Preconditioning protects endothelium by preventing ET-1-induced activation of NADPH oxidase and xanthine oxidase in post-ischemic heart.

Abstract The hypothesis was tested that endothelin-1 (ET-1)-induced superoxide (O 2 − ) generation mediates post-ischemic coronary endothelial injury, that ischemic preconditioning (IPC) affords endothelial protection by preventing post-ischemic ET-1, and thus O 2 − , generation, and that opening of the mitochondrial ATP-dependent potassium channel (mK ATP ) triggers the mechanism of IPC. Furthermore, the study was aimed at identifying the source of O 2 − mediating the endothelial injury. Langendorff-perfused guinea-pig hearts were subjected either to 30 min ischemia/35 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 washout of mK ATP opener diazoxide (0.5 mM). Coronary flow responses to acetylcholine (ACh) served as a measure of endothelium-dependent vascular function. Myocardial outflow of ET-1 and O 2 − and functional recoveries were followed during reperfusion. NADPH oxidase and xanthine oxidase (XO) activities were measured in cardiac homogenates. IR augmented ET-1 and O 2 − outflow and impaired ACh response. All these effects were attenuated or prevented by IPC and diazoxide, and 5-hydroxydecanoate (a selective mK ATP blocker) abolished the effects of IPC and diazoxide. Superoxide dismutase and tezosentan (a mixed ET-1-receptor antagonist) mimicked the effects of IPC, although they had no effect on the ET-1 generation. IR augmented also the activity of NADPH oxidase and XO. Apocynin treatment, that resulted in NADPH oxidase inhibition, prevented XO activation and O 2 − generation in IR hearts. The inhibition of XO, either by allopurinol or feeding the animals with tungsten-enriched chow, prevented post-ischemic O 2 − generation, although these interventions had no effect on the NADPH activity. In addition, the post-ischemic activation of NADPH oxidase and XO, and O 2 − generation were prevented by IPC, tezosentan, thenoyltrifluoroacetone (mitochondrial complex II inhibitor), and tempol (cell-membrane permeable O 2 − scavenger). In guinea-pig heart: (i) ET-1-induced O 2 − generation mediates post-ischemic endothelial dysfunction; (ii) IPC and diazoxide afford endothelial protection by attenuating the ET-1, and thus O 2 − generation, and the mK ATP opening triggers the protection; (iii) the NADPH oxidase maintains the activity of XO, and the XO-derived O 2 − mediates the endothelial injury, and (iv) ET-1 and O 2 − (probably of mitochondrial origin) are upstream activators of the NADPH oxidase–XO cascade, and IPC prevents the cascade activation and the endothelial dysfunction by preventing the ET-1 generation.