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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 9 4503-4512
Copyright © 2004 by The Endocrine Society

Two Types of Calcium Channels in Human Ovarian Endocrine Cells: Involvement in Steroidogenesis

Agnes Agoston, Lars Kunz, Annette Krieger and Artur Mayerhofer

Anatomisches Institut der Universität München, 80802 München, Germany

Address all correspondence and requests for reprints to: Artur Mayerhofer, Anatomisches Institut der Universität München, Biedersteiner Strasse 29, 80802 München, Germany. E-mail: mayerhofer{at}lrz.uni-muenchen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nature, regulation, and functional role of ion channels of human ovarian endocrine cells are not well known. In our present study, we show two types of voltage-activated Ca2+ currents (ICa) in cultured human luteinized granulosa cells (GCs), as assessed by whole-cell patch-clamp experiments. Electrophysiological properties, namely low threshold of activation, pronounced time-dependent inactivation, slow and voltage-dependent deactivation kinetics, insensitivity to SNX-482, and high sensitivity to Ni2+, defined the predominant ICa as a T-type Ca2+ current (ICa.T). In 4% of cells a Ni2+-insensitive ICa was measured alone or together with ICa.T. This Ca2+ current was high voltage activated and highly sensitive to dihydropyridine, indicative of an L-type Ca2+ current. RT-PCR analysis demonstrated the presence of mRNA coding for {alpha}1-subunits of two different Ca2+ channels (T-type Cav3.2 and L-type Cav1.2) in GCs. In addition, these two types were detected in the human corpus luteum by RT-PCR (Cav3.2) and immunohistochemistry (Cav1.2). Although stimulation of cultured GCs with human chorionic gonadotropin did not change the characteristics of recorded ICa.T, it markedly increased the percentage of cells displaying ICa from 29 to 63% and significantly increased (2.2-fold) the density of ICa.T. Furthermore, the stimulatory effect of human chorionic gonadotropin on progesterone production was diminished by pharmacological blockage of ICa.T by Ni2+ or flunarizine. Thus, our study provides evidence that human GCs in vivo and in vitro express T- and L-type Ca2+ channels and that the Cav3.2 (also called {alpha}1H) isoform is involved in a fundamental endocrine function of these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OVARY IS a dynamic organ in which steroid production and cell proliferation and differentiation occur constantly (1). Particularly, granulosa cells (GCs) of the follicle, which after ovulation luteinize and form the major component of the corpus luteum (CL), produce steroids. Whereas the important pituitary-derived hormones (LH/FSH) do not directly alter levels of intracellular-free Ca2+ concentration, [Ca2+]i (2), several locally produced ovarian signaling molecules (e.g. acetylcholine, oxytocin, prostaglandins) induce changes of [Ca2+]i in GCs (3, 4, 5). Results obtained by several groups suggest that the rapid elevation of [Ca2+]i after acute stimulation by most of these molecules mainly arises from release of Ca2+ from intracellular stores. However, extracellular Ca2+ may take part in the regulation of Ca2+ homeostasis in GCs, as was shown in several species. Studies with fluorescent dyes in rat GCs and radioisotopes in porcine GCs implied that extracellular Ca2+ enters these cells (6, 7). Patch-clamp experiments in porcine and avian GCs showed the presence of functional voltage-activated T- and L-type Ca2+ channels, which regulate influx of Ca2+ from the extracellular space in a voltage-dependent manner (8, 9, 10). Although several isoforms for different classes of voltage-gated Ca2+ channels (VGCCs) are known, the exact molecular identity of these Ca2+ channels has not been elucidated in these species. In contrast, nothing is known about VGCCs in human GCs.

Native Ca2+ channel currents have been classified into T, L, N, P/Q, and R type based on their electrophysiological and pharmacological characteristics. Even before the availability of selective toxins and blockers (e.g. {omega}-Agatoxin IVA, {omega}-Conotoxin GVIA, {omega}-Conotoxin MVIIA, SNX-482), the concept of low- and high-voltage-activated Ca2+ currents has been established based on the membrane potential at which they are activated. VGCCs are integral membrane protein complexes composed of an essential pore forming {alpha}1-subunit (11). Molecular cloning studies have defined 10 genes encoding pore-forming {alpha}1-subunits. According to the recently used new and old nomenclature (in brackets), the coded {alpha}1-subunits were named as follows: Cav1.1 ({alpha}1S), Cav1.2 ({alpha}1C), Cav1.3 ({alpha}1D), Cav1.4 ({alpha}1F), Cav2.1 ({alpha}1A), Cav2.2 ({alpha}1B), Cav2.3 ({alpha}1E), Cav3.1 ({alpha}1G), Cav3.2 ({alpha}1H), Cav3.1 ({alpha}1I). The new nomenclature divides the Ca2+ channel {alpha}1-subunits into three structurally and functionally related families (Cav1, Cav2, Cav3) based on sequence homology. (For a comprehensive review the reader is referred to Ref. 12 .) Inasmuch as {alpha}1-subunits are capable of interacting with a number of ancillary subunits, there is no absolute correspondence between {alpha}1-subunits and measured currents. To sum up, numerous nomenclatures are used in the literature for naming VGCCs. Bearing this in mind, we will employ the most appropriate terminology.

