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

Neuropeptides (1994) 21, 31-37 ,Q Longman Group Ltd 1994

Neuropeptide Y Inhibits Vasoactive Intestinal Peptide-stimulated Adenylyl Cyclase in Rat Ventral Prostate R. M. SOLANO,

M. J. CARMENA,

L. G. GUIJARRO

Departamento de Bioquimica y Biologia Molecular, Spain (Reprint requests to MJC)

and J. C. PRIETO Universidad

de Alcala, Alcala de Henares,

Abstract-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/effecter 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.

Introduction

localized with noradrenaline in sympathetic vasoconstrictor axons and with vasoactive intestinal peptide (VIP), dynorphin and somatostatin in vasodilator axons.2.6 NPY exerts many biological actions, the best established of which is vasoconstriction sometimes potentiating the effect of noradrenaline.‘.’ The diversity of NPY effects is mirrored in multiple NPY receptor subtypes that have been characterized by biochemical and pharmacological techniques in the central and peripheral nervous systems, and have been classified depending on their specificity for NPY fragments and analogs.8-‘0 The consequence is that NPY receptors activate various signal transduction path-

The neuropeptide Y (NPY) is a 36-amino acid member of the pancreatic polypeptide (PP) family of regulatory peptides. Initially isolated from porcine brain,’ NPY has subsequently been found widely distributed throughout the mammalian central and peripheral nervous systems.‘~’ NPY is coDate received 30 November 1993 Date accepted 31 January 1994 Correspondence to: Dr Maria J. Carmena, Unidad de Neuroendocrinologia Molecular, Departamento de Bioquimica y Biologia Molecuiar, Universidad de Alcala, E-28871 Alcala de Henares, Spain.

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ways (via guanine nucleotide regulatory G-proteins) including inhibition of adenylyl cyclase, generation of inositol phosphates or mobilization of intracellular Ca2+.‘&” A large number of nerves containing NPY and noradrenaline have been found in the male genital tract with the highest density in the prostate, seminal vesicle, corpus cavernosum and vas deferens suggesting that this active neuropeptide may play a role in the control of genital functions.‘3-15 In the prostate, NPY and VIP nerve fibres are localized in the subepithelial connective tissue and smooth muscle layers as well as in the walls of blood vesselsL4which support the involvement of these peptides in the physiological regulation of secretory activity, muscle contractility and blood supply at this level. We have previously shown the presence of specific VIP receptorsI coupled to cyclic AMP stimulation” in rat prostatic epithelial cells. The present study was designed to determine the interaction of NPY with the VIP receptor/effecter system in the rat prostate. Furthermore, we tested the ability of NPY to modify the effects of other adenylyl cyclase stimulatory agents such as the /Iadrenergic agonist isoproterenol or the diterpene forskolin.

Methods

Porcine VIP (chemically identical to rat VIP) and helodermin were obtained from Professor V. Mutt (Karolinska Institute, Stockholm, Sweden). Pituitary adenylyl cyclase activating peptide (PACAP27) was purchased from Peninsula Laboratories (Belmont, CA, USA). NPY, forskolin, isoproterenol, pertussis toxin, and 3-isobutyl-l-methylxanthine (IBMX) were from Sigma (St Louis, MO, USA). ‘*‘I-VIP was prepared by the chloramine-T method at a specific activity of about 250 Ci/g.‘* Adult male Wistar rats (30&400 g) were maintained under standardized environmental conditions (23°C artificial light between 0700-1900 h, and food and water available ad libitum. Epithelial cells were isolated from the ventral lobes of the prostate as described previously.16 A membrane preparation was obtained from ventral prostate by homogenization of the whole tissue in 25 mM tri-

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ethanolamine-HCl buffer (pH 7.5) containing 0.25 M sucrose, 0.5 mM EDTA, and 0.1 mM phenylmethylsulfonylfluoride. The homogenate was centrifuged for 10 min at 600 x g and 4°C. Plasma membranes were separated from the supernatant by centrifugation for 30 min at 25 000 x g and 4°C. Membranes were stored at -80°C until use. The protein concentration was determined by the method of Bradford” using bovine serum albumin as a standard. To study cyclic AMP accumulation, the cells were incubated at 0.3 mg protein/ml with VIP or other agents for 60 min at 15°C in a final volume of 0.5 ml of a medium consisting of 35 mM TrisHCl (pH 7.5) 50 mM NaCl, 1.4% bovine serum albumin, 1 mg/ml bacitracin, and 0.2 mM IBMX.” After the addition of 2.5 ml of methanol, the resulting suspension was centrifuged and the supernatant was evaporated and used for cyclic AMP determination.” The adenylyl cyclase assay was performed as described previously2’ with minor modifications. The incubation medium consisted of 25 mM triethanolamine-HCl buffer (pH 7.4) 1 mM IBMX, 5 mM MgS04, 1 mM EDTA, 1 mg/ml bacitracin, 10 PM GTP, 1.5 mM ATP, an ATP-regenerating system (7.4 mg/ml creatine phosphate and 1 mg/ml creatine kinase), and test substances in a final volume of 0.1 ml. After the addition of membranes (0.2 mg protein/ml), the mixture was incubated at 30°C for 10 min and then heated for 3 min. After refrigeration, 0.3 ml of an alumina slurry (0.25 g/ml) in triethanolamine buffer (pH 7.6) was added, the mixture centrifuged, and then cyclic AMP determined in the supernatant as above. The study of VIP binding was conducted as reported elsewhere.16 Prostatic membranes were incubated with 90 pM 1251-VIPand 0.1-100 unlabelled VIP in a medium similar to that of adenylyl cyclase experiments but excluding IBMX and the ATP-regenerating system. After 60 min at 15°C the reaction was stopped by centrifugation at 13 000 x g for 2 min. Membrane-bound radioactivity was determined and expressed as specific binding, i.e. total minus non-specific binding (about 20% of the total binding as estimated in the presence of 1 PM unlabelled VIP). Fluorescence polarization measurements were performed with a Perkin-Elmer LS-3B spectro-

