Quantitative identification of superoxide anion as a negative inotropic species

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Abstract
Reduced oxygen intermediates have been shown to directly depress cardiac muscle function of the subcellular, tissue, and whole animal levels. The exact species of reduced oxygen intermediate [superoxide anion radical (O2- .cntdot.), H2O2, hydroxyl free radical (HO .cntdot.)] and the concentrations necessary to depress cardiac muscle function have been quantified. To better understand the role of O2- .cntdot. and H2O2, we have studied rabbit right ventricular papillary muscle function in the presence of these reduced oxygen intermediates generated by a xanthine-xanthine oxidase system at 37.degree. C. In the presence of xanthine (0.1 mM) and xanthine oxidase (0.02 U/ml), 57.5 .+-. 0.85 nmol .cntdot. l-1 .cntdot. s-1 O2- .cntdot. and 69.25 .+-. 5.3 nmol .cntdot. l-1 .cntdot. s-1 H2O2 were produced. In the presence of superoxide dismutase (SOD), O2- .cntdot. was eliminated and H2O2 concentrated increased. Catalase effectively eliminated the accumulation of H2O2 without significantly changing the rate of O2- .cntdot. generation. When applied to isometrically contracting right ventricular papillary muscles, this system, with or without SOD and catalase, had no effect on peak developed tension or .+-. dT/dt derived either from length-tension or force-frequency studies. However, when the xanthine oxidase concentration was increased to 0.112 U/ml, the rate of O2- .cntdot. generation increased to 196.67 .+-. 3.26 nmol .cntdot. l-1 .cntdot. s-1 and H2O2 production increased to 142.19 .+-. 9.3 nmol .cntdot. l-1 .cntdot. s-1 with significant depression of papillary muscle tension development. SOD virtually eliminated O2- .cntdot. production, whereas H2O2 production increased to 199.48 .+-. 9.8 nmol .cntdot. l-1. s-1 with no effect on papillary muscle tension development. In the presence of catalase, O2- .cntdot. production increased to 238.86 .+-. 4.0 nmol .cntdot. l-1 .cntdot. s-1; H2O2 production was virtually eliminated, and this system resulted in a significant depression of peak developed tension. There was no depression of papillary muscle function in the presence of both superoxide dismutase and catalase. We conclude that the O2- .cntdot. is a negative inotropic species whose properties are concentration dependent. Hydrogen peroxide, in the concentrations measured, does not appear to be a negative inotropic species.