Endocrinology, Vol 135, 127-134, Copyright © 1994 by Endocrine Society

ARTICLES

Mechanisms by which endothelin-1 stimulates increased cytosolic free calcium ion concentrations in single rat Sertoli cells

OP Sharma, JA Flores, DA Leong and JD Veldhuis
Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908.

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.

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