Identification of key residues for the binding of glucagon to the N-terminal domain of its receptor: an alanine scan and modeling study.

Glucagon plays an essential role in the glycemia maintenance during fasting, but also aggravates hyperglycemia in diabetic patients. A series of analogues of glucagon were synthesized replacing each amino acid of the C-terminal region (residues 15-29) with alanine. The residues affecting the binding to the glucagon receptor are found to be located on one face of the glucagon helix. Several 3-dimensional models of the N-terminal domain of the glucagon receptor in complex with its ligand peptide were built and used to analyze the peptide-receptor interface in terms of the nature of the peptide residues and the interactions they form with the receptor. The models suggest that glucagon keeps its native helical structure upon binding, and that a large part of the interface formed with the receptor is hydrophobic. We find that in the C-terminal region, F22, V23, M27, and D15 are the most important residues for peptide binding. They bury a large portion of their solvent accessible surface area and make numerous interactions with the receptor mainly of the hydrophobic type.

Vasoactive intestinal peptide (VIP) stimulates [Ca2+]i and cyclic AMPin CHO cells expressing Galpha16.

The stimulatory effect of vasoactive intestinal peptide (VIP) and analogues on [Ca2+]i has been investigated in chinese hamster ovary (CHO) cells stably transfected with the reporter gene aequorin, and expressing either the human VPAC1or VPAC2 receptor in absence or in presence of the Galpha16. In cells that were not transfected with Galpha16 and expressed a similar density of receptors, the VIP induced [Ca2+]i ncrease was higher in VPAC1 than in VPAC2 receptor expressing cells. In aequorin/Galpha16 cotransfected cells, the VIP-induced response was higher, reaching 70 to 80% of the maximal calcium response, obtained after digitonin treatment, in response to both VPAC1 and VPAC2 receptor stimulation. The results suggest that in hematopoietic cells, which express both VIP receptors and Galpha16, the signalling pathway of VIP could be mediated through both cyclic AMP and [Ca2+]i increase.

Pituitary adenylate cyclase-activating polypeptide receptors of types I and II and glucagon-like peptide-I receptors are expressed in the rat medullary carcinoma of the thyroid cell line 6/23.

The expression of the messenger RNAs coding for glucagon-like peptide-I (GLP-I) receptor, VIP receptor, and pituitary adenylate cyclase-activating polypeptide (PACAP) receptor as well as the expression of the receptor proteins were demonstrated in the rat medullary carcinoma of thyroid cell line 6/23 by the following experiments: 1) RNA extraction, reverse transcriptase, and polymerase chain reaction with specific primers; 2) binding of the radiolabeled ligands [125I]GLP-I-(7-36)-NH2, [125I]PACAP-(1-27), and [125I]VIP and inhibition by, respectively, unlabeled GLP-I-(7-36)-NH2, PACAP-(1-27), and VIP; and 3) study of adenylate cyclase activation by the peptides and selective inhibition of the VIP/PACAP response by the antagonist [D-Phe2]VIP. Besides the highly selective GLP-I receptor, PACAP receptors of types I and II were present on the cell line and coupled to adenylate cyclase. PACAP stimulated the adenylate cyclase through type I and II receptors, whereas VIP interacted with type II receptors only. Messenger RNA analysis indicated that at least three splice variants of the PACAP type I receptor may be expressed in 6/23 cells.

Receptors for pituitary adenylate cyclase activating peptides in human pituitary adenomas.

The presence of pituitary adenylate cyclase activating polypeptide (PACAP) receptors coupled to adenylate cyclase was investigated in four types of human pituitary adenomas: three null adenomas and five gonadotropin-, three ACTH-, four GH-, and four PRL-producing adenomas. In all samples, except in prolactinomas, PACAP(1-27) and PACAP(1-38) stimulated adenylate cyclase activity equally well and potently (K(act) around 3 nmol). Vasoactive intestinal polypeptide (VIP) was systematically 100- to 300-fold less potent than both PACAPs. In prolactinomas, PACAP(1-27), PACAP(1-38), and VIP were inactive despite a response of the enzyme to guanosine 5'-triphosphate, Gpp(NH)p, forskolin, and fluoride. [125I-AcHis1]PACAP(1-27) binding was detected in all samples except in prolactinomas. In addition, a detailed analysis of receptors was feasible in all five gonadotropin- and in two ACTH-producing adenomas, confirming the existence of selective PACAP receptors that recognized PACAP(1-27) and PACAP(1-38) with similar high affinity (IC50 0.8-1.5 nmol) and VIP with a low affinity (IC50 100 nmol/L).

Pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide receptor subtypes are differently expressed in rat transplanted pituitary tumours (SMtTW) and in the normal gland.

The expression of the pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide (PACAP/VIP) receptor subtypes was evaluated in the normal rat pituitary gland and in different rat spontaneous transplantable SMtTW tumours (SMtTW2 which expresses prolactin (PRL), SMtTW10 which expresses GH and SMtTW3 which expresses both PRL and GH) by measurement of PACAP/VIP-stimulated adenylate cyclase activity and detection of the presence of mRNA coding for the different receptor forms. In normal glands, the order of potency of the peptides suggested that adenylate cyclase activity was mediated through interaction with PACAP selective receptors (PACAP I receptors); mRNAs coding for the PACAP I receptor, but also for the PACAP II VIP2 receptor, were detected. In SMtTW2 tumours, the functional response was close to that observed in the presence of PACAP II VIP2 receptors; mRNAs coding for PACAP I and PACAP II VIP1 and PACAP II VIP2 receptors were detected. In the SMtTW10 tumours, the functional response was complex but compatible with the involvement of PACAP I and PACAP II receptors; mRNAs coding for the PACAP I and PACAP II VIP1 receptors were detected. In the SMtTW3 tumour, the profile was similar to that of the normal pituitary gland and the mRNA coding for the PACAP I receptor only was detected. Thus, while the control of normal pituitary gland adenylate cyclase activity by PACAP and VIP was mediated by PACAP-selective receptors, in spontaneous transplantable tumours a variable profile was observed and PACAP, as well as VIP1 and VIP2 receptors, may contribute to the responses.

Lysine 195 and aspartate 196 in the first extracellular loop of the VPAC1 receptor are essential for high affinity binding of agonists but not of antagonists.

The role in ligand recognition and receptor activation of two adjacent charged residues (lysine 195 and aspartate 196) in the first extracellular loop of the human VPAC(1) receptor was investigated in stably transfected CHO cells expressing the wild type or point mutated receptors.Replacement of lysine 195 by glutamine or of aspartate 196 by asparagine reduced the agonists' ability to stimulate adenylate cyclase activity; VIP behaved like a partial agonist and a partial agonist behaved as an antagonist. The receptor's capacity to recognize agonists was reduced but antagonists' affinity was unaffected. Both results suggesting that the two charged residues are essential for VPAC(1) receptor activation. On the other hand, the double mutant was less severely affected than single mutants suggesting that hydrogen bonds may partially compensate the loss of charged residues. But the inversion of the residues affected receptor recognition and activation more markedly suggesting that the two charged residues do not interact directly.

Mutational analysis of the human vasoactive intestinal peptide receptor subtype VPAC(2): role of basic residues in the second transmembrane helix.

1. We investigated the role of two conserved basic residues in the second transmembrane helix arginine 172 (R172) and lysine 179 (K179) of the VPAC(2) receptor. 2. Vasoactive intestinal polypeptide (VIP) activated VPAC(2) receptors with an EC(50) value of 7 nM, as compared to 150, 190 and 4000 nM at R172L, R172Q and K179Q-VPAC(2) receptors, respectively. It was inactive at K179I mutated VPAC(2) receptors. These results suggested that both basic residues were probably implicated in receptor recognition and activation. 3. The VPAC(2)-selective VIP analogue, [hexanoyl-His(1)]-VIP (C(6)-VIP), had a higher affinity and efficacy as compared to VIP at the mutated receptors. 4. VIP, Asn(3)-VIP and Gln(3)-VIP activated adenylate cyclase through R172Q receptors with EC(50) values of 190, 2 and 2 nM, respectively, and through R172L receptors with EC(50) values of 150, 12 and 8 nM, respectively. Asn(3)-VIP and Gln(3)-VIP behaved as partial agonists at the wild type receptor, with E(max) values (in per cent of VIP) of 75 and 52%, respectively. In contrast, they were more efficient than VIP (E(max) values of 150 and 150% at the R172Q VPAC(2) receptors, and of 400 and 360% at the R172L receptors, respectively). These results suggested that the receptor's R172 and the ligand's aspartate 3 are brought in close proximity in the active ligand-receptor complex. 5. The K179I and K179Q mutated receptors had a lower affinity than the wild-type receptors for all the agonists tested in this work: we were unable to identify the VIP amino acid(s) that interact with K179.

