Susceptibility of caffeine- and Ins(1,4,5)P 3-induced contractions to oxidants in permeabilized vascular smooth muscle

Abstract

Two principal pathways of Ca 2+ release from the sarcoplasmic reticulum of excitable and non-excitable cells have been described: one pathway dependent on the second messenger d- myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3), and a second pathway sensitive to Ca 2+ and regulated by caffeine and ryanodine. It was found that the Ca 2+-pump activity of vascular smooth muscle sarcoplasmic reticulum is inhibited by superoxide anion radicals (O 2 ⋅−); however, the effects of reactive oxygen intermediates on sarcoplasmic reticulum Ca 2+ release in vascular muscle cells are not well defined. The purpose of the present study was to evaluate the effects of reactive oxygen intermediates generated from the hypoxanthine/xanthine oxidase reaction system on contractions induced by caffeine, Ins(1,4,5)P 3 and norepinephrine in staphylococcal α-toxin-permeabilized rabbit mesenteric arteries. This system generates O 2 ⋅−, H 2O 2, and hydroxyl radicals. We wished to identify which class of reactive oxygen intermediates is responsible for the associated loss of vascular smooth muscle contractile function. Caffeine and Ins(1,4,5)P 3 produced a transient contraction when the sarcoplasmic reticulum of the permeabilized preparations was preloaded with pCa 7.0 solution for 5 min before washing with 0.5 mM EGTA solution; norepinephrine also produced a transient contraction. Exposure of the preparations to hypoxanthine/xanthine oxidase (for 30 min) attenuated caffeine-induced contraction, but was without effect on Ins(1,4,5)P 3-induced contraction. The observed effect of hypoxanthine/xanthine oxidase exposure was superoxide dismutase-inhibitable, suggesting O 2 ⋅− involvement. Hypoxanthine/xanthine oxidase also inhibited norepinephrine-induced contraction. The effect of hypoxanthine/xanthine oxidase on norepinephrine contraction was protected by catalase, but not by superoxide dismutase and dimethyl sulfoxide; exogenously added H 2O 2 mimicked the effect of hypoxanthine/xanthine oxidase exposure. H 2O 2, added exogenously, was without effect on Ins(1,4,5)P 3-induced contraction. It is suggested that the pathway of Ca 2+ release from the sarcoplasmic reticulum dependent on Ins(1,4,5)P 3 is insensitive to O 2 ⋅−. Instead, caffeine-induced Ca 2+ release mechanisms may be susceptible to O 2 ⋅− and H 2O 2, rather than O 2 ⋅− and hydroxyl radicals, may be the active agent in the norepinephrine-induced contraction. Our results are also consistent with the view that the attenuation by H 2O 2 of the norepinephrine-induced contraction may be linked to the receptor-associated pathway of Ins(1,4,5)P 3 formation, but not to degradation processes of Ins(1,4,5)P 3.