The neuronal high voltage activated (HVA) Ca2+ channels (N, P/Q, and R type) are crucial for neurotransmitter release and inhibited by many G protein-coupled receptors (13, 14). Low-voltage-activated Ca2+ channels of the Cav3 family are involved in many cellular events. They mediate Ca2+ influx into excitable cells (neurons and muscle cells) and nonexcitable cells, including endocrine cells (of the adrenal cortex), osteoblasts, and germ cells (15, 16, 17, 18). They acutely contribute to action potential generation (19, 20, 21) but are also involved in processes including regulation of cell proliferation and differentiation (22, 23, 24). Importantly, T-type Ca2+ channels are required for steroid synthesis, as was shown in human, bovine, and rat adrenocortical cells (25, 26, 27). L-type Ca2+ channels are distinct from T-type channels by their pharmacological [dihydropyridine (DHP)-sensitive] and electrophysiological characteristics (HVA, slowly inactivating). Two isoforms (cardiac, Cav1.2, and endocrine, Cav1.3) of L-type Ca2+ channels are known to be widely expressed in many tissues in which they regulate heartbeat, muscle tonus, hormone secretion, and gene expression, whereas the other two isoforms (Cav1.1, Cav1.4) are expressed predominantly in the skeletal muscle and retina (11, 28). Interestingly, the patterns of tissue distribution of T- and L-type Ca2+ channels are partially overlapping; for example, both types can be found in steroid-producing endocrine cells of the adrenal cortex (16, 25).

To identify Ca2+ currents and underlying channels in human luteinized GCs, we used electrophysiological and molecular approaches. We performed patch-clamp studies on cultured human GCs that allowed us to identify two Ca2+ currents (ICa) based on detailed electrophysiological and pharmacological characterization complemented by RT-PCR and immunohistochemistry. The function of ICa in steroid production was investigated by measurements of the effects of Ca2+ channel blockers on progesterone production by cultured human GCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
GC preparation and culture

Human GCs were obtained from follicular aspirates of women undergoing in vitro fertilization. They were separated by centrifugation at 560 x g for 3 min and subsequent washing in serum-free DMEM/Ham’s F-12 medium (1:1, Sigma, Deisenhofen, Germany). Washed cells were resuspended in culture medium supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% fetal calf serum as previously described (3). The use of the cells for scientific experiments was approved by the local ethics committee, and written consent of the patients was obtained. For patch-clamp experiments cells were cultured on glass coverslips, otherwise in plastic dishes in a humidified atmosphere with 5% CO2 at 37 C.

Ovarian tissue samples

Human CL samples (n = 3) were obtained from women undergoing gynecological surgery. The use of these tissue samples was approved by the local ethics committees, and patients gave written consent. All tissue samples were fixed in Bouin's fixative, paraffin embedded, and sectioned (5 µm thick) for subsequent immunohistochemistry or isolation of mRNA (29, 30).

Electrophysiology

Voltage clamp recordings were conducted using the whole-cell configuration of the patch-clamp technique. ICa were recorded at room temperature with an EPC-9 amplifier and Pulse 8 software (HEKA, Lambrecht, Germany). The recording chamber was mounted on the stage of an Axiovert 135 TV microscope (Carl Zeiss, Jena, Germany). Single cells were observed at x40 magnification. To eliminate contamination from other ionic and exchange currents, the extracellular solution was Na+ and K+ free (millimoles): 117 tetraethylammonium-chloride, 20 BaCl2 or CaCl2, 10 HEPES, 1 MgCl2, 10 glucose, 30 sucrose (pH 7.4) adjusted with CsOH. Glass pipettes were filled with solution containing (millimoles) 120 CsCl, 20 tetraethylammonium-chloride, 1 CaCl2, 5 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid or 10 EGTA, 10 HEPES, 2 MgCl2, 4 Mg-ATP, 0.2 Na-GTP (pH 7.2) adjusted with CsOH and had a resistance of 1.5–3.0 M{Omega}. Patch pipettes were fabricated from thick-wall borosilicate glass capillaries (GB 150–8P; Science Products GmbH, Hofheim, Germany). Series resistance was continuously monitored and compensated by 70–80% using the EPC-9 correction routines (PULSE software, HEKA). The current output was filtered at 5 kHz with a four-pole Bessel filter, and data were acquired at 50 µsec intervals (20 kHz). If not otherwise stated, the holding potential was set to –80 mV, and current traces were elicited by different protocols with the step intervals of 5 sec (0.2 Hz). Ca2+ currents were elicited by step depolarization up to +50 mV with 10-mV increments of 100-msec duration. The voltage dependency of activation was obtained from the normalized I-V curve, fitted by Boltzmann function, I/Imax = 1/{1 + exp[(V – V50)/k)], where V50 is the midpoint of activation and k is the slope factor.

