Low extracellular Ca2+ activates a transient
Cl
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 |
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 |
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 |
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 M
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 |
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.

View larger version (23K):
[in this window]
[in a new window]
|
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+.

View larger version (9K):
[in this window]
[in a new window]
|
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.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
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.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
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.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
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.

View larger version (15K):
[in this window]
[in a new window]
|
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.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
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.

View larger version (10K):
[in this window]
[in a new window]
|
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 |
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 |
1.
Asem, EK,
and
Hertelendy F.
Role of calcium in luteinizing hormone induced progesterone and cyclic AMP production in granulosa cells of the hen (Gallus domesticus).
Gen Comp Endocrinol
62:
120-128,
1986[ISI][Medline].
2.
Asem, EK,
Schwartz JL,
Mealing GAR,
Tsang BK,
and
Whitfield JF.
Evidence for two distinct potassium channels in avian granulosa cells.
Biochem Biophys Res Commun
155:
761-766,
1988[ISI][Medline].
3.
Brown, EM,
Gamba G,
Riccardi D,
Lombardi M,
Butters R,
Kifor O,
Sun A,
Hediger MA,
Lytton J,
and
Hebert SC.
Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid.
Nature
366:
575-580,
1993[ISI][Medline].
4.
Brown, EM,
Pollak M,
Chou Y-HW,
Seidman CE,
Seidman JG,
and
Hebert SC.
The cloning of extracellular Ca2+-sensing receptors from parathyroid and kidney: molecular of mechanisms of extracellular Ca2+-sensing.
J Nutr
125:
1965S-1970S,
1995[Medline].
5.
Chiang, M,
Strong JA,
and
Asem EK.
Bovine serum albumin increases Ca2+ currents in chicken ovarian granulosa cells.
Mol Cell Endocrinol
94:
27-36,
1993[ISI][Medline].
6.
Chiang, M,
Strong JA,
and
Asem EK.
Luteinizing hormone activates chloride currents in hen ovarian granulosa cells.
Comp Biochem Physiol A Physiol
116:
361-368,
1997[ISI][Medline].
7.
Conkright, MD,
and
Asem EK.
Intracrine role of progesterone in fibronectin production and deposition by chicken ovarian granulosa cells in vitro: effect of extracellular calcium.
Biol Reprod
52:
683-689,
1995[Abstract].
8.
Eckstein, N,
Eshel A,
Eli Y,
Ayalon D,
and
Naor Z.
Calcium-dependent actions of gonadotropin releasing hormone agonist and luteinizing hormone upon cyclic AMP and progesterone production in rat ovarian granulosa cells.
Mol Cell Endocrinol
47:
91-98,
1986[ISI][Medline].
9.
Emanuel, RL,
Adler GK,
Kifor O,
Quinn SJ,
Fuller F,
Krapcho K,
and
Brown EM.
Calcium-sensing receptor expression and regulation by extracellular calcium in the AtT-20 pituitary cell line.
Mol Endocrinol
10:
555-565,
1996[Abstract].
10.
Gilbert, AB,
Evans AJ,
Perry MM,
and
Davidson MH.
A method for separating the granulosa cells, the basal lamina and the theca of the preovulatory ovarian follicle of the domestic fowl (Gallus domesticus).
J Reprod Fertil
50:
179-181,
1977[Medline].
11.
Kusaka, M,
Tohse N,
Nakaya H,
Tanaka T,
Kanno M,
and
Fujimoto S.
Membrane currents of porcine granulosa cells in primary culture: characterization and effects of luteinizing hormone.
Biol Reprod
49:
95-103,
1993[Abstract].
12.
Mattioli, M,
Barboni B,
and
Seren E.
Luteinizing hormone inhibits potassium outward currents in swine granulosa cells by intracellular calcium mobilization.
Endocrinology
129:
2740-2745,
1991[Abstract].
13.
Mealing, G,
Morley P,
Whitfield JF,
Tsang BK,
and
Schwartz JL.
Granulosa cells have calcium-dependent action potentials and a calcium-dependant chloride conductance.
Pflügers Arch
428:
307-314,
1994[ISI][Medline].
14.
Pillai, S,
Menon GK,
Bikle DD,
and
Elias PM.
Localization and quantitation of calcium pools and calcium binding sites in cultured human keratinocytes.
J Cell Physiol
154:
101-112,
1993[ISI][Medline].
15.
Pollak, MR,
Brown EM,
Chou Y-HW,
Hebert SC,
Marx SJ,
Steinmann B,
Levi T,
Seidman CE,
and
Seidman JG.
Mutations in the human Ca2+-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.
Cell
75:
1297-1303,
1993[ISI][Medline].
16.
Schwartz, JL,
Asem EK,
Mealing GAR,
Tsang BK,
Whitfield JF,
Rousseau E,
and
Payet MD.
T- and L-calcium channels in steroid-producing chicken granulosa cells in primary culture.
Endocrinology
125:
1973-1982,
1989[Abstract].
17.
Schwartz, JL,
Mealing GAR,
Asem EK,
Tsang BK,
and
Whitfield JF.
Ionic currents in avian granulosa cells.
FEBS Lett
241:
169-172,
1988[ISI][Medline].
18.
Tsang, BK,
and
Carnegie JA.
Calcium requirement in the gonadotropic regulation of rat granulosa cell progesterone production.
Endocrinology
113:
763-769,
1983[Abstract].
19.
Veldhuis, JD,
Klase PA,
Demers LM,
and
Chafouleas JG.
Mechanisms subserving calcium's modulation of luteinizing hormone action in isolated swine granulosa cells.
Endocrinology
14:
441-449,
1984.
20.
Wan, X,
Desilets M,
Soboloff J,
Morris C,
and
Tsang BK.
Muscarinic activation inhibits T-type Ca2+ current in hen granulosa cells.
Endocrinology
137:
2514-2521,
1996[Abstract].
21.
Weber, WM,
Liebold KM,
Reifarth FW,
Uhr U,
and
Clauss W.
Influence of extracellular Ca2+ on endogenous Cl
channels in Xenopus oocytes.
Pflügers Arch
429:
820-824,
1995[ISI][Medline].
Am J Physiol Cell Physiol 279(2):C319-C325
0363-6143/00 $5.00
Copyright © 2000 the American Physiological Society