Vasoactive Intestinal Polypeptide

Chapter 24

Vasoactive Intestinal Polypeptide EDWIN E. DANIEL and JO-ANN E. T. FOX-THRELKELD

Introduction Chemistry of VIP and Interaction with Receptors Locations of VIP in Nerves and VIP Release from Nerves Actions of VIP Localization and Action in the Immune System Localization and Action in the Cardiovascular System Localization and Action in the Central Nervous System Localization and Action in the Urogenital System Localization and Action in the Respiratory System Localization and Actions in the Gastrointestinal System Control of Enteric VIP Release Mucosal Function Interactions of VIP and NO Summary

Principles of Medical Biology,Volume 10B Molecular and Cellular Endocrinology, pages 517-530. Copyright 9 1997by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-815-3 517

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EDWIN E. DANIEL and JO-ANN E. T. FOX-THRELKELD INTRODUCTION

Vasoactive intestinal polypeptide (VIP) is a member of a family of homologous neuro- and endocrine peptides which has three separate groups distinguished by the receptors which recognize them. In the group to which VIP belongs, the receptors recognize with varying selectivity: VIP; its neighbor on the gene responsible for VIP synthesis; peptide histidine leucine (PHI, porcine) or peptide histidine methionine (PHM, human); the recently described pituitary adenylyl cyclase activating peptide fragments 1-27 and 1-38 (PACAP); secretin, rat, and human growth hormone releasing factor (GRF); and the Glia monster venom peptides, helospectrin I and II (II is shorter than I by one serine residue) and helodermin (see Table l a). A second set of receptors recognizes additional members of the family: glucagon, glicentin, oxyntomodulin, and the glucagon-like peptides 1 and 2. Gastric inhibitory peptide (GIP) is recognized by the third receptor type. This chapter will summarize the chemistry of VIP, locations of VIP nerves, proposed actions of VIP, nature of VIP receptors and receptor-response coupling, and physiological and pathophysiological roles of VIP.

C H E M I S T R Y OF VIP A N D I N T E R A C T I O N W I T H RECEPTORS VIP was first isolated by Said and Mutt in 1970 from porcine intestine (see McDonald 1990a,b). It contains 28 amino acids which are highly conserved phylogenetically with bovine, human, rat, canine, and porcine VIP being identical and only conservative substitutions occurring in opossum, chicken, guinea-pig, dogfish, and cod VIPs. Table lb shows the species differences in mammalian VIPs. Comparison of these structures shows that the N-terminal portion of VIP-related polypeptides is well-conserved with any variation being in length or amino acid composition of the C-terminal end. Thus amino acids 1 through 7 have only minor conservative variations. The N-terminal histidine is important for both receptor recognition and for activation of adenylyl cyclase. Additional amino acids in the entire VIP sequence are involved in receptor recognition and biological function. For example, VIP10_28 is a weak competitive antagonist at some VIP receptors. Rosselin (1986) suggested that one area of the molecule interacts with the receptor and another area constrains the molecule to a specific shape once VIP binds to the primary site. The prepro VIP/PHI gene has 9 kb with seven exons encoding the signal peptide, the VIP and PHI encoding regions, and a cAMP regulatory element. Increased cAMP and phorbol esters stimulate VIP mRNA production. Posttranslational processing of the VIP/PHI precursor leads to VIP, PHI, C- and N-terminal flanking peptides, and a bridging peptide, although alternative processing can lead to an extended PHI (PHV42) containing some of the bridging amino acid sequences (see Dockray, 1992). Heterogenicity of VIP receptors has been suggested on the basis of affinity and specificity characteristics in binding studies. A VIP receptor cloned from rat lung