Two tyrosine residues in the first transmembrane helix of the human vasoactive intestinal peptide receptors play a role in supporting the active conformation.

1: We investigated the human vasoactive intestinal polypeptide (VIP) receptors VPAC(1) and VPAC(2) mutated at conserved tyrosine residues in the first transmembrane helix (VPAC(1) receptor Y146A and Y150A and VPAC(2) receptor Y130A and Y134A). 2: [(125)I]-Acetyl-His(1) [D-Phe(2), K(15), R(16), L(27)]-VIP (1-7)/GRF (8-27) (referred to as [(125)I]-VPAC(1) antagonist) labelled VPAC(1) binding sites, that displayed high and low affinities for VIP (IC(50) values and per cent of high affinity binding sites: wild-type, 1 nM (57+/-9%) and 160 nM; Y146A, 30 nM (40+/-8%) and 800 nM; Y150A, 4 nM (27+/-8%) and 300 nM). [R(16)]-VIP behaved as a "super agonist" at both mutated VPAC(1) receptors and the efficacies of VIP analogues modified in positions 1, 3 and 6 were significantly decreased. 3: VIP was less potent at the Y130A and Y134A mutated VPAC(2) receptors (EC(50) 200 and 400 nM, respectively) than at the wild-type VPAC(2) receptor (EC(50) 7 nM). Furthermore, [hexanoyl-His(1)]-VIP behaved as a "super agonist" at the two mutated VPAC(2) receptors, and VIP analogues modified in positions 1, 3 and 6 were less potent and efficient at the mutated than at wild-type VPAC(2) receptors. However, the Y130A and Y134A mutants could not be studied in binding assays. 4: Our results suggest that the conserved tyrosine residues do not interact directly with the VIP His(1), Asp(3) or Phe(6) residues (that are necessary for receptor activation), but stabilize the correct active receptor conformation.

Relative quantitative analysis of glucagon receptor mRNA in rat tissues.

Total RNA prepared from nine rat tissues were analyzed for their content in glucagon receptor mRNA by two independent hybridization approaches: (1) simple dot blot analysis using labelled oligodeoxynucleotide; (2) highly specific RNase protection assay using labelled antisense RNA. Hybridization signal was quantified by laser densitometric scanning of autoradiographies. Results were expressed for each method relative to the liver content (100%) for either a constant amount of total RNA or for a constant amount of beta-actin mRNA. We obtained similar relative values of glucagon receptor mRNA per constant amount of total RNA by the two hybridization methods: in liver (100 and 100), in kidney (38 and 34), and in heart (12 and 11). The glucagon receptor mRNA was overestimated by the less specific dot assays, in adrenal glands (21 versus 10) and in adipose tissues (24 versus 5). In the stomach, brain, duodenum and lung, the signal was equal to or below the reliable quantification limit. Reverse transcription and polymerase chain reaction (RT-PCR) of glucagon receptor mRNA with limited cycle number were performed using two sets of primers: the first set amplified a single band at the 3' coding end, and the second, 3-6 bands at the 5' coding end, revealing tissue-specific polymorphism. RT-PCR data confirmed the presence of glucagon receptor mRNA in liver, kidney, heart, adrenal glands and adipose tissue, and allowed the detection of a very low amount of glucagon receptor mRNA in the stomach, the duodenum and brain but not in the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential alternative splicing of PACAP receptor in pituitary cell subpopulations.

The capability of rat pituitary cells to express receptors for pituitary adenylate cyclase activating polypeptide (PACAP) and VIP was evaluated by binding studies and measurement of adenylate cyclase activity on whole gland preparations and by reverse transcriptase-polymerase chain reaction (TR-PCR) using specific primers on preparations from isolated cell populations enriched in PRL- and GH-producing cells. Data obtained on whole gland preparations indicated that selective PACAP receptors (PACAP Type I) predominated. The mRNA coding for PACAP Type I and for the non-selective PACAP receptors Type II VIP2 (but not VIP1) were identified. The mRNA coding for four different spliced variants of the PACAP Type I receptor were detected. In PRL producing cells, three variants and the VIP2 mRNA were detected, whereas in GH-producing cells the mRNA coding for the variant having a 28-amino acid insert (termed HOP) in the third intracellular loop was the only present.

