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Biology of Reproduction 67, 88-98 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Potassium Channel Antagonists Influence Porcine Granulosa Cell Proliferation, Differentiation, and Apoptosis1

Mohan Manikkama, Yan Lia, Brianna M. Mitchella, Diane E. Masonb, and Lisa C. Freeman2,,a

a Departments of Anatomy and Physiology and b Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506-5802


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This investigation determined the effects of K+ channel antagonists on proliferation, differentiation, and apoptosis of porcine granulosa cells. The drugs screened for functional effects included the class III antiarrhythmic agents MK-499 and clofilium, the chromanol IKs antagonist 293B, the benzodiazepine IKs antagonists L-735,821 and L-768,673, and the peptidyl toxins charybdotoxin (CTX) and margatoxin (MTX). Granulosa cell proliferation and differentiation were assessed by serial measurements of cell number and progesterone accumulation in the culture media, respectively. Granulosa cell apoptosis was evaluated using flow cytometry. Additional information about drug effects was obtained by immunoblotting to detect expression of proliferating cell nuclear antigen, p27kip1 and the caspase-3 substrate poly(ADP-ribose) polymerase. The ERG channel antagonist MK-499 had no functional effects on cultured granulosa cells. However, the broad spectrum K+ channel antagonist clofilium decreased, in a concentration-dependent fashion, the number of viable granulosa cells cultured, and these effects were associated with induction of apoptosis. All three IKs antagonists (293B, L-735,821, and L-768,673) increased basal, but not FSH-enhanced progesterone accumulation on Day 1 after treatment without affecting the number of viable cells in culture, an effect that was blocked by pimozide. In contrast, CTX and MTX increased the number of viable cells in FSH-stimulated cultures on Day 3 after treatment without affecting progesterone output per cell. These data demonstrate that selective antagonism of granulosa cell K+ channels with distinct molecular correlates, electrophysiological properties, and expression patterns can influence differential granulosa cell proliferation, steroidogenic capability, and apoptosis. Thus, K+ channels may represent pharmacological targets for affecting Granulosa cell function and oocyte maturation, in vivo or in vitro.

follicle-stimulating hormone, granulosa cells, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ovarian follicular development reflects coordination of several processes known to be under endocrine and paracrine regulation. During the follicular phase of the estrous cycle, a finite number of preovulatory follicles are selected from a larger pool of antral follicles, the majority of which undergo atresia [1]. Granulosa cells surround the oocyte within the ovarian follicle and play an essential role in follicular development, ovulation, fertilization, and implantation [2]. During folliculogenesis, granulosa cells undergo a series of mitotic divisions (proliferation), then acquire gonadotropin receptors and enhanced steroidogenic activity (differentiation). Granulosa cells of ovulatory follicles contribute ultimately to formation of the corpus luteum, whereas granulosa cells of nonovulatory follicles undergo apoptosis. Factors that stimulate proliferation and differentiation of granulosa cells also are believed to suppress granulosa apoptosis [1]. Apoptosis underlies the naturally occurring process of follicular atresia, and represents the molecular mechanism by which a majority of ovarian follicles are eliminated from the ovary [1]. Atresia occurs not only in antral follicles, but at all stages of follicular development [1, 3]. In all species, the most common fate for granulosa cells is apoptosis.

Modulation of granulosa cell electrical activity may provide one means to regulate cell function [4–8]. Voltage-gated potassium (Kv) currents with distinct properties have been described in granulosa cells, and have been shown to regulate resting membrane potential [4–9]. Granulosa cell depolarization is an essential feature of follicular maturation. It has been hypothesized that electrical coupling between granulosa cells and oocytes may represent an important signaling pathway [5]. However, to date, no direct link between voltage-gated K+ channels and granulosa cell function has been demonstrated.

Changes in the activity and expression of Kv channels can influence proliferation [10–12], differentiation [13, 14], and apoptosis [15] of nonnerve, nonmuscle cells. K+ channel antagonists have been shown to suppress either cell proliferation [10–12, 16, 17] or differentiation [13], and to induce [18, 19] as well as inhibit [15, 20] apoptosis. The effects of K+ channel antagonists have not been examined previously in a model of ovarian granulosa cells development.

Porcine granulosa cells in primary culture are used commonly to study granulosa cell function, and their proliferation, differentiation, and apoptosis have been well characterized under a variety of culture conditions [21–25]. Porcine granulosa cell monolayers retain hormonal responsiveness and physiological functions resembling the in vivo condition. For example, these cells demonstrate gonadotropin sensitivity and steroidogenic capacity in vitro, in both serum-supplemented and defined serum-free media [21, 22]. During the course of 24–48 h in monolayer culture, porcine granulosa cells "luteinize" spontaneously, becoming primarily progesterone-secreting cells [25]. FSH treatment of cultured porcine granulosa cells augments progesterone secretion, and inhibits apoptosis [1, 21–25].

Our laboratory has identified previously two kinetically distinct delayed rectifier currents in granulosa cells: a slow current (IKs) associated with channels formed by the combination of KCNQ1 (KvLQT1) with KCNE1 (minK) channel proteins, and an ultrarapid current (IKur) associated with channels formed by coassembly of Kv1{alpha} and Kvß channel subunits [9]. However, the reasons for and consequences of the diversity of K+ channels in granulosa cells remain unknown. Our goal was to determine the effects of K+ channel antagonists on proliferation, differentiation, and apoptosis of porcine granulosa cells in short-term primary culture. To this end, we examined the functional effects of a variety of K+ channel antagonists with well-described effects on granulosa cell K+ currents [9]: MK-499, an inhibitor of neither IKs nor IKur; clofilium, a blocker of both IKs and IKur; 293B, L-735,821, and L-768,673, selective inhibitors of IKs; and charybdotoxin (CTX) and margatoxin (MTX), inhibitors of IKur components.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

Cell culture media, supplements, PBS (10x) and sera were obtained from Life Technologies (Gaithersburg, MD) unless stated otherwise. Chemicals were obtained from Sigma Chemical Company (St. Louis, MO) unless stated otherwise. Regular pork insulin was obtained from Eli Lilly (Indianapolis, IN). MTX and CTX were obtained from Alomone Laboratories (Jerusalem, Israel). BAPTA-AM and DiBAC4(3) were obtained from Molecular Probes, Inc. (Eugene, OR). Primary antibody to proliferating cell nuclear antigen (PCNA; Ab1-PC10) was obtained from Oncogene Science (Cambridge, MA). Antibodies to p27kip1 (C19) and poly(ADP-ribose) polymerase (PARP; F-2) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-{alpha}-tubulin (T9026) was purchased from Sigma. Nitrocellulose membranes (Hybond ECL), secondary antibodies, enhanced chemiluminescence (ECL) reagent, and film (Hyperfilm ECL) were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Porcine FSH was a gift from the National Hormone and Pituitary Program. Compound 293B was a gift from Aventis (Frankfurt, Germany). Compounds MK-499, L-735,821, and L-768,673 were obtained from Merck Research Laboratories (Westpoint, PA).

