Low extracellular Ca2+ activates a transient Clminus current in chicken ovarian granulosa cells

Wuxuan Qin1, Stanley G. Rane2, and Elikplimi K. Asem1

1 Department of Basic Medical Sciences, School of Veterinary Medicine and 2 Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1246


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of low Ca2+ on ion currents in hen ovarian granulosa cells were examined. A fast activating and inactivating transient outward current (TOC) and a slowly activating outward current (SOC) could be observed. In the presence of normal Ca2+ concentration (2.5 mM) and with a holding potential of -80 mV, SOC was activated in all cells with command pulses more positive than -20 mV. In 2.5 mM Ca2+, TOC appeared in 10% of cells at the command pulse of +80 mV and in 60-85% of cells at +100 to +120 mV. In low-Ca2+ solution and command potential of +80 mV (holding potential of -80 mV), the amplitude of TOC was enhanced in cells that expressed it in normal Ca2+, and TOC appeared in 43% of the cells that did not express it initially in normal Ca2+. At both normal and low Ca2+ levels, TOC decreased as the holding potential became more positive. TOC was reduced in Cl--deficient solution and in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid, a Cl- channel blocker. These findings suggest that chicken granulosa cells express a Ca2+-inactivated TOC carried by Cl-. This current may serve as a signal for some of the reduced metabolic functions of granulosa cells associated with Ca2+ deficiency.

patch clamp; ion channel; calcium-inactivated chloride current; ovary; ovarian follicle


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ELECTRICAL ACTIVITY HAS BEEN demonstrated in ovarian granulosa cells by several investigators. Granulosa cells express K+ channels (2, 11), Ca2+ channels (5, 11, 16, 17, 20), and Cl- channels (6, 13). In addition, action potentials have been observed in granulosa cells (13). Optimal functions of ovarian granulosa cells require the presence of adequate levels of Ca2+ in the extracellular compartment. In vitro studies with chicken, rat, and porcine granulosa cells have demonstrated that the full expression of the actions of gonadotropin luteinizing hormone (LH) and follicle-stimulating hormone require extracellular Ca2+. Significantly, the incubation of gonadotropin-stimulated porcine (19), rat (8, 18), and chicken (1) granulosa cells in Ca2+-deficient medium led to the suppression of steroidogenesis. Ca2+ deficiency affects early events such as cAMP production (1, 18, 19), as well as delayed/late events such as steroidogenesis (1, 8, 18, 19) and protein synthesis (7) in granulosa cells.

The presence of Ca2+ is required for the activation of members of some types of K+ or Cl- channel superfamilies. Evidence has been presented in support of the view that chicken and porcine granulosa cells express Ca2+-activated K+ (2, 11) and Cl- (6, 13) channels. The observation that, under physiological conditions, granulosa cells expressed Ca2+-activated ion channels causes one to wonder if these cells express Ca2+-inactivated ion channels as well. It has been reported that Xenopus oocytes express Ca2+-inactivated Cl- currents (21). If granulosa cells express Ca2+-inactivated ion channels, they would be maintained in an inactive state in the presence of physiological levels of the divalent cation. Ca2+ deficiency-associated changes in functions of granulosa cells, especially those of early events such as cyclic nucleotide second messenger generation (in seconds), could be the result of unidentified activities associated with plasma membrane of these cells, for example, the modulation of ion channel activities. It is hypothesized that ion channels (other than Ca2+ channels) are inactivated by physiological concentrations of Ca2+ and that these Ca2+-inactivated ion channels are released from inhibitory control in low Ca2+-containing medium and become activated. The aim of the present study was to study the effect of low extracellular Ca2+ on transmembrane ion currents in granulosa cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. EGTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid (BAPTA), HEPES, collagenase type 1A, soybean trypsin inhibitor, bovine serum albumin (BSA; fraction V), 4-aminopyridine (4-AP), and tetraethylammonium chloride (TEA) were from Sigma Chemical (St. Louis, MO). 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was obtained from Research Biochemicals International (Natick, MA). Medium 199 (M199) containing Hanks' salts was purchased from GIBCO (Grand Island, NY).

