,221). The influence of organic solvent such as TFE on the secondary structure of peptides is thought to be consistent with a role for the membrane lipid–water interface in stabilizing an active conformation of the peptide at the receptor level. Under these conditions, an amphiphilic a-helix conformation betweenresidues 8–18, two@urn structuresbetween residues 17–21 and 29–34 and a random-coil segment formed by residues 23–29 of CGRP have been suggested (146,300). Moreover, the structural characterization of various fragments of hCGRPa such as hCG~8.sT and hCGRPlg.37,as well as the linear analo e, [acetamidomethyl-cysteine2’7]hCGRPa([Cys(ACM)R‘ ]hCGRPa) lead to the suggestion that the residues 1–7 as well as the disulfide bridge have an important role in the stabilization of the a-helix whereas the C-terminal residues 19–37 do not (241). It thus appears that the amphiphilic a-helix identified between residues 8-18 plays a role in the interaction of the molecule with the receptor whereas the N-terminal loop region may principally be involved in triggering the ensuing signal transduction process (49,69,240).

652

I

VAN ROSSUM ET AL.

[IVSITU HYSRIDIZATK)N

II

IMMUNOHISTOCHEMISTRY

I

RECEPTOR BINDING

FIG. 3. a Summary of CGRP mRNA (+, first column), CGRP-like immunoreactive fibers (0, second column), CGRP-like immunoreactive cell body (A, second column) and CGRP binding sites in rat brain (~, tbird column). Abbreviations: Acb, accumbens nucIerrs;AOP, anterior olfactory nucleus; BST, bed nucleus of the stria terminals; Cg, cingulate cortex; CPU,caudate putamen (striatum); fmi, forceps minor of the corpus callosum; FStr, fundus striati; LS, laterat septat nucleus; MPA, medial preoptic area; Pir, piriform cortex; TT, tenia tecta.

1.2. Amylin Amylin, or islet amyloid polypeptide (IAPP), was isolated from amyloid fibrils of an insulin-secreting human tumor (377,378) and from pancreases of type 2 diabetic patients (55). It was found to be a major constituent of the amyloid deposits seen in islets of type 2 diabetic (noninstdin dependent) patients (55) [for recent reviews, see (25,57)]. Amylin is produced by pancreatic P-cells where it is co-localized (3,246) and co-released with insulin in response to glucose (87,171). In addition to the pancreas, amylin rnRNA and amylin-like immunoreactivity have been detected in the stomach, gastrointestinal (GI) tract, lung and dorsal root ganglia (15,89). Moreover, small but significant amounts were also detected in the brain (42). This recently isolated peptide is closely related to CGRP as the human form of amylin shares 4990 sequence homology with hCGRP/3. It thus seems likely to share a common ancestral gene with CGRP (292). Indeed, strong similarities are observed between amylin and CGRP, including a conserved intramolecular disulfide bridge at the 5’ coding region as well as an arnidated carboxyl terminal residue (Fig. 2). These structural features are essential, for the full biological activity of amylin on skeletal muscle, as a linearized, or nonamidated, analogue was much less active in this assay (292). Strong amyloidogenic properties have been associated with the human and cat forms of amylin but not with its

rodent counterparts (257,379). The interspecies variation in the amino acid sequence of residues 25–29 of amylin was shown to play a major role in amyloid deposition in the islets of humans. Secondary structure studies by circular dichroism spectroscopy revealed fundamental differences in the structures adopted by amylin from human and rat species as highly organized u-helical conformation vs. limited structural organization were observed, respectively (233). Indeed, human, but not rat amylininduced toxicity was reported in &cells of the adult pancreas of rats and humans (214) as well as in rat primary hippocampal cultures (230). However, the role of amylin in pathologies such as type 2 diabetes remains to be clarified.

1.3. Adrenomedullin Adrenomedtdlin (ADM) is a peptide discovered a few years ago by monitoring the elevation of activity of platelet cAMP inhuman pheochromocytoma (179). It belongs to the CGRP/calcitonin peptide family as it possesses approximately 2570 homology with CGRP (179). The human form consists of 52 amino acids and has one intramolecular disulfide bond between amino acid residues 16 and 21 (Fig. 2). Both the mRNA and the peptide were detected in several tissues including the adrenal medulla, ventricle, lung and kidney (149,180,182). Only low amounts (0.3 fmol

RECEPTORS

[

653

OF CGRP AND RELATED PEPTIDES

IN SfTIJHYBRIDIZATION

IMMWIOHISTOCHEMISTRY

I

I

-1,4C

FIG. 3. b See legend Figure 3a.Abbreviations: AHA, anterior hypotbatarnic area; APir, amygdatopiriform transition area; Arc, arcuatehypothalamic nucleus; BL, basolateral amygdrdoid nucleus; CG, centrrd periaqueductal gray; Cl%, caudate putamen (striatum); DG, dentate gyms; DM, dorsomediat hypothalamic nucleus; GP, globus patlidus; Hb, habcnular; La, lateral amygdaloid nucleus; Me, medial amygdaloid nucleus; MG, medial geniculate nucleus; MP, medial mammillarv. nucleus: PaAP...uaraventricular hyuotbalamic nucleus; Pir, ririform cortex; Re, reuniens thalamic nucleus; RPC, red nucleus; SNR, substantial .. nigra; Te, temporal cortex; VM, ventromediat tbalamic nucleus. “

mg–l tissue wet weight) of immunoreactive ADM have been detected thus far in human brain cortex (149). Moreover, significant amounts of ADM have been detected in blood circulation (181). The main biological effect reported thus far for ADM is its potent vasodilating properties. Indeed, intravenous injections of ADM elicit a potent and long lasting reduction in blood pressure and an increase in pulmonary blood flow in rats (158,179). This effect is mainly due to direct vasodilatation (158). The intravenous injections of the fragment human(h)ADM13.52 also adrenomedullin decreased systemic vascular resistance in a dose dependent manner, suggesting that the entire adrenomedullin molecule is not required to induce biological activit (210). However, 5? removal of the C-terminal residue (Tyr ) or the linearizalead to tion of hADM ([carbamoylmethyl-Cys IG’21]hADM) a significant decrease in both receptor affinity and cAMP response (81). Interestingly, the C-terminal fragment hADM22.52did not reveal any agonistic effect but showed weak antagonistic effects in vascular smooth muscle cells (81). Based on the peptide and tissue distribution, potential biological role(s) of ADM can be speculated in other organs such as lung and kidney but remains to be defined. LOCALIZATION 2. NEUROANATOMICAL

CGRP is one of the most broadly distributed peptides in

nerve tissues in both vertebrates and invertebrates. The detailed map of the discrete distribution of CGRP mRNA, CGRP immunoreactive cell bodies and nerve fibers have been reported in various species, including rat, cat and human brains, and is summarized in the following sections. 2.1. CGRP mRNA containing neurons as revealed by in situ hybridization Thus far, few laboratories have mapped the distribution of CGRP mRNA in neurons of the rat central nervous system using in situ hybridization histochemistry (8,191, 286,287). The distribution of neurons expressing CGRP mRNAs is described here according to these reports and summarized in Fig. 3a–c, (first column). The highest densities of neurons expressing the CGRP mRNA have been observed in the rostral part of the lateral hypothalamic area, as well as in the lateral portion of the ventral and dorsal parabrachial nuclei. Moreover, high amounts of CGRP mRNA positive neurons have been seen around the subparafascicular nucleus, the posterior thalamic nuclear group, the peripeduncular nucleus and in the lateral subparafascicular nucleus. The parabigeminal nucleus and the lateral superior olive nucleus are also densely labeled. Moderate numbers of CGRP mRNA-positive neurons have been found in the bed nucleus of the stria

654

VAN ROSSUM ET AL.

Mf SITU HYBRIDIZATION

RECEPTOR SINDING

-7.64

.$.30

-11.96 FIG. 3. c See legend Figure 3a. Abbreviations: 2, cerebehr lobules; 10, cerebella lobuIes; Amb, ambiguus nucleus; CG, central periaqueductal gray; CIC, central nucleus of the inferior colliculus; DR, dorsal raphe nucleus; DTg, dorsat tegmental nucleus; IO, inferior olive; LL, lateral lerrrniscus;LPGi, lateral paragigantocellular nucleus; M05, motor trigeminat nucleus; PB, parabrachial nuclei; PBG, parabigeminal nucleus; Pn, pontine nuclei; Pr5, principal sensory trigeminal nucleus; PrH, prepositus hypoglossal nucleus; RtTg, reticulotegmental nucleus of the pens; SO, supraoptic nucleus; Sp5, spinal trigeminal nucleus; SUG, superficial gray layer of the superior colliculus; Ve, vestibukwnucIei.

terminals, the nucleus of the lateral olfactory tract, the arcuate hypothalamic nucleus, the thalarnic ventroposterior nucleus and the parafascictdar nucleus. The lateral lemnisCUS,the reticular paragigantocellular nucleus and the external cuneate nucleus also contained a moderate number of cell bodies expressing CGRP rnRNA. Few CGRP mRNA expressing neurons were reported in the medial preoptic nucleus, zona incerta, ventromedial thalamic nucleus, ventromedial hypothalamic nucleus, paracentral thalamic nucleus, pontine nuclei, central gray and in the reticular nucleus. With the exception of the dorsal motor nucleus of the vagus nerve (X), the presence of CGRP mRNA has been reported in all cranial nuclei. CGRP mRNA signals in most neurons of the facial, hypoglossal and ambiguus nuclei are intense (286,287). However, in the motor nucleus of the trigeminal nerve (V), only a small population of neurons with low levels of expression was observed. Almost all large neurons in the ventral horn of the cervical and lumbar regions of the spinal cord were labeled with the CGRP probe (286) whereas up to 75Y0of the motoneurons of the ventral spinal cord (laminae VII–IX) at cervical and thoracic levels (C5,T8) were found to express CGRP mRNA.

A large number of small and medium size neurons of the rat dorsal root ganglion, at all spinal cord levels also showed intense hybridization signals. Sections of the trigeminal ganglion revealed that more than 50% of the large perikarya expressed the CGRP gene. 2.2. CGRP-immunoreactive structures in the brain The distribution of irnmunoreactive CGRP in the rat central nervous system has been examined in detail by immunocytochemistry (145,176,192,293,318,356). Similar to CGRP rnRNA distribution, CGRP-immunoreactive neurons and fibers are widely but unevenly distributed in the central nervous system, as described in the following sections. The second column of Fig. 3a–c provides a summary. 2.2.1. CGRP-immunoreactive cell bodies A widespread distribution of CGRP-positive cells has been reported throughout the central nervous system. CGRP-imrnunoreactive cell bodies are present in various nuclei of the hypothalamus including the medial preoptic, periventricular and anterior hypothalamic nuclei. The perifomical area and the lateral hypothalamus-medial forebrain

RECEPTORS

OF CGRP AND RELATED PEPTIDES

bundle area as well as in the premammillary nucleus, medial amygdaloid nucleus, dentate gyms of the ventral hippocampal formation, ventromedial nucleus of the thalarnus, periventricular gray and parafascicular area, extending laterally over the lemniscus medialis are also enriched with specific CGRP staining. Moreover, CGRP-positive cells are found in the peripeduncular area, ventral to the medial geniculate body and the parabigeminal nucleus as well as the superior colliculus. In the hindbrain, positive cells are found in the central portion of the lateral and medial parabrachial areas as well as the ventral tegmental nucleus, inferior colliculus, lateral lemniscus, superior olive and nucleus of the solitary tract. All cranial nuclei of the rat brain have shown positive CGRP-immunoreactive cell bodies including the oculomotor (III), trochlear (IV), trigeminal motor (V), facial motor (VII), ambiguus (X) and hypoglossal (XII) nuclei. CGRP-labeled somata as well as numerous perisomatic fibers have been reported in various autonomic ganglia such as the ciliary, sphenopalatine, otic, glossopharyngeal-vagal and submandibular ganglia, which compose the cranial postganglionic parasympathetic pathways (133,314). In contrast, only a small proportion ( < IYo)of sympathetic principal neurons in the stellate and lumbar sympathetic ganglia are positive for CGRP immunoreactive-like peptide. Some of the neurons in the stellate and lower lumbar ganglia, which contain both CGRP and vasoactive intestinal peptide, project to the sweat glands (197). Furthermore, the sympathetic superior cervical ganglion lacked stained somata but exhibited CGRP-immunoreactive perisomatic axons. Such axons are probably of sensory origin, as few autonomic parasympathetic preganglionic axons contain CGRP-immunoreactivity (IR) and since in other sympathetic ganglia, similar perisomatic peptidergic nerve terminals are of somatic sensory origin (314). In contrast to the rat, the human superior cervical ganglion cells contained CGRP colocalized with tyrosine hydroxylase-positive cells (16). 2.2.2. CGRP-immunoreactive jbers Limited numbers of CGRP nerve fiber terminals can be seen in the olfactory bulb, as well as in the medial prefrontal cortex, whereas very few fibers are found in most neocortical areas, except the ventral sector of the piriform cortex and in the perirhinal cortex. Moderately dense networks of CGRP-positive fibers have been observed in various thalamic areas, septum, the bed nucleus of the stria terminals, central amygdaloid nucleus and in the caudal and ventral portions of the striatum. Several hypothalamic nuclei are highly enriched with CGRP-positive fibers, including the medial preoptic area, periventricular area, dorsomedial nucleus, median eminence and medial forebrain bundle area. In the lower brainstem, the highest concentrations of fibers are seen in superficial layers of sensory trigeminal areas, with a moderately dense fiber network in the periaqueductal central gray, medial geniculate body, parabigeminal nucleus, lateral lemniscus, dorsal part of the interpeduncular nucleus, parabrachial nucleus, superior olive, cochlear nucleus, nucleus of the solitary tract and parts of the vestibular nuclei. Notable levels of CGRP-like immunoreactive fibers are present in the anterior pituitary in the rat (123). Doubleimmuno-staining experiments revealed the nearly complete co-localization with substance Pin these nerve fibers (166).

655

CGRP fiber terminals are heavily concentrated in laminae I–II and in the reticulated region of lamina V of the dorsal horn of the rat spinal cord (116,318,319). The CGRPcontaining axons are largely unrnyelinated or smalldiarneter myelinated and constitute almost 30% of the primary afferent axons of the Lissauer’s tract, the major afferent input to the superficial laminae of the dorsal horn (116,208). Furthermore, over 80% of spinal afferent neurons supplying visceral structures including urogenital and upper gastrointestinal tracts are CGRP-immunoreactive (329). In these and other systems, CGRP-containing primary afferent fibers are frequently found around blood vessels. CGRP-immunoreactive fibers have also been noted around the central canal and in a number of motoneurons of the ventral horn (116,318). In the human brainstem, CGRP-irnmunoreactive fibers are concentrated in the spinal trigerninal nucleus, nucleus of the solitary tract and principal sensory trigeminal nucleus (356). A moderately dense fiber network can also be seen in the locus coeruleus as well as in the parabrachial nucleus. Few scattered CGRP-immunoreactive fibers are found in the central periaqueductal gray, in the pars reticulate of the substantial nigra and in the medial and lateral lemnisci. In the ventral horn of the human spinal cord, CGRP-IR is concentrated in lamina IX, especially a-motoneurons (356), confirming findings obtained earlier in other species (116). A few positively stained fibers and nerve cells were also seen in the cuneate and gracilis nuclei (356). 2.3. Mismatches Generally, the widespread distribution of CGRP mRNA synthesizing neurons, as revealed by in situ hybridization, is in agreement with the localization of the reported CGRPimmunoreactive cells. However, no CGRP mRNA has been detected in the dentate gyms or in various regions of the arnygdaloid body whereas numerous CGRP-immunoreactive cells have been found in these regions. Moreover, in situ hybridization studies did not correlate with findings of CGRP irnmunoreactivity in the inferior colliculus, pedunculopontine, tegmental and medullary raphe nuclei. On the other hand, in situ hybridization data revealed the presence of CGRP mRNA expression in the nucleus of the lateral olfactory tract whereas no CGRP-immunoreactive cells have been reported in this area. These apparent mismatches could result from differential turnover rate of the CGRP mRNA and peptide, respectively. The ready availability of mature CGRP vs its mRNA transcript might suggest another level of regulation between the direct vs modulatory effects by this peptide in various brain areas. These apparent mismatches could also be due to the differential sensitivity and specificity of the respective techniques used. Additional detailed studies are required to differentiate between anatomical mismatches and technical pitfalls. 2.4. Amylin-like immunoreactivity in the brain Only a few studies have reported positive signals for arnylin-like immunoreactivity in the brain. Initial studies failed to detect any amylin-like peptide in the central nervous system. Subsequently, Chance et al. (42) could detect picomolar amounts of the peptide, essentially in the

656

VAN ROSSUM ET AL.

hypothalamus. Most recently, amylin-like immunoreactivity was reported in wide areas of the rat brain including the cortex, anterior striatum, septum, hippocampus, magnocellular hypothalamic and arcuate nuclei, the habenula, mamillary body, substantial nigra, red and dorsal raphe nuclei, locus coeruleus, peripeduncular, parabigeminal, pontine, ventral tegmental and parabrachial nuclei, the superior olive and the Purkinje cells of the cerebellum (321). Moreover, positive amylin-like immunoreactivity was observed in motoneurons and most deeper laminae of the spinal cord (321). This wide distribution of amylin-like immunoreactivity does not entirely correlate with the distribution of amylin binding sites thus far reported in the rat brain (see 3.3.1). Furthermore, with regard to the broad distribution observed for amylin-like immunoreactivity, the specificity of the antibody used remains to be fully established and the reported distribution should be confirmed and extended to other species. 2.5. Adrenomedullin-like immunoreactivity in the brain Little is known thus far with respect to the distribution of adrenomedullin in the brain. Only one study reported the presence of low amount of ADM-like immunoreactivity in the human brain cortex, as measured by radioimmunoassay (149). Interestingly, Wang et al. (373) recently reported that the ADM mRNA expression was significantly increased (up to 21-fold) in the rat cortex, following focal brain ischemia. Immunohistochemical analyses of the ischemic cortical area also revealed increases in ADM-like immunoreactivity. The ADM-like immunoreactivity was co-localized with intermediate filament markers for neurons which suggest localization of the peptide in nerve fibre processes (373). No immunoreactivity was observed in sham-operated rats or outside the cortical ischemic zone. These data thus support an overall low basal expression level of ADM mRNA as well as of ADM-like immunoreactivity in rat brain. 3. RECEFTORDISTRIBUTIONAND CHARACTERIZATION 3.1. CGRP receptors 3.1.1. Distribution of CGRP receptor sites in the brain Several studies have demonstrated the anatomically discrete distribution of binding sites for CGRP in the central nervous system of various species, including rat and human (135,150,192,349). These binding sites have a characteristic distribution (Fig. 3a–c, third column) which is distinct from those previously re orted for other neuropeptide receptors. High levels of [ 18I]hCGRP binding have been reported in the nucleus accumbens, ventral striatum and tail of the caudate putamen, central and basolateral nuclei of the amygdala, superior and inferior colliculi, molecular and Purkinje cell layers of the cerebellum and the inferior olive. Moderate levels of labeling have been seen in the mammillary body, habenula, substantialnigra, medial geniculate nuclei, central gray, laterodorsrd tegmental nucleus, pontine nuclei, reticular formation, locus coeruleus and the vestibular nuclei. The dorsal motor nucleus of the vagus (X), lateral cuneate nucleus and nucleus of the solitary tract also show moderate levels of CGRP binding sites. In contrast, most cortical areas, the hippocampus formation,

the thalamus and most of the hypothalamic nuclei exhibited relatively low levels of specific CGRP binding sites. In the rat spinal cord, high densities of []251]hCGRPbinding sites have been observed in laminae I and X and the medial portions of larninae 111and IV, as well as in the intermediolateral and intermediomedial nuclei. The substantialgelatinosa (lamina II) contains relatively lower densities of [1251] hCGRP binding sites whereas the ventral horn is overall not enriched with specific CGRP labeling (296,388). 3.1.2. Mismatches The distribution of CGRP binding sites corresponds rather well with that of CGRP-immunoreactive perikarya and fibers but apparent mismatches are also seen. For example, in the thalamus and hypothalamus, two areas enriched with CGRP fibers, only low levels of binding sites have been reported. Moreover, in the inferior olive complex and the molecular layer of the cerebella cortex, little or no immunoreactivity for CGRP has been found in the adult rat brain whereas very high densities of CGRP binding sites have been located in these two structures (192). However, transient expression of CGRP-like immunoreactivity has been observed in the olivo-cerebellar fibers during ontogeny suggesting a role for CGRP in the cerebellum, at least at the neonatal stages in the rat (242,277). Species differences also exist in regard to CGRP-like immunoreactivity in cerebella afferents. For example, positive CGRP-like staining in the cat cerebella cortex is localized in mossy fibers arising from neurons in the lateral reticular, external cuneate and inferior vestibular nuclei, as well as from the basilar pens (32,331). In humans, the density of [1251] hCGRP binding sites has been reported to be high in the ventral part of the spinal cord whereas correspondingly low levels of endogenous CGRP have been quantified except in lamina IX (ci-motoneurons) (356). In contrast, high concentrations of CGRP-like peptides have been detected in the pituitary gland which is apparently devoid of []251]hCGRPbinding (349). Finally, differences are seen between rat and human CGRP levels in subcortical nuclei, such as the nucleus accumbens, striatum and amygdaloid body. Indeed, high levels of CGRP binding were detected in the rat whereas only low levels were measured in humans in these brain areas. Moreover, the arcuate nucleus of the human medulla oblongata, the inferior olive nuclei and the cerebellum contain higher densities of CGRP binding as compared to the rat (71,344). Overall, the respective distribution of CGRP mRNA, CGRP-like immunoreactivity and CGRP binding sites throughout the brain correlated rather well. The source of some of the minor discrepancies may relate to the sensitivity of the various assays as well as the species specificity of the CGRP probes used. Major discrepancies such as in the adult rat cerebellum still remain to be resolved. 3.1.3. Pharmacology and biochemist The pharmacological characteristics of CGRP receptors have been studied in a variety of tissues. The existence of high affinity binding sites for CGRP have been reported in the brain (70,390) including the cerebellum (46,252,390) as well as in various peripheral organs such as heart (47,70,390), liver (252), spleen (70,252), skeletal muscle (161,290,341), lung (220,355) and lymphocytes (232,354).

