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Original Articles |
1
Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5; and Taipei Medical College Hospital (C.-J.T., C.-R.T.), 110 Taipei, Taiwan
Address all correspondence and requests for reprints to: Dr. Peter C. K. Leung, Department of Obstetrics and Gynecology, University of British Columbia, Room 2H30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca
Abstract
The presence of P2U purinoceptor in human granulosa-luteal cells
(hGLCs) indicates a potential role of ATP in regulating ovarian
function. In this study an inhibitory effect of ATP on hCG-induced cAMP
production was observed. Extracellular ATP has been shown to activate
protein kinase C (PKC) after binding to a purinoceptor. To understand
the role of PKC in mediating ATP action, hCG-stimulated cAMP level was
examined in the presence of the PKC activator, 1 µmol/L phorbol
12-myristate 13-acetate (PMA), or the PKC inhibitor, 1 µmol/L
staurosporin or 1 µmol/L bisindolylmaleimide I. PMA, like 10 µmol/L
ATP, significantly reduced hCG-evoked cAMP production. In addition, the
inhibitory effect of ATP was reversed by staurosporin and
bisindolylmaleimide I. To further investigate the involvement of PKC
isoforms in mediating the inhibitory effect of ATP, the presence of PKC
isoforms in cultured hGLCs was examined by Western blot using
monoclonal antibodies against specific isoforms. Translocation of PKC
isoforms from cytosolic fraction to membrane fraction was studied to
identify the active PKC isozymes subsequent to ATP treatment. The
change in PKC isoform in PKC-depleted cells (achieved by exposure to
PMA for 18 h) was also examined. Our results demonstrated the
presence of PKC
, -
, -
, and -
isoforms in hGLCs and the
translocation of PKC subsequent to ATP treatment. In
PKC-depleted cells the PKC level was reduced, and no significant
effect of ATP on hCG-stimulated cAMP production was observed. To our
knowledge, this is the first demonstration of PKC isoforms in hGLCs and
the involvement of activated PKC in mediating the antigonadotropic
effect of extracellular ATP. Taken together, these results further
support a role of this neurotransmitter in regulating human ovarian
function.
ATP IS RELEASED from cells such as platelets or coreleased with neurotransmitter granules from autonomic nerves by exocytosis (1). ATP has been shown to participate in various types of physiological responses, including secretion, membrane potential, cell proliferation, platelet aggregation, neurotransmission, cardiac function, and muscle contraction (2, 3, 4, 5, 6). It is tempting to speculate that the coreleased ATP from autonomic nerve endings in the ovary may play a role in regulating ovarian function. We have reported previously that the P2U purinoceptor was expressed in human granulosa-luteal cells (hGLCs) (7), further supporting a physiological role of ATP in the human ovary.
After binding to a G protein-coupled P2 purinoceptor, extracellular ATP
activates phospholipase C and phosphatidylinositol hydrolysis,
generating diacylglycerol and inositol 1,4,5-triphosphate, which
stimulate protein kinase C (PKC) and cytosolic calcium mobilization,
respectively (2, 7, 8). The PKC family, a group of widely
distributed serine/threonine kinases, mediates intracellular signaling
of numerous cellular regulators including hormones, neurotransmitters,
and growth factors (9, 10). Thirteen isozymes have been
identified and categorized into four subclasses: 1) conventional PKCs
(, ßI, ßII, and 
), which are regulated by diacylglycerol,
phosphatidylserine, and Ca2+; 2) novel PKCs (,
, 
, and 
), which are regulated by diacylglycerol and
phosphatidylserine; 3) atypical PKCs (
, , and ), whose
regulation has not been clearly established; and 4) a fourth subfamily,
µ and 
(11, 12, 13). It is noteworthy to address that
multiple and various PKC isoforms are present in the ovary of different
species. In the rabbit corpus luteum, , ß, and isoforms of PKC
are identified (14), whereas porcine corpora lutea
contains and ß (15). Western blot analysis reveals
that bovine corpus luteum expresses and (16).
