Phorbol ester induces loss of VIP stimulation of adenylate cyclase and VIP-binding sites in HT29 cells

Treatment of HT29 cells with the tumor promoting phorbol ester PMA resulted in an attenuation of VIP-stimulated cAMP production in intact cells and VIP-stimulated adenylate cyclase activity in cell membranes. PMA did not decrease the ability of cholera toxin and forskolin to elevate cAMP levels in intact cells. Fluoride-stimulated adenylate cyclase activity in HT29 cells homogenates was not affected by PMA. The maximal VIP binding capacity of homogenates prepared from HT29 cells treated with PMA was decreased by 50%. It is concluded that protein kinase C regulates VIP receptor function possibly through phosphorylation of the VIP receptor.     

HT 29, a model cell line: stimulation by the vasoactive intestinal peptide (VIP); VIP receptor structure and metabolism

HT 29, a cell line derived from a human colonic adenocarcinoma, is highly responsive to the vasoactive intestinal peptide (VIP) as shown by a more than 100-fold intracellular cAMP increase (Ka = 0.3 nM), the stimulations of protein kinase A (Ka = 0.1 nM) and the low-Km cAMP phosphodiesterase (Ka = 40 nM). Remarkably, adenylate cyclase, cAMP-dependent kinase and cAMP-specific phosphodiesterase are activated in a sequential manner. Binding studies with [125I]-labeled VIP indicate a high affinity site with a Kd value (0.5 nM) close to the activation constant value (Ka) of the three enzymes. The molecular structure of the VIP receptor was studied by immunological and chemical approaches. A monoclonal antibody (mAb 109-10-16) which partially decreased the binding of VIP to its receptor allowed the characterization of Mr = 53,000 and Mr = 48-49,000 polypeptides. More precise identification of protein components of the VIP receptor resulted from covalent cross-linking on intact HT 29 cells by four bifunctional reagents: dithiobis-(succinimidyl propionate) and its non-cleavable analog disuccinimidyl suberate, the photoactivable azido phenyl glyoxal and dimethylpimelimidate. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated a major band of Mr = 67,000 regardless of which cross-linker was used. The same band and an Mr = 49,000 species were found in experiments using a crude membrane fraction of HT 29 cells. Assuming one molecule of VIP (Mr = 3326) linked per polypeptide, these observations suggest that an Mr = 64,000 species belongs to the VIP specific plasma membrane receptor. This protein contains an Mr = 20,000 N-linked sialic acid rich oligosaccharidic moiety.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol modulation of membrane fluidity and VIP receptor/effector system in rat prostatic epithelial cells.

Treatment of rat prostatic epithelial cells with cholesteryl hemisuccinate (ChH) resulted in a time- and dose-dependent inhibition of the stimulatory effect of the neuropeptide vasoactive intestinal peptide (VIP) on cyclic AMP accumulation, with a 40% decrease in the response to a maximally effective VIP concentration. Cell treatment with ChH led also to a similar blocking of isoproterenol (a beta-adrenergic agonist) action but did not modify forskolin (which is assumed to act directly on the catalytic unit of adenylate cyclase) activity upon cyclic AMP levels. The levels of the transduction protein Gs were similar in membranes from both control and ChH-treated cells as suggested by experiments on cholera toxin-catalyzed ADP-ribosylation. The inhibitory effect of ChH was accompanied by an increase of membrane microviscosity as estimated by measurements of fluorescence polarization. Experiments on VIP binding indicated that increasing cholesterol concentration in the plasma membrane led to a higher VIP binding capacity without changes in the affinity of VIP receptors. These data suggest that membrane cholesterol incorporation diminishes the coupling efficiency between adenylate cyclase and the VIP-receptor complex or other receptor systems (i.e., desensitization) due to an increase of plasma membrane rigidity.     

