Novel peptides from the calcitonin gene: Expression, receptors and biological function

t'~ptid~. Vol. 6. Suppl. 3. pp. 265-271. 1985. Ankholnlcrnatiota=;Inc. Prinledin the U.S,A.

o1'46-9781/85$3.(~)

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Novel Peptides From the Calcitonin Gene: Expression, Receptors and Biological Function J A N A. F I S C H E W A N D W A L T E R B O R N

Re.~earck Lahoratm3" ,for Calcium Metabolism. Departments o f Ortkopedic Sttrge13" (Balgrist) and Medicine Forchstrassc 340, 8008 Zurich, Switzerland

FISCHER, J. A. AND W. BORN..Vovcl peptidc~ rr,,m tlw
Calcitonin gene-related peptide

Calcium

THE calcitonins are synthesized in parafollicular or C-cells of thyroid glands in mammals and in ultimobranchial organs in lower vertebrates. A second peptide also derived from the calcitonin gene. calcitonin gene-related peptide (CGRP) is predominantly a neuropeptide, but it has also been found outside the nervous system such as in the C-cells of the thyroid gland. In mammals both peptides are synthesized as larger precursors (Fig. I ). Calcitonin lowers blood calcium concentrations by inhibition of bone resorption in most species, including fishes. Interestingly. the peptide produces hypercalcemia in leopard sharks whose skeleton is composed of cartilage, and the effect does not require a bony skeleton [341. In the eel. on the other hand, a peptide related to parathyroid hormone, which raises serum calcium concentrations in mammals, lowers blood calcium [62]. In the salmon, calcitonin is a major gill hormone that stimulates the transport of calcium into the ambient sea water and by this means lowers the extracellular calcium concentration [631. The origin of the calcitonin producing C-cells from the neural crest [751 suggests a possible function of calcitonin in the nervous system. Besides the well known action on the skeleton, calcitonin has analgesic effects and inhibits food intake and gastric acid secretion and intestinal motility, probably via receptors in the central nervous system. Indeed, immunoreactive calcitonin has been detected in the nervous system of an ascidian, the sea quirt ciona intestinalis, of protochordates and a cyclostome, myxine, and of

Cardiovascular effects

PDN-21

the pond snail. Lymnaea stagnalis, all with no bony skeleton [28.33.89]. Calcitonin is, moreover, present in the brains of the bullfrog, the lizard, the pigeon, the rat and man [6, 23, 24, 29. 30. 56. 105]. Human and salmon calcitonin share only 16 of 32 amino acids (Fig. 2). Specific radioimmunoassays for the two peptides have been developed, and human as well as salmon calcitonin-like peptides have been recognized in the eel, salamander, pigeon, chicken, rat, ox. and in man [23, 78, 79]. This interesting observation suggests the presence of two genes encoding peptides with the same biological function in fish. reptiles and mammals. Human calcitonin gene-related peptide shares greater structural homology with salmon than with human calcitonin (Fig. 2). CGRP and calcitonin may also have arisen by gene duplication. Moreover, t ~ o closely related CGRP peptides, CGRP ! and II, have been discovered in man (92% homology) and rats 197% homology) by analysis of the sequence of the complementary DNA encoding the peptides [85,911. So far, the expression of CGRP II has not been verified by identification of the peptide in human and rat tissues. In the following, we only refer to human and rat CGRP I. abbreviated CGRP. CGRP has weak hypocalcemic effects which can be explained by the structural homology and crossreaction on receptor binding sites, but potent cardiovascular actions ~hich are only shared to a limited extent by calcitonin 131,83a}. In the present report the structure of calcitonin gene

'Requests for reprints should be addressed to Dr. J. A. Fischer, Klinik Balgrist. Forchstrasse 340. 8008 Zurich. Switzerland.

265

266

F I S C H E R A N D BORN

products, their receptors and biological effects are discussed in some detail. The large majority of results have been obtained in man and rats.

