Histamine and VIP interactions with receptor-cyclic AMP systems in the human gastric cancer cell line HGT-1

In HGT-1 cells incubated at 20 degrees C for 15 min with 1 mM 3-isobutyl-1-methylxanthine (IBMX), histamine (10(-4)M) increased basal cAMP levels from 2.12 +/- 0.14 to 22.9 +/- 2 pmol per 10(6) cells, with a potency of 6.4 X 10(-6)M. IBMX was added in order to inhibit cAMP degradation by low and high Km cAMP-phosphodiesterases (cAMP-PDE). The use of specific H1, H2 agonists or antagonists indicated that the histamine effect was due to an interaction with typical H2 -receptors that are involved in gastric acid secretion. Cyclic AMP levels were also increased (10-fold) by vasoactive intestinal peptide VIP (3 X 10(-11) - 10(-8)M). Porcine peptide having N-terminal histidine and C-terminal isoleucine amide (PHI) and secretin were respectively 80 and 3600 times less potent than VIP and did not produce additive effect when tested in combinations with VIP. This observation indicates that these two peptides, structurally related to VIP, are acting through the recognition sites for VIP. Combination of VIP and histamine results in additive stimulation on intact cells as well as on membrane-bound adenylate cyclase, suggesting the existence of two cell populations bearing respectively the two sets of receptors. Two other human cancer cell lines originating from nongastric tumors (HT-29 and HL-60) possess only VIP or histamine receptors, respectively, indicating the gastric cellular originality of the HGT-1 cells. It is concluded that HGT-1 cells possess both VIP and histamine H2 receptors with similar pharmacological properties to those characterized in normal human fundic glands (1,2). Therefore, this cell line can be a good model to study drugs used therapeutically during the treatment of patients for gastric ulcer or cancer.     

Demonstration of a functional receptor for vasoactive intestinal polypeptide on Molt 4b T lymphoblasts

Viable human T lymphoblasts derived from the "Molt 4b" cell line have been shown to possess functional plasma membrane receptors for vasoactive intestinal polypeptide (VIP). Specific binding of 125I-VIP to these lymphoblasts is rapid, reversible and linearly dependent on the number of cells present. Analysis of binding at 17 degrees C reveals a single class of high affinity binding sites over the concentration range of 10(-7) to 10(-11) M VIP (KD = 7.3 +/- 1.3 nM). The Bmax of 0.24 +/- 0.07 nM extrapolates to 15 000 +/- 4000 sites/cell. The binding of 125I-VIP to T lymphoblasts is highly specific; secretin and glucagon, peptides of similar molecular weight which show sequence homology with VIP, are unable to competitively inhibit binding of 125I-VIP to Molt 4b lymphoblasts. VIP activates adenylate cyclase in membrane preparations from Molt 4b lymphoblasts and increases cAMP in intact cells. Half maximal activation in both membrane preparations and intact cells occurs at 5 nM VIP. This demonstration of a functional receptor for VIP suggests that the Molt 4b lymphoblastic cell line may be a useful model system in which to study neuropeptide modulation of T lymphocyte function.     

The 38-amino acid form of pituitary adenylate cyclase-activating polypeptide stimulates dual signaling cascades in PC12 cells and promotes neurite outgrowth.

Vasoactive intestinal peptide (VIP) activates adenylylcyclase in sympathoadrenal cells at concentrations greater than 10(-6) M. We demonstrate here that two forms of a newly discovered peptide with homology to VIP named pituitary adenylate cyclase-activating polypeptide (PACAP) are much more potent activators of signal transduction in PC12 cells. Both the 27- and 38-amino acid forms of PACAP elevate cAMP levels in PC12 cells and stimulate adenylylcyclase in PC12 membranes, with an EC50 near 10(-9) M. PACAP38 additionally is a potent activator of the inositol lipid cascade in PC12 cells, elevating the content of inositol phosphates by 8-fold at 10(-8) M (EC50 = 7 x 10(-9) M). PACAP38 and PACAP27 have been thought to have essentially identical actions, but PACAP27 is 2-3 logs less potent in increasing inositol lipid levels. Moreover, PACAP38 at 10(-8) M is an effective inducer of neuronal morphology in PC12 cells, whereas PACAP27 is much less active in promoting neurite outgrowth. In contrast to the PACAP-preferring receptors on PC12 cells, another class of PACAP-binding sites with equal high affinities for VIP, PACAP38, and PACAP27 has been identified on several other cell types. We find that the cAMP content of rat CH3 pituitary cells, known to have high affinity VIP receptors, is in fact potently elevated by PACAP27 and PACAP38 as well as by VIP. However, PACAP38, even at 10(-6) M, is not capable of significant activation of inositol lipid turnover via these VIP/PACAP nondiscriminating sites.     

