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Endocrinology Vol. 147, No. 12 5798-5805
Copyright © 2006 by The Endocrine Society

Estradiol Inhibition of Voltage-Activated and Gonadotropin-Releasing Hormone-Induced Currents in Mouse Gonadotrophs

Dennis W. Waring and Judith L. Turgeon

Division of Endocrinology, Department of Medicine, School of Medicine, University of California, Davis, Davis, California 95616

Address all correspondence and requests for reprints to: Dennis W. Waring, Ph.D., Division of Endocrinology, Department of Medicine, University of California, One Shields Avenue, Davis, California 95616. E-mail: dwwaring{at}ucdavis.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We report the first study of voltage-activated and GnRH-induced plasma membrane currents and their modulation by estradiol (E2) in mouse gonadotrophs. In consideration of the pleiotropic effects of E2 on gonadotrophin secretion and the relationship between plasma membrane electrical excitability and secretion, our objective was to determine the role of E2 in modulating gonadotroph plasma membrane currents. We measured total voltage-activated and GnRH-induced currents using the perforated-patch configuration of the patch-clamp technique, which preserves signaling pathways, including GnRH-induced Ca2+ oscillations. We show that female mouse gonadotrophs are similar to those from other species in that the voltage-activated net current response exhibits an inward fast activating current that is inhibited by tetrodotoxin, which is characteristic of a Na+ current, and a larger magnitude outward current with a profile suggesting the presence of multiple K+ currents. Furthermore, in voltage-clamped mouse gonadotrophs, GnRH activates large amplitude current oscillations that are apamin sensitive and have a reversal potential of –90 mV, consistent with Ca2+-activated K+ currents. Significantly, E2 pretreatment for 2–5 d decreased the density of both the peak outward voltage-activated current and the peak GnRH-induced current. The specific linkage between the observed E2 effects on membrane currents and, ultimately, gonadotroph function remains to be established. However, because decreased K+ current density is associated with an increase in membrane electrical excitability, we postulate increased excitability is one of the modes of action of E2 in sensitizing the gonadotroph to GnRH, an event central to the regulation of cyclic gonadotrophin secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GONADOTROPHS OF THE anterior pituitary gland secrete the gonadotrophic hormones LH and FSH under the regulatory control of the hypothalamic hormone GnRH and the modulatory control of the steroid hormones estradiol-17ß (E2), progesterone, and testosterone. Through their receptors, steroid hormones affect gonadotroph function at many levels and on several time scales, i.e. receptor regulation, hormone production, and secretion modulation (1, 2).

Gonadotrophin secretion and plasma membrane electrical excitability are coupled through the GnRH-induced increase in intracellular [Ca2+], which is central to GnRH stimulated exocytosis. In response to GnRH, Ca2+ is released into the cytoplasm from intracellular Ca2+ stores and also enters from the extracellular space through voltage activated Ca2+ channels. Due to the characteristics of the Ca2+ release and reuptake mechanisms, the GnRH-stimulated increase in intracellular Ca2+ is inherently oscillatory. In turn, the intracellular Ca2+ oscillations drive oscillations in plasma membrane voltage (Vm) through cyclic activation of small conductance Ca2+-activated K+ channels (SK channels). The hyperpolarizing phase of these Vm oscillations removes inactivation from voltage-activated channels that are responsible, during the depolarizing phase, for action potential generation (reviewed in Ref. 3).

Gonadotroph electrophysiology primarily has been studied in rat (3) and sheep (4) anterior pituitary cells and the mouse-derived gonadotroph clonal cell line, {alpha}T3–1 (5, 6, 7, 8). Although E2 is known to modulate ion channel activity in electrically excitable cells, direct determination of E2 effects on membrane currents in gonadotrophs has received relatively little attention. In sheep gonadotrophs, E2 has been reported to increase voltage-activated Ca2+ and K+ currents (4). In other excitable cells, E2 has been found to increase electrical excitability by decreasing voltage-activated K+ currents. For example, in hypothalamic GnRH neurons, E2 decreases current densities for both an inactivating and a noninactivating voltage-activated K+ current (9), and in smooth muscle E2 decreased an inactivating voltage-activated K+ current but not a noninactivating one (10). E2 also can modulate SK channels, e.g. in the female guinea pig hypothalamus E2 increased SK3 mRNA, a SK channel subtype (11). The effect of E2 on gonadotroph SK channels has not been examined.

