Standardization of the homogeneous mobility shift assay protocol for evaluation of anti-infliximab antibodies. Application of the method to Crohn's disease patients treated with infliximab.

BACKGROUND: The availability of a quantitative method to measure anti-infliximab (IFX) antibodies (ATI) would facilitate the implementation of therapeutic drug monitoring in clinical decision-making. Our aim was to standardize the homogeneous mobility shift assay (HMSA) used in the measure of ATI levels. METHODS: In this prospective longitudinal multicenter study, 50 IFX-treated Crohn's disease (CD) patients were followed up for 54weeks. During this period 360 human serum samples were analysed. Monomeric ATI levels were measured by a quantitative HMSA-method using an anti-IFX calibrator. IFX trough levels measured by ELISA were correlated with ATI levels. RESULTS: Using HMSA and a pure anti-idiotypic monoclonal antibody specific for IFX (anti-IFX calibrator), we measured the levels of monomeric ATI generated in Crohn's disease patients treated with IFX. Anti-IFX calibrator allowed to quantify monomeric antibodies against IFX with a low limit of quantification (3nM). The threshold level of ATI in order to classify the immunogenicity of the patients was 10nM. We observed that 24% (12/50) of IFX-treated patients developed ATI (>10nM) during the observation period (54weeks). Serum concentration of ATI higher than 10nM dramatically increased the probability (OR=51.1; 95% CI: 20.4-128.0; p<0.0001) of presenting low levels of IFX (⩽1.5nM) in serum, as observed in some CD patients treated with standard doses of the drug. CONCLUSIONS: The HMSA-method described here allows an accurate quantification of ATI concentration in international units (IU) and therefore it could be useful in the study of the relationship between ATI concentration, infliximab level and the clinical response to the drug.

Characterization of adenylyl cyclase stimulated by VIP in rat and mouse peritoneal macrophage membranes.

Vasoactive intestinal peptide (VIP) stimulated adenylyl cyclase activity in rat and mouse peritoneal macrophage membranes. GTP potentiated the stimulatory effect of VIP so that it was routinely included at 10 microM GTP. Other agents like GTP, Gpp(NH)p, GTP-gamma-S, sodium fluoride, and forskolin, at a concentration of 0.1 mM, increased the basal activity of enzyme by 3.1, 5.7, 4.7, 3.6, and 7.8-fold, respectively. The stimulation of adenylyl cyclase by VIP was time, temperature, and membrane concentration dependent. Half-maximal enzyme activation (ED50) was very similar in rat and mouse peritoneal macrophage membranes (1.5 +/- 0.1 nM and 1.0 +/- 0.1 nM, respectively). However, VIP showed more efficacy in mouse macrophages membranes (about 3.1-fold basal values) than that in rat macrophage membranes (about 2.5-fold basal values). The relative potency of several peptides upon stimulation of adenylyl cyclase activity showed the following potency in both species: VIP = PACAP38 = PACAP27 > helodermin > PHI > secretin. On the other hand, a M(r)-45 kDa alpha s subunit of Gs protein was demonstrated by both ADP-ribosylation and immunoblot in mouse and rat peritoneal macrophage membranes. The present results, together other previous, strongly suggest that VIP play an important role in the regulation of macrophage function.

Characterization of vasoactive intestinal peptide receptors in human liver.

The stoichiometric, pharmacological and molecular properties of vasoactive intestinal peptide (VIP) receptors have been analyzed in human liver membranes and compared in parallel with those in rat liver membranes. The binding of [125I]VIP was rapid, saturable and specific. The stoichiometric data indicated the presence of two classes of binding sites in both human and rat liver membranes with Kd values of 0.22 (human) and 0.20 (rat) nM for the high-affinity site, and 27.3 (human) and 3.6 (rat) nM for the low-affinity site. Tracer binding was displaced by structurally related peptides with an order of potency: VIP = PACAP-27 > helodermin > secretin in human liver, and VIP = PACAP-27 = helodermin > secretin in rat liver. GTP inhibited [125I]VIP binding in a dose-dependent manner suggesting the involvement of a G protein in the signal transduction pathway. Cross-linking experiments revealed an apparent molecular mass for the VIP-receptor complex that was 67,500 +/- 2700 and 50,500 +/- 900 in human and rat preparations, respectively. VIP receptors were functional, since VIP stimulated adenylyl cyclase activity in a dose dependent manner with similar efficacy but different potency in human (ED50 = 1.2 nM) and rat (ED50 = 5.8 nM) liver membranes.

