INFLUENCE OF TUMOR-NECROSIS-FACTOR-ALPHA ON INTRACELLULAR CA2-CELLS IN-VITRO DURING FOLLICULAR DEVELOPMENT( IN HEN GRANULOSA)

In the present study, we have determined the influence of tumor necros is 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 ce lls (24%; n = 41). These responses were completely abolished in Ca2+-f ree media containing 5 mM EGTA and 2.5 mM Mg2+ or 1 mM Mn2+. Cch induc ed large increases (> 250 nM) in [Ca2+](i) via mobilization of Ca2+ fr om intracellular stores in approximately 50% of Cch-responsive F1 gran ulosa cells but only about 15% of Cch-responsive F5,6 cells. Pretreatm ent with TNF alpha (4-5 min) increased the magnitude of the Cch respon se in both F1 and F5,6 granulosa cells previously incapable of produci ng 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 fr om the fast Cch-induced Ca2+ transient repotted previously. Furthermor e, the TNF alpha effect was reversible, as subsequent challenge with C ch in the absence of TNF alpha failed to produce the large Ca2+-transi ents observed earlier with the cytokine present. in conclusion, TNF al pha induces transient increases in [Ca2+](i) by transmembrane Ca2+ flu x, which are suppressed during cytodifferentiation. In addition, TNF a lpha appeared to enhance Cch-induced mobilization of Ca2+ from intrace llular stores, These studies demonstrate a complex, follicular stage-d ependent interaction between cytokine and cholinergic input in the con trol of the Ca2+ signaling pathway for the regulation of granulosa cel l function.