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide stimulate two signaling pathways in CHO cells stably transfected with the selective type I PACAP receptor.

The properties of the pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor were studied on a clone of Chinese hamster ovary cells (CHO) stably transfected with the recombinant receptor. PACAP(1-27), PACAP(1-38) and VIP inhibited [125I-acetyl-His1]PACAP (1-27) binding, stimulated cyclic AMP and inositol phosphates production and induced [Ca2+]i increase with the same order of potency: PACAP(1-27) = PACAP(1-38) > VIP. The concentrations required for half maximal receptor occupancy, IP3- and [Ca2+]i increase were not different for both PACAPs (1 nM) and 100-fold higher than those required for cyclic AMP increase (0.010 nM). These data suggest that the occupancy of a portion of the total receptors available was sufficient for maximal cyclic AMP production but not for maximal IP3 production. It is concluded that the possibility of the type I PACAP receptor being coupled to a transduction pathway is not located at the level of the ligand but rather at the level of the G-proteins.

Autoradiographic visualization of the receptor subclasses for vasoactive intestinal polypeptide (VIP) in rat brain.

Vasoactive Intestinal Polypeptide (VIP) exerts its biological effects through interaction with two high affinity receptors named the VIP1- and the VIP2 receptors. Their messenger RNAs have been mapped in rat brain by in situ hybridization. A cyclic peptide (RO 25-1553) and a secretion analogue ([R16]chicken secretin) were identified as selective agonist peptides for the VIP2- and VIP1 receptors, respectively. The iodinated peptides retained the high affinity and selectivity of the unlabelled peptides and were used for the mapping of each receptor subclass in rat brain. VIP1 receptors were present in the cerebral cortex, the piriform cortex, the claustrum, the caudate-putamen, the dentate gyrus, the lateral amygdaloïd nucleus, the anteroventral thalamic nucleus, the rhomboïd nucleus, the supraoptic nucleus and the choroïd plexus. VIP2 receptors were present in the cerebral cortex, the claustrum, the caudate-putamen, the nucleus accumbens, the lateral septal nuclei, the bed nucleus of the stria terminalis, the basolateral amygdaloïd nucleus, the Ammon's horn, the thalamic nuclei except some centromedial nuclei, the medial habenula, the suprachiasmatic nucleus, the periventricular nucleus, the mammilary nucleus, the superior colliculus and the choroïd plexus.

Chromogranin A(210-301) is the major form of pancreastatin-like material in human gut extracts and endocrine tumors.

A radioimmunoassay of human pancreastatin was developed using a rabbit antiserum that selectively recognized the C-terminal amidated end of the peptide, and it was used for the identification of the molecular forms of pancreastatin in human gut (stomach, duodenum, small intestine, colon) and endocrine tumor extracts (liver metastasis of a gastrinoma and a medullary carcinoma of thyroid, one nonsecreting pancreatic tumor, one recurrence of a gut carcinoid, one vipoma and one insulinoma). In all gut extracts, a gel filtration chromatography revealed the presence of three peaks of pancreastatin-like immunoreactivity. The predominant form eluted with an apparent molecular weight higher than that of pancreastatin. This form was also predominant in the endocrine tumors analyzed, except in the insulinoma, where a lower molecular weight form predominated. The high molecular form was further purified from a liver metastasis of a gastrinoma. The pancreastatin-like immunoreactivity eluted in all the chromatographical systems (reverse-phase, ion exchange) as a single peak that was finally purified to homogeneity and sequenced. The sequence of the first 29 N-terminal amino acids was obtained unambiguously and corresponded to the sequence 210-238 of chromogranin A. Considering the selectivity of the assay used for peptide identification, this major form was identified as the fragment 210-301 of chromogranin A. It is likely that the predominant form of pancreastatin in human gut extracts and noninsular tumors is a 92 amino acid peptide.

Expression of pituitary adenylate cyclase activating polypeptide and receptors in human brain tumors.