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STRUCTURE OF CALCITONIN GENE PRODUCTS

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The calcitonins are single chain polypeptides of 32 amino acids with 9 amino acids in invariant positions, including 6 in an amino-terminal 7-amino acid ring structure linked by a disulfide bridge and a carboxyl-termina! proline amide (Fig. 2). The structures of salmon and eel, and the mammalian human, murine, bovine, porcine and ovine calcitonins have been derived by analysis of the amino acid sequence and the sequence of chicken calcitonin deduced from the complementary DNA structure [51, 70, 80]. The amino acid sequence of human calcitonin has been confirmed by analysis of the complementary DNA encoding the precursor of the peptide [19,52]. In this way, carboxyl-terminal flanking peptides were predicted in rats and man (Fig. l) [19,47] and subsequently identified in tissue extracts [23, 40, 57, 84]. The rat carboxyl-terminal adjacent peptide has 16 and the human peptide 21 amino acids. According to the terminology of Tatemoto and Mutt [95] the human peptide is called PDN-2 I. PDN-21 is also termed katacalcin, although the hypocalcemic activity alluded to by the name has subsequently not been verified [I, 57, 58, 83a]. Amino-terminal flanking peptides have been predicted from analyses of the complementary DNA. In the rat they include a signal sequence and a socalled common region. In man the corresponding region has recently been fully sequenced [52]. By molecular hybridization it was shown that the human calcitonin gene is localized on the short arm of chromosome ii [41,81]. The existence of rat and human calcitonin gene-related peptide (CGRP) has been predicted by analysis of the nucleotide sequence of the complementary DNA encoding the calcitonin gene products (Figs. 1 and 2) [4,92]. The corresponding amino acid sequence of human CGRP has subsequently been derived from medullary thyroid carcinoma extracts [66]. The CGRP are unique 37 amino acid peptides. Human CGRP shares greater structural homology with salmon (24%) than with human calcitonin (16%) (Fig. 2). To this end, a salmon calcitonin-like peptide has been recognized in rat and human tissues besides the conventional human calcitonin and PDN-21 [23,79]. The precursors for CGRP and calcitonin are both encoded by exons of a common initial transcript of the calcitonin gene. The exons which encode the signal peptide and the amino-terminal common region of CGRP and of calcitonin are the same for both precursors (Fig. 1). Messenger RNA coding for CGRP is linked to these exons by appropriate splicing of the initial transcript. Alternative splice sites are used to produce messenger RNA that encodes the signal peptide, the common region, and calcitonin and PDN-21, and CGRP. Interestingly. second CGRP genes have been recognized in rats and man with 97c~ and 92% homology, respectively [85,91]. The detailed organization of the respective gene structures remains to be analyzed and possible second calcitonin genes to be discovered. CALCITONIN AND CGRP IN THE THYROID. PITUITARY AND NERVOUS SYSTEM Tissue specific processing of the rat calcitonin gene transcripts, resulting in the formation of calcitonin and its carboxyl-terminal adjacent peptides (in man PDN-21) in the

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thyroid gland and of CGRP in the nervous system has been proposed as a novel mechanism involving selective use of alternative polyadenylation sites [3,4]. Messenger R N A encoding calcitonin is present in large amounts in normal thyroid glands and medullary thyroid carcinoma and in lung cancer cells [49. 87. 106]. Cell-fiee synthesis of messenger R N A from rat medullary thyroid carcinoma in the presence of microsomal membranes reveals the presence of a glycoprotein which is processed into a precursor of 12'000 molecular ~veight [48]. A common precursor of calcitonin and of its flanking peptides of a similar size has been identified in rat medullary thyroid carcinoma by microsequencing of incorporated radioactive amino acids [7]. A precursor of human calcitonin and PDN-21 has been similarly recognized on H P L C and gel permeation chromatography in the plasma of normal human subjects and of patients with medullary thyroid carcinoma and in the incubation medium of cultured medullary thyroid and small-cell lung carcinoma cells [98]. Human calcitonin (and PDN-21) have not only been recognized in the thyroid, but also among neural tissues in the human hypothalamus, and in catecholamine storage vesicles of p h e o c h r o m o c y t o m a , and in the pituitary [2, 6. 8. 20, 24, 26. 37. 72, 101]. Messenger RNA encoding calcitonin is also present, albeit in small concentrations, in the terminal ganglion and the trigeminal tract nucleus of the rat brain, but apparently not in the pituitary [46,85]. CGRP is predominantly synthesized in the nervous system, as revealed by the presence of messenger RNA encoding the peptide, but also in the pituitary and in normal thyroid C-cells and medullary thyroid carcinoma [71.85-87. 92]. The presence of CGRP has been demonstrated by immunocytochemistry and specific radioimmunoassays in combination with HPLC and gel permeation chromatography. In the central nervous system high levels are encountered in the dorsal spinal cord [32.97]. Rhizotomy caused depletion of immunoreactive CGRP in the dorsal spinal cord, suggesting that it is transported from dorsal root ganglia. Here CGRP and substance P are present in the same neurons [32.97. 103]. CGRP has also been recognized in the pituitary, the thyroid gland, the esophagus, and in the heart where it is predominantly present in the atrium [55. 83. 97, 99]. There CGRP is frequently associated with peripheral nerves and blood vessels [55.83.86]. Depletion of CGRP by capsaicin in the esophagus and the heart suggests important functions in afferent nerve fibres [55.83]. REGULATION OF SECRETION OF CALCITONIN AND CGRP With no exception so tar calcitonin and its carboxylterminal flanking peptides IPDN-21 in manl are cosecreted.

