Identification and Functional Properties of the Pituitary Adenylate Cyclase Activating Peptide (PAC1) Receptor in Human Benign Hyperplastic Prostate

Cell. Signal. Vol. 11, No. 11, pp. 813–819, 1999 Copyright  1999 Elsevier Science Inc.

ISSN 0898-6568/99 $ – see front matter PII S0898-6568(99)00052-2

Identification and Functional Properties of the Pituitary Adenylate Cyclase Activating Peptide (PAC1) Receptor in Human Benign Hyperplastic Prostate R. M. Solano,†§ M. J. Carmena,†§ R. Busto,† M. Sa´nchez-Chapado,‡ L. G. Guijarro† and J. C. Prieto*† †Unidad de Neuroendocrinologi´a Molecular, Departamento de Bioqui´mica y Biologi´a Molecular, and ‡Departamento de Ciencias Morfolo´gicas y Cirugi´a, Universidad de Alcala´, E-28871 Alcala´ de Henares, Spain

ABSTRACT. 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 < 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. cell signal 11;11:813–819, 1999.  1999 Elsevier Science Inc. KEY WORDS. PACAP, VIP, VIP/PACAP receptors, Adenylate cyclase, Phosphoinositide, Human prostate

INTRODUCTION Pituitary adenylate cyclase activating peptide (PACAP) is a member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family of structurally related peptides. This neuropeptide was initially isolated from ovine hypothalamus for its ability to stimulate adenylate cyclase activity and further shown to exist in two biologically active forms with 27 amino acids (PACAP-27) and 38 amino acids (PACAP38, showing the same NH2-terminal 27 residues) [1]. PACAP binds to membrane receptors that are positively coupled to G-proteins. At least three distinct types of PACAP receptors have been cloned and characterized by pharmacological means, showing that they are shared by VIP to different extents in a variety of tissues [2]. The nomenclature of VIP/PACAP receptors has been revised recently in accordance with the International Union of Pharmacology *Author to whom correspondence should be addressed. Tel.: 134-91-8854527; fax: 134-91-885-4585; e-mail: [email protected] §R. M. Solano and M. J. Carmena contributed equally to this work. Received 28 March 1999; accepted 27 May 1999.

[3]. The PAC1 receptor binds PACAP with high affinity but binds VIP with lower affinity and presents splice variants leading to variability in tissue expression and activation of either adenylate cyclase or phospholipase C [4–6]. The common VIP/PACAP receptors bind VIP and PACAP with similar affinity, act through adenylate cyclase stimulation, and are identical to VIP receptors: the VPAC1 receptor is the “classic” VIP binding site and shows low binding affinity for secretin, whereas the VPAC2 receptor is a “helodermin-preferring” site that exhibits no secretin binding but binds helodermin more potently than VIP and PACAP [3, 7, 8]. PACAP is widely distributed, occurring in the central nervous system and many peripheral tissues, with high abundance in brain and testes [1]. Both PACAP-27 and PACAP-38 are expressed in rat testis and play a regulatory role on Leydig and Sertoli cell functions [9–11]. However, reports regarding PACAP receptors in the human genitourinary tract are scarce. Preliminary results from our group point to the possibility of a PACAP-preferring receptor in human hyperplastic prostate [12]. Additional data suggest