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(Ishihara et al., 1992) was found to have 429 amino acids with a calculated M r of 48,964 and 7 transmembrane segments. This receptor was found to be homologous to the rat secretin, porcine calcitonin, and opossum parathyroid receptors. When transfected into COS cells, the receptor bound VIP at high- and low-affinity binding sites with dissociation constants of 0.2 and 21 nM, respectively, and exogenous VIP stimulated accumulation of cAMP. This receptor was slightly more sensitive to PACAP than VIP but less sensitive to Helodermin, PHM, or secretin and insensitive to glucagon. Binding studies have demonstrated two other classes of receptors. One has a similar affinity for VIP and PACAP, and the other has a greater affinity for VIP than PACAP (see for example Mao et al., 1991; Shivers et al., 1991; Katsoulis et al., 1993). Neither of these putative receptor subtypes has been cloned. VIP is degraded extracellularly by bond-specific enzymes (see Checler, 1990). VIP-receptor complexes have also been shown to undergo endocytosis and intracellular degradation in some systems.

L O C A T I O N S OF VIP IN NERVES A N D VIP RELEASE F R O M NERVES In nerves, VIP is primarily located within large granular vesicles (LGV) (e.g. see Berezin et al., 1987). So far as is known, VIP release is by Ca2§ exocytosis of these LGV, but does not utilize the same synaptophysin-related mechanisms thought to be utilized by small clear synaptic vesicles (usually containing acetylcholine) or by small granular vesicles (usually containing catecholamines or 5-hydroxydopamine). The details of release of neuropeptides from nerves is unknown, but may involve a requirement for higher frequency firing of nerve action potentials. Nerves which release VIP are widespread in the central (CNS), enteric (ENS), and peripheral (PNS) nervous systems (see Furness and Costa, 1987; Ekblad et al., 1990). The localization of VIP and other neuropeptides can be conveniently studied by immunohistochemistry. Immunohistochemistry depends on the specificity of the antiserum. Most studies of VIP localization were carried out before the recent discovery of PACAP with antisera which may identify both peptides. The few studies that distinguish between the two peptides find them colocalized in the same nerves as with VIP and PHI/PHM. The location of the gene coding for PACAP relative to that for VIP and PHI/PHM is unknown. Therefore the identity of the VIP immunoreactive compounds in nerves may require reinvestigation. VIP immunoreactivity in various nerves is colocalized with other neuropeptides, amines, and/or nitric oxide synthase. VIP-postganglionic-containing nerves are present in the airway and supply most visceral organs: liver, bladder, uterus, kidney, adrenals, pancreas, and blood vessels. Some are sensory, providing input from the intestine to celiac and other prevertebral ganglia.

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ACTIONS OF VIP Information concerning the action of VIP is usually based on studies in which VIP is added exogenously and thus represents an analysis of the pharmacological actions of VIP. There are no potent peptide or non-peptide antagonists available to study the physiological actions of VIP. There have been studies using anti-VIP antibodies but these have major limitations (limited diffusion etc.) and the substituted peptide antagonist for VIP has a Pa 2 of 6.5. VIPI0_28 has a Ki in binding studies of about 6.5 nm (Mao et al., 1991). Studies which show actions occurring concurrently with the release of VIP as measured by RIA should not lead to a presumption of causation since NO, PHI, galanin, and possibly PACAP and other colocalized mediators are also released. Until selective antagonists to VIP are demonstrated to block actions of VIP, its physiological functions will be hard to prove. Table 2 gives the major actions of VIP which have been determined by exogenous addition of VIP.

LOCALIZATION AND ACTION IN THE IMMUNE SYSTEM VIP has been identified in lymphocytes, eosinophils, polymorphonuclear cells, and mast cells, and VIP receptors have been identified on lymphocytes, monocytes, macrophages, and mast cells (see Wenger et al., 1990). T-helper cells (CD4+) have VIP receptors which when occupied inhibit these cells. The T-suppressor CD8 cell has few VIP receptors and is resistant to VIP. When VIP receptors are occupied cAMP is increased. Lymphocyte, monocyte, macrophage, and mast cell proliferation are all inhibited by VIP. VIP reduces IgA synthesis in lymphocytes, inhibits natural Killer cell activity, and inhibits lymphocyte migration. In astrocytes, VIP stimulates IL-6 secretion which appears to downregulate other immune cell activities. Thus, VIP appears to inhibit immune responses.

