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Vasoactive intestinal peptide has been identified as an important regulator of submandibular salivary gland function, consistent with its localization with acetylcholine in parasympathetic neurones innervating this gland. Enzymatically dispersed acini from rat submandibular gland are a useful system in which to study gland regulation at the cellular level. Here, three aspects of vasoactive intestinal peptide interactions with acini were examined: inhibition of binding of [125I]-vasoactive intestinal peptide, stimulation of cyclic AMP production and enhancement of mucin release. Vasoactive intestinal peptide and peptide histidineisoleucineamide inhibited [125I]-vasoactive intestinal peptide binding to intact acini, with IC50 values of 16 +/- 3 and 46 +/- 17 nM, respectively. This rank order of potency agrees with that observed previously in assays using rat submandibular gland membranes and is similar to values obtained in assays measuring increases in cyclic AMP production in which the ED50 values for vasoactive intestinal peptide and peptide histidineisoleucineamide were 3.1 +/- 1.8 and 29 +/- 13 nM, respectively. Although vasoactive intestinal peptide stimulation of cyclic AMP production was only about 10% of that seen in response to isoproterenol, the levels of mucin release induced by the two agents were more similar. The ED50 for vasoactive intestinal peptide-stimulated mucin release was 0.12 +/- 0.05 nM, thus suggesting an activation anomaly in the vasoactive intestinal peptide receptor-coupled signal transduction pathway at a point between cyclic AMP production and mucin release.

Receptors for several polypeptide hormones have been demonstrated in cells of the gastrointestinal tract and pancreas. Some of these polypeptides stimulate or inhibit cell division. Therefore, these receptors have been sought in carcinomas arising from these tissues, and several examples have been reported. In some instances, stimulation of tumor growth by the corresponding ligand has been demonstrated or blocked by a receptor antagonist. In experimental carcinogenesis, the incidence of tumors has been influenced by some of the same hormones. Receptors and binding proteins for steroid hormones also have been reported in carcinomas of the pancreas. Manipulation of sex steroid hormones has yielded both inhibition and enhancement of growth of human and rat pancreatic cancers. These observations imply that trophic or growth inhibiting polypeptide and steroid hormones may serve as promoters or inhibitors of carcinogenesis in gastrointestinal organs and may influence the growth of established carcinomas. Receptor blockers may provide a clinical approach for slowing the growth of some gastrointestinal cancers.

The purpose of this investigation was to characterize the nature of peptide histidine isoleucine (PHI) and vasoactive intestinal polypeptide (VIP) receptors, and to examine the role of PHI in internal anal sphincter (IAS) relaxation. The studies were performed on opossums anesthetized with alpha-chloralose. The pressures in the IAS were recorded using continuously perfused catheters. The IAS responses to PHI analogues, PHI-27, PHM-27, PHI-(14-27)-NH2, PHI-(1-13), to VIP, to rectal balloon distention, sacral nerve stimulation, and local intramural stimulation were evaluated before and after PHI-(14-27)-NH2, PHI tachyphylaxis, and the VIP antagonists [4 Cl-D-Phe6, Leu17] VIP (VIP analogue) and (N-Ac-Tyr1, D-Phe2)-GRF(1-29)-NH2 (growth hormone releasing factor analogue). The inhibitory responses by all of the PHI analogues and VIP were not modified by tetrodotoxin. PHI-(14-27)-NH2 and PHI tachyphylaxis caused significant antagonism of the fall in internal anal sphincter pressure by PHI-27 and PHM-27 without modifying the IAS responses to VIP and rectal balloon distention, sacral nerve stimulation, and local intramural stimulation. On the other hand, VIP and growth hormone releasing factor analogues caused significant antagonism of VIP responses without modifying the responses to PHI-27. We conclude that distinct PHI and VIP receptors are present in the IAS smooth muscle and that PHI may not play a significant role in the IAS relaxation via the rectoanal reflex.

Motilins purified from porcine and canine intestine differ in their amino acid composition in positions 7-8-12-13-14. We studied in vitro the contractile response of longitudinal duodenal muscles from various animals (guinea pig, rabbit, dog) to porcine and canine synthetic motilins. Both substances failed to elicit contraction of the guinea pig duodenum but were active and equally potent on rabbit muscle. In dogs, porcine motilin was inactive at the concentrations tested (up to 10(-4) M) whereas canine motilin induced duodenal contractions in a dose-response fashion (mean dose required to induce half-maximal response: 4.82 +/- 0.25 X 10(-5) M). The contraction generated by synthetic canine motilin (10(-5) M) was not influenced by atropine, hexamethonium, tetrodotoxin, naloxone, or sodium nitroprusside (all used at 10(-4) M) but was blocked by verapamil (10(-4)). Our study shows that species-related structural alterations in motilin molecules generate different bioactive capacities in some animal species, suggests that the middle portion of the molecule is important for its bioactive expression, suggests the presence of motilin receptors on canine duodenal muscle, and suggests that an influx of extracellular calcium is involved in the canine duodenal muscle contraction elicited by canine motilin.