Vasoactive intestinal peptide receptor regulation of cAMP accumulation and glycogen hydrolysis in the human Ewing's sarcoma cell line WE-68

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)     

Vasoactive intestinal peptide and forskolin regulate proliferation of the HT29 human colon adenocarcinoma cell line.

Although several lines of evidence implicate cAMP in the regulation of intestinal cell proliferation, the precise role of this second messenger in the control of the human colon cancer cell cycle is still unclear. In order to investigate the role of cAMP in HT29 cell proliferation, we have tested the effect of vasoactive intestinal peptide (VIP) and forskolin on DNA synthesis and cell number, focusing on the time-dependent efficacy of the treatment. The cells were arrested in G0/G1 phase by incubation for 24 h in serum-free medium and proliferation was re-initiated by addition of either 85 nM insulin or 0.5% fetal calf serum. In the presence of fetal calf serum, G1/S transition was found to occur earlier than with insulin. Exposure of the HT29 cells to 10(-5) M forskolin in the early stages of growth induction (within 12 h from FCS addition or within 14 h from insulin treatment) resulted in a significant inhibition of DNA synthesis and a delayed entry in the S phase. By contrast, VIP (10(-7) M) was inhibitory only when added within a narrow window (10 to 12 h or 12 to 14 h following FCS or insulin addition, respectively). The difference in efficiency of forskolin and VIP to inhibit cell proliferation may be correlated with their own potency to promote long-lasting cAMP accumulation. The combination of VIP plus forskolin had synergistic effects on both cAMP accumulation and cell-growth inhibition. Taken together, our data indicate that cAMP may act at a step in the late G1 or G1/S transition.     

Identification of high affinity receptors for vasoactive intestinal peptide on human lymphocytes of B cell lineage

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.     

Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation. Comparison with peptide histidine isoleucine and growth hormone-releasing factor

The vasoactive intestinal polypeptide (VIP) receptor was characterized on the GH3 rat pituitary tumor cell line using competitive binding studies with peptides having sequence homology with VIP. Further studies investigated receptor coupling to the adenylate cyclase complex by measurement of cAMP levels. Finally, the molecular weight of the receptor was estimated by affinity labeling techniques. Studies using 125I-VIP and unlabeled competing peptides revealed a single class of high affinity binding sites with a dissociation constant (KD) of 17 +/- 2 nM (mean +/- S.E.M.) for VIP, 275 +/- 46 nM for peptide histidine isoleucine (PHI), and 1380 +/- 800 nM for human pancreatic growth hormone releasing factor (GHRF). VIP and PHI each stimulated intracellular cAMP accumulation in a dose-dependent manner; both peptides demonstrated synergism with forskolin. In contrast, GHRF neither stimulated accumulation of cAMP nor demonstrated synergism with forskolin. VIP plus PHI (1 microM each) caused no significant increase in cAMP over either VIP or PHI alone, implying that the two peptides act through the same receptor. Covalent crosslinking of 125I-VIP to its binding site using either disuccinimidyl suberate (DSS) or ethylene glycol bis(succinimidyl succinate) (EGS) was followed by SDS-PAGE and autoradiography. The result is consistent with an Mr 47 000 VIP-binding subunit comprising or being associated with the VIP receptor of GH3 pituitary tumor cells.     

Cyclic AMP-stimulating effect of vasoactive intestinal peptide in isolated epithelial cells of rat ventral prostate

Vasoactive intestinal peptide (VIP) has been shown to increase cyclic AMP content in isolated epithelial cells of rat ventral prostate. The stimulatory effect of VIP was dependent on time and temperature and was potentiated by a phosphodiesterase inhibitor. At 15 degrees C, the response occurred in the 1 X 10(-10)-10(-7)M range of VIP concentrations. Half-maximal stimulation of cellular cyclic AMP was obtained at 1.4 nM and maximal stimulation (3-fold basal level) at about 100 nM VIP. Chicken VIP and porcine secretin were agonists of porcine VIP but exhibited a 2-times higher and a 170-times lower potency, respectively. A high concentration (1 X 10(-6)M) of glucagon, somatostatin, neurotensin, substance P, Met-enkephalin or Leu-enkephalin did not modify cAMP levels. The finding of a VIP-stimulated cAMP system in rat prostatic epithelial cells together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, as well as the presence of VIP-containing neurones innervating the male genitourinary tract, strongly suggest that VIP may be involved in prostatic growth regulation and function.     