Ontogenic development of the adenylyl cyclase enzyme and the alpha s, alpha i1 and alpha i2 G-protein regulatory subunits from rat prostate.

The expression of alpha s, alpha i1 and alpha i2 G-protein subunits measured by immunoblot increased in the rat prostate during sexual maturation, supporting their involvement in proliferation/differentiation. Northern blotting gave transcripts of 1.8 and 4 kb for alpha s, 1.4 and 4.5 kb (mainly) for alpha i1, and 2.4 kb for alpha i2 with levels suggesting a differential regulation (at transcription or post-transcription for alpha s, transcription for alpha i1, and translation for alpha i2). The stimulatory effects of forskolin, vasoactive intestinal peptide (VIP) and isoproterenol on adenylyl cyclase activity increased between 0.5-3 mo, remained constant up to 12 mo and decreased thereafter, conceivably following the expression of VIP and beta-adrenergic receptors. However, G-protein activation of adenylyl cyclase (by GTP and Gpp[NH]p) was maximal at 0.5 mo and then decreased as it occurred with toxin-catalyzed ADP-ribose incorporation to alpha subunits suggesting that other factors are also involved in the regulation of G-protein activity during rat prostatic development.

Identification and functional properties of the pituitary adenylate cyclase activating peptide (PAC1) receptor in human benign hyperplastic prostate.

Pituitary adenylate cyclase activating peptide (PACAP) is a novel neuropeptide with regulatory and trophic functions that is related to vasoactive intestinal peptide (VIP). Here we investigate the expression of specific PACAP receptors (PAC1) and common VIP/PACAP receptors (VPAC1 and VPAC2) in the human hyperplastic prostate by immunological methods. The PAC1 receptor corresponded to a 60-KDa protein whereas the already known VPAC1 and VPAC2 receptors possessed molecular masses of 58 and 68 KDa, respectively. The heterogeneity of VIP/PACAP receptors in this tissue was confirmed by radioligand binding studies using [125I]PACAP-27 by means of stoichiometric and pharmacological experiments. At least two classes of PACAP binding sites showing different affinities could be resolved, with Kd values of 0.81 and 51.4 nM, respectively. The order of potency in displacing [125I]PACAP-27 binding was PACAP-27 approximately equal to PACAP-38 > VIP. PACAP-27 and VIP stimulated similarly adenylate cyclase activity, presumably through common VIP/PACAP receptors. The PAC1 receptor was not coupled to activation of either adenylate cyclase, nitric oxide synthase, or phospholipase C. It appears to be a novel subtype of PAC1 receptor because PACAP-27 (but not PACAP-38 or VIP) led to increased phosphoinositide synthesis, an interesting feature because phosphoinositides are involved via receptor mechanisms in the regulation of cell proliferation.

Lindane decreases forskolin-stimulated cyclic AMP accumulation but does not modify Gs in rat enterocytes.

Treatment of isolated rat enterocytes with the halogenated insecticide lindane (the gamma-isomer of hexachlorocyclohexane, HCCH) did not modify the general membrane fluidity (as estimated by a fluorescence polarization technique) nor the guanine nucleotide binding regulatory protein Gs (as studied by both ADP-ribosylation of its alpha subunit by cholera toxin and Gpp[NH]p stimulation of membrane adenylate cyclase activity). However, lindane decreased in a dose-dependent manner the effect of the diterpene forskolin on direct activation of the adenylate cyclase catalytic subunit. After 5 min of cell treatment with 0.5 mM lindane, the maximal stimulatory effect of forskolin (at 100 microM) decreased by about 50%. There was a certain degree of specificity since delta-HCCH was indeed more potent, whereas dieldrin and endrin (non-lindane related halogenated compounds) behaved as lindane, and alpha- and beta-HCCH were poorly efficient on the inhibition of forskolin stimulation of adenylate cyclase activity. A similar effect of lindane was observed on receptor-stimulated cyclic AMP accumulation by using vasoactive intestinal peptide instead of forskolin. The results on a non-receptor mediated effect of lindane on the adenylate cyclase catalytic subunit itself could be related to: (i) alterations of membrane microdomains surrounding this and other integral proteins which would result in modifications of their activities; and/or (ii) a reciprocal relation between the two main routes of signal transduction so that the activation of protein kinase C (or other Ca(2+)-dependent protein kinases) by lindane would lead to phosphorylation of the adenylate cyclase catalytic subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of vasoactive intestinal peptide receptors and the G protein regulation of adenylyl cyclase in seminal vesicle membranes from streptozotocin-diabetic rats.