The steady-state inactivation was analyzed using 2-sec conditioning prepulses to various voltages (from –100 to –20 mV) followed by a test pulse to –10 mV of 100-msec duration. Each experimental data set was normalized to the peak current obtained after the –100 mV prepulse, plotted vs. conditioning prepulse potentials (–100 to –20 mV), and then fitted with a Boltzmann distribution. Deactivating tail currents were measured at different repolarization potentials (–100 to –20 mV) for 50 msec after a 15-msec prepulse to –10 mV. Tail currents were fitted with a single exponential function. A double-pulse protocol (–10 mV, 100 msec), applied from a holding potential of –90 mV, was used to estimate the recovery from short-time inactivation. From the ratio of the peak current (I2) elicited by the second pulse to the peak current (I1) during the first pulse, the fractional recovery was calculated and plotted as function of interpulse duration ({Delta}t): 10, 30, 50, 90, 170, 330, 650, 1290, and 2570 msec). The relationship was fitted by a monoexponential function and the half-time was determined. L-type Ca2+ channel modulators, nifedipine and BayK 8644, were dissolved in dimethylsulfoxide (DMSO) to make stock solutions of 10 and 1 mM, respectively. These stocks were stored in the dark at –20 C and diluted in the bath solution immediately before use. The final concentration of DMSO in the bath was 0.05–0.1%, which alone had no effect on the L-type current. SNX-482 (100 µM) and NiSO4 (200 mM) were dissolved in distilled water. Calcium channel modulators, except NiSO4, were purchased from Alomone Labs (Jerusalem, Israel), and all other chemicals were from Sigma.

Immunohistochemistry

As previously described (29, 30), deparaffinized tissue sections of human ovaries were treated with 3% H2O2 in methanol for 20 min to block endogen peroxidase activity and then incubated with 5% normal goat serum for 30 min to prevent nonspecific antibody binding. The sections were incubated overnight at 4 C with rabbit polyclonal antiserum (anti-Cav1.2, 1:200 or anti-Cav1.3, 1:500, Alomone Labs) and probed with biotin-coupled goat antirabbit antibody (1:500). The sites of immunoreaction were visualized by the ABC method (Vectastain Elite kit; Vector Laboratories, Burlingame, CA) and addition of 3'3'-diaminobenzidine tetrahydrochloride solution containing H2O2. To confirm the specificity of the immunoreaction, the antisera were preadsorbed with the peptide antigens. Further controls consisted of nonimmune rabbit normal serum (1:5,000 or 1:10,000) or omission of the antiserum.

RT-PCR

Human ovarian tissue sections were deparaffinized and CLs (0.5–1 cm in diameter), clearly identified by light microscopy, were scratched from the slides. Total RNA was extracted using the PUREscript RNA isolation kit (BIOzym Diagnostik, Hessisch Oldendorf, Germany) according to the manufacturer’s protocol. As previously described (31), mRNA was subjected to reverse transcription and PCR.

Total RNA from several batches of human GCs harvested on d 1–10 of culture was extracted and reverse transcribed, as described (29, 30). Commercial human ovarian cDNA (human MTC Panel II, BD CLONTECH, Inc., Heidelberg, Germany) was used in addition (32). Primer pairs were chosen to span at least one intron of the genomic sequence (25). PCR amplification for identification of Ca2+ channel subunits was performed with the oligodeoxynucleotide primer pairs listed in Table 1Go. The following protocol was used: denaturation at 94 C (30 sec), annealing at 54 C (30 sec), and elongation at 72 C (60 sec). The protocol was repeated 45 times for Cav3.1, Cav3.2, Cav1.2, and Cav1.3 and 35 times with the nested Cav3.2 primers, respectively. The identity of all PCR products was verified by direct sequencing using one of the specific primers.


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  		TABLE 1. Oligodeoxynucleotide primers used in PCRs

 
Progesterone assay

Human GCs were cultivated for 3–5 d in 24-well plates, as described previously (33). Thereafter, they were treated in triplicates for 24 h without or with 10 IU/ml human chorionic gonadotropin (hCG) alone or in combination with Ca2+ channel blockers [stocks: 2 mM nifedipine and 20 mM flunarizine in 10% DMSO (Sigma), 100 µM SNX-482, and 100 mM NiSO4 in water]. To examine the effect of toxins on the basal progesterone production, cells were treated with blockers for 24 h. As control 1 µl distilled water or DMSO (10%) was added to the culture media (1 ml). The supernatants were stored at –20 C until measurement of progesterone concentrations using a commercially available ELISA (DRG Instruments, Marburg, Germany).

Cell morphology

Viability of cells treated with Ca2+ channel blockers alone or in combination with hCG was assessed by trypan blue exclusion assay. After respective treatments, cells were incubated at room temperature for 10 min with 0.04% trypan blue (Sigma). Viable cells (unstained) and nonviable cells (stained) were counted, and the percentage of viable cells was calculated.

Cells used in progesterone assays were fixed with 5% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.4), postfixed with 1% osmium tetroxide/potassium hexacyanoferrate (II), and embedded in Epon. Sections (1 µm thick) were cut and inspected by light microscopy after staining with Azure II/methylene blue (1:1), as described (33).