Genetically manipulated mice increasingly are used in studies of anterior pituitary cell function. For GnRH signaling and gonadotrophin synthesis and secretion, data primarily have been obtained from murine-derived clonal cell lines (e.g. Refs. 12, 13, 14, 15, 16, 17, 18); a smaller number of studies have used mouse pituitary gland tissue or primary cell cultures to examine LH secretion or LH synthesis (19, 20, 21, 22, 23, 24). We are aware of no published reports of the study of mouse gonadotroph electrophysiology. For electrophysiological studies, the mouse and rat present similar challenges: in both, the anterior pituitary gland has a low number of gonadotrophs (≤10%) relative to the other secretory cell types (25, 26, 27). Because of the pleiotropic effects of E2 on gonadotrophin secretion and the relationship between secretion and electrical excitability, the objective of this study was to determine the role of E2 in the modulation of female mouse gonadotroph plasma membrane currents by measuring the total voltage-activated and GnRH-induced membrane current (Im) in single gonadotrophs from ovariectomized mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Media, trypsin/EDTA, and sera for cell culture were obtained from Invitrogen-GIBCO (Grand Island, NY). Kanamycin sulfate, BSA fraction V, dimethyl sulfoxide (DMSO), amphotericin B, apamin, tribromoethanol, E2, and GnRH were from Sigma-Aldrich (St. Louis, MO). HEPES-free acid and tetrodotoxin were from Calbiochem (San Diego, CA), buprenorphine hydrochloride was from Cardinal Health (Elk Grove, CA), and R6010 was from K. R. Anderson (Santa Clara, CA). General chemicals were from either Sigma-Aldrich or Fisher Scientific (Pittsburgh, PA).

Animals and cell culture
The surgical protocol was reviewed and approved by the University of California, Davis, Institutional Animal Care and Use Committee. The anesthetic tribromoethanol was prepared as a stock solution in amyl alcohol and diluted in PBS on the day of use. Adult female wild-type mice [C57/6/129sv hybrid as described (28)] were maintained in controlled light conditions (12 h light, 12 h dark). Mice were ovariectomized under ip tribromoethanol anesthesia and treated with sc buprenorphine (0.1 mg kg–1) as analgesic in the immediate postoperative period and maintained for 2 wk after ovariectomy before use.

Pituitary glands were removed after CO2 narcosis and decapitation. Anterior pituitary tissue was enzymatically dispersed (trypsin/EDTA) and prepared for cell culture as described (29). Cells were plated in 35 x 10 mm dishes incorporating a 12-mm glass-bottomed well (Bioscience Tools, San Diego, CA) at low density (15,000–20,000 cells/well) and flooded with MEM (Glutamax, Earl’s salts) supplemented with 200 µM kanamycin sulfate, 10% fetal bovine serum that had been charcoal treated to removed endogenous steroids (30), and with or without 0.2 nM E2 (prepared as a stock solution in ethanol). Residual E2 concentration in the charcoal-treated serum was below the detection limit of the RIA, and thus, the final concentration was less than 1 pM. Pituitary cells were maintained in a humidified atmosphere (37 C) of 5% CO2 in air and used on d 2–5 (day of plating = d 0). As reported by Stutzin et al. (31), gonadotrophs in cultures obtained from ovariectomized rats are readily identified on morphological criteria. We used similar morphological criteria in the present study. Putative mouse gonadotrophs so identified were subsequently challenged with GnRH. Only data from GnRH-responsive cells are reported. In this study 58% of tested mouse anterior pituitary cells responded to 1 nM GnRH. This is considerably lower than our success rate for rat anterior pituitary cells under similar experimental conditions (>95%, data not shown). The reduced success in identification of mouse gonadotrophs was due to their less distinctive and more variable morphology compared with rat gonadotrophs.

