A novel mechanism by which hydrogen peroxide decreases calcium sensitivity in airway smooth muscle

The purpose of this study was to test the hypothesis that H₂O₂ decreases the amount of force produced by a given intracellular Ca²⁺ concentration (i.e., the Ca²⁺ sensitivity) in airway smooth muscle (ASM) in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. A new preparation was developed and validated in which canine ASM strips were first exposed to H₂O₂ and then permeabilized with 10% Triton X-100 to assess the persistent effects of H₂O₂ on Ca²⁺ sensitivity. Experiments in which H₂O₂ was administered before permeabilization revealed a novel mechanism that contributed to reduced Ca²⁺ sensitivity independently of changes in rMLC phosphorylation, in addition to an rMLC phosphorylation-dependent mechanism. The mechanism depended on factors not available in the permeabilized ASM strip or in the buffer to which the strip was exposed, since there was no effect when H₂O₂ was added to permeabilized strips. H₂O₂treatment of a maximally thiophosphorylated purified myosin subfragment (heavy meromyosin) significantly reduced actomyosin ATPase activity, suggesting one mechanism by which the phosphorylation-independent reduction in Ca²⁺ sensitivity may occur.