Pituitary Adenylate Cyclase Activating Polypeptide Stimulates Rat ...

0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society

Vol. 138, No. 8 Printed in U.S.A.

Pituitary Adenylate Cyclase Activating Polypeptide Stimulates Rat Leydig Cell Steroidogenesis Through a Novel Transduction Pathway MARCO ROSSATO, ANDREA NOGARA, FRANCESCO GOTTARDELLO, PAOLA BORDON, AND CARLO FORESTA Patologia Medica III, University of Padova, Padova 35128, Italy ABSTRACT The aim of the present study was to evaluate the effects of pituitary adenylate cyclase activating polypeptide (PACAP) on testosterone production in isolated adult rat Leydig cells and its possible mechanisms of action. PACAP-38 stimulated testosterone secretion in a dose-dependent manner with a minimal and a maximal efficacious dose of 1.0 nM and 100 nM, respectively. PACAP-27 was without effect on testosterone secretion at any dose tested. Similarly, vasoactive intestinal peptide did not stimulate steroidogenesis nor interfere with PACAP-38 activity, as well as preincubation of Leydig cells with the vasoactive intestinal peptide-antagonist [Lys1, Pro2,5, Arg3,4, Tyr6]vasoactive intestinal peptide. Removal of extracellular Ca21 did not inhibit the stimulatory effects of PACAP-38 on Leydig cell testosterone production. Neither PACAP-38 nor PACAP-27 modified intracellular free Ca21 and cAMP levels at any dose tested thus excluding a role for Ca21 and cAMP in the stimulatory effects of PACAP. PACAP-38 was able to induce a plasma membrane depolarization

that was dependent on an influx of Na1 from the extracellular medium as confirmed by the monitoring of intracellular Na1 with the Na1-sensitive fluorescent dye sodium benzofuran isophtalate. When Na1 was removed from the extracellular medium, PACAP-38 did not stimulate testosterone production, demonstrating that Na1 influx through the plasma membrane is strictly related to the stimulatory effects of this peptide. In addition, preincubation of Leydig cells in the presence of pertussis-toxin (500 ng/ml for 5 h) significantly reduced PACAP-38-stimulated effects both on plasma membrane depolarization and testosterone secretion. These results demonstrate that PACAP-38 stimulates testosterone secretion in isolated adult rat Leydig cells through the interaction with a novel PACAP receptor subtype coupled to a pertussis toxin sensitive G protein whose activation induces a Na1-dependent depolarization of the plasma membrane and testosterone production. (Endocrinology 138: 3228 –3235, 1997)

P

ITUITARY adenylate cyclase activating peptide (PACAP) is a novel member of the vasoactive intestinal peptide (VIP)/glucagon/secretin/family of peptides first isolated from ovine hypothalamic tissue for its ability to stimulate adenylate cyclase (1, 2). Two forms of PACAP, derived from a single 176-amino acid precursor, are known: a longer form, named PACAP-38, constituted by 38 amino acids, and a C-terminally truncated form named PACAP-27 that comprises the first 27 amino acids of PACAP 38 (1, 2). The possible physiological roles of PACAP have been examined mainly at the pituitary level, where this peptide stimulates the secretion of several hormones including GH from somatotrophs (3) and LH from gonadotrophs (4). In somatotrophs PACAP has been shown to increase intracellular free Ca21 ([Ca21]i) by an influx of Ca21 through the plasma membrane, dependent on an increase of cAMP (5), whereas in gonadotrophs PACAP induces an increase of [Ca21]i through phospholipase C stimulation and 1,4,5trisphosphate-dependent Ca21 release from internal stores, an effect that is independent of cAMP production (6). All these findings support the hypothesis that PACAP is a novel regulator of hypothalamic-pituitary axis. Recently, a role for PACAP outside the brain has been proposed: it has been demonstrated that PACAP controls cathecolamine secretion from the adrenal medulla (7) and

regulates endocrine pancreas activity (8, 9). These observations strengthen the hypothesis that this peptide may have a regulatory effect outside of the pituitary. Beside brain, the testis also contains large amounts of PACAP, mainly represented by PACAP-38, although PACAP-27 is present at very low concentrations (10). Although PACAP-38 concentrations in the testis are comparable with or greater than those found in the pituitary, its role in testicular cell physiology is poorly understood. Only recently Heindel and colleagues (11) demonstrated that PACAP-38 can modulate rat Sertoli cell functions in vitro through adenylate cyclase activation. Furthermore, other authors (12, 13), demonstrating PACAP messenger RNA expression in rat seminiferous tubules, have hypothized that this peptide may act as a paracrine or autocrine modulatory factor for germ cells, with a specific role during early spermiogenesis. Finally, very recently Monts and colleagues (14) detected the presence of PACAP type I receptor messenger RNA in Leydig cells but to date there are no data about the role, if any, of PACAP on Leydig cell functions. In the present study we evaluated the effects of PACAP on Leydig cell steroidogenesis, investigating the signal transduction pathways involved in the action of this peptide.

Received October 24, 1996. Address all correspondence and requests for reprints to: Carlo Foresta, Patologia Medica III, University of Padova, Via Ospedale 105, 35128 Padova, Italy. E-mail: [email protected].

Materials

Materials and Methods Medium-199 with Hanks’ salts and l-glutamine, penicillin, and streptomycin were obtained from GIBCO (Grand Island, NY); collagenase

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PACAP STIMULATES STEROIDOGENESIS IN LEYDIG CELLS (type II), BSAe (BSA fraction V), HEPES, and soybean trypsin inhibitor (type 1s) were from Sigma (St. Louis, MO); Percoll and density marker beads were from Pharmacia Fine Chemicals AB (Uppsala, Sweden); silicone fluid was from Serva (Heidelberg, Germany). Fura-2/AM, bisoxonol, sodium benzofuran isophtalate acetomethylester (SBFI/AM), and pluronic acid were obtained from Molecular Probes (Eugene, OR). PACAP-38 and -27, VIP, [Lys1, Pro2,5, Arg3,4, Tyr6]-VIP (VIP-antagonist), gramicidin D, nifedipine, 8-Br-cAMP, 3-isobutyl-1-methylxantine, and pertussis toxin were obtained from Sigma. Highly purified human CG (hCG) was from Serono (Rome, Italy). Verapamil was purchased from Knoll AG (Liestal, Switzerland), and ATP was from Boehringer Mannheim (Mannheim, Germany). All other chemicals were of analytical grade.

Isolation and purification of Leydig cells Adult male rats of the Sprague-Dawley strain (280 –310 g) were used. The animals were housed in a controlled environment (22 C with 14 h light and 10 h dark). Food and water were available ad libitum. Interstitial cells were prepared from testes through decapsulation and collagenase digestion as previously described (15). Briefly, 12–16 testes were incubated with M-199 (3 ml/testis) with Hanks’ salts and l-glutamine, 0.2% BSA (fraction V), and 1 g/liter of collagenase (type II), at 34 C, in a shaking (90 cycles/min) water bath with controlled atmosphere [partial pressure of oxygen (pO2) 95%-partial pressure of carbon dioxide (pCO2) 5%]. After 15–20 min, the suspension was filtered through sterile nylon gauze (mesh 0.5– 0.8 mm), and erythrocytes were removed (about 75– 80%) by the addition of 5 ml 60% (vol/vol) Percoll to the bottom of each tube, followed by centrifugation at 800 3 g for 10 min at 22 C. After washing twice, cells were resuspended in M-199 and 5 ml interstitial cell suspension (20 –25 3 106 cells/ml) were layered on the top of each vial containing a previously prepared discontinuous density gradient of Percoll (0 – 60% vol/vol) and then centrifuged at 800 3 g for 20 min at room temperature. The fractions were collected from the bottom of the tubes with a peristaltic pump and then washed twice with isotonic M-199 (1:1; vol/vol) to remove any residual Percoll. The cells were then resuspended in M-199 and Leydig cells [92–95% staining positively for 3-b-OH-steroid-dehydrogenase activity (16)] were distributed in a band at the density corresponding to 40 –55% of Percoll. Cell concentrations (1.0 3 106 Leydig cells/ml) and viability (.90%) were determined using a hemocitometer and the trypan blue method, respectively.

Incubation of purified Leydig cells Aliquots (0.5 ml) of cell suspensions (1.0 3 106 cells/ml) were incubated in M-199 with Hanks’ salts, l-glutamine, HEPES, Tris(hydroxymethyl)-aminomethan, 0.2% BSA (fraction V), penicillin (10 U/liter), streptomycin (1 g/liter), pH 7.4, in polyethylene sterile tubes containing PACAP-38 and -27 at the doses ranging from 1029 to 1026 m, in a shaking (90 cycles/min) bath at 34 C in controlled atmosphere (pO2 95%-pCO2 5%). In parallel experiments aliquots (0.5 ml) of Leydig cell suspensions were incubated in the presence and absence of extracellular calcium (no added calcium and 0.1 mm EGTA) and stimulated with PACAP. After 3 h, the incubation was stopped by immersion of all tubes in an icecooled water bath immediately followed by centrifugation at 1500 3 g for 15 min at 4 C. Supernatants were stored at 220 C until assayed. In some experiments NaCl was replaced with choline chloride, N-methylglucamine, or sucrose.

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mm KH2PO, 5.6 mm glucose, 1.7 mmCaCl2, 104 IU/ml penicillin, 10 mg/ml streptomycin, and 20 HEPES (pH 7.4 at 37 C). Fura-2/AM was added at the concentration of 5 mm, and the incubation carried out for 45 min at 37 C. After loading, cells were washed free of extracellular dye by being centrifuged three times (250 3 g for 10 min at room temperature) in standard saline. Cells were kept at room temperature until used. Fura-2 was alternatively excited at 340 and 380 nm and the fluorescence was measured at 505 nm. All experiments were completed within 2 h of fura-2/AM loading. [Ca21]i was determined as previously described (17).

Measurement of [Na1]i in Leydig cell suspensions Intracellular free Na1 ([Na1]i) was evaluated using the fluorescent sodium-binding dye SBFI/AM. Leydig cells, suspended in standard saline, were incubated with 5 mm SBFI/AM in the presence of the nonionic detergent pluronic acid (20% in dimethylsulfoxide, 1:1 to SBFI/ AM) for 60 min at 37 C in continuous stirring. Cells were then centrifuged at 500 3 g for 10 min at room temperature. After centrifugation, the supernatant was discarded and cells were resuspended in standard saline and kept at room temperature until used. All experiments were performed within 90 min of the dye loading. SBFI fluorescence was monitored at the wavelength pair of 345 and 490 nm for excitation and emission, respectively (18).

Determination of manganese (Mn21) influx Mn21 uptake was measured by monitoring the rate of fluorescence quenching at the excitation wavelength of 360 nm (isosbestic point). When measured at the isosbestic wavelength, the rate of fura-2 fluorescence decrease is insensitive to [Ca21]i changes and proportional to the rate of Mn21 influx (19).

Measurement of plasma membrane potential in rat Leydig cells Plasma membrane potential variations were monitored with the fluorescent potential sensitive probe bis-oxonol, using the wavelength pair of 540 and 580 nm, as previously described (20). In some experiments NaCl was replaced by an iso-osmotic concentration of choline chloride, methylglucamine, or sucrose, as previously described (20). Fluorescence measurements were performed in a LS 50B PerkinElmer fluorimeter (Norwalk, CT) equipped with a thermostatted and magnetically stirred cuvette holder.

Hormone measurements Testosterone was determined by RIA method, using [3H]testosterone (Radim, Rome, Italy), as previously described (21). Sensitivity was estimated as 0.28 nmol/liter and intra- and interassay coefficients of variation were 7.8% and 7.0%, respectively.

cAMP measurement

In some experiments aliquots of Leydig cells were preincubated in the presence and absence of pertussis toxin (500 ng/ml) for 5 h at 37 C in controlled atmosphere (pO2 95%-pCO2 5%). After incubation, PACAP-38 (100 nm) was added to evaluate testosterone secretion. In parallel experiments, the effects of pertussis toxin preincubation on PACAP-38-induced depolarizing effects were determined.

After 3 h of incubation with the appropriate stimulus in the presence of the inhibitor of phosphodiesterase 3-isobutyl-1-methylxantine (0.1 mm), Leydig cell suspensions were quickly transferred to an ice-cold bath. After 10 min, each tube was centrifuged at 1000 3 g for 5 min at 4 C, and the supernatants were collected for assay of extracellular cAMP. The cell pellets were washed with ice-cold medium and processed for intracellular cAMP evaluation as previously described (22). cAMP concentrations were determined by RIA by the method of Steiner et al. (23) using kits supplied by Becton and Dickinson Immunodiagnostics (Rutherford, NJ). The sensitivity was estimated as 1.1 fmol/liter; the intra- and interassay coefficients of variation were 13.3% and 10.4%, respectively.

Measurement of [Ca21]i in Leydig cell suspensions

Statistical analysis

Leydig cell [Ca21]i was measured with the fluorescent probe fura2/AM as previously described (17). Briefly, cells were suspended in a standard saline containing: 125 NaCl, 4.8 mm KCl, 1.2 mm MgSO4, 1.2

Results of five independent experiments were considered and expressed as mean 6 sd. Statistical analysis was carried out using ANOVA, Duncan’s multiple range test, and Student’s t test for unpaired

Incubation with pertussis toxin

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data. A P value of less than 0.05 was chosen as the limit for statistical significance.

Results

Figure 1 shows that the addition of PACAP-38 to suspended rat Leydig cells stimulates testosterone secretion in a dose-dependent manner, with maximal effect at 100 nm. In the same experimental conditions, PACAP-27 was without effect on testosterone secretion at any dose tested (Fig. 1). It is well known that PACAP can interact with VIP receptors in a number of different cells (24). In Fig. 2 it is shown that preincubation of Leydig cells with the VIP-receptor antagonist did not modify the stimulatory effects of PACAP-38. According with these findings VIP did not evoke any significant testosterone secretion at the dose of 1.0 mm and did not interfere with PACAP-38 stimulating effects of testosterone production (Fig. 2). PACAP actions in the different cell types studied so far are mediated by elevations of cAMP or [Ca21]i (24). Therefore, we investigated the effects of this peptide on cAMP production and [Ca21]i in rat Leydig cells. As reported in Table 1, neither PACAP-38 nor PACAP-27 were able to induce any significant increase in cAMP at any dose tested. On the other hand, hCG stimulated an important rise of this cyclic nucleotide (Table 1). PACAP-38 addition to Leydig cell suspensions did not modify [Ca21]i as evaluated with the fluorescent probe fura-2 (Fig. 3, trace a). Also, PACAP-27 was without effect on [Ca21]i (Fig. 3, trace b), whereas extracellular ATP induced a prompt rise of [Ca21]i (Fig. 3 traces a and b) as demonstrated previously (25). These results were confirmed by the failure of PACAP-38 and -27 to induce fura-2 quenching by Mn21, a cation permeable through Ca21 channels, whereas ATP caused a prompt reduction of fura-2 fluorescence (Fig. 4). Leydig cell steroidogenesis is believed to be in part a Ca21-dependent process (26). The lack of any effect of PACAP on [Ca21]i prompted us to examine the effects of Ca21 on testosterone secretion stimulated by this peptide. As shown in Table 2, PACAP-38-stimulated testosterone secretion was not modified by Leydig cell incubation in Ca21-free medium. To further investigate the possible ionic mechanisms underlying the stimulatory effects of PACAP-38 in Leydig cells, plasma membrane potential was monitored

FIG. 1. Dose-dependent secretion of testosterone by PACAP-38 and PACAP-27 in isolated rat Leydig cells: PACAP-38 (f); PACAP-27 (o). Data are mean 6 SD of duplicate determinations from three separate experiments. *, P , 0.01; #, P , 0.001.

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using the membrane sensitive fluorescent dye bis-oxonol. Figure 5 shows that PACAP-38 induced a rapid (Fig. 5A, trace a) and dose-dependent (Fig. 5B) plasma membrane depolarization that was not maximal, because the Na1-channel forming ionophore gramicidin D was still able to induce a further collapse of the plasma membrane potential. On the contrary, PACAP-27 was without effect on plasma membrane potential at any dose tested (Fig. 5A, trace b and data not shown). Similarly, VIP did not modify Leydig cell plasma membrane at the dose of 1.0 mm and did not interfere with PACAP-induced depolarizing effects (Fig. 6, trace a). Furthermore, preincubation of cells with the VIP-antagonist did not alter the depolarizing effects of PACAP-38 (Fig. 6, trace b). It has been previously reported that PACAP6 –38 acts as an antagonist of PACAP receptors (27). In our experimental conditions, PACAP6 –38 stimulated testosterone secretion and depolarized plasma membrane potential (not shown). These results did not allow us to use this peptide as PACAP-antagonist. Addition of the cAMP analog 8-Br-cAMP (1 mm) or forskolin (10 mm), a well-known activator of adenylate cyclase as well as of hCG, did not induce any modification of the plasma membrane potential, further strengthening the hypothesis that the stimulatory effects of PACAP-38 are not mediated by adenylate cyclase activation (Fig. 7). In the presence of physiological ion gradients, the main charged ion carrier would be expected to be Na1. In the next series of experiments we examined the effects of PACAP-38 on plasma membrane potential in rat Leydig cells suspended in a medium in which Na1 was isotonically substituted by choline chloride. In these experimental conditions, PACAPinduced plasma membrane depolarization was completely inhibited (Fig. 8, trace b). Similar results were obtained when Leydig cells were suspended in medium in which Na1 was isotonically substituted by methylglucamine or sucrose (Fig. 8, traces c and d, respectively). Experiments showing PACAP effects in Na1-free media did not demonstrate unequivocally that the putative PACAP-38 activated channel allows Na1 influx. Then we determined [Na1]i with the Na1-sensitive fluorescent probe SBFI. Figure 9, trace a, shows that Leydig cell stimulation with PACAP-38 (100 nm) caused a rapid increase in the [Na1]i followed by a progressive decline probably due to activation of Na1 extrusion mechanisms and/or receptor/channel inactivation. When extracellular Na1 was reduced from 125 to 10 mm, the effects of PACAP-38 on [Na1]i were greatly reduced (Fig. 9, trace b). In both experimental conditions, the specific Na1 carrier monensin induced an important increase in [Na1]i, albeit obviously reduced in Fig. 9, trace b. The analysis of testosterone secretion from Leydig cells incubated in Na1-free medium demonstrated that PACAP-38 was not able to stimulate steroidogenesis when extracellular Na1 was absent in the external medium, whereas 8-Br-cAMP was fully competent in stimulating testosterone production (Table 3). In rat gonadotrophs, PACAP-38 effects are mediated by a G protein (28). Different G proteins are known and can be distinguished by their sensitivity to pertussis toxin: G proteins of the Gi/Go family are inhibited by pertussis toxin, whereas G proteins of the Gq/G11 family are not (29). We evaluated the sensitivity to pertussis toxin of PACAP-38-


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FIG. 2. Effects of VIP and VIP-antagonist preincubation on PACAP-38- stimulated testosterone secretion in isolated adult rat Leydig cells. VIP (1.0 mM) and VIP-antagonist (VIP/AT, 1.0 mM) were preincubated for 15 min before PACAP-38 addition. Data are mean 6 SD of duplicate determinations from three separate experiments. *, P , 0.01; #, P , 0.001.

TABLE 1. Effects of PACAP-38 and PACAP-27 on cAMP accumulation in rat Leydig cells cAMP (pmol/106 cells/3 h)

Control PACAP-38 1 nM 10 nM 100 nM 1000 nM PACAP-27 1 nM 10 nM 100 nM 1000 nM hCG 10 ng/ml

Intracellular

Extracellular

6.9 6 0.9

13.4 6 1.5

7.0 6 0.8 6.8 6 0.9 6.8 6 1.0 7.2 6 1.1

14.0 6 1.3 14.2 6 1.4 14.9 6 1.0 15.1 6 1.1

6.7 6 1.0 6.9 6 0.8 7.0 6 1.1 6.8 6 1.0 75.1 6 9.7a

13.2 6 0.8 13.8 6 0.9 14.1 6 1.1 14.6 6 1.2 168.8 6 12.9b

Leydig cell suspensions (1.0 3 106 cells) were incubated in the presence of 0.1 mM IBMX and stimulated with PACAP-38, PACAP-27, and hCG for 3 h. Control samples were treated with saline (M-199) alone. Data represent mean 6 SD of duplicate determinations from three separate experiments. a P , 0.01; b P , 0.001 vs. control and PACAP-treated samples.

induced effects by incubating aliquots of isolated adult rat Leydig cells in the presence of pertussis toxin (500 ng/ml for 5 h). Figures 10 and 11 show that pertussis-toxin treatment inhibited PACAP-38-stimulated steroidogenesis and plasma membrane depolarizing effects, demonstrating that the biological effects of this peptide in Leydig cells are mediated by a pertussis toxin-sensitive G protein (Gi/Go family). These results suggest that in rat Leydig cells PACAP-38 may activate a receptor coupled to a pertussis toxin-sensitive G protein that induces a Na1-dependent plasma membrane depolarization resulting in the stimulation of testosterone secretion. Discussion

PACAP is a novel peptide originally isolated from hypothalamic tissue and thought to possess an important regulatory function as hypophysiotropic hormone (1– 4). However several lines of evidence demonstrate that PACAP may influence extrapituitaric cell functions (7–9), in particular within the testis, where PACAP has been found at the highest concentrations outside the hypothalamus (10 –13). The

FIG. 3. Time course of changes in [Ca21]i in response to PACAP-38 and PACAP-27. Leydig cell suspensions (4.0 –5.0 3 105 cells) were loaded with fura-2/AM as described in Materials and Methods. Where indicated, PACAP-38 (P-38, 100 nM, trace a), PACAP-27 (P-27, 100 nM, trace b), and ATP (ATP, 100 mM) were added. Traces are representative of a typical experiment of five.

present study demonstrates that PACAP-38 stimulates rat Leydig cell steroidogenesis in a dose-dependent manner through the activation of a pertussis toxin-sensitive G protein. In the different cellular systems studied so far, the effects of PACAP are mediated by adenylate cyclase and/or phospholipase C activation thus involving cAMP and [Ca21]i increases (5, 6, 24). In rat Leydig cells, PACAP-38- stimulated testosterone secretion was not dependent on cAMP nor [Ca21]i rises, suggesting the activation of a novel transduction pathway. The monitoring of plasma membrane potential of Leydig cells in Na1- and Na1-free medium, as well as the evaluation of [Na1]i, demonstrate that PACAP-38 induces a rapid plasma membrane depolarization dependent on an influx of Na1 from the extracellular medium. These effects seem strictly related to PACAP-induced testosterone secretion, because they were completely blunted when Leydig cells were incubated in Na1-free medium. In the same experimental conditions, the cAMP analog 8-Br-cAMP was still able to stimulate testosterone secretion, ruling out any aspecific effect of the absence of Na1 from the extracellular medium on Leydig cell steroidogenesis. Recently, it has been demonstrated that in bovine adrenal

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FIG. 4. Effects of PACAP-38 and PACAP-27 on Mn21 influx through plasma membrane in rat Leydig cells. Leydig cell suspensions (4.0 – 5.0 3 105 cells) were loaded with fura-2/AM as described in Materials and Methods and then resuspended in medium containing MnCl2 (200 mM). Fluorescence was monitored at the Ca21-insensitive excitation wavelength (360 nm). Where indicated, 100 nM PACAP-38 (P-38, trace a), 100 nM PACAP-27 (P-27, trace b), and ATP (ATP, 100 mM) were added. Traces are representative of three similar experiments. TABLE 2. PACAP-stimulated testosterone production is not dependent on extracellular Ca21 Testosterone (pmol/106 cells/3 h)

Plus Ca21 Control PACAP-38 (100 nM) No Ca21 Control PACAP-38 (100 nM)

9.9 6 2.3 43.5 6 4.9* 11.9 6 2.3 41.7 6 3.0a

Leydig cells isolated as described in Materials and Methods were treated with PACAP-38 or vehicle alone in presence or absence of extracellular Ca21. Results are expressed as mean 6 SD of duplicate determinations from three separate experiments. a P , 0.001 vs. control (vehicle).

chromaffin (30) and HIT-T15 insulinoma cells (31) PACAP-38 induces a Na1-dependent plasma membrane depolarization. In these cells, Na1-dependent current results from cAMP activation of ion channels similar to the cAMPgated nonspecific cation channels of CRI-G1 insulinoma cells (32). In Leydig cells both the cell permeant cAMP analog 8-Br-cAMP and forskolin did not induce plasma membrane depolarization. Therefore the effects of PACAP-38 in Leydig cells could be related to the activation of specific receptors coupled to the induction of an influx of Na1 from the extracellular medium. At present it is not known whether the mechanism of action of PACAP-38 involves a plasma membrane Na1-selective channel, Na1 pumps, or Na1 antiporters. The Na1/Ca21 antiport has low affinity for Ca21 and begins to operate only when [Ca21]i reach values at least 10 times higher than basal levels. In our experimental conditions PACAP-38 did not modify Leydig cell [Ca21]i, thus ruling out any role for Na1/Ca21 antiporter in Na1 accumulation induced by this peptide. Further studies involving the direct measurement of Na1 currents will be necessary to clarify the precise mechanism of PACAP-induced Na1 entry in rat Leydig cells. In bovine adrenal chromaffin and HIT-T15 insulinoma

FIG. 5. Effects of PACAP-38 and PACAP-27 on plasma membrane potential in isolated rat Leydig cells. A) Leydig cell (4.0 –5.0 3 105 cells) were suspended in presence of 200 nM bis-oxonol as described in Materials and Methods. Where indicated, 100 nM PACAP-38 (P-38, trace a), 100 nM PACAP-27 (P-27, trace b), and gramicidin D (Gr, 0.1 mg/ml) were added. Traces represent result of a single experiment from five similar experiments. B) Dose-dependent effects of PACAP-38 on plasma membrane potential in isolated rat Leydig cells in presence of 200 nM bis-oxonol. Depolarization is expressed as arbitrary units of fluorescence increase. Data are means 6 SD of triplicate determinations for five separate experiments.

FIG. 6. VIP and VIP-antagonist do not interfere with PACAP-38 effects on plasma membrane potential in isolated rat Leydig cells. Leydig cells in suspension (4.0 –5.0 3 105 cells) in presence of 200 nM bis-oxonol, were preincubated with VIP (1.0 mM, trace a) and VIPantagonist (VIP-AT, 1.0 mM, trace b) for 15 min. Then PACAP-38 (P-38, 100 nM) and gramicidin D (Gr, 0.1 mg/ml) were added. Traces are representative of five similar experiments.

cells, cathecolamine and insulin secretion were strictly dependent on an influx of Ca21 through the plasma membrane induced by the activation of voltage-dependent Ca21 channels (VOCs) (30, 31). In Leydig cells, PACAP-38 did not


FIG. 7. Effects of adenylate-cyclase system activation on plasma membrane potential in isolated rat Leydig cells. Leydig cells (4.0 – 5.0 3 105 cells) were suspended in presence of 200 nM bis-oxonol as described in Materials and Methods. Where indicated, 1 mm 8-BrcAMP (8-Br, trace a), 10 mM forskolin (Fk, trace b), 10 ng/ml hCG (trace c) and gramicidin D (0.1 mg/ml) were added. Traces are representative of a typical of three similar experiments.

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FIG. 9. Effects of PACAP-38 on Leydig cell [Ca21]i. Sodium-binding benzofuran isophtalate-loaded rat Leydig cells (1.0 3 106) were incubated in standard saline (NaCl 125 mM, trace a) or in sucrosesupplemented medium (residual Na1 concentration 10 mM, trace b). Where indicated, PACAP-38 (P-38, 100 nM) and monensin (Mon, 1.0 mM) were added. Traces are representative of three similar experiments. TABLE 3. Effects of PACAP-38 on testosterone production in presence and absence of extracellular Na1 Testosterone production (pmol/106 cells/3 h)

Plus Na1 Control PACAP-38 (100 nM) 8-Br-cAMP (1.0 mM) No Na1 Control PACAP-38 (100 nM) 8-Br-cAMP (1.0 mM)

FIG. 8. Effects of PACAP-38 on plasma membrane potential in absence of extracellular Na1. Leydig cells (4.0 –5.0 3 105 cells) were suspended in presence of 200 nM bis-oxonol in Na1-containing saline (trace a) or in saline in which Na1 was iso-osmotically replaced by choline chloride (trace b), methylglucamine (trace c), or sucrose (trace d). Where indicated, PACAP-38 (P-38, 100 nM) and KCl (30 mM) were added. Traces are representative of three similar experiments.

induce any rise of [Ca21]i, and PACAP-induced testosterone secretion was completely Ca21 independent. The absence of any [Ca21]i rise after stimulation by PACAP-38 is very intriguing, because plasma membrane depolarization should induce VOCs activation and Ca21 influx. The present observations are in agreement with our previous results, showing that plasma membrane depolarization induced by gramicidin D and KCl was not able to induce any rise in [Ca21]i in rat Leydig cells (M. Rossato and C. Foresta, our unpublished results) and with the recent demonstration that rat Leydig cells do not possess VOCs (33). It is well known that PACAP is physiologically present in two forms, PACAP-38 and PACAP-27 (1, 2). In Leydig cells only PACAP-38 was active, whereas PACAP-27 was completely ineffective both on plasma membrane potential and testosterone secretion. These results are in agreement with the results by Shivers and colleagues (34) that demonstrated the absence of PACAP-27 binding sites in rat testicular interstitial cells.

11.2 6 0.9 43.6 6 9.8a 58.4 6 7.8a 11.7 6 1.0 12.6 6 1.5 53.2 6 5.9a

Leydig cell suspensions (1.0 3 106 cells) were suspended in Na1medium and Na1-free/choline chloride replaced medium and then stimulated with 100 nM PACAP-38 and 1.0 mM 8-Br-cAMP. Control samples were treated with Na1- or Na1-free saline alone, respectively. Data are expressed as mean 6 SD of duplicate determinations from three separate experiments. a P , 0.001 vs. controls.

PACAP has 68% homology with VIP (1), and it was previously demonstrated that VIP is able to stimulate testosterone secretion in neonatal rat Leydig cells (35). Our experimental data show that VIP is not able to induce neither plasma membrane depolarization or testosterone secretion in isolated adult rat Leydig cells and does not interfere with the effects induced by PACAP. PACAP receptors are classified into at least two subtypes (24): type I, found in the brain, pituitary, testis, adrenal medulla, and some tumor lines, and which binds specifically PACAP; and type II receptors, which are probably VIP receptors, found in heart, kidney, lung, and liver, and which binds PACAP and VIP with equal affinity (24 and references herein). Type I receptors can be further divided into type IA, which possess approximately equal high affinity for PACAP-38 and -27, and type IB, to which PACAP-38 binds with 1000 times higher affinity than PACAP-27 (24, 34). The results of the present study support the hypothesis that rat Leydig cells possess type IB receptors for PACAP. In fact, the lack of any effect of VIP and PACAP-27 on Leydig cell functions and the lack of any interference of these peptides on

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Leydig cells through the activation of a novel putative type IB receptor subtype coupled to an influx of Na1 from the external medium that induces the depolarization of the plasma membrane. References

FIG. 10. Pertussis toxin inhibits testosterone production stimulated by PACAP. Leydig cells isolated as described in Materials and Methods were incubated in absence (M-199) and presence of pertussis toxin (PTX, 500 ng/ml for 5 h) in a controlled atmosphere. After incubation, Leydig cells were stimulated with 100 nM PACAP-38 for 3 h before testosterone secretion evaluation: vehicle o; PACAP-38 f. Results are expressed as means 6 SD of duplicate determinations from three separate experiments. *, P , 0.001 vs. pertussis toxin treated samples.

FIG. 11. PACAP effects on plasma membrane potential are mediated by a pertussis toxin-sensitive G protein. Leydig cells (4.0 –5.0 3 105) were preincubated in absence (trace a) and presence (trace b) of pertussis toxin (500 ng/ml for 5 h) and then suspended in presence of 200 nM bis-oxonol. Where indicated, PACAP-38 (P-38, 100 nM) and gramicidin D (Gr, 0.1 mg/ml) were added. Traces are representative of three similar experiments.

PACAP-38 activity seems to exclude the presence of type IA and type II PACAP receptors in these cells. It has been reported that PACAP activation of type I receptors on target cells is mediated by elevation of cAMP and [Ca21]i (5, 6, 24). The observations of present study, demonstrating that PACAP-38 does not induce any cAMP nor [Ca21]i rise, are particularly intriguing, and the mechanism of action of this peptide in Leydig cells is at the moment unknown. One possible hypothesis is that PACAP-38 could activate a novel type IB receptor subtype coupled to the induction of an influx of Na1 from the extracellular medium. PACAP is highly conserved along the philogenesis, a characteristic that is considered a hallmark of biological importance for a given molecule. The observation that PACAP-38 induces a stimulatory effect in Leydig cells may be relevant to the physiology of these cells, although its actual role is unknown. In conclusion, the results of the present study demonstrate that PACAP-38 stimulates testosterone secretion in adult rat

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