RECEPTORS

OF CGRP AND RELATED PEPTIDES

Apparent affinity (K~) values varied from 9 pM in whole brain (390) to 6.3 nM in skeletal muscle (341). The nature of the tissue appears to be the main source of variation in K~ values as the species apparently had little influence [e.g. (383)]. Cross-linking studies have been used in combination with various enzymatic digestions, to provide insights into the structure of the CGRP receptors in various tissue preparations. The first purification of a CGRP receptor, from human placenta (97), revealed a membrane-bound receptor with an estimated molecular mass of 240 kD composed of multiple 62 to 68 kD subunits. Thereafter, single binding components without apparent multi-units were isolated from the porcine spinal cord (70 kD; (301)), cultured rat vascular smooth muscle cells and bovine endothelial cells (60 kD; (138)), guinea pig gastric smooth muscle and pancreatic acinar cells (57 kD; (144)), rat cerebellum (67 kD; (48)) and rat lung (64 kD; (28)). Recently, Stangl et al. (323) solubilized CGRP binding proteins from the cerebellum, brainstem, spinal cord, liver and spleen, each having apparent molecular masses varying from 68 to 90 kD. An enzymatic N-deglycosylation converted all these CGRP labeled binding proteins into a common solubilized protein of 44 kD suggesting that post-transcriptional modifications were responsible for the apparent differential molecular weights (323). Other studies have reported the existence of more than one cross-linked molecular weight band in several tissues: porcine coronary arteries (90 and 70 kD; (301)), rat atrium (120 and 70 kD; (301)), porcine kidney (30,58 and 78 kD; (4)), rat liver (70 and 44 kD; (45)) and skeletal muscle (110 and 70 kD; (45)). In the human cerebellum, two CGRP binding proteins of 50 and 13.7 kD were identified by DottiSigrist et al. (73) whereas three specifically labeled binding proteins with apparent molecular masses of 60, 54 and 17 kD were reported by Stangl et al. (322). The latter study also revealed that the solubilized proteins were glycosylated since following treatments with endoglycosidase, it converted the 60 and 54 kD to 46 and 41 kD binding components respectively (322). It remains to be demonstrated if these various proteins correspond to differential post-translational processing of the same protein or to multiple CGRP receptor subtypes. Indeed, the comparative potencies of CGRP and analogs in various in vitro and in vivo functional assays have led to the suggestion of the existence of multiple classes of CGRP receptors (see sections 3.1.4; 3.1.5; 3.1.6). Full receptor cloning will provide definite proof in this regard. To our knowledge up to now, one CGRP receptor was most recently successfully amplified from canine genomic DNA (174). The isolated cDNA is 1.1 kb long and predicts a 362 amino acid protein of 41 kD as moleculm mass, in the unglycosylated state (Table 1; see Note added in proof). Evidence from several biochemical and pharmacological studies suggests that the CGRP receptors belong to the family of G protein-coupled receptors. Various reports have described the effect of GTP or its analogs on CGRP binding affinity. In the presence of guanine nucleotides, a reduction in agonist affinity is predicted for G proteincoupled receptors as these nucleotides induce the dissociation of G proteins from the receptor and hence promote the formation of a lower affinity state. Indeed, CGRP binding affinity is sensitive to GTP or analogs in a variety of tissue

657

preparations such as the cerebellum (46,357), atrium (357), lung (355), vas deferens (357) and skeletal muscle (290,341). Furthermore, CGRP was shown to activate muscarinic K+ channels in rat atrial cells (178) and to enhance Ca++ currents in nodose ganglion neurons (382) via a G protein as these effects were pertussis toxinsensitive. Similarly, CGRP increased transmembrane Ca++ currents in guinea pig myocytes and bullfrog atria via a G protein since the effects of CGRP were potentiated in the presence of GTP~S (264). More recently, following the solubilization of the CGRP receptors from rat cerebellum and Western blots of receptor preparations with antisera against a fragment of the G,a subunit, Chatterjee et al. (48) provided direct evidence for the association of these receptors with G,o. Most recently, the cDNA sequence of a CGRP receptor predicts a seven-transmembrane-domain protein somewhat similar to other G protein-linked receptors, hence confirming the biochemical data (174). It revealed about 30% sequence homology with the rat ADM receptor (see 3.4; (173)). These two receptors may form a new subfamily of the seven-transmembrane rhodopsin type superfamily. Following transection in COS-7 cells, CGRP and to a lesser extent ADM, induce a dose-dependent increase of cAMP. The pharmacological properties of this newly cloned receptor correspond to the proposed CGRP1 receptor subtype (see 3.1.4). 3.1.4. The putative CGRP1 receptor subtype One of the most exciting findings with respect to CGRP structure-activity studies is undoubtedly the reported potent antagonistic properties of C-terminal fragments such as CGRP8.~T(49), CGRPg.gTand CGRP12.~T(69,240). Shorter C-terminal fragments including CGRP1g.~7(297),CGRPZM, and [Tyr0]CGRpz8.J7(40,229) also showed competitive antagonistic properties but with much lower potency as compared to CGRP8.ST.However, a wide range of potency was reported for these C-terminal fragments as they were highly potent in some in vitro and in vivo assays (109,132,148,369) while being mostly inactive in others (70,86). For example, CG~&JT is able to potently block the inotropic and chronotropic effects of CGRP in the guinea pig atrium (pA2 -7.4) but was significantly less potent in antagonizing the relaxing effects of CGRP in the electrically stimulated rat vas deferens (pA2 -6.5) (70,218,240,280). Moreover, concentrations up to 3 pM of CGRP8.~Tdid not affect the relaxation produced by either the a or /3forms of hCGRP in the guinea pig urinary bladder or the actions of CGRP on electrogenic ion transport in human adrenocarcinoma cell line (CO1-29)(60,119). Upon central administration in the rat, CGRP8.JT was able to antagonize the analgesic and anorexic effects of hCGRPa (163). However, no significant effects were observed with respect to the hyperthermia induced by central injections of CGRP (163). Taken together, these results suggest the existence of multiple CGRP receptor subtypes in both central and peripheral tissues. The CGRP1 receptor class was thus proposed as the subtype highly sensitive to the antagonistic properties of Cterminal fragments (Table 1) (70,280). Although various reports support this hypothesis, a recent study could not reproduce these data: the fragment CGRp&’jTcould only act as a relatively weak antagonist in both the guinea pig atria and rat vas deferens with pA2 values ranging between 7.1

658

VAN ROSSUM ET AL.

and 6.6, respectively (213). These assays were performed in the presence of neutral endopeptidase inhibitor, thiorphan, which selectively increased the potency of the CGRP antagonist in the vas deferens tissue preparation. These data thus suggest that differential metabolism rate of these peptides in the absence of the endopeptidase inhibitor underly the wide range of antagonistic potency for the fragment CGRP8.~Tin different tissue preparations. On the other hand, a pA2 value of 9.3 in a neuroblastoma cell line was reported by the same group, which would also support a wide range of potency for the CGRP C-terminal fragment (213). Further studies are required to clearly discriminate between differential potencies and metabolic rates for Cterminal antagonistic peptides and to provide definite evidence as to the existence of the defined CGRP receptor subtypes. In an attempt to further characterize these putative receptor subtypes, we recently investigated the binding profile of the C-terminal CGRP receptor antagonist hCGRP8.37in its iodinated form, [1251-Tyr]hCGRP8.37, in membrane preparations from the atrium, vas de erens and brain of the rat (359). The binding of [ IZ$f I-Tyr]hCGRP8.s7 in these tissue preparations was saturable, and best fitted to a sin le class of high affinity binding sites (KD 7.5 to 21 X 10-?, M). Although revealing typical binding features for antagonist radioligand, like an apparent insensitivity to buffer constituents, such as cyclic nucleotides, [1251Tyr]hCGRP8.s7 apparently labeled all classes of CGRP receptor with high affinity (359). The pharmacological selectivity of this C-terminal fragment thus seems to be limited to functional assays as little preferential binding affinity was observed in the various tissue preparations studied. New CGRP antagonists with higher differential affinities for the various CGRP receptor subtypes are thus essential to establish the pharmacological characteristics and anatomical distribution of putative CGRP receptor subtypes in brain and peripheral tissues. Moreover, the study of CGRP receptors in tissue preparations such as the urinary bladder and adrenocarcinoma cell line which seem to be highly enriched with one population of CGRP receptor subtype would also be of interest, in order to define the pharmacological characteristics of each receptor class. Finally, the recent cloning of a CGRP receptor, sharing a

CGRP1-like profile of ligand selectivity, should help to verify and demonstrate the existence of an additional receptor subtype (174). 3.1.5. The putative CGRP2 receptor subtype The evaluation of the biological activity in various in vitro and in vivo assays of the linear CGRP analogue, [Cys(ACM)2’7]hCGRPrxalso generated interesting findings. Indeed, this CGRP analogue behaves as a relatively potent agonist in the vas deferens (EC5070 nM) while being less effective in the atrial preparations (EC50 >700 nM) (69, 280). Similary, Stangl et al. (323) recently reported that this linear analogue and hCGRPa produced hCGRP8.~7-insensitive increases in cAMP formation in the liver but failed to alter cyclase activity in the spleen, a well established effect of the full ‘cyclic’ CGRP peptides (323). [Cys(ACM)2’7]hCGRPa also failed to produce vasodilatation of the perfused renal vascular bed compare to pD2 values of 10 for both rat and hCGRPa (51). In addition, following its central administration, the CGRP linear analogue induced hyperthermia whereas no analgesic effects could be measured, in contrast to hCGRPa itself (163). The differential sensitivity to the linear agonist as well as the relatively low potency of the antagonist CGRP8.STin these preparations were thus suggested as the key properties of the CGRP2 receptor subtype (Table 1) (69,280). Cloning is still awaited to confirm the existence of this unique receptor subtype. 3.1.6. The putative atypical receptor subtype The existence of a unique population of CGRP/salmon calcitonin-sensitive binding sites in a few brain areas including the nucleus accumbens,jimdus striati, parts of the lateral bed nucleus of the stria terminals and of the central arnygdaloid nucleus of the rat brain have been reported (Table 1) (71,280,305). Salmon calcitonin (sCT) revealed nanomolar affinity for [1251]hCGRPabinding in these selected brain areas whereas micromolar affinities were observed in all other brain areas investigated (280). In a similar manner, rat am lin-NH2 competed with rather low affinity for most [ 12I]hCGRPa binding sites in the rat brain (150-300 nM)s except in the nucleus accumbens and fundus striati, two regions where rat amylin-NH2 demonstrated higher

TABLE 1 PHARMACOLOGICAL CHARACTERISTICS OF PUTATIVE CGRP AND RELATED PEPTIDES RECEPTOR SUBTYPES

Relatively selective agonists Relatively selective antagonists Prototypical assays

Receptor cDNA

Amylin

ADM

amylin

ADM

none

AC187

ADMZZ.SZ

nucleus accumbens

skeletat muscle

vascular smooth muscle

(?)

(’7)

395 amino acids 45 kD 7 putative transmembrane domains K~: 8 X 10-9M EC50:7 X 10”9M

Atypical

CGRP,

CGRPZ

none

[Cys(ACM)2’7]hCGRPa hCGRPci sCT amylin

hCGRP8.~T (pA2 5.5-6.5) vas deferens liver, kidney urinary bladder colonic epitheliums (HCA-7) hyperthermia (in vivo) 362 amino acids 41 kD 7 putative (?) (see Note added in prooj) transmembrane domains K~: 9 X 10-9M EC50:3 X 10”9M hCGRP8.37(pA2 7.5-8.5) hCGRP9.~7 atrium, spleen mesenteric artery SK-N-MC cells analgesia (in vivo)

RECEPTORS

OF CGRP AND RELATED PEPTIDES

affinities (20–50 nM) for specific [1251] hCGRP~ binding (358). In contrast to sCT and arnylin, adrenomedullin demonstrated its lowest affinity (0.9-18 pM) for both CGRP and amylin labeling in the nucleus accumbens (360). These binding data thus support the existence of an atypical class of CGRP receptors in the nucleus accumbens and thefindus striati as both sCT and arnylin demonstrated high affinity for these sites while comparable competition profiles failed to be observed in all other brain regions investigated. The nature of the endogenous ligand for these atypical receptors remains to be clarified as all three neuropeptides, CGRP, sCT and amylin possess high affinity for these sites. The cloning of the atypical receptor(s) is thus awaited to confirm the present binding data, as none of the receptors cloned thus far revealed these pharmacological characteristics. Overall, the differential biological activity of CGRP through its various putative receptor subtypes still largely remains to be defined. One could speculate that the effects of CGRP on the cardiovascular system are mainly through CGRPI as its action on the heart as well as blood vessels could be potently antagonized by the CGRp&qTfragment. Furthermore, the relaxation induced by CGRP of smooth muscle such as the urinary bladder and vas deferens appears to be largely mediated through the CGRP2 receptor subtype (69,119). Pharmacological tools specific for each putative receptor subtype would be of importance especially in clinical applications (see 5.5.1) where focused biological activity is desired. 3.2. Calcitonin receptors Few subtypes of the calcitonin receptor have already been cloned. Lin et al. (211) first reported the cloning of a calcitonin receptor from a porcine kidney epithelial cell line. Subsequently, calcitonin receptors were cloned from human ovarian carcinoma cell line (124), rat hypothalamus (258,306), rat nucleus accumbens (5) and mouse brain (387). Hydropathy plots analysis revealed multiple hydrophobic regions that could generate seven transmembrane spanning domains, similar to other G protein-coupled receptors (122,21 1). CGRP and amylin showed no or little affinity for any of these receptors, except for the porcine calcitonin receptor, where arnylin had similar or even higher efficacy than salmon and porcine calcitonin in stimulating the production of cAMP (52,307). Most recently, a novel calcitonin receptor-like protein has been cloned from the human cerebellum (94). Interestingly, despite sharing over 5090 sequence similarity with the various calcitonin receptors reported thus far, neither sCT or CGRP bound significantly to this receptor and none of the peptides tested including human amylin and adrenomedullin increased cAMP levels in the transected cells. The endogenous ligand of this receptor thus remains to be identified (94). Although the deduced amino acid sequences of the cloned calcitonin receptors suggest some characteristics of the G protein-coupled receptors such as the presence of seven hydrophobic regions, cloned calcitonin receptors do not possess significant sequence similarity ( < 12%) with previously reported members of this superfamily. On the other hand, a significant degree of homology (26–51%) was observed with the recently cloned parathyroid hormone and parathyroid hormone-related peptide (167), secretin

659

(155) and vasoactive intestinal peptide receptors (156). Hence, these receptors may constitute members of an emerging subfamily of G protein-coupled receptors. One of the most striking differences of this subfamily resides in the third cytosoplasmic loop (between helices V and VI) which is markedly shorter compared to corresponding regions of other adenylate cyclase-coupled receptors (122,211). This segment of the receptor is believed to be implicated in the capacity to interact with specific G proteins. Indeed, the calcitonin receptors activate two independent signal transduction pathways (adenylate cyclase and phospholipase C) presumably by interacting with distinct G proteins (39,41,98,122). These findings further support the unique properties of this new subfamily of G protein-coupled receptors. 3.3. Amylin receptors On the basis of the similarities between some of the biological actions of amylin and CGRP, the existence of common receptors for these two peptides was initially hypothesized. The affinity of amylin for [1251]CGRP binding was thus evaluated in a variety of preparations including the liver (45,105,243,395), skeletal muscle (45,105), brain (105,358,363,395) and L6 myocytes (276,395). However, in most preparations, the affinity of arnylin was much lower (30 to 100-fold) than that of CGRP, for specific [’251]CGRP binding. Moreover, while hCGRPg.37can potently antagonize the hypotensive and tachycardiac effects induced by arnylin and CGRP (51,110), their inhibitory activity on glycogen turnover in the soleus muscle (66) and their positive inotropic actions in the isolated guinea pig atrium (119), only relatively high concentrations (10-100 PM) of this antagonist were able to block the action of amylin in liver (243) and skeletal muscle (370) preparations. Furthermore, hCGRp8.37failed to antagonize arnylin’s actions on the isolated guinea pig urinary bladder and rat vas deferens (119). Rat amylin also showed relatively low potencies (micromolar concentrations) to induce a positive inotropic effect in the guinea pig atrium and to inhibit the electrically stimulated contraction of the rat vas deferens, two prototypical CGRP bioassays (Table 1) (280,358). Most recently, Beaumont et al. (21) reported the potent anta onistic properties of a new analogue, AC187 (ac-30Asn,4 yr,SCTg.qz). The inhibition by amylin of insulin-stimulated glucose incorporation into the rat soleus muscle glycogen was shown to be potently antagonized by AC187 and with much lower potency by salmon calcitonin&qz(sCT8.32)and CGRp&qT(21). This IM2Wpharmacological tool should thus be most helpful in delineating the effects of arnylin that are mediated through genuine amylin receptors. Few reports thus far have described the characterization of [1251] amylin binding sites (20,26,310,358,363). Specific [1251] amylin binding sites in lung membrane preparations from various species as well as in the stomach, spleen, liver and various regions of the brain have been characterized (26). Moreover, the recent solubilization and partial purification of a [1251] amylin receptor/binding protein from rat lung membranes further support the existence of a distinct (from CT or CGRP) class of amylin receptors (27). Crosslinking analysis lead to the identification of a 66 kD protein from the rat lung (29). However, the full pharmacological characterization of this newly isolated protein is awaited.

660 Amylin was reported to stimulate adenylate cyclase in an ovary cell line (CHO cells) through receptors that are distinct from calcitonin and CGRP receptor sites (65). Moreover, amylin inhibited the insulin release in perfused rat pancreas by modulating adenylate cyclase activity via a pertussis toxin-sensitive Gi protein (316). Taken together, these findings support the existence of unique receptors for amylin which most likely also belong to the G proteincoupled receptor superfamily. Cloning is awaited to confirm this hypothesis. 3.3.1. Distribution andpharmacology of amylin receptors in the rat brain We (358) and others (20,308,363) have recently examined the distribution and pharmacological characteristics of binding sites for rat amylin in the rat brain. High affinity [1251] amylin binding sites (KD27 pM) were first identified in nucleus accumbens membrane homogenates (20). By using in vitro receptor autoradiography, the discrete distribution of [1251] amylin binding sites became apparent (308,358). High to moderate levels of amylin binding sites were detected in the nucleus accumbens, olfactory tubercle, ji.mdus striati, vascular organ of the lamina terrninalis, amygdalostriatal transition zone, central and medial amygdaloid nuclei, various hypothalamic nuclei including the medial preoptic, dorsomedial and medial tuberal nuclei, dorsal raphe, tegmental and parabrachial nuclei, locus coeruleus and the nucleus of the solitary tract. The cerebellum is devoid of specific amylin binding sites (308,358). Competition experiments on nucleus accumbens membrane homogenates revealed that in addition to amylin, sCT also has high affinity for [1251] amylin binding sites while CGRP showed significantly lower affiniy (-14fold) (20). The nanomolar affinity of sCT for [12I]amylin binding sites was later observed for all brain areas enriched with amylin binding sites thus far investigated, whereas hCGRPcx demonstrated 5–35 times lower affinity depending upon the region examined (358). Furthermore, it is noteworthy that both [1251] BH-amylin and [1251]sCT binding sites are very similarly distributed in the brain (320). Further studies will thus be required to clarify the precise nature of BH-amylin the endogenous ligand acting on these [ 1251] binding sites, as it would appear that at least some of the previously characterized sCT binding sites were, in fact, the amylin receptor. 3.4. Adrenomedullin receptors Only a few reports are available on the characteristics of the ADM receptor. However, the recent cloning of an ADM receptor should allow for detailed molecular and pharmacological characterization of this protein in the near future (173). As anticipated, the cloned ADM receptor protein revealed seven putative transmembrane domains and belongs to the G protein-coupled receptor superfamily. Northern blot analysis of ADM receptor transcripts showed positive signals from the lung, adrenal, ovary, heart, spleen, brain cortex and cerebellum (173). This distribution correlates well with the distribution of ADM binding sites reported thus far (266). In addition to these tissues, high-affinity ADM binding sites were also detected in vascular smooth muscle cells (82), liver, skeletal muscle and spinal cord membranes (266).

VAN ROSSUM ET AL. The CGRP receptor antagonist, hCGRP8.37was shown to inhibit in a dose-dependent manner the ADM-induced increases in cAMP formation in vascular smooth muscle cells (82). In addition, the CGRP antagonist could inhibit the in vivo effects of ADMIs.sz on blood flow (131). Adrenomedullin also revealed high affinity (0.37 uM) for [’251]hCGRPabinding in neuroblastoma cells (SK-N-MC) (84). However, we could only detect limited affinity (0.218 PM) of hADM for either [1251]hCGRPaor [’251]BH-rat amylin binding sites in rat brain sections (360). It thus appears that ADM and CGRP receptors share several pharmacological features, at least in some tissues, but unique receptor classes for each peptide are also evident and now confirmed by cloning data (173,174). 4. SECONDMESSENGERS 4.1. CGRP Activation of adenylate cyclase and/or increases in intracellular cAMP induced by CGRP have been reported in many tissue preparations including rat liver (386), rat and guinea pig heart cells (93,157,371), rat skeletal muscle (10), rat astrocytic culture (201) and human neuroblastoma and gliomas (291,361). The relaxant effects of CGRP on various smooth muscle preparations such as rat thoracic and abdominal aorta (91,126,372), rat intracerebral arterioles (80), porcine coronary arteries (170) as well as the guinea pig gastric muscle cells (50) also appear to be partly or fully mediated through raises in cAMP. The precise mechanism underlying the relaxation of smooth muscle by intracellular cAMP production remains to be elucidated as cGMP is generally believed to be the major second messenger involved in vasorelaxation. Indeed, CGRP has been shown to induce both endothelium-independent and endotheliumdependent vasorelaxation. Reports on CGRP-induced relaxation indicate that in several tissue preparations, such as in cat middle cerebral artery (76), rat mesentery (9,209) and rat skin rnicrovasculature (281), the effects of CGRP are endothelium-independent. In contrast, CGRP effects in the rat aorta were reported to be, at least partly, endotheliumdependent (35,91,125,126). In agreement with CGRP endothelium-dependent effects, inhibitors of nitric oxide synthase such as NG-nitro-L-arginine methyl ester (LNAME) antagonized CGRP-induced vasorelaxation of the rat aorta (91,126). In addition, L-NAME antagonized CGIW-induced hypotension in conscious rats (1) and the anti-ulceric activity of CGRP in the rat (54), suggesting that some effects of CGRP may be mediated through the induction of nitric oxide synthesis and hence possibly cGMP. CGRP was also shown to act on Kim channels in rabbit arterial smooth muscle cells (254,279). Glibenclamide, a blocker of Kxm channels, antagonized CGRP induced hypotension in rabbit (11). However, it appears that CGRP does so indirectly, through the elevation of cAMP levels which stimulate protein kinase A, leading in turn to the activation of KIW channels (279). CGRP-mediated vasorelaxation hence involves multiple second messengers, including cAMP, nitric oxide-cGMP and K+ channels. The precise role of cAMP remains unclear with respect to the relaxant effects of CGRP. In the rat aorta, CGRP-induced increase in cAMP levels was shown not to play a direct role

RECEPTORS

OF CGRP AND RELATED PEPTIDES

in vasorelaxation. While the presence of nitric oxide synthase inhibitors blocked the relaxant effects of CGRP, it did not alter the increase in cAMP levels (126,226). The stimulation of adenylate cyclase in rat aorta thus probably precedes the activation of nitric oxide synthase, or is simply not directly involved in this cascade of events. CGRP has also been shown to increase Ca++ inward currents in guinea pig atria (260,264), rat vas deferens (253) and rat nodose neurons (382) as well as to elevate cytosolic Ca++ in guinea pig cardiac myocytes (117). In contrast, CGRP attenuated both voltage-activated Ca++ and Na+ currents in rat cortical neurons through an increase in cAMP (396). Different neuronal populations thus appear to react differently upon the addition of CGRP. In macrophages, CGRP was shown to increase intracellular cAMP content and to enhance the activity of the Na+-H+ exchanger. The latter was shown to be dependent, at least in part, on the activity of protein kinase C (365). These results thus suggest that CGRP receptors might be directly coupled to multiple signaling pathways to regulate microphage functions. However, these two pathways might also interact and complement each other. Indeed, in skeletal muscle, CGRP was shown to stimulate phosphoinositide turnover, most likely through the CGRPinduced increased levels of cAMP as this effect was mimicked by other cAMP-mobilizing agents such as forskolin (199). In a similar fashion, CGRP was reported to increase intracellular cAMP levels in human ocular ciliary epithelial cells (62). This increase was apparently modulated by protein kinase C since the interaction of the latter with CGRP receptors prevented a further increase in cAMP levels. Overall, it thus appears that CGRP can activate various transduction signaling pathways. The activation of multiple intracellular pathways might be due to the existence and stimulation of various CGRP receptor subtypes coupled to different transduction mechanisms or to the close interaction between the different second messenger pathways measured, since crosstalk between these is now well established. However, the clear interactions between the second messengers involved here, i.e. cAMP with phospholipase C-inositol phosphates-protein kinase C, or cAMP with nitric oxide-cGMP and CGRP receptor, remain to be fully clarified. 4.2. Amylin and adrenomedullin Thus far, few studies have reported on the second messengers involved in the mediation of either amylin or ADM biological effects. Modulation of adenylate cyclase activity was reported for both peptides in several peripheral tissue preparations, including pancreas, vascular smooth muscle and mesangial cells (82,159,184,316). Following expression of the rat ADM receptor in COS-7 cells, ADM produced increases in cAMP (173). Similar to CGRP, multiple signal transduction pathways also appear likely to be involved for these peptides. Indeed, a recent study showed both the accumulation of cAMP and Ca++ mobilization upon ADM application on aortic endothelial cells (311). The full evahation of the second messenger cascades potentially involved in the wide variety of biological activities mediated by these peptides thus largely remains to be investigated.