Granulosa Cell Isolation and Culture

Sow ovaries were collected at a local slaughterhouse, and granulosa cells were isolated using techniques described previously in detail [9, 21, 22]. Briefly, small (1–3 mm diameter) to medium (4–6 mm diameter) follicles were aspirated by hand using a 19-gauge needle attached to a 10-cc syringe. Granulosa cells were separated from follicular fluid by centrifugation at 500 x g for 5 min. Cells were washed twice with a 1:1 mixture of Ham F10 nutrient medium and Dulbecco modified Eagle medium (DMEM) containing Hepes (25 mM), penicillin (50 U/ml), and streptomycin (50 µg/ml).

Culture conditions for granulosa cells were similar to those described previously by Barano and Hammond [21]. The basic culture medium consisted of DMEM:F10 (1:1) supplemented with fetal bovine serum (FBS, 10%), penicillin (50 U/ml), streptomycin (50 µg/ml), gentamicin (57 ng/ml), and amphotericin (2.5 µg/ml). The defined serum-free medium consisted of DMEM:F10 (1:1) containing insulin (300 mU/ml), hydrocortisone (40 ng/ml), transferrin (5 µg/ml), BSA (4 mg/ml) with gentamicin (57 µg/ml), penicillin (50 U/ml), streptomycin (50 µg/ml), and amphotericin (2.5 µg/ml). This defined serum-free medium significantly increases the magnitude and duration of basal and FSH-stimulated progesterone output by granulosa cells; moreover, this enhanced differentiated response is accompanied by only a moderate decrease in cell proliferation [21].

Freshly isolated granulosa cells were plated in the basic serum-supplemented culture media on collagen-coated 24-well (500 µl/well) or 6-well (2 ml/well) culture dishes, and incubated at 37°C in a humidified atmosphere of 5% CO2 and air. In every experiment, cells were plated at equal density for each treatment group. Plating density between experiments varied from 2 x 104 cells/well in 24 multiwells to 1 x 106 cells/well in 6-multiwells. The culture media were changed 12–24 h after plating, to either fresh serum-supplemented media or defined serum-free media that contained vehicle or treatments. Media were collected and replaced at 24-h intervals thereafter.

Effects of Potassium Channel Antagonists on Granulosa Cell Cultures

Drugs were added to the culture media from aqueous stock solutions of FSH (800 µM), clofilium (5 mM), and MK-499 (1 mM); media containing these drugs were prepared weekly. Stock solutions of compounds 293B (100 mM), L-735,821 (1 mM), L-768,673 (1 mM), pimozide (30 mM), and 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) (10 mM) were prepared in DMSO, and media containing these drugs were made up daily. Stock solutions of peptide toxins (1 mM) were prepared in the recommended buffer (0.1% BSA, 100 mM NaCl, 10 mM Tris pH 7.5) and stored at -20°C for less than 3 mo. Toxin-containing media were stored at 4°C and discarded after 1 wk. In one series of experiments in which the effects of clofilium were examined in the presence and absence of extracellular calcium, granulosa cells were incubated for 4–6 h in defined media described previously by Jayes et al. [26]. These solutions contained 127 mM NaCl, 5 mM KCl, 2 mM MgCl, 0.5 mM KH2PO4, 10 mM Hepes, 10 mM glucose, 5 mM NaHCO3, 0.1% BSA, and either 1.8 mM CaCl2 or 0.1 mM EGTA.

Measurement of Membrane Potential

The resting membrane potentials of granulosa cells were measured in the presence and absence of clofilium using whole-cell patch clamp techniques in current clamp mode (I = 0), as described previously in detail [9]. Clofilium-induced changes in membrane potential were also assessed by spectrophotometric determination of bis(1,3-dibarbituric acid)-trimethine oxanol (DiBAC4(3)) fluorescence (excitation, 485 nM; emission, 527 nM; Fluoroskan Ascent FL, LabSystems Inc., Helsinki, Finland). Cells were equilibrated with 2 µM DiBAC4(3) for 30 min at 37°C prior to fluorescence measurement.

Preparation of Granulosa Cell Lysates

Whole cell lysates were made from granulosa cell monolayers by standard techniques using a lysis buffer consisting of PBS with 1% (v/v) Nonidet P40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, and protease inhibitor cocktail (1:500) (P8340; Sigma). Lysis buffer was added to the culture dish after washing three times with cold PBS. The culture dishes were scraped and the lysate was aspirated into a syringe with a 21-gauge needle to shear DNA. The lysates were rocked in the cold for 1 h and centrifuged for 10 min at 10 000 x g. Protein concentrations of granulosa cell lysates were determined by the bicinchoninic acid method (Micro BCA Protein Assay, Pierce, Rockford, IL).

Immunoblotting

For direct comparisons of protein expression between drug-treated and untreated cells, equal amounts of protein (15 or 50 µg/lane, as indicated in figure legends) were loaded in adjacent lanes on a single SDS-PAGE gel, separated by SDS-PAGE under reducing conditions, and transferred to nitrocellulose membranes by the semidry transfer method. The membranes were blocked for 1 h at room temperature with 5% nonfat milk in Tris-buffered saline (TBS; 100 mM Tris, 0.9% NaCl pH 7.5) containing 0.1% Tween 20, then incubated overnight at 4°C with primary antibody diluted in the blocking solution. Primary antibody dilutions were {alpha}-PCNA (1:200), {alpha}-p27kip1 (1:400), {alpha}-PARP (1:100), and {alpha}-tubulin (1:1000). After 3 washes with 0.1% Tween/TBS, membranes were incubated for 1 h at room temperature with the appropriate horseradish peroxidase-conjugated secondary antibody diluted 1:1500 in 0.1% Tween/TBS. After 4 additional washes with 0.1% Tween/TBS, bound primary antibodies were visualized using an ECL detection system (Amersham Pharmacia Biotech), and recorded on radiographic film. Densitometric analysis was performed using Scion Image (Scion Corporation, Frederick, MD). Equal loading was confirmed by either immunoblotting for {alpha}-tubulin (Fig. 7) or Coomassie-blue staining of gel lanes loaded and subjected to SDS-PAGE in a manner identical to those used for immunoblotting (Fig. 2).