In order to determine the role of glucagon in cold acclimation, the changes in glucagon receptor were investigated in white adipocytes from cold-acclimated rats by establishing a glucagon radioreceptor assay method for isolated white adipocytes. The following conditions were found to be appropriate for specific glucagon receptor binding assay; cell concentration of about 1 X 10(5) cells/ml, 15 min preincubation with glucagon and 30 min-reaction at 25 degrees C in the presence of bacitracin (1 mg/ml). Cold acclimation decreased the size and increased the number of epididymal white adipocytes. Cold acclimation increased the number of glucagon receptors of white adipocytes, resulting in 140% in terms of unit cell, and 260% increase per unit surface area and 210% increase per whole tissue. However, the affinity of the binding site for glucagon was not affected. The results suggested that an enhanced metabolic response of cold-acclimated rats to glucagon could be partly explained by the increased number of glucagon receptor in white adipocytes.

1. Based on radioligand binding and adenylate cyclase activation, functional receptors to vasoactive intestinal peptide(VIP)/helodermin, were shown to coexist with beta 2-adrenoceptors and prostaglandin receptors in membranes from a cultured cloned BL/VL3 cell line of murine T-cell lymphoma induced by a radiation leukemia virus. 2. The relative potency of VIP-related peptides to stimulate adenylate cyclase activity was: helodermin greater than VIP greater than peptide histidine isoleucinamide. Five VIP analogs inhibited 125I-iodo-VIP binding and stimulated adenylate cyclase activity, their decreasing order of potency being: VIP greater than [D-Asp3]VIP greater than [D-Ser2]VIP greater than [D-Ala4]VIP = [D-His1]VIP = [D-Phe2]VIP. [D-Phe2]VIP acted as a partial agonist (with an intrinsic activity of 0.1 as compared to that of VIP = 1.0) and competitively inhibited helodermin- and VIP-stimulated adenylate cyclase activity with a similar Ki (0.07-0.10 microM). These data suggest the existence, in this murine T-cell lymphoma, of VIP receptors of the 'helodermin-preferring' subtype that are coupled to adenylate cyclase.

The effects of propylthiouracil (PTU) treatment on vasopressin, angiotensin II, glucagon and alpha 1-adrenergic receptors in both developing and adult rats were studied in liver membrane preparations by measuring the binding of the following ligands: [3H][8-lysine]vasopressin, [3H]Sar-angiotensin II, [125I]glucagon and [3H]prazosin, and in the case of glucagon, by measuring adenylate cyclase activation. Whatever the ligand used, in young as well as in adult animals, PTU treatment led to a similar reduction (about 50%) in the maximal number of binding sites (Bmax), without significant changes in the apparent dissociation constant (KD) of labeled hormone for its specific receptor. In normal adult animals, thyroxine treatment, i.e. hyperthyroidism, had an opposite effect on the Bmax (25-50% increase), without changes in the KD. In developing PTU-treated rats, the abnormalities completely disappeared after therapy with increasing physiological doses of thyroxine; consequently they were directly related to thyroid deficiency and not to toxic effects of PTU. Moreover, the abnormalities resulting from induced hypothyroidism were reversible. In developing and adult hypothyroid rats, neither basal, NaF-, nor Gpp(NH)p-stimulated adenylate cyclase activities were significantly affected. Glucagon-sensitive adenylate cyclase activity seemed to be slightly increased (by about 15%), without changes in the apparent activation constant (Kact). These results are considered in parallel with findings on plasmatic glucagon and vasopressin levels, compared with similar previous reports related to renal vasopressin receptors, and discussed with respect to unpublished observations concerning hepatic responsiveness to glycogenolytic hormones in young and adult rats with induced hypothyroidism.

At the maximally effective concentration of 10 nM, VIP induced a marked (12.5-fold stimulation above basal), and sustained increase in short circuit current in the human intestinal epithelial cell line Cl.19A grown on permeable filters and placed in Ussing chambers. Half-maximal increase of Isc was observed for 0.1 nM VIP. This was well correlated with the VIP-stimulated adenylate cyclase activity (ED50:0.07 nM). Binding studies using 125I-VIP indicated that Cl.19A cells express a peptide-specific VIP receptor with a dissociation constant of 0.07 nM. Covalent labeling of receptors followed by SDS-PAGE analysis of membrane proteins resulted in the identification of a 63,000 dalton binding protein in Cl.19A cells.

