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The Journal of Clinical Endocrinology & Metabolism 87(12):5566 –5574
Copyright © 2002 by The Endocrine Society
doi: 10.1210/jc.2002-020841
Ca2ⴙ-Activated, Large Conductance Kⴙ Channel in the
Ovary: Identification, Characterization, and Functional
Involvement in Steroidogenesis
LARS KUNZ, ANDREA THALHAMMER, FRANK D. BERG, ULRIKE BERG, DIANE M. DUFFY,
RICHARD L. STOUFFER, GREGORY A. DISSEN, SERGIO R. OJEDA, AND ARTUR MAYERHOFER
Anatomical Institute, University of Munich (L.K., A.T., A.M.), D-80802 Munich, Germany; Fertility Center Munich (F.D.B.,
U.B.), D-81675 Munich, Germany; and Divisions of Reproductive Sciences (D.M.D., R.L.S.) and Neuroscience (G.A.D.,
S.R.O.), Oregon National Primate Research Center, Oregon Health Sciences University, Beaverton, Oregon 97006
Progesterone production by the corpus luteum is a process
vital for reproduction. In humans its secretion is stimulated
by the placental hormone human chorionic gonadotropin
(hCG), and this stimulatory action can also be observed in
cultured human luteinized granulosa cells (GCs). We now provide evidence that opening of a Ca2ⴙ-activated Kⴙ channel, the
BKCa, is crucially involved in this process. Immunohistochemistry and RT-PCR revealed the presence of the pore-forming
␣-subunit in human luteinized GCs and in luteal cells of human, macaque, and rat, implying that BKCa channels are important throughout species. Blocking of BKCa channels by
iberiotoxin attenuated hCG-induced progesterone secretion.
The inhibitory action of iberiotoxin suggests that BKCa
channels are activated in the course of hCG-induced steroido-
F
ORMATION, ENDOCRINE FUNCTION, and regression
of the corpus luteum (CL) are processes crucial for
normal reproduction (1–3). During the life span of the CL, a
variety of cellular processes occur, comprising cell proliferation, differentiation, and, in particular, the onset of progesterone secretion by luteinized granulosa cells (GCs). This
process is stimulated by LH and/or by the placental hormone human chorionic gonadotropin (hCG) and is also observed in cultured human luteinized GCs, an accepted in
vitro model for ovarian endocrine cells (4). Over the past
years, a novel regulatory system in the ovary was discovered
that acts via locally produced signaling molecules, including
neurotransmitters (e.g. acetylcholine) and peptide hormones
(e.g. oxytocin). Human luteinized GCs produce several of
these molecules and possess receptors for some of them as
well (5– 8). These observations may account for a variety of
cellular responses to muscarinic agents in GCs, e.g. effects on
cell proliferation (9) and gap junction communication (10).
The presence and action of neurotransmitters and other signaling molecules in the ovary render participation of ion
channels in the control of endocrine function a likely, albeit
Abbreviations: ACh, Acetylcholine; BKCa, Ca2⫹-dependent, large
conductance K⫹ channel; 8-Br-cAMP, 8-bromo-cAMP; carb, carbachol;
CL, corpus luteum; DMSO, dimethylsulfoxide; EC, extracellular bath
solution; FCS, fetal calf serum; GC, granulosa cell; gsc, single channel
conductance; hCG, human chorionic gonadotropin; IbTx, iberiotoxin;
MR, muscarinic receptor; ORPRC, Oregon Regional Primate Research
Center; OT, oxytocin; StAR, steroid acute regulatory protein; Vh, holding
potential; Vr, reversal potential;V0, zero current potential.