661

5. BIOLOGICALACTIVITIES 5.1. Fiber pathways containing CGRP CGRP-immunoreactive structures are widely distributed in the brain, which suggests the possible involvement of this peptide in various brain functions, especially in specific sensory, motor and integrative systems. The main CGRP pathways are described in the following sections and summarized in Fig. 4. For an extensive review on immunohistochemical localization and functional aspects of CGRP in peripheral sensory branches, see Ishida-Yamamoto and Tohyama (154). 5.1.1. CGRP and olfaction A group of primary olfactory fibers that terminate in the glomerular layer of the olfactory bulb express CGRP-like immunoreactivity, suggesting that CGRP may play a role in olfaction. High densities of CGRP binding sites are observed in the olfactory system including the mitral and plexiform cell layers of the bulb and the accessory and anterior olfactory nuclei, in addition to related areas such as the diagonal band, olfacto~ tubercle, lateral and basolateral amygdaloid nuclei, primary olfactory, periamygdaloid and entorhinal cortices. Interestingly, when applied to cultured mouse olfactory bulbs, CGRP was reported to increase the number of tyrosine hydroxylase-expressing neurons in vitro (68). On the other hand, although few CGRP-stained receptor cells have been observed in some regions of the epitheliums,the peptide was hardly detectable within most olfactory neurons during development (17), or in response to lesions capable of increasing the number of maturing receptor cells (31). It thus remains unclear if CGRP is directly involved in the differentiation of dopaminergic olfactory bulb neurons (90). 5.1.2. CGRP and audition Positive CGRP cells were detected in the superior olive nucleus, in the area medial to the medial geniculate body and at a lower density, in the lateral lemniscus. In addition, positive fibers were seen in the inferior colliculus and the lateral lemniscus suggesting a role for CGRP in the processing of auditive information although no direct functional data are available at present. Following the injection of a retrograde tracer into the cochlea, the labeling of CGRP neurons was observed in the ipsilateral lateral superior olive nucleus (314,317). Most of these fibers were found in the inner spiral bundle under the inner hair cells and formed synaptic contacts with afferent terminals on inner hair cells (262). In addition, the presence of a cholinergic/CGRP vestibular efferent system that feeds back on primary sensory fibers and receptor hair cells in the cochlea was reported (261,262). Indeed, injection of a retrograde tracer into the vestibular end-organs labeled CGRP cells located just dorsolateral to the genus of the facial nerve which corresponds to one of the three main origins of this vestibular efferent system. Furthermore, CGRP and acetylcholine were shown to coexist in neurons of the facial nerve projecting to the vestibular end-organs (261). The functional interactions between CGRP and acetylcholine in this system is still unclear (261,304). The role of CGRP-immunoreactive fibers innervating cochlear as well as vestibular sensory receptor cells is so far not apparent.

662

VAN ROSSUM ET AL.

5.1.3. CGRP and learning The numerous CGRP-immunoreactive fibers in the caudal portion of the caudate-putamen seem to be of extrinsic origin, as few if any CGRP-immunoreactive perikarya are present in this region (151). By the uses of immunohistochemistry, selective knife cuts, retrograde tracer and electrolytic lesions, the main origin of the CGRP-thalamostriatal projection to the caudal portion of the caudate-putamen was located within the posterior thalamus, with a caudal extension toward the ventral and medial borders of the medial geniculate nucleus (194). In addition, a few CGRP neurons located in the thalamus project to central amygdaloid subnuclei (389). However, most CGRP terminals projecting to central amygdaloid subnuclei originated from cell bodies located in the parabrachial nucleus of the pens. It has been suggested that the overlap between acoustic and somesthetic inputs in the posterior thalamus, and the projection from this region to the amygdala and the striatum is the anatomical substrate for the formation of learnt associations between acoustic stimuli and pain (389). Indeed, similar to the interruptions of the pathways connecting the posterior thalamus to the amygdala and striatum, it has been observed that bilateral destruction in the region of the medial geniculate nucleus or of the amygdala and striatum prevents changes in autonomic activity and behavior elicited by acoustic conditioned stimuli (151). It is thus likely that the CGRP-immunoreactive projections from the posterior intralaminar complex to the amygdala and striatum may play a role in the mediation of these autonomic and behavioral responses to acoustic stimuli. In support of this hypothesis, CGRP was reported to be involved in the facilitation of learning and memory processing as it enhanced the acquisition, consolidation and retrieval of a passive learning task

(185,187). The effect of CGRP on learning could be blocked by pre-treatment with either a serotonergic, /3-adrenergic and opiate receptor antagonists (185,186,188), suggesting that CGRP modulates several systems that are involved in these behavioral effects. 5.1.4. CGRP and feeding CGRP is expressed in various neurons associated with taste, including the sensory fiber endings in taste buds, as well as in the central projections of these fibers terminating in the rostral part of the nucleus of the solitary tract. CGRP was also found to be expressed in the relay system originating from the parabrachial nucleus and projecting to the thalamic nucleus and in posterior agranular insular area. Moreover, most motor neurons in the hypoglossal nucleus (XII) are CGRP-positive. Similar to the peptide distribution, CGRP binding sites were observed in the multisynaptic gustatory pathway, including the nucleus of the solitary tract, the parabrachial nuclei, the central amygdaloid nucleus, the ventromedial posterior nucleus of the thakunus and the insular cortex. The presence of both CGRP-like immunoreactivity and CGRP binding sites in olfactory and gustatory systems suggests that this peptide may have a functional effect in ingestive behavior. Indeed, intracerebroventricuhw administration of CGRP decreased food intake (163,190,342). Some areas of the brain known to be involved in feeding such as the lateral and paraventricular nuclei of the hypothahunus and the perifomical and zona incerta areas, are also known to contain low to moderate levels of CGRP binding sites. As illustrated in Fig. 4, the lateral portion of the dorsal parabrachial area contains a significant amount of CGRP neurons that project to the ventromedial hypothalamic nucleus, an area consid-

A primary olfactory fibers

vestibular end organs

cochlea

vagus nerve

gloaaopharyngeal nerve

FIG. 4. Summary of the main CGRP-containing pathways depicted in a sagittal section diagram of the rat brain. Abbreviations: AI, agramdar insular cortex; Amb, ambiguus nucleus; BST, bed nucleus of the stria terminals; CeL, central amygdafoid nucleus; CPU,caudate putamen (striatum); g7, genu of the facial nerve; Gl, glomerular layer of the olfactory bulb; GP, globus pallidus; LH, lateral hypothafarnic area; MGV, medial geniculate nucleus; PB, parabrachial nuclei; PBG, parabigeminaf nucleus; Po, posterior thafamic nuclear group; PP, peripeduncular nucleus; SC, superior colliculus; Sol, nucleus of the solitary tract; SO, superior olive; VM, ventromedial thakmric nucleus; VMH, ventromedial hypothalamic nucleus.

RECEPTORS

OF CGRP AND RELATED PEPTIDES

ered as one of the major brain satiety centers (332). The lesion of the lateral portion of the parabrachial area, which contains CGRP and CCK-expressing neurons, induced hyperphagia and obesity whereas its stimulation caused hyperglycemia (250). Moreover, the inhibition of gastric acid secretion was observed following intracerebroventricular injection of CGRP (189,206,335). Gastric acid secretion appears to be influenced by the lateral and ventromedial regions of the hypothalamus as well as the central arnygdaloid nucleus (320), supporting a role for CGRP in these pathways. 5.1.5. CGRP and autonomic finctions Numerous CGRP-immunoreactive neurons are located in the parabrachial area which is an important relay center for processing autonomic-related information such as cardiovascular, respiratory and sleep regulations. The lateral parabrachial nucleus is particularly implicated in the regulation of cardiovascular functions and projects to a variety of forebrain structures including the central nucleus of the amygdala and bed nucleus of the stria terminals (165). Neurotensin- and CGRP-containing neurons have been described in these pathways (312). Recently, Harrigan et al. (134) showed that over 35?Z0of the corticotropin releasing factor neurons in the central amygdaloid nucleus are innervated by CGRP terminals which mainly originate from the parabrachial nucleus. Consistent with the anatomical distribution, rnicroinjections of CGRP into the central nucleus of the amygdala elicited increases in arterial blood pressure and heart rate in the rat (256). Moreover, CGRP neurons that were identified within the parabrachial area, make synaptic contacts with serotoninergic and non-serotoninergic neurons within the dorsal raphe (269). The latter are known to be implicated in the modulation of autonomic functions. As described earlier, CGRP is present throughout the parabrachial nucleus as well as in the caudal and intermediate part of the nucleus of the solitary tract, suggestive of its role in the relay of visceral sensory information from the vagus and glossopharyngeal nerves (333). This pathway appears to mostly arise from the parabrachial and peripeduncular nuclei and to project to various areas including the lateral hypothalamus, the central nucleus of the amygdala, caudal parts of the caudate-putamen and globus pallidus, the lateral septal nucleus and bed nucleus of the stria terminals as well as to layer III of the agranular insular and penrhinal cortices (295,339). The presence of CGRP in vagal sensory afferents was revealed by marked reductions in CGRP-like immunoreactivity seen in the nucleus of the solitary tract and area postrema following unilateral nodose ganglionectomy in the cat (346). However, the ascending fibers form the nucleus of the solitary tract to the parabrachial nucleus and are probably modulated by a non CGRP-containing pathway as significant amounts of CGRP have thus far not been reported in this pathway. In addition, CGRP-stained motoneurons in the rostral part of the nucleus ambiguus project through the vagus nerve which might innervate the heart and the branchial muscles in the pharynx. CGRP-containing cells in the inferior ganglia of the vagal nerve, the glossopharyngeal nerve geniculate ganglion terminate in the medial nucleus of the nucleus of the solitary tract. In addition, a number of CGRP neurons were located in the caudal portion of the nucleus of the solitary tract suggesting their potential association with vagal and

663

glossophagmgeal nerve afferents. This relationship could relate to the effects of CGRP on cardiovascular functions including baroreceptor reflexes. 5.1.6. CGRP and vision CGRP neurons are present throughout the parabigeminal nucleus which projects essentially to the superior colliculus. Together with the high density of CGRP binding sites in the superficial layers of the superior colliculus and its lower level in the lateral geniculate nucleus, this may suggest a role for CGRP in the visual system. 5.2. Neurobehavioral projZe of CGRP The central administration of CGRP produces a unique profile of behavioral effects. In addition to the potential and/ or reported central effects mediated by CGRP described in the previous section (see Fiber pathways containing CGRP, 5.1), CGRP was shown to induce a reduction in the frequency and amplitude of spontaneous growth hormone secretory pulses (342), hyperthermia (163), catalepsy (53,163) and reduced motor activity (53,163). Moreover, CGRP increased haloperidol-induced catalepsy and decreased apomorphine-induced hypermotility (53). Although these behaviors are known to be related to dopamhe, no significant change of striatal dopamine or DOPAC concentrations were observed after the central administration of CGRP (53). However, the direct administration of CGRP into the rat ventral tegmental area resulted in a dose-related selective increase in dopamine utilization in the medial mefrontal cortex but not in other mesocortical, mesolimbic’or striatal dopaminergic terminal field regions (72). These effects of CGRP on doparnine turnover are likely to be indirect, as the density of [1251] hCGRP binding sites in the nucleus accumbens was not modified following an injection of 6-hydroxydopamine into the ventral tegmental area, hence suggesting that CGRP binding sites are not located on the dopaminergic nerve terminals (227). Moreover, Orazzo et al. (265) detected large CGRPimmunoreactive neurons in the ventral tegmental area that were not tyrosine hydroxylase immunoreactive. On the other hand, CGRP-like immunoreactivity was observed in doparnine neurons of the Al 1 cell group. These dopaminergic neurons are located in the periventricular gray matter at the border between the hypothalamus and the mesencephalon and project to the spinal cord (265). The precise interaction between CGRP and various dopaminergic neuronal populations thus remains to be fully established. Following an iontophoretical application, CGRP predominantly depressed neuronal firing in the rat forebrain (352). Likewise, central injections of CGRP were effective in inhibiting nociceptive responses in the rat by blocking neuronal thalarnic firing evoked by peripheral noxious mechanical stimuli (267). In agreement with a modulatory role of CGRP on these neurons, primary afferent CGRP fibers make synaptic contacts with spinothalamic tract cells in laminae I–V (38). It is also known that central administration of CGRP produces antinociception in the mouse as measured in the hot plate and formalin tests (37,375). Finally, the intracerebroventricular administration of CGRP, in contrast to peripheral injections, produced concentration-dependent elevations of mean arterial pressure and heart rate by inducing a prompt rise in plasma

VAN ROSSUM ET AL.

noradrenaline levels (92,195). On the other hand, CGRP was reported to inhibit the electrical stimulation-evoked release of noradrenaline in rat hypothalamic slices (350). It thus seems that depending upon the brain area investigated, CGRP can have opposite effects with respect to the release of noradrenaline. Taken together, these various anatomical and functional data suggest a key role for brain CGRP innervation in the modulation and processing of various types of information, ranging from audition to olfaction, nociception and autonomic functions. 5.3. CGRP and motoneurons: development and finctions Immunocytochemical detection of CGRP shows that this peptide is present in motoneurons in embryonic chicks and rats as well as in adulthood (99,116,366). Motoneurons themselves synthesize CGRP which is then transported to the nerve terminal of the neuromuscular junction where it co-exists with acetylcholine (274,340). In rat phrenic nervehemidiaphragm preparations, CGRP increased in a dosedependent manner the twitch contraction induced by nerve or transmural stimulation with a concomitant increase in cAMP levels (353). However, CGRP by itself has no effect in such preparation (340). Several reports suggest that CGRP might play an active role in the formation of a functional synapse at the level of the neuromuscular junction. During chick development, the time course of appearance of CGRP-immunoreactive motoneurons parallels that of the formation of neuromuscular synapses (255,366). High affinity CGRP binding sites have also been characterized in both chick myotubes in culture (161) and chick skeletal muscle membrane preparations (290). Several effects of CGRP on striated muscle have been reported, including the induction of an increased level of surface acetylcholine receptors in cultured chick myotubes mainly by enhancing the rate of synthesis and insertion of new acetylcholine receptors into the plasma membrane (96,255), an increase in the rate of desensitization of nicotinic receptors produced by a decrease of the acetylcholine-activated channel opening frequency (249) and a regulation of the phosphorylation of the nicotinic acetylcholine receptor in rat myotubes (238). All these effects are possibly mediated by increased levels of intracellular cAMP. More recently, CGRP was reported to enhance the postsynaptic response at developing neuromuscular junctions by increasing the burst duration of acetylcholine channels in l-day-old Xenopus nerve-muscle cultures (215). This CGRP-induced potentiation of the acetylcholine response was mediated by protein kinase A. However, the potentiating effect of CGRP may occur only during an early phase of synaptic development as no effect of CGRP on acetylcholine channel burst duration was observed in 3-day-old culture (215). Finally, CGRP was reported to inhibit the insulin-stimulated synthesis of glycogen and to stimulate glycolysis in mamrnalian skeletal muscles (205). Taken together, these findings suggest that CGRP probably plays a role in the formation, maintenance and normal functioning of the neuromuscular junction. Levels of CGRP may also be influenced by axotomy of motoneurons. Sciatic nerve section or facial nerve crushing in rat induce a transient increase in the levels of immunoreactive CGRP and CGRP rnRNA in axotomized

motoneurons (12,270,299). This increase could indicate that CGRP is important for the acute survival of severed motoneurons or for the early stages of regeneration, by exerting a trophic action on the damaged motoneurons. In a similar manner, blockade of axorud flow increased the expression of CGRPa mRNA in motoneurons (175). Interestingly, levels of CGRP in motoneurons are also sensitive to deafferentation. Low thoracic spinal cord transection which deprived lumbar motoneurons of supraspinal inputs resulted in decreased CGRP-like immunoreactivity (223). In contrast, following neurotoxin-induced denervation of bulbospinal raphe neuronal inputs, in which CGRP is colocalized with serotonin (13,14), increases in CGRP-like immunoreactivity were observed in the ventral thoracolumbar cord (95). Globally, these studies suggest the existence of supraspinal influence on the expression of CGRP in the motoneurons. Despite relatively considerable basic knowledge about CGRP at the neuromuscular junction, little is known of its role in motoneuron diseases, such as amyotrophic lateral sclerosis (ALS). One report described a significant loss of CGRP-like immunoreactivity in the cervical spinal cord and in motor and sensory cranial nuclei in Wobbler mouse (394). The latter serves as a model to study motoneuron diseases such as ALS and infantile spinal muscular atrophy. It is speculated that CGRP might act as a trophic factor that would be essential for the survival of motoneurons. It might even be involved more directly in the evolution of this disease, as losses were also observed in non-motor regions of the CNS, including the spinal trigeminal tract (394). It is thus essential to further pursue these issues in this as well as in other motoneuron disease models in order to delineate the physiological role of CGRP at the neuromuscular junction. 5.4. CGRP and sensory neurons CGRP-immunoreactive cells constitute 40–50% of dorsrdroot ganglia neurons (116,164,208). Primary somatosensory systems containing CGRP originating from the trigeminal ganglion, terminate in the spinal trigeminal nucleus and those from the dorsal root ganglion project to the dorsal horn of the spinal cord and the dorsal column nuclei (6,193,273,367). Dorsal rhizotomy induces CGRP depletion (85%) within the ipsilateral dorsal zone of the spinal cord (273) whereas CGRP mRNA and immunoreactivity are increased in the dorsal root ganglia. These increases may be due to the effects of nerve growth factor supplied from the peripheral target (152). In support of this hypothesis, the expression of CGRP is reduced in dorsal root ganglia in response to peripheral axotomy (152,367). Since a neonatal capsaicin treatment destroys a large proportion of primary afferent nerves, it is not surprising that it also significantly reduces the level of CGRP in the dorsal horn of the spinal cord (114,273). While CGRP-positive cells are mainly sensory neurons with fibers of slow conduction velocities (unmyelinated C and small myelinated Ati fibers), 10 to 15% of CGRP-positive cells were identified as Aa/6 neurons. At the ultrastructural level, CGRP-labeled varicosities formed asymmetric synapses on unlabeled dendritic spines or neuronal somata and rarely established glomerular synaptic complexes, at least in cat and monkey spinal cord (6,137,288,348). Moreover, it appears that the CGRP content in primary afferent neurons is largely

RECEPTORS

OF CGRP AND RELATED PEPTIDES

independent of the nature of the innervated target tissue (139,208). In several instances, CGRP was shown to coexist with other peptides in sensory afferents (114,164,393). For example, CGRP was found with up to three other peptides (substance P, cholecystokinin, dynorphin) in the same neuron of the guinea pig dorsal root ganglia (114). CGRPand substance P-like immunoreactivity are even co-localized in the same large dense-core vesicles in dorsal root ganglia neurons (129). Co-existence with substance P occurs in about 25% of the CGRP-immunoreactive neurons of the human trigeminal sensory system (278). Finally, more than 75% of CGRP-like immunoreactive sensory fibers associated with the epidermis were also shown to contain somatostatin (115). The localization of CGRP in small dorsal root ganglion neurons and in the major site of termination of nociceptors suggests that CGRP may participate in nociceptive transmission. Amongst the many peptides that are located in dorsal root ganglion neurons, CGRP provides one of the best examples of a neuromodulator, in the sense of a molecule that exerts limited effects by itself, and yet significantly potentates the effects of other substance such as substance P. Indeed, Biella et al. (30) reported that concentrations of CGRP that by themselves had little or no consistent effect, markedly potentate the excitatory action of substance P or a noxious stimulation in the rat dorsal horn neurons in vivo. Likewise, intrathecal administration of CGRP in rat lumbar, while not evoking a caudally-directed scratching-biting behavior suggestive of nociceptive effect, potentates this behavior following the intrathecal administration of either substance P (380) or somatostatin (381) in the rat. Although the mechanism by which CGRP can potentate the effects of substance P is not clear, there is evidence that CGRP may retard the enzymatic degradation of substance P (202,203,222). CGRP was also shown to enhance the release of substance P (263) as well as of excitatory amino acids (172) from primary afferent fibers, hence possibly leading to the increased activity of these transmitters. Mrathecal administration of CGRP was reported to facilitate the spinal nociceptive flexor reflex in rats (385) whereas at low doses, an attenuation of the facilitation of the tail flick reflex induced by either substance P or noxious cutaneous stimulation was reported by Cridland and Henry (61). Consistent with the notion that CGRP is contributing to nociceptive processing in the dorsal horn, Morton and Hutchison (245) using the antibody microprobe technique, found that noxious thermal, mechanical, or electrical stimulation evoked the release of CGRP in the superficial dorsal horn. Moreover, the intrathecal administration of antisera to CGRP had an analgesic effect on thermal and mechanical noxious stimuli (177). Accordingly, iontophoretically applied CGRP produced a slower onset but prolonged excitation of nociceptive dorsal horn neurons in vivo (239). In vitro studies have also demonstrated that CGRP produces a slow depolarization by a direct action on nociceptive dorsal horn cells (298). In contrast, in mice no pain-related behavioral effects were measured following the intrathecal injection of CGRP (106,376). Taken together, these data indicate that CGRP possibly plays a role in the transmission of nociception in the rat spinal cord. The precise mechanism of interactions with other nociceptive neurotransrnitters in the spinal cord,

665

such as substance P, glutamate and opioids (see 5.4.1) remains however to be defined. 5.4.1. CGRP and opioids CGRP was reported to decrease the analgesia produced by the acute administration of either mu (morphine) or delta (DPDPE) opioid agonists (376). Moreover, morphine was shown to inhibit the acute release of CGRP in the spinal cord (272). We have recentiy investigated further potential interactions between opioids and CGRP, especially during the development of tolerance to the antinociceptive effects of opioids (234,235,237). It is well known that chronic exposure to opioids such as morphine, results into a marked decrease in its pharmacological effects, including antinociception. Indeed, following 5 days of chronic infusion of morphine in the rat lumbar spinal cord, tolerance to the antinociceptive effects of morphine clearly developed as monitored by the tail-immersion task (234). In parallel to the development of tolerance, we measured significant chan es in both CGRP-like irnmunoreactivity as well as of [1l?51]hCGRPa binding sites in the substantialgelatinosa of the spinal cord (234,235). Furthermore, the co-infusion of the CGRP antagonist, CGRP8.3T with morphine, was most effective in preventing the development of tolerance to the antinoceptive effects of the spinally infused mu opioid agonist as measured by the tail flickhil immersion, as well as in the paw pressure tests (237). Interactions with spinal CGRPs stem were also seen with the delta opioid agonist (D-Pen 15 D-Pen enkephalin) but to a much lesser extent with a kappa opioid agonist (U 50488H) (235). The mechanism by which CGRP interacts with opioids during the development of tolerance still remains to be clarified. Potential mediators include nitric oxide which was proposed as a modulator of spinal nociception. Initial data revealed the potentiation of the hyperalgesic response induced by CGRP by a nitric oxide donor (sodium nitroprusside), as monitored in the tail-immersion test (236). In addition, CGRP markers in the rat spinal cord were altered following subchronic infusion of sodium nitroprusside or the nitric oxide synthesis inhibitor (L-NAME) (236). These data thus reveal the existence of close interaction between CGRP and opioids as well as CGRP and nitric oxide in the transmission of nociception in the rat spinal cord. 5.5. CGRP effects in the cardiovascular system CGRP-like immunoreactivity was shown in various species, including humans, to be broadly distributed within the cardiovascular system, consisting of a dense peripheral sensory network innervating the arteries, veins and heart (77,78,101,231,247). In the heart, the atrial myocardium revealed the highest density of CGRP fibers. Moreover, the trigeminal ganglion contains a large number of CGRPimmunoreactive nerve cell bodies. Some of these neurons project to cerebral arteries as following unilateral section of the trigeminal nerve, a pronounced reduction in the levels of CGRP and substance P was observed in the middle cerebral arteries (231). Several studies showed that CGRP and substance P co-exist in a sub-population of sensory neurons innervating the cerebral circulation (114,216,231). Furthermore, there is ultrastmctural evidence for the co-localization of CGRP and substance P in large granular vesicles in

666

trigeminal ganglion cells and in peripheral nerve fibers around blood vessels (129,130), Similar to the distribution of CGRP-like immunoreactivity, specific binding sites for CGRP were detected in heart and blood vessels (59,183,313). [1251]CGRP receptor autoradiography revealed the presence of specific sites in the intima and media of the aorta, coronary arteries and heart valves (313). In rat heart membrane homogenates, specific [1251]CGRP binding was highest in the atria with only small amounts detected in the ventricles. CGRP is one of the most potent vasodilating substances known thus far. Indeed, following intravenous infusion of as little as femtomolar concentrations, this peptide induce profound and long lasting vascular response in various species, including human (35,107,118,225,328). Beside a decrease in vascular resistance, CGRP increases the rate and force of contraction of the heart (112,224,345,371). However, during infusion of low doses of CGRP (0.06 nmolh) in conscious rats, decreases in carotid, renal and mesenteric vascular resistance were observed whereas mean arterial pressure was not significantly affected. Higher doses of CGRP (6.0 nmol/h) were required to decrease mean arterial pressure and increase heart rate (107,109). In addition, atrial contractile effects of CGRP were not significant when these preparations were previously treated with CGRP, indicating that desensitization to CGRP occurred (93,313), in contrast to vasodilatory effects of CGRP, which were apparently not accompanied by significant tachyphylaxis (103). CGRP also potentates the local effects of inflammatory agents including histamine, leukotriene BJ, N-formylmethionyl-leucyl-phenylakmine (FMLP), platelet-activating factor (Paf,), bradykinin and substance P (34,63,148), essentially through its vasodilating properties, On the other hand, CGRP was also shown to modulate immune cells such as T-helper cells (369), B cells (232) and neutrophils (289). In addition to its direct effects on B cells and neutrophils, CGRP may mediate indirect influences via an inhibitory action on T-helper cells that subsequently modulate B cells and neutrophils (369). Hence, CGRP could have a role in inflammation by increasing vasodilation and modulating the activity of various immune cell types. 5.5.1. Clinical applications Despite considerable debate on the pathophysiology of migraine, there is a general agreement that cranial vessels play some role either in the pathogenesis or the expression of the migraine syndrome (79). The trigeminal innervation of the cerebral circulation with sensory fibers provides the substrate for pain of intracranial origin. Substance P and CGRP are believed to be the main two known sensory neuropeptides in the cerebral circulation. During a migraine attack, CGRP levels were elevated in the cranial circulation but not in peripheral blood, while no significant changes were reported for either substance P or neuropeptide Y (120,121). The selective elevation of CGRP in patients suffering from migraine provides a link between basic anatomical and physiological observations on the trigeminal vascular system and migraine. More recently, in agreement with a role of CGRP in the genesis of migraine, Wahl et al. (368) reported the involvement of both CGRP and nitric oxide in the first and transient increase in cerebral blood flow observed during migraine.