Retinoic acid (RA) induces partial differentiation of neuroblastoma (NB) cells in vitro. In the human NB line, SH-SY5Y (a neuroblastic subclone of SK-N-SH), RA was previously shown to enhance the stimulatory (PGE1) and inhibitory (opioid) regulation of adenylyl cyclase. Since these cells are also sensitive to cAMP stimulation by vasoactive intestinal peptide (VIP), we have tested the effects of RA on VIP receptor expression and function. Pretreatment of SH-SY5Y cells with 10 microM RA over 6 days dramatically increased VIP receptor number from approximately 3,000 to approximately 70,000 sites per cell and enhanced threefold the cAMP accumulation after external VIP addition, while VIP immunoreactive content in the cells increased 2-3-fold. In the light of the recently proposed autocrine function of VIP in this cell lineage, the strong enhancement of the VIP system may contribute to the differentiation effects of RA.

A bovine milk diet (BM) resulted in remarkable changes in histamine H2 receptor activity (sensitization) and PGE2 receptor activity (desensitization) in gastric glands isolated from adult rats. In contrast, the receptor-cAMP systems sensitive to glucagon(s) and secretin in parietal cells and muco-peptic cells were unaffected. In the two experimental groups, cimetidine produced a parallel displacement of the histamine dose-response curve suggesting competitive inhibition between this classical H2 receptor antagonist and histamine. The BM diet reduced the histidine decarboxylase activity in rat gastric mucosa; the histamine content was not significantly different in control and BM-fed rats. There was no alteration of the circadian rhythm of the parietal cell (ultrastructural changes: microvilli, tubulo-vesicles) determined at intervals of 6 hours in milk-fed rats. Prostaglandins and other components in milk (EGF, somatostatin, etc.) might therefore protect gastric mucosa by a differential control of PGE2 and histamine H2 receptor activity, either directly (PGE2 and EGF in milk) or indirectly (inhibition of endogeneous histamine synthesis/release and stimulation of prostaglandin synthesis/release).

Guanine nucleotide and Mg2+ ion regulation of [125I-Tyr10]monoiodoglucagon ([125I]MIG) binding to liver plasma membranes from chicken, rat, and rabbit was studied. It was found that [125I]MIG binding to chicken liver membranes was increased by the addition of Mg2+ ion, while binding to rat and rabbit liver membranes was unaffected. In the chicken liver membranes, the Mg2+ ion induced high affinity binding which was sensitive to guanine nucleotides, while the low affinity binding in the absence of Mg2+ ion was not. Maximal effects of Mg2+ ion were observed at 1 mM. Glucagon binding to rat liver membrane receptors was GTP sensitive regardless of whether Mg2+ ion was added. Glucagon binding to rabbit liver membranes was insensitive to both Mg2+ ions and GTP. This lack of GTP effect was not due to degradation of GTP; no effect of the nonhydrolyzable analog guanyl-5'-yl-imidodiphosphate was observable. Glucagon stimulation of rabbit liver adenylyl cyclase, however, was dependent on GTP, as was the case with all of the other liver adenylyl cyclases studied here. The Kact of GTP for the rabbit liver system was very similar to that for rat liver membranes. The glucagon receptor was covalently labeled with [125I]MIG using p-hydroxysuccinimidyl azidobenzoate and analyzed by sodium dodecyl sulfate-gel electrophoresis. In all cases, a major labeled band at 63,000 daltons was observed. The levels of glucagon receptor and stimulatory (Ns) and inhibitory (Ni) regulatory proteins of adenylyl cyclase were measured. The highest levels of glucagon receptor were measured in rat liver membranes, while the levels in chicken and rabbit membranes were 30-40% lower. Rabbit liver membrane had the highest levels of Ns, while rat liver membranes had 2-fold lower and chick liver membrane 4-fold lower levels than rabbit liver membranes. The levels of Ni was similar in the three systems. Thus, the ratio of Ns to glucagon receptor was highest in the rabbit. In the rat, this ratio was 3-fold lower than that in the rabbit. In the chicken membranes, the ratio was about 60% of that in the rat. These data suggest that the observed differences in effects of GTP on hormone binding can be explained by alterations in the ratio of the receptor and Ns proteins among the various species.

Nervous control of gastrointestinal motility is extremely complex, is regulated by the enteric system, the "brain of the gut", and modulated by extrinsic nerves. This system with its multiplicity of transmitters and receptors does not always allow a clear interpretation of experimental data, especially with compounds lacking specificity. In this review the complex situation is described particularly in relation to receptor populations (cholinergic, adrenergic, dopamine, histamine, 5-hydroxytryptamine, opioid, gamma-aminobutyric acid (GABA), prostanoid and dihydropyridine receptors), therapeutic aspects of drugs and their usefulness in children. Newer principles with known drugs and promising new compounds with a more appropriate kinetic or fewer side-effects, deriving from distinct pharmacological groups, as candidates for the treatment of gastrointestinal disorders are considered e.g. anticholinergics (prifinium or actilonium bromide), adrenergic alpha 2-agonists (clonidine, lidamidine) for diarrhoea in diabetic neuropathy, adrenergic beta-blockers for shortening postoperative ileus (propranolol), dopamine receptor antagonists (metoclopramide, domperidone, alizapride) and another prokinetic substance (cisapride) which may be useful for a number of applications as gastro-oesophageal reflux, gastro-paresis, intestinal pseudo-obstruction, cystic fibrosis and constipation, morphine derivatives (e.g. loperamide) for intractable diarrhoea and calcium antagonists (e.g. nifedipine) for achalasia. Increasing experience in digestive tract pharmacology and reliable clinical studies will furthermore be the basis for a more specific and better tolerated therapy of gastrointestinal motility disorders in adults and children.

