Endothelial mediators of the acetylcholine-induced relaxation of the rat femoral artery

Abstract

This study examined endothelium-derived mediators of acetylcholine-induced relaxation in male rat femoral arteries. Arterial rings were suspended in a myograph for the measurement of isometric force. The generation of hydrogen peroxide (H 2O 2) in endothelial cells was detected using the fluorescent probe, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester. N G-nitro- l-arginine methyl ester (L-NAME, NOS inhibitor) and 1H-[1,2,4]oxadiazolo[4,2-α]quinoxalin-1-one (ODQ, guanylate cyclase inhibitor) alone or in combination with indomethacin (cycloxygenase inhibitor) diminished acetylcholine-induced endothelium-dependent relaxation to a similar extent. A small relaxation to acetylcholine in 60 mM KCl-constricted rings was abolished by L-NAME. Acetylcholine-induced relaxation was reduced by charybdotoxin plus apamin (intermediate- and small-conductance Ca 2+-activated K + channel blockers, respectively) or by 30 mM KCl. Both ouabain (Na +/K + ATPase inhibitor) and BaCl 2 (K IR channel blocker) also inhibited the relaxation albeit to a lesser degree. In the presence of L-NAME, ODQ plus indomethacin, charybdotoxin plus apamin or ouabain plus BaCl 2 produced further inhibition. Catalase attenuated acetylcholine-induced relaxations and this attenuation was prevented by 3-amino-1,2,4-triazole (catalase inhibitor). Catalase did not affect acetylcholine-induced relaxations in rings treated with L-NAME or ODQ. Acetylcholine increased the dichlorofluorescein fluorescence intensity in native endothelial cells and this effect was abolished by catalase and by L-NAME. Exogenous H 2O 2 caused endothelium-independent relaxation that was slightly inhibited by iberiotoxin, ODQ or significantly reduced by elevated KCl, and abolished by catalase. The present results indicate that in addition to nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF, sensitive to charybdotoxin plus apamin, ouabain, and BaCl 2), the endothelium of rat femoral artery can release H 2O 2 in response to acetylcholine, which was sensitive to L-NAME. Thus, the eNOS-dependent H 2O 2 is likely to be the third mediator of acetylcholine-mediated relaxations in rat femoral arteries.