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University of Padova, Clinica Medica 3, 35128 Padova, Italy
Address all correspondence and requests for reprints to: Prof. Carlo Foresta, University of Padova, Clinica Medica 3, Via Ospedale 105, 35128 Padova, Italy. E-mail: forestac{at}protec.it
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thapsigargin, a sesquiterpene lactone produced from the plant thapsia garganica, causes a rapid and pronounced increase in [Ca2+]i in a variety of different cells [mast cells (8), platelets (9), lymphocytes (10), HeLa cells (11), GH4C1 pituitary cells (12), rat Sertoli cell (13), human keratinocytes (14), adrenal chromaffin cells (15), and chicken granulosa cells (14)], mimicking the agonist-evoked responses. The mechanism of action of this drug is related to the inhibition of Ca2+-adenosine triphosphatase (Ca2+-ATPase) pumps of nonmitochondrial IP3-sensitive intracellular pools, thus inducing Ca2+ leakage from these stores (16). In the different cell types studied to date, thapsigargin produces only a transient increase in [Ca2+]i in the absence of extracellular Ca2+, whereas in the presence of Ca2+ in the external medium, this drug produces a sustained increase in [Ca2+]i. This is in keeping with the capacitative model of Ca2+ entry suggested by Putney in 1986 (17), who hypothesized that emptying of intracellular Ca2+ stores sends a signal to the plasma membrane that opens Ca2+-permeable channels. Thapsigargin does not stimulate the hydrolysis of inositol phospholipids or PKC activity (18), nor has it been reported to have a direct action on plasma membrane Ca2+ channels or to act like a Ca2+ ionophore (19). Thus, it offers a method to study the release of intracellular calcium without concomitant IP3 production and PKC activation, and several researchers have used thapsigargin to investigate the nature of the Ca2+ pools involved in agonist-evoked Ca2+ oscillation and the physiological role of the [Ca2+]i rise. The aim of this study was to determine the effects of thapsigargin on ionic homeostasis and testosterone production in isolated adult rat Leydig cells.
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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,
14 h of light and 10 h of darkness). Food and water were
available ad libitum. Interstitial cells were prepared from
testes through decapsulation and collagenase digestion. Briefly, 12–14
testes were incubated in medium 199 (3 ml/testis) with Hanks’ salts
and L-glutamine, 0,2% BSA (fraction V), and 1
g/liter collagenase (type II) at 34 C in a shaking (90 cycles/min)
water bath under a controlled atmosphere (95%
pO2-5% pCO2). After 15–20
min the suspension was filtered through sterile nylon gauze (0.5–0.8
mm pore size mesh), and erythrocytes were removed (
75–80%) by the
introduction of 5 ml 60% (vol/vol) Percoll into 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 medium 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), as described previously (7),
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 medium 199
(1:1, vol/vol) to remove any residual Percoll. The cells then were
resuspended in medium 199, and Leydig cells, more than 90% staining
positively for 3ß-hydroxysteroid-dehydrogenase activity
(7), were distributed in a band at density corresponding
to 40–55% Percoll. Cells were suspended in medium 199 to a final
concentration of 1.0 x 106 cells/ml (to a
total of 22–25 x 106 Leydig cells for
each experiment) with a viability higher than 95%, as determined using
the trypan blue method.
Measurement of [Ca2+]i in Leydig cell
suspensions
Leydig cell
[Ca2+]i was measured with
the fluorescent probe fura-2/AM as previously described
(20). Briefly, Leydig cell suspensions were incubated with
5 µM fura-2/AM for 45 min at 37 C in standard saline
containing 125 mM NaCl, 4.8 mM KCl, 1.2
mM MgSO4, 1.2 mM
K2HPO4, 5.6 mM
glucose, 25 mM NaHCO3, 2.0
mM CaCl2, and 20 mM HEPES
(pH 7.4) supplemented with 0.3% BSA. After loading, cells were washed
free of extracellular dye by being centrifuged three times (25 x
g for 2 min at room temperature) in standard saline
supplemented with 0.3% BSA and were maintained at room temperature
until used. All experiments were completed within 3 h of loading
with fura-2.