Experimental procedure
For experiments, culture medium was replaced with an extracellular (EC) medium, without added E2, consisting of (in mM): 135 NaCl, 5 KCl, 5 CaCl2, 1.3 MgCl2, 4 NaHCO3, 5 glucose, 10 HEPES, 1 mg ml–1 BSA, and was adjusted to pH 7.4 with 5 N NaOH. Dishes with attached cells were transferred to an inverted microscope with a temperature regulated stage maintained at 22 C and superfused with EC medium using a peristaltic pump (~1 ml min–1). After current voltage (I-V) curve data were acquired, the cells were voltage clamped at the indicated potential and solution flow was switched between control EC medium and GnRH-containing EC medium using a valve placed close to the microscope stage. GnRH, 1 nM, was applied to the cells for 2 min. The presence of GnRH-activated membrane current in voltage clamped cells was taken as confirmation that the cell was a gonadotroph.

Electrophysiology.
Electrophysiological measurements were carried out using the perforated-patch configuration of the tight-seal patch-clamp recording technique (32). The pipette filling solution consisted of (in mM): 130 KAspartate, 20 KCl, 10 NaCl, 5 MgSO4, 10 HEPES, and adjusted to pH 7.2 with 5 N KOH. Perforation of the patch was achieved by inclusion of the pore-forming antibiotic amphotericin B. The antibiotic stock solution, 10 mg/ml in dry DMSO, was prepared fresh each day. This stock (25 µl) was mixed with 20% pluronic F-127 in DMSO (2.5 µl) and then added to the pipette solution (5 ml) for a final concentration of 50 µg amphotericin B per milliliter (0.02% pluronic F-127, 0.6% DMSO). This solution was used to backfill the pipette, the tip of which had been filled with antibiotic-free pipette medium by gentle suction. Pipettes were pulled from Corning pyrex 7740 glass (Garner Glass, Claremont, CA) and coated with R6010 to minimize the capacitance between pipette and bath; the remaining capacitance was compensated electronically. Pipettes had resistances of 5–9 M{Omega} (7.0 ± 0.1, n = 37) and attained an average access resistance of 26 ± 2 M{Omega} (n = 37); series resistance compensation was not used.

The pipette potential (Vp) was corrected for junction (13.6 mV) and Donnan (–2.8 mV) potentials as shown in the equation to give the reported Vm. The values for the correction were calculated using the junction potential calculator in Clampex (pClamp software suite; Axon Instruments/Molecular Devices, Sunnyvale, CA) together with a separate estimate of the Donnan potential (33). The latter was calculated assuming that intracellular impermeant/immobile ion concentration = 163 mM (33) and that for amphotericin B perforated patches, only monovalent ions are permeant.

Formula
Recordings were made with an Axopatch 1C patch-clamp amplifier via a DigiData 1320A computer interface using Clampex. Data were filtered at 5 kHz and sampled at 51 kHz. I-V curve acquisition used a P/4 protocol to subtract linear capacitance and leak currents. The P/4 protocol used a holding potential of –91 mV, which for the I-V pulse range of –106 to +74 mV, gave a P/4 voltage range of –118 to –73 mV. The latter value is well below the Vm at which activated current was observed. GnRH-induced currents were recorded from cells voltage clamped at –50, –66, or –116 mV. Reported currents were normalized to membrane capacitance (picoampere/picofarad) to correct for differences in cell size; there was no difference in membrane capacitance between the control and E2-pretreated cells [control: 7.20 ± 0.24 pF (n = 20), E2: 8.17 ± 0.48 pF (n = 17), P = 0.07]. These values are somewhat less than the mean membrane capacitance we reported previously for rat gonadotrophs under similar experimental conditions, 9.0 pF (34).

Data analysis
Voltage-activated current.
To compare the data from control and E2-pretreated cells, I-V curves were generated from three regions of the Im response for each cell using Clampfit (pClamp software suite). The I-V curve for the peak inward inactivating current was obtained from the negative peak between 5 and 8 msec using five smoothing points. The curve for peak outward current was obtained from the positive peak between 5 and 8 msec, again, with five smoothing points. Because of the activation profiles of these two currents, it was possible to separate them over part but not all of the voltage range of the pulse protocol. Finally, the sustained portion of the outward current was evaluated near the end of the voltage pulse at 102 msec by averaging over a 3-msec period. Curve fitting of voltage activated currents used Clampfit for single exponential fits to peak outward current and Origin (OriginLab Corp., Northampton, MA) to fit Boltzmann equations to inward current data to obtain an estimate of the Vm at half-activation.