The capacity of brain tumor samples to synthesize pituitary adenylate cyclase activating polypeptide (PACAP) was evaluated by the reverse transcriptase-polymerase chain reaction technique (RT-PCR). The expression of PACAP receptors was assessed by a combination of RT-PCR techniques, conventional binding techniques, and also by the ability of PACAP to stimulate adenylate cyclase activity. A weak PACAP mRNA and PACAP receptor mRNA expression was detected in only 3 of 16 meningiomas. A weak PACAP-stimulated adenylate cyclase activity (+20%) was detected in 10 of the 16 samples but binding of labeled PACAP was never observed. In the 16 gliomas studied (including two oligodendrogliomas and two ependymomas), PACAP mRNA was identified in 13 samples and PACAP receptor mRNA in 15 samples. PACAP receptors were identified in all the samples by binding studies and/or by PACAP stimulation of the adenylate cyclase activity. PACAP mRNA was never detected in pituitary adenomas (three prolactinomas, two mixed PRL-GH-producing tumors, three GH-secreting tumors, three gonadotrophinomas, one ACTH-producing tumor, two nonsecreting tumors) whereas PACAP receptor mRNA was highly expressed in all the tumors except prolactinomas, where it was at the limit of detection, confirming the binding and adenylate cyclase activation results. Thus, it is unlikely that the neuropeptide PACAP could influence meningioma's cell growth; PACAP secreted from extratumoral areas may influence pituitary tumors and PACAP could participate to gliomas development.

Proline residue 280 in the second extracellular loop (EC2) of the VPAC2 receptor is essential for the receptor structure.

Inspection of the amino acid sequence of the human VPAC1 and the VPAC2 receptors after alignment of the conserved residues indicates that the second extracellular loop (EC2) is one amino acid shorter in the VPAC1 receptor due to the lack of a proline residue in position 294. We hypothesized that this could be of importance for receptor structure and/or for ligand recognition. Insertion by directed mutagenesis of a proline in that position (294 VPAC1) had little consequence on the binding of several agonists but reduced the affinity for the VPAC1 antagonist. Coupling of the 294 VPAC1 receptor to adenylate cyclase was improved, as demonstrated by an increased affinity for VIP and other agonists, and by a shift of the VPAC1 antagonist to partial agonist behavior. Deletion of the proline 280 (DeltaPro280 VPAC2) in the VPAC2 receptor markedly reduced the apparent affinity for all the agonists tested. Replacement of the proline by a glycine residue had a smaller effect on the ligands affinities. The proline residue in the VPAC2 receptor EC2 is thus essential for the receptor structure, and the EC2 domain is involved in ligand recognition and receptor functionality.

Expression of pituitary adenylate cyclase activating polypeptide (PACAP) receptors in human glial cell tumors.

Twenty-three human gliomas were analyzed: 13 astroglial neoplasms including three grade II, four grade III, and six grade IV tumors; seven ependymomas; and three oligodendrogliomas. A crude membrane fraction was prepared within 30 min after surgical removal of the tumors and was immediately tested for the presence of pituitary adenylate cyclase activating polypeptide (PACAP) receptors. PACAP stimulated adenylate cyclase activity in 23 tumors, but a specific binding of [125I-acetyl-His1]PACAP-27 was detected in only 16 tumors. In all cases, PACAP-27 and -38 were equipotent (Kd or Kact of 1-3 nM) and were 100- to 1000-fold more potent than VIP. PACAP stimulated threefold the adenylate cyclase activity in the presence of GTP. The results were compatible with an interaction of PACAP with a highly selective type I PACAP receptor and not with a high-affinity VIP/PACAP type II receptor. The presence of PACAP receptors on glial neoplasic opens the possibility of a control of the tumor growth by this family of peptides.

The long-acting vasoactive intestinal polypeptide agonist RO 25-1553 is highly selective of the VIP2 receptor subclass.

