CARDIAC CYTOCHROME P450 ARACHIDONIC ACID EPOXYGENASE PATHWAY

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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