VAN ROSSUM ET AL.

CGRP and substance P contained in the trigeminovascular system are also believed to be involved in the vasomotor events occurring after a subarachnoid hemorrhage. A large increase in cerebrospinal fluid content of both peptides was detected 30 min after the onset of a subarachno;d hemorrhage (169,347). Interestingly, infusion of CGRP improved, with no adverse effects, neurological deficits seen after intracranial aneurysm surgery for subarachnoid hemorrhage in 9 out of 15 patients (85,162). It would thus appear that further studies on the use of CGRP-related agonists (ideally non-peptide) in subarachnoid hemorrhage are warranted. In patients with chronic congestive heart failure, the shortterm or prolonged infusions of CGRPhadbeneficial effects by increasing cardiac output and lowering blood pressure without changes in heart rate (74,113,309). Acute myocardial infarction is another situation where there is a demand for counteracting vasoconstriction. A significant increase (almost 2-fold) in plasma CGRP-like immunoreactivity was seen in patients suffering from acute myocardial infarction, suggesting the potential release of CGRP in response to myocardial ischernia or constriction of peripheral vessels (219). Accordingly, CGRP-like immunoreactivity release was evoked by low pH and lactic acid in the guinea pig heart (102,104). Moreover, CGRP was shown to decrease vascular resistance and increase renal blood flow in a rat model of reversible renal ischemic insult (24). Taken together, these results suggest the potential beneficial role(s) of CGRP and related agonists in various ischemic conditions. Serum concentrations of CGRP measured in patients with untreated mild to moderate essential hypertension was not significantly different from those seen in normotensive controls (302). It would thus seem that CGRP is not directly involved in the development of essential hypertension. On the other hand, CGRP might be responsible for changes in peripheral vasculature resistance in response to increased plasma volume such as in pregnancy and in haemodialysed patients. Indeed, CGRP plasma levels were significantly increased in normal pregnancy in contrast to a dramatic decrease (-50%) in patients with pregnancy-induced hypertension (327,343). Similarly, plasma CGRP concentrations were positively correlated with fluid excess being significantly higher in five patients with severe fluid overload (259). Accordingly, by inducing strong vasodilatation, CGRP might be an important defense mechanism against the serious consequences of increased plasma volume including oedema, hypertension and increased cardiac workload. 5.6. CGRP effects in the gastrointestinal tract-sensory components CGRP-like immunoreactivity is present in varying concentrations throughout the mammalian gastrointestinal tract, with highest amounts found in the stomach (127,248,325). CGRP-positive fibers arise from a set of neurons in the myentetic and submucous plexuses as well as from sensory neurons whose cell bodies are located in the nodose and dorsal root ganglia (325). Up to 9570 of spinal afferent neurons innervating the stomach contain CGRP immunoreactivity whereas less than 10Yoof vagal afferent neurons innervating the stomach are positive for CGRP (127,330). Systemic capsaicin treatments in adult or neonatal rats resulted in an almost complete loss of CGRP

RECEPTORS

OF CGRP AND RELATED PEPTIDES

immunostaining in the stomach (325,330,362). However, in contrast to CGRI%, CGRP6 was still present in the intestines of capsaicin-treated rats suggesting that distinct neuronal populations, i.e. sensory neurons and enteric autonomic neurons, could preferentially express CGIU% or CGRP/3,respectively (248). In agreement with an extrinsic site of synthesis of intestinal CGRPa, the analysis of CGRPa or CGRP(3mRNAs revealed that only the CGRP6 rnRNA was expressed in the intestine where it was localized to entenc neurons. On the other hand, both peptides rnRNAs were detected in the dorsal root ganglia (248). Moreover, the presence of specific CGRP receptor binding sites has been shown throughout the gastrointestinal tract (111,228). Over the years, the use of capsaicin has demonstrated the importance of sensory innervation in the regulation of gastric mucosal blood flow, gastric motility and gastric acid secretion (140,142,283). Similar effects were measured following either peripheral or central administration of CGRP including the suppression of acid secretion (147,189,206,336) and the inhibition of gastric motor functions (168,282,337). Interestingly in one study in humans, CGRP6 but not CGRPa could inhibit gastric acid secretion (22). Moreover, the protection of the gastric mucosa against damage induced by a variety of factors, such as ethanol following intragastric application of capsaicin, is essentially associated with a marked rise in gastric mucosal blood flow (143). This increase in blood flow is thought to be induced by CGRP. Accordingly, CGRP was reported to modulate gastric blood flow (19,108,141). Furthermore, capsaicininduced gastroprotection from ethanol injury is known to be inhibited by the CGRP receptor antagonist, CGRp&’3T (196) as well as by a CGRP monoclinal antibody (268). These findings thus support an important role for CGRP in capsaicin-induced gastric protection. It appears that CGRP mediates its gastric-related effects by various mechanisms (86). For example, the inhibition of gastric emptying is thought to be primarily through the activation of the sympathoadrenal axis as adrenalectomy or celiac ganglionectomy abolished the actions of CGRP (284). In contrast, the central action of CGRP to inhibit acid secretion does not involve sympathetic outflow but is mainly mediated by a decreased vagal outflow to the stomach (207,335). Moreover, the mechanisms by which peripheral CGRP injections inhibit acid secretion are still not fully understood but probably involve an increase in somatostatin release as the intravenous injection of somatostatin antibody significantly blocked the effects of CGRP (75). Another possibility is the modulation of cholinergic inputs as a decrease in acetylcholine release by CGRP was reported in the small intestine and antral mucosal fragments (285,303). In the latter, however, the inhibitory effects of CGRP on cholinergic discharge were mediated through the increased release of somatostatin (285). In the gastrointestinal tract, the functional implications of CGRP-containing sensory neurons in decreasing acid secretion and by increasing gastric blood flow resulting in gastric protection may thus provide a new target for the development of novel therapeutic strategies in the treatment of, for example, gastric ulcers. 5.7. Central and peripheral effects of amylin Little is known so far with regard to the potential role of

667

amylin in the brain. The discrete distribution of [1251] BH-rat arnylin binding throughout the rat brain suggests that this peptide may play a role in the modulation of food and water intakes and the control of body temperature as well as in various sensory and neuroendocrine functions. Indeed, amylin was already reported to have anorexic and adipic effects on injection into the hypothalamus (42,43). Moreover, both central and peripheral injection of arnylin inhibited gastric acid secretion (128). Systemic injection of amylin was also shown to induce both hyperglycemia and anorexia (44) suggesting that this peptide could serve as a link between the peripheral and central modulation of appetite. Amylin was also shown to alter the metabolism of dopamine and serotonin in the hypothalamus and striatum (42). However, the role of amylin in other brain regions remains to be established. Extensive reviews on the physiology of arnylin are already available (57,271). Briefly, the best documented effects of amylin are its opposing effects compared to those of insulin. These include decreased basal and insulin-stimulated rates of glycogen synthesis (205) and glucose uptake (56,334), as well as increased muscle glycogenolysis (391,392) via the modulation of glycogen synthase and phosphorylase enzymatic activities (67,200). However, there are conflicting reports with respect to the effects of amylin on insulin secretion, as decreased (315,374) and no effect, (153,251) as well as increased insulin secretion (88) have been reported. Because of its opposing effects to insulin and its amyloidogenic and neurotoxic properties, amylin was proposed to play a prominent role in the pathogenesis of type 2 diabetes [for recent reviews, see (25,57)]. In an attempt to demonstrate this possibility, several groups have developed transgenic mice that overexpress the human amylin gene (64,100,364). These transgenic animals revealed increased amylin as well as insulin content in the pancreas. Moreover, secretion of both peptides from the pancreas was elevated upon stimulation by glucose (64,364). However, arnyloid deposits were not detected in these animals up to 19 months, suggesting that other co-existing abnormalities in type 2 diabetes are required for the formation of amyloid deposits in the islets of Langerhans. The precise role of amylin in the pathology of this disease thus remains unclear. 5.8. Central and peripheral effects of adrenomedullin The role(s) of ADM in the brain is thus far not well defined. Only a low amount of ADM-like immunoreactivity in the brain cortex was reported (149,373). In contrast to its vasodepressor effects upon peripheral injection, Takahashi et al. (339) showed that intracerebroventricular injections of ADM (l–3 nmol/kg) in the rat brain produced an increase in blood pressure. These data suggest the existence of specific sites of action for ADM in the brain. However, it was not clear from this study whether these sites represented unique ADM receptors or if ADM acted through CGRP receptors to produce this hypertensive effect, especially since CGRP was shown to induce similar effects upon intracerebroventricular injection (92). On the other hand, both the mRNA for the ADM receptor as well as [1251]ADMspecific binding sites were recently reported in various areas of the CNS including the hypothalamus, thalarnus, cerebellum and spinal cord (173,266). Moreover, recent studies

668

VAN ROSSUM ET AL.

fitrther support a unique role for ADM in the brain. For example, the central administration of ADM was reported to exacerbate ischemic brain damage following focal ischemic brain injury (373). Indeed, similar to its effects on non-CNS vasculature, ADM induced vasodilation of cerebral vessels (18,373). On the other hand, ADM vasodilatory effects were abluminal compared to intrahnninal for CGRP (373). These data thus support a differential role for the two peptides. The dissociation between ADM-mediated effects through unique ADM vs. CGRP receptors thus still remain to be clarified but a unique role for ADM in the brain is most likely. 6. CONCLUS1ON ANDPERSPECTIVE

of peptidomimetic analogs and antagonists would greatly facilitate the efforts to delineate the pathophysiological implications of CGRP and related peptides. These new compounds could also offer great potential for CGRP research towards clinical manipulations. The complete isolation and cloning of the CGRP and amylin receptor subtypes is also anxiously awaited and should greatly help to provide key insights as to the molecular pharmacology of CGRP and related peptides receptors. 7. NOTEADDEDIN PROOF Following the acceptance of the present manuscript, we became aware of a report on the cloning of a novel protein that interacts with CGRP (Luebke, A.E.; Dahl, G.P.; Roos, B.A.; Dickerson, I.M. Proc. Natl. Acad. Sci. USA, in press). This small (146 amino acids), largely hydrophilic protein is believed to be either a CGRP receptor or a component of a CGRP receptor complex. This novel protein does not belong to the typical seven transmembrane domains G protein-coupled receptor superfamily, as it possesses a single transmembrane domain and a very short intracellular C-tail. Further biochemical and pharmacological characterization is required in order to relate this novel protein to the previously defined CGRP receptor subtypes.

CGRP and related peptides possess a broad variety of biological effects possibly mediated by various specific receptor subtypes. Suqmisingly, limited data are thus far available with respect to the molecular characteristics and topography of the respective receptors. Clearly, the ultimate pharmacological characterization of these receptor classes will follow their isolation, purification and cloning. Success is now apparent through this approach, as most recently few forms of the receptors belonging to this family were cloned (173,174,211). However, in parallel to these molecular approaches, future research should focus on the identification of new tissue preparations enriched with a single receptor subtype. The development of highly subtype-specific peptide and non-peptide analogs is also crucial for the successful characterization of these various receptor classes as the selectivity of currently available molecules is limited and does not allow for detailed pharmacological and clinical studies. In that regard, the novel antagonist AC187 (20), a selective amylin receptor blocker, should be useful in providing evidence in regard to the uniqueness and functional role of amylin and its receptors in brain and peripheral tissues. Taken together, future research aimed at the development

This research is supported by the Medical Research Council of Canada (MRCC). DvR is an awardee of the MRCC and of the Human Frontier Science Program Organisation (HFSPO). RQ is Chercheur Boursier from Fends de la Recherche en Sant6 du Qu6bec. The authors also wish to thank Dr. Daniel P. M6nard for numerous helpful discussions.

1. Abdelrahmarr, A.; Wang, Y.-X.; Chang, S. D.; Pang, C. C. Y., Mechanism of the vasodilator action of crdcitonin gene-related peptide in conscious rats. Br. J F’harnrucoL,106,45-481992. 2. Adema, G. J.; van Hulst, K. L.; Baas, P. D., Uridine branch acceptor is a cis-acting element involved in regulation of the alternative processing of calcitonirr/CGRP-I pre-mRNA. Nucleic Acids Res., 18,5365-53731990. 3. Ahr6n, B.; Sundler, F., Localization of calcitonin gene-related peptide and islet amyloid polypeptide in the rat and mouse pancreas. Cell Tissue Res., 269, 315–322 1992. 4. Aiyar, N.; Nambi, P.; Griffin, E.; Bhatnagar, P.; Feuerstein, G., Identification and characterization of calcitonin gene-related peptide receptors in porcine renal medulkuy membranes. Endocrinology, 129,965-9691991. 5. Albrandt, K.; Mull, E.; Brady, E. M. G.; Herich, J.; Moore, C. X.; Beaumont, K., Molecular cloning of two receptors from rat brain with high affinity for srdmon cafcitonin. F.EBSLetI., 325, 225–232 1993. 6. Alvarez, F. J.; Kavookjian, A. M.; Light, A. R., UftrastmcturaI morphology, synaptic relationships, and CGRP immunoreactivity of physiologically identified C-fiber terminals in the monkey spinal cord. J. Comp. Neurol., 329, 472–490 1993. 7. Amara, S. G.; Jonas, V.; Rosenfeld, M. G.; Ong, E. S.; Evans, R. M., Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature, 296,2402441982.

8. Amara, S. G.; Arriza, J. L.; Leff, S. E.; Swanson, L. W.; Evans, R. M.; Rosenfeld, M. G., Expression in brain of a messenger RNA encoding a novel neuropeptide homologous to cafcitonin generelated peptide. Science, 229, 1094–1097 1985. 9. Amerini, S.; Mantelli, L.; Ledda, F., Nitric oxide is not involved in the effects induced by non-adrenergic non-cholinergic stimulation and calcitonin gene-related peptide in the rat mesenteric vascular bed. Neuropeptides, 25, 51–56 1993. 10. Arrdersen, S. L. V.; Clausen, T., Calcitonin gene-related peptide stimulates active Na+-K+ transport in rat soleus muscle. Am. J. Physiol. Cell Physiol, 264,419-4291993. 11. Andersson, S. E., Glibenclarnide and L-NG-nitro-arginine methyl ester modulate the ocular and hypotensive effects of calcitonin generelated peptide. Eur. J. Pharnracol., 224, 89–91 1992. 12. Arvidsson, U.; Johnson, H.; Piehl, F.; Cullheim, S.; Hokfelt, T.; Risling, M.; Terenius, L.; Uftlmke, B., PenpheraI nerve section induces increased levels of cafcitonin gene-related peptide (CGRP)-like immunoreactivity in axotomized motoneurons. .Exp. Brain Res., 79, 212–216 1990. 13. Arvidsson, U.; Schafling, M.; Cullheim, S.; Ultkdce, B.; Terenius, L.; Verhofstad, A.; Hokfelt, T., Evidence for coexistence between cafcitonin gene-related peptide and serotonin in the bufbospinal pathway in the monkey. Brain Res., 532,47-571990. 14. Arvidsson, U.; Ultltake, B.; Cullheim, S.; Terenius, L.; Hokfelt, T., Cafcitonin gene-related peptide in monkey spinal cord and medulla oblongata. Brain Res., 558, 330–334 1991.

ACRNOWLEDGEMENTS

RECEPTORS

OF CGRP AND RELATED PEPTIDES

15. Asai, J.; Nakazato, M.; Miyazato, M.; Kangawa, K.; Matsuo, H.; Matsukora, S., Regional distribution and molecular forms of rat islet amyloid polypeptide. Biochem. Biophys. Res. Cornmun.,169, 788– 7951990. 16. Bafti, J.; Gores, T.; Slowik, F.; Horwith, M.; Lekka, N.; Piiaztor, E.; Palkovits, M., Neuropeptides in the human superior cervical ganglion. Brain Res., 570, 272–278 1992. 17. Baker, H., Calcitonin gene-related peptide in the developing mouse olfactory system. Dev. Brain Res., 54, 295–298 1990. 18. Baskaya, M. K.; Suzuki, Y.; Anzai, M.; Sefri,Y.; Saito, K.; Takayasu, M.; Shibuya, M.; Sugita, K., Effects of adrenomedullin, calcitonin gene-related peptide, and arnylin on cerebral circulation in dogs. J Cerebr. Blood Flow Metab., 15, 827-8341995. 19. Bauerfeind, P.; Hof, R.; Hof, A.; Cucafa, M.; Siegrist, S.; von Ritter, C.; Fischer, J. A.; Blum, A. L., Effects of hCGRP I and II on gastric blood flow and acid secretion in anesthetized rabbits. Am. J Physiol. Gastrointest. Liver Physiol, 256, 145-1491989. 20. Beaumont, K.; Kenney, M. A.; Young, A. A.; Rink, T. J., High affinity arnylin binding sites in rat brain. Mol. Pharnracol.,44,4934971993. 21. Beaumont, K.; Moore, C. X.; Pittner, R.; Prickett, K.; Gaeta, L. S. L.; Rink, T. J.; Young, A. A., Differential antagonism of amylin’s metabolic and vascular actions with amylin receptor antagonists. Can. J. Physiol. Pharmacol. (Abstract), 72,311994. 22. Beglinger, C.; Born, W.; Hildebrand, P.; Ensinck, J. W.; Burkhardt, F.; Fischer, J. A.; Gyr, K., Cafcitonin gene related peptides I and 11 and calcitonin: Distinct effects on gastric acid secretion in humans. Gastroenterology, 95, 958–965 1988. 23. Beglinger, C.; Born, W.; Miinch, R.; Kurtz, A.; Gutzwiller, J.-P.; Jager, K.; Fischer, J. A., Distinct hemodyrramicand gastric effects of human CGRP I and II in man. Peptides, 12, 1347-13511991. 24. Bergman, A. S. F.; FaJt, K.; Odar-Cederloef, I.; Westman, L.; Takolrmder, R., Calcitonin gene-related peptide attenuates experimental ischernic renal failore in a rat model of reversible renal ischemic insult. Renal Failure, 16, 351–357 1994. 25. Betsholtz, C.; Christianson, L.; Gebre-Medhin, S.; Westermark, P., Islet arnyloid polypeptide-hen of egg in type 2 diabetes pathogenesis?. Acta OncoZ.,32, 149-1541993. 26. Bhogal, R.; Smith, D. M.; Bloom, S. R., Investigation and characterization of binding sites for islet amyloid polypeptide in rat membranes. Endocrinology, 130, 906-9131992. 27. Bhogal, R.; Smith, D. M.; Bloom, S. R., Solubilisation of CGRP and IAPP receptors from rat lung. Neuropeptides (Abstract), 24, 186 1993. 28. BhogaJ, R.; Smith, D. M.; Porkiss, P.; Bloom, S. R., Molecular identification of binding sites for calcitonin gene-related peptide (CGRP) and islet arnyloid polypeptide (IAPP) in mammalian lung: species variation and binding of truncated CGRP and IAPP.. Endocrinology, 133,2351-23611993. 29. Bhogrd, R.; Smith, D. M.; BIoom, S. R., Characterization of islet amyloid polypeptide (L4PP)/calcitonin gene-related peptide (CGRP) receptors in mammalian lung. Can. J. Physiol. Pharmacol., 73, 1030–1036 1995. 30. Biella, G.; Panara, C.; Pecile, A.; Sotgiu, M. L., Facilitator role of calcitonin gene-related peptide (CGRP) on excitation induced by substance P (SP) and noxious stimuli in rat spinal dorssdhorn neurons. An iontophoretic study in vivo. Brain Res., 559, 352–356 1991. 31. Biffo, S.; DeLucia, R.; Mulatero, B.; Margolis, F.; Fasolo, A., Carrrosine-, calcitonin gene-related peptide- and tyrosine hydroxylase-imrmrnoreactivity in the mouse olfactory bulb following peripheral denervation. Brain Res., 528, 353–357 1990. 32. Bishop, G. A., Calcitonin gene-related peptide in afferents to the cat’s cerebella cortex: distribution and ongin. J. Comp.Neurol., 322, 201-2121992. 33. Bovenberg, R. A. L.; Moen, T. C.; Jansz, H. S.; Baas, P. D., In vitro spicing analysis of mini-gene constructs of the alternatively processed human calcitonin/CGRP-I pre-mRNA.. Biochim. Biophys. Acta, 1008, 223–233 1989. 34. Brain, S. D.; Williams, T. J., Inflammatory oedema induced by synergism between calcitonin gene-related peptide (CGRP) and mediators of increased vasclar permeability. Br. J. Pharmacol., 86, 855-8601985. 35. Brain, S. D.; Williams, T. J.; Tippins, J. R.; Morns, H. R.; MacIntyre, L, Calcitonin gene-related peptide is a potent vasodilator. Nature, 313,54-561985. 36. Breeze, A. L.; Harvey, T. S.; Bazzo, R.; Campbell, I. D., Solution

669

37. 38.

39.

40.

41. 42. 43. 44. 45. 46. 47.

48.

49.

50.

51.

52.

53. 54. 55.