The present report describes the status of the vasoactive intestinal peptide (VIP) receptor/effector system of signal transduction in seminal vesicle from streptozotocin (STZ)-treated rats. STZ-treatment modified the binding parameters of the high-affinity sites for VIP in seminal vesicle: 0.78 +/- 0.10 and 2.54 +/- 0.30 nM for the dissociation constant (Kd) in control and diabetic rats, respectively; 0.07 +/- 0.01 and 0.15 +/- 0.03 pmol VIP/mg protein for the maximum binding capacity (Bmax) in control and diabetic rats, respectively. It was associated with a reduced potency of VIP on the stimulation of adenylyl cyclase activity in the diabetic state (ED50 = 64.0 +/- 20.0 nM) as compared to control (ED50 = 9.5 +/- 4.3 nM). In contrast, the stimulatory effects of GTP, Gpp[NH]p and forskolin on the enzyme activity were not modified in diabetic rats. The levels of G-protein subunits in rat seminal vesicle were studied by immunoblot of alpha s and alpha i subunits: whereas alpha i-subunit levels did not vary, those corresponding to alpha s subunit decreased after STZ treatment. In diabetic rats, low concentrations of Gpp[NH]p failed to inhibit forskolin-stimulated adenylyl cyclase activity, suggesting the absence of functional Gi in this condition. In conclusion, present results show a decrease in the sensitivity of the VIP receptor/effector system in seminal vesicle membranes from STZ-treated rats suggesting a physiopathological role for VIP in the seminal neuropathy observed in diabetes.

The effect of streptozotocin diabetes on the vasoactive intestinal peptide receptor/effector system in membranes from rat ventral prostate.

All of the components of the neuropeptide vasoactive intestinal peptide (VIP) signal transduction system were underexpressed in rat prostatic membranes 6 weeks after streptozotocin-induced diabetes. Binding studies with [125I]VIP showed decreases of 86% and 62% in the binding capacity of the high and low affinity classes of VIP receptors in diabetes. Affinity labeling experiments indicated that the main form of VIP receptor was 51 kilodaltons in control rats and 45 kilodaltons in diabetic animals. The efficacy of VIP and forskolin in stimulation of adenylyl cyclase activity as well as the potentiating effect of GTP on VIP action were also reduced in diabetes, as was the expression of the alpha-subunit of the guanine nucleotide-binding regulatory proteins Gs and Gi (studied by ADP ribosylation with cholera and pertussis toxins). Gi function was lost in diabetes, as assessed with experiments on guanyl-5'-yl-imidodiphosphate potentiation of forskolin activity. These disturbances together with previous findings argue for VIP playing a role in the diabetic neuropathy of the genitourinary tract.

Characterization of vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptors in human benign hyperplastic prostate.

