Role of superoxide anions in the mediation of endothelium-dependent contractions

We designed experiments to characterize the role of superoxide anions in the mediation of endothelium-dependent contractions in isolated canine basilar arteries. Rings with and without endothelium were suspended for isometric tension recording in Krebs-Ringer bicarbonate solution bubbled with 94% O₂-6% CO₂ (37°C, pH 7.4). Radioimmunoassay was used to determine the levels of cyclic GMP and cyclic AMP. Calcium ionophore A23187 (10^-9 to 10^{- 6} mol/L) caused concentration-dependent contractions. The removal of endothelium abolished the effect of A23187. Contractions to A23187 were reversed into relaxations in the presence of superoxide dismutase (150 U/mL) or the prostaglandin H₂/thromboxane A₂ receptor antagonist SQ29548 (10^-6 mol/L). N(G)-nitro-L-arginine methyl ester (3 x 10^-4 mol/L) augmented contractions to A23187. In rings with endothelium, A23187 (3 x 10^-7 mol/L) significantly increased levels of both cyclic AMP and cyclic GMP. Indomethacin (10^-5 mol/L) inhibited stimulatory effects of A23187 on cyclic AMP production. In contrast, indomethacin augmented A23187-induced production of cyclic GMP. Selective augmentation of cyclic GMP production by indomethacin appears to be due to protection of nitric oxide or a closely related molecule released following translocation of calcium into endothelial cells. Our findings suggest that (1) an increased concentration of calcium in endothelial cells may activate both cyclooxygenase and the L-arginine/nitric oxide pathway, (2) arachidonic acid metabolism via cyclooxygenase is a source of superoxide anions, and (3) superoxide anions may be responsible for impairment of balance between relaxing and contracting factors leading to contraction of underlying smooth muscle cells.