In the present study we demonstrated that ATP reduced hCG-induced cAMP accumulation in hGLCs. To further define the mechanism of action of ATP, we examined the effect of PKC on hCG-induced cAMP production, the expression of PKC isozymes, and the translocation of PKC isozymes subsequent to ATP treatment.
Materials and Methods
Reagents and materials
ATP, staurosporin, hCG, and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma (St. Louis, MO). DMEM, penicillin-streptomycin, and FBS were purchased from Life Technologies, Inc. (Burlington, Canada). Bisindolylmaleimide I, a PKC inhibitor, was obtained from Calbiochem (Cedarlane, Hornby, Ontario, Canada).
hGLC culture
hGLCs were collected from patients undergoing an in vitro fertilization-embryo transfer program. The use of hGLCs was approved by University of British Columbia clinical screening committee for research and other studies involving human subjects. Granulosa cells were separated from red blood cells in follicular aspirates by centrifugation through Ficoll-Paque, washed, and suspended in DMEM containing 100 U penicillin G/mL, 100 µg streptomycin/mL, and 10% FBS as described previously (7). The cells were plated in culture dishes. Cells were then incubated at 37 C under a water-saturated atmosphere of 5% CO2 in air for 3 days.
RIA for intracellular cAMP
To determine the effect of ATP on hCG-induced intracellular cAMP accumulation, hGLCs were incubated in serum-free medium containing 0.1% BSA and 0.5 mmol/L 3-isobutyl-1-methylxanthine (Sigma-Aldrich Corp., St. Louis, MO) for 30 min. hGLCs were then treated with hCG (1 IU/mL) in the absence or presence of increasing concentrations of ATP (0.1–100 µmol/L) for 20 min. hGLCs were lysed with 100% ethanol. Intracellular cAMP levels were measured using the [3H]cAMP assay system according to the manufacturer’s suggested protocol (Amersham Pharmacia Biotech, Oakville, Canada).
Treatment for cAMP assay
To investigate the role of PKC in hCG-evoked cAMP accumulation, hGLCs were treated with 1 IU/mL hCG in the presence or absence of 1 µmol/L PMA, a PKC activator, for 20 min. To understand the involvement of PKC in the effect of ATP on hCG-induced cAMP production, hGLCs were treated with ATP plus hCG in the presence or absence of PKC inhibitors (1 µmol/L staurosporin or 1 µmol/L bisindolylmaleimide I) for 20 min. In this study hGLCs were treated with staurosporin or bisindolylmaleimide I for 15 min before the administration of ATP.
Western blot analysis
To establish the expression of PKC isozymes, hGLCs were washed with ice-cold PBS and lysed with 100 µmol/L cell lysis buffer [RIPA; 150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.5), 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1.0 mmol/L phenylmethylsulfonylfluoride (PMSF), 10 µg/mL leupeptin, and 100 µg/mL aprotinin] at 4 C for 30 min. The cell lysate was centrifuged at 10,000 x g for 5 min, and the supernatant was collected for Western blot analysis. The amount of protein was quantified using a protein assay kit (Bio-Rad Laboratories, Inc., Richmond, CA) following the manufacturer’s protocol. Aliquots (30 µg) were subjected to 10% SDS-PAGE under reducing conditions, as previously described (17). The proteins were then electrophoretically transferred from the gels onto nitrocellulose membranes (Amersham Pharmacia Biotech) according to the procedures of Towbin et al. (18). These nitrocellulose membranes were probed with mouse monoclonal antibodies directed against specific PKC isozymes (Transduction Laboratories, Lexington, KY) at 4 C for 16 h. After washing, the membranes were incubated with horseradish peroxidase-conjugated goat antimouse secondary antibody, and the signal was visualized using the ECL system (Amersham Pharmacia Biotech), followed by autoradiography.
RT-PCR
In view of the presence of PKC, expressed mainly in the
nervous system (19), and as the monoclonal antibody for
PKC may cross-react with PKC according to the manufacturer, a set
of primers was used to examine the expression of PKC, as reported
previously (20). Total ribonucleic acid (RNA) was isolated
from hGLCs as previously described (7). As a positive
control, the RNA from human antral gastrin cells was provided by Dr.