Determination of Mn2+ influx
Mn2+ uptake was measured by monitoring the
rate of fluorescence quenching at the excitation wavelength of 360 nm
(isosbestic point). (The isosbestic point for fura-2 is the
wavelength at which fura-2 fluorescence is insensitive on intracellular
Ca2+ concentrations and then the fluorescent signal emitted
is independent on variations in intracellular Ca2+
concentrations.) When measured at the isosbestic wavelength, the rate
of decrease in fura-2 fluorescence is insensitive to
[Ca2+]i changes and is
proportional to the rate of Mn2+ influx
(21).
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 with an isoosmotic concentration of
choline chloride, as previously described (22).
Measurement of intracellular free Na+
([Na+]i) in Leydig cell suspensions
[Na+]i was
evaluated using the fluorescent 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 with continuous
stirring. Cells were then washed by centrifugation (twice at 250
x g for 10 min each time at room temperature) in standard
saline. 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 345 and 490 nm
for excitation and emission, respectively (23).
Incubation of Leydig cells
Aliquots (0.5 ml) of Leydig cell suspension (1.0 x
106 cells/ml) were incubated in medium 199 with
Hanks’ salts, L-glutamine, HEPES,
Tris(hydroxymethyl)-aminomethane, 0.3% BSA (fraction V), penicillin
(10 U/liter), and streptomycin (1 g/liter), pH 7.4, in polyethylene
sterile tubes containing thapsigargin and CPA at doses ranging from
0.001–1 µM for thapsigargin and from 0.1–100
µM for CPA in a shaking bath (90 cycles/min) at 34 C in a
controlled atmosphere (5% pCO2). In parallel
experiments evaluating the role of external calcium, aliquots of Leydig
cell suspension were incubated in the absence of extracellular calcium
(no added calcium and 1.0 mM EGTA) and stimulated with the
same thapsigargin and CPA concentrations as those reported above. After
3-h 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.
To evaluate the role of K+ channels in thapsigargin-induced effects, Leydig cells were preincubated for 15 min in the presence of the K+ channels inhibitors tetraethyl ammonium (TEA) (10 mM) and charybdotoxin (100 nM) before thapsigargin and CPA addition. At these concentrations neither TEA nor charybdotoxin exerted any toxic or unspecific effect on Leydig cell [Ca2+]i, plasma membrane potential, or testosterone secretion.
Hormone measurement
Testosterone was determined by RIA using
[3H]testosterone (Radim, Rome, Italy). The
sensitivity was estimated as 0.28 nmol/liter, and intra- and interassay
coefficients of variation were 7.8% and 7.0%, respectively.
Statistical analysis
Data were analyzed using the StatView (Abacus Concepts, Inc.,
Berkeley, CA) statistical package. Statistical analysis was carried out
using ANOVA and t test. P < 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 thapsigargin
induced a rapid and dose-dependent rise in
[Ca2+]i, with maximal
effect at 100 nM (Fig. 1A). The increase in
[Ca2+]i was rapid, with a
peak and a small decline occurring with time and a following sustained
phase with kinetics that were not as expected if the
Ca2+ rise was dependent only on internal calcium
store depletion, but were in keeping with the presence of
Ca2+ influx from the external medium because they
were rapidly reversed by the addition of the
Ca2+-chelating agent EGTA (Fig. 1A). In the
absence of external Ca2+, thapsigargin still
produced a dose-dependent increase in
[Ca2+]i that was lower
than that obtained in Ca2+ medium (Fig. 1B) and
that was transient, as
[Ca2+]i declined to basal
levels after a few minutes, probably due to Ca2+
extrusion from the cytoplasm by plasma membrane
Ca2+ pumps, which are not inhibited by
thapsigargin. When Ca2+ was added back to the
medium, a rapid restoration of the sustained
[Ca2+]i was observed
(Fig. 1B).
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In the presence of Ca2+ in the extracellular
medium, CPA induced a dose-dependent increase in
[Ca2+]i that resembled
that produced by thapsigargin, although at higher concentrations, with
a maximal effect at 10 µM (Fig. 2A). As observed for thapsigargin in the
absence of extracellular Ca2+, CPA still produced
a [Ca2+]i rise, although
it was lower and transient, confirming that this drug releases
Ca2+ from intracellular stores (Fig. 2B).
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Extracellular Ca2+ plays an important role in the
rise in [Ca2+]i after
thapsigargin and CPA addition. This evidence is further supported by
the following experiments in which we evaluated the influx of
Mn2+, a divalent cation normally permeable
through Ca2+ channels and used as an indicator of
unidirectional Ca2+ influx (21).