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  		FIG. 7. FSH (200 ng/ml) alone or in combination with toxin was added to serum-supplemented (10% FBS) granulosa cell cultures 24 h after plating, and media were replaced at 24-h intervals thereafter. In all panels, asterisks indicate significant difference from FSH on same day after treatment. A) Progesterone accumulation by cultures exposed to FSH in the absence (n = 4) and presence (n = 4) of CTX (10 nM). B) Progesterone accumulation by cultures exposed to FSH in the absence (n = 10) and presence (n = 11) of MTX (50 nM). C) The number of viable cells on Day 3 after treatment in granulosa cell cultures exposed to FSH, alone (n = 16) or in combination with CTX (50 nM, n = 16) or MTX (50 nM, n = 16). D) MTX (50 nM) also enhanced expression of PCNA on Day 3 after treatment. Immunoblots (15 µg protein/lane) show expression of p27kip1, PCNA and {alpha}-tubulin (loading control) in lysates from granulosa cell cultures treated for 72 h with FSH, alone or in combination with MTX. Densitometric analysis of similar data (n = 3) demonstrates an approximately 4-fold increase in expression of PCNA and 3-fold decrease in p27kip1 in cultures treated with FSH and MTX compared to FSH alone



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  		FIG. 2. Top: Immunoblotting (50 µg protein/lane) indicates that 48-h exposure to clofilium (25 µM) is associated with increased cleavage of Poly(ADP-ribose) polymerase (PARP) in serum-supplemented (FBS) and serum-free (SF) primary cultures of porcine GC. Numeric labels (right) indicate Mr x 103 (results are representative of 4 experiments). Bottom: Caspase-3 activity in lysates of granulosa cells cultured for 24 h in basic, serum-supplemented medium in the absence (BM) and presence (CLO) of clofilium (25 µM). Positive controls shown are purified caspase-3 (CPP32) and lysate from Jurkat cells treated for 6–7 h with camptothecin (Jurkat Induced). Negative controls are CPP32 plus caspase inhibitor I (CPP32 + Inhibitor) and untreated Jurkat lysate (Jurkat Not Induced). Results are representative of two independent experiments

Steroid Hormone Assay

Aliquots of culture media were stored at -20°C for up to 60 days before the progesterone assay was performed. Progesterone concentrations were determined by a solid-phase radioimmunoassay technique (CAC Progesterone; Diagnostic Products Corp., Los Angeles, CA) validated for measuring progesterone in the culture media. The sensitivity of the radioimmunoassay was approximately 0.3 ng/ml. The within and between assay coefficients of variation for the progesterone assay were 4.3% and 11.7%, respectively. Estradiol-17ß was measured using commercially available reagents (TKE2; Diagnostic Products). This assay had been validated previously for porcine serum [27], and was further modified and validated for use with culture media containing FBS. In brief, estradiol-17ß could be quantitatively recovered when added to culture media, and increasing dilutions of media spiked with estradiol-17ß produced similar concentrations when evaluated in the assay. The sensitivity was 4.9 pg/ml. The intraassay and interassay coefficients of variation for estradiol assay were 4.8% and 8.3%, respectively.

Determination of Cell Number

At various time points, the number of viable granulosa cells in the monolayer cultures was determined by harvesting the attached cells with a trypsin-containing solution of 8 mg/ml NaCL, 0.4 mg/ml KCl, 1 mg/ml dextrose, 0.6 mg/ml NaHCO3, 0.2 mg/ml Na-EDTA, and 0.5 mg/ml trypsin, then directly counting the number of viable cells using trypan blue-exclusion and hemacytometry.

Caspase-3 Assay

Caspase-3 activity was assayed colorimetrically using Ac(N-acetyl)-(Asp-Glu-Val-Asp)-p-nitroaniline (Ac-DEVD-pNA; Calbiochem, San Diego, CA) as a substrate. Granulosa cells cultured for 24 h in serum-supplemented media in the presence and absence of 50 µM clofilium were harvested and counted as described above, then lysed in a buffer containing 50 mM Hepes pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM dithiothreitol (DTT), and 0.1 mM EDTA (4 x 104 cells/µl). Cytosolic extracts were flash-frozen and stored at -70°C. Control and clofilium-treated extracts from five independent experiments (1.5 x 106 cells/treatment group) were pooled for assay. The assay buffer contained 50 mM Hepes pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 0.1 mM EDTA, and 10% glycerol (DTT was added immediately before use). The assay volume was 200 µl. Positive controls included purified recombinant caspase-3 (BD PharMingen, San Diego, CA) and caspase-induced lysate (GenoTechnology, Inc., St. Louis, MO). Caspase uninduced lysate (GenoTechnology) and capase-3 plus inhibitor served as negative controls. Assay blanks included assay buffer, assay buffer plus substrate, and assay buffer plus inhibitor. Optical density at 405 nM was measured in a microtiter plate reader after 60 min of incubation at 37°C (Multiskan Ascent, Labsystems, Inc.).

Flow Cytometry

Flow cytometry was used to assess cell death characteristics (cell shrinkage and phosphatidylserine exposure) in clofilium-treated granulosa cells and concurrent control cultures. Apoptotic cells exhibit a decrease in forward scatter that corresponds to a decrease in cell size [28]. On this basis, changes in the light-scattering properties of granulosa cells were used to infer cell shrinkage. Briefly, 10 000 untreated granulosa cells were excited with a 488 nM argon laser to generate a forward scatter vs. side scatter dot plot and a forward scatter histogram. A gate based on the properties of the control cells was set to separate the normal and shrunken (apoptotic) cells, and remained constant throughout the analysis (Figs. 3 and 5).



