CELLULAR BASIS FOR FOLLICLE-STIMULATING HORMONE-STIMULATED CALCIUM SIGNALING IN SINGLE-RAT SERTOLI CELLS - POSSIBLE DISSOCIATION FROM EFFECTS OF ADENOSINE-3',5'-MONOPHOSPHATE

To study the cellular basis for FSH-stimulated dose-dependent graded i ncreases in intracellular Ca2+ concentrations in populations of Sertol i cells, we investigated the effects of FSH on free Ca2+ ion concentra tions ([Ca2+]i) in individual rat Sertoli cells using the Ca2+-sensiti ve dye fura-2/AM and digital fluorescent videomicroscopy. Ovine or rat FSH elicited a hormone-specific rise in [Ca2+]i within 20-140 sec, wi th 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, increasin g doses of FSH recruited a higher percentage of responding cells. Chel ation of extracellular Ca2+ or cotreatment with verapamil or cobalt ab olished 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+ increase s were mimicked by forskolin or protein kinase-A type I activators [ 8 -(6-amino-hexyl)amino-cAMP and N6-benzoyl-cAMP (N6B)]. However, the cA MP analogs, 8-bromo-cAMP, N6,2'-O-dibutyryl cAMP, or protein kinase-A type II activators (8-thiomethyl-cAMP and N6B), induced [Ca2+]i increa ses 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 indu ced by FSH. Similarly, the uninhibited rise in [Ca2+]i induced by FSH in pertussis toxin-pretreated Sertoli celts suggests that PT-sensitive G-proteins are not involved in the action of FSH on [Ca2+]i. In summa ry, we demonstrate that FSH evokes sustained [Ca2+]i increases in sing le Sertoli cells in a nongraded fashion and recruits increasing number s of responding cells in a dose-dependent fashion. We also provide exp licit evidence that FSH induces Ca2+ influx. Mimicry of the FSH-induce d [Ca2+]i rise by certain cAMP analogs [8-(6-amino-hexyl)amino-cAMP an d N6B; protein kinase-A type I activator] or forskolin suggests that C a2+ may be part of a dual pathway of cAMP-initiated intracellular sign aling.