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that this receptor is present in human prostate cancer cells and that prostate cancer cell growth is inhibited by the antagonist PACAP (6-38) [13]. VIP-immunoreactive nerve fibres are present in the human prostate and other sexual accessory glands that are under the primary control of testicular hormones [14, 15]. Our group has recently shown the existence of VIP/PACAP common receptors in human benign hyperplastic prostate that are positively coupled to adenylate cyclase [16]. Moreover, VIP potentiates the invasive capacity of the androgen-responsive human prosthetic carcinoma cell line LNCaP by a cAMP-dependent mechanism [17]. In addition to this, the role of other neuropeptides such as bombesin, TSH-like, PTH-like, and calcitonin-like peptides on growth and/or tumour development of the prostate has been suggested [15, 18, 19]. As an approach to ascertain the role of PACAP receptors in the pathogenesis of human prostate diseases, we have studied the expression as well as the characteristics of PACAP receptors and their signalling pathways in human benign hyperplastic prostate at the molecular and cellular levels. MATERIALS AND METHODS Prostate Tissue Processing Prostate tissue samples were obtained from 20 patients (age range, 65–85 years) who underwent surgery for transurethral or open resection for benign prostatic hyperplasia. Tissues were immediately used for immunohistochemistry or frozen in liquid nitrogen and stored at 2808C for other purposes. Prostate membranes were prepared as previously described [20] by homogenization of tissue samples at 48C in 25 mM triethanolamine-HCl buffer (pH 7.5) containing 0.25 mM sucrose, 0.1 mM phenylmethylsulfonilfluoride (PMSF) and 0.5 mM EDTA, using a glass/Teflon homogenizer. After centrifugation at 600 3 g for 10 minutes at 48C, the low gravity supernatant was collected and centrifuged again at 25,000 3 g for 30 min at 48C. Membranes were collected and stored at 2808C until use. The protein concentration was determined [21] using bovine serum albumin (BSA) as a standard. Western Blot Analysis of PAC1, VPAC1, and VPAC2 Receptors The primary antibody used for detection of human PAC1 receptor was kindly provided by Dr. A. Arimura (Hebert Center, Tulane University, Belle Chase, LA, USA) and was raised in rabbits against a synthetic peptide corresponding to the 25 amino acid sequence from Lys411 to Ala435 of human PAC1 receptor residing in the C-terminal intracellular domain [22]. The primary antibodies used for detection of human VPAC1 and VPAC2 receptors were kindly supplied by Dr. E.J. Goetzl (University of California, San Francisco, CA, USA) and were raised in rabbits against the 391–457 amino acid sequence at the C-terminal cytoplasmatic tail and the 19–37 amino acid sequence at the N-terminal extracellular

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domain, respectively [23]. To allow the recognition of nonspecific staining, the primary antibodies were preadsorbed with the synthetic peptide used for immunization. Prostate membranes were either directly used for immunodetection of PAC1 receptor or solubilized for immunodetection of VPAC1 and VPAC2 receptors. Membrane solubilization was performed in 50 mM Tris-HCl buffer (pH 7.4), 1% (v/v) Triton X-100 and 0.01% trypsin inhibitor for 30 min at 48C. After centrifugation at 38,000 3 g for 15 min at 48C, the supernatant (for VPAC1 and VPAC2 receptors) or the membrane suspension (for PAC1 receptors) were mixed with the same volume of 50 mM Tris-HCl buffer (pH 6.8) containing 20% glycerol, 6% SDS, 10% b-mercaptoethanol and 0.05% bromophenol blue. Proteins were run on 10% SDS-PAGE and then transferred to nitrocellulose sheets (Hybond, Amersham Pharmacia Biotech, Barcelona, Spain) for immunodetection with the corresponding primary antibodies (1:1000 dilution for PAC1 receptor and 1:200 dilution for VPAC1 and VPAC2 receptors, respectively). The immunoreactive proteins were revealed using peroxidase-conjugated goat anti-rabbit IgG and analysed by luminescence according to a standard protocol from Pierce (Rockford, IL, USA). Molecular weight markers (Bio-Rad, Hercules, CA, USA) were used to determine the molecular weights of receptor immunoreactivities. Receptor Binding Studies Binding experiments for membrane-bound receptors were performed as described previously [16]. Competitive binding assays were carried out on membranes (0.1–0.2 mg/ml) with 124 pM [125I]PACAP-27 and various amounts of the unlabelled peptides PACAP-27, PACAP-38, or VIP (Peninsula Laboratories, St. Helens, UK) in 0.25 ml of 50 mM Tris-HCl buffer containing 1% BSA, 0.5 mg/ml bacitracin, 5 mM MgCl2, 200 KIU/ml aprotinin and 2 mg/ml PMSF. After incubation for 60 min at 258C, membrane-bound peptide was separated by centrifugation and counted for radioactivity. Nonspecific binding was determined by incubation in the presence of an excess (0.3 mM) of unlabelled PACAP-27. Some experiments were performed with [125I]VIP in similar conditions. Results are expressed as means 6 S.E.M. Each determination was carried out in duplicate or triplicate. Adenylate Cyclase Assay Adenylate cyclase activity was measured as described previously [16]. PACAP-27, PACAP-38, or VIP were added to 0.1 ml of membrane suspension containing 1.5 mM ATP, an ATP-regenerating system, 2 mM 3-isobutyl-1-methylxanthine, 2 mM EDTA and 2 mg/ml bacitracin. The reaction was stopped after 10 min at 308C by boiling for 3 min and refrigeration. After addition of 0.2 ml of an alumina slurry (0.25 g/ml in triethanolamine-HCl buffer, pH 7.6), the supernatant was taken for cAMP assay [24].