Membrane currents were recorded from voltage-clamped Xenopus laevis oocytes, surrounded by their enveloping follicular and epithelial cells. Porcine vasoactive intestinal peptide (VIP) generated a membrane current due to an increase in membrane conductance to K+. The VIP current was mimicked by the adenylate cyclase activator forskolin and was potentiated by phosphodiesterase inhibitors, suggesting that adenosine 3',5'-cyclic monophosphate (cyclic AMP) plays a role in mediating the response. Though resembling the follicle's responses to catecholamines and adenosine in ionic basis and apparent mechanism, the response to VIP was not blocked by catecholaminergic or purinergic antagonists, indicating the presence of a specific VIP receptor in the follicle. Among the VIP related peptides, PHM-27 generated similar but smaller K+ currents and porcine secretin and glucagon neither elicited a response nor blocked that to VIP. After treating follicles with collagenase to remove the epithelial and follicular cells the responses to VIP were either substantially reduced or abolished, suggesting that the VIP receptors and K+ channels are both located in the follicular cells.

Incubation of human SUP T1 lymphoblasts with VIP, helodermin and related peptides induced homologous desensitization within 5 min as indicated by: 1) a secondary decrease in cellular cyclic AMP levels, even in the presence of phosphodiesterase inhibitors, 2) a reduced capacity of cells to bind [125I]helodermin, 3) decreased helodermin stimulation of adenylate cyclase activity in membranes, and 4) unaffected NaF- and Gpp[NH]p-stimulated adenylate cyclase activities. The desensitizing ability of all peptides correlated with their efficacy to occupy cell receptors, except for [D-Phe2]VIP, a partial VIP agonist with low intrinsic activity, that did not desensitize.

Electrophysiological studies have been made of the actions of vasoactive intestinal peptide (VIP) and angiotensin II (Ang II) on astrocytes in explant cultures of rat brain stem and spinal cord. Addition of VIP to the bathing solution at concentrations of 10(-8) to 10(-6) M caused hyperpolarizations. Ang II at the same concentrations depolarized the majority of astrocytes tested. At 10(-9) M, both peptides had no action or only small effects. In approximately one third of the cells, the depolarizations by Ang II were accompanied by rhythmic oscillations. The Ang II antagonist saralasin reversibly blocked the depolarizations by Ang II. These findings, together with autoradiographic binding studies from our laboratory strongly suggest the existence of VIP and Ang II receptors on astrocytes.

The time course of recovery of beta-adrenergic and VIP/helodermin receptors after homologous and heterologous down regulation was studied in the murine lymphoma cell line BL/VL3, a neoplastic equivalent of immature T cells. The heterologous part of isoproterenol down regulation was rapidly reversed, even in the presence of cycloheximide, suggesting that down regulation was linked to ligand-receptor interaction and/or cyclic AMP increase. Homologous down regulations of beta-adrenoceptors and VIP/helodermin receptors were less rapidly reversible and depended on protein synthesis as they were inhibited by cycloheximide: beta-adrenoceptors recovered faster than VIP/helodermin receptors.

It has been suggested that vasoactive intestinal polypeptide (VIP) has a role as a neurotransmitter and that it participates in the control of hormone release from thyroid follicles. Many hormones and neurotransmitters alter the ionic permeability properties of the plasma membrane of their target cells when they bind to their receptors. Concentrations of VIP able to elicit complex time-dependent changes in rat thyroidal cyclic nucleotide levels did not affect follicular cell membrane potential or input resistance. The suggestion is made that the findings may indicate that Ca2+ is not involved in the initial stages of stimulus-secretion coupling following VIP binding to its receptor on the follicular cell.

The processing of glucagon by circulating human mononuclear cells was examined. Glucagon bound to the membrane with a turnover time of 4.4 minutes per site after 15 minutes of incubation and 8 minutes per site after 90 minutes. The amount of intact intracellular hormone increased by 3-fold by 90 minutes suggesting a slowing of intracellular processing with prolonged incubation. Excess unlabelled insulin also slowed the processing of glucagon at the degradative step with no effect on binding or internalization of glucagon. Subcellular fractionation of the cells showed that most hormone accumulated in the 500xg pellet and in the 100,000xg supernatant. N-ethylmaleimide blocked intracellular glucagon degradation suggesting a role for intracellular sulfhydryl-dependent enzymes. Kinetic analysis of the dissociation of glucagon revealed a second order process with K values of 2.2 X 10(-2) fm-1 min-1 and 1.4 X 10(-2) fm-1 min-1 for dissociation from membranes and from membranes + intracellular sites, respectively. T 1/2 values were 6 min. for membrane dissociation and 9 min for membranes + cells. These findings suggest that glucagon interaction with mononuclear cells has characteristics similar to other receptor bound ligands including internalization processing and metabolism.