LOCALIZATION AND ACTION IN THE CARDIOVASCULAR SYSTEM VIP-containing nerves are found in the adventitia or adventitia-media border muscle of blood vessels both in the cranium and in the periphery (Edvinsson and Uddman, 1988). Stimulation of VIP receptors results in increases in adenylyl cyclase activity and relaxation of the blood vessels. Evidence for VIP as a neurotransmitter is most complete for cerebral and enteric blood vessels. In the cat, VIP has been shown to be present in adventitia blood vessels, to be released on nerve stimulation, and to cause vasodilatation (like nerve stimulation) in an endotheliumindependent fashion. However, VIP tachyphylaxis in dog cerebral arteries has been reported not to block neural vasodilation. Some of these VIP-immunoreactive nerves also contain NO synthase and release NO together with VIP. Moreover, NO produced in the endothelial cell is released in response to intraluminal stimuli and relaxes the vessels by stimulating guanylyl cyclase in response to local events such

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EDWIN E. DANIEL and JO-ANN E. T. FOX-THRELKELD

Table 2. Actions of VIP System

Organ

Cardiovascular

Blood vessels Heart

Respiratory

Bronchus Mucosa Salivary glands

Exocrine Glands

Endocrine and Metabolism

Pancreas Metabolism Pancreas Thyroid Kidney Pitu itary/Hypothalamus

Central Nervous System

Cortex Spinal cord Pineal gland

U rogen ital System

Fallopian tube Vagina Penis

Gastrointestinal System

Esophageal body LES Stomach

Small intestine

Large intestine Gallbladder Liver

Action Vasodilation. Ionotrophic and chronotrophic. Bronchodilation. Stimulates secretion. Vasodilation and amplifies cholinergic secretion. H20 & HCO~ secretion. Lypolysis and glygogenolysis. Releases insulin, glucagon, somatostatin & PP. Releases hormones. Releases renin. Release prolactin, oxytocin and vasopressin. Couples energy metabolism, blood flow and neuronal activity. Excites nerves. Stimulates melatonin production and release. Facilitates ovum transport. Increases blood flow and secretion. Vascular and cavernosa relaxation. Stimulates contractions, VlP released by catecholamines. Slow hyperpolarization and relaxation. Relaxes muscle without hypolarization, may uncouple mechanical and electrical events. Releases ACH and Substance P, relaxes some muscles, stimulates mucosal secretion. Relaxes muscles, stimulates mucosal secretion. Relaxes, inhibits water and NaCI absorption. Stimulates bile secretion.

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as stretch or altered flow. Current studies are aimed at finding if and how these two relaxing agents may work in concert. It should be kept in mind that vascular smooth muscle cells can readily induce NO synthase in response to various stimuli and may have some constitutive NO synthase. How vascular muscle might contribute NO to vasodilation obviously depends on the nature of the NO synthase and the availability of agonists to raise Ca 2§ near the enzyme. The heart has a sparse VIP innervation and VIP produces positive ionotrophic effects. However, PACAP is more effective than VIP in producing ionotrophism but equipotent in relaxing coronary blood vessels in neonatal pig hearts (see Ross-Ascuitto et al., 1993). Elevation of cAMP may mediate these effects.

LOCALIZATION A N D ACTION IN THE CENTRAL NERVOUS SYSTEM In the CNS, VIP is found in highest concentrations in hypothalamic nuclei and in a homogenous population of bipolar, radially oriented interneurons in the cerebral cortex with a 30% colocalization with GABA and an 80% colocalization with acetylcholine (Magistretti, 1990). VIP is released from neocortical slices in a Ca2§ fashion by depolarization. Neocortical release in vivo is inhibited by GABA, ~t-opioid agonists, and norepinephrine acting at ct2-adrenoceptors and stimulated by acetylcholine and glutamate. The major function of VIP in the cortex appears to be regulation of energy metabolism since VIP stimulation of adenylyl cyclase promotes glycogenolysis. Glycogen degradation by VIP was markedly potentiated by norepinephrine stimulation of Otl-adrenoceptors involving prostaglandin formation, by histamine via an indomethacin-insensitive mechanism, and by GABA Breceptors through a C1--sensitive, indomethacin-insensitive mechanism. Cerebral blood vessels are innervated by VIP-containing nerves and vasodilation results from VIP stimulation of adenylyl cyclase.

