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The mechanism underlying the inhibition by H2O2 of acetylcholine-induced contraction was investigated in epithelium-denuded strips of rabbit trachea. Acetylcholine (10 μM) generated a phasic, followed by a tonic increase in both the intracellular Ca2+ concentration ([Ca2+]i) and force. Although the acetylcholine-induced tonic contraction was around 9 times the high K+(80 mM)-induced one, the two stimulants induced similar [Ca2+]i increases (around 0.2 μM), indicating that acetylcholine generates tonic contraction via increases in both [Ca2+]i and myofilament Ca2+-sensitivity. H2O2 (30 μM) (a) enhanced the acetylcholine-induced tonic (not phasic) increase in [Ca2+]i but attenuated both phases of the acetylcholine-induced contraction and (b) enhanced the high K+-induced increase in [Ca2+]i but did not modify the high K+-induced contraction. In β-escin-skinned strips, application of acetylcholine in the presence of GTP enhanced the contraction induced by 0.3 μM Ca2+ so that its amplitude became similar to that induced by 1 μM Ca2+. H2O2 (30 μM) attenuated the contraction induced by 0.3 μM Ca2+ (alone or in the presence of acetylcholine) but not those induced by higher concentrations of Ca2+ alone (0.5 μM and 1 μM). These results indicate that H2O2 acts directly on contractile proteins in rabbit tracheal smooth muscle to inhibit the contraction induced by low concentrations of Ca2+ (< 0.5 μM). An action of H2O2 that increases [Ca2+]i (and thereby masks this reactive-oxygen-induced inhibition of myofilament Ca2+-sensitivity) is apparent in the presence of high K+ but not of acetylcholine. Thus, in rabbit tracheal smooth muscle H2O2 downregulates myofilament Ca2+-sensitivity more potently during acetylcholine-induced contraction than during high-K+-induced contraction, leading to an effective inhibition of the former contraction.