Specific, high affinity receptors for vasoactive intestinal peptide (VIP) have been identified on a human pre-B cell line, Nalm 6, and on a human plasma cell line, Dakiki. The single class of high affinity sites exhibited a KD of 12.6 +/- 2.9 nM for VIP in Nalm 6 cells and 9.1 +/- 2.7 nM in Dakiki plasma cells. The homologous peptides, peptide histidine methionine (PHM), growth hormone releasing factor (GHRF), and secretin were all less effective than VIP in competitively inhibiting binding of 125I-VIP to Nalm 6 and Dakiki plasma membranes. The putative receptor was characterized as a 47-kDa protein using covalent cross-linking techniques and VIP stimulated adenylate cyclase in pre-B cells. Human lymphocytes of B cell lineage thus appear to express functional VIP receptors homologous to the receptor identified in T lymphoblasts, brain, pituitary, and intestine.

This review focusses on the roles that membrane receptors and their transducers play in the physiology and pathology of the gastrointestinal tract. The multifactorial regulation of [correction] mucosal growth and function is discussed in relation to the heterogeneity of exocrine and endocrine populations that originate from progenitor cells in stomach and intestine.

Desensitization to the hemodynamic effects of vasoactive intestinal polypeptide (VIP) was examined in 12 anesthetized, open-chest dogs in which cardiac output, systemic arterial resistance, and heart rate were fixed. VIP was administered by intracoronary infusion, and the effects were compared with isoproterenol and forskolin. Measurements of left ventricular maximum rate of pressure development (dP/dt), coronary blood flow, and myocardial oxygen consumption were made before and after a 90-min infusion of either isoproterenol (6 dogs) or VIP (6 dogs). After isoproterenol infusion, there was a significant decrease in the effect of isoproterenol on left ventricular dP/dt and coronary blood flow. The effects of VIP and forskolin were not changed. After VIP infusion, there was a significant decrease in the effect of VIP on left ventricular dP/dt with no change in the effects of isoproterenol and forskolin. In this group, a significant increase in coronary blood flow with minimal change in myocardial oxygen could still be elicited by VIP injection after VIP infusion. The agonist infusion time to achieve a decrease in inotropic effect was less for VIP when compared with isoproterenol. Thus these data demonstrate acute homologous desensitization of myocardial VIP and beta-adrenergic receptors in canine myocardium with no development of heterologous desensitization, desensitization involving the catalytic subunit of adenylate cyclase, or desensitization of the VIP-mediated primary coronary vasodilator response.

This study describes functional characteristics of receptors for vasoactive intestinal peptide (VIP) on human Ewing's sarcoma WE-68 cells. These characteristics include 125I-VIP binding capacity, cellular cAMP generation, glycogen hydrolysis, and pharmacological specificity. Binding studies with 125I-VIP showed specific, saturable, binding sites for VIP in WE-68 cells. Scatchard analysis revealed the presence of a single class of high-affinity binding sites that exhibited a dissociation constant (Kd) of 90 pM and a maximal binding capacity (Bmax) of 24 fmol/mg of protein. VIP and VIP-related peptides competed for 125I-VIP binding in the following order of potency: human (h) VIP greater than human peptide with N-terminal histidine and C-terminal methionine (PHM) greater than chicken secretin much greater than porcine secretin. Glucagon and the C-terminal fragments VIP[10-28] and VIP[16-28] and the VIP analogue (D-Phe2)VIP did not inhibit 125I-VIP binding. Addition of hVIP to WE-68 cells provoked marked stimulation of cAMP accumulation, hVIP stimulated increases in cAMP content were rapid, concentration-dependent, and potentiated by 3-isobutyl-l-methylxanthine (IBMX). Half-maximal stimulation (EC50) occurred at 150 nM hVIP. The ability of hVIP and analogues to stimulate cAMP generation paralleled their potencies in displacing 125I-VIP binding. (D-Phe2)VIP, VIP[10-28], VIP[16-28], and (p-Cl-D-Phe6, Leu17)VIP, a putative VIP receptor antagonist, affected neither basal cAMP levels nor hVIP-induced cAMP accumulation. WE-68 cell responses to hVIP were desensitized by prior exposure to hVIP. Desensitization to hVIP did not modify the cAMP response to beta-adrenergic stimulation, and beta-adrenergic agonist desensitization did not modify responses to hVIP. hVIP also induced a time- and concentration-dependent hydrolysis of 3H-glycogen newly formed from 3H-glucose in WE-68 cultures. hVIP maximally decreased 3H-glycogen content by 36% with an EC50 value of about 8 nM. The order of potency of structurally related peptides of hVIP for stimulation of glycogenolysis correlated with their order of potency for inhibition of 125I-VIP binding. IBMX potentiated the glycogenolytic action of hVIP and PHM. The simultaneous presence of the calcium channel antagonist verapamil or the calcium ionophore A 23187 did not influence the glycogenolytic and cAMP stimulatory effects of hVIP. Collectively, these data indicate that Ewing's sarcoma (WE-68) cells are endowed with genuine VIP receptors which are coupled to the formation of cAMP that probably serves a second messenger role in stimulating glycogen hydrolysis in these cells in response to VIP.(ABSTRACT TRUNCATED AT 400 WORDS)