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  		FIG. 3. Flow cytometric determination of the light scattering properties of granulosa cells cultured for 18 h in basic media (DMEM:F10, 10% FBS), then an additional 48 h in either the presence or absence of clofilium (CLO, 50 µM). Forward-scatter and side-scatter characteristics are depicted in dot plots (left panels). Numbers on the forward-scatter histograms (right panels) indicate the percentage of cells that are not shrunken (not apoptotic)



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  		FIG. 5. Fourteen hours after plating, basic media in granulosa cell cultures were replaced with Hepes-buffered incubation media containing either 1.8 mM [Ca2+]o (+[Ca2+]o) or 100 µM EGTA (-[Ca2+]o), with (+CLO) or without (-CLO) 50 µM clofilium. Flow cytometry was performed 4 h later. Numbers on the forward-scatter histograms indicate the percentage of cells under each experimental condition that are not shrunken (not apoptotic)

Exposed phosphatidylserine in the plasma membrane, another indicator of apoptosis, was also measured. Granulosa cells were double-stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide for flow cytometry (ApoAlert; Annexin V, protocol PT3050-1, Clontech Laboratories, Palo Alto, CA). Adherent granulosa cells were washed with PBS and then lifted using the tryspin-containing solution described previously, washed once with serum-containing media, and once with the 1x Annexin binding buffer supplied by the manufacturer, before resuspension in the binding buffer. FITC-Annexin V (0.5 µg/ml) and propidium iodide (1.2 µg/ml) were added, and the mixture was incubated in the dark at room temperature for 10 min. Flow cytometry was performed using a Becton-Dickinson FACScan and the CellQuest software (Becton-Dickinson, Mount View, CA). Cells that were negative for Annexin V and propidium iodide (PI-/V-) were assessed as viable, whereas those that were positive for Annexin V-FITC (PI-/V+) were assessed as apoptotic. Double-stained cells (Annexin V and propidium iodide, PI+/V+) were considered dead (late apoptotic/necrotic). In these experiments at least 10 000 cells were counted and no gate was employed.

Statistical Analysis

Data are expressed as mean ± SEM. Significant differences between treatment groups were identified by ANOVA using appropriate general linear models. Multiple comparisons were made using the least significant difference procedure (Statistix, Analytical Software, Tallahassee, FL). Differences were considered to be significant when P <= 0.05. The numbers of replicates per treatment group and independent experiments associated with specific studies are provided in the figures or accompanying legends.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MK-499

The methanesulfonanilide class III antiarrhythmic agent MK-499 (5 µM), at a concentration known to block ether-a-go-go-related gene (ERG) (KCNH) channels selectively [29], had no effect on either progesterone accumulation or the number of viable granulosa cells in primary culture on any day after treatment, in the absence or presence of FSH (n = 8, data not shown). For example, on Day 2 after treatment, media progesterone concentrations (expressed as % Day 0) were 56% ± 2%, 51% ± 5%, 139% ± 17%, and 140% ± 16% for controls, MK-499-treated, FSH-treated, and FSH+MK-499-treated granulosa cells cultures, respectively. These results are consistent with our previous finding that porcine granulosa cells do not express ERG channel proteins nor do they exhibit rapid delayed rectifier currents similar to cardiac IKr [9].

Clofilium

Clofilium (25–100 µM) consistently decreased the number of viable granulosa cells in monolayer culture. The concentration-dependent deleterious effect of 48-h exposure to clofilium is shown in Figure 1A for both serum-supplemented and serum-free cultures. In fact, less than 48 h of exposure to clofilium was sufficient to decrease the number of viable granulosa cells. For example, in one experiment after 24 h of exposure to 25 µM clofilium, the number of viable cells (n = 3, wells/treatment) were 1.1 ± 0.8 x 106 vs. 5.2 ± 0.4 x 105 for cultures maintained in the serum-supplemented media without and with drug, and 1.1 ± 0.1 x 106 vs. 6.0 ± 0.4 x 105 for cultures maintained in the defined serum-free media without and with drug, respectively. Figure 1B illustrates the effect of 14 h of exposure to 25 µM clofilium.



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  		FIG. 1. A) The number of viable granulosa cells in serum-supplemented media (FBS, n = 5) and defined serum-free media (SF, n = 4) on Day 2 after treatment with clofilium (Clo) in the presence or absence of FSH (200 ng/ml). Clofilium (0, 10, 25, or 50 µM, as indicated) was added to cultures after 24 h; media were replaced at 24-h intervals thereafter. Asterisks indicate significant difference from controls or FSH on the same day after treatment. B) Granulosa cell monolayers maintained in serum-supplemented media, in the absence (control) and presence of 25 µM clofilium (14 h after treatment)

To gain insight into the mechanism by which clofilium accelerates granulosa cell apoptosis, we first examined the expression the caspase-3 substrate PARP. Activation of caspase-3 has been associated not only with follicular atresia and apoptosis of human luteinized granulosa cells [30–32], but also with clofilium-induced apoptosis of HL-60 cells [18, 31]. Caspase inhibitors have also been shown to decrease apoptosis in cultured porcine granulosa cells [24]. However, recent data suggest that clofilium inhibition of K+ efflux via voltage-gated channels might inhibit rather than activate caspase-3-like proteases [20, 33]. The immunoblot in Figure 2 suggests strongly that clofilium treatment of granulosa cells is associated with activation of caspase-3. The 117-kDa native form of PARP is present in control granulosa cell cultures but undetectable in cultures exposed to 25 µM clofilium for 48 h, which is consistent with a higher level of apoptosis in the latter. An 87-kDa band consistent with cleaved PARP is present in both the control and clofilium-treated cultures. Its expression in control cultures is consistent with previous descriptions of spontaneous apoptosis in cultured granulosa cells [23, 24]. To confirm that clofilium enhances caspase-3 activation in primary granulosa cell cultures, enzyme activity was evaluated in granulosa cell lysates using an assay based on cleavage of the chromophore p-(nitroaniline) from a synthetic substrate based on the PARP cleavage site (Fig. 2, bottom). Untreated granulosa cell lysates showed caspase-3 activity above background, which is consistent with the expected baseline of apoptotic cells. Clofilium treatment was associated with enhanced caspase-3 activity.