Anti-cell surface monoclonal antibodies which antagonize the action of VIP in a human adenocarcinoma cell line (HT 29 cells)

Hybridoma cells have been obtained by fusing RCY 3 Ag 1-2-3 rat myeloma cells with spleen cells from a rat hyperimmunized with human adenocarcinoma cells (HT 29 cell line) grown in serum-free medium. Immunoglobulins secreted by hybridomas were screened for: (i) specific binding to HT 29 cells; (ii) their ability to inhibit the binding of [125I]-vasoactive intestinal peptide (VIP) to HT 29 cells; (iii) their capacity to modulate the cAMP production induced by VIP. The monoclonal antibodies we have obtained from clones 109-10-16 and 109-10-19 compete for the binding of radiolabelled VIP to HT 29 cells and partially inhibit the production of cAMP induced by VIP while they are ineffective in reducing the intracellular level of cAMP attained after stimulation of HT 29 cells by isoproterenol. We never found antibodies which increase the cAMP level in HT 29 cells. The binding of the purified monoclonal antibody 109-10-16 Ig gamma 2c to HT 29 cells was visualized by indirect immunofluorescence and was not present at the surface of all cells. These observations strongly support the hypothesis that the monoclonal antibodies we have characterized interact with an antigenic determinant which belongs to the VIP receptor or at least to a cell surface component closely associated with the receptor.     

Vasoactive intestinal peptide enhances dopamine accumulation in primary cell culture of rat hypothalamus.

The effect of vasoactive intestinal peptide (VIP) and PHI-27 on dopamine accumulation in cultured rat hypothalamic cells was investigated. VIP enhanced [3H]dopamine accumulation dose dependently. This effect was significant at 10(-8)-10(-5) M VIP with a concomitant increase in intracellular cyclic AMP (cAMP), and reached its plateau level at 10(-6) M VIP. VIP increased [3H]dopamine accumulation significantly within 15 min. PHI-27 and dibutyryl cAMP ((Bu)2-cAMP) also enhanced [3H]dopamine accumulation. These results suggest that VIP enhances dopamine accumulation in hypothalamic cells by increasing intracellular cAMP.     

Vasoactive intestinal peptide receptor activity and specificity during enterocyte-like differentiation and retrodifferentiation of the human colonic cancerous subclone HT29-18

Commitment of HT29-18 cells to enterocyte-like differentiation by glucose removal is related to a decreased capacity to generate cAMP after treatment with vasoactive intestinal peptide (VIP), forskolin or sodium fluoride. In contrast, the potency of VIP (EC50 = 1.1 - 1.3 X 10(-10) M) and the pharmacological specificity of the VIP receptor (VIP greater than rh GRF 1-43 greater than PHI greater than secretin) are unchanged during differentiation and retrodifferentiation. These results indicate that disturbances in VIP receptor-post-receptor activity, involving cell surface VIP receptors, membrane and intracellular transducers of hormonal information, occur during enterocyte-like differentiation of the HT29-18 subclone.     

Fibroblast growth factor regulates the expression of luteinizing hormone receptors in cultured rat granulosa cells

We have investigated the effects of bFGF on both the FSH-induced LH receptor expression and cAMP production in cultured rat granulosa cells. Concentrations of pure FGF, from 10(-12) M to 10(-10) M, progressively inhibit the stimulatory actions of FSH with an ED50 of approximately 4 x 10(-12) M for both parameters. Higher FGF concentrations, from 4 x 10(-10) M to 10(-8) M, lead to a gradual reduction of the growth factor inhibitory effect. The effects of FGF are more prominent on the modulation of LH receptors than on the FSH-induced cAMP production. Moreover, FGF impairs the LH receptor formation induced by cholera toxin or 8-Bromo-cAMP, indicating that the growth factor also acts at a step distal to cAMP formation. The inhibitory effect of FGF on LH receptor expression increases during the entire course of granulosa cell differentiation, from 24 to 96 h, and is not due to variations in cell number or viability, but rather to a change in the content of LH receptors with no significant modification of binding affinity (KD congruent to 0.8 x 10(-10) M). These results suggest that bFGF may acutely regulate the capacity of granulosa cells to differentiate upon FSH stimulation and to respond to LH during the ovarian follicular maturation.     