The ability of members of the secretin-glucagon family of peptides to modulate the responses of mouse lymphoid cells stimulated with Concanavalin A (Con A), Lipopolysaccharide (LPS) and alloantigens was determined. It was observed that vasoactive intestinal peptide (VIP) and peptide having NH2-terminal histidine and COOH-terminal Isoleucine (PHI) inhibited the incorporation of 3H-methyl-thymidine by cells stimulated with Con A (55% inhibition) or alloantigen-bearing cells (40% inhibition). Secretin was approximately 10,000 less effective as an immunomodulator. Other members of the neuropeptide family, including glucagon and gastric inhibitory peptide, were ineffective in affecting mitogenesis elicited by Con A (20% inhibition). Lipopolysaccharide stimulated spleen cells were refractory to modulation by all members of the secretin-glucagon family of peptides (less than 5% modulation). The inhibition measured was concentration dependent over the range of 10(-6) to 10(-16) M. A peptide fragment of VIP encompassing amino acid residues 10-28, although capable of modulating in vitro responses, was 30-50% less effective than intact VIP. In addition, a VIP specific binding assay for mouse lymphoid cells was described. The binding of 125I-VIP to lymph node cells was rapid, saturable and reversible. Apparent equilibrium was reached within 15 minutes and nonspecific binding, measured as 125I-VIP binding in the presence of an excess (2 x 10(-7) M) of native VIP, did not exceed 25% of the total binding. In competitive experiments using VIP related peptides, PHI but not gastric inhibitory peptide, glucagon or secretin was able to significantly inhibit 125I-VIP binding. PHI had only one-eighth of the competitive capacity of native VIP. Scatchard analyses indicated the existence of a single class of high affinity receptors on lymph node cells (KD = 3.46 nM; 26,000 sites/cell). 125I-VIP specific binding to purified T cells (14%) was markedly higher than to B cells (3% binding). Thymocytes bound less than 2% of the label and had relatively few VIP binding sites (8,000) as compared with purified T cells (45,000 sites/cell). There was variability in the ability of various T cells tumors and functional T cell clones to bind 125I-VIP. The role of VIP as a physiological modulator of T cell activation is discussed.

Neuropeptides have recently been shown to modulate the immune response. Vasoactive intestinal polypeptide (VIP) modulates lymphocyte migration to Peyer's patches and inhibits natural killer cell activity. VIP receptors have been characterized on lymphocytes and have been compared with the VIP receptor in nervous tissue and in tissues of the gastrointestinal tract. The evidence supports the existence of at least two classes of high-affinity VIP receptors as well as a low-affinity receptor. The development of tissue-specific agonists and antagonists to the VIP receptor may thus be feasible.

Vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) are homologous neuropeptides which share vasodilatory properties. This paper addresses the question of whether PHI exerts its vascular action via a receptor distinct from that for VIP. Radioligand binding experiments were done using [Tyr(125I)10]VIP, [Tyr(125I)22]porcine PHI, [Tyr(125I)10]rat PHI and arterial preparations from rat, bovine and porcine species. The radioiodination of rat PHI by the lactoperoxidase-glucose oxidase method and analysis of the structure of the major radiolabeled derivatives were described. All the receptor binding experiments identified a VIP-preferring receptor irrespective of which radioligand or arterial preparation was utilized. VIP and PHI peptides demonstrated cross-desensitization in studies of relaxation of porcine coronary arterial strips in vitro. The present results favor the conclusion that the vascular actions of the PHI peptides are best explained by binding to a VIP-preferring receptor.

We studied the functional properties of hepatic glucagon receptors during rat development. Glucagon binding to liver membranes and isolated hepatocytes was significantly less in foetuses and weaning rats than in adult animals, reflecting changes in the number of receptors rather than any change in receptor affinity for the hormone. After correcting for the smaller surface area of foetal hepatocytes, glucagon receptor concentrations were still lower in foetuses than in adult rats. The time courses of glucagon association to liver membranes and of glucagon receptor degradation of prenatal and postnatal rats were similar, while inactivation of glucagon by liver membranes was significantly lower in foetal than in adult rats. Also, glucagon-stimulated production of cAMP was smaller in younger rats. These findings suggest that, according to several criteria, glucagon receptors in the foetus are functionally normal and their delayed development may be important for the metabolic processes occurring during the perinatal age.

