Stimulation of cAMP formation in fetal human non-pigmented ciliary epithelial cells by 10 microM prostaglandin E1 was inhibited by 30-50% by 15 min prior exposure to 1 microM phorbol 12-myristate, 13-acetate. Evidence that this inhibition was due to activation of protein kinase C is the following. First, inhibition was also caused by 10 microM dioctanoylglycerol, a diacylglycerol analog. Second, no inhibition was observed using 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C, whereas phorbol didecanoate was effective. And third, prior exposure of cells to staurosporine, an inhibitor of protein kinase C, blocked phorbol ester-induced inhibition of cAMP stimulation. Phorbol esters also inhibited stimulation of cAMP formation by 10 nM vasoactive intestinal peptide and by 1 microM isoproterenol. Stimulation of cAMP formation by either 1 microM cholera toxin or 10 microM forskolin was not inhibited by prior exposure of cells to phorbol esters. This suggests that protein kinase C acts neither at the level of GS activation of adenylyl cyclase, nor by inhibiting adenylyl cyclase directly. The possibility that protein kinase C acts on adenylyl cyclase-linked receptors was assessed by measuring the effect of phorbol esters on specific binding of [125I]vasoactive intestinal peptide to intact cells. Treatment of cells with either 1 microM phorbol 12-myristate,13-acetate or phorbol didecanoate resulted in a 25-40% reduction in the number of binding sites for [125I]vasoactive intestinal peptide, with little change in dissociation constants.(ABSTRACT TRUNCATED AT 250 WORDS)