Statistics

Data are expressed as means ± SEM in the text and graphs. Statistical significance of changes in current density was determined using unpaired t test with the help of the SigmaPlot 8.0 software package (Wavemetrics, Lake Oswego, OR). For progesterone assay, paired t test or ANOVA followed by Newman-Keuls test was performed using GraphPad Prism 3.02 software (GraphPad Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Electrophysiological characteristics of the dominant Ca2+ current (ICa.T) in human GCs

Activation and inactivation. To examine the expression of Ca2+ channels in cultured human luteinizing GCs, whole-cell patch-clamp recordings were carried out. Recordings were performed on 162 cells cultured without hCG. Of these, 29% possessed measurable inward ICa. Currents from 14 cells were selected for detailed analysis based on low and stable series resistance, high input resistance, and high signal-to-noise ratio. In 25% of all cells examined, a transient Ca2+ current was measured, which in our study represented the predominant current. Figure 1AGo illustrates a set of inward current traces of dominant ICa. Typically, currents recorded by voltage step protocol had an activation threshold at around –50 mV and peaked at –10 mV (Fig. 1AGo). The voltage dependency of the kinetics of activation and inactivation were compared in Fig. 1AGo. In both cases, increasing the amplitude of depolarization sped the rate of activation and inactivation until the time constants approached the lowest values. Activation rate accelerated at least 8-fold with increasing depolarization and inactivation became voltage independent above 0 mV. We recorded currents in the same percentage (20–29%) of cells irrespective of culture day (from d 3 to d 6). The characteristics of this dominant ICa were similar during this period, and there were no significant differences in the current densities (not shown). The average current density was –0.8 ± 0.1 pA/pF at –10 mV (n = 14). This finding indicates that the dominant current represents only one isoform, and there is no change in its expression during this period of culture.



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  		FIG. 1. Electrophysiological characterization of the predominant Ca2+ current (ICa.T) in human GCs. A, Top, left, Inward current traces were elicited by depolarizing voltage step pulses (see B, top, right). Note the typical criss-crossing pattern of ICa.T. Bottom, right, Voltage dependency of time constants of activation and inactivation, ({tau}act., {blacktriangleup} and {tau}inact., {blacksquare}, n = 12). B, Middle, Steady-state activation ({blacktriangledown}) and inactivation ({blacksquare}) properties of ICa.T (n = 8). Current traces (inset) and test pulses (top) are shown. Calculated relative (Rel.) window current (bottom) is expressed as percent of the peak current (n = 8). C, Deactivation properties of ICa.T. The deactivating tail currents (middle) were measured at different repolarization potentials (see pulse protocol, top). Plot of {tau}deact. as a function of the repolarization potentials (bottom, right, n = 4). D, Recovery kinetics of ICa.T was studied using a paired pulse-protocol (top). Current traces (middle) are shown for 30, 50, 90, 170, 330, 650, 1290, and 2570 msec ({Delta}t). Solid curve (bottom) shows the result of monoexponential fitting of I2/I1 ratios plotted against {Delta}t (n = 4).

 
Steady-state activation and inactivation. The voltage dependence of activation was assessed by normalizing the peak current amplitudes to the maximum peak current for each cell (see Materials and Methods) (Fig. 1BGo). The midpoint of activation (V50) was –34.1 ± 0.7 mV, and the slope factor (k) was 4.6 ± 0.6 mV (n = 12). The voltage-dependent inactivation behavior of ICa in human GCs was analyzed using conditioning prepulses followed by a test pulse (see Materials and Methods). Typical traces obtained are shown in Fig. 1BGo(inset). The k was –5.2 ± 0.4 mV and the V50 was –50.6 ± 0.5 mV (n = 8). To determine the putative window current, which is determined by the overlap of the activation and inactivation curves (Fig. 1BGo), the normalized current was multiplied by the steady-state inactivation values. The calculated window current peaked at –42 mV with an amplitude of 3% of the peak current (n = 8).

Deactivation. Deactivation properties of ICa were characterized by the kinetics of channel closing (deactivation) after a short activation (15 msec). This parameter was determined by the time course of tail current relaxation (Fig. 1CGo). The deactivation time constant ({tau}deact.) was strongly voltage dependent; for example, {tau}deact. was 1.5 ± 0.2 msec at –100 mV and 4.7 ± 0.5 msec at –50 mV (Fig. 1CGo, n = 4), suggesting the presence of ICa.T in GCs.

Recovery from short inactivation. The recovery from short-time inactivation was estimated by the use of a double-pulse protocol applied from a holding potential of –90 mV (Fig. 1DGo). The inactivation of ICa was induced by the first pulse. The channels were relieved from the inactivation by repolarization to –90 mV of variable duration (from 10 to 2570 msec). As interpulse interval increased, the current activated by the second pulse became larger. The fractional recovery (I2/I1) was calculated and plotted as a function of interpulse duration ({Delta}t). The relationship was fitted by a monoexponential function, given that the half-time for recovery is 223.2 ± 17.6 msec (n = 4).

Pharmacology. Electrophysiological properties of ICa in the majority of cases suggested an ICa.T. To rule out the involvement of R-type currents, which can inactivate rapidly and are also sensitive to the divalent cation Ni2+ (34), we applied SNX-482 (100 nM), a specific blocker of some R-type and other Cav2 channels (35, 36, 37). We found that SNX-482 had no effect on ICa.T (Fig. 2AGo).



