MECHANISMS BY WHICH ENDOTHELIN-1 STIMULATES INCREASED CYTOSOLIC-FREE CALCIUM-ION CONCENTRATIONS IN SINGLE-RAT SERTOLI CELLS

The regulation by endothelin-1 (ET-1) of cytosolic free calcium ion co ncentrations ([Ca2+](i)) was investigated in single immature rat (test icular) Sertoli cells. [Ca2+](i) was estimated in individual gonadal c ells by digital imaging videomicroscopy using the calcium indicator dy e fura-2/AM. Two concentration-dependent types of ET-1-induced [Ca2+]( i) signals were observed. Responses to high ET-1 concentrations (1.0-1 000 nM) were characterized by a biphasic, rapid, and transient [Ca2+]( i) rise (spike) within 10 sec, followed by an exponential decrease tow ard a new steady state level (plateau phase) in 98% of responsive cell s. At low concentrations of ET-1 (0.001 or 0.1 nM), the [Ca2+](i) incr ease was slower, reaching peak values 40-100 sec after stimulation and remaining elevated for 2-3 min of observation. There was cell-cell he terogeneity in the amplitude and kinetics of the [Ca2+](i) response to the same concentration of ET-1. However, there was a significant ET-1 concentration-dependent increase in the total percentage of cells res ponding to ET-1. Removal of extracellular Ca2+ or use of Ca2+ channel blockers (verapamil or cobalt) did not affect the ET-1-induced [Ca2+]( i) spike phase, but abolished the plateau phase, suggesting that ET-1 induces the mobilization of Ca2+ from internal stores, followed by cal cium influx from extracellular sources. In cell population experiments , ET-1 attenuated FSH-stimulated cAMP and estradiol accumulation by Se rtoli cells. These inhibitory effects were mimicked by phorbol 12-myri state 13-acetate, an activator of protein kinase-C, suggesting that ET -1 action on Sertoli cells might be linked to the protein kinase-C pat hway. In conclusion, the present investigations demonstrate that ET-1 activates an intracellular signaling pathway involving [Ca2+](i) in si ngle rat Sertoli cells. The sources of the biphasic [Ca2+](i) response include mobilization of Ca2+ from internal stores, followed by Ca2+ i nflux via verapamil- and cobalt-sensitive Ca2+ channels. Increasing ET -1 concentrations recruit an increasing number of individual Sertoli c ells responding with a spike-plateau [Ca2+](i) signal, thus offering a mechanism at the single cell level for the ET dose-response curve.