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Patologia Medica III, University of Padova, Padova 35128, Italy
Address all correspondence and requests for reprints to: Carlo Foresta, Patologia Medica III, University of Padova, Via Ospedale 105, 35128 Padova, Italy. E-mail: forestac{at}protec.it
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Abstract |
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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 Na+-dependent depolarization of the plasma membrane and testosterone production.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x
g for 10 min at 22 C. After washing twice, cells were
resuspended in M-199 and 5 ml interstitial cell suspension (20–25
x 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 x
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-ß-OH-steroid-dehydrogenase
activity (16)] were distributed in a band at the density corresponding
to 40–55% of Percoll. Cell concentrations (1.0 x
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 x
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 10-9 to 10-6
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 ice-cooled
water bath immediately followed by centrifugation at 1500 x
g for 15 min at 4 C. Supernatants were stored at -20 C
until assayed. In some experiments NaCl was replaced with choline
chloride, N-methylglucamine, or sucrose.
Incubation with pertussis toxin
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.
Measurement of [Ca2+]i in Leydig cell
suspensions
Leydig cell [Ca2+]i was measured with
the fluorescent probe fura-2/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
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 µM, 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 x
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. [Ca2+]i was determined as
previously described (17).
Measurement of [Na+]i in Leydig cell
suspensions
Intracellular free Na+
([Na+]i) was evaluated using the fluorescent
sodium-binding dye SBFI/AM. Leydig cells, suspended in standard saline,
were incubated with 5 µM 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 x 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 (Mn2+) influx
Mn2+ 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
[Ca2+]i changes and proportional to the rate
of Mn2+ 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 Perkin-Elmer 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
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 x 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.
Statistical analysis
Results of five independent experiments were considered and
expressed as mean ± SD. Statistical analysis was
carried out using ANOVA, Duncan’s multiple range test, and Student’s
t test for unpaired data. A P value of less than
0.05 was chosen as the limit for statistical significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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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 µM 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 [Ca2+]i (24). Therefore, we
investigated the effects of this peptide on cAMP production and
[Ca2+]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
[Ca2+]i as evaluated with the fluorescent
probe fura-2 (Fig. 3, trace a). Also,
PACAP-27 was without effect on [Ca2+]i (Fig. 3, trace b), whereas extracellular ATP induced a prompt rise
of [Ca2+]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 Mn2+, a cation permeable through Ca2+
channels, whereas ATP caused a prompt reduction of fura-2 fluorescence
(Fig. 4).
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, PACAP-38-stimulated
testosterone secretion was not modified by Leydig cell incubation in
Ca2+-free medium. To further investigate the possible ionic
mechanisms underlying the stimulatory effects of PACAP-38 in Leydig
cells, plasma membrane potential was monitored 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 Na+-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 µM 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.
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). In the presence of physiological ion
gradients, the main charged ion carrier would be expected to be
Na+. 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 Na+ was isotonically
substituted by choline chloride. In these experimental conditions,
PACAP-induced plasma membrane depolarization was completely inhibited
(Fig. 8, trace b). Similar results were
obtained when Leydig cells were suspended in medium in which
Na+ was isotonically substituted by methylglucamine or
sucrose (Fig. 8, traces c and d, respectively).
Experiments showing PACAP effects in Na+-free media did not
demonstrate unequivocally that the putative PACAP-38 activated channel
allows Na+ influx. Then we determined
[Na+]i with the Na+-sensitive
fluorescent probe SBFI. Figure 9, trace a,
shows that Leydig cell stimulation with PACAP-38 (100 nM)
caused a rapid increase in the [Na+]i
followed by a progressive decline probably due to activation of
Na+ extrusion mechanisms and/or receptor/channel
inactivation. When extracellular Na+ was reduced from 125
to 10 mM, the effects of PACAP-38 on
[Na+]i were greatly reduced (Fig. 9, trace
b). In both experimental conditions, the specific
Na+ carrier monensin induced an important increase in
[Na+]i, albeit obviously reduced in Fig. 9, trace b. The analysis of testosterone secretion from Leydig
cells incubated in Na+-free medium demonstrated that
PACAP-38 was not able to stimulate steroidogenesis when extracellular
Na+ was absent in the external medium, whereas 8-Br-cAMP
was fully competent in stimulating testosterone production (Table 3).
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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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, it has been demonstrated that in bovine adrenal chromaffin (30) and HIT-T15 insulinoma cells (31) PACAP-38 induces a Na+-dependent plasma membrane depolarization. In these cells, Na+-dependent current results from cAMP activation of ion channels similar to the cAMP-gated 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 Na+ from the extracellular medium. At present it is not known whether the mechanism of action of PACAP-38 involves a plasma membrane Na+-selective channel, Na+ pumps, or Na+ antiporters. The Na+/Ca2+ antiport has low affinity for Ca2+ and begins to operate only when [Ca2+]i reach values at least 10 times higher than basal levels. In our experimental conditions PACAP-38 did not modify Leydig cell [Ca2+]i, thus ruling out any role for Na+/Ca2+ antiporter in Na+ accumulation induced by this peptide. Further studies involving the direct measurement of Na+ currents will be necessary to clarify the precise mechanism of PACAP-induced Na+ entry in rat Leydig cells.
In bovine adrenal chromaffin and HIT-T15 insulinoma cells, cathecolamine and insulin secretion were strictly dependent on an influx of Ca2+ through the plasma membrane induced by the activation of voltage-dependent Ca2+ channels (VOCs) (30, 31). In Leydig cells, PACAP-38 did not induce any rise of [Ca2+]i, and PACAP-induced testosterone secretion was completely Ca2+ independent. The absence of any [Ca2+]i rise after stimulation by PACAP-38 is very intriguing, because plasma membrane depolarization should induce VOCs activation and Ca2+ 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 [Ca2+]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.
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 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 [Ca2+]i (5, 6, 24). The observations of present study, demonstrating that PACAP-38 does not induce any cAMP nor [Ca2+]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 Na+ 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 Leydig cells through the activation of a novel putative type IB receptor subtype coupled to an influx of Na+ from the external medium that induces the depolarization of the plasma membrane.
Received October 24, 1996.
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);
PACAP-27 (
). Data are mean ± SD of duplicate
determinations from three separate experiments. *,
P < 0.01; #, P < 0.001.
