| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Article |
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
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: lars.kunz{at}lrz.uni-muenchen.de.
Abstract
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 Ca2+-activated K+ 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 steroidogenesis. 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 17ß-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 Ca2+ 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.
FORMATION, ENDOCRINE FUNCTION, and regression of the corpus luteum (CL) are processes crucial for normal reproduction (1, 2, 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, 6, 7, 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 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, 17, 18, 19, 20, 21, 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, 25, 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 ß-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, 33, 34, 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 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 17ß-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 µM 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 17ß-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 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 µl; 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-µm 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.
|
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.
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 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.
|
-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 to the published sequence data (see Materials and Methods for databank accession number).
|
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 investigated 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).
|
|
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 17ß-estradiol and progesterone were investigated in the whole-cell configuration. Carb (100 µM) 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 carb-induced 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 µM) was also shown (Fig. 6, G and F).
|
). 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 current or membrane zero current potential (n = 15). This was observed in cell preparations yielding OT- or carb-sensitive cells and even in the very cells that did respond to OT before or after hCG application (n = 3; Fig. 7A). The steroid hormones progesterone (1 µM; n = 22; Fig. 7B) and 17ß-estradiol (1 µM; n = 9; data not shown) also showed no activation of BKCa currents, even in cells exhibiting BKCa currents induced by subsequent application of 100 µM carb (Fig. 7B). The steroid solutions (17ß-estradiol and progesterone) contained 0.01% DMSO, which did not cause any electrophysiological effects (not shown).
|
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 permeating ion (11, 18, 19, 20, 21, 22, 43, 44, 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 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. 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 17ß-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 we found, activates BKCa channels in human GCs. The transient character of BKCa activation probably reflects the short-lasting Ca2+ rise observed in MR and OT receptor activation (24, 25). Granulosa cells are part of intraovarian signaling systems, as they are both a source of and a target for locally produced ACh and OT (2, 6, 7, 8). Cultured human luteinized GCs share these characteristics with granulosa-luteal cells. They produce ACh and OT and also possess functional receptors for these intraovarian signaling factors (9, 25, 38). The release of OT can be increased by hCG/LH (48), and a similar stimulatory action may be assumed for ACh release as well. OT is well known to promote progesterone production in GCs (26, 49), whereas the results are controversial in the case of ACh. We did not observe any muscarinic effect on steroidogenesis, but Kornya et al. (50) reported such a stimulatory action in serum-free medium.
In summary, we provide evidence that a Ca2+-activated 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, including 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.
Footnotes
This work was supported by Deutsche Forschungsgemeinschaft (Ma 1080/12), Volkswagenstiftung, and the NIH (Grants RR-00163 and HD-24870).
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.
Received May 30, 2002.
Accepted August 26, 2002.
References
-adrenergic receptor. J Clin Endocrinol Metab 76:367–373[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  M. Greiner, A. Paredes, V. Rey-Ares, S. Saller, A. Mayerhofer, and H. E. Lara Catecholamine Uptake, Storage, and Regulated Release by Ovarian Granulosa Cells Endocrinology, October 1, 2008; 149(10): 4988 - 4996. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Olivero, E. Leiva-Salcedo, L. Devoto, and A. Stutzin Activation of Cl- Channels by Human Chorionic Gonadotropin in Luteinized Granulosa Cells of the Human Ovary Modulates Progesterone Biosynthesis Endocrinology, September 1, 2008; 149(9): 4680 - 4687. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Kunz, J. S. Richter, and A. Mayerhofer The Adenosine 5'-Triphosphate-Sensitive Potassium Channel in Endocrine Cells of the Human Ovary: Role in Membrane Potential Generation and Steroidogenesis J. Clin. Endocrinol. Metab., May 1, 2006; 91(5): 1950 - 1955. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Wu, W. G Foster, and E. V Younglai Rapid effects of pesticides on human granulosa-lutein cells Reproduction, February 1, 2006; 131(2): 299 - 310. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Platano, M. C. Magli, A. P. Ferraretti, L. Gianaroli, and G. Aicardi L- and T-Type Voltage-Gated Ca2+ Channels in Human Granulosa Cells: Functional Characterization and Cholinergic Regulation J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 2192 - 2197. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Agoston, L. Kunz, A. Krieger, and A. Mayerhofer Two Types of Calcium Channels in Human Ovarian Endocrine Cells: Involvement in Steroidogenesis J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4503 - 4512. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Trkulja, V. Crljen-Manestar, H. Banfic, and Z. Lackovic Involvement of the Peripheral Cholinergic Muscarinic System in the Compensatory Ovarian Hypertrophy in the Rat Experimental Biology and Medicine, September 1, 2004; 229(8): 793 - 805. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. V. Younglai, A. C. Holloway, G. E. Lim, and W. G. Foster Synergistic effects between FSH and 1,1-dichloro-2,2-bis(P-chlorophenyl)ethylene (P,P'-DDE) on human granulosa cell aromatase activity Hum. Reprod., May 1, 2004; 19(5): 1089 - 1093. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-N. Wu, S.-I. Liu, and M.-H. Huang Cilostazol, an Inhibitor of Type 3 Phosphodiesterase, Stimulates Large-Conductance, Calcium-Activated Potassium Channels in Pituitary GH3 Cells and Pheochromocytoma PC12 Cells Endocrinology, March 1, 2004; 145(3): 1175 - 1184. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
