DOWN-REGULATION OF VASOACTIVE-INTESTINAL-PEPTIDE RECEPTORS BY PROTEIN-KINASE-C IN FETAL HUMAN NONPIGMENTED CILIARY EPITHELIAL-CELLS

Stimulation of cAMP formation in fetal human non-pigmented ciliary epithelial cells by 10 mu M prostaglandin E(1) was inhibited by 30-50% by 15 min prior exposure to 1 mu M 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 mu M 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 mu M isoproterenol. Stimulation of cAMP formation by either 1 mu M cholera toxin or 10 mu M forskolin was not inhibited by prior exposure of cells to phorbol esters. This suggests that protein kinase C acts neither at the level of G(s) 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 [I-125]vasoactive intestinal peptide to intact cells. Treatment of cells with either 1 mu M phorbol 12-myristate, 13-acetate or phorbol didecanoate resulted in a 25-40% reduction in the number of binding sites for [I-125]vasoactive intestinal peptide, with little change in dissociation constants. 4 alpha phorbol didecanoate had little effect. The half-maximal concentration for inhibition of binding by phorbol 12-myristate, 13-acetate was similar to 5 nM, similar to its potency for inhibition of cAMP stimulation by prostaglandin E(1) or vasoactive intestinal peptide. These data suggest that VIP receptor number is down-regulated by a protein-kinase-C-dependent mechanism in fetal human non-pigmented ciliary epithelial cells.