Acyl chain length-specific ceramide-induced changes in intracellular Ca2+ concentration and progesterone production are not regulated by tumor necrosis factor alpha in hen granulosa cells

Although tumor necrosis factor alpha (TNF-alpha) has long been known to be a potent inhibitor of gonadotropin-induced cytodifferentiation in the ovari es of a variety of mammalian species, its early signal transduction events are poorly understood. We previously demonstrated that TNF- alpha induces a small, delayed follicular stage-dependent increase in intracellular Ca2+ c oncentration ([Ca2+](i)) in hen granulosa cells and promotes carbachol (Cch )-induced mobilization of Ca2+ from intracellular stores in cells otherwise unresponsive to the cytokine. The focus of the current study was to examin e the role of ceramide in TNF-alpha-induced Ca2+ regulation. Treatment with exogenous sphingomyelinase (SMase; 50 mU/ml) failed to influence basal [Ca 2+](i) but increased the magnitude of Cch-induced Ca2+ transients. While C8 -ceramide (0.03-30 mu M), but not C2-ceramide (0.03-30 mu M), mimicked this effect of SMase, challenge with sphingosine (3 mu M) resulted in a slow an d delayed increase in basal [Ca2+](i). In order to determine whether SMase is activated by TNF-alpha action, changes in sphingomyelin and ceramide con centrations in F1 and F5,6 granulosa cells were determined. SMase activatio n was not observed after 1-, 5-, 15-, and 60-min incubations with TNF-alpha (1-50 ng/ml) in either F1 or F5,6 cells. Exogenous SMase and C2-ceramide b oth inhibited LH-induced progesterone production in F1 and F5,6 cells; howe ver, incubation with C8-ceramide resulted in increases in both basal and LH -induced progesterone. In contrast, incubation with TNF-alpha had no effect on either basal or LH-induced steroidogenesis. In conclusion, our findings indicate that although ceramide regulates [Ca2+](i) and progesterone secre tion, the sphingolipid does not appear to play a role in the action of TNF- alpha in avian granulosa cells. Furthermore, ceramide-mediated responses ar e highly dependent on acyl chain length, potentially reflecting differences in the abilities of these ceramides to access, bind to, and/or activate ce ramide-dependent signal transduction mechanisms. Nonetheless, since TNF-alp ha did not increase the production of ceramide, the physiological regulator (s) of these responses remain unknown.