ENDOTHELIUM-DERIVED HYPERPOLARIZING FACTOR, BUT NOT NITRIC-OXIDE, IS REVERSIBLY INHIBITED BY BREFELDIN-A

The subcellular localization of the enzymes synthesizing endothelium-d erived vasodilator autacoids has been proposed to play a role in deter mining the ability of endothelial cells to enhance autacoid production in response to stimulation. We therefore investigated the effects of brefeldin A-induced disruption of the Golgi apparatus and Golgi-plasma membrane trafficking on the production of nitric oxide (NO), prostacy clin, and the endothelium-derived hyperpolarizing factor (EDHF) by nat ive and cultured endothelial cells. In porcine coronary artery segment s, brefeldin A (35 mu mol/L, 90 minutes) did not affect relaxations to sodium nitroprusside or the K+ channel opener cromakalim but elicited a rightward shift in the concentration-response curve to bradykinin w ithout altering the maximum vasodilator response (R-max). Brefeldin A failed to attenuate the bradykinin-induced, NO-mediated relaxation und er depolarizing conditions but inhibited the bradykinin response under conditions of combined cyclooxygenase/NO synthase blockade, suggestin g that this agent selectively interferes with the production of EDHF. Indeed, incubation of porcine coronary arteries with brefeldin A, whic h did not affect the bradykinin-induced accumulation of either cyclic GMP or 6-keto-prostaglandin F-1 alpha, markedly and reversibly attenua ted the EDHF-mediated hyperpolarization of detector smooth muscle cell s in a patch-clamp bioassay system. The microtubule destabilizer nocod azole also affected both the EC50 and R-max, to bradykinin in porcine coronary arteries. Since EDHF is thought to be a cytochrome P450-deriv ed metabolite of arachidonic acid and both brefeldin A and nocodazole are known to interfere with the targeting of cytochrome P450 from the Golgi apparatus to the plasma membrane, it is conceivable that brefeld in A inhibits EDHF formation by preventing the targeting of the EDHF-s ynthesizing enzymes to the plasma membrane.