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Inositol Phosphate Assay For measurement of inositol phosphates (IPs), prostate tissue slices were labeled with 5 mCi myo-[2-3H]inositol solution (DuPont NEN) for 90 min at 378C in a Hank’s/Hepes solution (pH 7.4) supplemented with 6 mM glucose and 0.5% BSA under 95% O2-5% CO2. Following centrifugation and aspiration of the supernatant, the tissue pieces were incubated in 0.3 ml of the same buffer supplemented with 10 mM LiCl in the presence of 1 mM peptide. After 1 min at 378C, 0.3 ml of cold 15% trichloroacetic acid was added. The precipitate was recovered by centrifugation and used for extraction of total IPs using strong anion exchange chromatography (Dowex AG 1-X8) [25]. Phosphatidylinositol Synthesis Incorporation of myo-[2-3H]inositol into phosphoinositides was studied as previously described [26], in both the absence and the presence of PACAP-related peptides. Minced prostate tissue was incubated with 5 mCi myo-[2-3H]inositol and the corresponding peptide in a Hank’s-Hepes solution (pH 7.4) prepared as for the inositol phosphate assay. After 90 min at 378C, the reaction was terminated as above and lipids were extracted as described elsewhere [27] for measurement of the phosphoinositide-associated radioactivity. Measurement of NOS Activity NOS activity was quantitated by monitoring the accumulation of nitrate in the incubation media from the prostate tissue, as described elsewhere [28]. Finely minced prostate tissue was homogenized in a Hank’s-Hepes solution (pH 7.4) and centrifuged for 10 min at 2000 3 g. An aliquot (0.15 ml) of the supernatant was incubated in a 0.4 final volume of a mixture containing the same buffer, 0.12 mM NADPH, 1 mM CaCl2 and the effectors. After 30 min at 378C, the reaction was stopped by the addition of 0.6 ml of Griess reagent. The reaction of NO22 with this reagent produces a pink colour, which was quantified at 540 nm against standards in the same buffer. RESULTS Immunodetection of PACAP Receptors To determine the expression of the PACAP-preferring PAC1 receptor in the human prostate gland, we used a previously characterized polyclonal antibody raised against their C-terminal intracellular domain [22]. As shown by Western blot analysis (Fig. 1), this PACAP receptor antibody specifically reacted against a protein of about 60 KDa, consistent with the molecular weight of the native PAC1 receptor [1, 2]. Another band of 72 KDa was evident under the reducing conditions used but it disappeared in non-reducing experiments (not shown), suggesting the existence of intramolecular disulfide bridges in the PAC1 receptor. Figure 1 also shows Western blots of common VIP/PACAP (VPAC1 and VPAC2) receptors in human prostate tissue as

FIGURE 1. Immunoblotting of human prostate PACAP/VIP re-

ceptors. Membranes were resolved and immunoblotted using specific antisera against specific PACAP receptors (PAC1) and common VIP/PACAP receptors (VPAC1 and VPAC2) as described in Materials and Methods. Lanes 1 correspond to specificity controls using preincubation of each antibody with the corresponding immunizing peptide. Reference protein-size markers are shown.

developed with previously characterized antibodies [23]. A major protein of approximately 58 KDa was observed for VPAC1 receptor whereas the parallel Western blot for VPAC2 receptor showed a major band of about 68 KDa. The specificity of the three antibodies was estimated by their preincubation with the corresponding immunizing peptides (see Materials and Methods). As shown in Figure 1, this preadsorption step completely abolished the immunostaining. Binding of [125I]PACAP-27 to Human Prostate Membranes In competition studies, the specific binding of [125I]PACAP27 to membranes from human benign hyperplastic prostate was inhibited by increasing concentrations of unlabelled PACAP-27 (Fig. 2). An important variability was seen in the values obtained for the individual patients. The corresponding analysis [29] resulted in upwardly concave curves suggesting receptor heterogeneity. The interpretation by a nonlinear curve-fitting program was resolved in terms of two classes of binding sites. The Kd and maximum binding capacity (Bmax) values were 0.81 6 0.07 nM and 1.09 6 0.36 pmol/mg of protein for the high-affinity sites whereas the corresponding parameters for the low-affinity sites were 51.4 6 6.6 nM and 11.0 6 1.1 pmol/mg of protein, respectively. The pharmacological characterization of [125I]PACAP-27 binding to prostate membranes was performed by competition experiments with various PACAP-related peptides (Fig. 3). PACAP-38 and PACAP-27 were nearly equipotent (IC50 values between 0.8–1.0 nM) but they were approximately 3-fold more potent than VIP in inhibiting tracer binding. Signal Transduction Through PACAP Receptors The possibility of PACAP receptors being coupled to different signal transduction pathways in human prostate tissue

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FIGURE 2. Competitive inhibition of [125I]PACAP-27 binding to human prostate membranes. Membranes were incubated with tracer

and increasing concentrations of unlabelled PACAP-27 as described in Materials and Methods. Left: The specific [125I]PACAP-27 binding was expressed as a percentage of total radioactivity added for each of the seven patients studied. Each curve corresponds to a different patient and represents the mean of triplicate determinations. Right: Scatchard plot of the same data (results are means 6 S.E.M. of the seven patients and were calculated from the mean stoichiometric results obtained for each individual subject).

was analysed by studying the effects of high concentrations of the structurally related peptides PACAP-27, PACAP-38, and VIP on main steps of cell signalling as those defined by enzyme activities such as adenylate cyclase, phospholipase C, and NOS. Figure 4 indicates that the three peptides stimulated adenylate cyclase activity in a dose-dependent manner. The effects of VIP and PACAP-27 were equipotent, whereas PACAP-38 had lesser effects at this level. Interestingly, the synthesis of phosphoinositides was significantly stimulated by PACAP-27 (1 mM) as studied by experiments on myo-[2-H3]inositol incorporation into human prostate tissue followed by lipid extraction (Figure 5). The increases observed at lower PACAP-27 doses (0.01 and

FIGURE 3. Effect of PACAP-related peptides on [125I]PACAP-

27 binding to human prostate membranes. Membranes were incubated with tracer and increasing concentrations of the indicated unlabelled peptides as described in Materials and Methods. Results are means of triplicates and correspond to a representative experiment of three performed for different patients.

0.1 mM) lacked statistical significance, whereas PACAP-38 and VIP were practically ineffective at this level up to the highest concentration studied. As shown in Table 1, a high dose (1 mM) of the three peptides did practically not modify either total inositol phosphate production or NOS activity. DISCUSSION In the present report, we clearly show the coexistence of the PAC1, VPAC1, and VPAC2 subtypes of the VIP/PACAP receptor family in the human prostate by means of immunological methods, using the corresponding specific antibodies and tissue pieces obtained from patients with benign hyperplasia of the gland. These experiments, together with the use of [125I]PACAP-27 as tracer in binding experiments, allowed us to establish a set of biochemical and pharmacological properties of the three classes of binding sites that extend previous data from our laboratory on VPAC1 and VPAC2 receptor molecules (common VIP/PACAP receptors) in this tissue [16] and define the presence and characteristics of the PAC1 receptor, which is specific for PACAP as shown in other tissues [2, 3]. Previous reports have demonstrated the presence of various neuropeptides and biogenic amines in human prostate epithelium, prostatic benign hyperplasia, and prostate adenocarcinoma [14, 15]. Interestingly, neuroendocrine differentiation in prostate adenocarcinomas has been associated with a poor prognosis and androgen withdrawal therapy results in enrichment in neuroendocrine cells of prostate tumour cell populations [30, 31]. Indeed, VIP exerts a potentiating effect on the invasive capacity of the androgen responsive human prostate carcinoma cell line LNCaP [17], whereas PACAP and VIP stimulate the proliferation of rat prostate cells in culture [32]. Furthermore, the antagonist

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FIGURE 5. Coupling of VIP/PACAP receptors to phosphoino-

sitide synthesis in human prostate tissue. The incubation with increasing concentrations of PACAP-27, PACAP-38, or VIP as described in Materials and Methods. The absolute basal level corresponded to 584 6 205 dpm and was expressed as 100%. Data represent means 6 S.E.M. of four experiments performed in triplicate. **P , 0.01 when comparing with basal value (statistical comparisons were made by ANOVA analysis followed by Dunnett test).

