Vasoactive intestinal peptide: Involvement of calmodulin and catalytic antibodies

Neurochem. Int. Vol. 23, No 3, pp. 215-219, 1993

0197-0186/93 $6.00+0.00 Pergamon Press Ltd

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CRITIQUE VASOACTIVE I N T E S T I N A L PEPTIDE : I N V O L V E M E N T OF C A L M O D U L I N A N D CATALYTIC ANTIBODIES SAMI | . SAID Departments of Medicine and Physiology, State University of New York at Stony Brook and

Northport V.A, Medical Center, Stony Brook, NY 11794-8172, U.S.A.

Over two decades after the discovery, isolation and chemical characterization of the vasoactive intestinal peptide (VIP), much has been learned about its biological properties, its relationship to other neurotransmitters, its mode of action, and ~ts importance in physiological regulation and disease. Not all questions have been answered, however, Our knowledge of the VIP receptors and the secondmessenger pathways, e.g. remains incomplete, and the place of VIP in human physiology and disease needs further definition. The work summarized by Paul and Ebadi (1993) addresses two specific facets of the biology of VIP: its binding to calmodulin or calmodulin-like molecules, and its catalysis by anti-VIP antibodies. This critique is meant to complement the accompanying review, amplifying some of the comments, and present them in a broader perspective.

since VIP biosynthesis is promoted by higher cyclic AMP levels (Gozes and Brenneman, 1989), the peptide is potentially capable of stimulating its own generation. Calcium and phosphoinositides

With few exceptions, the evidence is less compelling for other mechanisms of action. In some tissues, including the superior cervical ganglion (Audigier et al., 1988) and adrenal chromaffin cells (Malhorta et al., 1988), VIP, in relatively high (10 - 6 M) concentrations, increased the breakdown of phosphoinositides to inositol phosphates, enhancing intracellular mobilization of Ca :+ . The effect on adrenal medulla was linked to the induction of catecholamine secretion (Malhorta et al., 1988) In related experiments, VIP, in concentrations as low as 10 to M, increased intracellular [Ca 2÷] in at least some rat cortical astrocytes (Brenneman, personal communiSECOND MESSENGERS FOR VIP ACTION cation), and, at higher concentrations, in cultured rat hippocampal neurons (Tatsuno et al., 1992). In the Cyclic A M P latter preparation, PACAP, a VIP-related peptide, Paul and Ebadi highlight the multiple actions of exerted the same action but was more potent (Tatsuno VIP and emphasize its ability to act via different et al., 1992). The VIP-mduced rise in intracellular second-messenger pathways. At present, however, the [Ca 2+] in astrocytes was correlated with increased weight of the evidence points to adenylyl cyclase secretory activity of these cells, including the prostimulation and cyclic A M P production as the domi- duction of trophic factors (Brenneman). Similar, nant mode of action of VIP in most instances. This though more subtle, changes in intracellular [Ca -~+] certainly applies to the relaxation of vascular and due to VIP have been described in prolactin-prononvascular smooth muscle (Shreeve et al., 1992), the ducing rat anterior pituitary cells (Sand et al., 1989). stimulation of pancreatic exocrine (Robberecht et al., The full significance of intracellular [Ca 2+] and 1976), and intestinal secretion (Dupont et al., 1980) inositol phosphates in mediating the physiological or as well as of prolactin secretion (Gourdi et al., 1979), pharmacological actions of VIP in different cell types and steroidogenesis (Birnbaum et al., 1980), the remains largely unexplored. Further, the actions of modulation of pineal function (Chik et al., 1988), and VIP (as of other agents) on target cells are often the inhibition of certain immune functions (Ottaway, mediated by more than one signal transduction path1987). Studies on the gene expression of VIP suggest way. Interactions between these pathways have been a two-way relationship between VIP and cyclic AMP : described (Meisheri and Rfiegg, 1985; Pfitzer et al., 215

