RELEASE OF ENDOTHELIUM-DERIVED RELAXING FACTOR FROM PIG CULTURED AORTIC ENDOTHELIAL-CELLS, AS ASSESSED BY CHANGES IN ENDOTHELIAL-CELL CYCLIC-GMP CONTENT, IS INHIBITED BY A PHORBOL ESTER

Cultured aortic endothelial cells of the pig respond to the endothelium-derived relaxing factor (EDRF) they release with an increase in cyclic GMP content. This response is inhibited by haemoglobin or by L-N(G)-monomethyl-arginine (L-NMMA), and has been used to investigate the effects of phorbol esters on EDRF release. Pretreatment with phorbol-12,13-dibutyrate (PDB) but not the inactive 4α-phorbol-12,13-didecanoate (PDD), inhibited increases in cyclic GMP induced by substance P (10-8 M) in a time and concentration-dependent manner, PDB did not affect basal cyclic GMP levels. PDB (3 x 10-7 M), but not PDD (3 x 10-7 M), also inhibited ATP (10-5 M)-induced increases in cyclic GMP, but did not affect those induced by bradykinin (10-7 M). Increases in cyclic GMP induced by low (10-7 M) but not high (10-6 M) concentrations of the calcium ionophore A23187 were inhibited by PDB (3 x 10-7 M). This inhibitory effect was due to enhanced destruction of EDRF by superoxide anions rather than inhibition of EDRF release, as the inhibition was abolished in the presence of superoxide dismutase (SOD, 30 u ml-1) and catalase (CAT, 100 u ml-1). SOD and CAT did not affect the inhibitory action of PDB on substance P or ATP-induced increases in cyclic GMP. Increases in endothelial cell cyclic GMP content induced by sodium nitroprusside (10-5 M) were unaffected by PDB pretreatment. The inhibitory effects of PDB are probably a result of an action of protein kinase C on the steps between receptor occupation and phospholipase C activation.