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High Expression of the R-Type Voltage-Gated Ca²⁺ Channel and Its Involvement in Ca²⁺-Dependent Gonadotropin-Releasing Hormone Release in GT1–7 Cells

Department of Physiology, Nippon Medical School, Tokyo 113-8602, Japan

Address all correspondence and requests for reprints to: Dr. Masakatsu Kato, Department of Physiology, Nippon Medical School, Sendagi 1, Bunkyo, Tokyo 113-8602 Japan. E-mail: address: mkato{at}nms.ac.jp.

	Abstract

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The GT1 cell has been widely used as a model cell to study cellular functions of GnRH neurons. Despite the importance of Ca²⁺ channels, little is known except for L- and T-type Ca²⁺ channels in GT1 cells. Therefore, we studied the diversity of voltage-gated Ca²⁺ channels in GT1–7 cells with perforated-patch clamp and RT-PCR. An R-type Ca²⁺ channel blocker, SNX-482, inhibited the Ca²⁺ currents by 75.6% in all cells examined (n = 9). A T-type Ca²⁺ channel blocker, Ni²⁺, inhibited the Ca²⁺ currents by 12.6% in all cells examined (n = 9). An L-type Ca²⁺ channel blocker, nimodipine, inhibited the Ca²⁺ currents by 17.9% in five of 11 cells examined. When using Ba²⁺ as a charge carrier, another dihydropyridine antagonist, nifedipine, clearly inhibited the currents by 12.1% in all cells examined (n = 16). An N-type Ca²⁺ channel blocker, -conotoxin-GVIA, inhibited the Ca²⁺ currents by 13.8% in three of 20 cells examined. A P/Q type Ca²⁺ channel blocker, -agatoxin-IVA, had no effect on the currents (n = 9). RT-PCR revealed that GT1–7 cells expressed the _1B, _1D, _1E, and _1H subunit mRNA. Furthermore, SNX-482 and nifedipine inhibited the high K⁺-induced increase in the intracellular Ca²⁺ concentration and GnRH release. -Conotoxin-GVIA and -agatoxin-IVA had no effect. These results suggest that GT1–7 cells express R-, L-, N-, and T-type voltage-gated Ca²⁺ channels; the R-type was a major current component, and the L-, N-, and T-types were minor ones. The R- and L-type Ca²⁺ channels play a critical role in the regulation of Ca²⁺-dependent GnRH release.

	Introduction

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THE GnRH NEUROSECRETORY system constitutes the final common pathway in the neuroendocrine control of reproduction. Because the hypothalamus contains a relatively small number of GnRH neurons that are diffusely scattered, a considerable amount of work has been carried out with the immortalized GnRH neuronal cell line (GT1 cell) generated by genetically targeted tumorigenesis in transgenic mice (1). The GT1 cells are thought to preserve many characteristics of the native GnRH neurons. They generate spontaneous action potentials, exhibit transient oscillations of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) (2, 3), and secrete GnRH in a pulsatile manner (4, 5). Therefore, GT1 cells are still useful, especially in biochemical and molecular biology experiments, which often require more than 1000 cells with uniform characteristics, although enhanced green fluorescent protein-tagged GnRH neurons have recently become available (6, 7). The molecular biology techniques are routinely used to study the functional significance of cellular machineries, such as ion channels, receptors, transporters, and enzymes. These experiments are generally carried out by using clonal cells including GT1 cells because it is not easy to perform such experiments in primary cultured neurons. Therefore, further characterization of GT1 cells is necessary. In particular, precise analysis of the expression of the voltage-gated Ca²⁺ channels will help the understanding of how these channels relate critically to cellular functions, including GnRH release.