Buchan (20). Briefly, 1 µg RNA was reverse transcribed
into complementary DNA (cDNA) using the First Strand cDNA Synthesis Kit
(Pharmacia Biotech, Morgan, Canada). One set of
oligonucleotide primers (5-CCCGGCGTAGGCGATTCAGA-3 and
5-TACGTGGATCTCATCTGCTGT-3) (20) was used for PCR to
amplify the PKC isoform from hGLCs. PCR reactions were performed in
the presence of 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L
MgCl2, 400 µmol/L deoxy-NTPs, 0.25 U
Taq DNA polymerase, 2 µmol/L primers, and 1 µL cDNA
template/25 µL reaction. Amplification was carried out for 35 cycles
with denaturation at 94 C for 45 s, annealing at 59 C for 35
s, extension at 72 C for 45 s, and a final extension at 72 C for
15 min.
Translocation experiment of PKC isozymes
hGLCs were incubated in serum-free medium for 4 h before
treatment. To examine the translocation of activated PKC, hGLCs were
treated with 10 µmol/L ATP for 1 or 5 min. Fractionation of cytosolic
and membrane proteins was performed as described previously
(21). In brief, cells were harvested in test buffer [10
mmol/L Tris-HCl (pH 7.4), 250 mmol/L sucrose, 2 mmol/L ethylenediamine
tetraacetate, 10 mmol/L ethyleneglycol-bis-(ß-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid, 2 mmol/L dithiothreitol, 1000 U/mL aprotinin, 0.8 µg/mL
leupeptin, and 2 mmol/L PMSF], disrupted by three freeze/thaw steps,
and centrifuged at 17,000 x g for 30 min. The
supernatant was collected as the cytosolic fraction. The pellet was
redissolved in lysis buffer [20 mmol/L HEPES/NaOH (pH 7.4), 150 mmol/L
NaCl, 1% Triton X-100, 10% glycerol, 8 mmol/L
ethyleneglycol-bis-(ß-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid, 15 mmol/L MgCl2, and 2 mmol/L PMSF] and
centrifuged at 17,000 x g at 4 C for 30 min. The
supernatant was collected as the membrane fraction. Equal amounts of
cytosolic and membrane protein (20 µg) were loaded for Western blot
analysis. The translocation of PKC isoforms was detected using
monoclonal antibodies against PKC, -, -, or -.
PKC depletion
Long-term treatment with PMA (16 h) results in PKC depletion
(22). In this study hGLCs were pretreated with 1 µmol/L
PMA for 18 h. Separate studies were then performed to examine the
expression of PKC and the effect of ATP on hCG-induced cAMP
accumulation in PKC-depleted hGLCs using Western blot analysis and RIA,
respectively.
Statistical analysis
Intracellular cAMP levels were shown as picomoles per 2 x 105 cells/dish. Data were presented as the mean ± SE. Statistical analysis was performed by one-way ANOVA, followed by Tukey’s multiple comparison test. Differences were considered significant at P < 0.05.
Results
Effect of ATP on hCG-induced cAMP production
ATP has been demonstrated to increase intracellular cAMP
production by activating adenylate cyclase in several cell systems
(23, 24). To examine the effect of ATP on intracellular
cAMP production, hGLCs were treated with hCG in the absence or presence
of increasing concentrations of ATP for 20 min. As demonstrated in Fig. 1
, hCG markedly increased the
intracellular cAMP level. In contrast, 10 µmol/L ATP did not increase
intracellular cAMP accumulation in hGLCs compared with that in the
control group. This indicates that the P2U purinoceptor expressed in
hGLCs is not coupled to adenylate cyclase. Instead, ATP reduced
hCG-evoked cAMP production in a dose-dependent manner, whereas the
maximal effect was reached at 10 µmol/L. ATP at 10 µmol/L reduced
hCG-stimulated cAMP production by 40% compared with hCG treatment
alone. Besides, no significant difference was noted between cells
treated with 10 and 100 µmol/L ATP.