Effects of Vasoactive Intestinal Peptide and Other Peptides on Cyclic AMP Accumulation in Intact Pieces and Isolated Horizontal Cells of the Teleost Retina

The effects of vasoactive intestinal peptide (VIP) and several other peptides have been examined on cyclic AMP accumulation in intact pieces and isolated horizontal cells of the teleost (carp) retina. VIP was the most effective peptide examined, inducing a dose-related response, and an approximately fivefold increase in cyclic AMP production when used at a concentration of 10 microM. Porcine histidine isoleucine-containing peptide and secretin, peptides structurally related to VIP, also stimulated cyclic AMP accumulation, but at concentrations of 10 microM induced responses which were only approximately 40% and 10%, respectively, of the response observed with 10 microM VIP. In contrast, several other peptides, including glucagon, neurotensin, somatostatin, luteinizing hormone-releasing hormone, alpha-melanocyte-stimulating hormone, cholecystokinin octapeptide26-33, gastrin-releasing peptide, thyrotropin-releasing hormone, and VIP10-28 were totally inactive. The response to 10 microM VIP was not antagonized by several dopamine antagonists, indicating the presence of a population of specific VIP receptors coupled to adenylate cyclase, distinct from the population of dopamine receptors coupled to adenylate cyclase also known to be present in this tissue. Finally, experiments involving the use of fractions of isolated horizontal cells indicate that these neurons possess a population of VIP receptors coupled to cyclic AMP production which would appear to share a common pool of adenylate cyclase with a population of similarly coupled dopamine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that helodermin, a newly extracted peptide from Gila monster venom, is a member of the secretin/VIP/PHI family of peptides with an original pattern of biological properties

Helodermin, a newly isolated peptide from the venom of Gila monster (Heloderma suspectum) was shown to stimulate the adenylate cyclase activity of rat pancreatic membranes as efficiently as secretin and VIP. It also increased cyclic AMP levels and inhibited [125I]VIP binding in rat pancreatic acini. Finally, helodermin activated adenylate cyclase in membranes from rat heart, rat brain, and human heart, showing properties analogous yet distinct from those of secretin, VIP and PHI.     

Secretin Stimulates Cyclic AMP Formation in the Rat Brain

The effects of secretin on cyclic AMP levels in the rat brain were determined. Incubation of rat brain frontal cortex slices with secretin or the structurally related peptides peptide histidine leucine (PHI) or vasoactive intestinal polypeptide (VIP) in the presence of 10 mM theophylline resulted in a dose-dependent increase in the cyclic AMP levels. The half-maximal increase in cyclic AMP occurred using a 1 microM dose of secretin or a 2 microM dose of PHI or VIP. Preincubation of slices with secretin-(5-27) produced a dose-dependent inhibition of the secretin but not VIP- or PHI-stimulated increase in the cyclic AMP content. Also, in receptor binding studies, secretin-(5-27) produced a dose-dependent inhibition (Ki = 400 nM) of 125I-secretin but not of 125I-VIP binding to rat brain membranes. Guanyl-5'-yl imidodiphosphate decreased the affinity of radiolabelled secretin binding as a result of an increased rate of dissociation of bound 125I-secretin. These data suggest that secretin receptors in the rat brain may be coupled to adenylate cyclase in a stimulatory manner and that secretin-(5-27) may function as a central secretin receptor antagonist.     

The interaction of vasoactive intestinal peptide (VIP) with isolated bovine thyroid plasma membranes

The binding of vasoactive intestinal peptide (VIP) and stimulation of adenylate cyclase were studied in bovine thyroid plasma membranes. The binding depended on time, temperature and was saturable and specific. Binding studies suggested the presence of two classes of binding sites: a class with high affinity (Kd = 13 nM) and low capacity (6411 sites/pg), and a class with low affinity (Kd = 480 nm) and high capacity (105,300 sites/pg) at 15 degrees C. Secretin, glucagon, insulin and somatostatin did not displace the tracer from the membranes. VIP stimulated cyclic AMP production. Maximal cyclic AMP production (2-fold above basal values) was observed with 100 nM VIP and half-maximal response was obtained at 5 nM VIP at 15 degrees C.     

Elevation of Intracellular Cyclic AMP and Stimulation of Adenylate Cyclase Activity by Vasoactive Intestinal Peptide and Glucaeon in the Retinal Pigment Epithelium

Both vasoactive intestinal peptide (VIP) and glucagon rapidly elevated cyclic AMP levels in embryonic chick retinal pigment epithelium (RPE), in culture as well as in freshly dissected tissue. In cultured cells, the half-maximal activities of VIP and glucagon were 5 X 10(-8) M and 3 X 10(-8) M, respectively. After 3 min of reaction, VIP elevated intracellular cyclic AMP by 100-fold; elevation with glucagon was up to 10-fold. Both neuropeptides stimulated adenylate cyclase activity in RPE membranes. Glucagon showed a half-maximal activity of 1 X 10(-8) M. VIP remained more effective than glucagon in stimulating adenylate cyclase activity, but the dose-response curve was shifted to a higher concentration range when compared to that of the VIP-elevated intracellular cyclic AMP.     

