Direct effects of hydrogen peroxide on airway smooth muscle tone: Roles of Ca2+ influx and Rho-kinase

Reactive oxidant species are implicated in the chronic airway inflammation related to asthma and chronic obstructive pulmonary disease. This study was designed to determine mechanisms underlying contraction induced by hydrogen peroxide (H2O2), a clinical marker of oxidative stress, in airway smooth muscle. Isometric tension and fluorescent intensities of fura-2, an index of intracellular Ca2+ concentrations ([Ca2+]i), were measured in epithelium-denuded tracheal smooth muscle tissues isolated from guinea pigs. H2O2 (0.01–1 mM) caused contraction with an augmentation of [Ca2+]i in a concentration-dependent manner in the normal physiological solution containing 2.4 mM of extracellular Ca2+ concentrations. The contractile force and [Ca2+]i by H2O2 (1 mM) were approximately half of those in response to 1 μM methacholine. However, contraction by H2O2 was not generated under the condition that extracellular Ca2+ concentrations were less than 0.15 mM. Verapamil (10 μM), an inhibitor of voltage-operated Ca2+ channels, partially but significantly inhibited the H2O2-induced contraction. In contrast, SKF-96365 (1-{β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride) (100 μM), a non-selective inhibitor of Ca2+ channels, completely abolished both the contraction and the increase in [Ca2+]i elicited by H2O2. Moreover, Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide) (0.03–10 μM), an inhibitor of Rho-kinase, caused a concentration-dependent inhibition of the H2O2-induced contraction. In conclusion, both the Ca2+ influx from the extracellular side and the Ca2+ sensitization by Rho-kinase are involved in the regulation of airway smooth muscle tone induced by H2O2. An inhibition of the Rho/Rho-kinase pathway may be beneficial for the treatment of airflow limitation mediated by oxidative stress.