A TRANSFERABLE, BETA-NAPHTHOFLAVONE-INDUCIBLE, HYPERPOLARIZING FACTORIS SYNTHESIZED BY NATIVE AND CULTURED PORCINE CORONARY ENDOTHELIAL-CELLS

1. The vascular endothelium releases a hyperpolarizing factor (endothe lium-derived hyperpolarizing factor, EDHF) tentatively identified as a cytochrome P450-derived arachidonic acid metabolite. However, there i s still controversy concerning its transferability and identity. We de signed a bioassay system for assessing EDHF release in which the membr ane potential was recorded in cultured vascular smooth muscle cells lo cated downstream from donor endothelial cells. 2. Under combined nitri c oxide (NO) synthase and cyclo-oxygenase blockade with N-G-nitro-L-ar ginine (100 mu mol l(-1)) and diclofenac (10 mu mol l(-1)), the superf usate from bradykinin (30 nmol l(-1))-stimulated, cultured porcine cor onary endothelial cells induced a distinct hyperpolarization followed by a depolarization. Direct application of bradykinin to the smooth mu scle cells resulted solely in membrane depolarization. Similar results were obtained using bradykinin-stimulated porcine coronary arteries a s donor. 3. Single-channel current measurements suggest that this EDHF -induced hyperpolarization was elicited by the activation of Ca2+-depe ndent K+ channels. 4. Increasing the transmural pressure within the do nor segment significantly enhanced the duration, but not the amplitude of the hyperpolarization induced by the effluate from bradykinin-stim ulated donor segments. 5. Inhibition of P450 oxygenase activity with c lotrimazole (3 mu mol l(-1)) or 17-octadecynoic acid (3 mu mol l(-1)) abolished EDHF release from the coronary endothelium, while the P450-d erived arachidonic acid metabolite, 5,6-epoxyeicosatrienoic acid, indu ced a hyperpolarization of detector smooth muscle cells almost identic al to that induced by EDHF. Moreover, induction of P450 activity by be ta-naphthoflavone (3 mu mol l(-4), 48 h), significantly increased the bradykinin-induced release of EDHF. 6. These findings suggest that the vascular endothelium releases a transferable hyperpolarizing factor, chemically distinct from NO and prostacyclin, in response to agonists and mechanical stimulation. This beta-naphthoflavone-inducible EDHP ap pears to be a cytochrome P450-derived metabolite of arachidonic acid, which elicits hyperpolarization by activation of Ca2+-dependent K+ cha nnels.