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.

Differential effect of arachidonic acid on the vasoactive intestinal peptide receptor/effector system in rat prostatic epithelium during sexual maturation.

The effects of alterations in the membrane lipid environment on vasoactive intestinal peptide (VIP) binding and VIP-stimulated cyclic AMP accumulation have been analyzed by arachidonic acid treatment of prostatic epithelial cells from rats at puberty and maturity, two critical developmental periods with characteristic lipidic and androgenic statuses. Treating cells with 0.1 mM arachidonic acid for 15 min at 37 degrees C increased the affinity of VIP receptors and the potency of the neuropeptide (up to five times) in the formation of cyclic AMP at maturity, but not at puberty. The average plasma membrane fluidity (as measured by fluorescence polarization of diphenylhexatriene) remained unmodified after arachidonic acid treatment of cells. The modifications observed in mature rats were specific for the VIP receptor/effector system, since cyclic AMP stimulation by isoproterenol or forskolin was not affected by cell treatment with arachidonic acid. These results are compatible with the existence of a particular lipidic microdomain surrounding the VIP receptor in the cell membrane that would be altered by exposure to arachidonic acid (either directly or through conversion of arachidonic acid to its metabolites, as suggested by experiments on inhibition of the arachidonic acid cascade). This would make it possible for the activation of protein kinase C to phosphorylate VIP receptors in cells from mature rats, but not in those from pubertal animals with a very different membrane lipid composition (as suggested by the corresponding values of membrane fluidity and transition temperature).

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.

Beta-adrenergic stimulation of cyclic AMP accumulation in rat prostatic epithelial cells during sexual maturation.

The effect of age, castration and androgen-replacement therapy upon the stimulatory activity of the beta-adrenergic agonist isoproterenol on cyclic AMP accumulation was determined in rat prostatic epithelial cells. The potency of isoproterenol was similar in all the experimental models (ED50 = 0.3-0.4 microM). Mature animals showed a lower efficiency of isoproterenol than that of pubertal rats. Pubertal rats (which are absolutely dependent on androgens) exhibited an increase of the responsiveness of prostatic cyclic AMP to isoproterenol after castration, normalization being reached after subsequent testosterone treatment; this feature could be reproduced by in vitro incubation of the corresponding cells with testosterone or dihydrotestosterone. Mature rats (which are relatively dependent on androgens) maintained the efficiency of isoproterenol upon prostatic cyclic AMP after castration, and testosterone therapy elicited an increase of this activity. The present study contributes original observations mainly in the puberty period and supports the importance of the androgenic status not only for direct actions of androgens but also for the regulation of many other hormone/neurotransmitter/growth factor receptor-effector systems.

Modulation of the beta-adrenergic stimulation of cyclic AMP accumulation in rat prostatic epithelial cells by membrane fluidity.

1. The influence of membrane lipid composition on the beta-adrenergic stimulation of cyclic AMP accumulation in rat prostatic epithelial cells was assessed after treating the cells with cholesteryl hemisuccinate (ChH). 2. ChH treatment resulted in a 40% inhibition of the beta-adrenergic response after 30 min of lipid preincubation at 37 degrees C. 3. The inhibitory effect of ChH was dose-dependent and was accompanied by an increase of microviscosity as measured by a fluorescence polarization technique with the probe diphenylhexatriene. 4. Experiments with increasing concentrations of isoproterenol indicated that the efficiency, but not the potency, of the beta-adrenergic response was affected by the increasing of the cholesterol content in the cell membrane.

Identification of specific binding sites for vasoactive intestinal peptide in rat testis Leydig cells and study of developmental changes.

The interaction of vasoactive intestinal peptide (VIP) with isolated Leydig cells from rat testis was time- and temperature-dependent, as well as saturable and specific. Scatchard analysis suggested the presence of both high- and low-affinity binding sites with KD values of 1.7 and 43 nM, respectively, and receptor concentrations of 35 and 1394 fmol VIP bound/mg protein in mature (3- to 6-month old) rats. When considering pubertal (45-day old) rats, the affinities were similar but the binding capacities showed considerably lower values (25 and 193 fmol VIP bound/mg protein) indicating that VIP receptors are subject to developmental changes during animal maturation.

Characterization and age dependence of the stimulatory effect of VIP on cyclic AMP accumulation in rat Leydig cells.

The neuropeptide vasoactive intestinal peptide (VIP) has been shown to stimulate cyclic AMP accumulation in Leydig cells isolated from rat testis. The effect was dependent on time, temperature and cell concentration. At 15 degrees C, half-maximal and maximal stimulation were observed at about 1 and 100 nM VIP, respectively. The interaction was specific since an order of potencies chicken VIP greater than rat VIP greater than secretin greater than glucagon and no effect of neurotensin and substance P were obtained. The efficiency of VIP was lower in pubertal rats and then increased in young-adult and adult animals. These results together with the known presence of VIP in the testis support the idea that VIP may be involved in the regulation and function of Leydig cells during development.