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FIG. 2. Amino acid sequences of human CGRP thCGRP) i [92], and II [91], rat CGRP (rCGRP) I [4] and 11 [85]. salmon calcitonin (sCT) I [80] and human calcitonin chCTI [70]. Horizontal lines indicate amino acids in common with human CGRP !.

The most important regulator of calcitonin (and probably also of PDN-21) secretion is the extracellular calcium concentration. In response to an acute increase of blood calcium, calcitonin specific messenger RNA extracted from rat thyroids is increased within 2 min [90]. Similarly, a transient rise of circulating calcitonin and PDN-21 occurs in normal human subjects and in patients with medullary thyroid carcinoma 19.40.45a. 611. Besides calcium, potassium, catecholamines, glucagon, pentagastrin and other gastrointestinal peptides stimulate the secretion of calcitonin and PDN-21 [5, 17, 61, 84. 102]. Their physiological relevance remains to be elucidated. Much less is known about the regulation of secretion of CGRP. Circulating CGRP appears not to be stimulated in response to intravenous calcium, whereas a rise of extracellular calcium in the incubation medium of cultured medullary thyroid carcinoma cells enhances the release of calcitonin and CGRP [9]. As expected for a neuropeptide, potassium stimulates the release of CGRP in calcium dependent manner from rat trigeminal ganglion cells, but also from cultured medullary thyroid carcinoma cells [9,60]. CALCITONIN AND CGRP RECEPTORS Calcitonin binding sites and stimulation of adenylate cyclase activity have originally been discovered in bone and kidney cells which are classical end-organs of the hormone in the peripheral circulation [59]. Salmon calcitonin binding sites not linked to stimulation of adenylate cyclase activity have been recognized in membranes and by receptor autoradiography of the central nervous system of rats and man [22, 24, 35.38, 39.67.73, 82]. The level of salmon calcitonin binding sites is highest in the hypothalamus. Circumventricular localization and deficiency of calcitonin receptors in'rats with hereditary diabetes insipidus represents an abnormality whose importance remains to be elucidated [43]. Human calcitonin and analogues thereof and PDN-21 do not bind to human brain membranes [39]. In cultured rat brain cells human calcitonin apparently stimulates cyclic AMP accumulation; salmon calcitonin has not been tested [54]. Distinct CGRP binding sites apparently not linked to stimulation of adenylate cyclase activity have recently been discovered in the central nervous system of man and rats [35, 39, 97]. In man, the levels are highest in the cerebellum and spinal cord. Binding was visualized by receptor autoradiography over the molecular and Purkinje cell layers of the cerebral cortex and over the substantia gelatinosa posterior of the spinal cord which showed minimal binding of [l~:'I]salmon calcitonin [39,97]. Specific binding sites for CGRP are also present in the heart and they are predominantly associated with blood vessels, where CGRP exerts potent cardiovascular effects (see belowl. In kidney membranes and cultured kidney cells