Flow cytometry allows multiple cell death characteristics to be analyzed at the single-cell level in different cell populations, and we have used this technique to assess clofilium-induced cell shrinkage and phosphatidyl serine exposure. Apoptotic cells have been demonstrated to exhibit a decrease in forward scatter (reviewed by Gomez-Angelats et al. [28]), and this phenomenon was evident in clofilium-treated granulosa cells. In Figure 3, forward scatter was decreased in clofilium-treated cells (bottom panels) compared with controls (top panels), which is consistent with cell shrinkage and acceleration of apoptosis. Similarly, the percentage of shrunken granulosa cells exhibiting decreased forward scatter was increased from 11% ± 3% to 37% ± 4%, when serum-supplemented granulosa cells were exposed to 25 µM clofilium for 18–24 h (n = 4 trials). Comparable results were obtained for granulosa cells cultured in the serum-free defined media in which the shrunken population averaged 5% ± 1% and 16% ± 3% respectively, in the absence and presence and of clofilium.

Flow cytometric analysis of cells double-stained with FITC-Annexin V and propidium iodide was used to determine the relative percentages of viable, early apoptotic (live), and dead (late-stage apoptotic/necrotic) granulosa cells in untreated and clofilium-treated populations. Figure 4 shows an exemplary experiment (n = 3) in which 50 µM clofilium significantly decreased the number of viable cells and increased the percentage of apoptotic cells. In 4 additional experiments in which serum-supplemented granulosa cell monolayers were exposed to 25 µM clofilium for 18–24 h, the percentages of viable granulosa cells (PI-V-, lower left quadrant) were 74% ± 8% and 44% ± 13% for controls and clofilium-treated cultures. The percentages of apoptotic cells (PI-V+; Fig. 4, lower right quadrant) were 15% ± 5% for controls, and 30% ± 10% for clofilium treatment, whereas the percentages of dead cells (PI+V+; Fig. 4, upper right quadrant) were 9% ± 4% for controls, and 21% ± 8% for clofilium treatment. Thus, clofilium significantly accelerated apoptosis of porcine granulosa cells in serum-supplemented monolayer cultures. Comparable results were obtained when granulosa cells in serum-free defined medium were exposed to 25 µM clofilium for 12–18 h (n = 2 trials); the percentages of viable granulosa cells were 84% ± 2% and 73% ± 1% in control and clofilium-treated cultures, respectively.



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  		FIG. 4. The fraction of apoptotic granulosa cells with exposed phosphatidylserine on the outer plasma membrane determined by staining cells with FITC-Annexin V (V, 0.5 µg/ml) and propidium iodide (PI, 1.2 µg/ml). Viable cells, negative for PI uptake and FITC-Annexin V binding (PI-/ V-), are represented in the left lower quadrants. Early stage (live) apoptotic cells (PI-/ V+) are in the right lower quadrants. Dead (late-stage apoptotic and necrotic) cells (PI+/ V+) are displayed in the upper right quadrants. Numbers indicate the percentage of cells in the left or right lower quadrants. (Porcine granulosa cells plated as described in Figure 3)

Suppression of proliferation and induction of apoptosis by K+ channel antagonists in a variety of cell types has been linked to cell depolarization and calcium influx [19]. To determine whether these mechanisms could be involved in clofilium-induced granulosa cell apoptosis, we first examined the effect of clofilium on membrane potential using whole-cell patch clamp techniques to measure the resting membrane potentials of individual granulosa cells. Clofilium (25 µM) significantly decreased granulosa cell resting membrane potential by causing a +22 ± 0.5 mV depolarization (n = 3); similar to clofilium antagonism of K+ channels [9, 34], the drug's effect on resting membrane potential was irreversible. Fluorometric assessment of clofilium-induced depolarization was consistent with the electrophysiological data. Acute exposure to clofilium (50 µM) was associated with a 30% ± 5% increase in DiBAC4(3) fluorescence (n = 12), whereas 6 h of exposure to clofilium (50 µM) was associated with a 29% ± 3% increase in DiBAC4(3) fluorescence (n = 12). This voltage-sensitive dye has been shown previously to exhibit a fluorescence response of about 1% per millivolt [35].

To determine whether influx of extracellular calcium through T-type calcium channels mediates clofilium-induced apoptosis, we examined the effects of clofilium in the presence and absence of the T-channel antagonist, pimozide. In the absence of FSH, pimozide had no effect on either progesterone accumulation nor the number of viable granulosa cells in serum-supplemented cultures under our experimental conditions. Progesterone concentrations for controls and pimozide-treated (µM) granulosa cells were 24.2 ± 3.6 ng/ml and 20.2 ± 2.5 ng/ml at 24 h after treatment (n = 8). The percentage of live (PI-/V-), apoptotic (PI-/V+), and dead (PI+/V+) cells determined by flow cytometry after 24 h of treatment with 3 µM pimozide were 68% live, 28% apoptotic, and 4% dead, compared with 72% live, 24% apoptotic, and 3% dead in the basic serum-supplemented media. Twenty-four-hour exposure to 25 µM clofilium decreased the percentage of live cells to 54% and increased the percentage of apoptotic and dead cells to 39% and 6%, whereas 24 h of treatment with 3 µM pimozide and 25 µM clofilium decreased the number of live cells to 33% and increased the percentage of apoptotic and dead cells to 58% and 8%, respectively. Thus, pimozide did not inhibit but rather enhanced clofilium-induced acceleration of apoptosis. It was unclear whether this effect resulted from inhibition of calcium influx via T channels because a proapoptotic effect of pimozide related to sigma receptor binding has been described [36] and sigma receptors have been identified in ovarian follicles [37, 38].

As an alternative strategy, we examined the effects of clofilium in cells pretreated with the intracellular calcium chelator BAPTA-AM (1 µM) for 4 h before 24 h of exposure to drug [19]. BAPTA-AM alone had no effect on the percentage of live granulosa cells. The percentages of live (PI-/V-) granulosa cells after 14–18 h of culture in serum-supplemented media with and without BAPTA were 79% ± 1% and 83% ± 11%, whereas the percentages of apoptotic (PI-/V+) granulosa cells were 13% ± 6% and 16% ± 6% (n = 3). The percentages of shrunken granulosa cells in the basic media with and without BAPTA-AM were, respectively, 19% ± 10% and 19% ± 11%.