Ca²⁺ influx through voltage-gated Ca²⁺ channels triggers neurosecretion and influences neuronal membrane excitability, gene expression, and developmental events (8). In GT1 cells, several studies have shown that depolarization induces influx of Ca²⁺ via voltage-gated Ca²⁺ channels and stimulates GnRH secretion (3, 9). An increase in the [Ca²⁺]_i inhibits GnRH gene transcription and its mRNA stability (10). Voltage-gated Ca²⁺ channels have been classified into P-, Q-, N-, L-, R-, and T-types based on their molecular, biophysical, and pharmacological properties (8). The ₁ subunit for each voltage-gated Ca²⁺ channel has been cloned: _1A for P/Q-type (11); _1B for N-type (12); _1C, _1D, and _1F for L-type (13, 14); _1E for R-type (15, 16, 17, 18); and _1G and _1H for T-type (19, 20). The diversity of Ca²⁺ channel subtypes allows many possibilities for multiplicity of function. L-type Ca²⁺ channel blocker dihydropyridine has been reported to inhibit spontaneous Ca²⁺ oscillations and pulsatile release of GnRH in GT1–1 and GT1–7 cells (21, 22, 23, 24). The dihydropyridine inhibitor noticeably attenuates an increase in [Ca²⁺]_i induced by -aminobutyric acid (GABA), glutamate, N-methyl-D-aspartate, or kainate, thereby reducing the evoked GnRH release from GT1–7 cells (25). The Ca²⁺ current is blocked by dihydropyridine inhibitor in GT1–1 and GT1–7 cells (23, 24). T-type Ca²⁺ channel blocker Ni²⁺ also inhibits the Ca²⁺ current and spontaneous Ca²⁺ oscillations in GT1–7 cells (3, 24). It is therefore assumed that GT1 cells express L- and T-type Ca²⁺ channels, although their study does not exclude possible expression of the other subtypes of Ca²⁺ channels. Mouse GnRH neurons in olfactory pit explant cultures express both low and high voltage-gated Ca²⁺ currents (26). Recently, we showed that rat GnRH neurons express P/Q-, N-, L-, R-, and T-type Ca²⁺ channels (27), so that a precise investigation in GT1 cells will be useful for comparison to the expression of the voltage-gated Ca²⁺ channels.

In the present study, therefore, we carried out pharmacological identification of voltage-gated Ca²⁺ currents in GT1–7 cells by means of the perforated-patch clamp technique and examined the contribution of each Ca²⁺ channel subtype in Ca²⁺ signaling and GnRH release by using intracellular ion imaging and GnRH assay. Furthermore, expression of ₁ subunit of each Ca²⁺ channel subtype was also analyzed by the RT-PCR method.

	Materials and Methods

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Cell culture
GT1–7 cells (provided by Dr. R. Weiner, University of California at San Francisco, CA) were cultured in DMEM (Irvine Scientific, Santa Ana, CA) with 10% fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin (Life Technologies, Inc., Rockville, MD), 1 mM sodium pyruvate, 24 mM NaHCO₃, and 4 mM L-glutamine. The cultures were maintained at 37 C in a water-saturated atmosphere of 95% air and 5% CO₂. The culture medium was changed every 3–4 d, and cells within 15 passages were used for the experiments. For patch clamp experiments and intracellular Ca²⁺ imaging, the cells were plated on poly-L-lysine-coated coverslips for 3–10 d before use. For static incubation experiments, cells were cultured in 12-well plates.

Electrophysiology
The Ca²⁺ currents were recorded by the method of perforated-patch clamp configuration with amphotericin B. Patch electrodes were made of borosilicate glass capillaries and had a resistance of 6–9 M. The pipette solution consisted of the following (in mM): 95 CsOH, 95 L-aspartic acid, 47.5 CsCl, 1 MgCl₂, 0.1 EGTA, 10 HEPES, 2 ATP-Mg, and 50 µg/ml amphotericin B (pH 7.2; osmolality, 270 mOsm). The extracellular solution consisted of the following (in mM): 106.5 NaCl, 5 CsCl, 0.8 MgCl₂, 10 CaCl₂, 10 glucose, 20 HEPES, 0.6 NaHCO₃, 10 tetraethylammonium chloride, 0.1% BSA (fraction V, Sigma Chemical Co., St. Louis, MO) (pH 7.4; osmolality, 300 mOsm). In some experiments, CaCl₂ was replaced by BaCl₂. The sodium currents were blocked by 1 µM tetrodotoxin (Sankyo Co., Ltd., Tokyo, Japan). The indifferent electrode consisted of an Ag-AgCl wire connected to the bath solution via an agar bridge. An EPC-9 patch clamp amplifier (Physio-Tech Co., Ltd., Tokyo, Japan) in combination with PULSE software (HEKA Electronic, Lambrecht, Germany) was used. Currents were filtered at 2.3 kHz, digitized at 10 kHz, and recorded. Cells with series resistance less than 30 M were chosen for study. Series resistance was electronically compensated by 70%. Capacitive and leak currents were subtracted by the P/4 method (28). Cell capacitance was 17.1 ± 4.4 pF (n = 157). The liquid junction potential was not compensated. Recordings were made at room temperature.