56.

structure of human calcitonin gene-related peptide by ‘H NMR and distance geometry with restrained molecular dynamics. Biochemistry, 30, 575–582 1991. Carrdeletii, S.; Fern, S., Antinociceptive profile of intracerebroventricular salmon calcitonirr and calcitonin gene-related peptide in the mouse formalin test. Neuropeptides, 17, 93–98 1990. Carlton, S. M.; Westlund, K. N.; Zhang, D.; Sorkin, L. S.; Willis, W. D., Calcitonin gene-related peptide containing primary afferent fibers synapse on primate spinothrdamic tract cells. Neurosci. Lett., 109, 76-811990. Chabre, O.; Conkfin, B. R.; Lin, H. Y.; Lodish, H. F.; Wilson, E.; Ives, H. E.; Catanzariti, L.; Hemmings, B. A.; Boome, H. R., A recombinant calcitonin receptor independerrtfy stimulates 3’, 5’cyclic adenosine monophosphate and Ca2+/inositol phosphate signafing pathways. Mol. Endocrinol., 6, 551–556 1992. Chakder, S.; Rattan, S., [Tyri)]-calcitonin gene-related peptide 28-37 (rat) as a putative antagonist of calcitonin gene-related peptide responses on opossum intemaI amd sphincter smooth muscIe. J. Pharrnacol.Exp. Ther., 253,200-2061990. Chakraborty, M.; Chatterjee, D.; Kellokumpu, S.; Rasmussen, H.; Baron, R., Cell cycle-dependent coupling of the cafcitonin receptor to different G proteins. Science, 251, 1078–1082 1991. Chance, W. T.; Balasubramaniam, A.; Zharrg, F. S.; Wimafawarrsa, S. J.; Fischer, J. E., Anorexia following the intrabypothakunic administration of amylin. Brain Res., 539, 352–354 1991. Chance, W. T.; Balasubramaniarn, A.; Chen, X.; Fischer, J. E., Tests of adipsia and conditioned taste aversion following the intrafrypotfralamic injection of arnylin. Peptides, 13,961-9641992. Chance, W. T.; Balasubramarriarn, A.; Stallion, A.; Fischer, J. E., Anorexia following the systemic injection of arnylin. BrainRes., 607, 185-1881993. Chantry, A.; Leighton, B.; Day, A. J., Cross-reactivity ofamylin with calcitouin-gene-related peptide binding sites in rat liver and skeletal muscle membranes. Biochem. J., 277, 139–143 1991. Chatterjee, T. K.; Fisher, R. A., Multiple affinity forms of the cafcitonin gene-related peptide receptor in rat cerebellum. Mol. Pharmacol., 39, 798–804 1991. Chatterjee, T. K.; Moy, J. A.; Fisher, R. A., Characterization and regulation of high affinity cafcitonin gene-related peptide receptors in cultured neonatal rat cardiac myocytes. Endocrinology, 128, 2731– 27381991. Chatterjee, T. K.; Moy, J. A.; Cai, J. J.; Lee, H. C.; Fisher, R. A., Solubilization and characterization of a guanine nucleotide-sensitive form of the calcitonin gene-related peptide receptor. Mol. Phar?rl(lcol., 43, 167–175 1993. Chiba, T.; Yamaguchi, A.; Yamatani, T.; Nakamura, A.; Morishita, T.; Inui, T.; Fokase, M.; Noda, T.; Fujita, T., Calcitonin gene-related peptide receptor antagonist human CGRP-(8-37). Am. J. Physiol. Endocn”nol.Metab, 256, 331–335 1989. Chijiiwa, Y.; Kabemura, T.; Misawa, T.; Kawakami, O.; Nawata, H., Direct inhibitory effect of crdcitonin gene-related peptide and atrial natriuretic peptide on gastric smooth muscle cells via different mechanisms. Life Sci., 50, 1615–1623 1992. Chin, S. Y.; Hall, J. M.; Brain, S. D.; Morton, I. K. M., Vasodilator responses to clacitonin gene-related peptide (CGRP) and amylin in the rat isolated perfused kidney are mediated via CGRP1 receptors. J. Pharmacol. Exp. Ther., 269,989-9921994. Christmatrson, L.; Westermark, P.; Betsholtz, C., Islet amyloid polypeptide stimulates cyclic AMP accumulation via the porcine calcitonin receptor. Biochem. Biophys. Res. Comrnun.,205, 1226– 12351994. Clementi, G.; Grassi, M.; Valeno, C.; Prato, A.; Fiore, C. E.; Drago, F., Effects of cafcitonin gene-related peptide on extrapyramidaf motor system. Pharmacol. Biochem. Behav., 42, 545–548 1992. Clementi, G.; Caruso, A.; Prato, A.; De Bemardis, E.; Fiore, C. E.; Amico-Roxas, M., A role for nitric oxide in the anti-ulcer activity of calcitonin gene-related peptide. Eur. J. Pharrnacol,256, 7–8 1994. Cooper, G. J. S.; Willis, A. C.; Clark, A.; Turner, R. C.; Sire, R. B.; Reid, B. M., Purification and characterization of a peptide from amyloid-nch pancreases of type 2 diabetic patients. Proc. Natl. Acad. Sci. USA, 84, 8628–8632 1987. Cooper, G. J. S.; Leighton, B.; Dimitriadis, G. D.; Parry-Billings, M.; KowaJchuk,J. M.; Howkmd, K.; Rothbard, J. B.; Willis, A. C.; Reid, K. B. M., Amylin found in amyloid deposits in human type 2 diabetes mellitns may be a hormone that regulates glycogen metabolism in skeletal muscle. Proc. Natl. Acad. Sci. USA, 85, 7763–7766 1988.

670 57. Cooper, G. J. S., Amylin compared with cafcitonin gene-related peptide: structure, biology, and relevance to metabolic disease. Endocrine Rev., 15, 163–201 1994. 58. Cote,G. J.; Stolow, D. T.; Peleg, S.; Berget, S. M.; Gage], R. F., Identification of exon sequences and an exon binding protein involved in alternative RNA splicing of calcitonirr/CGRP. Nucleic Acids Res., 20, 2361–2366 1992. 59. Coupe, M. O.; Mak, J. C.; Yacoub, M.; Oldershaw, P. J.; Barnes, P. J., Autoradiographic mapping of calcitonin gene-related peptide receptors in human and guinea pig hearts. Circulation, 81, 741–747 1990. 60. Cox, H. M.; Tough, I. R., Calcitonin gene-related peptide receptors in human gastrointestinal epithelia. Br. J Pharrrraol., 113, 1243–1248 1994. 61. Crirfland, R. A.; Henry, J. L., Intrathecrd administration of CGRP in the rat attenuates a facilitation of the tail ilickreflex induced by either substance P or noxious cutaneous stimulation. Neurosci. Lett., 102, 241–246 1989. 62. Crook, R. B.; Yabu, J. M., CaIcitonin gene-reIated peptide stimulates intracellulm cAMP via a protein kinase C-controlled mechanism in human ocular ciliary epithelial cells. Biochem. Biophys. Res. Commun., 188,662-6701992. 63. Crnwys, S. C.; Kidd, B. L.; Mapp, P. I.; Walsh, D. A.; Blake, D. R., The effects of calcitonin gene-related peptide on formation of intraarticular oedema by inflammatory mediators. Br. J. Pharrrracol.,107, 116-1191992. 64. D’Alessio, D. A.; Verchere, C. B.; Kahn, S. E.; Hoagland, V.; Baskin, D. G.; Palmiter, R. D.; Ensinck, J. W., Pancreatic expression and secretion of human islet amyloid polypeptide in a trarrsgenicmouse. Diabetes, 43, 1457–1461 1994. 65. D’Santos, C. S.; Gatti, A.; Poyner, D. R.; Hanley, M. R., Stimulation of adenylate cyclase by amylin in CHO-K1 cells. Mol. Pharmacol., 41,894-8991992. 66. Deems, R. O.; Cardinaux, F.; Deacon, R. W.; Young, D. A., Amylin or CGRP(8-37) fragments reverse srrnylin-inducedinhibition of 14Cglycogen accumulation. Biochem. Biophys. Res. Commun., 181, 116–120 1991. 67. Deems, R. O.; Deacon, R. W.; Young, D. A., Amylin activates glycogen phosphorylase and inactivates glycogen synthase via a cAMP-independent mechanism. Bicrchem.Biophys. Res. Commun., 174,716-7201991. 68. Denis-Donini, S., Expression of dopaminergic phenotypes in the mouse olfactory bulb induced by the calcitonin gene-related peptide. Nature, 339, 701–703 1989. 69. Dennis, T.; Foumier, A.; St-Pierre, S.; Quirion, R., Structure-activity profile of calcitonin gene-related peptide in peripheral and brain tissues. Evidence for receptor multiplicity. J. Pharmacol. Exp. Ther., 251,718-7251989. 70. Dennis, T.; Foumier, A.; Cadieux, A.; Pomerleau, F.; Jolicoeur, F. B.; St-Pierre, S.; Quirion, R., hCGRP8-37, a cafcitonin gene-related peptide antagonist revealing calcitonin gene-relatedpeptide receptor heterogeneity in brain and periphery. J. Pharmacol. Exp. Ther., 254, 123–128 1990. 71. Dennis, T.; Foumier, A.; Guard, S.; St-Pierre, S.; Quirion, R., Cafcitonin gene-related peptide (hCGRPalpha) binding sites in the nucleus accumbens. Atypical structural requirements and marked phylogenic differences. Brain Res., 539, 59–66 1991. 72. Deutch, A. Y.; Roth, R. H., Calcitonin gene-related peptide in the ventral tegmental area: selective modulation of prefrontal cortical dopamine metabolism. Neurosci. Lat., 74, 169-1741987. 73. Dotti-Sigrist, S.; Born, W.; Fischer, J. A., Identification of a receptor for cafcitouirr gene-related peptides I and II in humsm cerebellum. Biochem. Biophys. Res. Commun., 151, 1081-10871988. 74. Dubois-Rand6, J. L.; Merlet, P.; Benvenuti, C.; Sediame, S.; Macquin-Mavier, I.; Chabner, E.; Braquet, P.; Castaigne, A.; Adnot, S., Effects of cafcitonin gene-related peptide on cardiac contractility, coronary hemodymurrics and myocardial energetic in idiopathic dilated cardiomyopathy. Am. J. Cardiol., 70, 906–912 1992. 75. Dunning, B. E.; Taborsky, G. J. J., Calcitonin gene-related peptide: a potent and selective stimulator of gastrointestinal somatostatin secretion. Endocrinology, 120, 1774–1781 1987. 76. Edvinsson, L.; Fredholm, B. B.; Hamel, E.; Jansen, I.; Verrecchia, C., Penvascrdar peptides relax cerebral arteries concomitant with stimulation of cyclic adenosine monophosphate accumulation or release of 58, an endothelium-derived relaxing factor in the cat. Neurosci. Mt., 213-2171985.

VAN ROSSUM ET AL. 77. Edvinsson, L.; Ekman, R.; Jansen, L; McCulloch, J.; Uddman, R., Calcitonin gene-related peptide and cerebral blood vessels: distribution and vasomotor effects. J. Cerebr. Blood Flow Metab., 7, 720– 7281987. 78. Edvinsson, L.; Ekman, R.; Jansen, I.; Ottosson, A.; Uddrrran, R., Peptide-containing nerve fibers in human cerebral arteries: immunocytochemistry, radioimmunoassay, and in vitro pharmacology. Ann Neurol, 21, 431–437 1987. 79. Edvinsson, L., Innervation and effects of dilatory neuropeptides on cerebral vessels. Blood Vessels, 28, 35–45 1991. 80. Edwards, R. M.; Stack, E. J., Trizna, W., Csdcitonin gene-related peptide stimulates adenylate cyclase and relaxes intracerebral arterioles. J. Pharrrraol. Exp. Ther., 257, 1020–1024 1991. 81. Eguchi, S.; Hirata, Y.; Iwasaki, H.; Sate, K.; Watanabe, T. X.; Inui, T.; Nakajima, K.; Sakakibma, S.; Marumo, F., Structure-activity relationship of adrenomedullin, a novel vasodilatory peptide, in cultured rat vascular smooth muscle cells. Endocrinology, 135, 2454-24581994. 82. Eguchi, S.; Hirata, Y.; Kane, H.; Sate, K.; Watanabe, Y.; Watanabe, T. X.; Nakajima, K.; Sakakibara, S.; Marumo, F., Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells. FEBS Lat., 340, 226–230 1994. 83. Emeson, R. B.; Hedjran, F.; Yeakley, J. M.; Guise, J. W.; Rosenfeld, M. G., Alternative production of calcitonin and CGRP mRNA is regulated at the calcitonin-specific splice acceptor. Nature, 341, 76– 801989. 84. Entzeroth, M.; Doods, H. N.; Wieland, H. A.; Wienen, W., Adrenomedullin mediates vasodilation via CGRP1 receptors. Life Sci, 56, L19-L25 1994. 85. European CGRP in Subarachnoid Hemorrhage Study Group. Effect of calcitonin-gene-related peptide in patients with delayed postoperative cerebral ischaemia after aneurysmal subarachnoid hemorrhage. Lancet, 339, 831-8341992. 86. Evangelista, S.; Tramontana, M.; Maggi, C. A., Pharmacological evidence for the involvement of multiple calcitonin gene-related peptide (CGRP) receptors in the antisecretory and rmtiulcer effect of CGRP in rat stomach. Z#e Sci, 50, 13-181992. 87. Febmann, H.-C.; Weber, V.; Goke, R.; Goke, B.; Arnold, R., Cosecretion of amylin and insulin from isolated rat pancreas. FEBS La., 262, 279–281 1990. 88. Fehmann, H.-C.; Weber, V.; Goke, R.; Goke, B.; Eissele, R.; Arnold, R., Islet amyloid polypeptide (IAPP; amylin) influences the endocrine but not the exocnne rat pancreas. Biochem. Biophys. Res. Commwr., 167, 1102–1108 1990. 89. Ferrier, G. J. M.; Pierson, A. M.; Jones, P. M.; Bloom, S. R.; Girgis, S. I.; Legon, S., Expression of the rat amylin (IAPP/DAP) gene. J. Mol. Endocr, 3, 1–4 1989. 90. Finger, T. E.; Bottger, B., Expression of the dopaminergic phenotype in the olfactory bulb: Neither calcitonin gene-related peptide nor olfactory input is necessary. Neurosci. Z.ett., 143, 15–18 1992. 91. Fiscus, R. R.; Zhou, H.-L.; Wang, X.; Han, C.; Ali, S.; Joyce, C. D.; Murad, F., Calcitonin gene-related peptide (CGRP)-induced cyclic AMP, cyclic GMP and vasorelaxant responses in rat thoracic aorta are antagonized by blockers of endothelium-derived relaxant factor (EDRF). Neuropeptides, 20, 133-1431991. 92. Fisher, L. A.; Kikkawa, D. O.; Rivier, J. E.; Amara, S. G.; Evans, R. M.; Rosenfeld, M. G.; Vale, W. W.; Brown, M. R., Stimulation of noradrenergic sympathetic outflow by calcitonin gene-related peptide. Nature, 305,534-5361983. 93. Fisher, R. A.; Robertson, S. M.; Olson, M. S., Stimulation and homologous desensitization of cafcitonin gene-related peptide receptors in cultured beating rat heart cells. Endocrinology, 123, 106–112 1988. 94. Ffuhmarm, B.; Muff, R.; Hunziker, W.; Fischer, J. A.; Born, W., A human orphan calcitonin receptor-like structure. Biochem. Biophys. Res. Commun.,206, 341-3471995. 95. Fone, K. C. F., Distribution of cafcitonin gene-related peptide in rat and rabbit spinal cords: Effect of intrathecal 5,7dihyrfroxytryptarnine.J. Neurochem., 59, 1251-12561992. 96. Fontaine, B.; Changeux, J. P.; Hokfelt, T.; Kfarsfeld, A., Calcitonin gene-related peptide, a peptide present in spinal cord motoneurons, increases the number of acetylcholine receptors in primary cultures of chick embryo myotubes. Neurosci. La., 71, 59–65 1986. 97. Foord, S. M.; Craig, R. K., Isolation and characterisation of a human calcitonin-gene-related peptide receptor. Eur. J. Biochem., 170, 373– 3791987.

RECEPTORS

OF CGRP AND RELATED PEPTIDES

98. Force, T.; Bonventxe,J. V.; Ffarmery,M. R.; Gem, A. H.; Yarnin, M.; Goldring, S. R., A cloned porcine renal calcitonin receptor couples to adenylyl cyclase and phospholipase C.. Am. J F%ysiol.Renal Fluid Electrolyte Physiol, 262, 1110–1 1151992. 99. Forger, N. G.; Hodges, L. L.; Breedlove, S. M., Ontogeny of calcitonin gene-related peptide immunoreactivity in rat lumbar motoneurons: delayed appearance and sexual dimorphism in the spinal nucleus of the bulbocavemosus. J. Cornp. Neurol., 330, 514-5201993. 100. Fox, N.; Schrementi, J.; Nishi, M.; Ohagi, S.; Chan, S. J.; Heissermarr, J. A.; Westermark, G. T.; Lecksttom, A.; Westerrmuk, P.; Steiner, D. F., Human islet arnyloid polypeptide transgenic mice as a model of non-insulin-dependent diabetes mellitus (NIDDM). FEBS Lett, 323, 40–44 1993. 101. Frarrco Cereceda, A.; Henke, H.; Lundberg, J. M.; Peterrnann, J. B.; Hokfelt, T.; Fischer, J. A., Calcitonin gene-related peptide (CGRP) in capsaicin-sensitive substance P-immunoreactive sensory neurons in animafs and man: distribution and release by capsaicin. Peptides, 8, 399-4101987. 102. Fmnco Cereceda, A.; Saria, A.; Lundberg, J. M., Differential release of calcitonin gene-related peptide and neuropeptide Y from the isolated heart by capsaicin, ischaernia, nicotine, bradykinin and ouabain. Acta Physiol. Scand., 135, 173–187 1989. 103. Franco Cereeeda, A., Cafcitonin gene-related peptide and human epicardial coronary arteries: presence, release and vasodilator effects. B-. .f. Pharrnacol., 102,506-5101991. 104. Franco-Cereceda, A.; Kallner, G.; Lundberg, J. M., Capsazepinesensitive release of calcitonin gene-related peptide from C-fibre afferents in the guinea-pig heart by low pH and lactic acid. Eur. J. Pharmacol., 238, 31 1–316 1993. 105. Galeazza, M. T.; O’Brien, T. D.; Johnson, K. H.; Seybold, V. S., Islet amyloid polypeptide (L4PP) competes for two binding sites of CGRP. Peptides, 12,585-5911991. 106. Gamse, R.; Saria, A., Nociceptive behavior after intrathecal injections of substance P, neurokinin A and calcitonin gene-related peptide in mice. Neurosci. Lett., 70, 143-1471986. 107. Gardiner, S. M.; Compton, A. M.; Bennett, T., Regional hemodynarnic effects of calcitonin gene-related peptide. Am. J. Physiol. Regulatory Integrative Comp. Physiol, 256,332-3381989. 108. Gardiner, S. M.; Compton, A. M.; Bennett, T., Differential effects of neuropeptides on coeliac and superior mesenteric blood flows in conscious rats. Regrd. Pept., 29, 215–227 1990. 109. Gardiner, S. M.; Compton, A. M.; Kemp, P. A.; Bennett, T.; Bose, C.; Foulkes, R.; Hughes, B., Antagonistic effect of human afpha-CGRP (8-37) on the in vivo regional haemodynamic actions of human alphaCGRP.. Biochem. Biophys. Res. Commun., 171,938-9431990. 110. Gardiner, S. M.; Compton, A. M.; Kemp, P. A.; Bennett, T.; Bose, C.; Foulkes, R.; Hughes, B., Antagonistic effect of human afpha-cafcitonin gene-related peptide (8-37) on regional hemodynarnic actions of rat islet arnyloid polypeptide in conscious Long-Evans rats. Diabetes, 40, 948–951 1991. 111. Gates, T. S.; Zimmerman, R. P.; Mantyh, C. R.; Vigna, R.; Mantyh, P. W., Calcitonin gene-related peptide-afpha receptor binding sites in the gastrointestinal tract. Neuroscience, 31,757-7701989. 112. Gennari, C.; Fischer, J. A., Cardiovascular action of cafcitonin generelated peptide in humans. Calc~ Tissue Znt.,37, 581–584 1985. 113. Gennari, C.; Nami, R.; Agnusdei, D.; Fischer, J. A., Improved cardiac performance with human calcitortin gene related peptide in patients with congestive heart failure. Cardiovasc. Res., 24, 239–241 1990. 114. Gibbins, I. L.; Fumess, J. B.; Costa, M., Pathway-specific patterns of the co-existence of substance P, cafcitonin gene-related peptide, cholecystokinin and dynorphin in neurons of the dorsal root ganglia of the guinea-pig. Cell Tissue Res., 248, 417–437 1987. 115. Gibbins, I. L.; Wattchow, D.; Coventry, B., Two immunohistochernically identified populations of calcitonin gene-related peptide (CGRP)-immunoreactive axons in human skin. Brain Res., 414, 143-1481987. 116. Gibson, S. J.; Polak, J. M.; Bloom, S. R.; Sabate, I. M.; Mulderry, P. K.; Ghatei, M. A.; Morrison, J. F. B.; Kelly, J. S.; Evans, R. M.; Rosenfeld, M. G., Calcitonin gene-related peptide (CGRP) immunoreactivity in the spinal cord of man and eight other species. J. Neurosci., 4, 3101–3111 1984. 117. Gill, J. S.; Moonga, B. S.; Huang, C. L. H.; Lu, F.; Zaidi, M.; Carom, A. J., Voltage-sensitive elevation of cytosolic [Ca2+] in guinea-pig cardiac myocytes elicited by calcitonin gene-related peptide. Exp. Physiol., 77,925-9281992.