genesis. In search of physiological activators we used an electrophysiological approach and could preclude a direct regulation
of channel activity by hCG or GC-derived steroids (progesterone
and 17␤-estradiol). Instead, the peptide hormone oxytocin and
an acetylcholine (ACh) agonist, carbachol, evoked transient
BKCa currents and membrane hyperpolarization. These two
molecules are both secreted by GCs and act via raised intracellular Ca2ⴙ levels. The release of oxytocin is stimulated by
hCG, and a similar mechanism is likely in the case of ACh. We
conclude that BKCa channel activity in GCs is mediated by components of the intraovarian signaling system, thereby interlinking a systemic hormonal and a local neuroendocrine system
in control of steroidogenesis. (J Clin Endocrinol Metab 87:
5566 –5574, 2002)
barely explored, possibility. For instance, in chicken GCs the
participation of a Cl⫺ current in LH-stimulated progesterone
secretion was described (11). We have now focused on potassium channels because of their crucial part in various
cellular functions, including control of membrane potential
and proliferation (12, 13). Furthermore, cellular K⫹ levels
were reported to affect progesterone secretion by luteal cells
(14, 15). Functional K⫹ currents/channels have been observed in avian, porcine, and human GCs to date (16 –22), but
as yet their molecular identity has been revealed only for two
channels in porcine GCs (23). A possible activation of Ca2⫹activated K⫹ channels attracted our attention, as some of the
signaling molecules, e.g. acetylcholine (ACh) and oxytocin
(OT), cause transient rises of intracellular Ca2⫹ levels in GCs
(8, 24 –26). One member of this class of K⫹ channels, the BKCa
(maxiKCa), is characterized by a high single channel conductance (27). It consists of a pore-forming ␣-subunit and one of
four possible modulatory ␤-subunits (28, 29). This channel
has a prominent role in the cessation of Ca2⫹-induced cellular
responses by repolarizing the plasma membrane and thus is
recognized to be involved in a variety of physiological processes. Among the best documented of these processes are
termination of synaptic neurotransmitter release and control
of vessel tone (30, 31). Although BKCa currents were described in mammalian endocrine cells (32, 33), and muscarinic stimulation was shown to activate BKCa channels in
some cell types (32–35), it is not known whether BKCa channels are involved in the regulation of endocrine cell function.
In the present study we addressed the question of whether
5566
Kunz et al. • Ovarian BKCa Channel and Steroidogenesis
the BKCa channel is present and functional in ovarian endocrine cells. To this end we have used various techniques and
examined the ovarian BKCa channel, which was found to be
active in cultured human GCs. We have studied its localization in ovarian cells of several species, including humans
and nonhuman primates. Besides the molecular identification, we aimed at the functional characterization in human
GCs using an electrophysiological approach. To examine a
potential physiological role of the ovarian BKCa, we investigated its involvement in the hCG-induced progesterone
production, the prime endocrine function of GCs in the active
CL. In search of physiological regulators of its activity, we
used the patch-clamp technique to determine the actions of
ovarian signaling molecules (ACh and OT), of GC steroid
products (progesterone and 17␤-estradiol), and of peptide
hormones (hCG) related to reproductive function.
Materials and Methods
Granulosa cell preparation and culture
Human luteinized GCs were derived from follicular aspirates of
women undergoing in vitro fertilization (36). Written consent was obtained, and experimental procedures and use of the cells were approved
by the local ethics committee. Isolation of the cells and cultivation in
DMEM/Ham’s F-12 (1:1; Sigma, Deisenhofen, Germany; 10% FCS) were
previously described (36). Nonluteinizing and luteinizing rhesus monkey GCs used for RNA extraction were retrieved by follicle aspiration
from anesthetized monkeys before and after hCG-induced ovulation,
respectively, in controlled ovarian stimulation cycles (37).
Ovarian tissue samples
Monkey ovaries were collected from adult rhesus macaques (Macaca
mulatta) undergoing ovariectomy for other purposes or were obtained
at necropsy through the tissue distribution program at the Oregon
National Primate Research Center (ONPRC) (17, 37, 38). The care and
housing of the monkeys at the ONPRC and the experiments were approved by the ONPRC animal care and use committee and were conducted in accordance with the NIH guide for the Care and Use of
Laboratory Animals. CL were excised from anesthetized monkeys on d
3, 10, and 14 after the LH surge, which corresponds to the early, mid,
and late luteal phases of the menstrual cycle, respectively (37). Human
ovaries (n ⫽ 3) containing CL were obtained from patients undergoing
gynecological surgery. The procedures were approved by the local ethics
committees, and patients gave written consent for the use of tissue
samples. Female rats (Sprague Dawley; colony at Technical University
Munich) were killed by decapitation under diethyl ether anesthesia for
removal of ovaries and brains. Upon collection, all tissue samples were
either rapidly frozen on dry ice for RNA extraction or were fixed in
Bouin’s fixative for subsequent paraffin embedding and immunohistochemistry (17, 37, 38).
Chemicals and solutions
The following stock solutions were prepared in distilled water unless
otherwise stated and further diluted into the extracellular solution (EC;
see electrophysiology): 10 ␮m iberiotoxin (IbTx; Tocris/Biotrend, Köln,
Germany), 50 mm NS 1619 (in ethanol), 10 mm niflumic acid, 5 mm
phloretin (in 50% ethanol), 1000 IU/ml hCG, 100 mm carbachol (carb),
1 mm OT, 10 mm 17␤-estradiol [0.01% dimethylsulfoxide (DMSO)], and
10 mm progesterone (0.01% DMSO; all purchased from Sigma). Carb,
OT, and hCG were applied in concentrations shown to be effective in
previous studies (25, 26, 39).