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  		FIG. 2. Pharmacological and molecular identity of ICa.T and ICa.L in human GCs. A, The predominant ICa.T was not affected by SNX-482 (100 nM). B, Effect of 100 µM Ni2+ on a cell displaying ICa.T and ICa.L (one representative experiment). Top, Superimposed current traces (left) measured at different test potentials (–70, –30, –10, +10, and +30 mV) in control ({blacksquare}) and after 100 µM Ni2+ application ({blacktriangleup}). Corresponding current traces (except at –70 mV, right) before and after Ni2+ application are shown. Note the strong effect of Ni2+ at negative voltages (–30, –10 mV). Bottom, Peak (left) and mean (right) current-voltage relationship in control and after 100 µM Ni2+ application are shown. C, BayK 8644 (1 µM) increased and nifedipine (5 µM) blocked the ICa.L. Top, right, Current traces are measured at +10 mV. D, Left, Immunohistochemical detection of Cav1.2 (L-type Ca2+ channel) subunit protein in human CL. Right, Preadsorption control (Co.) is shown (scale bar, 50 µm). E, Top, Nested RT-PCR revealed the presence of mRNA for Cav3.2 in cultured luteinized GCs from different ages in culture (3, 5, 6 d) in the human ovary and two human CL samples. Bottom, By RT-PCR we also detected mRNA for Cav1.2 in human GCs. As negative control (Neg. Co.), RT-PCR was performed without template.

 
Previous reports (15, 38) described specific features of three isoforms of T-type currents generated by recombinant Cav3.1, Cav3.2, and Cav3.3 subunits. It is well known that Cav3.3 subunit-generated currents show the slowest activation and inactivation kinetics and the fastest deactivation of the T-type channel family (39). Thus, in our study in GCs, this isoform can be ruled out. It is known that the expression of Cav3.1 or Cav3.2 subunits generates Ca2+ currents with similar hallmarks of T-type currents (21) but that can be distinguished by the sensitivity to Ni2+ (40). The specific feature of Cav3.2 currents is their high sensitivity to Ni2+. We therefore studied the effect of 100 µM Ni2+ on ICa.T. We found that 100 µM Ni2+ reversibly and quickly blocked the peak ICa.T by 92 ± 3% (n = 4, not shown), implying that the ICa.T is due to a T-type Ca2+ channel composed of Cav3.2.

DHP-sensitive, HVA Ca2+ current in human GCs

Whereas 25% of GCs cultured without hCG displayed only ICa.T, additional 4% of GCs showed a Ni2+-insensitive Ca2+ current, which was recorded in cells also displaying (Fig. 2BGo) or lacking ICa.T (Fig. 2CGo). This second, Ni2+-insensitive Ca2+ current was HVA, peaked at +10 mV, and showed slow time-dependent inactivation (Fig. 2CGo). Because of the slow time-dependent inactivation, the peak and mean current-voltage relationship of this second current in GCs peaks at the same test potential (in our case at +10 mV) (Fig. 2BGo). The mean current that was measured at the end (93–98 msec) of the step protocol represents the current amplitude, which does not inactivate during long (100 msec) depolarization. In contrast, the time-dependent inactivation of T-type currents are much slower below –20 mV than above –10 mV, at which potential it peaks (Fig. 1AGo), as was shown in our study. Consistently, the Ni2+-sensitive mean T-type current could be measured only between –50 and –10 mV (Fig. 2BGo). Thus, the mean current-voltage relationship is an informative way to show the simultaneous presence of a transient T-type and a slowly inactivating HVA Ca2+ current in GCs. Importantly, we found that the HVA current was DHP sensitive because BayK 8644 (1 µM) increased and nifedipine (5 µM) blocked this current (Fig. 2CGo). When Ca2+ was used as a charge carrier, this HVA current showed a pronounced calcium-dependent inactivation, inactivating faster than the Ba2+ current. In some experiments we changed the holding potential from –80 to –40 mV, and we found that this Ni2+-insensitive current could still be elicited (not shown). These results strongly suggest that the L-type Ca2+ current (ICa.L) is another, second type of ICa in cultured human GCs.

Molecular identity of {alpha}1-subunits underlying ICa.T and ICa.L

To determine the molecular nature of the T- and L-type Ca2+ channel {alpha}1-subunits, we performed RT-PCR analyses using RNA of human luteinized GCs and luteal tissue from human ovarian slices. Using nested primer pairs and subsequent sequencing, we demonstrated the presence of Cav3.2 mRNA in either sample (Fig. 2EGo). A faint band of expected size was already obtained after the first PCR in GCs (not shown). We also performed RT-PCR using specific primers, and we detected mRNA for Cav1.2 in GCs but not in samples of the CL. We did not detect mRNA for other isoforms (Cav3.1 or Cav1.3) of Ca2+ channels in GCs (not shown).

Unfortunately, antisera recognizing Cav3.2 Ca2+ channel {alpha}1-subunit were not available to us. However, by using specific antisera against {alpha}1-subunits of two L-type isoforms (Cav1.2, Cav1.3), we examined protein expression of these Ca2+ channels in the CL. Immunohistochemical staining with anti-Cav1.2 antiserum was specific and could be prevented after preadsorption of the antiserum with the corresponding peptide. In agreement with our patch-clamp experiments, Cav1.2 was present only in a small proportion of large luteal cells of the human CL (Fig. 2DGo). We also performed immunostaining with an anti-Cav1.3 antiserum. The staining (not shown) was, however, not abolished after preadsorption with the antigen.

