Mechanisms underlying the hydrogen peroxide-induced, endothelium-independent relaxation of the norepinephrine-contraction in guinea-pig aorta

Abstract

The mechanisms underlying the hydrogen peroxide-induced relaxation of the norepinephrine-contraction were studied by measuring isometric force, myosin light chain (MLC 20) phosphorylation and cyclic GMP in endothelium-denuded muscle from the guinea-pig aorta. Norepinephrine (5.2±1.3 μM) produced a phasic, followed by a tonic contraction. Hydrogen peroxide (10 and 100 μM), glyceryl trinitrate (30 and 300 nM) and 8-bromo cyclic GMP (30 and 100 μM) did not change the basal tone, but reduced the norepinephrine-induced contraction. Phosphorylation of MLC 20 (percentage of phosphorylated to total MLC 20) was increased 1 min (5.9±1.0% vs. 35.9±4.9%) and, to a lesser extent, 20 min (3.7±1.7% vs. 13.9±1.6%) after the addition of norepinephrine. Hydrogen peroxide (100 μM) did not modify basal MLC 20 phosphorylation, but reduced the increase in MLC 20 phosphorylation induced by 1-min exposure to norepinephrine (20.9±4.1%). Its effect was abolished by catalase. When the tissue was incubated for 20 min with norepinephrine in the presence of hydrogen peroxide, norepinephrine-induced MLC 20 phosphorylation was not changed (13.6±1.5%), as compared to that in the absence of hydrogen peroxide. Hydrogen peroxide relaxed norepinephrine-stimulated aortas in a concentration-dependent fashion with EC 50 values of 5.9±0.2 μM. The relaxation was inhibited by soluble guanylate cyclase inhibitors and increased by an inhibitor of cyclic GMP-selective phosphodiesterase. In aorta precontracted with norepinephrine, hydrogen peroxide (100 μM) relaxed the tissue by 89±11% and almost doubled tissue concentrations of cyclic GMP, whereas sodium nitroprusside (1 μM) relaxed the tissue by 100% and increased cyclic GMP concentrations 30-fold. It is suggested that the inhibitory effects of hydrogen peroxide on the norepinephrine-induced phasic and sustained contractions are explained by a decrease in MLC 20 phosphorylation and by an alteration in MLC 20 phosphorylation-independent mechanisms, respectively. The effects of hydrogen peroxide were in part mediated by cyclic GMP.