GnRH-induced current.
Peak current during GnRH application was determined on records that had been filtered at 100 Hz after acquisition; the current values over approximately 500 msec centered on the peak value were averaged to obtain the peak current response for each cell.

Statistical analyses.
Differences in the results between control and E2-pretreated cells were analyzed with the Wilcoxon rank-sum test using S-Plus (Insightful Corp., Seattle, WA). The differences in slopes and reversal potentials for the GnRH-induced current I-V curves were determined using the linear regression model in S-Plus. Data are presented as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To evaluate a potential role for E2 in the modulation of gonadotroph electrical excitability, we examined, in control and E2-pretreated cells under voltage clamp, the change in net membrane current, Im, elicited by step changes in Vm or in response to GnRH.

Voltage-activated membrane current
Im elicited under voltage-clamp in response to test voltage pulses is shown in Fig. 1Go. Based on the current responses, we considered three aspects of Im as shown in Fig. 1Go, B and C: a transient inward current, an outward current that partially inactivated, and a sustained outward current.


Figure 1
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  		FIG. 1. Voltage-activated Im in mouse gonadotrophs. A, Pulse protocol. From a holding potential of –66 mV and after a 211-msec prepulse to –116 mV, Im traces were elicited by 105-msec voltage steps from –106 to +74 mV in 5-mV increments. B, Averaged currents for control [gray traces (n = 20)] and E2-pretreated gonadotrophs [red traces (n = 17)]. At the beginning and end of each test pulse, a 100- to 900-µsec section has been blanked to remove capacitance transient artifacts from records not completely compensated by the P/4 protocol. C, Im response on an expanded time scale to show the inward current time-course. D, I-V plots of the sustained outward Im from the indicated region of the current response. Values were obtained by averaging the response over 3 msec. E, I-V plots of the peak inward Im (peak negative current). F, I-V plots of the peak outward Im (peak positive current).

 
The peak inward and outward currents were partially separable because of their different activation profiles. The mean peak inward Im occurred at –11 mV (Fig. 1EGo). Maximum outward Im, peak and sustained, occurred at the most depolarized potential tested, +74 mV (Fig. 1Go, D and F).

The peak of the transient component of the outward current was dramatically and significantly inhibited by E2 pretreatment (Fig. 1Go, B and F, and Table 1Go). However, E2 pretreatment did not affect the peak of the inward current (Fig. 1Go, C and E, and Table 1Go). The sustained component of the outward current, measured at the end of the test pulse, was not different between control and E2-pretreated cells (Fig. 1Go, B and D, and Table 1Go).


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  		TABLE 1. Voltage-activated membrane currents in mouse gonadotrophs

 
The I-V curve for the peak inward Im (Fig. 1EGo) suggests a possible difference in the activation profile between the control and E2-pretreated cells. We further examined this over a selected voltage range, –106 to +6 mV, during which the outward current was minimally activated (Fig. 1Go, D and F). For each cell we calculated the fractional inward current (the peak value at each potential divided by the maximum peak value) and fit Boltzmann curves to the fractional inward current; the averaged values are plotted in Fig. 2Go. The half-maximal values for each cell in the control or E2-pretreated groups were averaged as shown in Table 2Go. In the E2-pretreated group, the inward current reached its half-maximal value at a significantly more negative potential, compared with control (Table 2Go and Fig. 2Go). Importantly, the activation of the inward current below half-maximal for both control and E2-pretreated groups occurred at test potentials for which outward current was low or not detectable (Fig 1Go, D and F).


Figure 2
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  		FIG. 2. Inward Im activation. Fractional inward current (mean ± SEM): dotted and solid curves represent the fit of the Boltzmann equation to the control and E2-pretreated data, respectively. The vertical lines indicate the significantly different Vm at which the control and E2-pretreated inward currents were half maximal.

