Watanabe Miho, Sakuma Yasuo, Kato Masakatsu
Department of Physiology, Nippon Medical School, Bunkyo, Tokyo 113-8602 Japan.
Endocrinology. 2004 May;145(5):2375-83. doi: 10.1210/en.2003-1257. Epub 2004 Jan 21.
The GT1 cell has been widely used as a model cell to study cellular functions of GnRH neurons. Despite the importance of Ca(2+) channels, little is known except for L- and T-type Ca(2+) channels in GT1 cells. Therefore, we studied the diversity of voltage-gated Ca(2+) channels in GT1-7 cells with perforated-patch clamp and RT-PCR. An R-type Ca(2+) channel blocker, SNX-482, inhibited the Ca(2+) currents by 75.6% in all cells examined (n = 9). A T-type Ca(2+) channel blocker, Ni(2+), inhibited the Ca(2+) currents by 12.6% in all cells examined (n = 9). An L-type Ca(2+) channel blocker, nimodipine, inhibited the Ca(2+) currents by 17.9% in five of 11 cells examined. When using Ba(2+) as a charge carrier, another dihydropyridine antagonist, nifedipine, clearly inhibited the currents by 12.1% in all cells examined (n = 16). An N-type Ca(2+) channel blocker, omega-conotoxin-GVIA, inhibited the Ca(2+) currents by 13.8% in three of 20 cells examined. A P/Q type Ca(2+) channel blocker, omega-agatoxin-IVA, had no effect on the currents (n = 9). RT-PCR revealed that GT1-7 cells expressed the alpha(1B), alpha(1D), alpha(1E), and alpha(1H) subunit mRNA. Furthermore, SNX-482 and nifedipine inhibited the high K(+)-induced increase in the intracellular Ca(2+) concentration and GnRH release. omega-Conotoxin-GVIA and omega-agatoxin-IVA had no effect. These results suggest that GT1-7 cells express R-, L-, N-, and T-type voltage-gated Ca(2+) channels; the R-type was a major current component, and the L-, N-, and T-types were minor ones. The R- and L-type Ca(2+) channels play a critical role in the regulation of Ca(2+)-dependent GnRH release.
GT1细胞已被广泛用作研究GnRH神经元细胞功能的模型细胞。尽管钙通道很重要，但除了GT1细胞中的L型和T型钙通道外，人们对其他类型的了解甚少。因此，我们用穿孔膜片钳和RT-PCR研究了GT1-7细胞中电压门控钙通道的多样性。R型钙通道阻滞剂SNX-482在所有检测细胞（n = 9）中使钙电流抑制了75.6%。T型钙通道阻滞剂Ni(2+)在所有检测细胞（n = 9）中使钙电流抑制了12.6%。L型钙通道阻滞剂尼莫地平在11个检测细胞中的5个中使钙电流抑制了17.9%。当使用Ba(2+)作为载流子时，另一种二氢吡啶拮抗剂硝苯地平在所有检测细胞（n = 16）中使电流明显抑制了12.1%。N型钙通道阻滞剂ω-芋螺毒素-GVIA在20个检测细胞中的3个中使钙电流抑制了13.8%。P/Q型钙通道阻滞剂ω-蛛毒素-IVA对电流无影响（n = 9）。RT-PCR显示GT1-7细胞表达α(1B)、α(1D)、α(1E)和α(1H)亚基mRNA。此外，SNX-482和硝苯地平抑制了高钾诱导的细胞内钙浓度升高和GnRH释放。ω-芋螺毒素-GVIA和ω-蛛毒素-IVA无影响。这些结果表明，GT1-7细胞表达R型、L型、N型和T型电压门控钙通道；R型是主要电流成分，L型、N型和T型是次要成分。R型和L型钙通道在钙依赖性GnRH释放的调节中起关键作用。
