IDENTIFICATION OF MOUSE CYP2C INVOLVED IN ARACHIDONIC ACID

目的：对CYP 2C的内源性功能知之甚少。我们推测这些酶在生理上重要的内源性底物如花生四烯酸和视黄酸（维生素A）的代谢中具有重要作用。我们的目标是鉴定小鼠中的所有CYP 2C，确定它们对特定类花生酸代谢物产生的贡献，并鉴定它们在这些组织中的生理作用。已知P450衍生的AA代谢物在许多组织中具有强效的生物学作用。这些效应取决于产物的区域和立体化学。我们正试图确定这些异构体的器官和细胞特异性定位。一个长期的目标是产生敲除小鼠来研究这类酶的生理重要性，并在细胞系中过表达这些酶以进一步建立它们的功能。我们已经在小鼠中鉴定了五种（可能是六种）CYP 2C。我们在cDNA表达系统中表达了其中的五种，检测了它们在形成环氧二十碳三烯酸（epoxyeicosatrienoic acids，ETES）和羟基二十碳四烯酸（hydroxyyeicosatrienoic acids，HETES）中的区域和立体特异性。发现它们产生非常特异的区域特异性和立体特异性产物。CYP 2C 29是主要的肝脏形式，产生14，15-EET。Western blotting和RT-PCR检测结果表明，肺组织中含有相对大量的CYP 2C 29。CYP 2C 40在结肠、盲肠、肠道中含量较高，在肾脏和心脏中也有发现。发现CYP 2C 40将花生四烯酸（AA）代谢为16 R-HETE（66%）和16 S-HETE（34%）。这是发现的第一个生产这种HETE作为主要产品的P450。16 R-HETE被认为在诸如肾血管舒张和抑制中性粒细胞聚集和粘附的过程中是重要的。肾脏含有CYP 2C 40和一种新的未鉴别的CYP 2C亚型。动脉内皮细胞含有CYP 2C 29和部分CYP 2C 40。- 内源性化合物，花生四烯酸，维甲酸，维生素A，环氧二十碳三烯酸

AIMS: Little is known about the endogenous function of the CYP2Cs. We postulate that these enzymes have important roles in the metabolism of physiologically important endogenous substrates such as arachidonic acid and retinoic acid (Vitamin A). Our goal has been to identify all CYP2Cs in the mouse, determine their contribution to the production of particular eicosanoid metabolites and to identify their physiological role in these tissues. The P450-derived AA metabolites are known to possess potent biological actions in numerous tissues. These effects depend on the regio- and stereochemistry of the products. We are attempting to determine the organ and cell-specific localization of these isoforms. A long-range goal is to produce knockout mice to study the physiological importance of this class of enzymes, and to overexpress these enzymes in cell lines to further establish their function. ACCOMPLISHMENTS We have identified five (potentially six) CYP2Cs in the mouse. We expressed five of these in cDNA expression systems examined their regio and stereospecificity in the formation of epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETES). They were found to produce very specific regiospecific and stereospecific products. CYP2C29 is the predominant hepatic form and produces 14,15-EET. Western blotting and RT-PCR indicted that lung contained relatively large amounts of CYP2C29. CYP2C40 was found in high amounts in colon, cecum, gut, and was also found in kidney and heart. CYP2C40 was found to metabolize arachidonic acid (AA) to 16R- HETE (66%) and 16S-HETE (34%). This is the first P450 found to produce this HETE as a primary product. 16R-HETE is thought to be important in processes such as renal vasodilation and inhibiting neutrophil aggregation and adhesion. Kidney contained CYP2C40 and a new unidentified CYP2C isoform. Endothelial cells of arteries contained CYP2C29 and some CYP2C40. - endogenous compound, arachidonic acid, retinoic acid, Vitamin A, epoxyeicosatrienoic acid