Measurement of [Ca²⁺]_i
Cells were loaded with 2 µM fura-PE-3-acetoxymethyl ester (TefLabs, Austin, TX) for 2 h at 37 C. The coverslip was placed in a small superfusion chamber on the stage of an IX70 inverted microscope (Olympus, Tokyo, Japan). [Ca²⁺]_i was recorded by using the QuantiCell 700 system (Applied Imaging, Sunderland, UK). The cells were illuminated alternately at 340- and 380-nm excitation wavelengths, and then 510-nm emission light images were captured by an image-intensifying, charge-coupled device camera (Photonics Science, Turnbridge Wells, UK). The time interval of each 340- to 380-nm ratio frame was 3 sec. Ratios were converted to Ca²⁺ concentrations as previously described (29, 30). Normal perfusion medium consisted of (in mM): 126.3 NaCl, 5 KCl, 10 CaCl₂, 0.8 MgCl₂, 10 glucose, 20 HEPES, 0.6 NaHCO₃, 0.1% BSA (pH 7.4; osmolality, 300 mOsm). K⁺ medium (50 mM) was prepared by replacing Na⁺ with K⁺ in the normal perfusion medium. Cells were continuously superfused at 37 C throughout the experiment. All drugs were applied through superfusion. A 50 mM K⁺-induced increase in [Ca²⁺]_i was considered significant when its peak value exceeded 20 nM.

Measurement of GnRH secretion
For measurement of GnRH secretion, the culture medium was replaced by normal perfusion medium supplemented with 0.2% BSA. The cells were incubated with each drug for 5 min at 37 C and then stimulated with 50 mM K⁺ medium containing each drug for 5 min at 37 C. The medium was collected, and the GnRH concentration in the medium was measured by means of an RIA kit (Peninsula Laboratories, San Carlos, CA) according to the manufacturer’s protocol.