|
Phorbol ester was shown to activate PKC in human ovarian tissue
(25). When hGLCs were treated with 1 µmol/L PMA,
hCG-stimulated cAMP production was reduced by 30% (Fig. 2).
|
, the inhibitory effect of ATP on
hCG-evoked cAMP production was reversed by PKC inhibitors, supporting
the active involvement of PKC in the regulation of cAMP production.
|
Eight antibodies against various PKC isozymes were used for the
Western blot analysis. Compared with the positive control, five (,
, , , and ) isoforms were identified by showing bands of
the expected sizes (Fig. 4). PKCß,
-, and - were absent in hGLCs. In the present study Jurkat cells
were used as the positive control for PKC, whereas rat brain was
used as the positive control for the rest of the PKC isoforms.
|
may cross-react
with PKC, and PKC is found mainly in the nervous system
(19), RT-PCR was performed to examine the
presence of PKC in hGLCs. Our results demonstrated the absence
of PKC in hGLCs. Primers specific for PKC amplified a band of the
expected size from the positive control, human antral gastrin cells,
but not from hGLCs (data not shown). This observation ruled out the
existence of PKC in hGLCs.
Translocation of PKC from cytosolic to membrane fraction
Activation of PKC is associated with a translocation of the enzyme
from the cytosolic fraction to the plasma membrane (21).
In the present study hGLCs were treated with 10 µmol/L ATP for 1 or 5
min. Of the four PKC isoforms, only PKC, which is found mainly in
the cytoplasm, was noted to have increased expression in the membrane
fraction and reduced expression in the cytosolic fraction after
treatment (Fig. 5). The translocation of
the PKC isoform to the plasma membrane was accompanied by a decrease
in the amount of PKC in the cytosolic fraction.
|
After treatment with PMA for 18 h, the PKC isozyme in
hGLCs was significantly down-regulated compared with that in the
control group (Fig. 6A). There was no
significant effect of ATP on hCG-stimulated cAMP production after PMA
pretreatment for 18 h (Fig. 6B).
|
The presence of P2U purinoceptor in hGLCs suggests a role of
extracellular ATP in the human ovary (7). We reported
previously that ATP exerted an antigonadotropic action by reducing
hCG-induced progesterone levels (26). To examine the
underlying mechanism, we demonstrated in this study that PKC was
associated with the inhibitory effect of ATP on hCG-stimulated cAMP
accumulation. The presence of PKC in hGLCs and its translocation
from the cytosolic fraction to the membrane fraction indicate a role of
activated PKC in mediating the action of ATP on hCG-induced cAMP
production.
cAMP is well established in mediating hCG actions such as
progesterone production in the ovary. PGF2, an
antigonadotropic agent, inhibits gonadotropin-induced
progesterone production in hGLCs via reducing
gonadotropin-stimulated cAMP accumulation (27). In
this study we demonstrated that ATP has an inhibitory effect on
reducing hCG-induced cAMP production, further supporting a regulatory
role of extracellular ATP in ovarian function.
Extracellular ATP has been shown to regulate cellular function through
activation of PKC (1, 2, 3). It appears that PKC may have
dual actions by providing positive forward actions as well as negative
feedback in controlling various signaling steps (28, 29).
We have recently reported that ATP is able to induce cytosolic calcium
oscillations, and that activated PKC can negatively regulate ATP-evoked
calcium mobilization from both intracellular stores and extracellular
influx in hGLCs (30). In this study the forward action of
PKC in mediating the ATP effect on hCG-induced cAMP accumulation was
demonstrated using a PKC activator and PKC inhibitors. PKC isozymes
consist of single polypeptide chains, each containing an amino-terminal
regulatory region and a carboxyl-terminal kinase domain
(31). Phorbol esters cause activation of conventional and
novel PKC isozymes through binding to the regulatory region
(28). In the present study PMA mimicked the effect of ATP
by reducing the hCG-induced cAMP production. Staurosporin, which is a
potent PKC inhibitor (32), and bisindolylmaleimide I
(33), which is a selective PKC inhibitor for PKC, -ß,
and -, effectively reversed the inhibitory action of ATP in
hCG-evoked cAMP production. These observations support the idea that
PKC plays a role in mediating ATP action in the human ovary.
