Lai Tsung-Hsuan, Lin Yuan-Feng, Wu Feng-Chang, Tsai Yu-Hui
Division of Reproduction Medicine, Department of Obstetrics and Gynecology, Cathay General Hospital, Taipei, Taiwan 106, Republic of China.
Endocrinology. 2008 Mar;149(3):1031-7. doi: 10.1210/en.2007-1244. Epub 2007 Dec 6.
Our previous study demonstrated that FSH-induced immediate Ca(2+) influx in rat Sertoli cells (SCs) is mediated by the Galphah/phospholipase C-delta1 (PLC-delta1) signaling pathway. As to which Ca(2+) channel is responsible for such Ca(2+) influx was not understood. In this study, thapsigargin triggered an in-store calcium release and evoked a 1.5-fold elevation of intracellular Ca(2+) in Ca(2+)-free media, whereas FSH exhibited no effect. The readdition of CaCl(2) (2.5 mm) to FSH-pretreated or thapsigargin-sensitized SCs in Ca(2+)-free media immediately elicited a rapid Ca(2+) influx or a 2-fold increase of second intracellular Ca(2+) elevation, respectively. The addition of Ca(2+) chelator EGTA (0.2 mm) reduced the FSH-induced elevation of intracellular Ca(2+) in SCs incubated with CaCl(2). However, pretreatment with dantrolene (25 microM), which inhibits in-store calcium release, did not affect the FSH-induced elevation of intracellular Ca(2+). NiCl(2) (10 microM), a T-type calcium channel blocker, abolished the FSH-induced SC Ca(2+) influx. Furthermore, mibefradil (10 and 100 microm), another specific blocker for T-type Ca(2+) channels, dose-dependently suppressed the FSH-induced Ca(2+) influx. In contrast, nifedipine (10 and 50 microm) or omega-conotoxin GVIA (100 and 500 nm), blocker of L- or N-type Ca(2+) channels, respectively, did not affect the FSH-induced SC Ca(2+) influx. On the other hand, FSH-induced Ca(2+) influx was significantly reduced by pretreatment of SCs with myristoylated synthetic peptide (0.1 and 1 microm) of PLC-delta1 fragment TIPWNSLKQGYRHVHLL but not affected by 2',5'-dideoxyadenosine (3 and 15 microm), a selective inhibitor of adenylate cyclase. In conclusion, the FSH-induced Galphah/PLC-delta1 pathway-dependent Ca(2+) influx of rat SCs is mediated by T-type Ca(2+) channels and independent of in-store calcium release.
我们之前的研究表明，促卵泡激素（FSH）诱导的大鼠支持细胞（SCs）中钙离子（Ca(2+)）的快速内流是由Gαh/磷脂酶C-δ1（PLC-δ1）信号通路介导的。但尚不清楚哪种Ca(2+)通道负责这种Ca(2+)内流。在本研究中，毒胡萝卜素引发了细胞内钙释放，并在无钙培养基中使细胞内Ca(2+)升高了1.5倍，而FSH则无此作用。向无钙培养基中经FSH预处理或对毒胡萝卜素敏感的SCs中重新添加氯化钙（2.5 mM），立即分别引发了快速的Ca(2+)内流或细胞内Ca(2+)的第二次升高增加了2倍。添加钙离子螯合剂乙二醇双四乙酸（EGTA，0.2 mM）可降低在氯化钙中孵育的SCs中FSH诱导的细胞内Ca(2+)升高。然而，用丹曲林（25 μM）预处理抑制细胞内钙释放，并不影响FSH诱导的细胞内Ca(2+)升高。镍离子（NiCl(2)，10 μM），一种T型钙通道阻滞剂，消除了FSH诱导的SCs中Ca(2+)内流。此外，米贝拉地尔（10和100 μM），另一种T型Ca(2+)通道的特异性阻滞剂，剂量依赖性地抑制了FSH诱导的Ca(2+)内流。相比之下，硝苯地平（10和50 μM）或ω-芋螺毒素GVIA（100和500 nM），分别为L型或N型Ca(2+)通道的阻滞剂，并不影响FSH诱导的SCs中Ca(2+)内流。另一方面，用PLC-δ1片段TIPWNSLKQGYRHVHLL的肉豆蔻酰化合成肽（0.1和1 μM）预处理SCs可显著降低FSH诱导的Ca(2+)内流，但不受腺苷酸环化酶选择性抑制剂2',5'-二脱氧腺苷（3和15 μM）的影响。总之，FSH诱导的大鼠SCs中依赖Gαh/PLC-δ1途径的Ca(2+)内流是由T型Ca(2+)通道介导的，且与细胞内钙释放无关。