1. The involvement of second messengers and of other chemical mediators, in the modulation of the membrane potential of the Schwann cell of the giant nerve fiber of the Tropical squid Sepioteuthis sepioidea is described. 2. The involvement of the cyclic nucleotide adenosine 3', 5' monophosphate (cAMP) in mediating the actions of the nicotinic Ach receptors of the Schwann cells is suggested. 3. The presence of octopaminergic receptors in the Schwann cells, mediating their actions through the activation of adenylate cyclase, is also described. 3. Receptors for vasoactive intestinal peptide (VIP) are also present on the Schwann cells, and their actions are mediated via a second messenger system that does not involve the activation of adenylate cyclase. 5. The three independent receptor systems referred above are able to interact in a complex way, which involves both their direct actions on the Schwann cell membrane potential and modulatory effects between the systems.

Vasoactive intestinal peptide (VIP) is a neuropeptide with a broad range of biological activities in various tissues. After interaction with its membrane receptor, VIP generally induces a very large increase in the intracellular cyclic AMP level. Receptors for VIP have been described in numerous tissues and cell lines. The first results on VIP receptor structure have been obtained by covalent cross-linking using bifunctional reagents. The molecular mass of the different components characterized in this way differs greatly according to the species and the tissue used. This heterogeneity may reflect either a difference in the length of the cross-linked polypeptide backbone or differently glycosylated forms of the same polypeptide. The VIP binding site of intact human adenocarcinoma cells (HT29 cells) is an Mr 64,000 glycoprotein with 20kDa of N-linked oligosaccharide side chains containing sialic acid. The structure of the VIP binding site from HT29 cell is compared, first to the structure of the VIP receptor from other tissues, particularly that from rat liver, and second to the structure of the hepatic glucagon binding site. Recently, solubilization of the VIP receptor in an active form has provided a new way of studying this receptor. The HT29 cell line is an appropriate model to study the dynamics of the VIP receptor. After binding to its receptor, VIP is rapidly internalized, probably by receptor-mediated endocytosis. This internalization leads to a decrease in the cell surface receptor number and simultaneously to a homologous desensitization of adenylate cyclase. VIP is then degraded in the lysosomes, while most of the receptors are recycled back to the cell surface. The presence of an intracellular pool of unoccupied VIP receptors has been demonstrated after inactivation of the cell surface receptors by chymotrypsin. The kinetics of the receptor reappearance at the cell surface, after inactivation by chymotrypsin or after receptor-mediated endocytosis, indicate 2 possible intracellular pathways for occupied and unoccupied VIP receptors.

In purified rat pancreatic plasma membranes, (D-Phe4)PHI interacts as a selective VIP agonist for rat pancreatic VIP-preferring receptors, based on binding selectivity and adenylate cyclase activation, therefore allowing us to discriminate between the participation of VIP-preferring and secretin-preferring receptors in VIP stimulation. VIP-preferring receptors also bind GRF. They rely on disulfide bridges for their functional integrity. Their coupling with adenylate cyclase, based on the intrinsic activity of VIP analogues, is poor when compared to that of hepatic VIP receptors. In fresh rat liver plasma membranes, high-affinity VIP receptors are specifically labeled with [125I]helodermin and [125I]His1, D-Ala NLeu27)GRF and are well coupled to adenylate cyclase while low-affinity VIP receptors are not. The first subtype of VIP receptors is highly responsive to guanyl nucleotides and is easily altered by dithiothreitol. Only after freezing and thawing are low-affinity hepatic VIP receptors coupled to adenylate cyclase. Concerning the chemical characterization of VIP receptors, 66- and 35-kDa peptides are detected after specific [125I]VIP cross-linking with double agents in rat pancreatic membranes. In contrast, in intact pancreatic acini, the main source of radioactivity has a molecular mass of 130-180 kDa (with no contribution of intramolecular disulfide bridges), and an 80-kDa peptide is also detectable. The 66-kDa species in membranes can conceivably derive from the 80-kDa species observed in intact cells. Its molecular mass is higher than that of the 56-kDa [125I]VIP cross-linked protein previously observed in rat liver membranes. Besides, species differences between rat and guinea pig pancreas are also evident.