CGRP enhances adenylate cyclase activity, albeit at 500-1000-fold higher concentrations than salmon calcitonin [35.104]. in view of the 24% structural homology between CGRP and salmon calcitonm, the stimulation is probably mediated by calcitonin receptors exhibiting crossreactivity to CGRP. BIOLOGICAL IMPLICATIONS Calcitonin is the most important circulating peptide lowering serum calcium levels by inhibition of bone resorption. PDN-21 which is cosecreted with calcitonin was claimed to also inhibit bone resorption and have hypocalcemic activity and it was therefore called katacalcin [57]. The same authors and an independent group of investigators have been unable to confirm the findings subsequently [I, 58, 83a]. PDN-21 and the corresponding carboxyl-terminal adjacent peptide in rats have so far no known biological activity. CGRP in 500--1000fold higher concentrations than calcitonin also lowers blood calcium levels [83a,96], and the effect is probably mediated by calcitonin receptors. In the kidney, calcitonin stimulates urinary phosphate and sodium excretion and enhances 1,25dihydroxycholecalciferol production [36,42]: renal effects of CGRP have not been reported as of yet. CGRP is considered as a neuropeptide with receptors in the central nervous system, but distinct binding sites for calcitonin are also present in the brain at least in the hypothalamus, lntracerebroventricular administration of CGRP causes noradrenergic sympathetic outflow and a rise in blood pressure, whereas intravenous injections evoke hypotension [25]. Through both modes of administration the heart rate is increased. Peripheral administration of CGRP in man and rats and perfusion of the isolated rat atrium have potent cardiovascular effects which include positive chronotropic and inotropic action and vasodilatation [11, 31, 55, 96]. Most of these effects are not obliterated by adrenergic blocking agents and appear to be specific for CGRP, Interestingly, capsaicin exerts similar biological effects, and endogenous CGRP in the heart may represent the so far elusive mediator of the action of capsaicin on the cardiovascular system [55]. In man, facial flushing was very marked with CGRP, while equimolar amounts ofcalcitonin had little effect [31]. In view of the absence of recognizable calcitonin binding sites by receptor autoradiography in the cardiovascular system, the action of calcitonin may be mediated by CGRP receptors (Sigrist, S., H. Henke and J.A. Fischer, in preparation). Both centrally and peripherally injected CGRP and calcitonin inhibit gastric acid secretion [44, 53, 65, 88, 93, 94, 100]. For calcitonin, far higher amounts of subcutaneously than intracerebroventricularly administered peptide are required to suppress gastric secretion, and the effects may be mediated by receptors localized in the hypothalamus [65,88].

268

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lntracerebroventricular injections of CGRP and calcitonin inhibit ingestive behaviour [27,50]. The anorectic effect of calcitonin may be mediated by prostaglandin E~ [21]. Centrally administered calcitonin also inhibits intestinal motility, and the effect is abolished after vagotomy [12,t3]. Intracerebroventricular administration of calcitonin causes analgesia, an effect which was not suppressed by the opiate antagonist naloxone [10,76]. lntracerebroventricular injections of calcitonin in high amounts, unlike intravenous injections, stimulated prolactin secretion, while low amounts of calcitonin administered centrally and peripherally inhibit the release of prolactin [14, 15, 74]. The inhibition of prolactin secretion may be mediated via the nigro-striatal dopaminergic system [16]. However, calcitonin administered into the third ventricle is readily released into the peripheral blood [88]. Prolactin secretion may therefore be directly inhibited by calcitonin at the level of the pituitary, much like the inhibition of thyroliberin stimulated prolactin secretion during infusions of calcitonin [45]. Peripheral administration of calcitonin, moreover, suppresses the secretion of thyroliberin and thyrotropin, and the hypothalamus may also be involved [64]. Calcitonin and CGRP exert characteristic behavioral changes in rats [16,103]. The action of calcitonin may be mediated through stimulation of nigrostriatal dopaminergic activity which is in line with the central inhibition of prolactin secretion [16]. The effects of CGRP may be related to the presence of the peptide together with substance P in the same spinal neurons [32,103]. Intramuscular administration of calcitonin increases cerebral 5-hydroxytryptamine contents and acetylcholinesterase activity in rats [69]. The effects were suppressed in thyroidectomized animals receiving calcium supplements and may be influenced by the presence of endogenous calcitonin [68].