216

('r]l~q ue

1985 : Szewczak et al., 1990 : Calderano et al., 1993), but are not yet fully understood. Role o/cahm~&/lin

A discussion ofintracellular Ca-" brings us to the possible role of calmoduhn in mediating VIP action. Paul and Ebadi (1993) suggest that this regulatory protein may be the predominant VIP-binding protein, tit least m guinea-pig lung mcmbrancs. The evidence they present includes: porcine brain calmodulin and a VIP-bmdmg protein (plS) purified from guinea-pig lung membranes exhibit close similarmes, in electrophoretic and chromatographic behavior, highaffinity binding of VIP, and amino acid sequencc. These two proteins may indeed be identical, but it is probably too early to conclude that the VIP receptor, at least in the lung, is calmodulin. Underscoring this cautionary note, two arguments cited by the authors in support of a Oltical role for calmoduhn and a less-than-dominant role for cyclic AMP m mediating VIP acnons, necd further confirmation: (a) that a calmodulin antagonist mhiblt~ certain VIP actions, and (b) that phosphodiesterasc lnhibitors do not enhance tracheal smooth muscle relaxation by VIP. In preliminary experiments, we were unable to demonstrate attenuation with calmidazolium, a calmodulin inhibitor, of VIP-induced relaxation of strips of guinea-pig trachea or mesenteric artery (Sharaf and Said, unpubhshed). We have also observed potentiation of two VIP actions on addition of phosphodiesterase inhibltors. Thus, theophylline augmented VIP-induced tracheal relaxation (Said el al., 1974), and isobutyl methylxanlhmc enhanced the inhibition of small-cell lung cancer cell proliferation b~ VIP and, simultaneously, the increase m cellular levels of cyclic AMP (Maruno and Said, 1993a). With the identification of the nucleoude sequence and amino acid sequence of the VIP-receptor eDNA ( Ishlhara et al.. 1992). it should be possible to estabhsh beyond question whether and to what extent this molecule and calmodulin are alike. COEXISTENCE AND INTERACTIONS YklTH OTHER NEUROTRANSMITTERS

Numerous reports document the colocalization of VIP with other neuropeptldes or neurotransmltters {Furness el al., 1992). These neuropeptides include peptlde histidlne methlonine or peptlde hlstldme isoleucine, oploid peptides, galanln, substance P, calcHonin gene-related peptide and neuropeptide Y. Some of the nonpeptide neurotransmitters that ma>

coexist with r i P in the same neurons arc accDIcholine, norepinephrine and nitric oxide synthasc, the enzyme that catalyzes the lk)rmatlon of n l m c oxide from L-arginine. The physiological lmphcatlons of these colocahzatlons include (a) the innervauon of exocrine glands with acetylcholine and VIP ensures greater secretion and blood flow responses lhan ll" lhc two transmitters were present akme (Lundberg c t a / . 1980), (b) VIP modulates chohnergm translnlS~lon m respnatory allways (Ellis and Farmer, 19~;9. Marun e; a/.. 1990) (c) the co-presence of VIP and substancc P m cholinergic nerves to the airways (De3 cta/.. 1988) provides a balance betwccn the bronchoconstrictor, pro-inflammatory >ubstancc P and the bronchial-relaxant, antHnflamrnatory VIP and Ic) VIP and nitric oxide act cooperatively to bring about smooth muscle relaxation m blood vessels, respiratory airways, and other tissues (Said, 1992: Grider et aL, 1992).

f U N C T I O N S REG{_ I,ATEI) BY VIP

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Probably among the principal functions of VIP is the relaxation of smooth muscle tone. In particular, VIP has been considered a likely mediator of the dominant nonadrenergic, noncholinerglc component of neurogenic relaxaUon of airway, gastrointestinal, and vascular smooth muscle (Said, 1991). Within the last 2 yr, it has become increasingly apparent that another major transmitter of this relaxation is nlmc oxide, and that VIP and nitric oxide act together, lhrough different mechanisms, to effect the relaxation (Stud. [992). The contributions of these two transmitters and the interplay between them m different tissues is still under investigation (Gridcr, 1993). l "IP and cell prohleratum