671 118. Girgis, S. I.; MacIntyre, I.; Macdonafd, D. W. R.; Lynch, C.; Morns, H. R.; Wimalawansa, S. J.; Self, C. H.; Stevenson, J. C.; Bevis, P. J. R., Calcitonin gene-related peptide - potent vasodilator and major product of calcitonin gene. I.arrcet,2, 14–16 1985. 119. Giuliani, S.; Wimalawansa, S. J.; Maggi, C. A., Involvement of multiple receptors in the biological effects of cafcitonin gene-related peptide and amylin in rat and guinea-pig preparations. Br. J. Pharmacol., 107, 510–514 1992. 120. Goadsby, P. J.; Edvinsson, L.; Ekman, R., Vasoactive pcptide release in the extracerebral circulation of humans during migraine headache. Ann. Neurol., 28, 183–187 1990. 121. Goadsby, P. J.; Edvinsson, L., The trigeminovascular system and migrtine: studiescharacterizing cerebrovascular and neuropeptide changes seen in humans and cats. Ann. Neurol., 33, 48–56 1993. 122. Goldring, S. R.; Gem, A. H.; Yarnin, M.; Krane, S. M.; Wang, J.-T., Characterization of the stmcturaf and functional properties of cloned calcitonin receptor cDNAs. Horm. Metab. Res., 25, 477–480 1993. 123. Gon, G.; Giaid, A.; Steel, J. H.; O’Hallomn, D. J.; Van Noorden, S.; Ghatei, M. A.; Jones, P. M.; Amara, S. G.; Ishikawa, H.; Bloom, S. R.; Polak, J. M., Localization of immunoreactivity for cafcitonin gene-related peptide in the rat anterior pituitary during ontogeny and gonadal steroid manipulations and detection of its messenger ribonucleic acid. Endocrinology, 127, 2618–2629 1990. 124. Gem, A. H.; Lin, H. Y.; Yamin, M.; Auron, P. E.; Flarmery, M. R.; Tapp, D. R.; Manning, C. A.; Lodish, H. F.; Kmne, S. M.; Goldring, S. R., Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line. .f. Clin. Invest., 90, 1726-17351992. 125. Gray, D. W.; Marshafl, L, Nitric oxide synthesis inhibitors attenuate calcitonin gene-related peptide endothelium-dependent vasorelaxation in rat aorta. Eur. J. Pharmacol., 212, 37–42 1992. 126. Gray, D. W.; Marshall, I., Human a-calcitonin gene-related peptide stimulates adenylate cyclase and guanylate cyclase and relaxes rat thoracic aorta by releasing nitric oxide. Br. J. Pharrnacol.,107,6916961992. 127. Green, T.; Dockray, G. J., Characterization of the peptidergic afferent innervation of the stomach in the rat, mouse and guinea-pig. Neuroscience, 25, 181–193 1988. 128. Guidobono, F.; Coluzzi, M.; Pagani, F.; Pecile, A.; Netti, C., Amylin given by central and peripheral routes inhibits acid gastric secretion. Peptides, 15, 699–702 1994. 129. Gulbenkian, S.; Merighi, A.; Wharton, J.; Vamdell, I. M.; Polak, J. M., Ultrastmctural evidence for the coexistence of calcitonin generelated peptide and substance P in secretory vesicles of peripheral nerves in the guinea pig. J. Neurocytol., 15, 535–542 1986. 130. Gulbenkian, S.; Edvinsson, L.; Opgaard, O. S.; Wharton, J.; Polak, J. M.; David-Ferreira, J. F., Peptide-containing nerve fibres in guineapig coronary arteries: immunohistochemistry, ultrastructure and vasomotility. J. Auton. Nerv. Syst., 31, 153–168 1990. 131. Hall, J. M.; Siney, L.; Lippton, H.; Hyman, A.; Chrmg,J. K.; Brain, S. D., Interaction of human adrenomedullinl~.~~with calcitonin generelated peptide receptors in the rnicrovasculature of the rat and hamster. Br. J. Pharrrsacol.,114, 592–597 1995. 132. Han, S.-P.; Naes, L.; Westfafl, T. C., Inhibition of periarterial nerve stimulation-induced vasodilation of the mesenteric arterial bed by CGRP (8-37) and CGRPreceptor desensitization. Biochem.Biophys. Res. Commun.,168,786-7911990. 133. Hardebo, J. E.; Suzuki, N.; Ekblad, E.; Owman, C., Vasoactive intestinal polypeptide and acetylcholine coexist with neuropeptide Y, dopamine-b-hydroxylase, tyrosine hydroxylase, substance P or calcitonin gene-related peptide in neuronal subpopulations in cranial parasympathetic ganglia of rat. Cell TissueRes., 267,291-3001992. 134. Harrigrm, E. A.; Magnuson, D. J.; Thunstedt, G. M.; Gray, T. S., Corticotropin releasing factor neurons are imervated by cdcitonin gene-related peptide terminals in the rat central arnygdaloid nucleus. Brain Res. Bull., 33,529-5341994. 135. Henke, H.; Tschopp, F. A.; Fischer, J. A., Distinct binding sites for calcitonin gene-related peptide and safmon cafcitonin in rat central nervous system. Brain Res., 360, 165–171 1985. 136. Henke, H.; Sigrist, S.; Lang, W.; Schneider, J.; Fischer, J. A., Comparison of binding sites for the calcitonin gene-related peptides I and II in man. Brain Res., 410,404-4081987. 137. Henry, M. A.; Nousek-Goebl, N. A.; Weshum, L. E., Light and electron microscopic localization of calcitonin gene- related peptide immunoreactivity in lamina 11of the feline trigerninal pars caudafid medullarydorsrdhom: A qualitativestudy. Synapse, 13,99–107 1993.

672 138. Hirata, Y.; Takagi, Y.; Takata, S.; Fukuda, Y.; Yosfrirni,H.; Fujita, T., Calcitonin gene-related peptide receptor in cultured vascular smooth muscle and endothelial cells. Bioclrem. Bioplrys. Res. Commun., 151, 1113–1121 1988. 139. Hoheisel, U.; Mense, S.; Scherotzke, R., Crdcitonin gene-reIated peptide-imrnunoreactivity in functionally identified primary afferent neurones in the rat. Anal. EmbryoZ.,189, 41–49 1994. 140. Holzer, P., Local effecter functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, cafcitonin generelated peptide and other neuropeptides. Neuroscience, 24, 739– 7681988. 141. Holzer, P.; Guth, P. H., Nerrropeptide control of rat gastric mucosal blood flow. Increase by calcitonirr gene-related peptide and vasoactive intestinal polypeptide, but not substance P and neurokinin A.. Circ. Res., 68, 100-1051991. 142. Holzer, P.; Livingston, E. H.; Guth, P. H., Sensory neurons signal for an increase in rat gastric mucosal blood flow in the face of pending acid injury. Gastroerrterology,101, 416–423 1991. 143. Holzer, P.; Livingston, E. H.; Saria, A.; Guth, P. H., Sensory neurons mediate protective vasodilatation in rat gastric mucosa. Am. J. Physiol. Gastrointest. Liver Physiol, 260, 363–370 1991. 144. Honda, T.; Zhou, Z.-C.; Gu, Z.-F.; Kitsukawa, Y.; Mrozinski, J. E. J.; Jensen, R. T., Structural analysis of CGRP receptors on gastric smooth muscle and pancreatic acinar cells. Am. J. PhysioZ.Gastrointest. Liver Physiol, 27, 1142–11521993. 145. Hokfelt, T.; ~Arvidsson,U.; Ceccatelli, S.; Cork%, R.; Cullheim, S.; Dagerlind, A.; Johnson, H.; Orazzo, C.; Piehl, F.; Pieribone, V.; Schafling, M.; Terenius, L.; UIfbake, B.; Verge, V. M.; Vilktr, M.; Wiesenfeld-Hallin, Z.; Xu, X.-J., Xu, Z., Cafcitonin gene-related peptide in the brain, spinal cord, and some peripheral systems. Ann. NYAcad. Sci., 657, 119–134 1992. 146. Hubbard, J. A. M.; Martin, S. R.; Chaplin, L. C.; Bose, C.; Kelly, S. M.; Price, N. C., Solution structures of cafcitonin-gene-relatedpeptide analogues of cafcitonin-gene-related peptide and amylin. Biochem. J., 275, 785–788 1991. 147. Hughes, J. J.; Gosnell, B. A.; Levine, A. S.; Morley, J. E.; Silvis, S. E., Intraventricular cafcitonin gene-related peptide inhibits gastricacid secretion. Peptides, 5, 665–667 1984. 148. Hughes, S. R.; Brain, S. D., A cafcitonin gene-related peptide (CGRP) antagonist (CGRP8-37) inhibits microvascukir responses induced by CGRP and capsaicin in skin. B-. J. Pharmacol., 104, 738-7421991. 149. Ichiki, Y.; Kitatmrra, K.; Kangawa, K.; Kawamoto, M.; Matsuo, H.; Eto, T., Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Lett., 338, 6– 101994. 150. Inagaki, S.; Kite, S.; Kubota, Y.; Girgis, S.; Hillyard, C. J.; MacIntyre, L, Autoradiographic localization of cafcitonin generelated peptide binding sites in human and rat brains. Brain Res., 374,287-2981986. 151. Inagaki, S.; Matsuda, Y.; Nakai, Y.; Takagi, H., Cafcitonin generelated peptide (CGRP) immrrnoreactivity in the afferents to the caudate-putarnen and perirhinal cortex of rats. Brain Res., 537, 263– 2701990. 152. Inaishi, Y.; Kashihara, Y.; Sakaguchi, M.; Nawa, H.; Kuno, M., Cooperative regulation of calcitonin gene-related peptide levels in rat sensory neurons via their central and peripheral processes. J. Neurosci., 12, 518–524 1992. 153. Inoue, K.; Hirarnatsu, S.; Hisatomi, A.; Umeda, F.; Nawata, H., Effects of arnylirr on the release of insulin and glucagon from the perfused rat pancreas. Horrn.Metab. Res., 25, 135-1371993. 154. Ishida-Yamamoto, A.; Tohyama, M., Cafcitonin gene-related peptide in the nervous tissue. Prog. Neurobiol., 33, 335-3861989. 155. Isfrihara,T.; Nakarmrra,S.; Kaziro, Y.; Takahashi, T.; Takahashi, K.; Nagata, S., Moleculm cloning and expression of a cDNA encoding the secretin receptor. EMBO J., 10, 1635-16411991. 156. Ishihara, T.; Shigemoto, R.; Men, K.; Takaftashi, K.; Nagata, S., Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide. Neuron, 8, 811–819 1992. 157. Ishikawa, T.; Okamura, N.; Saito, A.; Masaki, T.; Goto, K., Positive inotropic effect of calcitonin gene-related peptide mediated by cyclic AMP in guinea pig heart. Circ. Res., 63, 726-7341988. 158. Ishiyama, Y.; Kitamura, K.; Ichiki, Y.; Nakamura, S.; Kida, O.; Kangawa, K.; Eto, T., Hemodynamic effects of a novel hypoterrsive peptide, human adrenomedullin, in rats. Eur. J. Pharmacol., 241, 271–273 1993.

VANROSSUMET AL. 159. Ishizaka, Y.; Tanaka, M.; Kitamnra, K.; Krmgawa,K.; Minamirro,N.; Matsuo, H.; Eto, T., Adrenomedullin stimulates cyclic AMP formation in rat vascular smooth muscle cells. Biochem. Biophys. Res. Commun., 200,642-6461994. 160. Jansen, L, Characterization of calcitonin gene-related peptide (CGRP) receptors in guinea pig basilar artery, Neuropeptides, 21, 73–79 1992. 161. Jennings, C. G. B.; Mudge, A. W., Chick myotubes in culture express high-affinity receptors for calcitonin gene-related peptide. Brain Res., 504, 199–205 1989. 162. Johnston, F. G.; Bell, B. A.; Robertson, I. J.; Miller, J. D.; Hrdibum, C.; O’Shaughnessy, D.; Riddell, A. J.; O’Laoire, S. A., Effect of cafcitonin-gene-related peptide on postoperative neuroIogicrddeficits after subarachnoid hemorrhage. I.ancet, 335, 869–872 1990. 163. Jolicoerrr, F. B.; Merrard, D.; Foumier, A.; St-Pieme, S., Stmctureactivity anafysis of CGRP’S neurobehavioral effects. Ann. NYAcad. Sci., 657, 155-1631992. 164. Ju, G.; Hokfelt, T.; Brodin, E.; Fabrenkmg, J.; Fischer, J. A.; Frey, P.; Elde, R. P.; Brown, J. C., Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecystokinirr-immrrnoreactive ganglion cells. Cell Tissue Res., 247, 417–431 1987. 165. Ju, G., Cafcitonin gene-related peptide-like immunoreactivity and its relation with neurotensin- and corricotropin-releasing hormone-like immunoreactive neurons in the bed nuclei of the stria terrninafis in the rat. Brain Res. Bull., 27, 617–624 1991. 166. Ju, G.; Liu, S.-J.; Ma, D., Calcitonin gene-related peptide- and substance P-like-immunoreactive innervation of the anterior pituitary in the rat. Neuroscience, 54, 981–989 1993. 167. Juppner, H.; Abou-Samra, A.-B.; Freeman, M.; Kong, X. F.; Schipani, E.; Richards, J.; Kolakowski, L. F.; Hock, J.; Potts, J. T.; Kronerrberg, H. M.; Segre, G. V., A G protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide. Science, 254, 1024–10261991. 168. Jurgen Lenz, H., CaIcitonin and CGRPirrhibit gastrointestinal transit via distinct neuronal pathways. Am. J. Physiol. Gastrointest. Liver Physiol, 254,920-9241988. 169. Juul, R.; Aakhus, S.; Bjomstak, K.; Gisvold, S. E.; Bmbakk, A. O.; Edvinsson, L., Cafcitonin gene-related peptide (human a-CGRP) counteracts vasoconstriction in human subaracfmoid hemorrhage. Neurosci. Lett., 170, 67–70 1994. 170. Kageyama, M.; Yanagisawa, T.; Taira, N., Calcitonin gene-related peptide relaxes porcine coronary arteries via cyclic AMP-dependent mechanisms, but not activation of ATP-sensitive potassium channels. J. Pharmacol. Exp. Ther., 265,490-4971993. 171. Kartatsuka,A.; Makino, H.; Ohsawa, H.; Tokuyama, Y.; Yarnaguchi, T.; Yoshida, S.; Adachi, M., Secretion of islet amyloid polypeptide in response to glucose. FEBSLett., 259, 199-201 1989. 172. Kangrga, L; Larew, J. S. A.; Randic, M., The effects of substance P and cafcitonin gene-related peptide on the efflux of endogenous glutamate and aspartate from the rat spinal dorsal horn in vitro. Neurosci. Lett., 108, 155–160 1990. 173. Kapas, S.; Catt, K. J.; Clark, A. J. L., Cloning and expression of cDNA encoding a rat adrenomedullin receptor. J. Biol. Chem., 270, 25344-253471995. 174. Kapas, S.; Clark, A. J. L., Identification ofarr orphan receptor gene as 1c~citonirr gene-related peptide receptor. Biochem. Biophys. a type Res. Commun.,217, 832–838 1995. 175. Katoh, K.; Tohyama, M.; Noguchi, K.; Senba, E., Axomd flow blockade induces alpha-CGRP mRNA expression in rat motonerrrons.Brain Res., 599, 153–157 1992. 176. Kawai, Y.; Takarni, K.; Sfriosaka, S.; Emson, P. C.; Hillyard, C. J.; Girgis, S.; MacIntyre, L; Tohyama, M., Topographic localization of calcitonirr gene-related peptide in the rat brain: an immunofristochemical analysis. Neuroscience, 15, 747–763 1985. 177. Kawarrmra, M.; Kuraishi, Y.; Mirrami, M.; Satoh, M., Antinociceptive effect of intrathecafly administered antisemm against cafcitonirr gene-related peptide on thermal and mechanical noxious stimuli in experimental hyperalgesic rats. Brain Res., 497, 199–203 1989. 178. Kim, D., Crdcitonin gene-realted peptide activates the muscarirricgated K+ current in atrial cells. Pjugers Arch., 418,338-3451991. 179. Kitarrmra, K.; Kartgawa, K.; Kawamoto, M.; Ichifd, Y.; Nakarmrra, S.; Matsuo, H.; Eto, T., Adrenomedullin: a novel hypoterrsivepeptide isolated from human pheochromocytoma. Biochem. Biophys. Res. Commun.,192, 553–560 1993.

RECEPTORS

OF CGRP AND RELATED PEPTIDES

180. Kitamura, K.; Sakata, J.; Kangawa, K.; Kojima, M.; Matsuo, H.; Eto, T., Cloning and characterization of cDNA encoding a precursor for human adrenomedullin. Biochem. Biophys. Res. Cmnmurz., 194, 720-7251993. 181. Kitamura, K.; Ichiki, Y.; Tanaka, M.; Kawamoto, M.; Emura, J.; Sakakibara, S.; Kangawa, K.; Matsuo, H.; Eto, T., Immunoreactive adrenomedullin in human plasma. FEBS Lett., 341, 288–290 1994. 182. Kitamura, K.; Kangawa, K.; Kojima, M.; Ichiki, Y.; Matsuo, H.; Eto, T., Complete amino acid sequence of porcine adrenomedullin and 338, 306–310 cloning of cDNA encoding its precursor. FEBS I.@., 1994. 183. Knock, G. A.; Wharton, J.; Gaer, J. A. R.; Yacoub, M. H.; Ta lor K. ‘ M.; Polak, J. M., Regional distribution and regulation of [ 12I]calcltonin gene-related peptide binding sites in coronary arteries. Eur. J. pha?nu2coi., 219, 415–425 1992. 184. Kohno, M.; Yokokawa, K.; Yasunari, K.; Kane, H,; Horio, T.; Takeda, T., Stimulation of cyclic adenosine monophosphate formation by the novel vasorelaxant peptide adrenomedullin in cultured rat mesangial cells. Metabolism, 44, 10–12 1995. 185. Kovics, A.; Telegdy, G., Effects of intracerebroventricular administration of calcitonin gene-related peptide on passive avoidance behaviour in rats. Neuropeptides, 23,51-541992. 186. Kovics, A.; Telegdy, G., Effect ofcalcitonin gene-related peptide on passive avoidance behavior in rats. Role of transmitters. Ann. NY Acad. Sci., 657,543-5451992. 187. Kovics, A.; Telegdy, G., Behavioral impairment induced by calcitonin gene-related peptide (CGRP) antiserum in passive avoidance reflex in rats. Neuropeptides, 26, 233–236 1994. 188. Kovhcs, A.; Telegdy, G., CGRP prevents electroconvulsive shockinduced amnesia in rats. Pharrnacol.Biochem. Behav., 47, 121–125 1994. 189. Kraenzlin, M. E.; Ch’ng, J. L. C.; Mulderry, P. K.; Ghatei, M. A.; Bloom, S. R., Infusion of a novel peptide, calcitonin gene-related peptide (CGRP) in man. Pharmacokinetics and effects on gastric acid secretion and on gastrointestinal hormones. Regul. Pept., 10, 189– 1971985. 190. Krafm, D. D.; Gosnell, B. A.; Levine, S.; Morley, J. E., Effects of calcitonin gene-related peptide on food intake. Peptides, 5, 861–864 1984. 191. Kresse, A.; Jacobowitz, D. M.; Skofitsch, G., Distribution of calcitonin gene-related peptide in the central nervous system of the rat by immrrnocytochemistryand in situ hybridization histochemistry. Ann. NY Acad. Sci., 657, 455–457 1992. 192. Kruger, L.; Mantyh, P. W.; Stemirri, C.; Brecha, C. N.; Mantyh, C. R., Calcitonin gene-related peptide (CGRP) in the rat central nervous system: patterns of immunoreactivity and receptor binding sites. Brain Res., 463, 223–244 1988. 193. Kruger, L.; Silverman, J. D.; Mrmtyh, P. W.; Stemini, C.; Brecha, N. C., Peripheral patterns of calcitonin gene-related peptide general somatic sensory innervation: cutaneous and deep terminations. J. Comp. Neurol., 280,291-3021989. 194. Kubota, Y.; Inagaki, S.; Shimada, S.; Takatsuji, K.; Tohyama, M.; Takagi, H., Striatal calcitonin gene-related peptide-like immunoreactive affenmts from the regions venraf and medial to the medial geniculate nucleus of rats. Neuroscience, 40, 423–428 1991. 195. Kuo, T.-f.; Ouchi, Y.; Kim, S.; Toba, K.; Orimo, H., The role of activation of the sympathetic nervous system in the central pressor action of calcitonin gene-related peptide in conscious rats. Naunyn SchmiedebergsArch. PharmacoL, 349,394-4001994. 196. Lambrecht, N.; Burchert, M.; Respondek, M.; Miiller, K.-M.; Peskar, B. M., Role of calcitonin gene-related peptide and nitric oxide in the gastroprotective effect of capsaicin in the rat. Gastroenterology, 104, 1371-13801993. 197. Landis, S. C.; Fredieu, J. R., Coexistence of cafcitorringene-related peptide and vasoactive intestinal peptide in cholinergic sympathetic innervation of rat sweat glands. Brain Res., 377, 177–181 1986. 198. Latchman, D. S., Cell-type-specific splicing factors and the regulation of alternative RNA splicing. New Biol., 2, 297–303 1990. 199. Laufer, R.; Changeux, J.-P., Cafcitorrin gene-related peptide and cyclic AMP stimulate phosphoinositide turnover in skeletal muscle cells. .1.Biol. Chem., 264, 2683–2689 1989. 200. Lawrence, J. C.; Zhang, J.-N., Control of glycogen synthase and phosphorylase by amylin in rat skeletal muscle. J. Biol. Chem., 269, 11595-116001994. 201. Lazar, P.; Reddington, M.; Streit, W.; Raivich, G.; Kreutzberg, G. W., The action of calcitonin gene-related peptide on astrocyte

673

202. 203. 204. 205. 206.

207. 208. 209. 210. 211.

212. 213.

214. 215. 216.

217. 218. 219. 220. 221.

222.

223.

morphology and cyclic AMP accumulation in astrocyte cultures from neonatal rat brain. Neurosci. Left., 130, 99–102 1991. Le Greves, P.; Nyberg, F.; Terenirrs,L.; Hokfelt, T., Crdcitonin generelated peptide is a potent inhibitor of substance P degradation. Eur. J. Pharrmzcol.,115, 309–311 1985. Le Greves, P.; Nyberg, F.; Hokfelt, T.; Terenius, L., Calcitonin generelated peptide is metabolized by an endopeptidase hydrolyzing substance P.. Regul. Pept., 25, 277–286 1989. Leff, S. E.; Evans, R. M.; Rosenfeld, M. G., Splice commitment dictates neuron-specific alternative RNA processing in calcitonirr/ CGRP gene expression. Cell, 48,517-5241987. Leighton, B.; Cooper, G. J. S., Pancreatic amylin arrdcalcitonirrgenerelated peptide cause resistance to insulin in skeletal muscle in vitro. Nature, 335, 632–635 1988. Lerrz,H. J.; Rivier, J. E.; Vale, W. W.; Brown, M. R.; Mortmd, M. T., Calcitonirr gene related peptide acts within the central nervous system to inhibit gastric-acid secretion. Regul. Pept., 9, 271–277 1984. Lerrz, H. J.; Mortmd, M. T.; Rivier, J. E.; Brown, M. R., Central nervous system actions of calcitonin gene-related peptide on gastric acid secretion in the rat. Gastroeruerdogy, 88, 539–544 1985. Levine, J. D.; Fields, H. L.; Basbaum, A. I., Peptides and the primary afferent nociceptor. J. Neurosci., 13, 2273–2286 1993. Li, Y.; Duckles, S. P., Effect of endothelium on the actions of sympathetic and sensory nerves in the perfused rat mesentery. Eur. 210, 23–30 1992. J. Phar?rracol., Lin, B.; Gao, Y.; Chang, J. K.; Heaton, J.; Hyman, A.; Lippton, H., An adrenomedullin fragment retains the systemic vasodepressor activity of rat adrenomedullin. Eur. J. Pharrruzcol.,260, 1–4 1994. Lin, H. Y.; Harris, T. L.; Flarmery, M. S.; Arrrffo, A.; Kaji, E. H.; Gem, A.; Kolakowski, L. F.; Lodish, H. F.; Gohfring, S. R., Expression cloning of an adenylate cyclase-coupled calcitonin receptor. Science, 254, 1022–1024 1991. Lips, C. J.; Stecnbergh, P. H.; Hoppener, J. W.; Bovenberg, R. A.; van der Sluys-Veer, J.; Jansz, H. S., Evolutionmy pathways of the calcitonin genes. Mol. Cell. ,%rdocrinol., 57, 1–6 1988. Longmore, J.; Hogg, J. E.; Hutson, P. H.; Hill, R. G., Effects of two truncated forms of human calcitonirr-gene related peptide: implications for receptor classification. Eur. J. PharrnacoL, 265, 53–59 1994. Lorenzo, A.; Razzaboni, B.; Weir, G. C.; Yankner, B. A., Pancreatic islet cell toxicity of amylin associated with type-2 diabetes mellitus. Nature, 368, 756–760 1994. Lu, B.; Fu, W.-m.; Greengard, P.; Poo, M.-m., Calcitonin generelated peptide potentates synaptic responses at developing neuromuscular junction. Nature, 363, 76–79 1993. Lundberg, J. M.; Frarrco-Cereceda,A.; Hua, X.; Hokfelt, T.; Fischer, J. A., Co-existence of substance P and calcitonin gene-related Pep’ide-like irmnunoreactivities in sensory nerves in relation to cardiovascular and bronchoconstrictor effects of capsaicin. Eur. J. pharrrracol.,108,315-3191985. Lynch, B.; Kaiser, T., Biologicrd properties of two models of calcitonin gene related peptide with idealized arnphiphilic rr-helices of different lengths. Biochemistry, 27, 7600-76071988. Maggi, C. A.; Chiba, T.; Giuliani, S., Human alpha-calcitonirrgenerelated peptide-(8-37) as an antagonist of exogenous and endogenous calcitorrirrgene-relatedpeptide. Eur. J. Pharmacol.,192, 85–88 1991. Mair, J.; Lechleitner, P.; Langle, T.; Wiedermarm, C.; Dienstl, F.; Saria, A., Plasma CGRP in acute myocardial infarction. ,@rcet, 335, 1681990. Mak, J. C. W.; Barnes, P. J., Autoradiographic localization of calcitonin gene-related peptide (CGRP) binding sites in human and guinea pig lung. Peptides, 9, 957–963 1988. Manning, M. C., Conformation of the alpha form ofhumancalcitonin gene-related peptide (CGRP) in aqueous solution as determined by circular dichroism spectroscopy. Biochem. Biophys. Res. Commun., 160,388-3921989. Mao, J.; Coghill, R. C.; Kellstein, D. E.; Frenk, H.; Mayer, D. J., Crdcitonin gene-related peptide enhances substance P-induced behaviors via metabolic inhibition: In vivo evidence for a new mechanism of neuromodulation. Brain Res., 574, 157–163 1992. Marlier, L.; Rajaofetra, N.; Peretti-Renucci, R.; Kachidian, P.; Poulat, P.; Feuerstein, C.; Privat, A., Calcitonin gene-related peptide staining intensity is reduced in rat lumbar motoneurons after spinaf cord transection: a quantitative irmmmocytochemical study. Exp. Brain Res., 82, 40–47 1990.