sible for the synthesis of the calcitonin precursor [19]. With the same technique carboxyl-terminal flanking peptides (PDN-21 in man) also encoded by the calcitonin gene were predicted and their presence localized in thyroid C-cells. medullary thyroid carcinoma and the human brain [2, 23, 66. 84]. An important discovery in the field of calcitonin gene products represents alternative splicing of the initial transcripts from the calcitonin gene [4]. Mature messenger RNA encoding calcitonin and its flanking peptides have been detected predominantly in C-cells of thyroid glands, and another messenger RNA encoding calcitonin gene-related peptide tCGRP) ~as recognized in neural tissues and in Io~ amounts also in thyroid C-cells. The amino acid sequence of human CGRP xvas obtained in medullary thyroid carcinoma extracts and corresponds exactly to the nucleotide sequence of the messenger RNA encoding the peptide [66,92]. Distinct binding sites for CGRP and for calcitonin have been recognized in human and rat brains [35.39, 97]. In the heart, onlt CGRP receptors are recognizable and in the kidney, onl.~ calcitonin receptors. Limited structural homology of CGRP and of calcitonin is responsible for crossreaction of receptor binding and of related biological properties of the two peptides. CGRP exerts potent cardiovascular effects lvasodilatation, hypotension, positive chronotropic and inotropic effects in the heart) which are only shared to a limited extent by calcitonin, xvhereas calcitonin is more active than CGRP in inhibiting bone resorption. Both peptides exert probably direct effects in the central nervous system, such as inhibition of gastric acid secretion and food intake. Caleitonin reduces pain perception and affects prolactin secretion. The recently discovered CGRP. and distinct receptors for CGRP and calcitonin mediating different but overlapping biological effects remain to be characterized in more detail.

SUMMARY AND CONCLUSIONS Calcitonin has been discovered by C o p p e t al. [18] as a hypocalcemic principle. The amino acid sequence of human calcitonin was obtained in medullary thyroid carcinoma extracts [70], and subsequently confirmed by analysis of the complementary DNA encoding the messenger RNA respon-

ACKNOWLEDGEMENTS This work ~as supported b.'. the Swiss National Science Foundation grant 3.95--0.84. the Kanton of Zurich. the Ciba-Geig.~ Company and the Schweizerische Verein Balgri-t.

REFERENCES 1. Alander, C. B., B. A. Roos, D. C. Aron and L. G. Raisz. Calcitonin carboxyl-terminal adjacent peptide does not inhibit bone resorption. ('alc(f TiA.~ut' Int 35: 664. 1983 (Abstract). 2. Ali-Rachedi, A., I. M. Varndell, P. Facer. C. J. Hillyard, R. K. Craig, I. Maclntyre and J. M. Polak. Immunocytochemical Iocalisation of katacalcin, a calcium-lowering hormone cleaved from the human calcitonin precursor. J C/in Emlocrinol Mctah 57: 680-682, 1983. 3. Amara, S. G., R. M. Evans and M. G. Rosenfeld. Calcitonin/calcitonin gene-related peptide transcription unit: Tissuespecific expression involves selective use of alternative polyadenylation sites. Mol ('ell Biol 4:215 I-2160. 1984. 4. Amara, S. G,, V. Jonas. M. G. Rosenfeld. E. S. Ong and R. M. Evans. Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Naturc 298: 240--244. 1982. 5. Avioli, L. V., W. Shieber and D. M. Kipnis. Role of glucagon and adrenergic receptors in thyrocalcitonin release in the dog. Endocrinoh~gy 88:1337-1340. 1971.

6. Becker. K. L.. R. H. Snider. C. F. Moore. K. G. Monaghan and O. L. Silva. Calcitonin in extrathyroidal tissues of m,m. Ac/u Eml,,crin,,I (('opcnhp 92: 746-751. 19-9. 7. Birnbaum. R. S.. W. C. Mahoney, D. M. Burns. J. A. O'Neil. R. E. Miller and B. A. Roos. Identification of procalcitonin in it rat medullary thyroid carcinoma cell line..I Bi,,I Chcm 259: 287t~2874. 19~4. 8. Bone. H. G.. Ill. B. D. Catherwood and L. J. Deftos. Extraction of a substance with calcitonin-like immunoreactivity from pituitary glands of intact and thyroidectomized rats. ('ahit 7i,.~uc htt 35: 620--623, 1983. 9. Born. W.. J. Ittner. M. A. Dambacher. J. P. Petermann. F A. Tschopp. R. S. Birnbaum. B. A. Roos and J. A. Fischer. Calcitonin gene products in normal man and medullary th~ roid carcinoma. C . h ' ( f 7)'.~suc lnt 37, in pres~. 1985 IAbstractl. 10. Braga. P.. S. Ferri, A. Santagostino. V. R. Olgiati and .-X Pecile. Lack of opiate receptor involvement in centrall3 induced calcitonin analgesia. L(I~. Sci 22: 9-1-978. 1978.