Paul and Ebadl refer to the abihty of VIP to snmulate mitosis in some cell types. This has been demonstrated in some types of cancer of the lung (Scholar and Paul, 1991) and breast (Gespach el a/., 1988). VIP also snmulates the proliferation of some types of normal, non-neoplastic cells, including keratinocytes (Haegerstrand et al., 1989). and neuronal cells (Pincus et a l , 1990) On the other hand, VIP has also been shown to mhibit the growth and proliferation of other cell types, including vascular smooth muscle (Hultgfirdh-Nilsson 1988), and bronchial smooth muscle (Maruno and Said, 1993b), t.s well as human gastric carcinoma (Klm el al., 1991 ) and small-cell lung cancer cells (Maruno and Said, 1993a). Why difl'erent

Critique cell types, normal or malignant, respond in opposite manners to VIP is an important question for which there is at present no good answer, Trophic action

VIP promotes the survival of neuronal cells and their precursors in culture, and protects neurons from the toxic effect of the envelope protein of the Human Immunodeficiency Virus (Brenneman et al., 1988). In a follow-up study, the same group of investigators reported that VIP, acting via specific receptors, has growth-promoting functions in whole cultured mouse embryos (Gressens et al., 1993). Anti-in[tammatory action

Recent data suggest that VIP has potent antiinflammatory activity in the lung (Said, 1993). The evidence is based on the ability of the peptide to (a) inhibit the function of the inflammatory cells, including lymphocytes, alveolar macrophages and mast cells; (b) antagonize major humoral mediators of inflammation, including histamine, prostaglandin F~, leukotrienes C4 and D 4, platelet activating factor, neurokinins A and B, and endothelin: and (c) attenuate acute edematous lung injury in several experimental models. In experimental models of acute lung injury that mimic the Adult Respiratory Distress Syndrome (ARDS), VIP dose-dependently protects perfused, ventilated rat or guinea-pig lungs against injury caused by (a) intra-tracheal instillation of HCI, (b) infusion of PAF or of the oxidant herbicide paraquat, (c) addition to the perfusate of xanthine and xanthine oxidase which react to produce toxic oxygen species, and (d) prolonged perfusion of the lungs, a form of Injury that also results from free oxygen radicals. The protective effect of VIP is selective, since of its related peptides only helodermin gave equal protection, while secretin and glucagon were ineffective. VIP DEFICIENCY IN DISEASE

Diseases in which VIP is thought to be a causative factor closely reflect its biological actions and postulated functions, either through the excessive release or the deficiency of VIP. The selective lack of VIP innervation in certain tissues has been linked to the pathogenesis of several disease processes. In achalasia of the esophagus, the loss of normal peristalsis of the esophageal body and the failure of the lower sphincter to relax with swallowing are correlated with the virtual absence in esophageal smooth muscle of VIP-containing nerves (Gridelli et al., 1982). In Hirschsprung's disease, or congenital megacolon, the normally rich