RT-PCR
Total RNA from several batches of human GCs harvested on d 2– 4
of culture and from monkey tissue were prepared and reverse transcribed as previously described (9, 36, 40). For identification of the BKCa
J Clin Endocrinol Metab, December 2002, 87(12):5566 –5574 5567
channel ␣-subunit, PCR amplification was performed with the following
oligodeoxynucleotide primer pairs constructed for a first (using outer
primers) and a second nested PCR step (using inner primers) (36, 38)
with matching sequences of human (GenBank accession no. U13913),
rhesus monkey (AF026001), and rat (AF135265): outer sense primer,
5⬘-GTGACCATGGAGGTGCC-3⬘; outer antisense primer, 5⬘-TAGAGAAGGAAGAACAC-3⬘; inner sense primer, 5⬘-CCTCTTCATCATCTTGCTC-3⬘; and inner antisense primer, 5⬘-CTGGCAGGATTCTATTGG-3⬘. In addition, a commercial human ovarian cDNA (2 ␮l;
Human MTC Panel II, BD CLONTECH Laboratories, Inc., Heidelberg,
Germany) (17) was used for PCR. The identity of the PCR products was
verified by sequencing either directly using one of the specific primers
or after subcloning into the pGEMT vector (Promega Corp., Mannheim,
Germany) (17, 36, 38).
Immunohistochemistry
Detection of the BKCa ␣-subunit protein was performed following
standard procedures using a polyclonal antiserum (rabbit antimouse;
Alamone Laboratories, Jerusalem, Israel; 1:1000) and the horseradish
peroxidase/diaminobenzidine staining reaction (36, 38). In addition,
monkey ovarian sections were immunostained using a fluorescence
protocol as previously described (9). An additional microwave treatment for antigen retrieval was employed (36, 38). For control purposes,
the first antibody was omitted, replaced by normal rabbit serum, or
preadsorbed with the peptide used for antiserum preparation.
Cell morphology
Cells grown on coverslips for 3 d were incubated with different
stimulants for 24 h. Thereafter, they were fixed with 5% glutaraldehyde
in 0.1 m sodium cacodylate buffer (pH 7.4) and postfixed with 1%
osmium tetroxide/potassium hexacyanoferrate II. After embedding in
Epon, 1-␮m thick sections were cut, stained with Azure II/methylene
blue (1:1), and inspected by light microscopy.
Electrophysiology
Patch-clamp measurements were performed with human GCs grown
on glass coverslips (d 2–11 of culture) using an EPC-9 amplifier (HEKA
Elektronik, Lambrecht, Germany) and an Axiovert 135 microscope (Carl
Zeiss, Oberkochen, Germany). Borosilicate glass pipettes (GB150-8P,
Science Products, Hofheim, Germany) were pulled and heat-polished
with a DMZ-Universal Puller (Zeitz, Augsburg, Germany). Filled with
electrolyte solution, they showed a resistance of 3–5 m⍀. The extracellular bath solution (EC) contained 140 mm NaCl, 3 mm KCl, 1 mm CaCl2,
10 mm HEPES, and 10 mm glucose (pH 7.4).
The pipette solution contained either 130 mm potassium gluconate or
130 mm KCl, and in both cases contained 5 mm NaCl, 1 mm MgCl2, 10
mm HEPES (pH 7.4), and 100 nm free Ca2⫹ (1 mm CaCl2 and 2 mm
EGTA). Current-voltage relationships were measured in the whole-cell
configuration (41) by application of a series of voltage pulses between
⫺100 and ⫹100 mV. Positive currents were defined as outward currents,
and the given potentials refer to the cytoplasmic side. Activation of BKCa
channels was shown as the change in steady state outward currents at
a positive holding potential of Vh ⫽ ⫹100 mV (I⫹100). At this voltage the
current activated by BKCa channel openers was markedly bigger than
the background current (see Fig. 3, C–E). Based on the activation by the
openers and the inhibition of the elicited current by IbTx, this current
(I⫹100) was equated with the BKCa current.
Single channel measurements were carried out in the inside-out and
the cell-attached configurations (41). The single channel current traces
were sampled with a rate of 10 kHz and low pass filtered at 2.5 kHz.
