CARDIAC CYTOCHROME P450 ARACHIDONIC ACID EPOXYGENASE PATHWAY

尽管我们将大部分精力集中在人类 CYP2J2 和小鼠 CYP2J5 上，但我们已经发现了许多哺乳动物 CYP2J 亚家族成员。人类 CYP2J2 cDNA 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表征。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，并且在 AA 到 EET 的代谢中很活跃。其中一种 EET (11,12-EET) 可改善长期全身缺血后的心脏功能，导致静息膜电位超极化和心脏动作电位缩短，并显着抑制心脏 L 型 Ca 通道活性。在稳定转染 CYP2J5 cDNA 的人脐静脉内皮细胞中，电容性 Ca 进入显着增加。生理浓度的 11,12-EET 通过抑制促炎转录因子 NF-B 的机制减弱内皮细胞活化（TNF 诱导的 VCAM-1 表达）。此外，用 CYP2J2 cDNA 瞬时转染内皮细胞可抑制 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发挥重要作用。人类 CYP2J2 基因的内含子/外显子组织已被克隆、测序和表征。 CYP2J2 基因似乎部分受到外显子 1/内含子 1 连接处的选择性剪接的调节。已经克隆了在多种组织中表达的两种可变剪接变体（CYP2J2-H2和CYP2J2-H5），并且重组蛋白被证明是不稳定的。我们还鉴定了几种 CYP2J2 多态性变体，这些变体导致 CYP2J2 蛋白的位置 143、158、192 和 404 处的氨基酸取代。初步建模研究表明，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 变体将使用定点诱变生成，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) 构建心肌中过度表达 CYP2J2 的转基因小鼠，以检查 CYP2J2 衍生的类二十烷酸对体内心脏功能的影响； (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

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