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.