Solutions. The pipette solution contained 132 mM KCl, 5 mM NaCl, 3 mM Na2ATP, 5 mM Na3GTP, 10 mM HEPES, 0.1 mM EGTA, 70 µM CaCl2, and 1 mM MgCl2, pH 7.2, with KOH. The free Ca2+ concentration was 0.3 µM. The bath or external solution contained 134.3 mM NaCl, 5.4 mM KCl, 2.5 mM CaCl2, 1.1 mM MgCl2, 5.6 mM glucose, and 10 mM HEPES, pH 7.4. When necessary, CaCl2 was omitted and substituted with equimolar concentrations of MgCl2.

Animals and granulosa cell culture. White Leghorn hens in their first year of reproductive activity were obtained from Purdue University Animal Farms (West Lafayette, IN) and were caged individually in a windowless, air-conditioned room with a 14-h light:10-h darkness cycle. They had free access to a layer ration and tap water. The animals were killed by cervical dislocation, and granulosa cells were isolated from the largest preovulatory follicle (F1) 10-12 h before ovulation. The granulosa cell layer was separated from the theca layer as described by Gilbert et al. (10), and the cells were dissociated in M199 containing NaHCO3 (350 mg/l), HEPES (10 mM), collagenase (500 U/ml), and trypsin inhibitor (200 µg/ml), pH 7.4 (1). Cell viability, determined by the trypan blue exclusion method, was routinely >95%. All recordings were made at 23°C from single cells that had adhered to the dish.

Voltage clamp. The conventional whole cell recording method was used for the experiments. Recording pipettes were fabricated from borosilicate filament glass (Warner Instrument, Hamden, CT). Electrode resistance was between 3 and 9 MOmega in the bath solution. The linear (leak) component of the total membrane current was subtracted by extrapolating the linear currents obtained during voltage steps in more negative potential regions (-100 to -80 mV), where no voltage-activated currents were seen. Current amplitudes were small enough that the series resistance error was <5 mV. Analog compensation was applied to attenuate capacitive current transients and to estimate cell capacitances, a measure of cell membrane area. Membrane currents were measured with an Axopatch 1-D patch-clamp amplifier (Axon Instruments, Foster City, CA) and filtered at 1 kHz. The currents were digitized and stored directly to disk (DigiData 1200 Interface, Axon Instruments). Data analyses were performed with pCLAMP 6.0.3 software (Axon Instruments).

The granulosa cells were cultured in M199 with 0.1% BSA for 30 min on a 12-mm round coverslip (Warner Instrument) before being transferred to a perfusion chamber (Warner Instrument) containing bath solution (see Solutions). The volume of the chamber was 180 µl. In the conventional whole cell mode, the membrane potential was held at -80 mV. Membrane currents were elicited by test pulses of 330 ms in duration from -60 mV to +120 mV in 20-mV steps. All experiments were conducted at room temperature (21-23°C). Cells were perfused such that complete bath exchange was accomplished within 1 min. All bath solutions were at pH 7.4.

Data analysis. The amplitude of the transient outward current (TOC) was determined after the currents obtained with -40-mV holding potential were subtracted from currents recorded with holding potential of -80 mV.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the conventional whole cell configuration, with a holding potential of -80 mV, two different types of outward currents could be observed in chicken granulosa cells under the present quasiphysiological conditions (Fig. 1A). One was a delayed current that was elicited at test potentials positive to -20 mV and displayed outward rectification. It was slowly activating and is referred to as slow outward current (SOC). The SOC that was observed in all successful experiments (>60 cells) showed no sign of inactivation during the depolarizing test pulses. The second type of outward current, TOC, was fast activating and fast inactivating. It was always superimposed on the SOC (Fig. 1A). At the holding potential of -80 mV, TOC could be activated at +80 mV or more positive voltages. The TOC was activated almost instantly after the onset of the command pulse, reached a peak in <2 ms, and was inactivated in a few to 100 ms. When the holding potential was changed to -40 mV, the TOC disappeared but the SOC was not affected (Fig. 1B). The characteristics of TOC were determined after the current recorded with a holding potential of -40 mV was subtracted from the current recorded with -80 mV holding potential (Fig. 1C). TOC was not observed in all the cells. The percentage of cells that showed TOC increased with command potential. With a holding potential of -80 mV, TOC was observed in only 10% of cells at command pulse of +80 mV. At the command potential of +120 mV, TOC was observed in 60-85% of the cells. No TOC could be elicited at a command pulse of +60 mV or less (Fig. 1A). The amplitude of TOC also increased with command potential (see Fig. 4C). These results indicate that TOC activation increases with command potential. In addition, the activation of TOC was dependent on the holding potential. As indicated above, TOC disappeared at a holding potential of -40 mV, even at a command potential of +120 mV. It seems that the more positive the holding potential, the less activation of TOC. It was never observed when the holding potential was less negative than -60 mV.