NPY EFFECTS

IN RAT PROSTATE

33

fluorometer. 3 ml of cell suspension in phosphate buffered saline (about 0.3 absorbance units at 365 nm) were mixed with 1 ~1 of 1.5 mM 1,6-diphenyl1,3,5_hexatriene (DPH, a fluorescent probe dissolved in tetrahydrofuran) and incubated for 15 min at 37°C. The fluorescence polarization (P) was computedz2 following the equation P = (Ivy I,,)/@,, + Ivh) where I,, and Ivh are the fluorescence intensities observed with the analyzing polarizer parallel and perpendicular to the polarizer excitation beam, respectively. Then, lipid microviscosity was estimated by the empirical relation: 2P/(O.46 - P). Each individual experiment was performed in triplicate. The results are expressed as the mean + SEM. For statistical evaluation, Student’s t-test was used. Results

NPY (100 nM) inhibited in a non-competitive manner the stimulating effect of VIP on cyclic AMP accumulation in rat prostatic epithelial cells whereas the basal values remained unaffected (Fig. I A). However, half-maximal stimulation (EDJ of

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cyclic AMP levels was elicited at the same VIP dose (2.5 nM) both in the absence and presence of the inhibitory neuropeptide. In contrast, NPY was ineffective for inhibiting the cyclic AMP accumulation induced by increasing doses of either the /Iadrenergic agonist isoproterenol (Fig. 1B) or the diterpene forskolin (Fig. 1C). Interestingly, the use of some VIP structurally-related peptides showed (Fig. 2) that NPY could reduce by 50% the PACAPstimulated adenylyl cyclase activity in rat prostatic membranes, whereas the stimulating response to helodermin remained unaffected by NPY. As shown in Figure 3, NPY (0.1 nM-1 PM) inhibited VIP-induced cyclic AMP accumulation in a concentration-dependent manner, with maximal inhibition (30%) attained at 30 nM NPY. NPY was ineffective for inhibiting either the /I-adrenergic stimulation or the basal values of the cyclic AMP levels. Additional experiments were performed to search for the possible cause of the selective inhibitory effect of NPY on VIP stimulation of cyclic AMP accumulation in rat prostatic epithelial cells. Fluorescence polarization measurements with DPH

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Fig. 1 Effect of NPY on cyclic AMP accumulation in rat prostatic epithelial cells. The cells were incubated with increasing concentrations of VIP (A), isoproterenol (B), and forskolin (C) in the absence (closed symbols) or presence (open symbols) of 0.1 pM NPY. Data are mean + SEM of 46 experiments. *p < 0.05.

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using cells previously incubated with increasing NPY concentrations did not support any significant modification of lipid membrane microviscosity even at the highest dose (1 ,uM) of neuropeptide tested (Fig. 4). On the other hand, NPY did not alter the interaction of VIP with its corresponding membrane receptors: as shown in Figure 5, the extent of ‘251-VIPbinding was competitively inhibited by increasing concentrations of unlabelled VIP but it remained unaffected by the presence of varying doses (0.1 nM-0.1 ,uM) of NPY. Finally, we analyzed the involvement of inhibitory guanine regulatory proteins (Gi) in the observed NPY inhibition of VIP-stimulated adenylyl cyclase activity. Rat prostatic membranes were pretreated with 100 nmol/ml pertussis toxin for 30 min at 25°C and then adenylyl cyclase activity was determined. Fig. 6 shows that pertussis toxin

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Fig. 2 Inhibitory effect of NPY on the stimulation of prostatic adenylyl cyclase activity by VIP and related peptides. Rat pros-

tatic membranes were incubated with 0.1 pM NPY and increasing concentrations of VIP (a), PACAP(A) or helodermin (+). The results are expressed in terms of percentage of inhibition of adenylyl cyclase activity in a representative experiment.