217

VIP innervation of the small intestine is strikingly reduced in the affected, aganglionic segments of the bowel, possibly contributing to their persistent contraction (Tsuto et al., 1989). In diabetic impotence, the abnormally sparse VIP innervation of penile cavernous tissue may be responsible for the failure of effective vasodilation required for penile erection (G u et al., 1984). In this, as in the preceding conditions, a simultaneous deficiency of nitric oxide synthase, and thus of nitric oxide, may well be an important pathogenetic factor. In cystic fibrosis, the marked deficiency of VIP nerves around sweat-gland aclni and ducts may be related to the defect in chloride ion transport and to other aspects of the exocrmopathy of this disorder (Hemz-Erian et al., 1985). In the acquired immune deficiency syndrome, the killing of neurons by glycoprotein gpl20, the envelope protein of the human immunodeficiency virus, may be related in some way to interference with the neurotrophic effects of VIP in the central nervous system (Brenneman et al., 1988). A deficiency of VIP may be a cause of bronchial hyperreactivity and possibly other changes in asthmatic airways. This hypothesis rests on the findings that (1) VIP is a likely neurotransmitter, with nitric oxide, of the dominant neurogenic component of airway relaxation in humans, the nonadrenergic relaxant system (Matsuzaki et al., 1980), and (2) sections of airways from subjects with asthma show a selective absence of nerves immunoreactive to VIP, m contrast to sections from control subjects without asthma, which have a rich distribution of VIP nerves (Ollerenshaw et al., 1989). The lack of VIP-containing nerves in asthmatic airways does not establish whether the VIP deficiency is a cause or a result of the disease. To prove that the VIP deficiency is a primary defect, the finding should be shown to antedate the onset of the disease, and direct evidence of impaired gene expression and biosynthesis of VIP should be demonstrated in VIP-containing neuronal cell bodies within the lung. If the lack of VIP in asthmatic airways is significantly related to the pathogenesis of the disease, then supplemental VIP may prove to be rational replacement therapy, much like insulin for type I diabetics. The possible significance in asthma of circulating high-affinity VIP-binding antibodies, some of which are capable of catalyzing the hydrolysis of the peptide (Paul et al., 1989), is discussed below. CATALYTIC ANTI-VIP ANTIBODIES

Apart from the inherent interest in it as a chemical reaction, the catalysis of VIP by antibodies may have

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Critique

slgmflcant pathophysiological a n d chnical implications. Catalytic antibodies could neutrahze VIP and thus eliminate or a t t e n u a t e ~ts physiological influence. To date, two conditions have been f o u n d to be associated with increased circulating levels and affinity of catalytic a n t i - V I P a n t i b o d i e s : muscular exercise in normal subjects, a n d bronchial asthma. It is difficult to explain why regular exercise in n o r m a l healthy subjects should p r o v o k e the presence of such a n t > bodies. N o r is it clear what, if any, u n t o w a r d effects the antibodies have under those conditions. In bronchial asthma, on the other hand, the presence o f high c o n c e n t r a t i o n s of high-affinity catalytic antibodms against VIP could conceivably c o n t r i b u t e to decreased VIP activity, and thereby to airway constriction and hypcrreactivity (Matsuzaki et al., 19801. Considering the two conditions together, could catalytic antibodies be held responsible for some o f the features of asthma, while they cause no ill effects in n o r m a l subjects? The a p p a r e n t discrepancy may be explained by q u a n titative differences in the binding affinity of the antibodies a n d in their local c o n c e n t r a t i o n s at the sites of VIP release in airway s m o o t h muscle and o t h e r sites. Alternatwely, the antibodies m a y have no pathogenetic significance even in asthmaUc subjects. This novel research area, pioneered by Paul et al., (19891 clearly deserves further investigation.