RNA isolation and RT-PCR
Total RNA from GT1–7 cells was isolated with ISOGEN (Nippon Gene Co., Ltd., Tokyo, Japan) according to the manufacturer’s protocol. Isolated RNA was treated with deoxyribonuclease (ribonuclease free) to prevent genomic DNA from serving as a template in the subsequent PCR. First strand cDNA was synthesized with 1 µg total RNA using random 9 mers by means of an RNA LA PCR kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s protocol. The reaction conditions were 30 C for 10 min, 50 C for 30 min, 99 C for 5 min, and 5 C for 5 min. PCR was performed in a 20-µl reaction mixture containing 1 µl of cDNA from the above reaction, 2.5 µM of each specific primer, PCR buffer (GenHunter, Nashville, TN), 0.2 mM dNTP mixture, and 1.25 U AmpliTaq Gold (Applied Biosystems, Foster City, CA). The PCR conditions were 95 C for 9 min, 29 cycles of 94 C for 30 sec, 60 C for 30 sec, 72 C for 90 sec, and finally, 72 C for 5 min. The following primers were used: _1A, 5'-CCCGAGAACAGCCTTATCGT-3' [GenBank accession no., U76716; location, nucleotide (nt) 3133–nt 3152] and 5'-CCACTGACCACTATGAAGTC-3' (nt 3677–nt 3658); _1B, 5'-ACAACCAGCGTAATGTCACC-3' (U04999; nt 3260–nt 3279) and 5'-CCACTGACAACGATGAAGTC-3' (nt 3770–nt 3751); _1C, 5'-AGACGTGCTGTACTGGATGC-3' (L01776; nt 2264–nt 2283) and 5'-CACTTCTGTGAGCCAGTGAG-3' (nt 2831–nt 2812); _1D, 5'-AGTGATTTCATGCCTCAAGG-3' (AK018426; nt 531–nt 550) and 5'-GGTTCTGATGTGTACCATTC-3' (nt 892–nt 873); _1E, 5'-GAAGGCAGCAGTGAACAAGC-3' (L29346; nt 3687–nt 3706) and 5'-AGTCCAGGATGTTCCAGAGG-3' (nt 4290–nt 4271); _1F, 5'-AAGCTGAGAACAACCCAGAAC-3' (AF192497; nt 5033–nt 5053) and 5'-CAAAGC G GG AA AGAATAGAC-3' (nt 5959–nt 5940); _1G, 5'-GATGCGGGTG-CTGAAGCTAG-3' (AJ012569; nt 2622–nt 2641) and 5'-TGACAGGCAGCTGAATACAG-3' (nt 3076–nt 3057); and _1H, 5'-AGCAACATCGTGTTCACCAG-3' (NM021415; nt 2473–nt 2492) and 5'-GAAGAGCATTAGCAGCATGC-3' (nt 2766–nt 2747). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control with primers 5'-TGCATCCTGCACCACCAACT-3' (M32599; nt 487–nt 506) and 5'-AACACGGAAGGCCATGCCAG-3' (nt 745–nt 726). The PCR products were separated by electrophoresis in 1% agarose gel and visualized by ethidium bromide staining under UV illumination.

Chemicals
-Agatoxin-IVA (Aga-IVA), -conotoxin-GVIA (GVIA), and SNX-482 were purchased from Peptide Institute, Inc. (Osaka, Japan). Nifedipine and nimodipine were purchased from Sigma. All drugs were prepared just before use.

Statistical analysis
The results are expressed as the mean ± SD. The differences were analyzed by paired t test or two-sample t test. P < 0.05 was considered statistically significant.

	Results

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Electrophysiological analysis of voltage-gated Ca²⁺ channels
The Ca²⁺ currents were activated by 100-msec voltage steps from –60 to 60 mV in 10-mV increments from the holding potential of –100 mV at 0.2 Hz. The maximum amplitude of –20.2 ± 12.4 pA/pF (n = 157) was observed at 0–10 mV (Fig. 1A). The low voltage-gated Ca²⁺ current was observed at –50 mV in 5 of 16 cells (Fig. 1B). The value was –1.1 ± 0.2 pA/pF (n = 5), and no current was activated in the remaining 11 cells.

View larger version (18K): [in this window] [in a new window]   FIG. 1. Voltage-gated Ca2+ currents in GT1–7 cells. A, The Ca2+ currents were evoked by 100-msec voltage pulses from –60 to 60 mV in 10-mV increments from the holding potential of –100 mV at 0.2 Hz (upper left panel). Amplitude of the voltage pulses is indicated by the numbers next to each trace. The Ca2+ current was activated from –40 mV and reached the maximum amplitude at 0 mV. Calibrations: 200 pA, 20 msec. B, T-type Ca2+ current in GT1–7 cells. The Ca2+ current was elicited by –60-, –50-, –40-, and –30-mV pulses from the holding potential of –100 mV at 0.2 Hz as shown in the upper panel. The Ca2+ current was activated at –50 mV. For clarity, the currents were filtered at 1 kHz. Calibrations: 40 pA, 20 msec.

