Influence of tumor necrosis factor alpha on intracellular Ca2+ in hen granulosa cells in vitro during follicular development.

Abstract

In the present study, we have determined the influence of tumor necrosis factor alpha (TNF alpha) on both basal and carbamylcholine chloride (Cch)-induced [Ca2+]i in granulosa cells from the largest (F1) and smallest (F5,6) preovulatory follicles. TNF alpha (10 ng/ml) induced a small (51-63 nM) and delayed (approximately 1 min) transient increase in [Ca2+]i. The percentage of cells that responded to the cytokine was greater in F5,6 granulosa cells (48%; n = 48) than F1 granulosa cells (24%; n = 41). These responses were completely abolished in Ca(2+)-free media containing 5 mM EGTA and 2.5 mM Mg2+ or 1 mM Mn2+. Cch induced large increases (> 250 nM) in [Ca2+]i via mobilization of Ca2+ from intracellular stores in approximately 50% of Cch-responsive F1 granulosa cells but only about 15% of Cch-responsive F5,6 cells. Pretreatment with TNF alpha (4-5 min) increased the magnitude of the Cch response in both F1 and F5,6 granulosa cells previously incapable of producing large Cch-induced changes in [Ca2+]i. In F1 cells, the effects of TNF alpha on Cch-induced [Ca2+]i were far more extensive, such that the Cch response in the presence of TNF alpha was indistinguishable from the fast Cch-induced Ca2+ transient reported previously. Furthermore, the TNF alpha effect was reversible, as subsequent challenge with Cch in the absence of TNF alpha failed to produce the large Ca(2+)-transients observed earlier with the cytokine present. In conclusion, TNF alpha induces transient increases in [Ca2+]i by transmembrane Ca2+ flux, which are suppressed during cytodifferentiation. In addition, TNF alpha appeared to enhance Cch-induced mobilization of Ca2+ from intracellular stores.(ABSTRACT TRUNCATED AT 250 WORDS)