Multiple and various PKC isoforms have been shown in the ovary of
different species (14, 15, 16). In the present study we
identified the presence of PKC, -, -, and - isoforms in
hGLCs. PKC subspecies are expressed specifically in certain tissue
(19). PKC appears to be present predominantly in the
nervous system, such as the brain and spinal cord (34).
Outside of the nervous system, PKC is identified in human antral
gastrin cells (20). Based on the RT-PCR results, we ruled
out the presence of this isoform in hGLCs. We reported previously that
10 µmol/L ATP induced cytosolic calcium oscillations in hGLCs
(7), implicating the activation of a calcium-dependent
PKC subsequent to ATP exposure. Our results demonstrated that the
PKC, a calcium-dependent PKC isoform, was translocated from the
cytosolic fraction to the membrane fraction after ATP treatment,
indicating that PKC is involved in the antigonadotropic action of
ATP in hGLCs.
Long-term exposure to phorbol esters causes down-regulation of PKC
activity associated with proteolysis of PKC. Proteases, such as calpain
or serine protease, act at the hinge region between the regulatory and
catalytic domains of PKC (35, 36, 37). Prolonged exposure to
PMA (16 h) has been demonstrated to down-regulate PKC activity in hGLCs
(22). In this study long-term treatment of hGLCs with PMA
down-regulated the expression of PKC (Fig. 6
), which was shown to be
activated by ATP (Fig. 5). The effect of ATP on hCG-stimulated cAMP
accumulation was lost in PKC-depleted cells, indicating the involvement
of PKC in reducing hCG-induced cAMP production.
The observation that ATP inhibited intracellular cAMP responses to hCG through activation of PKC leads us to speculate several potential action sites of activated PKC. Considering the presence of potential PKC phosphorylation sites in the third intracellular loop and at the C-terminal of the LH/hCG receptor, this receptor may be affected by activated PKC after ATP treatment. These intracellular regions of LH/hCG receptor are coupled to the Gs protein, suggesting that active PKC may be involved in the dissociation of the LH/hCG receptor from the Gs protein (38, 39). With respect to receptor-coupled G proteins, several studies have shown that phorbol ester can regulate G protein-mediated responses (40, 41, 42), indicating that the LH/hCG-coupled Gs protein may be inhibited by activated PKC in hGLCs. G protein-coupled adenylate cyclase is associated with cAMP accumulation. PKC has been demonstrated to alter the activity of adenylate cyclase (43), pointing out another possible site of action for activated PKC subsequent to ATP treatment in hGLCs. cAMP phosphodiesterase causes degradation of cAMP and thus alters intracellular cAMP accumulation. Stimulation of cAMP phosphodiesterase via PKC has been reported in cultured hGLCs (44), suggesting one more factor that may be associated with the regulation of cytosolic cAMP levels.
In conclusion, our results demonstrated that 1) extracellular ATP has
an inhibitory effect on hCG-stimulated cAMP accumulation; 2) the
PKC, -, -, and - isoforms are present in hGLCs; 3) PKC
is translocated from the cytosolic fraction to the membrane subsequent
to ATP treatment; and 4) PKC is involved in mediating the
antigonadotropic action of extracellular ATP. Taken together, these
results further support a role of this neurotransmitter in ovarian
steroidogenesis.
Acknowledgments
We thank Dr. Margo Fluker and the Genesis Fertility Center (Vancouver, Canada) for the provision of human granulosa-luteal cells.
Footnotes
1 This work was supported by the Canadian Institutes of Health
Research. 
2 Recipient of a studentship award from the British Columbia
Research Institute for Children’s and Women’s Health.
3 Recipient of a career investigator award.
Received November 6, 2000.
Revised February 14, 2001.
Accepted March 3, 2001.
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