Action of calcitonin gene-related peptide at the calcitonin receptor of the T47D cell line

Some effects of calcitonin (CT) can also be produced by calcitonin gene-related peptide (CGRP), an alternative product of the calcitonin gene. This might be mediated by interaction of CGRP at the CT-receptor site. The human breast cancer cell line T47D possesses well characterized CT-receptors (KD = 2.3 x 10(-10) M for 125I salmon CT). 50% inhibition of 125I-sCT binding was achieved with 10(-9) M sCT, 5 x 10(-6) M rat CGRP and 10(-5) M human CGRP. Half maximal cAMP production in T47D cells was seen with 6 x 10(-10) M sCT, 5 x 10(-6) M rCGRP and 10(-5) M hCGRP. Binding and displacement capacity as well as the biological activity of CT and CGRP seems to correlate well. These findings suggest that CGRP in pharmacological doses acts via the CT-receptor. This could be explained by the homology and conformational similarities between CT and CGRP.     

Molecular identification and structural requirement of vasoactive intestinal peptide (VIP) receptors in the human colon adenocarcinoma cell line, HT-29.

The human colon adenocarcinoma cell line HT-29 in culture exhibits a cyclic AMP production system highly sensitive to vasoactive intestinal peptide (VIP), making HT-29 cells a unique cultured cell system for studying the mechanism of VIP action [Laburthe, Rousset, Boissard, Chevalier, Zweibaum & Rosselin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2772-2775]. The quantitative characteristics of VIP receptors in HT-29 cells and their structural requirement and molecular size were studied. 125I-labeled VIP bound in a time-dependent manner to HT-29 cell homogenates. At equilibrium (60 min incubation at 30 degrees C), unlabelled VIP in the 0.01-10 nM concentration range competed with 125I-VIP for binding to cell homogenates. Scatchard analysis of binding data gave a straight line, indicating that VIP bound to a single population of sites with a KD of 0.12 +/- 0.02 nM and a capacity of 120 +/- 9 fmol/mg of protein. The structural requirement of these receptors was studied with peptides structurally related to VIP, either natural or synthetic. Several peptides inhibited 125I-VIP binding to HT-29 cell homogenates with the following order of potency, which is typical of the human VIP receptor: VIP (IC50 = 0.1 nM) greater than VIP-(2-28)-peptide (IC50 = 13 nM) greater than human growth hormone releasing factor (IC50 = 56 nM) greater than peptide histidine isoleucine amide (IC50 = 80 nM) greater than secretin (IC50 greater than 10 000 nM). To characterize the molecular component(s) of the VIP receptor in HT-29 cells, 125I-VIP was covalently bound to cell homogenates by using the cross-linker dithiobis(succinimidyl propionate). Sodium dodecyl sulphate/polyacrylamide-gel autoradiographic studies of affinity-labelled cell homogenates revealed two major bands, corresponding to 125I-VIP-protein complexes of Mr 66 000 and 16 000. The labelling of the Mr-66 000 component was specific, since it was abolished by native VIP, whereas that of the Mr-16 000 component was not. Densitometric scanning of autoradiographs indicated that the labelling of the Mr-66 000 complex was inhibited by low VIP concentrations in the 0.1-10 nM range (IC50 = 0.6 nM), but was unaffected by 1 microM-glucagon or octapeptide of cholecystokinin. It was also decreased by VIP-(2-28)-peptide with a potency 1% that of VIP. Assuming that one molecule of 125I-VIP bound per molecule of protein, one protein of Mr 63 000 was identified as a component of the VIP receptor in HT-29 cells.     

Influence of VIP on D-galactose transport across rabbit jejunum in vivo

D-galactose absorption during 1 min perfusion periods was not affected by the presence of 10(-7)-10(-8) M VIP in the sugar solution, but exposure of mucosa to VIP for 5 min inhibited sugar absorption in the subsequent periods of perfusion. This inhibition is reversed after washing with saline solution. The effect of VIP disappeared when 10(-6) M RMI 12330A was added to the incubation solution together with 10(-7) M VIP and 1 mM D-galactose solution. These results suggest the existence of VIP receptors on the brush border membrane. The action of VIP could be mediated through the cAMP system.     