Vasoactive intestinal peptide (VIP) is an important member of the group of neuropeptides that appears to be involved in the regulation of prostatic growth and function. Here we studied VIP receptors in membranes from human benign hyperplastic prostate. Accordingly to observations in rat prostatic membranes, [125I]VIP binding to human prostatic membranes suggested two classes of binding sites with high Kd = 0.22 nM) and low (Kd = 37.7 nM) affinities. VIP bound in human and rat membrane preparations to a common VIP/pituitary adenylate cyclase-activating peptide (PACAP) receptor, as VIP, PACAP-27, and PACAP-38 were equipotent for competition of [125I]VIP binding. A PACAP-preferring receptor appears to be expressed in human prostate, since [125I]PACAP binding was displaced with more potency by PACAP than by VIP, and a messenger RNA corresponding to type I PACAP receptor was found. Cross-linking experiments suggested a VIP receptor of about 71 kDa in human and 52 kDa in rat prostates. The binding of [125I]VIP to membranes and the labeling of the bands observed after electrophoresis were competitively inhibited by GTP, suggesting the coupling of VIP receptors to a G protein. Moreover, after solubilization and cross-linking, we observed a 120-kDa band that corresponded to the VIP receptor-alpha s association. VIP stimulated adenylyl cyclase activity in a dose-dependent manner, but the potency and/or the efficacy of VIP were lower in all human preparations studied than in rat prostatic membranes. In conclusion, this study clearly demonstrates the expression of VIP/PACAP common receptors associated with alpha s protein in human prostate and suggests that these neuropeptides could play an important and complex role in the physiology and pathophysiology of this human gland.

Receptors for pituitary adenylate cyclase-activating peptide in human liver.

The presence as well as the pharmacological, molecular, and functional properties of pituitary adenylate cyclase-activating peptide (PACAP) receptors have been analyzed in human liver membranes compared in parallel with vasoactive intestinal peptide (VIP) receptors. [125I]PACAP-27 bound to two classes of receptors with high [dissociation constant (Kd) = 0.47 nmol/L] and low (Kd = 8.0 nmol/L) affinities that represented about 34% and 66% of total binding sites, respectively. The pharmacological profile of [125I]VIP and [125I]PACAP-27 binding to membranes supported the coexistence with VIP receptors of specific receptors for PACAP with a mol wt equal to 67.7K. When [125I]PACAP-27 was used, the order of potency of various related peptides for competition of tracer binding was PACAP-27 greater than PACAP-38 greater than VIP. Both PACAP-27 and VIP stimulated adenylate cyclase activity with similar efficacy, although PACAP-27 showed a potency (half-maximal efficient concentration or EC50 = 0.5 nmol/L) greater than that of VIP (EC50 = 4.1 nmol/L). When the two peptides were present simultaneously in the incubation medium, no additive effect on the stimulation of adenylate cyclase activity was observed, which suggests a unique receptor coupled to this enzyme.

Alpha-2 adrenoceptors modulate the somatostatinergic system and G protein levels in the rat hippocampus.

alpha 2-Adrenoceptor agonists and somatostatin (SS) exert opposite effects on the spike discharge of pyramidal and granule cells in the rat hippocampus. We studied whether clonidine, an alpha 2-adrenoceptor agonist, and yohimbine, an alpha 2-adrenoceptor antagonist, can modulate somatostatin-like immunoreactivity (SSLI) levels, binding of 125I-Tyr11-somatostatin (125I-Tyr11-SS) to its specific receptors, SS-inhibited adenylyl cyclase (AC) activity, and the guanine-nucleotide binding regulatory proteins Gi and G(o) in the rat hippocampus. Clonidine (1 mg/kg, intraperitoneally (IP) or yohimbine (5 mg/kg, IP) injected at both 10 and 16 hours before decapitation did not affect SSLI content in the hippocampus. Clonidine administration decreased the number of specific SS receptors and increased the apparent affinity in hippocampal membranes. This change in SS binding was not the result of a direct effect of clonidine on these receptors because no effect in binding was produced by high concentrations of clonidine (10(-5) M) when added in vitro. Pretreatment with yohimbine prevented the clonidine-induced in SS binding. Yohimbine alone produced a significant increase in the number of 125I-Tyr11-SS receptors and a decrease in its apparent affinity. Clonidine decreased the ADP-ribosylation of a 41- and a 39-kDa G-protein by pertussis toxin (PTX), whereas yohimbine had no effect on the PTX-catalyzed ADP-ribosylation. No significant differences were seen for the basal or for the forskolin (FK)-stimulated AC enzyme activities in the control, clonidine- and/or yohimbine-treated groups. Somatostatin caused a significantly lower inhibition in AC activity in hippocampal membranes of clonidine-treated rats, whereas yohimbine led to an opposite effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of sulpiride on somatostatin receptors, somatostatin-like immunoreactivity and modulation of adenylyl cyclase activity in the rat brain.