Channel properties were determined using standard procedures (42).
Briefly, the open probability corresponds to the time fraction in which
the channel is active, multiplied by the number of simultaneously open
channels. Currents recorded at different holding potentials were taken
to calculate single channel conductance (gsc) and reversal potential (Vr)
from slope and x-intercept, respectively, of the current-voltage relationship. A previously described, fast pressurized perfusion system was
used for drug application (17). EC was applied to test for mechanical
interference by the mere approaching flow of solutions.
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J Clin Endocrinol Metab, December 2002, 87(12):5566 –5574
Progesterone assay
Cells were seeded in a 24-well plate coated with laminin (17). On d
3 of culture they were exposed to fresh culture medium alone (control)
or medium containing the stimulants, using triplicate wells for each
treatment (1 ml/well; for concentrations see Results). After 24 h the
culture media were collected and frozen at ⫺20 C until measurement of
progesterone concentrations using a progesterone ELISA (DRG Instruments, Marburg, Germany). Mean values were calculated from the triplicate determinations and normalized to the control treatment. The experiment was repeated four times with independent cell preparations.
Normalized mean values were averaged, and a paired t test was chosen
to evaluate the results.
Kunz et al. • Ovarian BKCa Channel and Steroidogenesis
a polyclonal antiserum directed against the C-terminal part
of the protein (Fig. 1). In all three species, pronounced immunoreactivity was found in the CL. In monkey CL, immunostaining was similar in both large and small luteal cells
(Fig. 1, C and D). In the human CL, thecal cell-derived small
luteal cells showed a higher degree of immunoreactivity than
the granulosa cell-derived large luteal cells (Fig. 1E). In all
three species there was a strong staining of thecal cells of
antral follicles and weak staining of follicular granulosa cells.
Staining was absent in all control experiments for each tissue
investigated.
Data analysis
Data acquisition, analysis, statistics, and presentation were performed using Pulse and TAC X4.1 (HEKA Elektronik, Lambrecht,
Germany), PRISM 3.0 (GraphPad Software, Inc., San Diego, CA), and
SigmaPlot (Wavemetrics, Lake Oswego, OR). Data represent the
mean ⫾ sem.
Results
BKCa ␣-subunit protein is expressed in the CL and other
ovarian compartments
The pore-forming BKCa ␣-subunit was detected by immunohistochemistry in human, monkey, and rat ovaries using
FIG. 1. Immunohistochemical detection of
BKCa ␣-subunit in ovarian tissues of different
species. Consecutive sections of the rat ovary
(*, granulosa cells; arrow, thecal cells) incubated with BKCa ␣-subunit antiserum (A) or
nonimmune rabbit antiserum (B). C and D,
Monkey (Macaca mulatta) antral follicle (C)
and CL (C and D). Staining of follicular GC
was weak, but clearly present, as shown by
immunofluorescence (see inset in C; TC, thecal
cells; bar, 50 ␮m). BKCa ␣-subunit was detected in human CL (E). Notice the intense
staining in the peripheral thecal-luteal cells of
the human CL. Rat brainstem served as a
positive control tissue expressing the BKCa
␣-subunit (F). Bars, 70 ␮m (C and D), 80 ␮m
(A, B, and F), and 125 ␮m (E).
Expression of BKCa ␣-subunit mRNA in ovary, CL, and
primate GCs
The BKCa ␣-subunit mRNA was detected by RT-PCR in
ovarian samples from humans, monkeys, and rats (Fig. 2A).
Moreover, ␣-subunit mRNA was found in luteinized GCs of
both human and monkey origins and, in addition, in nonluteinized granulosa cells of the monkey (Fig. 2B). Cells of the
CL and the preovulatory follicle, in vivo counterparts of GCs,
expressed the ␣-subunit throughout the luteal phase (Fig.
2B). Sequencing of cDNAs showed that they were identical
Kunz et al. • Ovarian BKCa Channel and Steroidogenesis
J Clin Endocrinol Metab, December 2002, 87(12):5566 –5574 5569
tigated by measuring the released progesterone in the cell
supernatant after 24-h stimulation using a commercial ELISA
(Fig. 4; n ⫽ 4). The stimulatory action of hCG (10 IU/ml) was
diminished by application of the specific BKCa channel
blocker IbTx (10 nm), whereas IbTx alone had no effect on the
basal progesterone secretion. IbTx also attenuated progesterone production induced by 8-bromo-cAMP (8-Br-cAMP),
a cell-permeable analog of cAMP, which is elevated in the cell
after binding of hCG to the LH receptor (Fig. 5A). Thereby,
a direct toxic effect of IbTx on the LH receptor could be
excluded. General cytotoxic effects of IbTx alone or in combination with hCG could also be ruled out, because cell
morphology was unaffected (Fig. 5B).