Effects of hCG on Ca2+ channels

We performed patch-clamp experiments on an additional 90 cells cultured with hCG for 24–36 h. The most conspicuous difference between cells cultured in the absence (control cells) or presence of hCG was that the percentage of cells displaying Ca2+ currents increased from 29% up to 63% (Fig. 3AGo). Forty percent of cells examined showed T-type, 17% both ICa.T and ICa.L, and 6% only ICa.L. We analyzed T- and L-type current densities in control and hCG-treated cells (Fig. 3BGo). Interestingly, ICa.L density did not change markedly after hCG-treatment (from –0.5 ± 0.2 pA/pF, n = 5, to –0.7 ± 0.3 pA/pF, n = 11). In contrast, incubation with hCG significantly (P = 0.027) increased ICa.T density from –0.8 ± 0.1 pA/pF (n = 14) to –1.8 ± 0.3 pA/pF (n = 29) without changes in voltage dependence and kinetics. Parameters for the voltage dependence of activation were V50 = –33.7 ± 1.9 mV, k = 4.0 ± 0.9 mV (n = 3), and for the steady-state inactivation V50 = –57.7 ± 1.4 mV, k = –5.0 ± 0.4 mV (n = 2). The half-time for the recovery was 223.0 ± 0.6 msec (n = 3). The {tau}deact. at –100 mV and –50 mV were 2.2 ± 0.6 msec and 7.7 ± 1.4 msec (n = 3), respectively. One hundred micromoles Ni2+ inhibited the current (ICa.T) by 94.8 ± 1.8% (n = 3).



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  		FIG. 3. Long-term effects of hCG (10 IU/ml) on ICa in human GCs. A, Left, Percentage of cells cultured without (control) or with hCG (+hCG), which showed measurable ICa. Right, Cells were grouped according to properties of their recorded ICa (control, white; +hCG, black). Total numbers of cells examined are given. B, Left, T-type current densities measured in hCG-treated cells (+hCG) was significantly higher, compared with values measured in nontreated (control) cells (unpaired t test). Right, Individual values of L-type current densities measured at +10 mV in control and hCG-treated cells (+hCG).

 
Physiological role of T-type Ca2+ channels in human GCs

To examine whether Ca2+ channel activity is involved in steroidogenic function of cultured human GCs, the effect of T-, L-, and R-type Ca2+ channel blockers (100 µM Ni2+, 2 µM nifedipine, 100 nM SNX-482) on the basal and hCG-stimulated progesterone production was measured (Fig. 4Go). For this purpose, cells were incubated with or without 10 IU/ml hCG alone or in combination with one of the blockers for 24 h. We found that none of the used Ca2+ channel blockers had any significant effect on the basal progesterone production. Treatment of GCs with 100 µM Ni2+, which is an effective T-type (Cav3.2) channel blocker, resulted in marked decrease in hCG-stimulated progesterone production (–30.7 ± 6.1%, P = 0.047, n = 6 independent experiments). Flunarizine, at a concentration (20 µM) that blocks all three isoforms of T-type Ca2+ channels, diminished the effect of hCG by 33.8% in two experiments. SNX-482 (+5.7 ± 9.7%, n = 5) and nifedipine (–0.1 ± 4.1%, n = 3) were ineffective on hCG-stimulated progesterone production. Cellular morphology and a cell viability assay (trypan-blue exclusion assay) revealed that 100 µM Ni2+ had no detectable toxic effect on GCs.



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  		FIG. 4. Summary of the effects of Ca2+ channel blockers on basal and hCG-stimulated progesterone production determined after 24 h (n = 3–6). Note that only the coadministration of 100 µM Ni2+ attenuated significantly hCG-stimulated (10 IU/ml) progesterone production but did not alter basal production. Data are means ± SEM. Changes were considered significant if P < 0.05 (ANOVA, Newman-Keuls test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
VGCCs are effective modulators of intracellular Ca2+ levels. Ten isotypes of {alpha}1-subunits of VGCCs have been cloned to date and were shown to display distinct kinetical and pharmacological properties (11, 12, 15). The present study provides the first description of functional VGCCs in human luteinized GCs. The detailed electrophysiological characterization of these currents (ICa) as well as RT-PCR and immunohistochemical analysis revealed that two types (T- and L- type) of VGCCs are present in human GCs and that the pore forming {alpha}1-subunits of the underlying channels are Cav3.2 (also called {alpha}1H) and Cav1.2 (also called {alpha}1C).

In our present investigation, we show that 29% of human luteinized GCs cultured in the absence of hCG display ICa, as assessed by patch-clamp experiments. To identify the recorded currents, we first performed a detailed electrophysiological and pharmacological characterization. Results obtained revealed two types of Ca2+ currents, a predominant current in 25% and another type of Ca2+ current in 4% of cells examined.

In case of the predominant type, the recorded Ca2+ currents had a low activation threshold, activated and inactivated rapidly, producing a criss-crossing pattern, and showed a high sensitivity to low concentration (100 µM) of Ni2+. These findings suggested the presence of ICa.T (15). However, under certain recording conditions, R-type Ca2+ currents may be mistaken for T-type currents because R-type currents can activate at more hyperpolarized threshold potentials than other HVA Ca2+ currents and inactivate rapidly during strong depolarization (34). Therefore, we used two major criteria for the identification of the predominant ICa. First, because both (T- and R-type) currents are DHP insensitive and highly sensitive to Ni2+ blockage, we applied SNX-482 (35), which blocks some R-type and also P/Q currents (37). However, SNX-482 had no effect on the recorded current. Second, we measured deactivation kinetics to discriminate ICa.T from R-type Ca2+ current traces (and, of course, from other HVA currents). Deactivation kinetics of ICa is an important criterion for the identification of low-voltage-activated T-type currents because T-type currents deactivate 10 times slower (with time constants of a few milliseconds) than HVA Ca2+ currents (21, 23, 34, 38). The major current recorded in GCs deactivated slowly and in a strong voltage-dependent manner. Collectively, these data indicated unambiguously that the major ICa recorded in human GCs is a ICa.T. Our results are consistent with previous results obtained from pig GCs (8, 9) and avian GCs (10), in which the presence of a T-type current was demonstrated.

