CARDIAC CYTOCHROME P450 ARACHIDONIC ACID EPOXYGENASE PATHWAY

心脏细胞色素 P450 花生四烯酸环氧化酶途径

• 批准号: 6289939
• 负责人: Darryl C Zeldin
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6289939
• 关键词: arachidonate; clinical research; cytochrome P450; eicosanoid metabolism; enzyme activity; epoxides; gene expression; genetic promoter element; genetically modified animals; heart cell; heart function; hemoprotein metabolism; human subject; laboratory mouse; myocardial ischemia /hypoxia; oxygenases; reperfusion

We have discovered a number of mammalian CYP2J subfamily members, although we have focused most of our efforts on human CYP2J2 and mouse CYP2J5. The human CYP2J2 cDNA has been cloned, expressed and extensively characterized with respect to its tissue distribution, cellular localization and function. This enzyme appears to be the major human P450 expressed in heart, where it is localized to cardiac myocytes and endothelial cells, and is active in the metabolism of AA to EETs. One of the EETs (11,12-EET) improves cardiac function following prolonged global ischemia, causes hyperpolarization of the resting membrane potential and shortening of the cardiac action potential, and markedly inhibits cardiac L-type Ca++ channel activity. Capacitative Ca++ entry is significantly increased in human umbilical vein endothelial cells stably transfected with the CYP2J5 cDNA. Physiologic concentrations of 11,12-EET attenuate endothelial cell activation (TNF -induced VCAM-1 expression) by a mechanism involving inhibition of the pro-inflammatory transcription factor, NF- B. Furthermore, transient transfection of endothelial cells with the CYP2J2 cDNA inhibits NF- B mediated gene transcription. Together, these results suggest that CYP2J-derived eicosanoids may be important in ischemic heart disease, vascular inflammation and atherogenesis in humans. The human CYP2J2 gene has been cloned, sequenced and characterized with respect to its intron/exon organization. The CYP2J2 gene appears to be regulated, in part, by alternative splicing at the exon 1/intron 1 junction. Two alternative splice variants (CYP2J2-H2 and CYP2J2-H5) which are expressed in multiple tissues have been cloned and the recombinant proteins were shown to be unstable. We have also identified several CYP2J2 polymorphic variants that result in amino acid substitutions at positions 143, 158, 192 and 404 of the CYP2J2 protein. Preliminary modeling studies show that the Ile192 Asn and Asn404 Tyr substitutions occur near the CYP2J2 active site and are predicted to affect catalytic efficiency. These CYP2J2 variants will be generated using site-directed mutagenesis and characterized as part of the NIEHS Environmental Genome Project. Efforts are also currently underway to: (a) examine the human CYP2J2 promoter including identification of relevant cis-acting elements; (b) examine the effect of CYP2J2 overexpression on cardiac myocyte function in vitro; (c) construct transgenic mice that overexpress CYP2J2 in heart muscle to examine the effects of CYP2J2-derived eicosanoids on cardiac function in vivo; and (d) clone the putative receptor which transduces EET signals in endothelial cells and myocytes. - arachidonic acid eicosanoid cytochrome 450 ischemic heart disease preconditioning - Human Subjects

我们已经发现了许多哺乳动物CYP 2 J亚家族成员，尽管我们的大部分努力集中在人CYP 2 J2和小鼠CYP 2 J5上。人CYP 2 J2 cDNA已被克隆、表达，并就其组织分布、细胞定位和功能进行了广泛表征。该酶似乎是心脏中表达的主要人P450，在心脏中其定位于心肌细胞和内皮细胞，并且在AA至E2的代谢中具有活性。其中一种EET（11，12-EET）可改善长时间全脑缺血后的心功能，引起静息膜电位超极化和心脏动作电位缩短，并显著抑制心脏L-型Ca++通道活性。稳定转染CYP 2 J5 cDNA的人脐静脉内皮细胞中容量性Ca++内流显著增加。生理浓度的11，12-EET通过涉及抑制促炎转录因子NF- B的机制减弱内皮细胞活化（TNF诱导的VCAM-1表达）。此外，用CYP 2 J2 cDNA瞬时转染内皮细胞抑制NF- B介导的基因转录。总之，这些结果表明，CYP 2 J衍生的类二十烷酸可能在人类缺血性心脏病、血管炎症和动脉粥样硬化形成中很重要。人CYP 2 J2基因已被克隆、测序，并对其内含子/外显子结构进行了表征。CYP 2 J2基因似乎部分受外显子1/内含子1连接处的选择性剪接调控。已克隆了两种在多种组织中表达的可变剪接变体（CYP 2 J2-H2和CYP 2 J2-H5），并且重组蛋白显示不稳定。我们还鉴定了几种CYP 2 J2多态性变体，这些变体导致CYP 2 J2蛋白质的143、158、192和404位的氨基酸取代。初步建模研究表明，Ile 192 Asn和Asn 404 Tyr取代发生在CYP 2 J2活性位点附近，并预测会影响催化效率。这些CYP 2 J2变体将使用定点诱变产生，并作为NIEHS环境基因组计划的一部分进行表征。目前还在努力：（a）检测人CYP 2 J2启动子，包括鉴定相关的顺式作用元件;（B）检测CYP 2 J2过表达对体外心肌细胞功能的影响;（c）构建在心肌中过表达CYP 2 J2的转基因小鼠，以检测CYP 2 J2衍生的类花生酸对体内心脏功能的影响;和（d）克隆在内皮细胞和肌细胞中转导EET信号的推定受体。- 花生四烯酸类二十烷酸细胞色素450缺血性心脏病预处理-人类受试者