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Fig. 1.   Whole cell membrane currents of granulosa cells in Ca2+-replete solution. The cell was stimulated from different holding potentials to test potentials between -20 mV and +120 mV in 20-mV steps with 330-ms duration. The voltage protocol is shown (inset). The bath solution contained 2.5 mM Ca2+. A: holding potential was -80 mV. B: holding potential was -40 mV. C: current traces following the subtraction of currents in B from currents in A. Vertical bar = 100 pA; horizontal bar = 50 ms.

Effect of extracellular Ca2+ on the TOC. As mentioned previously, at holding potential of -80 mV and command potential of +80 mV, 10% of the cells expressed TOC. In these cells, an increase in TOC amplitude was observed when Ca2+ was omitted from the external solution (free Ca2+ concentration was 100 nM; Fig. 2B and Fig. 3A). Interestingly, in Ca2+-deficient medium and at a command potential of +80 mV, TOC was activated in 43% of cells that did not express it initially in the normal (2.5 mM) Ca2+ bath (Fig. 3B). Cumulative data in Fig. 3 show the effect of extracellular Ca2+ on whole cell TOC and SOC density levels. Here membrane area was measured as the whole cell capacitance, as indicated in MATERIALS AND METHODS. When 5 mM EGTA was added to Ca2+-deficient medium to obtain essentially Ca2+-free solution (free Ca2+ concentration was 1 nM), TOC activation was greater than with either 2.5 mM Ca2+ or 100 nM Ca2+ (Figs. 2 and 4). In Ca2+-free solution, TOC could be activated immediately after the onset of a command pulse of +80 mV in almost all cells studied (n = 47 of 48 cells). Ca2+ depletion increased the average amplitude of TOC by 6-, 15-, and 200-fold at command potentials of +120, +100, and +80 mV, respectively (Fig. 4C). In Ca2+-free solution, TOC activation threshold was +20 mV, much more negative than that in cells in Ca2+-replete solution (+80 mV). The current-voltage relationships of TOC before (Fig. 4A) and after (Fig. 4B) the perfusion with 5 mM EGTA-containing bath solution is presented in Fig. 4C. The activation of TOC in Ca2+-free or Ca2+-deficient solutions was reversed with the replacement of bath solution containing 2.5 mM Ca2+ (Fig. 2E). In low-Ca2+ solution, the relationship between TOC and command potential remains the same as that in Ca2+-replete solution: TOC activation increases with command potential (Fig. 4C). Also, the relationship between TOC and holding potential is similar in Ca2+-replete and Ca2+-free solutions: TOC decreased when the holding potential was made more positive. At the holding potential of -40 mV, TOC disappeared in the Ca2+-free solution (Fig. 2D, compare with Fig. 2C). Therefore, in all cases, the peak of TOC (net current) was measured after the current recorded with -40 mV holding potential was subtracted from current recorded with -80 mV holding potential. Figure 5 shows the relationship between holding potential and TOC activation under Ca2+-free conditions. Experiments were conducted to determine if TOC activation is solely a function of the Ca2+ buffering capability of EGTA. When membrane currents were recorded from cells perfused with bath solution containing 5 mM EGTA and total Ca2+ concentration of 7.5 mM (free Ca2+ was 2.5 mM), no TOC could be activated with a holding potential of -80 mV and command pulses of +80 to +120 mV (data not shown). This finding indicated that EGTA activates TOC through deprivation of Ca2+.


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Fig. 2.   Whole cell membrane currents of granulosa cells perfused sequentially with solutions containing different concentrations of Ca2+. Currents were recorded from granulosa cells (with holding potential of -80 mV) either in bath solution replete with Ca2+ (2.5 mM Ca2+; A, control), deficient in Ca2+ (100 nM Ca2+; B), or containing 5 mM EGTA, free of Ca2+ (1 nM Ca2+; C). D: the current was recorded with a holding potential of -40 mV in Ca2+-free solution (compare with C). E: current recorded after the EGTA-containing solution was replaced with Ca2+-replete solution. Test potential was +80 mV for all currents. The voltage protocol is shown (inset). Vertical bar = 200 pA; horizontal bar = 50 ms.