In addition, pretreatment with BAPTA-AM neither attenuated nor enhanced the proapoptotic effect of clofilium (25 µM) in granulosa cells cultured in serum-supplemented media. The average percentages of live (PI-/V-) granulosa cells 14–18 h after treatment in serum-supplemented cultures exposed to clofilium in the absence and presence of BAPTA-AM (n = 4) were 63% ± 3% and 61% ± 4%, compared with 78% ± 3% for granulosa cells not exposed to clofilium. The average percentages of apoptotic (PI-/V+) granulosa cells 14–18 h after treatment in serum-supplemented cultures exposed to clofilium in the absence and presence of BAPTA-AM were, respectively, 49% ± 12% and 45% ± 9%, compared with 19% ± 6% for granulosa cells that were not exposed to clofilium. Similar results were obtained in serum-supplemented media using 1 h instead of 4 h of pretreatment with BAPTA-AM (n = 2); thus, the lack of a protective effect of BAPTA could not be attributed to compartmentalization associated with the 4-h loading time.

To control for the possibility that FBS in the basic culture media interfered with BAPTA-AM loading of granulosa cells by causing inappropriate extracellular hydrolysis of the BAPTA-AM, these experiments were repeated using granulosa cells cultured in the serum-free defined media; the results obtained were comparable to those from serum-supplemented cultures. The average percentages of shrunken granulosa cells cultured in serum-free media for 14 h (n = 2) in the absence or the presence of BAPTA-AM, clofilium, or both BAPTA-AM and clofilium were 6% ± 1% (serum-free media), 6% ± 1% (BAPTA-AM), 15% ± 4% (clofilium), and 16% ± 1% (BAPTA-AM + clofilium). The percentages of shrunken granulosa cells in serum-free cultures exposed to the same treatments for 48 h (n = 1) were 48% (serum-free media), 50% (BAPTA-AM), 73% (clofilium), and 83% (BAPTA-AM + clofilium).

Recent work has suggested that BAPTA-AM may have confounding effects in experiments designed to investigate calcium signaling in apoptotic pathways, because of its ability to chelate free calcium in not only the cytosol, but also the endoplasmic reticulum [39]. This possibility was of concern given our inability to demonstrate any effect of BAPTA-AM on clofilium-associated apoptosis in granulosa cells. To clarify the importance of extracellular calcium to the proapoptotic effects of clofilium, we examined the effects of clofilium on granulosa cells in calcium-replete (1.8 mM) and calcium-depleted (100 µM EGTA) incubation media. The solutions used were shown previously to support porcine granulosa cell function in vitro during a 4-h incubation period, without lasting adverse effects on the treated granulosa cells [26]. Results of three representative experiments appear in Figure 5. Incubation of granulosa cells in the calcium-depleted media for 4–6 h was associated with acceleration of apoptosis and a ~15%–20% increase in the percentage of shrunken cells. More importantly, clofilium significantly enhanced apoptosis and increased the percentage of shrunken granulosa cells under the calcium-depleted, as well as the calcium-replete, culture conditions. These data are inconsistent with the hypothesis that the proapoptotic action of clofilium on granulosa cells requires the influx of extracellular calcium.

IKs Antagonists

Three selective antagonists of IKs, 293B, L735,821, and L768,763, increased basal but not FSH-stimulated progesterone accumulation in serum-supplemented granulosa cell cultures on Day 1 after treatment (Fig. 6, A and C). These drugs were without any concomitant effect on cell number. For example, the numbers of viable cells after 18 h of culture for controls (n = 18), 50 nM L735,821 (n = 4), and 50 µM 293B (n = 17) were, respectively 2.9 ± 0.3 cells/well x 105, 2.9 ± 1.0 cells/well x 105, and 2.5 ± 0.1 cells/well x 105. The IKs antagonists also had no effect on accumulation of estradiol-17ß in the cell culture media. Concentrations of estradiol-17ß before treatment and 24 h after treatment were, respectively, 382.5 ± 21.3 pg/ml and 252.8 ± 18 pg/ml for controls (n = 6); 423.3 ± 42.9 pg/ml and 290.8 ± 21.3 pg/ml for L768,763 (n = 9); and 358.2 ± 27.6 pg/ml and 255.4 ± 21.0 pg/ml for 293B.



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  		FIG. 6. A) After 12 h of culture in basic media (DMEM:F10, 10% FBS), granulosa cells received fresh basic media containing vehicle (BM, n = 6), FSH (200 ng/ml, n = 6), L768,763 (50 nM, n = 9), or both (n = 9). Media were then replaced at 24-h intervals. *Significant difference (P <= 0.05) between control and 293B on Day 1 after treatment. B) After 12 h of culture, basic media were replaced with defined serum-free media containing vehicle (SF, n = 7), FSH (200 ng/ml, n = 7), 293B (10 µM, n = 8), or both (n = 8). Media were then replaced at 24-h intervals. C) Two benzodiazepine IKs antagonists, L735,821 (50 nM, n = 5) and L768,763 (50 nM, n = 15), and the chromanol compound 293B (50 µM, n = 6) have comparable stimulatory effects on basal progesterone accumulation by treated granulosa cell cultures on Day 1 after treatment. Asterisks indicate a significant difference from controls. D) The T-type Ca2+ channel antagonist pimozide (3 µM) significantly attenuated the stimulatory effect of 293B (50 µM) on progesterone accumulation (n = 8). Granulosa cells were cultured in basic media for 4 h before the introduction of media containing pimozide, 293B, or both. Samples were collected for progesterone assay 12 h after treatment. *Significant difference from pimozide alone

This stimulatory effect of the IKs antagonists on progesterone accumulation coincides with the limited time period when both IKs and T-type calcium current are expressed by granulosa cells in monolayer culture [7, 9]. To determine whether the stimulatory effect of IKs antagonists on progesterone accumulation was mediated by influx of calcium through T-type channels, we examined the effect of 293B in the presence and absence of the pimozide (3 µM). Pimozide significantly attenuated the stimulatory effect of 293B (50 µM) on progesterone accumulation (Fig. 6D).

It is interesting that the stimulatory effect of IKs antagonists on progesterone accumulation detected in the serum-supplemented granulosa cells cultures on Day 1 after treatment was also absent from serum-free cultures. Lack of 293B effect is shown in Figure 6B. L735,821 and L768,763 also failed to stimulate progesterone accumulation in serum-free cultures (n = 6, data not shown). Why IKs antagonists exert their stimulatory effect on progesterone accumulation in serum-supplemented but not defined serum-free media in unclear. Nonetheless, functional effects of K+ channel antagonists cells have been reported to vary between serum-supplemented and serum-free cultures [12], and serum has been shown to influence K+ channel expression [40].