Pharmacological characterization of the novel helodermin/VIP receptor present in human SUP-T1 lymphoma cell membranes

[Acetyl-His1]VIP stimulated adenylate cyclase with higher potency than VIP in membranes from human SUP-T1 lymphoblasts and was used as an efficient radioiodinated ligand with low non-specific binding to evaluate the relationship between receptor occupancy and adenylate cyclase activation and the possible interference of peptide T (an epitope derived from HIV envelope protein gp120). Various peptides inhibited [125I-acetyl-His1]VIP binding and activated the enzyme, their order of potency being: helodermin greater than [acetyl-His1]VIP greater than VIP = PHI = [Phe1]VIP greater than [D-Phe2]VIP = [D-Ala4]VIP = [D-Phe4]PHI greater than or equal to [D-Phe4]VIP greater than [D-His1]VIP giving further support for the existence of a novel subtype of helodermin/VIP receptors. [D-Ala1]peptide T and VIP-(10-28) did not recognize the binding site and did not inhibit, even at high concentration, VIP - or VIP analogue - stimulated adenylate cyclase activities.     

Muscarinic cholinergic inhibition of cyclic AMP formation and adrenocorticotropin secretion in mouse pituitary tumor cells

Cholinergic muscarinic receptors were identified in AtT-20/D16-16 (AtT-20) cell membranes by receptor binding techniques and the effect of carbachol on basal and stimulated cyclic AMP formation and ACTH release was investigated. Carbachol markedly decreased the stimulatory effect of the adenylate cyclase activator, forskolin, on both cyclic AMP formation and ACTH secretion. Carbachol also reduced forskolin-stimulated adenylate cyclase activity. The stimulatory effects of (-) isoproterenol on cyclic nucleotide formation and ACTH secretion were also blocked by carbachol. The inhibitory effects of carbachol on (-) isoproterenol-stimulated cyclic AMP synthesis and ACTH secretion were reversed by the muscarinic antagonist, atropine, and not by the nicotinic antagonist, gallamine. These data suggest that in AtT-20 cells, inhibition of ACTH secretion may be regulated by activation of muscarinic receptors coupled negatively to adenylate cyclase.     

Decreased Cyclic AMP Degradation in NG 108-15 Neuroblastoma × Glioma Hybrid Cells and S49 Lymphoma Cells Chronically Treated with Drugs that Inhibit Adenylate Cyclase

The increase in hormone-stimulated cyclic AMP accumulation observed in a variety of intact cells after chronic pretreatment with drugs that inhibit adenylate cyclase activity has been attributed to an increase in adenylate cyclase activity following withdrawal of the inhibitory drug. In NG 108-15 mouse neuroblastoma X rat glioma hybrid cells (NG cells) chronically treated with the muscarinic cholinergic agonist carbachol, we have found a significant decrease in the apparent degradation rate constant for cyclic AMP, in addition to an increase in the prostaglandin E1 (PGE1)-stimulated cyclic AMP synthesis rate in intact cells. In carbachol-pretreated NG cells that were stimulated with a maximally effective dose of PGE1, and that accumulated steady-state cyclic AMP concentrations fourfold or more higher than in control cells, the apparent rate constant for degradation was about 53% lower than the value for control cells. In carbachol-pretreated cells stimulated with a submaximal dose of PGE1 to yield a steady-state cyclic AMP concentration comparable to control cells, the apparent rate constant was 31% lower than the value for control cells. In S49 mouse lymphoma cells (S49 cells) chronically treated with an analog of the inhibitory agonist somatostatin, the first-order rate constant for cyclic AMP degradation in intact cells following isoproterenol stimulation was 29% lower than the value for control cells. Despite these changes in the kinetics of cyclic AMP degradation in intact NG cells and S49 cells, there was either no change or a minimal change (less than 10%) in phosphodiesterase activities assayed in extracts of cells chronically exposed to inhibitory drugs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasoactive-intestinal-polypeptide-stimulated adenosine 3'',5''-cyclic monophosphate accumulation in GH3 pituitary tumour cells. Reversal of desensitization by forskolin.

