J.C. Prieto

Receptors for vasoactive intestinal peptide on isolated epithelial cells of rat ventral prostate

Receptors for porcine vasoactive intestinal peptide have been characterized in isolated epithelial cells of rat ventral prostate. The interaction of 125I-labelled VIP with cells was rapid, reversible, specific, saturable and dependent on temperature. Degradation of peptide and receptors was minimized at 15 degrees C. At apparent equilibrium, the binding of 125I-labelled peptide was competitively inhibited by native VIP in the 1 X 10(-10)-10(-7)M range concentration. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 4.0 nM and a low binding capacity (0.12 pmol VIP/mg cell protein), and a low-affinity class with a Kd = 17.8 nM and a high binding capacity (1.6 pmol VIP/mg cell protein). Chicken VIP and porcine secretin exhibited a 7-fold higher and a 7-fold lower affinity than porcine VIP for binding sites, respectively. Glucagon, Leu-enkephalin, Met-enkephalin and somatostatin were ineffective. The presence of high-affinity receptors for VIP together with previous reports on the occurrence of VIP-containing neurones innervating the male genitourinary tract strongly suggest that this peptide may be important in the physiological regulation of the functions of prostatic epithelium.

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.

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.

Role of lindane in membranes. Effects on membrane fluidity and activity of membrane-bound proteins

The influence of lindane (gamma-hexachlorocyclohexane) on fluidity of plasma membranes from rat renal cortical tubules has been investigated. Preincubation with lindane increased membrane fluidity. This effect was accompanied by (i) a decrease in the transport of glucose with regard to the controls and (ii) an inhibition of the β-adrenergic stimulatory activity upon cyclic AMP accumulation. However, a significant decrease of the membrane fluidity was found when rats were injected with lindane for 12 days. The injection of lindane exerted the opposite effect on the membrane proteins, the glucose transporter and the β-adrenergic receptor, enhancing the glucose uptake and increasing the isoproterenol-stimulated cycle AMP accumulation. A possible explanation of the difference could involve a resistance to membrane disordering by lindane through a regulatory mechanism that would balance the activity of many lindane-sensitive proteins in insecticide-injected rats.

Effects of age and androgens upon functional vasoactive intestinal peptide receptors in rat prostatic epithelial cells

The specific binding of vasoactive intestinal peptide (VIP) and the stimulatory effect of VIP upon cyclic AMP accumulation in isolated epithelial cells of rat ventral prostate were age dependent. The number of VIP receptors decreased but the efficiency of VIP on cyclic AMP accumulation increased in prostatic epithelium when considering the periods 35-65 days and 3-6 months. Since these features could be related to the known age-related decrease of androgen and androgen-receptor levels, we studied the effect of testosterone and its 5 alpha-reduced metabolite dihydrotestosterone upon both steps of VIP action. The two steroid hormones exerted a non-competitive inhibition on VIP-induced cyclic AMP accumulation but did not modify VIP binding to its specific receptors. This modulatory effect of androgens might involve their interaction with specific sites on the cell membrane leading to modifications of membrane activities including adenylate cyclase, as has been suggested by an increasing number of recent reports.

Cyclic AMP response to vasoactive intestinal peptide andβ-adrenergic or cholinergic agonists in isolated epithelial cells of rat ventral prostate

Vasoactive intestinal peptide (VIP) and the β-adrenergic agonist isoproterenol stimulated cyclic AMP formation through independent receptors in isolated epithelial ceils of rat ventral prostate. The specific β-adrenergic antagonist propranolol inhibited the stimulatory effect of isoproterenol but not that of VIP. Besides small differences in the efficiency of both agents, results indicated that isoproterenol was 500 times less potent than VIP. Acetylcholine did not modify the basal cyclic AMP levels but inhibited the accumulation of the cyclic nucleotide in the presence of either VIP or isoproterenol. The inhibitory action of muscarinic receptors was calcium-dependent. The coexistence of receptors for cholinergic, adrenergic and peptidergic agents which can regulate cyclic AMP suggests that the functions of prostatic epithelium may be interdependently controlled by multiple neural effectors.

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.

Different sites of action of arachidonic acid on steroidogenesis in rat Leydig cells

The present study in purified rat Leydig cells shows that arachidonic acid may act as an intratesticular factor regulating LH-mediated testicular steroidogenesis. Arachidonic acid decreased, in a dose-dependent manner, the LH-stimulated cAMP and testosterone levels, over 2 h incubation. Incubation of Leydig cells with arachidonic acid did not modify 125I-hCG binding to the cells as compared to control, showing that the action of arachidonic acid is not related to a decrease of hCG binding to the cells. Forskolin-stimulated cAMP and testosterone production were inhibited by 51.65 and 70.9%, respectively, in the presence of arachidonic acid (100 microM), although the ED50 for the diterpene was not changed. When isobutyl-methyl-xanthine was added to the incubation medium, the same percentage of inhibition was found indicating that arachidonic acid inhibition of cAMP production is not due to stimulation of Leydig cell phosphodiesterase activity. Pretreatment of the cells with pertussis toxin, to inactivate Gi, was also without effect on arachidonic acid inhibition of LH-stimulated cAMP production, but pertussis toxin abolished the inhibitory effects of arachidonic acid when adenylate cyclase was stimulated with forskolin. However, arachidonic acid addition resulted in inhibition of LH- and forskolin-stimulated testosterone production, even if the cells were pretreated with pertussis toxin. It can be concluded that: (1) The inhibitory effect of arachidonic acid is neither due to a decrease of hCG binding to Leydig cells nor to a stimulation of cell phosphodiesterase activity; (2) arachidonic acid modulates cAMP production at two different levels, either by activation of Gi protein and by inhibition of Gs protein or adenylate cyclase; (3) the effect of arachidonic acid on steroidogenesis is also beyond cAMP formation.

5-Hydroxytryptamine1A Receptor-Mediated Effects on Adenylate Cyclase and Nitric Oxide Synthase Activities in Rat Ventral Prostate

The rat ventral prostate possesses specific 5-hydroxytryptamine (5-HT1A) receptors coupled to adenylate cyclase. In vivo treatment of rats or in vitro preincubation of minced prostatic tissue with the 5-HT1A receptor agonist 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH-DPAT) in different experimental conditions shows the possibility of desensitisation mechanisms with switching from inhibitory to stimulatory pattern on adenylate cyclase activity. As in the majority of systems, we observed the inhibition of forskolin-stimulated adenylate cyclase activity as a functional correlate of 5-HT1A receptor activation. A similar feature occurred when the direct stimulation of the enzyme by the diterpene was replaced by a receptor-mediated activation with the neuropeptide vasoactive intestinal peptide. Furthermore, 8-OH-DPAT stimulated nitric oxide synthase (NOS) activity in a dose-dependent manner. Thus, serotonin appears to be able to act in the rat prostate gland through specific 5-HT1A receptors coupled to a complex system of signal transduction involving an inhibitory response of adenylate cyclase that can become stimulatory, as well as an enhancement of NOS activity.

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.