FIGURE 4. Coupling of VIP/PACAP receptors to adenylate cy-

clase stimulation in human prostate membranes. Membranes were incubated with increasing concentrations of PACAP-27, PACAP-38, or VIP as described in Materials and Methods. The absolute basal level corresponded to 9.4 6 0.1 pmol cAMP/mg protein/min and was expressed as 100%. Data represent means 6 S.E.M. of four experiments performed in triplicate.

PACAP(6-38) inhibits the growth of human prostate carcinoma cells lines [13]. In the human prostate, VIP immunoreactivity has been detected in several regions innervating both epithelial cells and smooth muscle [33]. However, the presence of PACAP has not been described at present in this gland but it must be kept in mind that this peptide is located at high concentrations in the seminiferous tubules and that PACAP is able to cross the hematotesticular barrier [1], making conceivable its interaction with the receptors described in the present report. Binding experiments using [125I]PACAP-27 indicated that the PAC1 receptor is the preferred PACAP receptor subclass for this peptide in the human prostate because both PACAP-27 and PACAP-38 were high-affinity competitors, whereas VIP showed a lower affinity. However, as VIP exhibited an IC50 value that was approximately 3-fold higher than those of PACAP peptides, the possibility exists that [125I]PACAP-27 may be bound to VIP receptors in addition to PACAP receptors. The observed pharmacological profile was unequivocally repeated in prostate tissue from seven different patients and correlates with the different affinities of peptides for the human PAC1 receptor in other systems [34, 35]. Previous data on VPAC1 and VPAC2 receptors at this level using [125I]VIP gave an equipotent role for PACAP-27, PACAP-38, and VIP because these two subclasses behave as common VIP/PACAP receptors [16].

In immunodetection experiments using specific antibodies, we identified the human prostate PAC1 receptor as a 60KDa protein, which is comparable to the molecular mass reported for other human tissues [1, 36]. A bigger form of 72 KDa was also detected and it may be attributed to the reducing conditions of the experiments, allowing the appearance of free sulfhydryl groups as previously reported for this receptor molecule in pig brain in terms of a stabilizing role of intrachain disulfide bonds [37]. The VPAC1 and VPAC2 receptors were characterized by the corresponding antibodies as 58 and 68 KDa proteins, respectively, in agreement with previous studies on common VIP/PACAP receptors in various human tissues [23, 36]. In human tissues, the splice variants from PAC1 receptors cloned at present differ from each other by the presence or absence (short form) of either one or two 84-bp cassettes in the third intracellular loop and in the stimulation of adenylate cyclase and phospholipase C activities simultaneously [4]. However, the PAC1 receptor described here seems to be TABLE 1. Coupling of VIP/PACAP receptors to inositol phos-

phate (IPs) production and NOS activity in human prostate tissue Peptide PACAP-27 PACAP-38 VIP Basal

IPs 113.8 101.2 106.9 383

6 6 6 6

1.3 0.1 11.0 66 dpm

NOS 107.3 109.9 106.1 10.6

6 6 6 6

3.1 5.7 2.5 2 mmol/mg

The experiments were carried out as described in Materials and Methods. Peptide concentration was 1 mM. Absolute basal values are included at the bottom of the table with the corresponding units. The results obtained in the presence of peptide are expressed as percent of the corresponding basal value. Data represent means 6 S.E.M. of four experiments performed in triplicate.