Expression studies demonstrated that although the three isotypes (Cav3.1, Cav3.2, Cav3.3) of ICa.T have very similar threshold potentials and voltage dependencies of activation, differences in several electrophysiological characteristics allow their discrimination. Because the recorded ICa.T activated and inactivated rapidly, we excluded the contribution of Cav3.3 currents, which exhibit the slowest activation and inactivation and the most rapid deactivation kinetics of the T-type channel (Cav3) family (39). Although Cav3.1 and Cav3.2 currents exhibit very similar deactivation, activation and inactivation kinetics, the recovery from short-term inactivation of Cav3.2 currents, is 3-fold slower than for Cav3.1 currents (38). The ICa.T of human GCs recovered slowly from its short-time inactivation, which is consistent with the characteristics of Cav3.2 currents. Moreover, the observation that the recorded current was almost completely inhibited by 100 µM Ni2+ was in favor of the presence of Cav3.2 rather than Cav3.1 currents (40). Thus, the Ni2+-sensitive ICa.T of GCs displayed properties well comparable with ICa.T described in adrenal, kidney, liver, heart, and sensory ganglia, in which also mRNA for Cav3.2 was detected (15, 16, 17, 24, 41).

Previous studies revealed that Na+ and Ca2+ channels can be activated during small depolarizations and do not completely inactivate, producing a so-called window current (42, 43). The window current, which is determined by the overlap of activation and steady-state inactivation curves, is a further characteristic of ICa.T. This has important consequences considering [Ca2+]i, because small local Ca2+ influxes are possible over a wide range of physiologically relevant membrane potentials. According to our experiments, the calculated window current was maximal at a potential of –42 mV, which is close to the resting membrane potential of cultured human GCs (32).

Additionally, we observed a Ni2+-insensitive Ca2+ current in the minority (4%) of cells examined. This current was high voltage activated because it could be elicited when the holding potential is –40 mV. BayK 8644 (L-type channel activator) increased and nifedipine (L-type channel blocker) inhibited this Ni2+-insensitive Ca2+ current, proving clearly the presence of L-type Ca2+ channels in GCs (11, 12). Methodological limitations may have, however, affected our measurements. Thus, in some cases the current amplitude was very small (–15 to –20 pA) and currents could be measured only after BayK 8644 application. Therefore, it is possible that the percentage of cells exhibiting L-type currents was underestimated. Another possible limitation in our patch-clamp experiments could be related to the fact that two isoforms of ICa.L (Cav1.2 and Cav1.3 subunit-mediated currents) have similar electrophysiological properties when high concentrations of Ba2+ are used in the extracellular solution (44).

Besides identification of electrophysiological properties of recorded ICa, we examined gene expression of pore-forming subunits, which may give rise to slowly deactivating ICa.T and DHP-sensitive ICa.L. We detected mRNA for the Cav3.2 and the Cav1.2 subunit in cultured human luteinized GCs and, importantly, for Cav3.2 in human CL, supporting the identity and presence of Ca2+ channels in vitro and in vivo. The Cav1.2 mRNA could not be detected in fixed, paraffin-embedded human CL, which was likely due to mRNA degradation. However, immunohistochemical detection of Cav1.2 protein of L-type Ca2+ channels in human CL gave proof of its presence in vivo. Interestingly, we found that only a portion of large luteal cells of the active CL are immunopositive. This result is consistent with our patch-clamp experiments, which showed that after hCG treatment 23% of cells had ICa.L alone or in combination with ICa.T. Because antibodies recognizing Cav3.2 were unfortunately not available to us, we could not examine the localization of this type of Ca2+ channels in the CL, but RT-PCR studies indicated its presence in the human CL. Moreover, the absence of another T-type isoform, Cav3.1, in GCs was indicated by RT-PCR. Additional RT-PCR and immunohistochemical studies failed to detect another L-type isoform, which could not be excluded otherwise. Thus, we did not detect mRNA for Cav1.3 (endocrine L-type Ca2+ channel), and immunostaining on the CL using a commercial Cav1.3 antibody could not be blocked by preadsorption with a specific immunogenic peptide. Therefore, we concluded that from the L-type channel family, only the Cav1.2 isoform is present in human GCs in vivo and in vitro.

Based on our patch-clamp experiments, we found that GCs are heterogeneous with respect to the nature of ICa. Thus, cells posses ICa.T and/or ICa.L with the dominance of the T type. Only in a few cases, an L-type current was recorded alone. There is evidence that the expression of voltage-activated Ca2+ channels undergoes dynamic changes during cell proliferation and differentiation (22, 23, 24). Because human GCs in culture are dynamic cells, which also proliferate (45) and differentiate, it is possible that the expression of isoforms of Ca2+ channels is confined to certain phases of the cell cycle and/or differential stages. Our study, in which GCs were treated with hCG, further suggested this possibility and indicated regulation of Ca2+ channel expression.