The present study investigates the effects of the administration of an intracerebroventricular (i.c.v.) dose of 500 micrograms/rat of the neuroleptic (-) sulpiride on somatostatin-like immunoreactivity (SSLI) levels, 125I-Tyr11-SS binding to its specific receptors, SS-modulated adenylyl cyclase (AC) activity and the pertussis toxin (PTX) substrates measured by toxin-catalysed ADP ribosylation of the alpha-subunits from G-proteins. (-) Sulpiride significantly decreased the SSLI levels in the frontoparietal cortex at 30 min but was without effect on the SSLI concentration in the striatum. This decrease had disappeared within 24 hr. The administration of (-) sulpiride produced a significant increase in the number of 125I-Tyr11-SS receptors and a significant reduction in their affinity at 30 min after injection in the striatum without affecting the frontoparietal cortex. The effects of the (-) sulpiride injection had disappeared after 24 hr. This change in SS binding was not due to a direct effect of (-) sulpiride on these receptors since no effect on binding was produced by high concentrations of (-) sulpiride (10(-5) M) when added in vitro. No significant differences were seen in either brain region for the basal or the forskolin (FK)-stimulated AC enzyme activities in the control and (-) sulpiride groups. In the (-) sulpiride group, the capacity of SS to inhibit FK-stimulated AC in the frontoparietal cortex was significantly higher than in the control group with no significant difference in the striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Somatostatin levels and binding in duodenal mucosa of rabbits with ligation of the pancreatic duct.

Atrophy of the exocrine pancreas was induced in rabbits by pancreatic duct ligation. Somatostatin concentration and binding in cytosol from rabbit duodenal mucosa were studied after 6 and 14 weeks of pancreatic duct ligation. Somatostatin-like immunoreactivity was significantly increased in the duodenal mucosa in both periods. Scatchard analysis showed a parallel increase in the number of binding sites rather than a change in their affinity. The physiological significance of these findings remains to be clarified.

Adenylyl cyclase stimulation by VIP in rat seminal vesicle membranes.

Vasoactive intestinal peptide (VIP) stimulated adenylyl cyclase activity in membranes from rat seminal vesicle. GTP potentiated the stimulatory effect of VIP so that it was routinely included at 10 microM. The stimulation of adenylyl cyclase by VIP was time and temperature dependent. The response was linear with time up to 15 min at 30 degrees C. Half-maximal adenylyl cyclase activation (in the presence of 10 microM GTP) was achieved at 3.0 nM VIP. The enzyme activity increased about 150% with respect to basal values at the maximal VIP concentration tested (1 microM). The relative potency of peptides upon stimulation of adenylyl cyclase activity was: VIP greater than helodermin greater than peptide histidine isoleucinamide greater than rat growth hormone-releasing factor. Other agents like GTP (0.1 mM), GppNHp (0.1 mM), forskolin (0.1 mM) and sodium fluoride (10 mM) increased the adenylyl cyclase activity 1.8-, 4.4-, 6.7- and 2.4-fold, respectively. Taken together, the presence of VIP in nerve terminals innervating the seminal vesicle of rats and the existence of VIP receptors coupled to adenylyl cyclase strongly suggest a physiological role for this neuropeptide in the modulation of seminal vesicle cell function.

VIP receptor/effector system in liver membranes from cholestatic rats.

A reduced efficacy of VIP (43% of the control) without modification in its potency (ED50 = 2.2 nM) was observed in regenerating rat liver after cholestasis (bile duct ligation). The same occurred with glucagon-stimulated adenylyl cyclase activity because the efficacy of this VIP-related peptide was also reduced (53% of the control) without changes in its potency in this experimental model. The equilibrium binding data revealed no changes in either the affinity or the VIP binding capacity of liver membranes during cholestasis. Cross-linking experiments gave the same apparent molecular mass for the liver VIP-receptor complex (52 kDa) in control and cholestatic rats. The coupling between the VIP receptor and the Gs-protein was also unaffected because the sensitivity of VIP binding to GTP did not change after bile duct ligation. However, liver membranes from cholestatic rats showed a low extent of both ADP-ribosylation of the Gs-protein alpha subunit (as assessed with cholera toxin) and adenylyl cyclase stimulation by a direct effector such as forskolin. Thus, VIP-stimulated adenylyl cyclase activity is decreased in regenerating liver after cholestasis due probably to an impairment in the interaction between Gs-protein and adenylyl cyclase as well as a defect in the enzyme itself.