LOCALIZATION A N D ACTION IN THE UROGENITAL SYSTEM VIP is found in nerves supplying the blood vessels, smooth muscle, and epithelium of the genitourinary tract (see Fahrenkrug, 1990). Activation of adenylyl cyclase by VIP produces vasodilation, smooth muscle relaxation, and epithelial secretion. VIP has been suggested to participate in the propulsion of the ovum down the Fallopian tube and relaxation of the sphincter-like mechanism at the uterine tube isthmus, acting as a non-adrenergic non-cholinergic inhibitory transmitter. However, the role of NO in this process has not yet been studied. In women, exogenous VIP stimulates vaginal blood flow and vaginal lubrication. After menopause, replacement of sex hormones is required for this action to occur. VIP is released during sexual stimulation, suggesting that it plays a role in this action. In the male, the presence of VIP-containing nerves in the arterioles and

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EDWIN E. DANIEL and JO-ANN E. T. FOX-THRELKELD

vascular spaces of the corpus cavernosum and the ability of VIP to relax cavernosal tissue dose dependently, and produce a weak erection in dog and human penises, led to the proposal that VIP was responsible for penile erection. However, with the discovery of NO as the major inhibitory transmitter of the corpus cavernosum responsible for penile erection (see Bumett et al., 1992), VIP is no longer considered to be the major transmitter in penile erection. With the development of antagonists to VIP, it would be possible to determine if the presence of VIP facilitates or amplifies the action of NO.

LOCALIZATION AND ACTION IN THE RESPIRATORY SYSTEM

VIP-containing nerves arising from cell bodies in the ganglionic plexus of the adventitia of the major bronchi and trachea and others running in the vagus, have fibers within the smooth muscle bundles, around the pulmonary and bronchial blood vessels and around the submucosal glands of the tracheobronchial wall (Joss et al., 1988). In the airway as in the intestine (see below), stimulation of intrinsic nerves leads to relaxation in several species (man, guinea-pig, cat, but not dog). VIP release from intrinsic nerves has, as in the gastrointestinal tract, been postulated to mediate airway dilation (see Said, 1991). However, as with the non-adrenergic, non-cholinergic (NANC) inhibition of the gastrointestinal tract, NO has been suggested to be the main NANC bronchodilator. Again, in analogy with the gastrointestinal tract, the possibility exists that both VIP and NO are involved in several ways. These are: (1) either as co-mediators acting independently (VIP is usually suggested to be released when high-frequency and prolonged NANC stimulation is applied and to cause persistence of inhibition); (2) as co-mediators with NO providing feedback to enhance VIP release and vice versa; (3) as sequential mediators with VIP released from nerves to release NO from other structures, usually claimed to be smooth muscle cells, and (4) some combination of these possibilities. However, in the airway, NO may also be produced as an epithelialderived relaxing factor.

LOCALIZATION AND ACTIONS IN THE GASTROI NTESTINAL S Y S T E M In the enteric nervous system of species, VIP nerves are the most numerous among neuropeptide nerves and VIP quantitatively is present in the largest amount of any neuropeptide studied (see Furness and Costa, 1987; Ekblad et al., 1990; McDonald et al., 1990). VIP nerves, in the guinea pig intestine (the most widely studied model), as well as human, rat and canine intestine, have nerve cell bodies in the myenteric plexus and the submucous plexus. In the myenteric plexus most nerves project anally, going either to distal ganglia or to the underlying circular muscle. VIP has been found in the guinea pig ileum to be colocalized with cholecystokinin (CCK),