Mn2+ once added to fura-2-loaded cells gradually
enters the Leydig cell cytoplasm, resulting in a slow progressive
quenching of fura-2 fluorescence (Fig. 3), as evaluated at the
Ca2+ isosbestic point (21).
Thapsigargin (100 nM) greatly increased the rate of fura-2
fluorescence quenching, demonstrating the activation of
Ca2+ channels in the plasma membrane (Fig. 3, trace a). Under the same experimental conditions CPA induced similar
effects, although at higher concentrations (Fig. 3, trace b).
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, trace a). To confirm this
hypothesis we performed the same experiment in
Ca2+-free medium. As shown in Fig. 4 (trace b),
under these experimental conditions the first rapid phase of
hyperpolarization induced by thapsigargin was unaffected and lasted for
several minutes, with a progressive return to basal levels. The
readdition of Ca2+ to the bathing solution
induced a rapid membrane depolarization (Fig. 4, trace b). These
results demonstrate that the depolarizing current induced by
thapsigargin is carried by Ca2+, the influx of
which is activated by internal Ca2+ store
depletion. This hyperpolarization-repolarization phase was parallel to
the [Ca2+]i increase
after Ca2+ store depletion, as demonstrated in
Ca2+-free medium (Fig. 5). Furthermore, the plasma membrane
hyperpolarization induced by thapsigargin seems to be completely
dependent on Ca2+ store depletion, and, in fact,
the addition of CPA after the depletion of internal
Ca2+ stores in Ca2+-free
medium with a maximal dose of thapsigargin did not induce any
hyperpolarization of the plasma membrane, as shown for the
[Ca2+]i rise (not shown).
The absence of any depolarizing effect induced by thapsigargin in
Ca2+-free medium seems to exclude a role for
Na+ in thapsigargin-induced effects on plasma
membrane potential in Leydig cells. Experiments using the
Na+-sensitive dye SBFI demonstrated that
thapsigargin did not induce any rise in
[Na+]i (Fig. 4, inset). Furthermore, in Na+-free
medium (choline chloride-substituted medium), the depolarization
induced by thapsigargin was similar to that observed in
Na+-containing medium (not shown). Thus, these
results exclude a role for Na+ in the
depolarizing effect of thapsigargin in rat Leydig cells.
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). Under these experimental conditions
plasma membrane depolarization after Ca2+
readdition to the medium was significantly reduced with respect to that
observed in Ca2+-containing medium, reflecting a
reduced influx of Ca2+ from the extracellular
medium. To confirm these observations, the
[Ca2+]i rise induced by
thapsigargin under the same experimental conditions was reduced
compared with that observed in the absence of K+
channel inhibitors (Fig. 6, inset).
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Role of [Ca2+]i rise in the activation of
Ca2+ influx by thapsigargin
Next we investigated whether the elevation of
[Ca2+]i induced by
thapsigargin in Leydig cells could be a signal for
Ca2+ entry. The experiment reported in Fig. 7 demonstrates that persistent
[Ca2+]i elevation is not
required to maintain an open Ca2+ entry pathway,
because when Leydig cells were treated with thapsigargin in
Ca2+-free medium, the readdition of
Ca2+ once
[Ca2+]i had returned to
basal levels was still able to induce a sustained
Ca2+ influx. In these experimental conditions it
could be argued that the previous, albeit transient,
[Ca2+]i rise that
occurred after thapsigargin addition caused the persistent activation
of Ca2+ influx pathways on the plasma membrane. A
methodological approach to investigate this consists in the depletion
of intracellular Ca2+ stores without causing a
detectable rise in
[Ca2+]i by stimulating
cells with sequential additions of low doses of thapsigargin in the
absence of extracellular Ca2+ (Fig. 7). In this
situation the inhibition of SERCA-ATPase is slow, and the leakage of
Ca2+ from the internal stores allows cellular
Ca2+-buffering systems and plasma membrane
Ca2+ pumps (that are not inhibited by
thapsigargin) to counteract any rise in
[Ca2+]i. In these
experimental conditions, restoration of extracellular
Ca2+ concentrations results in a rapid rise in
[Ca2+]i, demonstrating
that plasma membrane Ca2+ pathways are also
active without previous detectable increases in
[Ca2+]i due to
intracellular Ca2+ store emptying.
