Effect of classic preconditioning and diazoxide on endothelial function and O2− and NO generation in the post-ischemic guinea-pig heart

Objectives: A hypothesis was tested that a reaction product between superoxide (O2−) and nitric oxide (NO) mediates post-ischemic coronary endothelial dysfunction that ischemic preconditioning (IPC) protects the endothelium by preventing post-ischemic cardiac O2− and/or NO formation, and that the opening of the mitochondrial ATP-dependent potassium channel (mKATP) plays a role in the mechanism of IPC. Methods: Langendorff-perfused guinea-pig hearts were subjected either to 30 min global ischemia/30 min reperfusion (IR) or were preconditioned prior to IR with three cycles of either 5 min ischemia/5 min reperfusion or 5 min infusion/5 min wash-out of mKATP opener, diazoxide (0.5 μM). Coronary flow responses to acetylcholine (ACh) and nitroprusside were used as measures of endothelium-dependent and -independent vascular function, respectively. Myocardial outflow of O2− and NO, and functional recoveries were followed during reperfusion. Results: IR impaired the ACh response by approximately 60% and augmented cardiac O2− and NO outflow. Superoxide dismutase (150 U/ml) and NO synthase inhibitor, l-NMMA (100 μM) inhibited the burst of O2− and NO, respectively, and afforded partial preservation of the ACh response in IR hearts. NO scavenger, oxyhemoglobin (25 μM), afforded similar endothelial protection. IPC and diazoxide preconditioning attenuated post-ischemic burst of O2−, but not of NO, and afforded a complete endothelial protection. Diazoxide given after 30-min ischemia increased the O2− burst and was not protective. The effects of IPC and diazoxide preconditioning were not affected by HMR-1098 (25 μM), a selective blocker of plasmalemmal KATP, and were abolished by glibenclamide (0.6 μM) and 5-hydroxydecanoate (100 μM), a nonselective and selective mKATP blocker, respectively. 5-Hydroxydecanoate produced similar effects, whether it was given as a continuous treatment or was washed out prior to IR. Conclusion: The results suggest that in guinea-pig heart: (i) a reaction product between O2− and NO mediates the post-ischemic endothelial dysfunction; (ii) the mKATP opening serves as a trigger of the IPC and diazoxide protection; and (iii) the mKATP opening protects the endothelium in the mechanism that involves the attenuation of the O2− burst at reperfusion.