View larger version (14K): [in this window] [in a new window]   FIG. 2. Effects of SNX-482 and Ni2+ on the Ca2+ current in GT1–7 cells. A, Representative Ca2+ currents evoked by 100-msec voltage pulses to 0 mV from the holding potential of –100 mV at 0.2 Hz. The Ca2+ current was significantly inhibited by 100 nM SNX-482 and was further inhibited by the addition of 50 µM Ni2+. Calibrations: 100 pA, 20 msec. B, Effect of 100 nM SNX-482 and 50 µM Ni2+ on the current-voltage relationship of GT1–7 cells. Closed circle, Control; open circle, SNX-482; closed triangle, SNX482 and Ni2+; open square, wash. C, Concentration-response relationship of SNX-482 to the inhibition of peak Ca2+ current on GT1–7 cells (n = 7) and summary of the effect of 100 nM SNX-482 and 50 µM Ni2+ (n = 9). The inhibitory effect of each blocker was evaluated by comparing the responses with and without the specific blocker in a paired t test. **, P < 0.01 vs. control; N.S., not significant vs. control; a, P < 0.01 vs. 100 nM SNX-482 alone.

View larger version (18K): [in this window] [in a new window]   FIG. 3. Steady-state inactivation and activation of the R-type Ca2+ current in GT1–7 cells. The experiments were conducted in the presence of nifedipine and GVIA. A, The upper panel shows the voltage protocol for the steady-state inactivation. Holding potentials varied from –100 to 0 mV, and the Ca2+ currents elicited by the test pulse (10 mV) were measured as indicated by the filled circle. Calibrations: 200 pA, 20 msec. B, The upper panel shows voltage protocol for the activation. The holding potential was –80 mV. Ten-millisecond prepulses of –60 to 30 mV were applied, and the tail currents at –80 mV were measured as indicated by the filled square. Calibrations: 100 pA, 2 msec. C, Steady-state inactivation (filled circle, n = 5) and activation (filled square, n = 9) are shown. The half-inactivation voltage was –45 ± 1.9 mV, and the half-activation voltage was –11 ± 3.6 mV, determined by the Boltzmann equation.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Effects of subtype-specific Ca2+ channel blockers on the peak Ca2+ currents of GT1–7 cells. A, Representative Ca2+ currents evoked by 100-msec voltage pulses to 10 mV from the holding potential of –80 mV at 0.2 Hz as shown at the upper panel. Representative current traces taken at times indicated with a, b, c, d, and e in panel B are shown. Traces a and b are overlapped. Calibrations: 100 pA, 20 msec. B, Plot of the peak Ca2+ currents evoked by 100-msec voltage pulses to 10 mV from the holding potential of –80 mV at 0.2 Hz. The cells were treated sequentially with 200 nM Aga-IVA, 1 µM GVIA, 10 µM nimodipine, and 100 nM SNX-482 at the times indicated with hori-zontal bars. Bath-applied drug reached the cells with a delay of approximately 1 min. C, The effects of the drugs are collectively shown as a percentage of the total current. The P/Q-type indicates the current blocked by 200 nM Aga-IVA (n = 9). The N-type indicates the current blocked by 1 µM GVIA (n = 20). The L-type indicates the current blocked by 10 µM nimodipine (n = 11). The R-type indicates the current blocked by 100 nM SNX-482 (n = 9). The T-type indicates the current blocked by 50 µM Ni2+ (n = 9). For the T-type, the data in Fig. 2C were used. *, P < 0.05; **, P < 0.01; N.S., not significant; by paired t test.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Effects of nifedipine and SNX-482 on the currents evoked by ramp depolarization. A, Representative currents evoked by 1-sec ramp depolarization from the holding potential of –80 mV to 60 mV as shown in the upper panel. Nifedipine (10 µM) slightly inhibited the current. Addition of 100 nM SNX-482 strongly inhibited the current. Calibrations: 50 pA, 0.1 sec. B, Representative currents evoked by 5-sec ramp depolarization from the holding potential of –80 mV to 60 mV as shown in the upper panel. Nifedipine clearly inhibited the current, and addition of SNX-482 further inhibited the current. Calibrations: 20 pA, 0.5 sec. C, The effects of the drugs are collectively shown as a percentage of the control current. The L-type indicates the current blocked by 10 µM nifedipine (n = 4 for 1-sec ramp; n = 5 for 5-sec ramp), and the R-type indicates the current blocked by 100 nM SNX-482 (n = 4). The inhibitory effect of each blocker was evaluated by comparing the responses with and without the specific blocker in a paired t test. *, P < 0.05; **, P < 0.01; N.S., not significant.

