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Enriched fractions of small and large luteal cells were incubated for 2 h with 1 or 10 microM calcium ionophore, A23187: unstimulated secretion of progesterone and viability in small cells were not affected but these measures were decreased (P less than 0.01) for unstimulated large cells and were significantly correlated (P less than 0.05). This effect in large cells was independent of extracellular calcium. Therefore, incubations of the two cell types were made in the presence of increasing concentrations of a protein kinase C activator, phorbol 12-myristate 13-acetate (TPA). Secretion of progesterone and viability were not augmented in unstimulated small cells, but TPA prevented (P less than 0.05) the full stimulation of secretion of progesterone by LH. Secretion of progesterone in unstimulated large cells was inhibited (P less than 0.01) by TPA (100 nM and 10 microM), although viability was unaffected. The non-tumour promoting phorbol ester, 4 alpha-phorbol didecanoate, had no effect on large cells. Extracellular calcium was not required for the observed effect of TPA. Sphingosine, an agent inhibitory to protein kinase C activity, inhibited (P less than 0.01) secretion of progesterone in small and large cells, and also reduced (P less than 0.01) cell viability. These values were significantly correlated (P less than 0.05) in both cell types. The above observations suggest that protein kinase C may invoke negative regulation on progesterone production in unstimulated large and hormone-stimulated small luteal cells of sheep. Since sphingosine significantly reduced viability in small and large cells and ionophore selectively inhibited viability in large cells, the ability of these agents to influence calcium-mediated intracellular regulation of steroidogenesis is still uncertain.

Influence of agents that affect intracellular calcium regulation on progesterone secretion in small and large luteal cells of the sheep