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). In Ca2+-free
medium, thapsigargin was still able to stimulate testosterone
production in a dose-dependent manner, although with a lesser effect
(Fig. 8A). In the same experimental conditions, CPA, when added to
Leydig cell suspensions, induced testosterone secretion similarly to
that produced by thapsigargin, although at higher doses, as
demonstrated for the
[Ca2+]i rise (Fig. 8B).
Testosterone secretion induced induced by thapsigargin and CPA was
similar to that stimulated by hCG (10 ng/ml) in both
Ca2+-containing and
Ca2+-free medium. Addition of hCG (10 ng/ml)
after Leydig cell stimulation with maximally active thapsigargin and
CPA concentrations did not stimulate further Leydig cell
steroidogenesis in Ca2+-containing or
Ca2+-free medium (Fig. 8, A and B). In Fig. 8C
are reported the effects of thapsigargin on testosterone secretion in
rat Leydig cells incubated with TEA and charybdotoxin. In the presence
of these K+ channels inhibitors, testosterone
secretion was lower than in their absence.
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Discussion |
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Leydig cell steroidogenesis is controlled in vivo by endocrine, paracrine, and autocrine interactions between different hormones and peptides (1). Several of these agonists operate via Ca2+-mobilizing receptors, but the precise roles of the different Ca2+ sources in testosterone production are not fully understood. LH is the physiological stimulator of Leydig cell steroidogenesis, activating its receptor coupled to adenylate cyclase to form cAMP (27), and it has been reported that in rat granulosa and bovine luteal cells LH can stimulate the turnover of phospholipids and IP3 formation, thus releasing Ca2+ from intracellular stores (28). To date there is no clear evidence for a similar effect of LH in rat Leydig cells, although Kumar et al. in 1994 (2) reported that hCG was able to stimulate a rise in Ca2+ in Leydig cells. Since then no other studies have confirmed those results; on the contrary, Tomic et al. (29) recently demonstrated that in these cells LH is without effect in Ca2+ homeostasis, confirming previous personal data (Rossato, M., and C. Foresta, unpublished observations). Other studies have reported that LHRH stimulates rat Leydig cell steroidogenesis through a mechanism involving Ca2+ and not cAMP, with an effect as high as 30–40% of that obtained by LH (4). Finally, we recently demonstrated that extracellular ATP stimulates testosterone production in isolated adult rat Leydig cells through the induction of a [Ca2+]i rise (6). In Ca2+-free medium, thapsigargin and CPA stimulated a rise in Ca2+ that was consistently transient. In these experimental conditions thapsigargin and CPA still stimulated testosterone secretion, but to a lesser extent with respect to that observed in Ca2+-containing medium. These data demonstrate that although the emptying of intracellular Ca2+ stores is, per se, able to induce the stimulation of steroidogenesis, the influx of Ca2+ from the external medium is necessary to activate testosterone secretion in rat Leydig cells to an extent similar to that induced by hCG (at a dose of 10 ng/ml). Furthermore, the data of the present study demonstrate that the influx of external Ca2+ induced by the emptying of internal Ca2+ stores does not occur through VOCCs, as the VOCC inhibitors nifedipine and verapamil did not modify thapsigargin and CPA stimulated-Ca2+ influx, confirming previous results obtained in other cell types (15). On the other hand, it has been recently demonstrated that rat Leydig cells do not possess VOCCs (29), and in previous experiments we did not observed any rise in [Ca2+]i after depolarization of Leydig cell plasma membrane with gramicidin or KCl (23). The monitoring of Leydig cell plasma membrane potential demonstrated that the depletion of internal Ca2+ stores induced a biphasic variation in the plasma membrane potential characterized by a first phase of hyperpolarization followed by a sustained depolarization. Plasma membrane depolarization is dependent on Ca2+ influx from the external medium activated by internal stores depletion, as it was reversed by the addition of EGTA, and it was completely absent in Ca2+-free medium. Ca2+ currents activated by depletion of intracellular Ca2+ stores have been described using the patch-clamp technique in a variety of nonexcitable cells and have been called Ca2+ release-activated Ca2+ current (26). On the contrary, it has been reported that external Na+ has no significant role in ionic currents activated by intracellular Ca2+ store depletion (30). The results of the present study confirm these reports, demonstrating that in Leydig cells intracellular Ca2+ store depletion induces ionic currents, and Na+ does not play any role in these events. Thus, although these results are only suggestive (as the plasma membrane potential has been monitored only with fluorescent probes), the ionic currents described in Leydig cells may resemble the Ca2+ release-activated Ca2+ current described in other cell types using patch-clamp (26).