View larger version (24K): [in this window] [in a new window]   FIG. 6. Effects of SNX-482, nifedipine, GVIA, and Aga-IVA on 50 mM K+induced [Ca2+]i increase and GnRH release. A, 50 mM K+ was applied as indicated with horizontal bars. SNX-482 (100 nM), nifedipine (10 µM), GVIA (1 µM), and Aga-IVA (200 nM) were applied as indicated with open bars. SNX-482 attenuated the 50 mM K+-induced increase in [Ca2+]i. Nifedipine also inhibited the response. GVIA and Aga-IVA did not inhibit but augmented the response. Calibrations: 10 nM, 2 min. In the last three traces, the plateau phase in [Ca2+]i was produced by the 16-min application of 50 mM K+. SNX-482 (100 nM) slightly inhibited the plateau phase, whereas nifedipine (10 µM) almost completely inhibited the response. B, The inhibitory effects of the SNX-482 (n = 68), nifedipine (n = 53), GVIA (n = 35), and Aga-IVA (n = 43) on 50 mM K+-induced increase in [Ca2+]i are collectively shown. The ordinate indicates the value of S2/S1 (%), where S1 is the response to the first application of 50 mM K+ and S2 that to the second application. **, P < 0.01, by paired t test. C and D, 50 mM K+ significantly increased the GnRH release (control). GT1–7 cells were incubated with drugs from 5 min before the stimulation with 50 mM K+. SNX-482 (100 nM) reduced 50 mM K+-induced increase in GnRH release (n = 3). Nifedipine (10 µM) also reduced GnRH release (n = 3). GVIA (1 µM) (n = 3) and Aga-IVA (400 nM) (n = 3) had no effect. Inhibition by SNX-482 and Ni2+ of 50-mM K+-induced increase in GnRH release (D). SNX-482 (100 nM) attenuated the GnRH release (n = 3) similarly to that in Fig. 6C. Ni2+ (50 µM), applied either alone (n = 3) or with SNX-482 (n = 3), did not attenuate the GnRH release. The results in C and D were obtained in independent experiments. The responses with or without a specific blocker were compared in two-sample t test. **, P < 0.01 vs. basal; *, P < 0.05 vs. basal; a, P < 0.05 vs. control; b, P < 0.01 vs. control.

RT-PCR analysis of ₁ subunit of the voltage-gated Ca²⁺ channels
To examine the expression of the voltage-gated Ca²⁺ channel ₁ subunit mRNA, RT-PCR analysis was performed for GT1–7 cells and whole mouse brain (Fig. 7). PCR of mRNA without reverse transcription was performed as a negative control. RT-PCR of GAPDH mRNA was used as an internal control. RT-PCR with a specific primer for _1E subunit cDNA yielded an amplified product of the predicted size from GT1–7 cells; _1B, _1D, and _1H subunits were also detected. These PCR products were sequenced, and it was confirmed that they corresponded to each subunit mRNA (data not shown). In the whole mouse brain, _1A, _1B, _1C, _1D, _1E, _1G, and _1H subunits were detected. There were no PCR products without the process of reverse transcription, indicating no contamination with genomic DNA in the PCR.

View larger version (37K): [in this window] [in a new window]   FIG. 7. RT-PCR analysis of the Ca2+ channel 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H subunit mRNA in GT1–7 cells and mouse whole brain. Total RNA was treated with (+) or without (–) reverse transcriptase and amplified by PCR with primers specific for each 1 subunit and GAPDH. The PCR products were electrophoresed on 1% agarose gel. M, Size marker.

