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 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.
Sharma
O P
OP
Department of Internal Medicine, and National Science Foundation for Center for Biological Timing, University of Virginia Health Sciences Center, Charlottesville 22908.
Flores
J A
JA
Leong
D A
DA
Veldhuis
J D
JD
D000249:Adenosine Monophosphate / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism; D008297:Male; D009994:Osmolar Concentration; D037342:Pertussis Toxin; D051381:Rats; D017207:Rats, Sprague-Dawley; D012708:Sertoli Cells / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D010566:Virulence Factors, Bordetella; D000249:Adenosine Monophosphate; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP; D037342:Pertussis Toxin; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium