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 concentrations ([Ca²⁺](i)) was investigated in single immature rat (testicular) Sertoli cells. [Ca²⁺](i) was estimated in individual gonadal cells by digital imaging videomicroscopy using the calcium indicator dye fura-2/AM. Two concentration-dependent types of ET-1-induced [Ca²⁺](i) signals were observed. Responses to high ET-1 concentrations (1.0-1000 nM) were characterized by a biphasic, rapid, and transient [Ca²⁺](i) rise (spike) within 10 sec, followed by an exponential decrease toward a new steady state level (plateau phase) in 98% of responsive cells. At low concentrations of ET-1 (0.001 or 0.1 nM), the [Ca²⁺](i) increase was slower, reaching peak values 40-100 sec after stimulation and remaining elevated for 2-3 min of observation. There was cell-cell heterogeneity 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 responding 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 calcium influx from extracellular sources. In cell population experiments, ET-1 attenuated FSH-stimulated cAMP and estradiol accumulation by Sertoli cells. These inhibitory effects were mimicked by phorbol 12-myristate 13-acetate, an activator of protein kinase- C, suggesting that ET-1 action on Sertoli cells might be linked to the protein kinase-C pathway. In conclusion, the present investigations demonstrate that ET-1 activates an intracellular signaling pathway involving [Ca²⁺](i) in single rat Sertoli cells. The sources of the biphasic [Ca²⁺](i) response include mobilization of Ca²⁺ from internal stores, followed by Ca²⁺ influx via verapamil- and cobalt-sensitive Ca²⁺ channels. Increasing ET-1 concentrations recruit an increasing number of individual Sertoli cells responding with a spike-plateau [Ca²⁺](i) signal, thus offering a mechanism at the single cell level for the ET dose-response curve.