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dynorphin, enkephalins, and gastrin-releasing peptide (GRP) in nerves with cell bodies in the myenteric plexus projecting to the superior mesenteric ganglia, with dynorphin, enkephalins, and neuropeptide-y (NPY) projecting directly to the underlying circular muscle and with GRP and dynorphin projecting distally to the circular muscle. A similar distribution also applies to other species (dog and rat). VIP is also colocalized in nerves with the enzymes producing the inhibitory neurotransmitter NO which have the same projections as the nerves which execute distal inhibition in response to local distention or contraction. VIP has been a major candidate for the role of mediator of distal inhibition, but NO has been identified as the major inhibitory transmitter. VIP nerve cell bodies in the myenteric plexus also have projections to the submucous plexus. In nerve cell bodies in the submucous plexus with projections to the villi, VIP is colocalized with dynorphin. In cells with projections to the mucosa which form dense fiber networks around the crypts and frequently are in close apposition to the villous epithelium, VIP is colocalized with either galanin or neuropeptide-y (NPY) (see Brown and Miller, 1990 for review). These nerves may play an important role in stimulation of enteric secretion (see below). Both the salivary glands and the pancreas are well supplied with VIP immunoreactive nerves. In the salivary gland, VIP has been shown to be coreleased on stimulation of nerves with acetylcholine and to produce the nonmuscarinic vasodilation which accompanies cholinergic-induced water and electrolyte secretion. VIP may also contribute to the nonmuscarinic secretion of salivary proteins (see Tobin et al., 1990). In the pancreas, VIP promotes water and bicarbonate secretion. In the gastrointestinal tract, the enteric nervous system contains many VIP nerves. For muscle, they are usually inhibitory, promoting relaxation. Nearly all sphincters in the gastrointestinal tract are relaxed by VIP, and VIP has been a candidate mediator of relaxation of sphincters. Also VIP has been proposed to be the non-adrenergic, non-cholinergic (NANC) mediator of distal inhibition in response to distentions. The evidence for VIP as the NANC inhibitory mediator can be summarized as follows: (1) VIP is located in the nerves with appropriate projections to provide distal inhibition; (2) VIP is released during stimulation leading to distal inhibition; (3) VIP (at least in some systems) can relax intestinal muscle, and (4) inhibitors of VIP action, antagonists, or VIP antiserum reduce the inhibitory responses to stimuli promoting distal inhibition (see Daniel et al., 1989). However, VIP does not reproduce the cellular response produced by stimulation of NANC inhibitory nerves. This in most systems consists of membrane hyperpolarization; that is, an inhibitory junction potential, an action reproduced by NO and NOreleasing compounds. In isolated circular muscle from canine intestine, pylorus, and colon, NANC inhibition and relaxation are associated with both fast and slow inhibitory junction potentials (ijps). These all appear to be mediated primarily by NO rather than VIP (Daniel et al., 1994a & b). In all cases, inhibitors of NO-synthase abolish or reduce ijps markedly, effects reversible by L-arginine but not by D-arginine. In canine small

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EDWIN E. DANIEL and JO-ANN E. T. FOX-THRELKELD

