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

In the present study, we have determined the influence of tumor necrosis factor alpha (TNFα) on both basal and carbamylcholine chloride (Cch)- induced [Ca²⁺](i) in granulosa cells from the largest (F1) and smallest (F5,6) preovulatory follicles. TNFα (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 Ca²⁺-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 TNFα (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 TNFα on Cch-induced [Ca²⁺](i) were far more extensive, such that the Cch response in the presence of TNFα was indistinguishable from the fast Cch-induced Ca²⁺ transient reported previously. Furthermore, the TNFα effect was reversible, as subsequent challenge with Cch in the absence of TNFα failed to produce the large Ca²⁺transients observed earlier with the cytokine present. In conclusion, TNFα induces transient increases in [Ca²⁺](i) by transmembrane Ca²⁺ flux, which are suppressed during cytodifferentiation. In addition, TNFα appeared to enhance Cch-induced mobilization of Ca²⁺ from intracellular stores. These studies demonstrate a complex, follicular stage-dependent interaction between cytokine and cholinergic input in the control of the Ca²⁺ signaling pathway for the regulation of granulosa cell function.