Molecular Cloning, Expression, and Functional Significance of a Cytochrome P450 Highly Expressed in Rat Heart Myocytes*
Molecular Cloning, Expression, and Functional Significance of a Cytochrome P450 Highly Expressed in Rat Heart Myocytes*
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DOI：
10.1074/jbc.272.19.12551
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发表时间：
1997-05
期刊：
影响因子：
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通讯作者：
Shu Wu;Weina Chen;E. Murphy;S. Gabel;K. Tomer;J. Foley;C. Steenbergen;J. Falck;C. Moomaw;D. Zeldin
中科院分区：
文献类型：
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作者：
Shu Wu;Weina Chen;E. Murphy;S. Gabel;K. Tomer;J. Foley;C. Steenbergen;J. Falck;C. Moomaw;D. Zeldin
根据大鼠假基因CYP 2 J3 P1和CYP 2 J3 P2的5′端和3′端序列设计引物，通过聚合酶链反应，从大鼠心脏和肝脏总RNA中扩增出编码P450单加氧酶的cDNA。序列分析表明，这1，778个碱基对的cDNA含有一个开放阅读框架，编码一个新的502个氨基酸的蛋白质，命名为CYP 2 J3。基于推导的氨基酸序列，CYP 2 J3与人CYP 2 J2和兔CYP 2 J1的同源性约为70%。利用杆状病毒表达系统在Sf 9昆虫细胞中共表达重组CYP 2 J3蛋白和NADPH-细胞色素P450氧化还原酶。CYP 2 J3/NADPH-细胞色素P450氧化还原酶转染细胞的微粒体部分将花生四烯酸代谢为14，15-、11，12-和8，9-环氧二十碳三烯酸和19-羟基二十碳四烯酸作为主要反应产物（催化转换，37 °C下0.2 nmol产物/nmol细胞色素P450/min）。使用抗重组CYP 2 J2（与CYP 2 J3交叉反应，但不与其他已知大鼠P450交叉反应）的多克隆抗体对从大鼠组织制备的微粒体组分进行免疫印迹，证明CYP 2 J3蛋白在心脏和肝脏中大量表达。使用抗CYP 2 J2 IgG和抗生物素蛋白-生物素-过氧化物酶检测法对福尔马林固定石蜡包埋的大鼠心脏组织切片进行免疫组织化学染色，发现CYP 2 J3主要表达于心房和心室肌细胞。在离体灌注大鼠心脏模型中，20 min全脑缺血后40 min复流仅恢复基线收缩功能的44 ± 6%。在全脑缺血前向灌流液中加入5 μm 11，12-环氧二十碳三烯酸导致心肌收缩力恢复显著改善1.6倍（基线的69 ± 5%，p = 0.01，与单独溶剂相比）。重要的是，无论是14，15-环氧二十碳三烯酸还是19-羟基二十碳四烯酸都没有显著改善全脑缺血后的功能恢复，证明了11，12-环氧二十碳三烯酸区域异构体的生物效应的特异性。基于这些数据，我们得出结论：（a）CYP 2 J3是负责大鼠心肌细胞中内源性花生四烯酸库氧化的主要酶之一，（B）11，12-环氧二十碳三烯酸可能在心脏对缺血的反应中发挥重要的功能作用。
A cDNA encoding a P450 monooxygenase was amplified from reverse transcribed rat heart and liver total RNA by polymerase chain reaction using primers based on the 5′- and 3′-end sequences of two rat pseudogenes, CYP2J3P1 andCYP2J3P2. Sequence analysis revealed that this 1,778-base pair cDNA contained an open reading frame and encoded a new 502 amino acid protein designated CYP2J3. Based on the deduced amino acid sequence, CYP2J3 was approximately 70% homologous to both human CYP2J2 and rabbit CYP2J1. Recombinant CYP2J3 protein was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 insect cells using a baculovirus expression system. Microsomal fractions of CYP2J3/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolized arachidonic acid to 14,15-, 11,12-, and 8,9-epoxyeicosatrienoic acids and 19-hydroxyeicosatetraenoic acid as the principal reaction products (catalytic turnover, 0.2 nmol of product/nmol of cytochrome P450/min at 37 °C). Immunoblotting of microsomal fractions prepared from rat tissues using a polyclonal antibody raised against recombinant CYP2J2 that cross-reacted with CYP2J3 but not with other known rat P450s demonstrated abundant expression of CYP2J3 protein in heart and liver. Immunohistochemical staining of formalin-fixed paraffin-embedded rat heart tissue sections using the anti-CYP2J2 IgG and avidin-biotin-peroxidase detection localized expression of CYP2J3 primarily to atrial and ventricular myocytes. In an isolated-perfused rat heart model, 20 min of global ischemia followed by 40 min of reflow resulted in recovery of only 44 ± 6% of base-line contractile function. The addition of 5 μm 11,12-epoxyeicosatrienoic acid to the perfusate prior to global ischemia resulted in a significant 1.6-fold improvement in recovery of cardiac contractility (69 ± 5% of base line,p = 0.01 versus vehicle alone). Importantly, neither 14,15-epoxyeicosatrienoic acid nor 19-hydroxyeicosatetraenoic acid significantly improved functional recovery following global ischemia, demonstrating the specificity of the biological effect for the 11,12-epoxyeicosatrienoic acid regioisomer. Based on these data, we conclude that (a) CYP2J3 is one of the predominant enzymes responsible for the oxidation of endogenous arachidonic acid pools in rat heart myocytes and (b) 11,12-epoxyeicosatrienoic acid may play an important functional role in the response of the heart to ischemia.