REFERENCES

Audlgmr S., Barberls C. and Jard S (1988) Va~oactlve intestinal polypeptide increases inosltol phosphate breakdown in the rat superior cervical ganghon Ann NY Acad. S~t 527, 579 581 Blrnbaum R S., Alfonzo M and Kowal J. 119801Vasoactwe mteslmal peptidc- and adrenocorticotropin-sttmulated adenyl cyclase in cultured adrenal tumor cells: evidence for a specific vasoactive intestinal peptlde receptor Endocrinology 106, 1270 1275. Brenneman D. E., Westbrook G. L, Fitzgerald S. P., Enmst D. L., Elkms K. L., Ruff M. R and Pert C B. (1988) Neuronal cell killing by the envelope protein of HIV and its preventmn by vasoactwe intestinal peptide. Nature 335, 639 642. Calderano V., Chlosl E , Greco R , Spma A. M., Giovanc A , Quaghuolo L., Servillo L., Balestneri C and Ilhano G (19931 Role of calcium in chloride secretion mediated by cAMP pathway activation in rabbit distal colon mucosa Am. J. Physud. 264~ (Gastromtest Liter Physiol. 271. G252 G260. Chik C L,, Ho A K and Klein D C (19881cq-Adrenergic potentiation of vasoactlvc intestinal peptlde stnnulation of rat plnealocyte adenosine 3',5'-monophosphate and guanosme 3',5'-monophosphate' evidence for a role for calcium and protein kinase-C, Endocrinolo,qr 122, 702 708. Dey R D , Hoffpauir J and Stud S. I (1988)Co-locahzatlon

o1 vasoactlve intestinal peptide- and substance P-timtaming nerves in cat bronchi. Neuroscience 24, 275 28 I Dupont C., Laburthe M., Broyart J. P., Batallle D and Rosselin G. (1980) Cyclic AMP production m isolated colonic epithelial crypts, a highly sensitive model for thc evaluation ofwisoactive intestinal peptide action in human intestine. Eur. J. din. ltwe,st. 10, 67 76. Ellis J. L and Farmer S. G. (1989) Modulatmn ofchohnerglc neurotransmisszon by vasoactwe intestinal peptide and peptide histamine lsoleucine In guinea-pig tracheal smooth muscle. Pulmon. Pharmac 2, 107 112. Furness J. B, Bornsteln J C., Murphy R. and Pompolo S 11992) Roles of peptides m transmission in the enteric nervous system, Trend,~ Neuro,w~ 15, 66 71. Gespach C , Bawab W , de Cremoux P. and Calco l: (1988) Pharmacology, molecular identllicatlon and functlomd character[sims of vasoactlve intestinal peptlde receptors m human breast cancer cells Cancer Res. 48, 5079 5083 Gourdi D., Batadle D,, Vauchn N . Grouselle D , Rossehn G, and Tixier-Vidal A (1979) Vasoactwe intestinal pephde (VIP) stimulates prolactln (PRL) release and cAMP production in a rat pitmtary cell line (GH3/B6) Additivc effects of VlP and TRH on PRL release. FEBS Lett 104, 165 168. Gozes I. and Brcnneman D. E [1989) VIP" molecular biology and neuroblologmal function. Moh'c Neurohzol 3, 201 236 Gressens P, Hill J. M., Gozes l., Fndkln M. and Brenneman D E (19931 Growth factor function of vasoacm'e mte,,,tlnal peptide in whole cultured mouse embryos Nalltrc In press Gndelh B., Buffa R, Salwm P., Ral D., Uggen F , Flocca R and Said S I. (1982) Lack of VIPqmmunoreactive nerves in oesophageal achalasia, Ital J Gastroentet 14, 211 213 Gnder J. R (1993) Interplay of VIP and nitric oMdc m regulation of the descending relaxation phase of peristal~l~. Am J. Ph3mol. 264 (Ga~trom/e,~t Lwer Phv~'lol, 27L G334 G340. Gnder J R., Murthy K. S, Jm J -G and Makhlouf G. M (19921 Stimulation of nitric oxide from muscle cells b~ VIP preluncyional enhancement of VIP release. 4m J, P]tvstol 262 (Ga.~lroitllest. Lit cr Physiol. 251. G774 G778 Gu J , Polak J. M., Lazandes M , Morgan R., Pryor J P., Marangos P J , Blank M. A and Bloom S R. (1984) Decrease of vasoactive intestinal polypeptide (VIP) in the peruses from impotent men Lam'et 2, 315 318 Haegerstrand A , Jonzon B., Dalsgaard C. J. and NiIsson J 119891 Vasoactive intestinal potypeptlde stimulates cell proliferatmn and adenylate cyclase activity of cultured human keratmocytes. Proc. man 4cad Set U.S,I 86, 5993 5996 Hemz-Erlan P., Dcy R. D., Flux M, and Said S I. (19851 Deficient vasoactlve intestinal peptlde lnnervatlon m sweat glands of cystic fibrosis patients Scwnce 229, 1407 1408 Hultgfirdh-Nilsson A., Nitsson J., Jonzon B, and Dalsgaard C-,I ([988) Growth-inhibitory properties of vasoactlve intestinal polypeptide Re,q. Peptutes 22. 267 274 Ishlhara T., Shlgemoto R., Mort K , Takahashl K. and Nagata S (1992) Functional expression and tissue dlstllbution ofa no',el receptor for vasoactive intestinal pol',peptlde. Neuron 8, 811 819 KHn S W., Beauchamp R. D , Townsend Jr 12' M and Thompson J C (1991) Vasoacme intestinal pol~peptide

