Endothelium-derived hyperpolarizing factor activates Ca2+-activated K+ channels in porcine coronary artery smooth muscle cells

Although endothelium-derived hyperpolarizing factor (EDHF) activity has been demonstrated in arteries from various species, EDHF has not been chemically identified, nor its mechanism of action characterized. To elucidate this mechanism, we tested the effect of EDHF on lage-conductance Ca2+-activated K+ (K-Ca) channels in porcine coronary artery smooth muscle cells. By using a patch-clamp technique, single-channel currents were recorded in cultured smooth muscle cells; the organ bath also contained a strip of porcine coronary with endothelium, which served as the source of endothelium-derived relaxing factor(s) including EDHF. Exposure of en dothelium to 10(-6) M bradykinin activated K-Ca channels in cultured smooth muscle cells in cell-attached patches. When the experiment was performed in the presence of 10 mu M indomethacin and 30 mu M N-G-nitro-L-arginine (L-NNA), which block the generation of prostaglandin I-2 (PGI(2)) and NO, respectively, K-Ca channel activity was stimulated by bradykinin, indicating the direct involvement of EDHF in K-Ca channel stimulation. Neither 10 mu M methylene blue nor 25 mu M Rp-cAMPS inhibited bradykinin-induced K-Ca channel activity. In inside-out patches, the addition of bradykinin to the solution was without effect on K-Ca channel activation. However, in the presence of 0.5 mM guanosine triphosphate (GTP) and 1.0 mM adenosine triphosphate (ATP) in the bath solution, K-Ca channels was activated by bradykinin. In outside-out patches, the addition of bradykinin also increased K-Ca channel activity, when GTP and ATP were added to the pipette solution. The addition of GDP-beta-S (100 mu M) in the cytosolic solution completely blocked the activation K-Ca channels induced by bradykinin in inside-out and outside-out patches. Pretreatment with 30 mu M quinacrine, a phospholipase A(2) inhibitor, or 3 mu M 17-octadecynoic acid (17-ODYA), a cytochrome P-450 inhibitor, in addition to indomethacin and L-NNA, abolished bradykinin-stimulated K-Ca channel activity in cell-attached patches. Both 14,15-epoxyeicosatrienoic acid (EET) and 11,12-EET increased the open probabilities of K-Ca channels in cell-attached patches. These results suggest that EDHF, released from endothelial cells in response to bradykinin, hyperpolarizes smooth muscle cells by opening K-Ca channels. Furthermore, our data suggest that EDHF is an endothelium-derived cytochrome P-450 metabolite of arachidonic acid. The effect of EDHF on K-Ca channels is not associated with an increase of cAMP and cGMP. The activation of K-Ca channels appears to be due to the activation of GTP-binding protein.