674 224. Marshall, I.; Adarns, M.; Alkazwini, S. J.; Craig, R. K.; Roberts, P. M.; Shepperson, N. B,, Cardiovascular effects of human and rat CGRP compared in the rat and other species. Eur. J. Pharrnacol., 123,207-2161986. 225. Marshafl, I.; Alkazwini, S. J.; Craig, R. K.; Holman, J. J., Human and rat alpha-CGRP but not calcitonin cause mesenteric vasodilatation in rats. Eur. J. Pharmacol., 123, 217–222 1986. 226. Marshafl, I., Mechanism of vascular rekixation by the cafcitonin gene-related peptide. Ann. NY Acad. Sci., 657, 204-2151992. 227. Masuo, Y.; Giscard-Dartevelie, S.; Bouizar, Z.; Rostene, W., Effects of cerebral lesions on binding sites for calcitonin and cafcitoningenerelated peptide in the rat nucleus accumbens and ventral tegmental area. J. Chem. Neuroanat., 4, 249-2571991. 228. Maton, P, N.; Sutliff, V. E.; Zhou, Z.-C.; Collins, S. M.; Gardner, J. D.; Jensen, R. T., Characterization of receptors for crdcitonin generelated peptide on gastric smooth muscle cells. Am. J. Physiol. Gastrointest. Liver Physiol, 254,789-7941988. 229. Maton, P. N.; Pradharr, T.; Zbou, Z.-C.; Gardner, J. D.; Jensen, R. T., Activities of calcitonin gene-related peptide (CGRP) and related peptides at the CGRP receptor. Peptides, 11,485-4891990. 230. May, P. C.; Boggs, L. N.; Fuson, K. S., Neurotoxicity of human amylin in rat primary hippocarnpal cultures: similarity to Alzheimer’s disease arnyloid-(3neurotoxicity. J. Neurochem., 61, 2330– 23331993. 231. McCuIIoch, J.; Uddman, R.; Kingman, T.; Edvinsson, L., Cafcitonin gene-related peptide: functional role in cerebrovascular regulation. Proc. Natl. Acad. Sci. USA, 83, 5731–5735 1986. 232. McGillis, J. P.; Humphreys, S.; Rangnekar, V.; CiaJlella, J., Modulation of B lymphocyte differentiation by calcitonin gene-related peptide (CGRP). I. Characterization of high-affinity CGRP receptors on munne 70Z13cells. Cell. Immunol., 150, 391–404 1993. 233. McLean, L. R.; Balasubrarnaniam, A., Promotion of &stmcture by interaction of diabetes associated polypeptide (arnylin) with phosphatidylcholine. Biochim. Biophys. Acta, 1122,317-3201992. 234. Menard, D. P.; van Rossum, D.; Kar, S.; Jolicoeur, F. B.; Jhamarrdas, K.; Quirion, R., Tolerance to the antinociceptive properties of morphine in the rat spinal cord: alteration of calcitonin gene-related peptide-like immunostaining and receptor binding sites. J. Pharrnacol. Exp. Ther., 273, 887–894 1995. 235. Menard, D. P.; van Rossum, D.; Kar, S.; Qrririon, R., Alteration of calcitonin gene-related peptide and its receptor binding sites during the development of tolerance to mu and delta opioids. Can. J. Physiol. Pharrnacol.,73, 1089–1095 1995. 236. Menard, D. P.; van Rossum, D.; Kar, S.; Quirion, R., Chronic intrathecal treatment with nitric oxide (NO) modulators afters calcitonin gene-related peptide (CGRP) markers in the rat spinal cord. Soc. Neurosci. Abst. (Abstract), 21,3771995. 237. Menard, D. P.; van Rossum, D.; Kar, S.; St-Pien’e, S.; Sutak, M.; Jhamandas, J. H.; Quirion, R., A cafcitonin gene-related peptide receptor antagonist prevents the development of tolerance to spinal morphine anafgesia. J. Neurosci., 16, 2342–2351 1996. 238. Miles, K.; Greengard, P.; Hugaoir, R. L., Cafcitonin gene-related peptide regulates phosphorylation of the nicotinic acetylcholine receptor in rat myotubes. Neuron, 2, 1517–1524 1989. 239. Miletic, V.; Tan, H., Iontophoretic application of calcitonin generelated peptide produces a slow and prolonged excitation of neurons in the cat lumbar dorsal horn. Brain Res., 446, 169-1721988. 240. Mimeault, M.; Foumier, A.; Dumont, Y.; St-Pieme, S.; Quirion, R., Comparative affinities and antagonistic potencies of various human calcitonin gene-related peptide fragments on calcitonin gene-related peptide receptors in brain and periphery. J. Pharmacol. Exp. Ther., 258, 1084–1090 1991. 241. Mimeault, M.; St-Pieme, S.; Fournier, A., Confirmational characterization by circular-dichroism spectroscopy of various fragments and analogs of calcitonin gene-related peptide. Eur. J. Biochem., 213, 927–934 1993. 242. Morara, S.; Provini, L.; Rosina, A., CGRP expression in the rat olivocerebellar system during postnatal development. Brain Res., 504,315-3191989. 243. Morishita, T.; Yamaguchi, A.; Fujita, T.; Chiba, T., Activation of adenylate cyclase by islet amyloid polypeptide with COOH-terminal amide via calcitonin gene-related peptide receptors on rat liver plasma membranes. Diabetes, 39,875-8771990. 244. Morns, H. R.; Panico, M.; Etienne, T.; Tippins, J.; Girgis, S. I.; MacIntyre, I., Isolation and characterization of human calcitonin gene-related peptide. Nature, 308,746-7481984.

VANROSSUMET AL. 245. Morton, C. R.; Hutchison, W. D., Release of sensory neuropeptides in the spinal cord: studies with calcitonin gene-related peptide and gafarrin.Neuroscience, 31,807-8151989. 246. Mulder, H.; Lindh, A.-C.; Sundler, F., Islet amyloidpolypeptide gene expression in the endocrine pancreas of the rat: a combined in situ hybridization and immunocytochemical study. Cell TissueRes., 274, 467-4741993. 247. Mulderry, P. K.; Rodrigo, J.; Rosenfeld, M. G.; Pokdc,J. M.; Allen, J. M.; Ghatei, M. A.; Bloom, S. R., Calcitonin gene-related peptide in cardiovascular tissues of the rat. Neuroscience, 14, 947-9541985. 248. Mulderry, P. K.; Ghatel, M. A.; Spokes, R. A.; Jones, P. M.; Pierson, A. M.; Hamid, Q. A.; Kanse, S.; Amara, S. G.; Burnn, J. M.; Legon, S.; Polak, J. M.; Bloom, S. R., Differential expression of afpha-CGRP and beta-CGRP by primary sensory neurons and enteric autonomic neurons of the rat. Neuroscience, 25, 195–205 1988. 249. Mulle, C.; Benoit, P.; Pinset, C.; Roa, M.; Changeux, J.-P., Crdcitonin gene-related peptide enhances the rate of desensitization of the nicotinic acetylcholine receptor in cuftrrred mouse muscle cells. Proc. Natl. Acad. Sci. USA, 85, 5728–5732 1988. 250. Nagai, K.; Ino, H.; Yamamoto, H.; Nakagawa, H.; Yamrmo, M.; Tohyama, M.; Sfriosaka, S.; Inagaki, S.; Kite, S., Lesions of the lateral part of the dorsal parabrachial nucleus caused hyperglycemia and obesity. J. Clin. Biochem. Nutr., 3, 103–112 1987. 251. Nagarnatsu, S.; Carroll, R. J.; Grodsky, G. M.; Steiner, D. F., Lack of islet amyIoid polypeptide regulation of insulin biosynthesis or secretion in normrd rat islets. Diabetes, 39, 871–874 1990. 252. Nakarnuta, H.; Fnkrrda, Y.; Koida, M.; Fujii, N.; Otaka, A.; Funakoshi, S.; Yajima, H.; Mitsuyasu, N.; Orlowski, R. C., Binding sites of calcitonin gene-related peptide (CGRP): abundant occurrence in visceral organs. Jpn. J. PharrnacoL,42, 175–180 1986. 253. Nakazawa, K.; Saito, H.; Matsuki, N., Effects of calcitonin genereIated peptide (CGRP) on Ca Z+-chmne~ Cumentof isolated smooth muscle cells from rat vas deferens. Naunyn Schmiedebergs Arch. Pharmacol., 346, 515–522 1992. 254. Nelson, M. T.; Huang, Y.; Brayden, J. E.; Hescheler, J.; Standen, N. B., Arterial dilations in response to calcitonin gene-related peptide involve activation of K+ channels. Nature, 344, 770–773 1990. 255. New, H. V.; Mudge, A. W., Cafcitonin gene-related peptide regulates muscle acetylcholine receptor synthesis. Nature, 323, 809– 8111986. 256. Nguyen, K. Q.; Jacobowitz, D. M.; Sills, M. A., Cardiovascular effects produced by microinjection of calcitonin gene-related peptide into the rat central amygdaloid nucleus. Peptides, 7, 337– 3391986. 257. Nishi, M.; Chan, S. J.; Nagamatsu, S.; Bell, G. I.; Steiner, D. F., Conservation of the sequence of islet amyloid polypeptide in five marnrnafs is consistent with its putative role as an islet hormone. Proc. Natl. Acad. Sci. USA, 86, 5738–5742 1989. 258. Njuki, F.; Nicholl, C. G.; Howard, A.; Mak, J. C. W.; Barnes, P. J.; Girgis, S. I.; Legon, S., A new cafcitonin-receptor-like sequence in rat pulmonary blood vessels. Clin. Sci., 85, 385–388 1993. 259. Odar-Cederloef, L; Theodorsson, E.; Ericsson, F.; Kjellstrand, C. M., Plasma concentrations of cafcitonin gene-related peptide in fluid overload. Z.zmcet,338, 41 1–412 1991. 260. Ohmura, T.; Nishio, M.; Kigoshi, S.; Murarnatsu, I., ElectrophysiologicaJ and mechanical effects of calcitonin gene-related peptide on guinea-pig atria. Br. J. Pharmacol., 100, 27–30 1990. 261. Ohno, K.; Takeda, N.; Yamarro, M.; Matsunaga, T.; Tohyama, M., Coexistence of acetylcholine and calcitonin gene-related peptide in the vestibular efferent neurons in the rat. Brain Res., 566, 103–107 1991. 262. Ohno, K.; Takeda, N.; Tanaka-Tsuji, M.; Matsunaga, T., Cafcitonin gene-related peptide in the efferent system of the inner ear. A review. Acta Otolaryngol. (Stockh.), 501, 16-201993. 263. Oku, R.; Satoh, M.; Fujii, N.; Otaka, A.; Yajima, H.; Takagi, H., Cafcitonin gene-related peptide promotes mechanical nociception by potentiating release of substance Pfromthe spinal dorsal homin rats. Brain Res., 403,350-3541987. 264. One, K.; Giles, W. R., Electrophysiological effects of cafcitonin gene-related peptide in bull-frog and guinea-pig atrkd myocytes. J. Physiol., 436, 195-2171991. 265. Orazzo, C,; Pienbone, V. A.; Ceccatelli, S.; Terenius, L.; Hokfelt, T., CGRP-like immunoreactivity in All doparnine neurons projecting to the spinal cord and a note on CGRP-CCK cross-reactivity. Brain Res., 600,39-481993.

RECEPTORS

OF CGRP AND RELATED PEPTIDES

266. Owji, A. A.; Smith, D. M.; Coppock, H. A.; Morgan, D. G. A.; Bhogal, R.; Ghatei, M. A.; Bloom, S. R., An abundant and specific binding site for the novel vasodialtor adrenomedullin in the rat. Endocrinology, 136,2127-21341995. 267. Pecile, A.; Grddobono, F.; Netti, C.; Sibilia, V.; Biella, G.; Braga, P. C., Calcitonin gene-related peptide: antinociceptive activity in rats, comparison with calcitonin. Regul. Pep?., 18, 189–199 1987. 268. Peskar, B. M.; Wong, H. C.; Walsh, J. H.; Holzer, P., A monoclomd antibody to crdcitonin gene-related peptide abolishes capsaicininduced gastroprotection. Eur. J. Pharmacol., 250, 201–203 1993. 269. Petrov, T.; Jhamandas, J. H.; Krukoff, T. L., Characterization of peptidergic efferents from the lateral parabrachial nucleus to identified nenrons in the rat dorsal raphe nucleus. J. Cherrr.Neuroanar., 5, 367-3731992. 270. Piehl, F.; Arvidsson, U.; Johnson, H.; Culheim, S.; Dagerlind, A.; Ulfhake, B.; Cao, Y.; Elde, R.; Pettersson, R. F.; Terenius, L.; Hokfelt, T., GAP-43, aFGF, CCK and ct. and (J-CGRP in rat spinal motoneurons subjected to axotomy ardor dorsal root severance. Eur. J Neurosci., 5, 1321–1333 1993. 271. Pittrrer, R. A.; Albrarrdt, K.; Beaumont, K.; Gaeta, L. S. L.; Koala,J. E.; Moore, C. X.; Rittenhouse, J.; Rink, T. J., Molecular physiology of amylin. .f. Cell. Biochem, 55S, 19–28 1994. 272. Pohl, M.; Lombard, M. C.; Bourgoin, S.; Carayon, A.; Benoliel, J. J.; Mauborgne, A.; Besson, J. M.; Hamon, M.; Cesselin, F., Opioid control of the in vitro release of calcitonin gene-related peptide (CGRP) from primary afferent fibres projecting in the rat cervical cord. Neuropeptides, 14, 151–159 1989. 273. Pohl, M.; Benoliel, J. J.; Bourgoin, S.; Lombard, M. C.; Mauborgne, A.; Taquet, H.; Carayon, A.; Besson, J. M.; Cesselin, F.; Harnon, M., Regional distribution of calcitonin gene-related peptide-, substance P-, cholecystokinin-, met5-enkephalin-, and dynorphin A (1-8)-like materials in the spinal cord and dorsal root ganglia of adult rats: effects of dorsal rhizotomy and neonatal capsaicin. J. Neurochem., 55, 1122-11301990. 274. Popper, P.; Micevych, P. E., Localization of calcitonin gene-related peptide and its receptors in a striated muscle. Brain Res., 496, 180– 1861989. 275. Poyner, D. R., Pharmacology of receptors for calcitonin gene-related peptide and arnylin. TIPS, 16,424-4281995. 276. Poyner, D. R.; Andrew, D. P.; Brown, D.; Bose, C.; Hanley, M. R., Pharmacological characterization of a receptor for calcitonin generelated peptide on rat, L6 myocytes. Br. J. Pharrnacol.,105,441-447 1992. 277. Provini, L.; Morara, S.; Rosina, A.; Forloni, G., Expression of CGRP binding sites in the developing rat cerebellum. Ann. NY Acad. Sci., 657,423-4251992. 278. Quartrr, M.; Diaz, G.; Floris, A.; Lai, M. L.; Priestley, J. V.; Del Fiacco, M., Calcitonirr gene-related peptide in the human trigernirral sensory system at developmental and adult life stages: Imrrnrnohistochemistry, neuronal morphometry and coexistence with substance P. J. Chem. Neuroanat., 5, 143–157 1992. 279. Quayle, J. M.; Bonev, A. D.; Brayden, J. E.; Nelson, M. T., Calcitonin gene-related peptide activated ATP-sensitive K+ currents in rabbit arterial smooth muscle via protein kinase A. J. PhysioG.,475, 9-131994. 280. Quirion, R.; van Rossum, D.; Dumont, Y.; St-Pierre, S.; Foumier, A., Characterization of CGRPI and CGRP2 receptor subtypes. Ann. NY Acad. Sci., 657, 88–105 1992. 281. Ralevic, V.; Khalil, Z.; Dusting, G. J.; Helme, R. D., Nitric oxide and sensory nerves are involved in the vasodilator response to acetylcholine but not calcitonin gene-related peptide in rat skin rnicrovasculature. Br. J. PharnracoL,106, 650–655 1992. 282. Raybould, H. E.; Kolve, E.; Tache, Y., Central nervous system action of calcitonirr gene-related peptide to inhibit gastric emptying in the conscious rat. Peptides, 9, 735–737 1988. 283. Raybould, H. E.; Holzer, P.; Reddy, S. N.; Yang, H.; Tache, Y., Capsaicin-sensitive vagal afferents contribute to gastric acid and vascular responses to intracistemal TRH analog. Peptides, 11,789–

7951990. 284.Raybotdd, H. E., Inhibitory effects of calcitonin gene-related peptide on gastrointestinal motility. Ann. NYAcad. Sci., 657, 2482571992. 285. Ren, J.; Young, R. L.; Lassiter, D. C.; Rings, M. C.; Harty, R. F., Calcitonin gene-related peptide: Mechanisms of modulation of antral endocrine cells and cholinergic neurons.Am. J. Physiol. Gastrointest Liver Physiol, 262, 732–G739 1992.

675 286. Rethelyi, M.; Metz, C. B.; Lund, P. K., Dishibution of neurons expressing calcitonin gene-related peptide rnRNAS in the brain stem, spinal cord and dorsal root ganglia of rat and guinea-pig. Neuroscience, 29, 225–239 1989. 287. Rethelyi, M.; Mohapatra, N. K.; Metz, C. B.; Petmsz, P.; Lund, P. K., Colchicine enhances rnRNAs encoding the precursor of calcitonin gene-related peptide in brainstem motoneurons. Neuroscience, 42, 531-5391991. 288. Ribeiro da Silva, A., Ultrastmctural features of the co-localization of CGRP with substance P or somatostatin in the dorsal horn of the spinal cord. Can. J. Physiol. Pharmacol., 73, 940–944 1995. 289. Richter, J.; Andersson, R.; Edvinsson, L.; Gullberg, U., Calcitonin gene-related peptide (CGRP) activates human neutrophils-Inhibition by chemotactic peptide antagonist BOC-MLP.. Immunology, 77, 416-4211992. 290. Roa, M.; Charrgeux,J.-P., Characterization and developmental evolution of a high-affinity binding site for calcitonin gene-related peptide on chick skeletaJ muscle membrane. Neuroscience, 41, 563-5701991. 291. Robberecht, P.; De Neef, P.; Woussen-Colle, M.-C.; Vertongen, P.; De Witte, O.; Brotchi, J., Presence of calcitonin gene-related peptide receptors coupled to adenylate cyclase in human gliomas. Regul. Pept., 52, 53–60 1994. 292. Roberts, A. N.; Leighton, B.; Todd, J. A.; Cockbum, D.; Schofield, O. N.; Sutton, R.; Holt, S.; Boyd, Y.; Day, A. J.; Foot, E. A.; Willis, A. C.; Reid, K. B. M.; Cooper, G. J. S., Molecula and functional characterization of amylin, a peptide associated with type 2 diabetes mellitrrs. Proc. Natl. Acad. Sci. USA, 86, 9662–9666 1989. 293. Rosenfeld, M. G.; Merrnod, J.-J.; Amara, S. G.; Swanson, L. W.; Sawchenko, P. E.; Rivier, J.; Vale, W. W.; Evans, R. M., Production of a novel nerrropeptide encoded by the calcitonin gene via tissues~ific RNA processing. Nature, 304, 129–135 1983. 294. Rosenfeld, M. G.; Amara, S. G.; Evans, R. M., Alternative RNA processing: determining nerrronal phenotype. Science, 225, 131513201984. 295. Rosenfeld, M. G.; Emeson, R. B.; Yeakfey, J. M.; Merillat, N.; Hedjran, F.; Lenz, J.; Delsert, C., Calcitonin gene-related peptide: A neuropeptide generated as a consequence of tissue-specific, developmentally regulated alternative RNA processing events. Ann. NY Acad. Sci., 657, 1–17 1992. 296. Rossler, W.; Gerstberger, R.; Sann, H.; Pierau, F.-K., Distribution and binding sites of substance P and calcitonin gene-related peptide and their capsaicin-sensitivity in the spinal cord of rats and chicken: a comparative study. Neuropeptides, 25, 241–253 1993. 297. Rovero, P,; Giuliani, S.; Maggi, C. A., CGRP antagonist activity of short C-terminal fragments of human aCGRP, CGRP(19-37), CGRP(23-37). Peptides, 13, 1025-10271992. 298. Ryu, P. D.; Gerber, G.; Murase, K.; Randic, M., Actions of calcitonin gene-related peptide on rat spired dorsal horn neurons. Brain Res.,

441,357-3611988. 299. Saika, T.; Senba, E.; Noguchi, K.; Sate, M.; Kubo, T.; Matsunaga, T.; Tohyarna, M., Changes in expression of peptides in rat facial motoneurons after facial nerve crushing and resection. Mol. Brain Res., 11,187–196 1991. 300. Srddanha, J.; Mahadevarr, D., Molecular model-building of arnylin and a-calcitonin gene-related polypeptide hormones using a combination of knowledge sources. Protein .Errgineering,4, 539–544 1991. 301. Sane, Y.; Hiroshima, O.; Yuzuriha, T.; Yamato, C.; Saito, A.; Kirrmra, S.; Hirabayashi, T.; Goto, K., Calcitonin gene-related peptide binding sites of porcine cardiac muscles and coronary arteries: solubilization and characterization. J. Neurochem., 52, 1919-19241989. 302. Schifter, S.; Krosell, L. R.; Sehested, J., Normal serum levels of calcitonin gene-related peptide (CGRP) in mild to moderate essential hypertension. Am. J. Hypertens., 4,565-5691991. 303. Schworer, H.; Schmidt, W. E.; Katsoulis, S.; Creutzfeldt, W., Calcitorrin gene-related peptide (CGRP) modulates cholinergic neurotransmission in the small intestine of man, pig and guineapig via presynaptic CGRP receptors. Regul. Pept., 36, 345-358 1991. 304. Sewell, W. F.; Starr, P. A., Effects of calcitonin gene-related peptide and efferent nerve stimulation on afferent transmission in the lateral line organ. J. Neurophysiol., 65, 1158-11691991. 305. Sexton, P. M.; McKenzie, J. S.; Mendelssohn,F. A. O., Evidence for a new subclass of calcitonin/calcitonin gene-related peptide binding site in rat brain. Neurochem.Znt.,12, 323–335 1988.