 

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  		TABLE 2. Mouse gonadotroph membrane current characteristics

 
To further examine the effect of E2 pretreatment on the peak outward current, we fit single exponential curves to the outward current between the half-maximal and peak values for each cell over a selected voltage range +54 to +74 mV, the region in which the Im data for control and E2-pretreated cells diverged (Fig. 1FGo). The averages of the time constants ({tau}) for the exponential fits at each voltage step are plotted in Fig. 3AGo. Fig. 3BGo shows the distribution of the individual {tau} for all voltage steps and demonstrates that the larger variation about the mean for the E2-pretreated cells, compared with control cells, is due to the presence of cells with a large {tau}. For the outward Im, there are two populations of cells evident for E2-pretreated cells. In one the outward Im is biphasic, an initial peak followed by a decline to a plateau or sustained current. The second exhibits only the plateau or sustained component. A distinguishing characteristic of these two populations is that {tau} for the single exponential fit is much slower, more than 1 msec, for the group exhibiting only the plateau component. The value of {tau} is less than 1 msec for cells exhibiting biphasic outward Im (Fig. 3BGo, inset).


Figure 3
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  		FIG. 3. Peak outward Im {tau}. A, Average {tau} (±SEM) at the indicated Vm for control (n = 20) and E2-pretreated gonadotrophs (n = 17). B, Histogram showing {tau} distribution for control and E2-pretreated data without regard to Vm. Inset, Representative control (top trace) and E2-pretreated (lower two traces) traces on an expanded scale. The bottom trace shows outward Im from an E2-pretreated gonadotroph in which the inactivating peak outward Im is absent, revealing the slow rate of activation for the remaining noninactivating Im.

 
Figure 4Go shows initial data for further identification of these voltage-activated currents. We examined the fast inward Im using the voltage-activated Na+ channel blocker tetrodotoxin. As shown in A2 and B2 of Fig. 4Go, tetrodotoxin inhibited the fast inward Im; A3 and B3 show the current obtained by subtracting the record acquired in the presence of tetrodotoxin from the control record to yield the tetrodotoxin inhibitable Na+ current (INa). Column B in Fig. 4Go shows the results of addition of tetraethylammonium-Cl to the EC medium and substitution of Cs+ for K+ in the pipette medium, conditions that block voltage-activated K+ channels. The absence of outward Im (Fig. 4Go, B1 and B2) identifies the outward Im as K+ current (IK).


Figure 4
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  		FIG. 4. Voltage-activated current isolation. Perforated-patch recordings from gonadotrophs; the P/4 pulse protocol was not used in these experiments. A, Panels in this column show data from a cell bathed in EC medium using a pipette solution with KCl (45 mM) and K2SO4 (75 mM). B, Panels in this column show data from a cell bathed in EC medium containing 10 mM tetraethylammonium-Cl (substitution for equimolar NaCl) using a pipette solution in which CsCl (62 mM) and Cs2SO4 (60 mM) replaced the K+ salts. 1, Panels in this row show data representing pretreatment control I-V acquisitions. 2, Panels in this row show data obtained after 2 min treatment of the cells with 1 µM tetrodotoxin. 3, Panels in this row were obtained by subtracting the corresponding data records in row 2 from row 1; the panels in this row, therefore, show the tetrodotoxin inhibitable current, INa.

 
GnRH-induced membrane current
For GnRH-induced current, the response to 1 nM GnRH was examined at membrane holding potentials of –50, –66, or –116 mV. Administration of 1 nM GnRH to voltage clamped cells elicited a spectrum of Im responses: oscillations from the baseline, plateau currents, and oscillations on a plateau current. A given cell could exhibit one to all of these patterns (Fig. 5Go). The current was markedly reduced in the presence of the bee venom toxin apamin (Fig. 5GGo), indicating that SK channels contribute to the GnRH-induced current. The current remaining in the presence of apamin was not examined further. To assess the effect of E2-pretreatment, we determined the peak current achieved during the time of GnRH exposure for each cell. The peak current I-V over the voltage range examined was linear with a reversal potential of approximately –90 mV, which is similar to the calculated K+ reversal potential of –88 mV (Fig. 5FGo). The slope of the response for E2-pretreated cells was significantly less than that for control cells (Table 2Go and Fig. 5FGo), indicating that E2 pretreatment decreased the density of the peak GnRH-induced current in gonadotrophs. Additionally, the estimated reversal potential was not altered by E2 pretreatment (Table 2Go).