	Discussion

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We identified the R-type Ca²⁺ current by its high sensitivity to SNX-482 (31) and found that 76% of total Ca²⁺ current was the R-type (Fig. 2). This percentage is higher than the 15–32% in several types of neurons (37, 38, 39). However, in rat GnRH neurons tagged with enhanced green fluorescent protein, the percentage of the R-type Ca²⁺ current was 55% in neonates and 40% around puberty (27), so that the high expression of the R-type Ca²⁺ channels might be characteristic of GnRH neurons. The presence of the R-type Ca²⁺ channel in GT1–7 cells was also confirmed by the expression of _1E subunit mRNA (Fig. 7). Properties of the voltage-dependent activation and inactivation of the R-type Ca²⁺ current in GT1–7 cells (Fig. 3) are similar to those in rat GnRH neurons (27) and other neurons (17, 39, 40, 41). These biophysical properties indicate that the slow depolarization inactivates the R-type Ca²⁺ current. We showed this clearly by means of the ramp voltage protocol (Fig. 5). The contribution of the R-type current was smaller in the current evoked by the ramp depolarization than in that evoked by the square voltage pulse. In addition, we showed here the contributions of the R-type Ca²⁺ channels in high K⁺-induced increase in [Ca²⁺]_i and GnRH release (Fig. 6). In these experiments, the extracellular K⁺ concentration ([K⁺]_o) changed over several seconds. The membrane potential change may follow this time course of [K⁺]_o change. The small contribution of the R-type Ca²⁺ channel in the high K⁺-induced responses is mainly due to the inactivation of the R-type Ca²⁺ channels by slow depolarization in these experimental conditions. Furthermore, the inactivation of the R-type Ca²⁺ channel may lead to a lesser contribution of the R-type channel in the plateau phase of [Ca²⁺]_i (Fig. 6). These results clearly indicate the expression of the R-type Ca²⁺ channels in GT1–7 cells and its contribution to the Ca²⁺-dependent GnRH release. The R-type Ca²⁺ channel has been reported to play a critical role in the release of neurotransmitters in calyx-type synapses of the medial nucleus of the trapezoid body, oxytocin neurons, and adrenal chromaffin cells (18, 40, 42, 43) and to be responsible for the dendritic Ca²⁺ influx induced by action potentials in CA1 pyramidal neurons of the hippocampus (44, 45).

On the other hand, the L-type Ca²⁺ channel may be slightly inactivated during slow depolarization. In fact, the effect of L-type Ca²⁺ channel blocker nifedipine (36) was very strong in ramp depolarization (Fig. 5), in high K⁺-induced increase in [Ca²⁺]_i, in the plateau phase of [Ca²⁺]_i, and in GnRH release (Fig. 6), although the contribution of the L-type Ca²⁺ channel was small in the total Ca²⁺ current evoked by the square pulse (Fig. 4). Nimodipine attenuates the high K⁺-induced increase in [Ca²⁺]_i by 30% in GT1–7 cells (22). Nifedipine inhibits high K⁺-induced GnRH release by 65% in GT1–1 cells (21), GABA-induced increase by 81% in [Ca²⁺]_i, and GABA-induced GnRH release by 92% in GT1–7 cells (25). In addition, nifedipine partially inhibits the pulsatile GnRH release from hypothalamic slice culture, primary culture of GnRH neurons, and GT1–1 cells (21). Therefore, the present results confirmed the contribution of the L-type Ca²⁺ channels in the Ca²⁺-dependent GnRH release in GT1–7 cells, which was further supported by the expression of _1D subunit mRNA (Fig. 7). The presence of the L-type Ca²⁺ current has been demonstrated not only in GT1 cells but in mouse (26) and rat GnRH neurons (27). Nimodipine (50 µM) inhibits the Ba²⁺ currents by 70% in GT1–1 cells with the conventional whole-cell patch clamp technique (23), and 1 µM nifedipine inhibits Ba²⁺ currents by 87% in GT1–7 cells with the perforated-patch clamp technique (24). In the present experiment, however, the inhibition by nifedipine of Ba²⁺ currents was only 12%. We cannot explain this quantitative difference in the L-type Ca²⁺ current. In rat GnRH neurons, the L-type Ca²⁺ current constitutes 20% of total Ca²⁺ current, both in neonates and around puberty (27).

