CARDIAC CYTOCHROME P450 ARACHIDONIC ACID EPOXYGENASE PATHWAY

我们已经发现了一些哺乳动物的CYP2J亚家族成员，尽管我们主要集中在人类的CYP2J2和小鼠的CYP2J5上。人CYP2J2基因已被克隆、表达，并对其组织分布、细胞定位和功能进行了广泛的研究。这个酶似乎是人类在心脏表达的主要P450，它定位于心肌细胞和内皮细胞，并在AA到EETs的代谢中发挥作用。其中一种内毒素(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，并显著抑制心肌L型钙通道的活动。稳定表达CYP2J5基因的人脐静脉内皮细胞电容性钙内流显著增加。生理浓度的11，12-EET通过抑制促炎转录因子NF-B来抑制内皮细胞的激活(肿瘤坏死因子诱导的VCAM-1表达)。此外，瞬时转染组内皮细胞可抑制NF-B介导的基因转录。综上所述，这些结果表明，CYP2J衍生的二十烷基类化合物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起重要作用。人类CYP2J2基因已被克隆、测序，并对其内含子/外显子的组织结构进行了研究。通过外显子1/内含子1交界处的选择性剪接，似乎部分地调控了CYP2J2基因。在多种组织中表达的两种选择性剪接变异体(CYP2J2-H2和CYP2J2-H5)已经被克隆，重组蛋白被证明是不稳定的。我们还发现了几个导致CYP2J2蛋白第143、158、192和404位氨基酸替换的多态变异。初步的模拟研究表明，Ile192Asn和Asn404Tyr替换发生在CYP2J2活性中心附近，预计会影响催化效率。这些CYP2J2变异体将使用定点突变产生，并被鉴定为NIEHS环境基因组计划的一部分。目前还在进行以下工作：(A)检测人的CYP2J2启动子，包括鉴定相关的顺式作用元件；(B)在体外检测CYP2J2过表达对心肌细胞功能的影响；(C)构建在心肌中过表达CYP2J2的转基因小鼠，以检测由CYP2J2衍生的二十烷类化合物对体内心脏功能的影响；以及(D)克隆在内皮细胞和心肌细胞中传导EET信号的可能的受体。-花生四烯酸二十烷类细胞色素450缺血性心脏病预适应-人类

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