The interaction between forskolin and vasoactive intestinal polypeptide (VIP) in the regulation of cyclic AMP production in GH3 pituitary tumour cells was investigated. Both forskolin (10nM-10 microns) and VIP (10pM-10nM) increased the cyclic AMP content of GH3 cells. Forskolin (50-100nM) was additive with VIP in stimulating cyclic AMP accumulation when low concentrations (less than 1 nM) of the peptide were used, but exhibited a synergistic interaction with higher VIP concentrations (10-100 nM). These effects on cyclic AMP accumulation were reflected in a leftward shift in the concentration-response curve for VIP-stimulated prolactin release from GH3 cells, a process known to be regulated by intracellular cyclic AMP concentrations. The synergy observed did not appear to be related to changes in cyclic nucleotide phosphodiesterase activity, since it was even more marked in the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor. Studies of the time-course of VIP-induced changes in GH3-cell cyclic AMP content revealed that, with high concentrations of VIP, production ceased within 2 min of addition. This attenuation of cyclic AMP synthesis was still observed in the presence of isobutylmethylxanthine, but was markedly inhibited by low concentrations of forskolin (50-100nM). The results suggest that VIP-induced cyclic AMP production rapidly becomes desensitized. This process, which is prevented by forskolin, may be related to changes in the ability of the guanine nucleotide regulatory protein to couple receptor occupancy to activation of adenylate cyclase.     

Effect of forskolin and VIP on the release of somatostatin and the cAMP content of cultured rat diencephalon neurons

Forskolin, a direct activator of adenylate cyclase, stimulates somatostatin release in dispersed fetal diencephalic cells in culture (10 j). It was found that concentrations ranging from 10(-8) M to 10(-4) M increase the release of somatostatin in a dose-dependent manner, as well as the formation of intracellular cyclic AMP. Furthermore, VIP (10(-6) M) which produces a significant (p less than 0.03) elevation of SRIF release at 30 min of incubation, also induces a prompt increase of intracellular cyclic AMP (10 min). These results suggest that VIP could stimulate the somatostatin release through a cyclic AMP-dependent mechanism.     

The hepatic angiotensin II receptor. II. Effect of guanine nucleotides and interaction with cyclic AMP production

Guanine nucleotides were observed to modify the binding of 125I-angiotensin II to rat hepatic plasma membrane receptors. GTP and its nonhydrolyzable analogues greatly increased the dissociation rate of bound 125I-angiotensin II and altered hormone binding to the receptor under equilibrium conditions. In the absence of GTP, 125I-angiotensin II labeled both high affinity sites (Kd1 = 0.46 nM, N1 = 650 fmol/mg) and low affinity sites (Kd2 = 4.1 nM, N2 = 1740 fmol/mg). In the presence of guanine nucleotides, the affinities of the two sites were unchanged, but the number of high affinity sites decreased markedly to 52 fmol/mg. In analogous experiments using the angiotensin II antagonist, 125I-sarcosine1,Ala8-angiotensin II (125I-saralasin), guanine nucleotides minimally affected the interaction of 125I-saralasin with its receptor, increasing the dissociation rate 1.9-fold and the Kd 1.4-fold. The guanine nucleotide inhibition of agonist binding required a cation such as Na+ or Mg2+, with a maximal effect occurring at about 1 mM Mg2+. In liver plasma membranes prepared in EDTA, angiotensin II inhibited basal and glucagon-stimulated adenylate cyclase activities by 30% and 10%, respectively. Angiotensin II also caused a 40% inhibition of glucagon-stimulated cyclic AMP accumulation in intact hepatocytes, with a half-maximal effect occurring at 1 nM. The inhibition by angiotensin II of adenylate cyclase in membranes and of cAMP levels in intact cells could be reversed by the antagonist sarcosine1,Ile8-angiotensin II. Vasopressin caused a smaller 26% inhibition of glucagon-stimulated cyclic AMP accumulation. The ability of angiotensin II to inhibit cyclic AMP synthesis may provide an explanation for the observed effects of guanine nucleotides on 125I-angiotensin II binding to plasma membranes.     

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.     

VIP and related peptides induce rapid homologous desensitization in the human lymphoma SUP T1 cell line

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.     

Adenovirus type 12 transformation involves loss of beta-adrenergic receptors and isoproterenol responsiveness.