219

Critique inhibits c-myc expression and growth of human gastric carcinoma cells. Surgery 110, 270 276. Laburthe M., Rousset M., Chevalier G., Boissard C., Dupont C., Zweibaum A. and Rosselin G. (1980) Vasoactire intestinal peptide control of cychc adenosine 3',5'monophosphate levels in seven human colorectal adenocarcinoma cell lines in culture. Cancer Res. 40, 2529 2533. Lundberg J. M., Anggfird A., Emson P., Fahrenkrug J., H6kfelt T. and Mutt V. (1980) Vasoactive intestinal polypeptide in chohnerglc neurons of exocrine glands: functional significance of coexisting transmitters of vasodilation and secretion. Proc nam. Acad. Sci. U.S.A. 77, 1651 1655. Malhotra R. K., Wakade T. D and Wakade A. R. (1988) Vasoactlve intestinal polypept~de and muscarme mobilize mtracellular Ca -'+ through breakdown of phosphomositides to induce catecholamine secretion. J. biol. Chem. 263, 2123 2126. Martin J. G., Wang A., Zacour M. and Blggs D. F. (1990) The effects of vasoactive intestinal polypeptide on cholmerglc neurotransmlssion in an isolated innervated guinea pig tracheal preparation. Respiratton Physiol. 79, 111 122. Maruno K. and Said S. I. (1993a) Small cell lung carcinoma: inhibition of proliferation by vasoactive intestinal peptlde and helodermin and enhancement of inhibition by antibombesin antibody. Li]e Sci. (Pharmac. Lett.) 52, PL 267271. Maruno K. and Said S. 1 (1993b) Inhibition of human airway smooth muscle cell proliferation by vasoactive intestinal peptide (VIP). Am. Rev. Respir. Dis. 147, A253. Matsuzakl Y., Hamasakl Y. and Said S. I. (1980) Vasoactlve intestinal peptide, a possible transmitter of nonadrenergic relaxation of guinea pig airways. Science 210, 1252 1253. Meisheri K. D and Rfiegg J C. (1983) Dependence of cyclicAMP reduced relaxation on Ca -'+ and calmodulin in skinned smooth muscle of guinea pig Taema coll. Pfliigers Arch. 399, 315 320. Ollerenshaw S,, Jarvls D., Woolcock A., Sullivan C. and Schelbner T. (1989) Absence of immunoreactive vasoact~ve intestinal polypeptlde in tissue from the lungs of patients with asthma. New Engl. J. Med. 320, 1244 1248 Ottaway C. A. (1987) Selective effects of vasoactive intestinal peptide on the mltogemc response ofmurine T cells. Immunology 62, 291 297. Paul S. and Eba& M. (1993) Vasoactive intestinal peptide: its interactions with calmoduhn and catalytic antibodies Neurochem. htt 23, preceedmg paper. Paul S, Voile D. J., Beach C. M., Johnson D. R., Powell M. J. and Massey R. J. (1989) Catalytic hydrolysis of vasoactive intestinal pephde by human autoantibody. Science244, 1158 1162 Pfitzer G., Rfiegg J. C., Zlmmer M. and Hofmann F. (1985) Relaxation of skinned coronary arteries depends on the relative concentrations of Ca -'+, calmodulin and active cAMP-dependent protein kinase Pfliigers Arch. 405, 70 76. Pmcus D. W., Dlclceo-Bloom E. M. and Black I B. (1990) Vasoactive intestinal peptide regulates mitosis, differ-

