Sharma O P, Flores J A, Leong D A, Veldhuis J D
Department of Internal Medicine, and National Science Foundation for Center for Biological Timing, University of Virginia Health Sciences Center, Charlottesville 22908.
Endocrinology. 1994 Apr;134(4):1915-23. doi: 10.1210/endo.134.4.8137759.
To study the cellular basis for FSH-stimulated dose-dependent graded increases in intracellular Ca2+ concentrations in populations of Sertoli cells, we investigated the effects of FSH on free Ca2+ ion concentrations ([Ca2+]i) in individual rat Sertoli cells using the Ca(2+)-sensitive dye fura-2/AM and digital fluorescent videomicroscopy. Ovine or rat FSH elicited a hormone-specific rise in [Ca2+]i within 20-140 sec, with a peak level 2.7 +/- 0.9-fold greater than the basal value (mean +/- SEM; n = 8) lasting for 4-16 min. The amplitude and kinetics of the FSH-induced [Ca2+]i signal were not dose dependent. Instead, increasing doses of FSH recruited a higher percentage of responding cells. Chelation of extracellular Ca2+ or cotreatment with verapamil or cobalt abolished FSH-induced [Ca2+]i increases. Furthermore, in the presence of extracellular Mn2+, direct evidence for FSH-mediated Ca2+ influx was obtained from the quench of fura-2 fluorescence. Induced Ca2+ increases were mimicked by forskolin or protein kinase-A type I activators [8-(6-amino-hexyl)amino-cAMP and N6-benzoyl-cAMP (N6B)]. However, the cAMP analogs, 8-bromo-cAMP, N6,2'-O-dibutyryl cAMP, or protein kinase-A type II activators (8-thiomethyl-cAMP and N6B), induced [Ca2+]i increases even in the absence of extracellular Ca2+, and the time course of the [Ca2+]i rise induced by cAMP analogs was more rapid than that induced by FSH. Similarly, the uninhibited rise in [Ca2+]i induced by FSH in pertussis toxin-pretreated Sertoli cells suggests that PT-sensitive G-proteins are not involved in the action of FSH on [Ca2+]i. In summary, we demonstrate that FSH evokes sustained [Ca2+]i increases in single Sertoli cells in a nongraded fashion and recruits increasing numbers of responding cells in a dose-dependent fashion. We also provide explicit evidence that FSH induces Ca2+ influx. Mimicry of the FSH-induced [Ca2+]i rise by certain cAMP analogs [8-(6-amino-hexyl)amino-cAMP and N6B; protein kinase-A type I activator] or forskolin suggests that Ca2+ may be part of a dual pathway of cAMP-initiated intracellular signaling.
为了研究促卵泡激素（FSH）刺激下支持细胞群体中细胞内钙离子浓度呈剂量依赖性分级增加的细胞基础，我们使用钙离子敏感染料fura - 2/AM和数字荧光视频显微镜，研究了FSH对单个大鼠支持细胞中游离钙离子浓度（[Ca2+]i）的影响。绵羊或大鼠FSH在20 - 140秒内引起[Ca2+]i激素特异性升高，峰值水平比基础值高2.7±0.9倍（平均值±标准误；n = 8），持续4 - 16分钟。FSH诱导的[Ca2+]i信号的幅度和动力学不依赖于剂量。相反，FSH剂量增加时，有反应的细胞百分比增加。螯合细胞外钙离子或与维拉帕米或钴共同处理可消除FSH诱导的[Ca2+]i增加。此外，在细胞外存在锰离子的情况下，通过fura - 2荧光淬灭获得了FSH介导的钙离子内流的直接证据。毛喉素或蛋白激酶A I型激活剂[8 -（6 - 氨基己基）氨基 - cAMP和N6 - 苯甲酰 - cAMP（N6B）]可模拟诱导的钙离子增加。然而，环磷酸腺苷类似物8 - 溴 - cAMP、N6,2'-O - 二丁酰环磷酸腺苷或蛋白激酶A II型激活剂（8 - 硫代甲基 - cAMP和N6B）即使在没有细胞外钙离子的情况下也能诱导[Ca2+]i增加，且环磷酸腺苷类似物诱导的[Ca2+]i升高的时间进程比FSH诱导的更快。同样，在百日咳毒素预处理的支持细胞中，FSH诱导的[Ca2+]i不受抑制的升高表明，百日咳毒素敏感的G蛋白不参与FSH对[Ca2+]i的作用。总之，我们证明FSH以非分级方式引起单个支持细胞中[Ca2+]i持续增加，并以剂量依赖性方式募集越来越多的有反应细胞。我们还提供了明确的证据表明FSH诱导钙离子内流。某些环磷酸腺苷类似物[8 -（6 - 氨基己基）氨基 - cAMP和N6B；蛋白激酶A I型激活剂]或毛喉素模拟FSH诱导的[Ca2+]i升高，表明钙离子可能是环磷酸腺苷启动的细胞内信号传导双重途径的一部分。