MTX and CTX

MTX and CTX enhanced FSH-stimulated progesterone accumulation and the number of viable cells in granulosa cell cultures on Day 3 after treatment (Fig. 7). CTX and MTX exhibited these effects when added to the serum-supplemented basic culture media at concentrations of either 10 nM or 50 nM. The increased concentrations of progesterone in the media of toxin-containing cultures could be completely accounted for by the increased number of viable cells; on Day 3 after treatment, progesterone accumulation expressed in terms of picograms per cell did not differ significantly between cultures treated with 200 ng/ml FSH (1.2 ± 0.2 pg/cell, n = 3) and cultures treated with 200 ng/ml FSH plus 50 nM MTX (1.1 ± 0.1 pg/cell, n = 3). The toxin-induced increases in the number of viable cells on Day 3 were associated with increased expression of proliferating cell nuclear antigen (PCNA, Fig. 7D), a sensitive marker of granulosa cell proliferation [41]. Similar results were obtained for CTX (n = 2, data not shown). The MTX-induced increases in the number of viable cells on Day 3 were also associated with decreased expression of the cyclin-dependent kinase inhibitor p27kip1 (Fig. 7D). Expression of p27kip1 was not examined in CTX-treated granulosa cells cultures.

In contrast to their effects on FSH-stimulated granulosa cell cultures, neither CTX nor MTX enhanced progesterone accumulation or cell viability in the absence of FSH. For example, in one experiment (n = 6), progesterone concentrations 3 days after the initiation of MTX treatment were 48.1 ± 9.3 ng/ml and 43.8 ± 11.7 ng/ml, respectively for untreated and MTX-treated granulosa cell cultures, but 589.8 ± 137.9 ng/ml and 1229.2 ± 332.5 ng/ml, respectively, for cultures treated with FSH and FSH + MTX. In the absence of FSH, treatment with either CTX or MTX also failed to increase expression of PCNA (n = 2, data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The presence of overlapping, delayed rectifier K+ currents associated with different molecular correlates is common. In neurons and myocytes, K+ channel diversity and redundancy provide a means to maintain membrane potential in the face of insults, and to fine tune cellular responses to repetitive stimuli of different frequencies and magnitudes. In nonnerve, nonmuscle cells that express delayed rectifier K+ currents, the gating properties of the contributing channels influence not only resting membrane potential, but also the range of membrane potentials over which outward current is available to oppose depolarizing stimuli that may induce growth arrest, differentiation, or apoptosis [15, 17,19, 42]. Expression of a variety of Kv channel subunits may also provide a means for both excitable and nonexcitable cells to respond differentially to second messengers; different Kv channels are subject to different forms of regulation [43].

We report that 3 classes of K+ channel antagonists with distinct pharmacological profiles (Table 1) differentially influence granulosa cell function. Our data demonstrate that K+ channel blockade can influence granulosa cells apoptosis, steroidogenic capability, and proliferation, and that these drug effects may depend on the duration of primary culture and the presence or absence of gonadotropins and serum factors. Expression of K+ channels by granulosa cells changes with time in monolayer culture [7, 9]. It is likely that the qualitatively and temporally distinct drug effects described here reflect the precise complement of voltage-sensitive channels in the plasma membrane of granulosa cells at different times under different culture conditions. In the following paragraphs, we discuss individually the effects of K+ channel antagonists with 3 distinct pharmacological profiles, and the potential mechanisms of action.


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  		TABLE 1. Specificity of K+ channel antagonists that affect granulosa cells

Clofilium

At concentrations capable of blocking granulosa K+ channels by >=80% and decreasing granulosa cells resting membrane potential by +20–30 mV [9], clofilium accelerated granulosa cell apoptosis. Clofilium enhancement of granulosa cell apoptosis was associated with enhanced caspase-3 activity. This profile is consistent with the molecular mechanisms described previously for clofilium-induced granulosa cell apoptosis [18, 24, 30–32, 44]. Clofilium appears to interact with the granulosa cell death pathways involved in the physiological process of follicular development, atresia, and luteal regression.

Clofilium exposure is not universally proapoptotic. Whereas clofilium has been shown to induce apoptosis in HL-60 and Schwann cells, it attenuated apoptosis in cortical neurons [11, 18, 20]. The cellular response to clofilium likely depends on the precise complement of ion channels and pumps expressed in the plasma membrane. For example, membrane depolarization would be expected to have opposite effects on Ca2+ influx and Ca2+-dependent functions in cells that do and do not express voltage-gated calcium channels; in the former, depolarization decreases the driving force for calcium entry (Em–ECa), whereas in the latter, a pathway for Ca2+ entry is enhanced.

Although sustained depolarization has been recognized recently as a general component of the apoptotic process, intracellular calcium has not been shown to act as a mediator of depolarization-induced cell death in all instances [19, 42]. In some cells, K+ efflux through potassium channels is an essential component of depolarization-mediated apoptosis [29, 42]. Thus, the relative balance of K+- and Ca2+-dependent and -independent components of apoptotic machinery can play a role in determining the fate of a particular cell type after clofilium exposure. Granulosa cells have been shown to possess apoptotic pathways that are both dependent on and independent of K+ efflux [33].

Our data suggest that the proapoptotic effects of clofilium on granulosa cells were not mediated by calcium, because neither BAPTA-AM nor removal of extracellular calcium prevented clofilium from accelerating apoptosis. Thus, the drug's enhancement of apoptosis must be mediated by mechanisms that are distinct from those previously described for nonspecific K+ channel antagonists in HepG2 cells [19]. Further investigations are required to establish a causal relationship between clofilium-induced depolarization and granulosa cell apoptosis, and to identify the events that link these processes. Clofilium-induced depolarization of granulosa cell resting potential may directly trigger an opening of the mitochondrial permeability transition pore and subsequent initiation of apoptosis. Alternatively, K+ channel blockade by clofilium may enhance the activity of mammalian proteins that interfere with inhibitor of apoptosis proteins (IAPs); K+ channel block contributes to the proapoptotic effects of Drosophila proteins that activate caspases by antagonizing IAPs [45].