PEPTIDES FROM THE CALCITONIN

GENE

11. Brain. S. D.. T. J. Williams, J. R. Tippins, H. R. Morris and I. Maclntyre. Calcitonin gene-related peptide is a potent vasodilator. Nature 313: 54-56, 1985. 12. Bueno. L., J. P. Ferre, J. Fioramonti and C. Honde. Effects of intracerebroventricular administration of neurotensin, substance P and calcitonin on gastrointestinal motility in normal and vagotomized rats. Regtd Pept 6: 197-205. 1983. 13. Bueno, L., J. Fioramonti and J. P. Ferre. Calcitonin--C.N.S. action to control the pattern of intestinal motility in rats. Peptide~ 4: 63-66. 1983. 14. Chihara, K.. J. Iwasaki, Y. Iwasaki, N. Minamitani, H. Kaji and T. Fujita. Central nervous system effect of calcitonin: Stimulation of prolactin release in rats. Brain Res 248:331-339, 1982. 15. Clementi, G., F. Nicoletti, F. Patacchioli, A. Prato, F. Patti, C. E. Fiore. M. Matera and U. Scapagnini. Hypoprolactinemic action of calcitonin and the tuberoinfundibular dopaminergic system. J Neurochem 40: 885-886, 1983. 16. Clementi, G.. A. Prato. R. Bernardini, F. Nicoletti, F. Patti, D. De Simone and U. Scapagnini. Effects of calcitonin on the brain of aged rats. Neurobiol Aging 4: 229-232, 1983. 17. Cooper. C. W., W. H. Schwesinger, A. M. Mahgoub and D. A. Ontjes. Thyrocalcitonin: Stimulation of secretion by pentagastrin. Science 172: 1238-1240, 1971. 18. Copp. D. H.. A. G. F. Davidson and B. Cheney. Evidence fora nexv parathyroid hormone which lowers blood calcium. Proc Anm~ Meet Can Fed Biol Soc 4: 17, 1961 (Abstract). 19. Craig. R. K.. L. Hall. M. R. Edbrooke, J. Allison and I. Maclntyre. Partial nucleotide sequence of human calcitonin precursor mRNA identifies flanking cryptic peptides. Nature 295: 345347. 1982. 20. Deftos, L. J.. D. Burton, B. D. Catherwood, H. G. Bone, J. G. Panhemore. R. Guillemin, W. B. Watkins and R. Y. Moore. Demonstration by immunoperoxidase histochemistry of calcitonin in the anterior lobe of the rat pituitary. J Clhz End,,~'rinol Metab 47: 457--460, 1978. 21. Fargeas, M. J., J. Fioramonti and L. Bu4no. Prostaglandin E2: A neuromodulator in the central control of gastrointestinal motility and feeding behavior by calcitonin. Science 225: 1050--1052, 1984. 22. Fischer, J. A., S. M. Sagar and J. B. Martin. Characterization and regional distribution of calcitonin binding sites in the rat brain. Life Sci 29: 663-671, 1981. 23. Fischer, J. A., P. H. Tobler, H. Henke and F. A. Tschopp. Salmon and human calcitonin-like peptides coexist in the human thyroid and brain. J Clin Endocrinol Metab 57: 13141316. 1983. 24. Fischer, J. A.. P. H. Tobler, M. Kaufmann, W. Born, H. Henke. P. E. Cooper. S. M. Sagar and J. B. Martin. Calcitonin: Regional distribution of the hormone and its binding sites in the human brain and pituitary. Proc Natl Ac'ad Sci USA 78: 7801-7805, 1981. 25. Fisher, L. A.. D. O. Kikkawa, J. E. Rivier, S. G. Amara, R. M. Evans. M . G . Rosenfeld, W . W . Vale and M . R . Brown. Stimulation of noradrenergic sympathetic outflow by calcitonin gene-related peptide..Vature 305: 534-536, 1983. 26. Flynn. J. J., D. L. Margules and C. W. Cooper. Presence of immunoreactive calcitonin in the hypothalamus and pituitary lobes of rats. Brain Res Bull 6: 547-549, 1981. 27. Freed, W. J., M. J. Perlow and R. J. Wyatt. Calcitonin: Inhibitory effect on eating in rats. Science 206: 850-852, 1979. 28. Fritsch, H. A. R., S. Van Noorden and A. G. E. Pearse. Localization of somatostatin-, substance P- and calcitonin-like immunoreactivity in the neural ganglion of Ciona intestinalis L. (Ascidiaceae). Cell Tissue Res 202: 263-274, 1979. 29. Gatan Galan, F., R. M. Rogers, S. I. Girgis, T. R. Arnett, M. Ravazzola, L. Orci and I. MacIntyre. lmmunochemical characterization and distribution of calcitonin in the lizard. Acta Emlocrinol (Copenh~ 97: 427-432, 1981. 30. Galan Galan, F., R. M. Rogers, S. I. Girgis and I. Maclntyre lmmunoreactive calcitonin in the central nervous system of the pigeon. Brain Res 212: 59-66, 1981.