Figure 5
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  		FIG. 5. GnRH-induced Im in mouse gonadotrophs. A–E, Representative current traces showing the response to 1 nM GnRH, 2 min, indicated by the horizontal black line. For presentation, the number of data points has been reduced by a factor of 100. The voltage clamp potential is shown next to each trace. Control, gray traces; E2-pretreated, red traces. F, I-V curves for the peak Im during GnRH exposure. G, Upper trace, Response to 1 nM GnRH, 2 min (horizontal black line), at the indicated voltage clamp potential; lower trace, response of the same cell to another 2 min exposure to GnRH but in the presence of 1 µM apamin (dark green line). The responses in G are shown on an expanded scale; for both traces the start of GnRH exposure is not shown. Apamin exposure was started 4 min before initiation of GnRH. For presentation the number of data points has been reduced by averaging 200 point segments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here the first study of voltage-activated and GnRH-induced plasma membrane currents, Im, and the modulation of Im by E2 in the female mouse gonadotroph. This also is the first report of an effect of E2 on GnRH-induced Im. The objective of this study was to assess the effect of E2 on Im under conditions that optimally preserve intracellular integrity. Consequently, we used the perforated-patch configuration of the patch-clamp technique, which limits diffusional exchange between the patch pipette and the cell interior to monovalent ions and therefore preserves signaling pathways, including GnRH-induced Ca2+ oscillations. We chose to examine total net current to allow assessment of the effect of E2 on multiple potential targets. In the species and cell lines studied previously, gonadotrophs have been found to express Na+, K+, and Ca2+ voltage-activated plasma membrane ionic currents as well as Ca2+-activated K+ currents. Each of these currents is linked in varying degrees to gonadotroph electrical excitability, gonadotrophin secretion, or both (reviewed in Ref. 3).

We show here that mouse gonadotrophs are similar to those from other species in that the voltage-activated net current response exhibits characteristics of an inward fast activating INa and a larger magnitude outward current with a profile that suggests the presence of multiple IK. Furthermore, as shown in other species, we find in voltage-clamped mouse gonadotrophs that GnRH activates large amplitude current oscillations consistent with a Ca2+-activated K+ current, SK. Significantly, we found that E2 pretreatment of mouse gonadotrophs affected both the voltage-activated Im and the GnRH-induced Im.

Based on our results and previous studies in gonadotrophs, the voltage-activated outward currents we observe in mouse gonadotrophs can be tentatively identified as K+ currents. Inhibition of the inactivating outward current by E2 was dramatic. Analysis of the kinetics of activation of this current revealed that E2 shifted the distribution of rate constants to larger values. Because E2 did not affect the sustained outward current, we interpret these results as indicating that E2 inhibits an inactivating IK revealing, to varying degrees, the slower time constant for activation of a different, sustained IK. The slower rate of activation of the sustained current is evident in records in which the inactivating current is absent. For gonadotrophs, the effect of E2 on IK has been previously reported only in sheep, which exhibit only a sustained delayed rectifier IK; E2 was found to increase this current (35). The apparently different IK targets for E2 modulation in mouse and ovine gonadotrophs could be the result of experimental or species differences, but a resolution requires as a minimum identification of the K+ channel types responsible for the seemingly more complex outward Im profile in the mouse. In other systems E2 has been shown to decrease IK. There is considerable evidence that E2 increases neuronal and smooth muscle excitability by decreasing voltage-activated K+ current density (e.g. Refs. 9, 10). Our results suggest that, as in neurons and smooth muscle, an increase in excitability could result from E2 inhibition of IK in the mouse gonadotroph.

The fast activating inward Im in mouse gonadotrophs is tentatively identified as a voltage-activated Na+ current, INa, based on electrophysiological and pharmacological characteristics. For this presumptive INa, the Vm for half-maximal activation of E2-pretreated cells was shifted to the left indicating this current would activate at more negative Vm. Thus, in E2-pretreated cells, Na+-dependent action potentials would be elicited sooner, i.e. at a more negative potential, during Vm depolarization. Examination of the effect of E2 on INa has been reported only in ovine gonadotrophs, and E2 was found to be without effect (35). More generally, the effect of E2 on INa has been little studied, and there are few reports of E2 affecting this current. However, E2 was found to increase INa density in rat cortical neurons in primary culture (36). Examination of isolated currents will be required to establish that the inward current affected by E2 is a Na+ current and, thus, if there is a difference between mouse and sheep gonadotrophs in the E2 regulation of INa.