676 306. Sexton, P. M.; Houssami, S.; Hilton, J. M.; O’Keeffe, L. M.; Center, R. J.; Gillespie, M. T.; Darcy, P.; Findlay, D. M., Identification of brain isoforms of the rat calcitonin receptor. Mol. Endocrinol., 7, 815-8211993. 307. Sexton, P. M.; Houssarni, S.; Brady, C. L.; Myers, D. E.; Findlay, D. M., Amylin is an agonist of the renal porcine calcitonin receptor. Endocrinology, 134,2103-21071994. 308. Sexton, P. M.; Paxinos, G.; Kenney, M. A.; Wookey, P. J.; Beaumont, K., In vitro autoradiographic localization of arnylin binding sites in rat brain. Neuroscience, 62,553-5671994. 309. Shekhar, Y. C.; Anand, I. S.; Sarma, R.; Ferrari, R.; Wahi, P. L.; Poole Wilson, P. A., Effects of prolonged infusion of human alpha calcitonin gene-related peptide on hemodyrramics,renal blood flow and hormone levels in congestive heart failure. Am. J. Cardiol., 67, 732-7361991. 310. Sheriff, S.; Fischer, J. E.; Balasubramaniam, A., Characterization of arnylin binding sites in a human hepatoblastoma cell line. Peptides, 13, 1193-11991992. 311. Shimekake, Y.; Nagata, K.; Ohta, S.; Kambayashi, Y.; Teraoka, H.; Kitarnura, K.; Eto, T.; Kangawa, K.; Matsuo, H., Adrenomedrrllin stimulates two signal transduction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic endothelial cells. J. Biol. Chem., 270,4412-44171995. 312. Shinohara, Y.; Yarnano, M.; Matsuzaki, T.; Tohyama, M., Evidence for the coexistence of substance P, neurotensin and calcitonin generelated peptide in single neurons of the extemrd subdivision of the lateral parabrachial nucleus of the rat. Brain Res. Bull., 20, 257–260 1988. 313. Sigrist, S.; Frrmco-Cereceda, A.; Muff, R.; Herrke, H.; Lundberg, J. M.; Fischer, J. A., Specific receptor and cardiovascular effects of calcitonin gene-related peptide. Endocrinology, 119, 381–389 1986. 314. Silverman, J. D.; Kruger, L., Calcitonin gene-related peptide-immunoreactive innervation of the rat head with emphasis on specialized sensory structures. J. Comp. Neurol., 280, 303–330 1989. 315. Silvestre, R. A.; Salas, M.; Degano, P.; Peiro, E.; Marco, J., Reversal of the inhibitory effects of calcitonin gene-related peptide (CGRP) and amylin on insulin secretion by the 8-37 fragment of human CGRP. Biochem. Pharmacol., 45,2343-23471993. 316. Silvestre, R. A.; Salas, M.; Garcia-Herrnida, O.; Fontela, T.; Degarro, P.; Marco, J., Amylin (islet amyloidpolypeptide) inhibition of insulin reIease in the perfused rat pancreas: implication of the adenylate cyclase/cAMP system. Regul. Pept., 50, 193–199 1994. 317. Simmons, D. D.; Raji-Kubba, J., Postnatal calcitonin gene-related peptide in the superior olivary complex. J. Chem. Neuroanat., 6, 407-4181993. 318. Skofitsch, G.; Jacobowitz, D. M., Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system. F’eptides,6, 721–745 1985. 319. Skofitsch, G.; Jacobowitz, D. M., Quantitative distribution of calcitonin gene-related peptide in the rat central nemous system. Peptides, 6, 1069-10731985. 320. Skofitsch, G.; Jacobowitz, D. M., Calcitonin- and calcitorrirrgenerelated peptide: receptor binding sites in the central nervous system. In: Bjorldund, A.; Hokfelt T.; Kuhar, M. J. eds. Handbook of Chemical Neuroarratomy, Vol.11: Neuropeptide Receptors in the CNS., Amsterdam: Elsevier Science publishers B. V.; 97-144; 1992. 321. Skotitsch, G.; Wimalawansa, S. J.; Gubiscb, W., Comparative immunohistochemical distribution of amylin-like and calcitonin gene-related peptide-like inrmunoreactivity in the rat cerrtraInervous system. Can. J. Physiol. Pharmacol., 73, 945–956 1995. 322. Stangl, D.; Born, W.; Fischer, J. A., Characterization and photoaffinity labeling of a calcitonin gene-related peptide receptor solubilized from human cerebellum. Biochemistry, 30,8605-86111991. 323. Stangl, D.; Muff, R.; Schmolck, C.; Fischer, J. A., Photoaffirrity labeling of rat calcitonin gene-related peptide receptors and adenyIate cycIase activation: Identification of receptor subtypes. Endocrinology, 132,744-7501993. 324. Steenbergh, P. H.; Hoppener, J. W. M.; Zandberg, J.; Visser, A.; Lips, C. J. M.; Jansz, H. S., Structure and expression of the human calcitonin/CGRP genes. FEB.SI.ztt., 209, 97–103 1986. 325. Sternini, C.; Reeve, J. R. Jr.; Brecha, N., Distribution and characterization of calcitonin gene-related peptide irmnrrnoreactivity in the digestive system of normal and capsaicin-treated rats. Gastroenterology, 93, 852-8621987. 326. Stemirri, C.; Anderson, K., Calcitouin gene-related peptide-corrtaining neurons supplying the rat digestive system: differential

VANROSSUMET AL. distribution and expression pattern. Somatosens. Mot. Res., 9, 45591992. 327. Stevenson, J. C.; Booker, M. W.; Macdomdd, D. W.R.; Warren, R. C.; Whitehead, M. I., Increased concentration of circulating calcitonin gene related peptide during normal human-pregnancy. Br. Med. J., 293, 1329–1330 1986. 328. Stmthers, A. D.; Beacham, J. L.; Brown, M. J.; Macdonald, D. W. R.; MacIntyre, L; Morns, H. R.; Stevenson, J. C., Human calcitonin gene related reptide - a potent endogenous vasodilator in man. Clin. Sci., 70,389-3931986. 329. Su, H. C.; Ballesta, J.; Bloom, S. R.; Ghatei, M. A.; Gibson, S. J.; Morrison, J. F. B.; Mulderry, P. K.; Polak, J. M.; Terenghi, G.; Wharton, J., Calcitonin gene-related peptide immunoreactivity in afferent neurons supplying the urinary tract: combined retrograde tracing and immunohistochemistry. Neuroscience, 18, 727–747 1986. 330. Su, H. C.; Bishop, A. E.; Power, R. F.; Harnada, Y.; Polak, J. M., Dual intrinsic and extrinsic ongins of CGRP- and NPY-irnmunoreactive nerves of rat gut and pancreas. J. Neurosci., 7, 2674–2687 1987. 331. Sugimoto, T.; Itoh, K.; Mizuno, N., Calcitonin gene-related peptidelike immrrnoreactivity in neuronal elements of the cat cerebellum. Brain Res., 439, 147–154 1988. 332. Swanson, L. W. The hypothalamus. In: Bjorkhmd, A., Hokfelt, T. and Swanson, L. W., eds. Handbook of Chemical Nenroanatomy, VO1.5:Integrated Systems of the CNS, Part 1. Amsterdam: Elsevier Science PnbIishers B. V.; 1-124: 1987. 333. Sykes, R. M.; Spyer, K. M.; Izzo, P. N., Central distribution of substance, calcitonin gene-related peptide and 5-hydroxytryptamine in vagal sensory afferents in the rat dorsal medulla. Neuroscience, 59, 195-2101994. 334. Tabata, H.; Hirayarna, J.; Sowa, R.; Frrrrrta,H.; Negoro, T.; Sanke, T.; Nanjo, K., Islet amyloid polypeptide (IAPP/arnylin) causes insulin resistance in perfused rat hindlimb muscle. Diabetes Res. Clin. Pract., 15, 57–62 1992. 335. Tache, Y.; Goto, Y.; Gunion, M.; Lauffenberger, M., Inhibition of gastric-acid secretion by intracerebral injection of calcitonin generelated peptide in rats. Life Sci., 35, 871–878 1984. 336. Tache, Y.; Pappas, M.; Lauffenburger, M.; Goto, Y.; Walsh, J. H.; Debas, H., Calcitonin gene-related peptide: potent peripheral inhibitor of gastic acid secretion in rats and dogs. Gastroenrerology,87, 344-3491984. 337. Tache, Y.; Raybould, H.; Wei, J. Y., Central and peripheral actions of calcitonin gene-related peptide on gastric secretory and motor function. Adv. Exp. Med. Biol., 298, 183–198 1991. 338. Tache, Y.; Holzer, P. M.; Rosenfeld, G., Calcitonin gene-related peptide. The first decade of a novel pleiotropic nenropeptide. New York: Annals of the New York Academy of Sciences, vol 657; 1992. 339. Takahashi, H.; Watanabe, T. X.; Nishimura, M.; Nakanishi, T.; Sakamoto, M.; Yosfrimura, M.; Korniyama, Y.; Masuda, M.; Murakarni, T., Centrally induced vasopressor and sympathetic responses to a noveI endogenous peptide, adrenomedullin, in anesthetized rats. Am. J. Hypertens., 7, 478–482 1994. 340. Takarni, K.; Tohyarna, M.; Uchida, S.; Yoshida, H.; Shiotaui, Y.; Emson, P. C.; Girgis, S.; MacIntyre, I.; Hillyard, C. J.; Kawai, Y., Effect of calcitonin gene-related peptide on contraction of striatedmuscle in the mouse. Neurosci. L@., 60, 227-2301985. 341. Takamon, M.; Yoshikawa, H., Effect of calcitonin gene-related peptide on skeletal muscle via specific binding site and G protein. J. Neurol. Sci., 90, 99–109 1989. 342. Tannenbaum, G. S.; Goltzman, D., Calcitonin gene-related peptide mimics calcitonin actions in brain on growth hormone release and feeding. Endocrinology, 116,2685-26871985. 343. Taquet, H.; Uzan, S., Plasma calcitouirr gene-related peptide during gestation. Lancet, 340, 11701992. 344. Tiller-Borcich, J. K.; Capili, H.; Gordarr, G. S., Human brain calcitonirr gene-related peptide (CGRP) is concentrated in the locus coeruleus. Neuropeptides, 11,55–611988. 345.Tippins, J. R.; Morris, H. R.; Panico, M.; Etienne, T.; Bevis, P.; Girgis, S.; MacIntyre, I.; Azria, M.; Attirrger, M., The myotmpic aod plasma-calcium modulating effects of calcitonin gene-related peptide (CGRP). Neuropeptides, 4,425-4341984. 346. Torrealba, F., Calcitouin gene-related peptide immunoreactivity in the nucleus of the tractns solitarirrsand the carotid receptors of the cat originates from peripheral afferents. Neuroscience, 47, 165–173 1992.

RECEPTORS

OF CGRP AND RELATED PEPTIDES

347. Tran Dinh, Y. R.; Debdi, M.; Couraud, J.-Y.; Cremirron,C.; Seylaz, J.; Sercombe, R., Time course of variations in rabbit cerebrospinal fluid levels of crdcitonin gene-related peptide- and substance P-like immunoreactivity in experimental subarachnoid hemorrhage. Stroke, 25, 160–164 1994. 348. Traub, R. J.; Allen, B.; Humphrey, E.; Ruda, M. A., Analysis of calcitonin gene-related peptide-like immunoreactivity in the cat dorsaI spinal cord and dorsal root ganglia provide evidence for a multisegmentaf projection of nociceptive C-fiber primary afferents. 562–574 1990. J. Comp. ?feurol., 302, 349.Tschopp, F. A.; Henke, H.; Petemtann, J. B.; Tobler, P. H.; Jrmzer, R.; Hokfelt, T.; Lundberg, J. M.; Cuello, C.; Fischer, J. A., Calcitonirr gene-related peptide and its binding sites in the human central nervous system and pituitary. Proc. Natl. Acad. Sci. USA, 82, 248– 2521985. 350. Tsuda, K.; Tsuda, S.; Goldstein, M.; Nishio, I.; Masuyama, Y., Calcitonin gene-related peptide in noradrenergic transmission in rat hypothakurms. Hypertension, 19,639-6421992. 351. Tverberg, L. A.; Russo, A. F., Cell-specific glucocorticoid repression of calcitonin/calcitonirr gene-related peptide transcription. Localization to an 18-base pair basal enhancer element. J. Biol. Chem., 267, 17567–17573 1992. 352. Twery, M. J.; Moss, R. L., Calcitonin and cafcitonirr gene-related peptide after the excitability of neurons in rat forebrain. Peptides, 6, 373-3781985. 353. Uchida, S.; Yamamoto, H.; Ilio, S.; Matsumoto, N.; Wang, X. B.; Yonehara, N.; Imai, Y.; Irroki, R.; Yoshida, H., Release of calcitonin gene-related pcptide-like immunoreactive substance from neuromuscularjunction by nerve excitation and its action on striated muscle. J. Neurochem., 54, 1000–1003 1990. 354. Umeda, Y.; Arisawa, M., Characterization of the calcitonin generelated peptide receptor in mouse T lymphocytes. Neuropeptides, 14, 237-2421989. 355. Umeda, Y.; Arisawa, M., Characterization of calcitonin gene-related peptide (CGRP) receptors in guinea pig lung. Jpn. J. Pharrnacol.,51, 377-3841989. 356. Unger, J. W.; Lange, W,, Irrrmunohistochernical mapping of nerrrophysins and calcitonin gene-related peptide in the human bminstem and cervical spinal cord. J. Chem. Neuroanat., 4, 2993091991. 357. van Rossum, D.; Menard, D. P.; Quirion, R., Effect of guanine nucleotides and temperature on calcitonin gene-related peptide receptor binding sites in brain and peripheral tissues. Brain Res., 617, 249–257 1993. 358. van Rossum, D.; Merrard, D. P.; Foumier, A.; St-Pieme, S.; Quirion, R., Autoradiographic distribution and receptor binding profile of [lfiI]BH-rat amylin binding sites in the rat brain. J. Pharmacol. Exp. Ther., 270, 779–787 1994. 359. van Rossum, D.; Menard, D. P.; Foumier, A.; St-Pierre, S.; Quirion, R., Binding profile of a selective calcitonin gene-related peptide (CGRP) receptor antagonist ligand, [1151-Tyr]hCGRPs.gT, in rat brain and peripheral tissues. J. Pharrnacol. E.rp. Ther., 269, 8468531994. 360. van Rossum, D.; Menard, D. P.; Chang, J. K.; Quirion, R., Comparative affinities of human adrenomedullin for [1251] hCGRPa and [1251] BH-amylin specific binding sites in the rat brain. Can. J. Physiol. Pharmacol., 73, 1084-10881995. 361. Van Valen, F.; Piechot, G.; Jurgens, H., Calcitonirr gene-related peptide (CGRP) receptors are linked to cyclic adenosine monophosphate production in SK-N-MC human neuroblastoma cells. Neurosci. Lett., 119, 195–198 1990. 362. Varru, A.; Green, T.; Holmes, S.; Dockray, G. J., Calcitonirrgenerelated peptide in visceral afferent nerve fibres: quantification by radioimmunoassay and determination of axonal transport rates. Neuroscience, 26, 927–932 1988. 363. Veale, P. R.; Bhogal, R.; Morgan, D. G.; Smith, D. M.; Bloom, S. R., The presence of islet amyloid polypeptide/calcitonin gene-related peptide/salmon calcitonin binding sites in the rat nucleus accumbens. Eur. J. PhartrracoL,262, 133–141 1994. 364. Verchere, C. B.; D’Alessio, D. A.; Palmiter, R. D.; Kahn, S. E., Transgenic mice overproducing islet amyloid polypeptide have increased insulin storage and secretion in vitro. Diabetologia, 37, 725-7281994. 365. Vignery, A.; Wang, F.; Ganz, M. B., Macrophages express functional receptors for calcitonin gene-related peptide. J. Cell. PhysioL, 149, 301-3061991.

677 366. Villar, M. J.; Roa, M.; Huchet, M.; Hokfelt, T.; Changeux, J.-P.; Fahrenkmg, J.; Brown, J. C.; Epstein, M.; Hersh, L., Irnmunoreactive crdcitonin gene-related peptide, vasoactive intestinal polypeptide, and somatostatin in developing chicken spinal cord motoneurons: distribution and role in regulation of cAMP in cultured muscle cells. Eur. J. Neurosci., 1,269–287 1989. 367. Villar, M. J.; Wiesenfeld-Haflin, Z.; Xu, X.-J.; Theodorsson, E.; Emson, P. C.; Hokfelt, T., Further studies on gafanin-,substaace P-, and CGRP-like immunoreactivities in primary sensory neurons and spinal cord: effects of dorsal rhizotomies and sciatic nerve lesions. Exp. Neurol., 112,29-391991. 368. Wafd, M.; Schilling, L.; Parsons, A. A.; Kaumann, A., Involvement of calctionin gene-related peptide (CGRP) and nitric oxide (NO) in the pial artery dilatation elicited by cortical spreading depression. Brain Res., 637, 204–210 1994. 369. Wang, F.; Millet, I.; Bottornly, K.; Vignery, A., Calcitonin generelated peptide inhibits interlenkirr 2 production by marine T lymphocytes. J. Biol. Chem., 267, 21052–21057 1992. 370. Wang, M.-W.; Young, A. A.; Rink, T. J.; Cooper, G. J. S., ‘-37hCGRP antagonizes actions of amylin on carbohydrate metabolism in vitro and in vivo. FEBSLert., 291, 195–198 1991. 371. Wang, X.; Fiscus, R. R., Crdcitonin gene-related peptide increases cAMP, tension, and rate in rat atria. Am. J. Physiol. Regulatory Integrative Comp. Physiol, 256, 421–R428 1989. 372. Wang, X.; Han, C.; Fiscus, R. R., Calcitonin gene-related peptide (CGRP) causes endotbelium-deperrdent cyclic AMP, cyclic GMP and vasorelaxant responses in rat abdominal aorta. Neuropeptides, 20, 115-1241991. 373. Wang, X.; Yue, T.-L.; Barone, F. C.; White, R. F.; Clark, R. K.; Willette, R. N.; Sulpizio, A. C.; Aiyar, N. V.; Ruffolo, R. R.; Feuerstein, G. Z., Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage. Proc. Natl. Acad. Sci. USA, 92, 11480-114841995. 374. Wang, Z. L.; Bennet, W. M.; Ghatei, M. A.; Byfield, P. G. H.; Smith, D. M.; Bloom, S. R., Influence of islet amyloid polypeptide and the 837 tiagment of islet amyloid polypeptide on insulin release from penfused rat islets.. Diabetes, 42,330-3351993. 375. Welch, S. P.; Cooper, C. W.; Dewey, W. L., An investigation of the antinociceptive activity of calcitonin gene-related eptide alone and in combination with morphine: comelation to 4&a++ uptake by synaptosomes. J. Phartnacol. .Exp.Ther., 244, 28–33 1988. 376. WeIch, S. P.; Singha, A. K.; Dewey, W. L., The antinociception produced by intrathecal morphine, calcium, A23187, U50,488H, [DAlaz, N-Me-Phe4, Gly-ol]enkephalin and [D-Pen2, D-Pen5]enkephalin after intrathecal adminismationof cafcitonin gene-related peptide in mice. J. Pharrrracol.Exp. Ther., 251, 1–8 1989. 377. Westermark, P.; Wemstedt, C.; Wilander, E.; Sletten, K., A novel peptide in the calcitonin gene-related peptide family as an amyloid fibril protein in the endocrine pancreas. Biochem. Biophys. Res. Commun.,140, 827-8311986. 378. Westerrrwk, P.; Wemstedt, C.; Wilander, E.; Hayden, D. W.; O’Brien, T. D.; Johnson, K. H., Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a nenropeptide-like protein also present in normal islet cells. Proc. Natl. Acad. Sci. USA, 84, 3881–3885 1987. 379. Westermark, P.; Engstrom, U.; Johnson, K. H.; Westermark, G. T.; Betsholtz, C., Islet amyloid polypeptide: Pinpointing amino acid residues linked to amyloid fibril formation. Proc. Natl. Acad. Sci. USA, 87,5036-50401990. 380. Wiesenfeld-Hallin, Z.; Hokfelt, T.; Lundberg, J. M.; Forssmann, W. G.; Reinecke, M.; Tschopp, F. A.; Fischer, J. A., Immunoreactive cafcitonin gene-related peptide and substance P coexist in sensory neurons to the spinal cord and interact in spinal behavioral responses of the rat. Neurosci. Lett., 52, 199–204 1984. 381. Wiesenfeld-Hallin, Z., Somatostatirr and calcitonin gene-related peptide synergistically modulate spinal sensory and reflex mechanisms in the rat: behavioral and electrophysiological studies. Neurosci. Lett., 67,319-3231986. 382. Wiley, J. W.; Gross, R. A.; Macdonald, R. L., The peptide CGRP increases a high-threshold Ca2+ current in rat nodose nenrones via a pertussis toxin-sensitive pathway. J. Physiol. (Lard.), 455, 367–381 1992. 383. Wimalawansa, S. J.; E1-Kboly,A. A., Comparative study of distribution and biochemical characterization of brain calcitonin generelated peptide receptors in five different species. Neuroscience, 54,513-5191993.

678 384. Wind, J. C.; Born, W.; Rijnsent, A.; Boer, P.; Fischer, J. A., Stimulation of calcitonin/CGRP-I and CGRP-H gene expression by dibutyryl cAMP in a hyman medullary thyroid carcinoma (lT) cell line. Mol. Cell. Errdocrinol.,92, 25–31 1993. 385. Xu, X.-J.; Wiesenfeld-Hallin, Z.; Villar, M. J.; Fahrenkmg, J.; H okfelt, T., On the role of gafanin, substance P and other neuropeptides in primary sensory neurons of the rat: studies on spinal reflex excitability and peripheral axotomy. Eur. J. Neurosci., 2, 733–743 1990. 386. Yamaguchi, A.; Cfriba, T.; Yamatani, T.; Inui, T.; Morishita, T.; Nakamura, A.; Kadowaki, S.; Fukase, M.; Fujita, T., Calcitonin generelated peptide stimulates adenylate cyclase activation via a guanine nucleotide-dependent process in rat liver plasma membranes. Endocrinology, 123,2591-25961988. 387. Yamin, M.; Gem, A. H.; Ffannery, M. R.; Jenkins, N. A.; Gilbert, D. J.; Copeland, N. G.; Tapp, D. R.; Kmne, S. M.; Goldring, S. R., Cloning and characterization of a mouse brain cafcitonin receptor complementary deoxyribonucleic acid and mapping of the calcitonin receptor gene. Endocrinology, 135, 2635–2643 1994. 388. Yashpal, K.; Kar, S.; Dennis, T.; Quinon, R., Quantitative autoradiographic distribution of calcitonin gene-related peptide (hCGRPcx) binding sites in the rat and monkey spinal cord. J. Corrsp.Neurol., 322,224-2321992. 389. Yasui, Y.; Saper, C. B.; Cechetto, D. F., Calcitonin gene-related peptide (CGRP) immunoreactive projections from the thafarnus to the striatum and amygdafain the rat. J Conrp.Neurol., 308,293-310 1991.

VAN ROSSUM ET AL.

390.Yoshizaki, H.; Takamiya, M.; Okada, T., Characterization of picomolar affinity binding sites for [1251] -human cafcitonin gene-related peptide in rat brain and heart. Biochem.Biophys. Res. Conrmun.,146, 443-4511987. 391. Young, A. A.; Carlo, P.; Smith, P.; Wolfe-Lopez, D.; Pittner, R.; Wang, M.-W.; Rink, T., Evidence for release of free glucose from muscle during amylin-induced glycogenolysis in rats. FEBS Lett.,

334,317-3211993. 392.Young, A. A.; Cooper, G. J.S.; Carlo, P.; Rink, T. J.; Wang, M.-W.,

393.

394.

395. 396.

Response to intravenous injections of amylin and glucagon in fasted, fed, and hypoglycemic rats. Am. J. Physiol. Endocrinol. Metab, 264, 943-9501993. Zhang, X.; Nicholas, A. P.; Hokfelt, T., Ultrastmctural studies on peptides in the dorsal horn of the spinal cord-I., Co-existence of galanin with other peptides in primary afferents in normal rats. Neuroscience, 57, 365–384 1993. Zhang, Y. Q.; Vacca-Gafloway, L. L., Decreased irnrnunoreactive (IR) calcitonin gene-related peptide correlates with sprouting of IRpeptidergic and serotonergic neuronal processes in spinal cord and brain nuclei from the Wobbler mouse during motoneoron disease. Brain Res., 587, 169–177 1992. Zhu, G.; Dudley, D. T.; Saltiel, A. R., Amylin increases cyclic AMP formation in L6 myocytes through cafcitonin gene-related peptide receptors. Biochenr.Biophys. Res. Commun., 177, 771–776 1991. Zona, C.; Farini, D.; Palma, E.; Eusebi, F., Modulation of voltageactivated channels by calcitonin gene-related peptide in cultured rat neurones. J. PhysioL, 433, 631–643 1991.