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 (O-2(-)) and nitric oxide (NO) mediates post-ischemic coronary endothelial dysfunction that ischemic preconditioning (IPC) protects the endothelium by preventing post-ischemic cardiac O-2(-) and/or NO formation, and that the opening of the mitochondrial ATP-dependent potassium channel (mK(ATP)) 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 mK(ATP) opener, diazoxide (0.5 muM). Coronary flow responses to acetylcholine (ACh) and nitroprusside were used as measures of endothelium-dependent and -independent vascular function, respectively. Myocardial outflow of O-2(-) and NO, and functional recoveries were followed during reperfusion. Results: IR impaired the ACh response by approximately 60% and augmented cardiac O-2(-) and NO outflow. Superoxide dismutase (150 U/ml) and NO synthase inhibitor, L-NMMA (100 muM) inhibited the burst of O-2(-) and NO, respectively, and afforded partial preservation of the ACh response in IR hearts. NO scavenger, oxyhemoglobin (25 muM), afforded similar endothelial protection. IPC and diazoxide preconditioning attenuated post-ischemic burst of O-2(-), but not of NO, and afforded a complete endothelial protection. Diazoxide given after 30-min ischemia increased the O-2(-) burst and was not protective. The effects of IPC and diazoxide preconditioning were not affected by HMR-1098 (25 muM), a selective blocker of plasmalemmal K-ATP, and were abolished by glibenclamide (0.6 muM) and 5-hydroxydecanoate (100 PM), a nonselective and selective mK(ATP) 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 O-2(-) and NO mediates the post-ischemic endothelial dysfunction; (ii) the mK(ATP) opening serves as a trigger of the IPC and diazoxide protection; and (iii) the mK(ATP) opening protects the endothelium in the mechanism that involves the attenuation of the O-2(-) burst at reperfusion. (C) 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.