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poorly coupled to adenylate cyclase. This feature is not due to an absence of either the enzyme or the G-protein regulatory subunits [16]. The stimulatory effects of PACAP-27 and VIP on adenylate cyclase activity were similar, suggesting that these actions of both peptides were exerted through the common receptors (VPAC1/VPAC2) previously characterized in human benign hyperplastic prostate [16]. The study of distinct signalling pathways indicated that neither PACAP nor VIP were able to modify NOS activity in the human prostate. In fact, the coupling of VIP/PACAP receptors to this enzyme has been rarely found [6]. On the other hand, no effects could be seen on phospholipase C activity because high concentrations of these peptides did not modify the level of inositol phosphates in human prostate in spite of the fact that PAC1 receptors can dually activate adenylate cyclase and phospholipase C in various tissues [4]. However, we observed a significant increase in the synthesis of phosphoinositides in the presence of PACAP-27 (but not with PACAP-38 or VIP), which suggests that the human prostate could express a novel subtype of PAC1 PACAP-preferring receptor. This feature is interesting because phosphoinositides are cofactors of phosphatidylcholine-specific phospholipase D and substrates for phosphoinositide 3-kinase (both of which are also receptor activated), and are related to the regulatory mechanisms of cell proliferation [38, 39]. The lack of equipotency between PACAP-27 and PACAP-38 at this level does not have an apparent explanation. A possible explanation lies in the makeup of the PAC1 receptor/G-protein repertoire in human prostate that may give a special relevance to the difference in the length of the amino acid chain between both peptides. The multiplicity of the mechanisms of action of PACAP is an increasing field, as shown by the cloning from rat cerebellum of a PACAP-preferring receptor coupled to an L-type calcium channel [40], a signalling phenotype characteristic of the PACAP receptor involved in the regulation of insulin secretion from pancreatic islets. Moreover, in rat Leydig cells a novel PACAP-preferring subtype has been characterized coupled to a pertussis toxin-sensitive G-protein whose activation induces a Na1-dependent depolarization of the plasma membrane as well as testosterone production [11]. Although such a receptor subclass has not been cloned at present from human tissues, the PACAP-preferring (PAC1) receptor characterized here could be the human version of the PACAP receptors coupled to ionic inflow. In conclusion, this report shows the expression of a novel subtype of PAC1 receptor in the epithelium of human hyperplastic prostate that is not coupled to adenylate cyclase, NOS, or phospholipase C but leads to an increase in phosphoinositide synthesis.

We are greatly indebted to Dr. A. Arimura and Dr. E.J. Goetzl for generously providing the specific human PAC1 (A. A.), and VPAC1 and VPAC2 (E. J. G.) receptor antibodies used in this study. This work was supported by the Direccio´n General de Investigacio´n Cientı´fica y Te´cnica (Grants PB93/0492 and PB94/0360) and by the Direccio´n