II

Dose-dependence of NPY effect on cyclic AMP accumulation in rat prostatic epithelial cells. The cells were incubated in the absence (0) or presence of 0.1 PM VIP (0) or 0.1 mM isoproterenol (A) with increasing NPY concentrations. Data are mean f SEM of 4 experiments. Fig. 3

blocked the NPY inhibition adenylyl cyclase activity.

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Discussion

The present results show that NPY inhibits adenylyl cyclase activity in rat ventral prostate. The inhibitory effect was evidenced when using VIP as stimulator of the enzyme activity and it was repeated by studying cyclic AMP accumulation in isolated rat prostatic epithelial cells. NPY did not affect basal cyclic AMP levels, and the observed NPY activity was selective since the neuropeptide did not modify either the /I-adrenergic or the forskolin stimulation of cyclic AMP levels. It is the first indication of the presence of NPY receptors in the prostate gland. We chose VIP to stimulate rat prostatic adenylyl

35

NPY EFFECTS IN RAT PROSTATE

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Effect of NPY on lipid membrane microviscosity in rat prostatic epithelial cells. The cells were pretreated with NPY in the conditions of cyclic AMP experiments, then washed and assayed for microviscosity with DPH as indicated under Methods. Data are mean F SEM of 4 experiments. No significant differences were found when comparing basal and NPY-dependent values.

Fig. 4

cyclase activity since the effect of this neuropeptide has been well studied in this accessory sex gland and shown to be initiated after binding to specific membrane receptors coupled to a stimulating G, protein.‘6s17 The demonstration of the NPY effect in the presence of the phosphodiesterase inhibitor IBMX shows that NPY does not act through an increase of cyclic AMP degradation but through a direct effect on adenylyl cyclase activity, as also shown in this study. The inhibitory activity of NPY on adenylyl cyclase is a well-known feature as previously observed in other tissues23-2s using VIP and/or other agents as stimulators of the enzyme. It is assumed that all of the NPY signal-transduction pathways involve NPY receptors acting via G-proteins.10-‘2, 26In this regard, the present results obtained with pertussis toxin clearly show an abolishment of the inhibitory effect of NPY on VIPstimulated adenylyl cyclase thus suggesting the

involvement of a Gi protein in the NPY-mediated second messenger response. Among various adenylyl cyclase stimulating agents used, we found that NPY inhibited only the response specifically initiated through VIP membrane receptors in the rat prostate. The fact that the stimulating response elicited by the VIP-structurally related peptide PACAPwas blocked by NPY was not unexpected since this peptide binds to common VIP/PACAP receptors in various tissuesz7 On the other hand, the fact that NPY did not modify the action of helodermin (a VIP-related peptide isolated from Gila Monster venom) can be explained from the evidence on the existence of different helodermin-preferring and VIP-preferring receptors in several tissues.‘* The marked preference of the inhibitory effect of NPY for the VIP signal transduction system is further supported by the lack of activity of NPY in experiments with

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Lack ofeffect ofNPY on ‘*‘I-VIP binding to rat prostatic membranes. The membranes were incubated with 90 pM tracer in the absence and presence of increasing concentrations of VIP (0) or NPY (+). Data correspond to a representative experiment. Fig. 5

VIP-unrelated stimulators of adenylyl cyclase such as isoproterenol (which acts through specific Badrenergic membrane receptors) and the diterpene forskolin (which is assumed to act directly on the catalytic subunit of the enzyme). These results could be explained by bearing in mind that NPY contains an amphipathic cc-helical region that may interact with lipids resulting in alterations in the cell-membrane bilayer structurez9 that selectively affect some classes of receptors. Unfortunately, we did not find significant NPY-induced changes of lipid membrane microviscosity in our experimental system. However, in a heterogeneous preparation such as cell membranes the measured microviscosity values must be interpreted as a weighted average of cell lipid domains. 3oThus, although we were unable to directly prove our hypothesis, the present data are compatible with selective effects of NPY on deter-

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Log [VIP], M Effect of pertussis toxin on the inhibitory action of NPY on VIP-stimulated adenylyl cyclase activity. Rat prostatic membranes were preincubated in the absence (0) and presence (0) of 100 nmol/ml pertussis toxin for 30 min at 25°C and then exposed to 0.1 PM NPY and increasing VIP concentrations for the adenylyl cyclase study. The results are expressed in terms of percentage of inhibition of adenylyl cyclase activity in a representative experiment. Fig. 6

mined lipid membrane microdomains surrounding some receptor/effecter systems. Finally, it is clear from present results that NPY does not bind to prostatic VIP receptors since it did not modify the VIP-receptor interaction as expected from their unrelated amino acid sequences. Taken together, our data in the rat ventral prostate suggest the existence of specific NPY receptors coupled to adenylyl cyclase in an inhibitory manner, probably through a Gj protein.

Acknowledgements This work was supported by grants from the Comunidad Autonoma de Madrid (305/92), Fondo de Investigaciones Sanitarias

NPY EFFECTS

37

IN RAT PROSTATE

de la Seguridad Social (93/280), and Direction Investigation Cientifica y Tecnica (92/44).

General

de 16.

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