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Fig. 3.   The densities of whole cell membrane currents of granulosa cells perfused sequentially with Ca2+-replete and low-Ca2+ solutions. Ca2+-replete solution contained 2.5 mM free Ca2+, and Ca2+-deficient solution contained 100 nM free Ca2+. Holding potential = -80 mV. Step potential = +80 mV. A: cells that expressed transient outward current (TOC) in Ca2+-replete solution. B: cells that did not express TOC in Ca2+-replete solution. SOC, slow outward current. Data presented were obtained from experiments described for Fig. 2. Each point is the mean ±SE of 3 cells.



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Fig. 4.   Effect of EGTA on TOC. Whole cell membrane currents were recorded from granulosa cells perfused sequentially with Ca2+-replete (A) and 5 mM EGTA containing Ca2+-free (B) solutions. The current traces shown here were recorded with a holding potential of -80 mV and test potentials between -20 mV and +120 mV in 20-mV steps. Vertical bar = 500 pA; horizontal bar = 50 ms. The voltage protocol is shown (inset). C: current-voltage relationship of TOC. Each point is the mean ±SE of 7 cells.



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Fig. 5.   Voltage dependence of steady-state inactivation of TOC in Ca2+-free solution. The current amplitude at the peak of TOC was measured after the voltage was stepped to +80 mV from the indicated holding potential. The TOC at various holding potentials were normalized (I/Imax) to the current recorded at the holding potential of -120 mV (which is the maximum current or noninactivated current, Imax). The data were fitted with a curve defined by the Boltzman equation, I/Imax = 1/1+exp(Vhold - V1/2/K), where Vhold is the holding potential from which the command step was evoked, V1/2 is the voltage corresponding to half-inactivation of the current, and K is the slope constant. V1/2 = -84 mV, r2 = 0.99.

Ionic basis of TOC. To determine if the transient current was carried by K+, the effect of K+ channel blockers 4-AP and TEA were tested. Up to 20 mM 4-AP (n = 4) or 15 mM of TEA (n = 3) had no apparent effect on the activation or amplitude of TOC current (data not shown). In other experiments, the replacement of K+ in the pipette solution with cesium had no effect on the activation of TOC (n = 4, data not shown). These results demonstrate that TOC was not carried by K+. To determine if TOC was carried by Cl-, ion substitution experiments were conducted. After the replacement of extracellular Cl- with aspartate, the amplitude of TOC was reduced by 80% (n = 7 cells; Fig. 6). The replacement of Cl- in the bath solution resulted in the partial recovery of TOC (Fig. 6). Substitution of Cl- with gluconate resulted in similar effects observed with aspartate (n = 3, data not shown). The Cl- channel blocker, NPPB, suppressed TOC (Fig. 7). These results indicate that Cl- is a primary carrier of TOC.


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Fig. 6.   Dependence of TOC on extracellular Cl-. Whole cell membrane currents were recorded from granulosa cells perfused sequentially with Ca2+-free solutions containing different amounts of Cl-. A: the membrane potential was held at -80 mV and stepped to +80 mV. Trace 1 = 140 mM Cl-; trace 2 = 7.6 mM Cl-; trace 3 = 140 mM Cl- again. Vertical bar = 200 pA; horizontal bar = 50 ms. B: current-voltage relationship of TOC measured in Cl--replete (140 mM Cl-) or Cl--deficient (7.6 mM Cl-) solutions. The peak of TOC was obtained after current recorded at -40 mV holding potential was subtracted from current recorded at -80 mV holding potential. Each point is the mean ±SE of 7 cells.



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Fig. 7.   Effect of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) on TOC. Whole cell currents were recorded from granulosa cells in Ca2+-free solution containing 140 mM Cl- before and after the addition of NPPB (0.1 mM). A: the holding potential was -80 mV, and the step potential was +80 mV. Trace 1: control; trace 2: NPPB; trace 3: NPPB washed out. Vertical bar = 100 pA; horizontal bar = 50 ms. B: the current-voltage relationship of TOC recorded before and after the addition of NPPB. The peak of TOC was obtained after current recorded at -40 mV holding potential was subtracted from current recorded at -80 mV holding potential. Each point is the mean ±SE of 3 cells.