Properties of interleukin-1 and interferon-gamma receptors in B lymphoid cell line

We have previously shown that interleukin-1 alpha (IL-1 alpha) and interferon-gamma (IFN-gamma) induce surface IgM expression, stimulate Na+/H+ exchange, and activate protein kinase C in the murine pre-B lymphocyte cell line, 70Z/3. Because the two structurally different lymphokines induce similar effects, in this study we set out to compare the properties of the IL-1 and IFN-gamma surface receptors. In contrast to their similar cellular effects, we found that IL-1 alpha and IFN-gamma receptors have different properties. 70Z/3 have high (100 sites/cell) and low (900 sites/cell) affinity IL-1 receptors with dissociation constants (KD) 6 x 10(-11) and 10(-9) M, respectively. In contrast, IFN-gamma receptors are of one class with a KD of 3 x 10(-10) M and are at a higher number, 8000 sites/cell. After binding to their receptors both IL-1 alpha and IFN-gamma are internalized and intracellularly degraded, but the rate of internalization of IFN-gamma is greater than IL-1 alpha. The effective median concentrations (EC50) of IL-1 alpha- or IFN-gamma-induced surface IgM expression are similar (4-5 x 10(-12) M). However, at this concentration 10-fold more of IFN-gamma than IL-1 alpha molecules are bound per cell. Our studies indicate that structurally different lymphokines can induce similar biological events even though their signaling is mediated by surface receptors whose properties are different.     

Vasoactive intestinal polypeptide binds with high affinity to non-small cell lung cancer cells and elevates cyclic AMP levels

Vasoactive intestinal polypeptide (VIP) receptors were characterized on non-small cell lung cancer (NSCLC) cells. ¹²⁵I-VIP bound specifically to membranes derived from 6 NSCLC cell lines. Specific ¹²⁵I-VIP was time dependent and a linear function of EPLC-65H membrane concentration. ¹²⁵I-VIP bound with high (K_d=0.2 nM) and moderate (K_d=39 nM) affinity to two classes of sites. Pharmacology studies indicated that the order of peptide potency was VIP > rGHRH > PHI = helodermin > secretin > glucagon. Also VIP elevated the cAMP levels 10-fold using cell line ADLC-5M2. These data indicate that functional VIP receptors are present on NSCLC cells.     

Interaction of vasoactive intestinal peptide (VIP) with a mouse adrenal cell line (Y-1): specific binding and biological effects.

Y-1 cells specifically bind radiolabelled vasoactive intestinal peptide (VIP) with a dissociation constant of about 10^?9 M. [¹²⁵I]-VIP bound was not displaced by ACTH. VIP stimulates both steroid and cAMP production, with half-maximal stimulation at 10^?9 and 10^?8 M, respectively. At maximal concentration VIP produces the same stimulation of steroidogenesis as ACTH, but induced three times lower production of cAMP than ACTH. Y-1 DNA synthesis is inhibited by VIP in a dose-dependent manner with half-maximal inhibition at 10^?8 M. At submaximal concentrations the effects of VIP and ACTH on cAMP and steroid production and on inhibition of DNA synthesis are additive. Similar additive effects on cAMP production and on inhibition of DNA synthesis were observed at submaximal ACTH and maximal VIP concentration, but the phenomenon was no longer seen at maximal concentrations of both peptides. These data suggest that in Y-1 cells VIP stimulates, through its own distinct receptors, only a part of the pool of adenylate cyclase sensitive to ACTH.     

Dual effect of vasoactive intestinal peptide on the mitogenic response of rabbit spleen lymphocytes

The effects of vasoactive intestinal peptide (VIP) have been investigated on the mitogenic response of rabbit spleen cells. Specific binding of 125I-VIP to these mononuclear cells is rapid and saturable. Analysis of binding reveals two classes of binding sites, a class of high-affinity binding sites with KD = 0.93 +/- 0.11 nM and maximal binding capacity of 2000 +/- 560 sites/cell, and a class of low-affinity binding sites with KD = 225 +/- 58 nM and maximal binding capacity of 280,000 +/- 60,000 sites/cell. The VIP regulatory effect on mitogen-stimulated rabbit spleen cell proliferation appears to be time dependent and bimodal. When VIP was added simultaneously with mitogens, it induced an inhibition of the proliferative response. With concanavalin A (Con A) or pokeweed mitogen (PWM), addition of 10(-8) M VIP resulted in a maximal 30% inhibition of [3H]thymidine incorporation after 96 h of culture. This inhibitory effect was significant at concentrations from 10(-8)-10(-6) M and half-maximal inhibition was obtained with 1.2 x 10(-9) M VIP. By contrast, when rabbit spleen cells were preincubated for 18 h with VIP alone, the lymphocyte proliferative response to Con A was increased. However, this increase was mitogen-selective, since it was only observed when the T-cell mitogen Con A was used. The maximal response was obtained after 96 h of culture in the presence of Con A. The VIP stimulatory effect was dose-dependent with a maximal effect at 10(-7) M and a half-maximal effect at 1.7 x 10(-9) M VIP. The effect of VIP was also time-dependent, since a 6 h preincubation was sufficient to induce a significant increase in the proliferative response which was maximal after an 18 h preincubation.