intestine circular muscle, VIP does not affect the membrane potential and is an ineffective relaxant. However, in the canine colon circular muscle, VIP does relax, and hyperpolarize, and its effects are reduced by nerve blockade with tetrodotoxin or L-NAME, suggesting that VIP releases a nerve mediator such as NO which participates in relaxation. The ijp produced by NANC nerve stimulation is reduced by inhibition of NO-synthase and restored by L-arginine, suggesting that NO is the final mediator. Similar results have been obtained in the canine lower esophageal sphincter. However, here VIP is a relaxant without any detectable hyperpolarizing effect, and blockade of NO synthase does not block VIP-induced relaxation. In a number of other systems, VIP-induced sphincter relaxation or muscle inhibition is reduced and shifted to the fight on the concentration-effect curve by block of NO synthase. This has suggested that VIP in these tissues, for example, smooth muscle, releases NO to induce part of its effect (see Makhlouf and Grider, 1993). It is also conceivable that VIP releases NO from nerves. Indeed, in many systems, VIP raises cAMP levels in cells with VIP receptors. In enteric and other nerves, elevation of cAMP is associated with increased mediatory release. This means that VIP may also release excitatory as well as inhibitory mediators. Receptors with both a higher and lower affinity for VIP than PACAP have been identified on canine ileal myenteric, submucous and deep muscular plexus synaptosomes (Mao et al., 1991, 1993). VIP produces slow excitatory postsynaptic potentials in the guinea pig myenteric and submucous plexus. In the guinea pig ileum myenteric plexus-longitudinal muscle preparation, PACAP is more potent than VIP in stimulating spontaneous release of acetylcholine and in stimulating muscle contractions, while the two are equipotent in inhibiting stimulated acetylcholine release (see Katsoulis et al., 1993). The muscle relaxing and hyperpolarizing effects of VIP in this preparation only become apparent after both muscarinic and substance P effects are inhibited (He and Goyal, 1993). In canine intestine, VIP releases acetylcholine at a higher concentration than PACAP. Definitive investigation of physiological roles for VIP as an enteric neuromodulator await the availability of potent, highly selective, and stable competitive antagonists. It has also been reported that in some gastrointestinal tissues VIP acts on smooth musclecells to release NO, and that NO contributes to the direct effect of VIP on relaxation (see Makhlouf and Gilder, 1993). However, under normal (noninflamed, resting) conditions, there is no significant amount of NO synthase found in most intestinal muscles. The claims for release of NO by VIP from smooth muscle cells have been based on studies that failed to eliminate artifacts or contributions from nerves or nerve fragments (Daniel et al., 1994b). In brief, whole tissues or isolated smooth muscle cells have been used to claim constitutive NO production from smooth muscle cells Without establishing definitively that NO can be or is produced from smooth muscle cells experimentally. The methods for measuring NO produc-

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tion have been indirect and subject to artifacts associated with mechanisms for arginine uptake and compartmentalization of [3H]-L arginine.

CONTROL OF ENTERIC VIP RELEASE Sampling of small intestinal venous blood in vivo (cats) or effluent from an isolated arterially perfused segment (dogs) revealed that VIP was tonically released into the venous blood. Release was from nerves since it was abolished by tetrodotoxin, the absence of Ca 2§ and e0-conotoxin (GVIA) (an N-Ca 2§ channel blocker), and reduced by nifedipine. The elevated levels were the result of tonic prejunctional cholinergic activity, since acetylcholine stimulated VIP release and hexamethonium reduced and atropine abolished tonic output. The tonic VIP release was inhibited by norepinephrine acting at ~2-adrenoceptors (dog and cat), opioids acting at ~t- and ~5-but not ~c-opioid receptors (dog, cat, and rat), motilin acting through the release of opioids (dog), galanin (dog and pig), peptide yy (PYY) and NPY (dog and cat), and somatostatin (dog, cat, and man). Enteric VIP release was stimulated by electrical stimulation of intrinsic and extrinsic nerves (dog, cat, pig, rat), the NO donor Na nitroprusside (dog), neurotensin (dog and cat), and CCK 8 (dog), while the NK1 and NK2 selective tachykinins were without effect in the dog but stimulated VIP release in the rat stomach (see Daniel and Fox-Threlkeld, 1992; Jodal et al., 1993; Fox-Threlkeld et al., 1991, 1993, 1994).