Site of Ca2+ action. Because the removal of Ca2+ from the external medium resulted in the activation of the TOC, it was hypothesized that the reduction of cytosolic Ca2+ concentration was the cause of this phenomenon. In effect, the suppression of Ca2+ influx or transport may account for the activation of the TOC. Therefore, experiments were conducted in which Ca2+ influx was inhibited with cobalt, a Ca2+ channel blocker. Granulosa cells have an inward current that has been demonstrated to be carried by Ca2+ (5, 11, 16, 17, 20). In Fig. 8, cobalt blocked the inward current without having any effect on TOC (n = 4 cells). This finding demonstrates that the prevention of Ca2+ influx or transmembrane Ca2+ transport cannot account for the activation of TOC observed in Ca2+-deficient solution. In other experiments, the inclusion of 5 mM EGTA in the pipette solution did not itself activate TOC (n = 10, data not shown). Similarly, the inclusion of 5 mM BAPTA, another divalent cation chelator, in the pipette solution was without effect on the appearance of TOC (n = 4, data not shown). Moreover, the inclusion of 5 mM EGTA in the pipette did not affect the activation of TOC by Ca2+-free extracellular solution (n = 6, data not shown). These data indicate further that the activation of TOC is independent of intracellular Ca2+ levels.


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Fig. 8.   Effect of cobalt on TOC. Whole cell currents were measured in Ca2+-replete solution in the presence or absence of 5 mM cobalt. Holding potential was -80 mV. Step potentials were -20 mV and +120 mV. The voltage protocol is shown (inset). A: control; B: cobalt. Vertical bar = 50 pA; horizontal bar = 50 ms.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present results demonstrate that avian granulosa cells express a TOC that is carried by Cl- and is sensitive to low extracellular Ca2+. The current could be observed in the absence of known hormonal regulators of granulosa cell functions (such as gonadotropins, growth factors, and cytokines). The observation that the Cl--dependent TOC is activated in Ca2+-deficient medium supports the view that the channels responsible for the current are suppressed by physiological concentrations of Ca2+. A Ca2+-inactivated Cl- current was described in Xenopus oocytes (21). The Xenopus oocyte current inactivated slowly in contrast to the fast-inactivating current observed in the present studies. The voltage dependence of steady-state inactivation of TOC in the present study shows that the amplitude of the Cl- current is dependent on the holding potential. The more negative the holding potential, the more TOC carrying Cl- channels can be activated with depolarizing voltage steps.

Earlier studies have shown that chicken and pig granulosa cells express an outward current that is characteristic of the delayed rectifier K+ current and is inhibited by TEA (6, 11, 17). It is noteworthy that TEA-sensitive delayed rectifier K+ current is a major component of outward current of unstimulated chicken granulosa cells (6, 17). In this study, SOC is probably this delayed rectifier K+ current because it possessed similar characteristics and disappeared when pipette K+ was replaced by Cs+ (data not shown). In addition to the slow current, pig granulosa cells express a transient K+ current (11). The TOC observed in the present study is distinctly different from the one observed in pig granulosa cells because it is carried by Cl- and it is activated at +80 mV, compared with the -40 mV activation threshold for the pig granulosa cell TOC (11). In addition, the pig granulosa cell transient current was sensitive to 4-AP and cobalt, but the chicken granulosa cell current was not affected by 4-AP, TEA, or cobalt.

A Ca2+-dependent Cl- current was observed in chicken granulosa cells (13). The reasons why the Cl--carried TOC was not observed in earlier studies in chicken granulosa cells are unknown. In the present study, the protocol used (holding potential of -80 mV coupled with command pulses more positive than +80 mV) were different from those used in previous ones. For example, Chiang et al. (6) used a holding potential of -40 mV with command pulses up to +50 mV. Mealing et al. (13) used a holding potential of -80 mV with command pulses up to +40 mV.

In an earlier report, pituitary-derived LH activated a Cl- current in chicken granulosa cells (6). The LH-activated Cl- current showed outward rectification with no appreciable time or voltage dependence (6). Moreover, application of cobalt to the external solution had no effect on the transient Cl- current in the present study, whereas the Ca2+ channel blocker inhibited the LH-activated Cl- current (6). Taken together, the LH-activated Cl- current is different from the transient Cl- current monitored in the present study.