The nature of the first phase of hyperpolarization induced by intracellular Ca2+ store depletion is less clear. The dependence of this hyperpolarization on internal stores emptying and the reduction of hyperpolarizing effects incubating Leydig cells with TEA and charybdotoxin, two well known inhibitors of K+ channels, suggest that this hyperpolarizing phase may depend on the activation of K+ efflux through Ca2+-activated K+ channels whose presence has been demonstrated in a number of different endocrine cells (31, 32), including rat Leydig cells (25).
The physiological role of this first rapid hyperpolarization of the plasma membrane induced by Ca2+ store emptying is unknown, but it is possible that it could contribute to an increase in Ca2+ influx from the external medium after internal Ca2+ store depletion by increasing the electrochemical gradient for Ca2+. The results of the present study seem to support this hypothesis, as in the presence of K+ channel blocking agents, and then in the absence of the hyperpolarizing phase induced by Ca2+ store emptying, Ca2+ influx was significantly lower than that observed in control experiments. In these experimental conditions the steroidogenetic response to thapsigargin and CPA was lower. It can be concluded that the activation of K+ current hyperpolarizing the cell plasma membrane accelerates Ca2+ influx and thus the biological responses coupled to it.
Although the mobilization of Ca2+ from internal stores induced by thapsigargin and CPA is well understood, the mechanism of Ca2+ entry induction is still somewhat controversial. Current models suggest the existence of two main ways of coupling between Ca2+ store depletion and Ca2+ influx activation: direct and indirect. The direct coupling mechanism assumes the physical interaction between internal stores and plasma membrane to directly open Ca2+ channels (33). Indirect coupling proposes the release or production of a messenger molecule that triggers the opening of Ca2+ channels in the plasma membrane or activating a cascade of events that, in turn, gates these channels. During the last years many different mechanisms have been proposed to verify the hypothesis of an indirect activation of capacitative or store-operated Ca2+ entry: involvement of adenine and guanine nucleotides (34); small molecular weight and heterotrimeric G proteins (35), and a product of oxidative cytochrome P450 enzyme system (36). A role for tyrosine kinase (37) and PKC (38) was also suggested. More recently, Randriamampita and Tsien (39) suggested that the message released from internal Ca2+ stores to plasma membrane is transferred by means of a diffusable factor designated Ca2+ influx factor, although this hypothesis has been recently questioned (40). It has also been suggested that the increase in [Ca2+]i determined by store depletion causes the opening of Ca2+ channels in the plasma membrane (41). The results of the present study seem to exclude a fundamental role for transient or persistent Ca2+ rise in Ca2+ influx induction in rat Leydig cells, as demonstrated also in rat Sertoli and mouse lacrimal acinar cells (13, 41). At present, the precise nature of the message by which the intracellular Ca2+ pools communicate with plasma membrane to activate Ca2+ entry remains to be elucidated.
In conclusion, the data reported here demonstrate that thapsigargin and CPA, two well known SERCA-ATPase inhibitors, induce a depletion of a common intracellular Ca2+ pool that, in turn, activates Ca2+ influx through the plasma membrane and are consistent with the existence of the capacitative or store-operated Ca2+ entry model in rat Leydig cells. The rise of Ca2+ induced by thapsigargin and CPA is able to stimulate testosterone production, providing evidence for a role of internal Ca2+ store emptying in the regulation of steroidogenesis in these cells. Furthermore, internal Ca2+ store emptying by inhibition of SERCA-ATPase induces modifications of Leydig cell plasma membrane potential due to Ca2+ leakage from internal stores and to activation of Ca2+-dependent K+ channel modulating the influx of Ca2+ from the external medium and testosterone production by these cells.
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Acknowledgments |
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Footnotes |
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Received December 27, 2000.
Accepted for publication May 8, 2001.
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References |
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) and absence (
) of
extracellular Ca2+. On the right of A and B
are reported the effects of hCG (10 ng/ml) on testosterone secretion in
the presence (