The responsiveness of a growth-regulated rat 3Y1 cell line and five clones of 3Y1 cells transformed by the highly oncogenic human adenovirus type 12 to the catecholamine hormone (-)-isoproterenol was studied. The untransformed cells contained beta-adrenergic receptors characterized by specific binding of the beta-adrenergic receptor antagonist (-)-[3H]dihydroalprenolol, a 9- to 12-fold increase in cyclic AMP production in intact cells after incubation with 10 microM (-)-isoproterenol, and significantly increased adenylate cyclase (ATP pyrophosphatelyase [cyclizing], EC 4.6.1.1) activity in the presence of the hormone. In contrast, (-)-isoproterenol (10 to 100 microM) had no apparent effect on cyclic AMP production or the basal adenylate cyclase activity in the transformed cell lines. Binding studies revealed that untransformed cells contained approximately 19,400 beta-adrenergic receptor sites per cell. Three transformed cell clones tested showed a three- to fourfold loss of beta-adrenergic receptors.     

Pharmacological characterization of VIP receptors in human lung membranes

The ability of VIP, PHI, secretin, helodermin, and seven N-terminally D-amino monosubstituted VIP and PHI analogs to occupy (125I)iodo-VIP labeled receptors and to activate adenylate cyclase was tested on human lung membranes purified by the method of Schachter et al. Best fitted Kd, Kact and % of max. values suggested the coexistence, in near equal proportions, of two classes of VIP-preferring binding sites coupled to adenylate cyclase that showed similar decreasing affinity for: VIP greater than (D-Ala4)-VIP greater than (D-Asp3)-VIP = (D-Ser2)-VIP greater than (D-His1)-VIP greater than PHI greater than (D-Phe2)-VIP greater than (D-Phe4)-VIP. (D-Arg2)-VIP was a non-selective agonist. A third receptor type, coupled to adenylate cyclase and showing high affinity for secretin and helodermin but not for VIP, was also detected.     

"Spare" beta-adrenergic receptors of rat white adipocyte membranes

The apparent equilibrium dissociation constants for the interaction of isoproterenol with beta-receptors and adenylate cyclase were determined under the same conditions in rat adipocyte membranes and were compared with the apparent dissociation constant for the interaction of isoproterenol with cyclic AMP accumulation in the adipocyte. From these determinations, it was calculated that the occupancy of less than 4% of the receptor population is required for half-maximal stimulation of adenylate cyclase in membranes and cyclic AMP accumulation in intact cells, provided that receptor-binding and adenylate cyclase assays are performed in the presence of guanine nucleotides. Since guanine nucleotides are also required for adenylate cyclase activation in intact cells, it is concluded that the beta-receptors of rat adipocytes are "spare" receptors.     

Agonists differentiate muscarinic receptors that inhibit cyclic AMP formation from those that stimulate phosphoinositide metabolism

Muscarinic receptor stimulation elicits two distinct biochemical responses in embryonic chick heart cells: inhibition of catecholamine-stimulated cyclic AMP formation and stimulation of phosphoinositide (PhI) hydrolysis. We observe two major differences in the effects of agonists on these responses. First, carbachol and oxotremorine both inhibit cyclic AMP formation, but only carbachol stimulates PhI hydrolysis. Second, the dose-response relationships for the cyclic AMP and PhI responses differ; the half-maximal concentrations of carbachol needed to inhibit cAMP accumulation and stimulate PhI hydrolysis are 2 X 10(-7) and 2 X 10(-5) M, respectively. We carried out radioligand binding studies on intact chick heart cells to determine whether these data could be explained in terms of different agonist binding states of the muscarinic receptor. In intact cells, carbachol competes for [3H]quinuclidinyl benzilate-binding sites with high and low affinity, while oxotremorine shows only high affinity binding. We suggest that the receptor state common to both agonists is the state associated with inhibition of adenylate cyclase, while the very low affinity binding site seen only with carbachol is associated with the PhI response. We also consider the possibility that both responses are caused by a single receptor state that is efficiently coupled to adenylate cyclase inhibition and inefficiently coupled to PhI hydrolysis. Whichever mechanism is correct, our findings demonstrate that muscarinic receptors coupled to adenylate cyclase and the PhI response can be differentiated by virtue of their sensitivity to agonist and the efficiency with which some agonists induce receptor change and elicit receptor-mediated biochemical responses.