R. M. Solano et al. General de Ensen˜anza Superior e Investigacio´n Cientı´fica (Grant PM97/0069).

References 1. Arimura A. and Shioda S. (1995) Front. Neuroendocrinol. 16, 53–88. 2. Rawlings S. R. (1994) Mol. Cell. Endocrinol. 101, C5–C9. 3. Harmar A. J., Arimura A., Gozes I., Journot L., Laburthe M., Pisegna J. R., Rawlings S. R., Robberecht P., Said S. I., Sreedharan S. P., Wank S. A. and Wasche J. A. (1998) Pharmacol. Rev. 50, 265–270. 4. Pisegna J. R. and Wank S. A. (1996) J. Biol. Chem. 271, 17267–17274. 5. Muller A., Monnier D., Rene F., Larnet Y., Koch B. and Loeffler J. P. (1997) J. Neurochem. 68, 1696–1704. 6. Murthy K. S., Jin J. G., Grider J. R. and Makhlouf G. M. (1997) Am. J. Physiol. 272, G1391–G1399. 7. Gourlet P., Vertongen P., Vandermeers A., VandermeersPiret M. C., Rathe J., De Neef P., Waelbroeck M. and Robberecht P. (1997) Peptides 18, 403–408. 8. Jiang S., Kopres E., McMichael M., Bell R. H. and Ulrich D. (1997) Cancer Res. 57, 1475–1480. 9. Hannibal J. and Fahrenkrug J. (1995) Regul. Pept. 55, 111– 115. 10. Hurley J. D., Gardiner J. V., Jones P. M. and Bloom S. R. (1995) Endocrinology 136, 550–557. 11. Rossato M., Nogara A., Gottardello F., Bordon P. and Foresta C. (1997) Endocrinology 138, 3228–3235. 12. Carmena M. J., Solano R. M., Guijarro L. G. and Prieto J. C. (1996) Ann. NY Acad. Sci. 805, 708–712. 13. Leyton J., Coelho T., Coy D. H., Jakowlew S., Birrer M. J. and Moody T. W. (1998) Cancer Lett. 125, 131–139. 14. Jen P. Y. and Dixon J. S. (1995) J. Anat. 187, 169–179. 15. Tainio H. (1995) Acta Histochem. 97, 113–119. 16. Solano R. M., Carmena M. J., Carrero I., Cavallaro S., Roman F., Hueso C., Travali S., Lopez-Fraile N., Guijarro L. G. and Prieto J. C. (1996) Endocrinology 137, 2815–2822. 17. Hoosein N. M., Logothetis C. J. and Chung I. W. K. (1993) J. Urol. 149, 1209–1213. 18. Gkonos P. J., Lokeshwar B. L., Balkan W. and Roos B. A. (1995) Regul. Pept. 59, 43–51. 19. Han K., Viallet J., Chevalier S., Zheng W., Bazinet M. and Aprikian A. G. (1997) Prostate 31, 53–60. 20. Carmena M. J., Clemente C., Guijarro L. G. and Prieto J. C. (1992) Endocrinology 131, 1993–1998. 21. Bradford M. (1976) Anal. Biochem. 72, 248–254. 22. Cavallaro S., D’Agata V., Drago F., Musco S., Nuciforo G., Ricciardolo F., Travali S., Stivale F., Arimura A. and Canonico P. L. (1996) Ann. NY Acad. Sci. 805, 555–557. 23. Goetzl E. J., Patel D. R., Kishiyama J. L., Smoll A. C., Turck C. W., Law N. M., Rosenzweig S. A. and Sreedharan S. P. (1994) Mol. Cell. Neurosci. 5, 145–152. 24. Gilman A. G. (1970) Proc. Natl. Acad. Sci. USA 67, 305– 312. 25. Berridge M. J., Dawson R. M., Downes C. P., Heslop J. P. and Irvine R. F. (1983) Biochem. J. 212, 473–482. 26. Carrero I., Recio M. N., Prieto J. C. and Pe´rez-Albarsanz M. A. (1996) Pestic. Biochem. Physiol. 56, 79–87. 27. Downes C. P. and Wusteman M. M. (1983) Biochem. J. 216, 633–640. 28. Green L. C., Wagner D. A., Glogowski J., Skipper P. L., Wishnok J. S. and Tannenbaum S. R. (1982) Anal. Biochem. 126, 131–138. 29. Scatchard G. (1949) Ann. NY Acad. Sci. 51, 660–672. 30. Cohen R. J., Glezerson G., Taylor L. F., Grundle A. J. and Naude´ J. H. (1993) J. Urol. 150, 365–368.

PACAP Receptors in Human Prostate 31. Nakada S. Y., Di Sant’Agnese P. A., Moines R. A., Hiipakka R. A., Liao S., Cockett A. T. K. and Abrahamsson P. A. (1993) Cancer Res. 53, 1967–1970. 32. Guijarro L. G., Juarranz M. G., Marinero M. J., Pajuelo L., Carmena M. J. and Prieto J. C. (1996) Ann. NY Acad. Sci. 805, 723–728. 33. Crowe R., Chapple C. R. and Burnstock G. (1991) Br. J. Urol. 68, 53–61. 34. Ogi K., Miyamoto Y., Masuda Y., Habata Y., Hosoya M., Ohtaki T., Masuo Y., Onda H. and Fujino M. (1993) Biochem. Biophys. Res. Commun. 196, 1511–1521. 35. Olianas M. C., Ingianni A., Sogos V. and Onali P. (1997) J. Neurochem. 69, 1213–1218.

819 36. Guijarro L. G., Rodriguez-Henche N., Garcia-Lo´pez E., Noguerales F., Dapena M. A., Juarranz M. G. and Prieto J. C. (1995) J. Clin. Endocrinol. Metab. 80, 2451–2457. 37. Cao Y. J., Gimpl G. and Fahrenholz F. (1994) Biochim. Biophys. Acta 1222, 432–440. 38. Exton J. (1997) Eur. J. Biochem. 243, 10–20. 39. Franke T. F., Kaplan D. R. and Cantley L. C. (1997) Cell 88, 435–437. 40. Chatterjee T. K., Sharme R. V. and Fisher R. A. (1996) J. Biol. Chem. 271, 3226–3232.