Erythromycin A (EMA) is a potent stimulator of gastrointestinal motor activity. In vitro studies suggest that it mimics motilin, a peptide that stimulates motor activity in human and in rabbit via smooth muscle receptors. We have compared the in vitro contractile effect of EMA and two derivatives, 8,9-anhydro-EMA-6,9-hemiketal (EM201) and EMA N-oxide, on rabbit duodenal smooth-muscle strips with their ability to displace iodinated motilin bound to crude smooth-muscle membrane fractions. The concentrations required to induce 50% of the maximum contractile response to a supramaximal dose of acetylcholine were 5.0 x 10(-8), 2.0 x 10(-6), and 1.0 x 10(-4) M for, respectively, EM201, EMA, and EMA N-oxide. The concentrations required to displace 50% of the labeled motilin were, in the same order, 1.0 x 10(-8), 1.3 x 10(-7), and 4.0 x 10(-6) M. Both parameters were well correlated. The dose-response curve of the EMA was parallel to that of motilin and the effects of motilin and EMA were additive. Contractions induced by EMA were insensitive to pretreatment with tetrodotoxin or atropine. EMA had no effect on muscle strips of rat or dog duodenum but did induce contractions in human strips. EMA was totally ineffective on ileal preparations, which are also unresponsive to motilin and in which motilin binding is absent. EMA has therefore the same regional and species specificity as motilin. We conclude that EMA is a motilin receptor agonist.

When dispersed acini from guinea pig pancreas are first incubated with carbachol, the subsequent binding of 125I-vasoactive intestinal peptide (VIP) is inhibited during a second incubation. This inhibitory action of carbachol on binding of 125I-VIP depends on time, temperature, and the concentration of carbachol in the first incubation and can be blocked by atropine. First incubating acini with A23187, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), cholecystokinin octapeptide, bombesin, or 12-O-tetradecanoylphorbol-13-acetate does not alter binding of 125I-VIP. Adding EGTA to the first incubation medium abolishes the effect of carbachol on binding of 125I-VIP. In control acini or acini first incubated with carbachol, approximately half of the bound 125I-VIP can be stripped by acetic acid. 125I-VIP interacts with two distinct classes of receptors on pancreatic acini. One has a high affinity for VIP (Kd, 1 nM); the other has a low affinity for VIP (Kd, 2 microM). First incubating acini with carbachol decreases the number but not the affinity of high-affinity VIP receptors with no change in the number or affinity of low-affinity VIP receptors. Pancreatic acini possess two classes of muscarinic cholinergic receptors: one has a high affinity (Kd, 4 microM) and the other has a low affinity (Kd, 698 microM) for carbachol. The dose-response curve for carbachol-induced inhibition of binding of 125I-VIP and that for occupation of low-affinity muscarinic cholinergic receptors by carbachol are similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Using isolated submucosal glands from feline trachea, we examined the effect of vasoactive intestinal peptide (VIP) on mucus glycoprotein secretion and glandular contraction by measuring released radiolabeled glycoconjugates and induced tension, respectively. VIP (10(-10) to 10(-6) M) produced a dose-dependent increase in [3H]glycoconjugate release of up to 300% of controls, which was inhibited by VIP antiserum and not inhibited by atropine, propranolol, or phentolamine. VIP at a low concentration (10(-9) M), which did not produce any significant increases over controls, produced a 2.4- to 5-fold augmentation of the glycoconjugate release induced by 10(-9) to 10(-7) M methacholine (MCh). Atropine or VIP antiserum abolished the augmentation. VIP did not produce any alteration in isoproterenol- or phenylephrine-evoked glycoconjugate secretion. VIP (up to 10(-5) M) did not produce any alteration in the tension, even when the gland had contracted with MCh, or any augmentation of contraction induced by MCh (10(-9) to 10(-7) M). These results indicate that VIP induces mucus glycoprotein release from secretory cells and also that it potentiates the secretion induced by cholinergic stimulation.

1. The receptors for vasoactive intestinal peptide (VIP) present in dispersed acini and membranes from rat and guinea-pig pancreas differed in selectivity pattern, i.e. in the displacement of [125I]iodo-VIP by parent peptides, as revealed by a VIP:secretin IC50 ratio at least ten times higher in rat than in guinea-pig preparations. The molecular properties of these VIP receptors were therefore investigated. 2. When comparing six succinimidyl ester cross-linkers, bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone proved to be the most universal [125I]iodo-VIP cross-linker for all pancreatic preparations. 3. In intact rat acini the main labeled peptide had an Mr of 80,000, whereas the main labeled peptide in intact guinea-pig acini was a smear of Mr 160,000. In both rat and guinea-pig pancreatic membranes, the main labeled peptide ([125I]iodo-VIP-binding-protein complex) had an Mr of 66,000. In addition, variable proportions of an Mr-80,000 peptide and an Mr-83,000 peptide were visualized in, respectively, rat and guinea-pig membranes. The labeling of all peptides was suppressed by VIP and by GTP. Reducing conditions allowed only a better resolution, making the presence of intermolecular disulfide bridges unlikely. 4. Taking into account the Mr of VIP it is thus plausible that the main native Mr-77,000 VIP-binding site present in rat acini could be easily converted to an Mr-63,000 peptide during membrane preparation, while in guinea-pig acini Mr-80,000 and/or Mr-63,000 VIP-binding sites were often closely associated with another membrane component in the native state. These molecular differences between VIP receptors in intact rat and guinea-pig acini are in keeping with functional differences.