Expression of vasoactive intestinal peptide (VIP) receptors in human uterus.

We show the existence of functional vasoactive intestinal peptide (VIP) receptors in normal human female genital tract (endometrium, myometrium, ovary and Fallopian tube) as well as in leiomyoma (a frequent uterine pathology). The correlation between VIP binding and stimulation of adenylyl cyclase activity for all studied tissues was linear (r = 0.86) suggesting the expression of VIP receptors throughout the human female genital tract. Immunodetection of VIP receptor subtypes gave different molecular weights for VPAC(1) (47 kDa primarily) and VPAC(2) (65 kDa), which may be due to different glycosylation extents. In conclusion, this study demonstrates the expression of both subtypes of VIP receptors and their functionality in human female genital tract, suggesting that this neuropeptide could play an important physiological and pathophysiological role at this level.

Inhibitory effect of cyclosporin A peptide on rat hepatocytes proliferation induced by mitogens.

Treatment of cultured rat hepatocytes with cyclosporin A (0.01-1 microM) for 24, 48, or 72 h in the presence of insulin and epidermal growth factor induced an inhibition on cell proliferation in a time- and concentration-dependent manner, with an IC50 = 0.05 microM CsA corresponding to 48-h treatment. The inhibitory effect of CsA at < or = 0.1 microM doses for 48 h on [3H]thymidine uptake was reversed after withdrawal of the drug and subsequent addition of insulin plus EGF or serum; however, at 1 microM CsA the effect was irreversible and numerous bright small vesicles were observed. The molecular mechanism involved in CsA action in hepatocytes seems to be independent on cAMP and pertussis-toxin sensitive G proteins.

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.

Somatostatin binding sites in cytosolic fraction isolated from rabbit antral and fundic gastric mucosa.

Specific binding sites for somatostatin have been identified and characterized in cytosolic fraction of rabbit gastric mucosa at both antrum and fundus levels. The binding depended on time, temperature and pH, and was reversible and saturable. The stoichiometric data suggested the presence of two classes of binding sites: a class with high affinity (Kd = 26.7 and 37.0 nM in antrum and fundus, respectively) and low capacity (2.1 and 4.1 pmol somatostatin/mg protein in antrum and fundus, respectively), and a class with low affinity (Kd = 246.4 and 162.5 nM in antrum and fundus, respectively) and high capacity (134.1 and 110.9 pmol somatostatin/mg protein in antrum and fundus, respectively) at 25 degrees C and pH 7.4. The binding sites were shown to be highly specific for somatostatin since neuropeptides such as Leu-enkephalin, neurotensin and substance P behaved as ligands with very low affinity.

Neuropeptide Y inhibits vasoactive intestinal peptide-stimulated adenylyl cyclase in rat ventral prostate.

Neuropeptide Y (NPY), a peptide present in the prostate gland, was found to inhibit vasoactive intestinal peptide (VIP)-stimulated cyclic AMP accumulation in isolated rat prostatic epithelial cells as well as VIP-stimulated adenylyl cyclase activity in rat prostatic membranes. The inhibitory effect of NPY was selective for the VIP receptor/effector system since it was also observed when using pituitary adenylyl cyclase activating peptide (PACAP-27) which presumably recognizes VIP receptors in this gland, but not when using unrelated substances such as isoproterenol or forskolin. NPY did not modify either the general lipid membrane microviscosity or the VIP-receptor binding. The inhibitory effect of VIP was blocked by pretreatment of the prostatic membranes with pertussis toxin. These results suggest the presence of NPY receptors in rat ventral prostate coupled in an inhibitory manner to adenylyl cyclase through a guanine nucleotide regulatory Gi protein.