Transient activation of the BKCa channel by
physiological stimuli
FIG. 2. A, Expression of the BKCa ␣-subunit in the ovary of primates
(human and monkey) and the rat. B, Presence of BKCa ␣-subunit
mRNA in human luteinized GC, monkey luteinized (lGC) and nonluteinized (nlGC) GC, as well as monkey CL at all phases of its life
span during the menstrual cycle (E, early; M, middle; L, late). The
identity of the amplified cDNAs (319 bp) was confirmed by sequencing. In the PCR controls shown (Co), no template was added.
to the published sequence data (see Materials and Methods for
databank accession number).
Electrophysiological characterization of the BKCa in human
luteinized granulosa cells
Figure 3A depicts the typical BKCa single channel current
traces in the inside-out patch-clamp configuration measured
at different holding potentials (Vh). Analysis of these single
channel currents yielded the characteristically high conductance of about 150 –200 pS and a negative reversal potential
indicative of a channel mainly permeable to K⫹ (Fig. 3B). In
the cell-attached mode, BKCa channels were observed in
about 15% of all experiments and exhibited similar characteristics: gsc ⫽ 162 ⫾ 12 pS and Vr ⫽ V0 ⫹ (11 ⫾ 9) mV (n ⫽
16). V0 is equal to the zero current potential measured in
whole-cell recordings (V0 ⫽ ⫺25 ⫾ 2 mV; n ⫽ 88). Identification of the BKCa channel was possible by studying the
action of BKCa channel openers (NS1619, niflumic acid, and
phloretin) and of the blocker IbTx on whole cell currents. In
76% of all experiments the openers entailed either an increase
in the K⫹ outward current (I⫹100; n ⫽ 4) or hyperpolarization
of the plasma membrane (n ⫽ 7) as a consequence of the
induced current, or both (n ⫽ 2; Fig. 3, C–E), which are all
indicative of activation of a BKCa outward current. IbTx
blocked the currents elicited by the channel openers, either
completely (n ⫽ 8) or partially (n ⫽ 2), in all experiments in
which it was applied (n ⫽ 10).
Role of the BKCa channel in granulosa cell function
A possible part of BKCa channels in the major GC function,
i.e. the production and secretion of progesterone, was inves-
In search of physiological activators of the BKCa, the effects
of an ACh agonist (carb), the peptide hormones OT and hCG,
and the steroid hormones 17␤-estradiol and progesterone
were investigated in the whole-cell configuration. Carb (100
␮m) activated BKCa currents in 18 of 21 experiments, as
detected by the induced K⫹ outward current at a holding
potential of ⫹100 mV (I⫹100), the resulting hyperpolarization
of the plasma membrane, or both (Fig. 6). The incidence of
carb-induced BKCa activation (86%) was similar to the value
for the BKCa channel openers (76%). The induced BKCa current was composed of a predominant transient component
and a smaller steady state part and could be activated at least
twice in the same cell after a short recovery period of about
1–2 min. The current-voltage relationship of the carbinduced current was the same as that activated by the BKCa
channel openers (not shown). All recordings with IbTx application (n ⫽ 6) exhibited either a complete (n ⫽ 4) or partial
(n ⫽ 2) inhibitory effect of this specific BKCa channel blocker
on the induced K⫹ current. In the presence of 100 nm IbTx,
carb was not capable of eliciting the K⫹ outward current in
cells in which carb alone induced the current (Fig. 6, A and
C). In cells with a steady state component of the carb-induced
current, this steady state current could be blocked by IbTx
(not shown). This inhibitory effect of IbTx together with the
identical current-voltage relationships of the induced currents clearly proves that the carb-evoked I⫹100 corresponds
to the current elicited by the BKCa openers. In cell-attached
measurements, transient activation of single BKCa channels
by extracellularly applied carb (100 ␮m) was also shown (Fig.
6, G and F).
Similar to carb, 100 nm OT activated the BKCa current and
caused hyperpolarization in 6 of 11 cells investigated (Fig. 6).
The lower incidence of BKCa activation by OT (55%) compared with the effects of the BKCa channel openers (76%) and
carb (86%) most likely reflects the fact that the expression
maximum of OT receptors in human GCs is not reached
before d 5 of culture (26). OT induced the BKCa current in cells
that had been in culture for 3–11 d, but all nonresponding
cells had been in culture for 3 or 4 d, and on d 3 only 1 of 5
cells responded to the OT stimulus.