GT1–7 cells expressed the _1B subunit mRNA (Fig. 7), and the N-type Ca²⁺ channel blocker GVIA (35) weakly inhibited the Ca²⁺ current in 15% of GT1–7 cells examined (Fig. 4), indicating weak expression of the N-type Ca²⁺ channel in a small population of GT1–7 cells. In rat GnRH neurons, N-type Ca²⁺ current contributes 20% of total Ca²⁺ current, both in neonates and around puberty (27). It has been reported that GVIA has no effect on high K⁺-induced increase in [Ca²⁺]_i and GnRH release in GT1–7 cells (22). In high K⁺-induced increase in [Ca²⁺]_i and GnRH release, GVIA did not show any inhibitory effect (Fig. 6), so that N-type Ca²⁺ channels play little role in the Ca²⁺-dependent GnRH release in GT1–7 cells.

The P/Q-type Ca²⁺channel blocker Aga-IVA (33, 34) did not inhibit the Ca²⁺ current (Fig. 4), and no expression of the _1A subunit mRNA was detected (Fig. 7). No inhibitory effect of Aga-IVA was observed in high K⁺-induced increase in [Ca²⁺]_i and GnRH release (Fig. 6), so that we could conclude there is no expression or very little expression of P/Q-type Ca²⁺ channels in GT1–7 cells. In rat GnRH neurons, there is almost no expression of P/Q type Ca²⁺ current in neonates and weak expression around puberty (27).

GT1–7 cells expressed the _1H subunit mRNA (Fig. 7), and a low concentration of Ni²⁺ (50 µM) inhibited the SNX-482-insensitive Ca²⁺ currents by 51% (Fig. 2), indicating a small expression of the T-type Ca²⁺ channel. This was further confirmed by measuring the Ca²⁺ current activated by a –50 mV pulse from –100 mV (Fig. 1) in which the T-type current is solely activated. In this voltage protocol, 31% of GT1–7 cells elicited a small Ca²⁺ current. But T-type Ca²⁺ channels play little role in the Ca²⁺-dependent GnRH release in GT1–7 cells because Ni²⁺ did not inhibit the high K⁺-induced GnRH release (Fig. 6). Ni²⁺ (100 µM) has been reported to inhibit spontaneous Ca²⁺ oscillations in 76% of GT1–7 cells (3) and to inhibit the low voltage-gated Ca²⁺ current by 90% in GT1–7 cells (24). Ni²⁺ (100 µM) also reduces the spontaneous action potential frequency but does not abolish spontaneous spiking (24). This inhibition by Ni²⁺ is seen only in spontaneously active cells with baseline potentials more negative than –60 mV, so that the T-type Ca²⁺ channel in GT1–7 cells may function under certain conditions and contributes to cell excitability. In addition, the R-type Ca²⁺ channel may contribute to the cell excitability, because Ni²⁺ (100 µM) also inhibits the R-type Ca²⁺ current (46). In rat GnRH neurons, weak expression of the T-type Ca²⁺ current is seen in a small population of neurons in neonates but observed in all neurons around puberty (27).

In conclusion, the present results indicate the expression of the R-, L-, N-, and T-type Ca²⁺ channels in GT1–7 cells. The R-type was a major current component, and the L-, N-, and T-types were minor ones. The R- and L-type Ca²⁺ channels play a critical role in the regulation of Ca²⁺-dependent GnRH release.

	Acknowledgments

 
We are grateful to Drs. T. Sudo, K. Ui-Tei, T. Hamada, and Y. Wada-Kiyama for their technical suggestions.

	Footnotes

 
This work was supported in part by Grants-in-Aid for Scientific Research (C) 10670071 and 13680883 from the Japan Society for the Promotion of Science and by the Sasakawa Scientific Research Grant from The Japan Science Society.

Abbreviations: Aga-IVA, -Agatoxin-IVA; [Ca²⁺]_i, intracellular Ca²⁺ concentration; GABA, -aminobutyric acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GVIA, -conotoxin-GVIA; nt, nucleotide.

Received September 19, 2003.

Accepted for publication January 13, 2004.

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