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The objective of this study was to examine the effects of Y-27632, a selective Rho-kinase inhibitor, on ischemic preconditioning (IP) and carbachol preconditioning (CP) in anesthetized rats. Administration of Y-27632 (0.1 mg/kg) produced slight, but not significant, reduction in mean arterial blood pressure and suppressed the total number of ventricular ectopic beats (VEBs). IP, induced by 5 min coronary artery occlusion and 5 min reperfusion, decreased the incidence of ventricular tachycardia (VT) from 100 (n = 30) to 25% (n = 24) and abolished the occurrence of ventricular fibrillation (VF) (40% in control group) during 30 min of ischemia. The incidences of VT and VF in Y-27632 + IP group were found to be similar to IP group. Carbachol (4 μg/kg/min for 5 min) induced marked depressions in mean arterial blood pressure, heart rate and attenuated the total number of VEBs, but significant reductions in VT and VF incidences were noted in Y-27632 + CP group. Y-27632 infusion for 5 min abolished VF occurrence. Marked reductions in plasma lactate levels were observed in all treatment and preconditioning groups. IP led to marked decrease in malondialdehyde levels. Decreases in infarct size were also observed with all groups when compared to control. These results suggest that infusion of Y-27632 was able to produce cardioprotective effects on myocardium against arrhythmias, infarct size or biochemical parameters and mimic the effects of ischemic preconditioning in anesthetized rats. Therefore, it is likely that inhibition of Rho-kinase is involved in the signaling cascade of myocardial preconditioning.