CARDIAC CYTOCHROME P450 ARACHIDONIC ACID EPOXYGENASE PATHWAY

我们已经发现了许多哺乳动物CYP2J亚家族成员，尽管我们的大部分工作都集中在人类CYP2J2和小鼠CYP2J5上。人类CYP2J2 cDNA已被克隆、表达，并在组织分布、细胞定位和功能等方面得到了广泛的研究。这种酶似乎是在心脏中表达的主要人类P450，它定位于心肌细胞和内皮细胞，并在AA到eet的代谢中发挥活性。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。在稳定转染CYP2J5 cDNA的人脐静脉内皮细胞中，ca++进入能力显著增加。11,12- eet的生理浓度通过抑制促炎转录因子NF- B的机制减弱内皮细胞的活化（TNF -诱导的VCAM-1表达）。此外，短暂转染内皮细胞CYP2J2 cDNA可抑制NF- B介导的基因转录。总之，这些结果表明cyp2j衍生的类二十烷可能在人类缺血性心脏病、血管炎症和动脉粥样硬化中起重要作用。人类CYP2J2基因已被克隆，测序和表征其内含子/外显子的组织。CYP2J2基因似乎在一定程度上受到外显子1/内含子1连接处的选择性剪接的调节。两个备选剪接变体（CYP2J2-H2和CYP2J2-H5）在多个组织中表达，重组蛋白被证明是不稳定的。我们还发现了几个CYP2J2多态性变异，导致CYP2J2蛋白的143、158、192和404位置的氨基酸替换。初步的建模研究表明，Ile192 Asn和Asn404 Tyr取代发生在CYP2J2活性位点附近，预计会影响催化效率。这些CYP2J2变异将通过定点诱变产生，并作为NIEHS环境基因组计划的一部分进行表征。目前正在进行的工作还包括：(a)检查人类CYP2J2启动子，包括鉴定相关的顺式作用元件；(b)体外检测CYP2J2过表达对心肌细胞功能的影响；(c)构建心肌过表达CYP2J2的转基因小鼠，在体内检测CYP2J2来源的类二十烷醇对心功能的影响；(d)克隆内皮细胞和肌细胞中已知的传导EET信号的受体。-花生四烯酸类二十烷细胞色素450缺血性心脏病预处理-人类受试者

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