The profound effect of EGTA (when added to the external medium) on the TOC may be solely a function of its Ca2+ buffering capability, because EGTA had no effect on the transient current in the presence of supraphysiological levels of external Ca2+. The role of Ca2+ deficiency in the activation of TOC may be caused by the lack of Ca2+ influx through Ca2+-specific ion channels. The results of the cobalt experiment indicate that the activation of TOC cannot be explained by the lack of Ca2+ influx, because cobalt, a blocker of Ca2+ influx, had no effect on the Cl--carried TOC under conditions in which it blocked Ca2+-carried inward current. Of course, it is possible that there exist other pathways for Ca2+ influx that are not blocked by cobalt. However, a decrease in intracellular Ca2+ levels is not likely responsible for the activation of the TOC. If it was, experimental reduction of cytosolic Ca2+ levels would have resulted in activation of the transient current. When EGTA or BAPTA were included in the pipette solution, there was no change in the activation kinetics of the TOC (thus a reduction in the concentration of cytosolic Ca2+ is not the cause of activation of TOC). The cytosolic levels of Ca2+ were not determined in the experiments in which EGTA and BAPTA were included in the pipette solution; therefore, it could be argued that the cytosolic Ca2+ concentrations were not sufficiently reduced.

Activation of the Cl--carried TOC does not appear to be due to inhibition of Ca2+ influx or dependent on a reduction in cytosolic levels of Ca2+; therefore, the site of Ca2+ deficiency-induced activation of TOC may be at the external surface of the plasma membrane. What is the nature of the site of action of Ca2+ on the external plasma membrane? Could it be a Ca2+-sensing receptor? A 120-kDa, G protein-coupled Ca2+-sensing receptor has been discovered in cells of the parathyroid gland (3, 4, 15), kidney (4), pituitary, and AtT-20 pituitary cell line (9). Other studies have revealed specific Ca2+ binding sites on external membranes of cells such as keratinocytes (14). The existence of a Ca2+-sensing receptor or binding site in granulosa cells has yet to be demonstrated. Other questions remain to be answered. For example, what is (are) the physiological regulator(s) of the Cl--carried TOC? What is the functional role of this current? One possibility is that the transient Cl- current serves as a signal for some of the reduced metabolic functions of granulosa cells under Ca2+ deficient conditions.

Mealing et al. (13) reported that the resting potential of chicken granulosa cells is -62 mV. The experiment of the voltage dependence of steady-state inactivation showed that when holding potential was -60 mV, TOC could be activated, although its amplitude was about 25% of that at -80 mV holding potential (that was used predominantly in this study). The command potential required to activate TOC must be more positive than +20 mV. At this point, we do not know if TOC can be activated under physiological conditions. In vivo, there are gap junctions between granulosa cells and the oocyte, which make granulosa cells and the oocyte a syncitium of electrical and chemical communication. Therefore, granulosa cells are under great influence of the oocyte in vivo. As mentioned previously, granulosa cells have action potentials with unknown function. Obviously, the physiological conditions of granulosa cells are far from being understood. In addition, we do not know if any regulating factors such as hormones can activate TOC under physiological conditions. Clearly, additional work is needed to assess the physiological regulation of TOC and to determine the characteristics of single channels that carry it.

In summary, a TOC was observed in chicken granulosa cells in the absence of known regulators of granulosa cell function. The current is carried by Cl-, and it is activated when the extracellular solution is deficient in Ca2+. These results suggest that granulosa cells express Ca2+-inactivated Cl- channels. Furthermore, the data suggest that Ca2+ may act at an external site of the plasma membrane. Normal or high concentrations of Ca2+ inhibit the transient outward Cl- current, and low concentrations stimulate it.


    ACKNOWLEDGEMENTS

This work was supported by funds from Purdue Univ. School of Veterinary Medicine. W. Qin was supported by a Purdue Research Foundation research assistantship.


    FOOTNOTES

Address for reprint requests and other correspondence: E. K. Asem, Dept. of Basic Medical Sciences, School of Veterinary Medicine, Purdue Univ., 1246 Lynn Hall, West Lafayette, IN 47907-1246 (E-mail: eka{at}vet.purdue.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 6 October 1999; accepted in final form 2 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 279(2):C319-C325
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