In contrast, hCG, at a concentration that maximally stimulates progesterone release (10 IU/ml), did not activate the
BKCa current or induce any change in whole-cell steady state
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J Clin Endocrinol Metab, December 2002, 87(12):5566 –5574
Kunz et al. • Ovarian BKCa Channel and Steroidogenesis
FIG. 3. Functional characterization of
BKCa channels in human GCs. BKCa
channels were studied either as single
channel currents in the inside-out configuration (A and B) or as whole-cell
currents (C–E). In all experiments
physiological conditions were chosen,
i.e. [K]i ⫽ 125 mM and [K]a ⫽ 3 mM. A,
Single channel current traces measured
at different holding potentials (Vh) exhibiting the fluctuation of the channel
between the open (o) and the closed (c)
state. The bars represent the corresponding current levels. B, Typical
BKCa single channel current-voltage relationship showing the high single
channel conductance of gsc ⫽ 150 pS and
a reversal potential of Vr ⫽ ⫺45 mV.
C–E, At the whole-cell experiments,
cells were clamped at approximately
the measured zero current potential.
Activation of BKCa currents by BKCa
channel openers (50 ␮M NS 1619, C; 50
␮M phloretin (phlo.), D; 100 ␮M niflumic
acid, D) was detected as a rise in the
outward K⫹ current (I⫹100) and/or by
the subsequent hyperpolarization (C).
The activated current was blocked by
simultaneous application of the BKCa
channel blocker IbTx (100 nM). E, Original current traces (corresponding to D)
elicited by stepwise changes in test potentials over the range of ⫺100 to 100
mV (10 mV/step). The graphs exhibit
the activation of the BKCa current by
phloretin compared with the control
measurement, which can be blocked by
simultaneous application of IbTx and
reactivated by another BKCa channel
opener, niflumic acid.
steroid hormones progesterone (1 ␮m; n ⫽ 22; Fig. 7B) and
17␤-estradiol (1 ␮m; n ⫽ 9; data not shown) also showed no
activation of BKCa currents, even in cells exhibiting BKCa
currents induced by subsequent application of 100 ␮m carb
(Fig. 7B). The steroid solutions (17␤-estradiol and progesterone) contained 0.01% DMSO, which did not cause any
electrophysiological effects (not shown).
Discussion
FIG. 4. The BKCa channel is involved in hCG-stimulated progesterone secretion. The secretion of progesterone was measured by an
ELISA in the supernatant of GCs after 24 h of stimulation. The
presence of 10 nM IbTx markedly attenuated the stimulatory effect of
10 IU/ml hCG (P ⫽ 0.044, by paired t test), but exhibited no effect on
basal progesterone secretion (P ⫽ 0.459, compared with control without treatment). Data represent the mean ⫾ SEM progesterone levels
normalized to control values, which were measured in four independent experiments (triplicates in each experiment).
current or membrane zero current potential (n ⫽ 15). This
was observed in cell preparations yielding OT- or carbsensitive cells and even in the very cells that did respond to
OT before or after hCG application (n ⫽ 3; Fig. 7A). The
In the present communication we demonstrate the existence of functionally active BKCa channels in human luteinized GCs. As we located this channel to various endocrine
cells of the human, monkey, and rat ovary, these results are
probably of relevance throughout species. In human GCs the
BKCa channel has a prominent role in hCG-induced progesterone secretion, and it is activated by muscarinergic and
oxytocinergic signals, indicating for the first time that locally
produced signaling molecules are physiological regulators of
ion channel activity in ovarian endocrine cells.
Currently, limited information exists concerning the
presence of ion channels in ovarian endocrine cells, and
the available data were almost exclusively gained from
studies of cultured granulosa cells of avian (chicken) and
nonprimate mammalian (porcine) origins. These studies
described K⫹, Ca2⫹, and Cl⫺ currents/channels, which
were, however, characterized mainly with regard to the
Kunz et al. • Ovarian BKCa Channel and Steroidogenesis
FIG. 5. The action of IbTx on hCG-induced progesterone secretion by
human luteinized GCs is not due to cytotoxicity. A, IbTx has no direct
toxic effect on the LH/hCG receptor. The effect of a cell-permeable
cAMP analog, 8-Br-cAMP, on progesterone secretion was investigated
as described in Fig. 4. On d 3 of culture, cells were stimulated for 24 h
with medium alone (Co), 10 nM IbTx, 1 mM 8-Br-cAMP, and their
combination. IbTx attenuated the stimulatory effect of 8-Br-cAMP
(P ⫽ 0.047, by unpaired t test). Data represent the mean ⫾ SEM
progesterone levels normalized to control value (all in triplicate). B,
Cell morphology of human GCs appears to be unaffected by IbTx alone
and in combination with hCG. Cells were incubated for 24 h with
medium alone (Co), 10 IU/ml hCG, 10 nM IbTx, or both hCG and IbTx.