RO 25-1553 is a synthetic VIP analogue that induced a long-lasting relaxation of tracheal and bronchial smooth muscles as well as a reduction of edema and eosinophilic mobilization during pulmonary anaphylaxis. In the present study, we tested in vitro the capacity of RO 25-1553 to occupy the different VIP/PACAP receptor subclasses and to stimulate adenylate cyclase activity. The cellular models tested expressed one single receptor subtype: Chinese hamster ovary (CHO) cells transfected with the rat recombinant PACAP I, rat VIP1, and human VIP2 receptors; SUP T1 cells expressing the human VIP2 and HCT 15 and LoVo cells expressing the human VIP1 receptor. RO 25-1553 was threefold more potent than VIP on the human VIP2 receptor, 100- and 600-fold less potent than VIP on the rat and human VIP1 receptors, respectively, and 10-fold less potent than VIP and 3000-fold less potent than PACAP on the PACAP I receptor. RO 25-1553 was a full agonist on the VIP2, the PACAP I, and the rat recombinant VIP1 receptor but a partial agonist only on the human VIP1 receptor. Thus, RO 25-1553 is a highly selective agonist ligand for the VIP2 receptor subclass.

Development of high affinity selective VIP1 receptor agonists.

The biological effects of VIP are mediated by at least two VIP receptors: the VIP1 and the VIP2 receptors that were cloned in rat, human and mice. As the mRNA coding for each receptor are located in different tissues, it is likely that each receptor modulates different functions. It is therefore of interest to obtain selective agonists for each receptor subtype. In the present work, we achieved the synthesis of two VIP1 receptor selective agonsits derived from secretin and GRF. [R16]chicken secretin had IC50 values of binding of 1,10,000, 20, and 3000 nM for the rat VIP1-, VIP2-, secretion- and PACAP receptors, respectively. This peptide, however, had a weaker affinity for the human VIP1 receptor (IC50 of 60 nM). The chimeric, substituted peptide [K15, R16, L27]VIP(1-7)/GRF(8-27) had IC50 values of binding of 1,10,000, 10,000 and 30,000 nM for the rat VIP1-, VIP2-, secretin- and PACAP receptors, respectively. Furthermore, its also showed an IC50 of 0.8 nM for the human VIP1 receptor and a low affinity for the human VIP2 receptor. It is unlikely that this GRF analogue interacted with a high affinity to the pituitary GRF receptors as it did not stimulate rat pituitary adenylate cyclase activity. The two described analogues stimulated maximally the adenylate cyclase activity on membranes expressing each receptor subtype.

Presence of calcitonin gene-related peptide receptors coupled to adenylate cyclase in human gliomas.

Eleven surgical samples of gliomas (1 of grade II, 3 of grade III and 7 of grade IV) were analyzed. Calcitonin gene-related peptide (CGRP) receptors were identified by 125I-alpha h-CGRP binding in 9 cases and the presence of a CGRP-stimulated adenylate cyclase in all the 11 cases. Tracer binding was inhibited by unlabelled alpha h-CGRP (Kd of 0.3 nM), by (8-37) alpha h-CGRP (Kd of 30 nM), by (12-37) alpha h-CGRP (Kd of 3.000 nM) but not by human calcitonin. The mean density of CGRP receptors (120 fmol/mg membrane protein) was comparable to that of beta-adrenergic receptors. CGRP stimulated 1.4 to 4.7-fold (mean 2.7) the adenylate cyclase activity with a K(act) of 2.0 nM. The CGRP fragments had no intrinsic activity but inhibited the CGRP effect. The (8-37)CGRP fragment had a Ki of 30 nM. Thus, at variance with previous reports on rat and human brain membranes, that showed the presence of CGRP receptors not coupled to adenylate cyclase, we observed in human gliomas the presence of CGRP receptors that, when occupied, stimulated efficiently the adenylate cyclase activity.

VIP and pituitary adenylate cyclase activating polypeptide (PACAP) have an antiproliferative effect on the T98G human glioblastoma cell line through interaction with VIP2 receptor.

Functional VIP/PACAP receptors were identified in the human glioblastoma cell line T98G, based on the relative potency of VIP, PACAP and PACAP-38 to stimulate adenylate cyclase activity. Analysis of the T98G cells mRNA by reverse transcription followed by a polymerase chain reaction (RT-PCR) demonstrated the expression of the mRNA coding for the VIP2 receptor subclass only. VIP, PACAP-27 and PACAP-38 were potent and efficIent inhibitors of cell proliferation, assessed by the colorimetric MTT assay. VIP, PACAP-27 and PACAP-38 also reduced the incorporation of 3H-thymidine in T98G cells, but did not significantly alter the percentage of cells present at each stage of the cell cycle. Thus, VIP and PACAP, probably acting through a VIP2 receptor subtype, decreased cell proliferation.