Endothelium-derived hyperpolarizing factor, but not nitric oxide or prostacyclin release, is resistant to menadione-induced oxidative stress in the bovine coronary artery

Abstract

The interaction of superoxide anions (O₂ ^–) generated by menadione with the synthesis and/or action of nitric oxide (NO), prostacyclin (PGI₂) and endothelium-derived hyperpolarizing factor (EDHF) was investigated in segments of the left anterior descending coronary artery (LAD) isolated from bovine hearts. EDHF and NO release were monitored by superfusion bioassay in segments pre-constricted with the thromboxane mimetic, U46619, PGI₂ release in addition by enzyme immunoassay for 6-keto-prostaglandin F_1α, and generation of O₂ ^– by lucigenin-enhanced chemiluminescence. Bradykinin (1–1,000 pmol) elicited a prominent, endothelium-dependent, relaxant response, 50–60% of which was insensitive to combined blockade of cyclooxygenase with diclofenac (1 µM) and NO synthase with N ^G-nitro-l-arginine (50 µM). This diclofenac/N ^G-nitro-l-arginine-insensitive relaxant response was completely abrogated in the presence of tetrabutylammonium (3 mM), a non-selective inhibitor of Ca²⁺-dependent K⁺ channels, or when the segments were pre-constricted with potassium chloride (60 mM) instead of U46619, and thus most likely mediated by EDHF. Despite causing a two- to fourfold increase in the concentration of O₂ ^– in or at the vessel wall, menadione (30 µM) did not affect the diclofenac/N ^G-nitro-l-arginine-insensitive relaxant response to bradykinin. When administered in the absence of N ^G-nitro-l-arginine, however, menadione significantly inhibited the relaxant response to bradykinin, presumably by attenuating the NO-mediated relaxation. Menadione also abolished the bradykinin-stimulated release of PGI₂ from luminally perfused segments of the LAD. This effect was more pronounced in the absence of N ^G-nitro-l-arginine, indicating that PGI₂ release in this preparation may be more sensitive to inhibition by peroxynitrite, the reaction product of NO and O₂ ^–, than to O₂ ^– alone. These findings suggest that, in contrast to NO and PGI₂, the release, and presumably also the mechanism of action, of EDHF in the bovine LAD is resistant to an increase in the local concentration of O₂ ^– or peroxynitrite which is thought to play an important role in coronary heart disease.