Scale bar (Co), 20 ␮m.
permeating ion (11, 18 –22, 43– 45). Only recently were the
molecular identities of two K⫹ channels in porcine GCs
studied (23). The molecular characterization of ion channels is crucial, however, for a thorough comparison of the
data and for revelation of ion channel functions, especially
in view of pronounced species-dependent differences in
electrophysiological properties of GCs.
With respect to primate species, the first detailed study
was performed by Bulling and co-workers, who used human
luteinized granulosa cells as a model for the preovulatory
follicle and the CL (17, 36). They identified and characterized
a voltage-dependent sodium channel and revealed the molecular identity of this endocrine-type Na⫹ channel. Furthermore, their studies indicated the existence of at least two
J Clin Endocrinol Metab, December 2002, 87(12):5566 –5574 5571
different K⫹ currents. In the present study we focused on K⫹
channels and could unambiguously identify the BKCa channel based on its activation by the BKCa channel openers
NS1619, niflumic acid, and phloretin, and particularly by the
sensitivity of the induced K⫹ current to the highly specific
BKCa blocker, IbTx. The occurrence of the BKCa channel in
human luteinized GCs was further confirmed by RT-PCR
studies that detected the BKCa ␣-subunit mRNA. The presence of BKCa channels in ovarian endocrine cells may be
phylogenetically widespread, as Asem and co-workers (22)
described a Ca2⫹-activated K⫹ channel with a typically high
single channel conductance in chicken GCs, but did not further identify it as BKCa.
We did not only reveal the activity of the BKCa channel in
human luteinized GCs, but also studied its identity and tissue distribution by immunohistochemistry and RT-PCR in
primates and rodents. The identification in the ovary is in
accordance with other studies reporting expression of BKCa
␣-subunit mRNA in human ovarian samples (29, 46). However, these studies did not exclude the possibility that the
mRNA detected could have derived from nonendocrine cells
due to the heterogeneous composition of the ovary, especially from vascular smooth muscle cells, which are known
to express BKCa channels (30). We have now used RT-PCR
and immunohistochemistry to unequivocally prove that the
BKCa channel is expressed in ovarian endocrine cells. The
BKCa antibody decorated all major types of endocrine cells in
the ovary, e.g. luteal, thecal, and, albeit to a lower degree,
follicular granulosa cells. BKCa expression in monkey GCs
harvested immediately before and after induction of ovulation and in the CL of primates and rats supports the assumption that human luteinized GCs are an adequate model
for granulosa cells of both ovulatory follicles and newly
formed CLs.
The presence of BKCa protein in ovarian endocrine cells
suggests its physiological significance in the regulation of
follicular and luteal function. Although the full spectrum of
actions of the BKCa channel is currently unknown, we report
that the BKCa channel has a crucial part in the prime endocrine function of GCs. Its blockage by IbTx caused a marked
reduction in hCG-stimulated progesterone secretion. The detailed mechanisms of how the BKCa channel affects progesterone secretion are as yet unknown. General IbTx cytotoxicity is implausible because of the apparently unchanged cell
morphology. A possible direct toxic action of IbTx on the
LH/hCG receptor is also unlikely. 8-Br-cAMP bypassed the
hCG-induced rise in cAMP and stimulated progesterone secretion as well. This stimulatory effect was attenuated in both
cases by the BKCa channel blocker, indicating that the inhibiting effect on hCG action is not caused by direct IbTx toxicity
on the LH receptor. Another possible target of potential IbTx
toxicity would be steroid biosynthesis. The key enzyme of
this catabolic pathway is steroidogenic acute regulatory protein (StAR) (47). Although we did not study this pathway in
detail, a pilot study, applying Western blot analysis, did not
reveal any reduction of StAR protein levels upon treatment
with IbTx alone or in combination with hCG (Kunz et al.,
unpublished data).
Based on our results, we assumed that opening of BKCa
channels is normally required for endocrine GC function.