To analyze the GnRH-induced current, we generated I-V curves for control and E2-pretreated cells by recording the Im response to GnRH at three separate holding potentials. Each of the resulting I-V curves had a reversal potential near the K+ reversal potential, indicating that the GnRH-induced current is carried predominantly by K+. Furthermore, the slopes of the control and E2-pretreated curves were significantly different, indicating that E2 decreased K+ current density. Because these cells were voltage clamped, the current response cannot be attributed to a voltage-activated current and is, most reasonably, the result of Ca2+-activated K+ channel activation in response to GnRH-induced release of Ca2+ from intracellular stores. The observation that the reversal potential was not affected indicates that the relative contribution of IK to the GnRH-induced current was not altered by E2. Whereas the identity of the channel subtype(s) involved remains to be determined, we have tentatively identified SK channels (inhibition by apamin) as responsible for a major portion of the current. SK channels are responsible for similar GnRH-induced current oscillations in rat gonadotrophs (3). An effect of E2 on GnRH-induced current has not been reported. It is unlikely that the observed inhibition of GnRH-induced current is secondary to an E2-induced decrease in GnRH receptors. Although E2 can lead to a modest reduction in GnRH receptor number in the mouse-derived {alpha}T3–1 gonadotroph cell line (37), E2 has a slight stimulatory effect on GnRH receptor expression in the more differentiated mouse gonadotroph line LßT2 (12, 38) and is reported to increase GnRH receptor number in gonadotrophs from mice (37) and other species (4, 39, 40, 41).

The relative expression of ion channels and their kinetic behavior determine membrane excitability, and K+ channels are central in this regard. A decrease in K+ channel activity is generally associated with an increase in membrane excitability (42, 43, 44). Taken together the results reported here suggest that E2 pretreatment leads to modification of ion channel activity with a resulting increase in the electrical excitability of the gonadotroph. In the absence of GnRH, this could be manifest as a depolarization of the baseline Vm, an increase in action potential firing, or both. In the presence of GnRH, the E2-induced reduction in Ca2+-activated IK could lead to a decrease in the magnitude of Im oscillations. Whether E2-induced inhibition of plasma membrane ionic currents impacts GnRH-induced exocytosis remains to be determined. Interestingly, in single gonadotrophs in which exocytosis was measured as a change in membrane capacitance, i.e. a change in membrane surface area, E2 was found to augment secretion: in rat gonadotrophs E2 pretreatment increased both GnRH- and K+ depolarization-stimulated single-cell exocytosis (34), whereas in single LßT2 cells, only GnRH-stimulated exocytosis was augmented (45).

We have shown that E2 pretreatment inhibits both voltage-activated and GnRH-induced currents in female mouse gonadotrophs. Although additional experiments will be required to unequivocally establish the ion channels and currents modulated by E2, the experimental conditions we used as well as published data support the hypotheses that the outward currents are K+ currents, that the inward current is a Na+ current, and that GnRH activates a Ca2+-sensitive K+ current. Whether the regulation of IK by E2 is through down-regulation of K+ channel expression or via signaling pathways is to be determined. In electrically excitable cells, inhibition of IK is linked to increased plasma membrane excitability. For the gonadotroph, it will be important to establish the specific association between the observed E2 effects on membrane currents and excitability and, ultimately, gonadotroph function. However, we postulate that increased excitability is one of the modes of action of E2 in sensitizing the gonadotroph to GnRH, an event central to the regulation of cyclic gonadotrophin secretion.


   Acknowledgments
 
We thank Drs. A. Formina and N. Chiamvimonvat for their helpful comments and criticisms on a draft of this manuscript.


   Footnotes
 
This work was supported by National Institutes of Health Grant HD12137.

Disclosure statement: the authors have nothing to disclose.

First Published Online August 31, 2006

Abbreviations: DMSO, Dimethyl sulfoxide; E2, estradiol; EC, extracellular; Im, membrane current; IK, K+ current; INa, Na+ current; SK, small conductance Ca2+-activated K+; {tau}, time constant; Vm, plasma membrane voltage.

Received August 14, 2006.

Accepted for publication August 18, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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S. Wen, J. R. Schwarz, D. Niculescu, C. Dinu, C. K. Bauer, W. Hirdes, and U. Boehm
Functional Characterization of Genetically Labeled Gonadotropes
Endocrinology, June 1, 2008; 149(6): 2701 - 2711.
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