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FISCHER AND BORN 67. Nakamuta, H., S. Furukawa, M. Koida, H. Yajima. R. C. Orlowski and R. Schlueter. Specific binding of 12'~l-salmon calcitonin to rat brain: Regional variation and calcitonin specificity. Jpn J Pharmacol 31: 53-60, 1981. 68. Nakhla, A. M. and A. Latif. Response of rat brain to calcit,min alteration. Brain Res 238: 48%493, 1982. 69. Nakhla, A. M. and A. P. Nandi Majumdar. Calcitoninmediated changes in plasma tryptophan and brain 5-hydroxytryptamine and acetylcholinesterase activity in rats. Biochem J 170: 445-448, 1978. 70. Neher, R., B. Riniker, W. Rittel and H. Zuber. Menschliches Calcitonin. I11. Struktur yon Calcitonin M und D. Heir ('him Acta 51: 1900-1905, 1968. 71. Nelkin. B. D., K. I. Rosenfeld, A. de Bustros, S. S. Leong. B. A. Roos and S. B. Baylin. Structure and expression of a gene encoding human calcitonin and calcitonin gene related peptide. Bioclwm Biophys Res Cmmnttn 123: 648-655. 1984. 72. O'Connor, D. T.. R. P. Frigon and L. J. Deftos. lmmunoreactive calcitonin in catecholamine storage vesicles of human pheochromocytoma. J Clin Endocrimd 3h'tah 56: 582-585. 1983. 73. Olgiati, V. R., F. Guidobono. C. Netti and A. Pecile. Localization of calcitonin binding sites in rat central nervous s.~stem: Evidence of its neuroactivity. Brain Rc.~ 265: 209-215. 1983. 74. Olgiati, V. R.. C. Netti, F. Guidobono and A. Pecile. High sensitivity to calcitonin of prolactin-secreting control in lactating rats. Endocrinology i I 1: 641-644. 1982. 75. Pearse, A. G, E. and T. Takor Takor. Embryology of the diffuse neuroendocrine system and its relationship to the common peptides. Fed Proc 38: 2288--2294. 1979. 76. Pecile, A., S. Ferri, P. C. Braga and V. R. Olgiati. Effects of intracerebroventricular calcitonin in the conscious rabbit. L.rpcriemia 3 h 332-333, 1975. 77. Perez Cano. R.. F. Galan Galan. S. 1. Girgis. T. R. Arnett ~md 1. Maclntyre. A human calcitonin-like molecule in the ultimobranchial body of the amphibia (Rana pipiensJ. E.rpcricmia 37: 1116--I 118, 1981. 78. Perez Cano, R.. F. Galan Galan. S. I. Girgis. T. R. Arnett and Identification of both human and salmon calcitonin-like molecules in birds suggesting the existence of two calcitonin genes. J Emlocrinol 92: 351-355, 1982. 79. Perez Cano, R.. S. I. Girgis and I. Maclntyre. Further evidence for calcitonin gene duplication: the identification of t ~ o different calcitonins in a fish, a reptile and t~o mammals. Acta Endocrino/(Copenh) 100: 256-261, 1982. 80. Potts, J. T.. Jr.. H. D. Niall, H. T. Keutmann and R. M. Lequin. Chemistry of the calcitonins: Species variation plus structure-activity relations, and pharmacologic implications. In: Cahium. Parathyroid Hormone and the Cah'itonin.~. edited by R. V. Talmage and P. L. Munson. Amsterdam: Excerpta Medica, 1972, pp. 121-127. 81. Przepiorka0 D.. S. B. Baylin. O. W. McBride. J. R. Testa. A. de Bustros and B. D. Nelkin. The human calcitonin gene is located on the short arm of chromosome II. Biochcm Bi,,ph~ Rt's ('o/ttmlltt 120: 493-499, 1984. 82. Rizzo, A. J. and D. Goltzman. Calcitonin receptors in the central nervous system of the rat. Ettdo(ritttJh~gy 108: 1672-1677. 1981. 83. Rodrigo, J., J. M. Polak, L. Fernandez, M. A. Ghatei. P. Mulderry and S. R. Bloom. Calcitonin gene-related peptide immunoreactive sensory and motor nerves of the rat. cat. and monkey esophagus. Gastroenteroh~ey 88: 444-451. 1985. 83a. Roos. B. A., J. A. Fischer. W. Pignat. C. A. Alander and L. G. Raisz. Evaluation of the in viro and bz vitro calcium-regulating actions of noncalcitonin peptides produced via calcitonin gene expression. Endocrin,~h~gy 118: in press, 1986. 84. Roos, B. A., M. B. Huber, R. S. Birnbaum. D. C. Aron. A. W. Lindall, K. Lips and S. B. Baylin. Medullary thyroid carcinomas secrete a noncalcitonin peptide corresponding to the carboxyl-terminal region of preprocalcitonin..I ('lin E~d, ,~ ril~,,! Metab 56: 802-807. 1983.