5572
J Clin Endocrinol Metab, December 2002, 87(12):5566 –5574
Kunz et al. • Ovarian BKCa Channel and Steroidogenesis
FIG. 6. Induction of the BKCa current by
100 ␮M carb or 100 nM OT in the whole-cell
(A–E) or cell-attached (F and G) mode, respectively. The cells were clamped to the
actual zero current potential of each cell.
EC was applied to test for interference by
the mere approaching flow of solution. A,
Induction of a K⫹ outward current by carb,
which was unambiguously identified as
the BKCa current because it cannot by activated in the presence of IbTx. The current
traces were elicited by stepwise changes in
the test potentials in the range of ⫺100 to
100 mV (10 mV/step). B, OT causes a transient activation of the BKCa current similar to the carb effect. C, Time course of
transient BKCa current activation by carb
(100 ␮M; measured at a potential of Vh ⫽
⫹100 mV), which can be blocked by 100 nM
IbTx. D, The carb-induced BKCa activation
results in a transient hyperpolarization. E,
Time course of transient BKCa current activation by OT (measured at a potential of
Vh ⫽ ⫹100 mV). The traces in A correspond to C, whereas those in B are taken
from the same experiment as that in E. F,
Transient activation of the BKCa channel
in the cell-attached mode (Vh ⫽ ⫹140 mV)
measured before (control), briefly after (15
s), or a longer period after (50 s) the start
of carb application. The channel exhibited
a single channel conductance of gsc ⫽ 180
pS. G, Time course of the transient BKCa
activation monitored as modification of
channel open probability (NPO) in the
same experiment as that in F.
Potential physiological openers of BKCa channels might include hCG and/or steroid hormones produced by GCs.
However, we could not observe any direct activation of BKCa
currents in human GCs upon hCG stimulation. This finding
renders direct involvement of the BKCa channel in the regulation of human GC function by pituitary (LH) or placental
(hCG) hormones rather unlikely. Furthermore, the steroid
hormones 17␤-estradiol and progesterone did not activate
any BKCa currents in all of the human GCs studied with the
electrophysiological technique. In contrast to these results,
the endocrine Na⫹ channel of human GCs is regulated by
hCG (17), and likewise, some currents in nonprimate GCs
were affected by LH (11, 18, 21).
As neither hCG nor GC-derived steroids act as BKCa openers, we examined the possibility that neuroendocrine substances released from GCs could be involved, namely ACh
and OT. Indeed, we found that one molecular key event
associated with MR activation is opening of BKCa channels.
This is probably triggered by the well known rise in the
intracellular Ca2⫹ concentration elucidated by activation of
MRs (24, 25). Similarly, OT is a locally produced signaling
peptide that increases intracellular Ca2⫹ levels (8, 26) and, as
Kunz et al. • Ovarian BKCa Channel and Steroidogenesis
J Clin Endocrinol Metab, December 2002, 87(12):5566 –5574 5573
cluding ACh and OT. Thus, for the first time we demonstrate
the cooperation of a gonadotropin, an ion channel, and
local neuroendocrine factors in the control of ovarian
steroidogenesis.
Acknowledgments
We gratefully acknowledge technical help by R. Rämsch, A. Krieger,
M. Rauchfuss, B. Zschiesche, A. Mauermayer, and G. Prechtner. We also
thank C. Heiss (Klinik am Eichert, Göppingen, Germany) for providing
human ovaries, and D. M. Stocco (Texas Tech University Health Sciences
Center, Lubbock, TX) for StAR antiserum.
Received May 30, 2002. Accepted August 26, 2002.
Address all correspondence and requests for reprints to: Dr. Lars
Kunz, Anatomical Institute, University of Munich, Biedersteiner Strasse
29, D-80802 Munich, Germany. E-mail: [email protected].
This work was supported by Deutsche Forschungsgemeinschaft (Ma
1080/12), Volkswagenstiftung, and the NIH (Grants RR-00163 and
HD-24870).
References
FIG. 7. Steroid hormones and hCG do not directly activate BKCa
currents. The steady state current at a holding potential of Vh ⫽ ⫹100
mV did not show any change upon application of 10 IU/ml hCG (A; n ⫽
15) or 1 ␮M progesterone (B; n ⫽ 22). In some experiments the presence of functional BKCa channels was proven by preceding or subsequent application of either 1 nM OT or 100 ␮M carb.
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K channel, the BKCa, acts together with local signaling
molecules to mediate the stimulatory action of hCG on
steroidogenesis in GCs. The mechanism of action involves
activation of the channel by GC-derived substances, in-
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