IKs Antagonists

Antagonism of IKs was associated with increased basal progesterone but not FSH-stimulated progesterone accumulation on Day 1 after treatment. Structurally unrelated IKs antagonists, the chromanol 293B and the benzodiazepines, L735,821, and L768,763 stimulated progesterone accumulation. Furthermore, this effect coincided with the limited time period when primary cultures of pig granulosa cells express IKs and its molecular correlates, KCNQ1 and KCNE1 [7, 9]. Inhibition of granulosa cell IKs is associated with approximately a +10 mV decrease in resting membrane potential, and calcium current amplitude in freshly isolated granulosa cells was maximal at -30 mV, a test potential approximately 10 mV positive to normal granulosa cell resting membrane potential [4–7]. Depolarization-induced calcium entry could enhance granulosa cell progesterone production via induction of genes that encode P450scc and the steroidogenic acute regulatory protein (StAR) [26].

Pimozide (3 µM) had no effect on basal progesterone accumulation, but attenuated the stimulatory effects of IKs block. Although effects of pimozide on targets other than T channels cannot be ruled out completely on the basis of our experiments [38, 46], the ability of pimozide to block the stimulatory effects of the IKs antagonists is consistent with a transduction pathway involving depolarization-induced calcium influx via T-type channels. Comparable concentrations of pimozide have been shown to inhibit T-channel-dependent events in other cells; moreover, pimozide inhibition of steroid synthesis in adrenal cortical cells was specific for the steroidogenic response associated with calcium entry via voltage-gated channels [47, 48].

It is noteworthy that IKs antagonists significantly enhanced basal but not FSH-stimulated progesterone accumulation. This observed lack of additivity between the stimulatory effects of the drugs and FSH may reflect similarities in their underlying mechanisms of action. As described above, increased progesterone accumulation after granulosa cell exposure to IKs antagonists is likely to be mediated by a depolarization-induced increase in Ca2+ entry via voltage-sensitive channels. FSH-stimulated progesterone synthesis and steroidogenic gene induction in granulosa cells are also believed to be mediated in part by Ca2+ influx through voltage-gated channels [26, 49, 50]. It will be interesting to determine whether FSH-stimulated progesterone production is analogous to ACTH-stimulated cortisol production, and dependent on K+ channel blockade to cause depolarization-induced activation of T channels [47]. FSH stimulation of adenylate cyclase would be expected to inhibit granulosa cell IKs, although FSH treatment has not been associated previously with a decrease in granulosa cell resting membrane potential [5, 6].

CTX and MTX

Our laboratory and others have demonstrated a delayed rectifier K+ current with electrophysiological and pharmacological characteristics typical of Kv1.3 in cultured porcine granulosa cells and luteal cells [7, 9]. We have also determined that granulosa cell expression of Kv1.3 channel protein increases over 72 h of monolayer culture [9]. Here we show that both progesterone accumulation and the number of viable cells in culture are significantly increased 72 h after treatment with FSH in combination with either CTX or MTX, peptidyl toxins that antagonize Kv1.3 currents more effectively than those associated with the other subunits expressed by >=48 h granulosa cell cultures, Kv1.5 or Kv1.6 [9, 51].

Increased expression of Kv1.3 as granulosa cells luteinize in culture, and persistence of a Kv1.3-like current in luteal cells [7, 9] suggest that this current is important for granulosa cells in transition from a proliferative to a differentiated state. Our data suggest that Kv1.3 may also play a critical role in coordinating granulosa cell growth arrest and differentiation during the granulosa-to-luteal transition. We demonstrate that inhibition of Kv1.3 enhances cell proliferation without compromising the differentiated function (progesterone production) of granulosa cells cultured for 72 h in the presence of FSH. Assuming that depolarization associated with CTX and MTX inhibition of Kv1.3 would enhance calcium influx, our observation of toxin effects in the presence but not the absence of FSH is consistent with the mechanism proposed for the mitogenic action of FSH: activation of a growth factor type I FSH receptor, influx of Ca2+ via voltage-sensitive channels, and subsequent activation of extracellular-regulated kinase [49, 50, 52].

Previous work has shown that granulosa cell proliferation and differentiation can be uncoupled, but also that diminished expression of the cyclin-dependent kinase (CDK) inhibitor p27kip1 is critical to this uncoupling [53, 54]. The intracellular level of p27kip1 protein is regulated significantly by posttranslational control [55]. In our experiments, depolarization-mediated calcium influx associated with antagonism of granulosa cell Kv1.3 channels could contribute to the observed decrease in p27kip1, because this CDK inhibitor is a substrate for the calcium-activated protease calpain [56]. Validation of this working hypothesis will require definition of the calcium influx pathways present in luteinized granulosa cells; presently, the molecular correlates of these channels are poorly defined [7, 57].

The therapeutic and technological implications of this finding also remain to be determined. Kv1.3 channels are widely regarded as novel therapeutic targets because of their important role in T-lymphocyte activation [58]. Blockade of lymphocyte Kv1.3 channels is immunosuppressive, and selective antagonists are being investigated for treatment of delayed-type hypersensitivity reactions and autoimmune diseases, including multiple sclerosis [58, 59]. Our data suggest that it may be prudent to screen novel immunomodulators that antagonize Kv1.3 for their effects on ovarian function in vivo. Moreover, Kv1.3 could be a target for affecting granulosa cells function in vitro.

Summary

In conclusion, our data demonstrate that selective antagonism of granulosa cells K+ channels with distinct molecular correlates, electrophysiological properties, and expression patterns can influence differentially granulosa cells proliferation, steroidogenic capability, and apoptosis. These data suggest that K+ channels may contribute to the long-term aspects of cellular regulation, and thus may represent pharmacological targets for affecting granulosa cells function and oocyte maturation, in vivo or in vitro. The signaling pathways that transduce the effects of specific classes of K+ channel inhibitors are likely to involve depolarization and depolarization-mediated changes in intracellular calcium and/or other ions. Full definition of these pathways will require increased knowledge about the complement of voltage-sensitive channels and pumps present in the plasma membrane of granulosa cells at different times under different culture conditions.


   ACKNOWLEDGMENTS
 
We thank Suhasini Ganta for expert technical assistance with cell culture and protein chemistry, Chris Ross for help obtaining photomicrographs, and Wilma Shuman for contributing flow cytometry expertise.


   FOOTNOTES
 
First decision: 18 September 2001.

1 These studies were supported by National Institutes of Health grants HD-34235 and HD-36002 to L.F. Back

2 Correspondence: Lisa C. Freeman, Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Coles Hall 228, Manhattan, KS 66506-5802. FAX: 785 532 4557; freeman{at}vet.ksu.edu Back

Accepted: January 18, 2002.

Received: August 17, 2001.


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