Normally GCs are exposed to LH or hCG in vivo, and therefore we mimicked this physiological circumstance by treating cells with hCG. Importantly we found that hCG stimulation increased the proportion of cells displaying ICa from 29 up to 63%, and cells showing both ICa.T and ICa.L from 2 to 17%. Treatment with hCG also significantly increased the T type but not the L-type current density. These effects of hCG could be due to an increased transcription of Ca2+ channel genes or changed expression of channel proteins, thereby controlling trafficking or turnover of channel proteins.

The electrophysiological characterization of ICa and the molecular identification of the underlying channels in human GCs described here are prerequisites for the elucidation of Ca2+ current-dependent cellular processes. Cultured human luteinized GCs correspond to the large luteal cells of the CL derived from the granulosa cells of the preovulatory follicle (1). These cells produce large amounts of progesterone and express receptors for different locally produced substances (e.g. muscarinic acetylcholine receptors), which can modulate [Ca2+]i (3, 29). Kunz et al. (33) showed that activation of muscarinic receptors resulted in activation of a K+ channel, presumably via increasing [Ca2+]i. Previous studies indicated that this K+ and a Na+ channel of human luteinized GCs participate in the process of progesterone production, the main steroid of GCs (32, 33). Although currently details are unknown, it is possible that Ca2+-activated large conductance potassium channel and Ca2+ channels might cooperate in the control of [Ca2+]i (46) and possibly steroid production. In rat GCs, the Ca2+ channel blockers verapamil (L-type channel blocker) and La3+ (T-type channel blocker) inhibited GnRH- and FSH-stimulated progesterone synthesis, suggesting that L- and T-type Ca2+ channels are functionally coupled to steroid production, at least in this species (47, 48). In human GCs uptake of extracellular Ca2+ was reported to potentiate long-term hCG-stimulated progesterone synthesis (49), but the nature of the underlying channels has until now remained unknown.

In our present study, we found that hCG-stimulated progesterone synthesis and activity of T-type (Cav3.2) Ca2+ channels, but not of L-type (Cav1.2) channels, are linked. The hormone hCG, which exerts its effect via G protein-coupled LH receptors, increased both progesterone production and T-type current density after long-term stimulation. An effective T-type channel blocker (Ni2+) decreased the hCG-stimulated progesterone synthesis, which was not due to a general toxic effect of Ni2+. Because there is a possibility that Ni2+ might also block Na+-dependent Ca2+ uptake by Na+-Ca2+ exchanger into cells (50), the presence of which has not been examined in GCs up to date, we applied flunarizine, another T-type channel blocker (51). Results obtained indicated reduction of stimulated progesterone production as well. Our results in human GCs are therefore in agreement with other studies in human, bovine, and rat adrenocortical cells, which report that T-type Ca2+ channel blockage diminished steroid production (25, 26, 27).

How Ca2+ influx via T-type channels in human GCs is linked to steroidogenesis is currently unknown but may be related to regulation of gene transcription. A recent study provided evidence that extracellular Ca2+ could potentiate FSH-stimulated transcription of a steroidogenic enzyme (P450 side-chain cleavage) gene in porcine GCs (52). Similarly, numerous studies indicated that factors regulating gene expression work, at least in part, through modulation of Ca2+ influx (53). However, the majority of reports implicated L-type Ca2+ channels in modulation of [Ca2+]i, which in case of human luteinized GCs represents the minority of detected functional Ca2+ channels.

In summary, in the present study, we provide molecular and functional evidence for the presence of two voltage-activated Ca2+ channels (T-type Cav3.2 and L-type Cav1.2) in human luteinized GCs. We show that hCG stimulation markedly increased both the percentage of cultured GCs displaying Ca2+ currents and the current density of the dominant T-type Ca2+ currents. Taken together, our results support a role for hCG in the regulation of Ca2+ channel expression in human GCs and a role of the Cav3.2 channel in steroid production in human luteinized GCs.


   Acknowledgments
 
We gratefully acknowledge technical help by R. Rämsch, M. Rauchfuß, A. Mauermayer, and G. Prechtner. We also appreciate the supply of human GCs provided by Drs. F. D. Berg and U. Berg (Fertility Center Munich, Munich, Germany) and human ovaries by Dr. C. Heiss (Klinik am Eichert, Göppingen, Germany).


   Footnotes
 
This work was supported by Deutsche Forschungsgemeinschaft (Graduiertenkolleg 333, Biology of Human Diseases).

Abbreviations: [Ca2+]i, Intracellular free Ca2+ concentration; CL, corpus luteum; DHP, dihydropyridine; DMSO, dimethylsulfoxide; GC, granulosa cell; hCG, human chorionic gonadotropin; HVA, high voltage activated; I2, peak current during the first pulse; I1, peak current during the second pulse; ICa, voltage-activated Ca2+ current; ICa.L, L-type Ca2+ current; ICa.T, T-type Ca2+ current; k, slope factor; {Delta}t, interpulse duration; {tau}deact., deactivation time constant; V50, midpoint of activation; VGCC, voltage-gated Ca2+ channel.

Received December 29, 2003.

Accepted May 20, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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