M U C O S A L FUNCTION The localization of VIP nerves in the mucosa around blood vessels and the villous crypts is consistent with a major epithelial function (see Brown and Miller, 1990). VIP receptors have been identified on the basolateral membranes of enterocytes, and VIP activates adenylyl cyclase, thus increasing cAMP. The increase in cAMP is associated with an increase in short-circuit current (Isc) with no significant effect on ion conductances. The Isc change is attributed to decreases in Na § and C1mucosal fluxes and an increase in C1- secretion. Such C1- secretion is accompanied by net movement of water into the lumen. The response to VIP is not altered by removal of nerves nor by the presence of the neurotoxin, tetrodotoxin, suggesting a direct action of the peptide at the enterocyte. However, recent evidence (Jodal et al., 1993) suggests, that the secretory response to cholera toxin in vivo involves a mucosal reflex in which cholera toxin stimulates afferent serotonergic fibers to activate a cholinergic fiber originating in the myenteric plexus. This cholinergic fiber releases Ach to a submucosal nicotinic receptor on VIP nerves terminating on the enterocyte. VIP-secreting tumors are associated with massive intestinal secretion and diarrhea. One effective treatment of this condition (Wemer-Morrison

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EDWIN E. DANIEL and JO-ANN E. T. FOX-THRELKELD

syndrome) is administration of long-acting somatostatin analogues, presumably acting by inhibition of VIP release from tumor cells. INTERACTIONS

OF VIP AND

NO

Many autonomic nerves contain and release both NO and VIP and there is evidence that they may interact additively over time (VIP extending the transient effect of labile NO) or at the same time (to enhance the response to each of them). It is thus worthwhile to speculate how their second messenger systems might interact. Almost universally, NO raises cGMP levels by interacting with the Fe in guanylyl cyclase to cause activation. VIP very frequently leads to activation of adenylyl cyclase through a G s protein linked to its receptor. Possibly each of these signals (cGMP 1~ or cAMP ~) acts differently to lower intracellular Ca 2§ or decrease sensitivity to [Ca2+]i and cause relaxation. Elevation of cGMP frequently lowers intracellular Ca 2§ by enhanced pumping of Ca 2§ into SR stores and may also lead to phosphorylation at sites decreasing Ca 2§ sensitivity of myosin light-chain kinase. Elevation of cAMP also lowers [Ca2+]i, probably by affecting plasmalemma Ca 2§ pumps (or SR-Ca 2§ pumping by a mechanism different from that used as the result of cGMP elevation). It also decreases [Ca2+]i sensitivity by phosphorylation of sites on myosin light chain kinase. Also elevated cGMP can lead to inhibition of phosphodiesterases that degrade cAMP (and vice versa). Thus it is possible that VIP and NO mutually support the action of each other by operating different second messenger systems. SUMMARY VIP is a neuropeptide found in many organs. It appears to play a major role in vasodilation, epithelial secretion, as a neurotransmitter and neuromodulator, and as a relaxing agent in those smooth muscles which possess receptors and which relax when intemal levels of cAMP are raised. Many of the claims for a major role as the non-adrenergic, non-cholinergic inhibitory transmitter have recently been seriously disputed. The best candidate for this role at present is NO. However, since NO and VIP are frequently colocalized, the release and action of these two transmitters may be complementary. One further complicating feature of the studies on VIP is the locus and action of its homologue, pituitary adenylyl cyclase activating peptide (PACAP). Few studies on localization are available which use antisera distinguishing these two peptides. Careful pharmacological studies comparing the action describe at least three potential receptors, recognizing VIP more, equi- or less potently than PACAP. Identification of physiological functions of VIP output await the availability of a highly selective and potent competitive antagonist.

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ACKNOWLEDGMENTS The authors thank Dr. Steven Threlkeld for a critical reading of this manuscript and the Medical Research Council of Canada for support of their research on VIP.

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RECOMMENDED READINGS Volume 805 of Annals of the NY Academy of Sciences, published in 1996 (Arimura, A., & Said, S.I., Eds.) is devoted to the subject of PACAPNIP. Dela Fuente, M., Delgado, M., & Gomariz, R. (1996). VIP modulation of immune cell functions. Adv. Neuroimm. 6, 75-91. Miller, J.D., Morin, L.P., Schwartz, W.J., & Moore, R.Y. (1996). New insights into the mammalian circadian clock. Sleep 19, 641-667.