entiatlon and survival of cultured sympathetic neuroblasts. Nature 343, 564-567. Robberecht P. Conlon T. P. and Gardner J. D. (1976) Interaction of porcine vasoactive intestinal peptide with dispersed pancreatic acinar cells from the guinea pig : structural reqmrements for effects of VIP and secretin on cellular cyclic AMP. J. biol. Chem. 251, 4635 4639. Rodnguez-Pena M. S., Guijarro L. G. and Prieto J. C. ( 1991 ) Adenylyl cyclase stimulation by VIP in rat seminal vesicle membranes Peptides 12, 821 824. Said S. 1. (1991) Vasoactive intestinal polypeptide : biological role in health and disease. Trends Endocrin. Metah. 2, 107 112 Said S. i. (1992) Nitric oxide and vasoactive intestinal peptide as co-transmitters of smooth muscle relaxation. News Physiol. Sci. 7, 18l 183. Said S. 1. (1993) Vasoactlve intestinal peptlde (VIP) and related peptides as anti-asthma and anti-inflammatory agents. Biomed. Res. In press. Said S. I., Kitamura S., Yoshlda T., Preskltt J. and Holden L. D. (1974) Humoral control of the airways. Ann. N Y Acad. Sct. 221, 103 114. Satto K., Yamatani K., Manaka H., Takahashi K., Tominaga M. and Sasaki H. (1992) Role of Ca z+ on vasoactive intestinal peptide-induced glucose and adenosine Y,5'monophosphate production m the isolated perfused liver Endocrinology 130, 2267-2273. Sand O., Chert B., Li Q., Karlsen H. E., Bjoro T. and Haug E. (1989) Vasoachve intestinal peptide (VIP) may reduce the removal rate of cytosolic Ca 2+ after transient elevations in clonal rat lactotrophs. Acta Phystol. Stand 137, 113 123 Scholar E. and Paul S. (1991) Shmulatlon of tumor cell growth by vasoactlve intestinal peptide. Cancer 67, 1561 1564, Shreeve S. M., DeLuca A. W., Diehl N. L. and Kermode J. C. (1992) Molecular properties of the vasoactive intestinal peptlde receptor m aorta and other tissues. Peptides 13, 919 926. Szewczak S. M., Behar J. and Billett G. (1990) VlP-induced alterations in cAMP and inositol phosphates in the lower esophageal sphincter. Am. J. Physiol 259 (Gastrobttest. Liver Physiol. 22), G239 G244. Tatsuno I , Yada T., Vigh S, Hldaka H. and Arimura A. (1992) Pituitary adenylate cyclase activating polypeptide and vasoactive intestinal peptide cytosolic free calcium concentration in cultured rat hippocampal neurons. Endocrinology 131, 73 81. Tsuto T., Obata-Tsuto H. L., lwai N., Takahashi T. and Ibata Y. (1989) Fine structure of neurons synthesizing vasoactive intestinal peptlde in the human colon from patients with Hlrschsprung's disease. Htstochemist O" 93, 1 8.

Yu D., Seitz P. K., Selvanayagam P., Rajaraman S, Townsend Jr. C. M. and Cooper C. W. (1992) Effects of vasoactive intestinal peptide on adenosine 3',5'-monophosphate, ornithme decarboxylase, and cell growth m a human colon cell line. Endocrinology 131, 1188 1194.