Cytochrome P450 Eicosanoids and Myocardial Injury

描述(申请人提供)：我们实验室已经证明，花生四烯酸(AA)代谢的几种环氧合酶(COX)和脂氧合酶(LOX)产物在犬缺血再灌流心肌中产生心脏保护作用，例如PGI2、PGE1和12-HETE。然而，关于AA代谢的另一个主要途径，细胞色素P-450(CYP)途径的产物对心脏的影响的数据很少。在已建立的犬心肌梗死模型和分离的兔心肌细胞中，我们假设AA的CYP代谢物在缺血和再灌流期间被释放，并通过开放和/或阻断心肌细胞上的ATP敏感(KATP)或钙激活钾(KCA)通道来产生心肌保护或加强心脏损伤。初步结果表明，在冠状动脉闭塞和再灌流过程中，在闭塞结束和再灌流过程中，环氧二十碳三烯酸(EETs)、二羟基二十碳三烯酸(DHETs)和20-羟基二十碳四烯酸(20-HETE)含量增加，两种非特异性CYP抑制剂阻断它们的形成后，心肌梗死面积显著缩小。此外，我们有初步数据表明，外源性给予20-HETE会导致犬心脏梗塞面积的增加。因此，基于这些有趣的结果，我们将研究EETs、DHETs和20-HETE在体内心肌梗死模型和体外缺氧-复氧模型中调节心肌损伤的作用和细胞机制。具体地说，我们将识别和量化心脏产生的区域异构体EETs、DHETs和20-HETE，以及在犬心脏缺血再灌注损伤中表达的CYP亚型。其次，我们将研究CYP代谢产物对犬缺血再灌流心肌和缺氧-复氧的成年兔心肌细胞KATP通道和KCA通道的作用及其主要的CYP途径。最后，我们将研究在缺血-再灌注和缺氧-复氧过程中COX和/或LOX通路与CYP通路的相互作用。我们在成熟的细胞和动物模型中使用生理学、药理学、化学和分子生物学的综合方法，是确定CYP途径在缺血再灌注损伤中所起作用的有效方法。

DESCRIPTION (provided by applicant): Several cyclooxygenase (COX) and lipoxygenase (LOX) products of arachidonic acid (AA) metabolism have been shown by our laboratory to produce a cardioprotective effect in the ischemicreperfused canine myocardium, examples being PGI2, PGE1 and 12-HETE. However, very little data exist concerning the cardiac effects of the products of the other major pathway of AA metabolism, the cytochrome P-450 (CYP) pathway. In a well-established model of canine myocardial infarction and in isolated rabbit cardiomyocytes, we hypothesize that the CYP metabolites of AA are released during ischemia and reperfusion and produce cardioprotection or enhance cardiac injury via the opening and/or blockade of ATP-sensitive (KATP) or calcium-activated potassium (Kca) channels in cardiac myocytes. Preliminary results indicate that during coronary artery occlusion and following reperfusion that increases in epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acids (DHETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) occur at the end of occlusion and throughout reperfusion and that by blocking their formation by two nonspecific CYP inhibitors, a marked reduction in myocardial infarct size occurred. In addition, we have preliminary data to suggest that exogenous administration of 20-HETE results in an increase in infarct size in the canine heart. Therefore, based on these intriguing results, we will study the role and cellular mechanisms by which the EETs, DHETs and 20-HETE modulate myocardial injury in an in vivo model of infarction and an in vitro model of hypoxia-reoxygenation. Specifically, we will identify and quantify the regioisomeric EETs, DHETs and 20-HETE that are produced by the heart as well as the CYP isoforms that are expressed in response to ischemia-reperfusion injury in dog hearts. Secondly, we will investigate the roles that CYP metabolites have on KATP channels and Kca channels in the ischemic-reperfused dog myocardium and on isolated adult rabbit cardiac myocytes exposed to hypoxia-reoxygenation and the major CYP pathways involved. Finally, we will investigate the interaction of the COX and/or LOX pathways with the CYP pathway during ischemia-reperfusion and hypoxia-reoxygenation. Our use of integrative physiology, pharmacology, chemistry and molecular biology in well-established cell and animal models is a powerful approach for identifying the role that the CYP pathway serves in ischemia-reperfusion injury.