PEPTIDES FROM THE CALCITONIN

GENE

85. Rosenfeld, M. G.. S. G. Amara and R. M. Evans. Alternative RNA processing: Determining neuronal phenotype. St'iem'e 225: 1315-1320. 1984. ~6. Rosenfeld, M. G.. J.-J, Mermod. S. G. Amara. I.,. W. Swanson. P. E. Sawchenko, J. Rivier, W. W. Vale and R. M. Evans. Production of a no'.el neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Natto'~, 304: 12%135, 1983. ST. Sabate, M. I., L. S. Stolarsky, J. M. Polak. S. R. Bloom, I. M. Varndell. M. A. Ghatei. R. M. Evans and M. G. Rosenfeld. Regulation of neuroendocrine gene expression by alternative RNA processing. Colocalization of neuroendocrine gene expression by alternative RNA processing..1 Biol Chem 260: 2589-2592, 1985. 88. Sabbatini, F., C. J. Fimmel. F. Pace, P. H. Tobler. R. A. Hinder, A. L. Blum and J. A. Fischer. Distribution of intraventricular salmon calcitonin and suppression of gastric secretion. Di~,e~tion 32: 273-281. 1985. 89. Schot, L. P. C., H. H. Boer, D. F. Swaab and S. Van Noorden. lmmunocytochemical demonstration of peptidergic neurons in the central nervous system of the pond snail Lymnaea stagnalis with antisera raised to biologically active peptides of vertebrates. Cell Tisstw Re~ 216: 273-291, 1981. 90. Segond. N., A. Julienne. F. Lasmoles, C. Desplan, G. Milhaud and M . S . Moukhtar. Rapid increase of calcitonin-specific mRNA after acute hypercalcemia. Era" ,I Bioctwm 139: 209215. 1984. 9t. Steenbergh, P. H.. J. W. M. H6ppener. J. Zandberg, C. J. M. Lips and H. S. Jansz. A second human calcitonirffCGRP gene. FEBS Lett 183: 403-407. 1985. 92. Steenbergh. P. H.. J. W. M. H6ppener. J. Zandberg. W. J. M. Van de Ven. H. S. Jansz and C. J. M, Lips. Calcitonin gene related peptide coding sequence is conserved in the human genome and is expressed in medullary thyroid carcinoma..! Clio Endocrimd Me,tab 59: 358--360. 1984. 93. Tache. Y.. M. Gunion. M. Lauffenburger and Y. Goto. Inhibition of gastric acid secretion by intracerebral injection of calcitonin gene related peptide in rats, Lib" Sci 35: 871-878, 1984. 94. TachC Y.. T. Pappas. M. Lauffenburger. Y. Goto. J. H. Walsh and H. Debas. Calcitonin gene-related peptide: Potent peripheral inhibitor of gastric acid secretion in rats and dogs. Gastroenterology 87: 344-349. 1984. 95. Tatemoto, K. and V. Mutt. Isolation and characterization of the intestinal peptide porcine PHI (PHI-27). a new member of the glucagon-secretin feamily. Prow Natl Ac ad Sc i USA 78: 6603-6607, 1981. 96. Tippins. J. R., H. R. Morris. M. Panico, T. Etienne. P. Bevis. S. Girgis, I. Maclntyre. M. Azria and M. Attinger. The myotropic and plasma-calcium modulating effects of calcitonin gene-related peptide (CGRP). Net~r~peptid~,s 4: 425-434, 1984.

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