MOLECULAR-CLONING, EXPRESSION, AND FUNCTIONAL-SIGNIFICANCE OF A CYTOCHROME-P450 HIGHLY EXPRESSED IN RAT-HEART MYOCYTES

A cDNA encoding a P450 monooxygenase was amplified from reverse transc ribed 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 and CYP2J3P2. Sequence analysis revealed that this 1,778-bas e pair cDNA contained an open reading frame and encoded a new 502 amin o acid protein designated CYP2J3. Based on the deduced amino acid sequ ence, 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 baculoviru s expression system. Microsomal fractions of CYP2J3/NADPH-cytochrome P 450 oxidoreductase-transfected cells metabolized arachidonic acid to 1 4,15-, 11,12-, and 8,9-epoxyeicosatrienoic acids and 19-hydroxyeicosat etraenoic acid as the principal reaction products (catalytic turnover, 0.2 nmol of product/nmol of cytochrome P450/min at 37 degrees C). Imm unoblotting of microsomal fractions prepared from rat tissues using a polyclonal antibody raised against recombinant CYP2J2 that cross-react ed with CYP2J3 but not with other known rat P450s demonstrated abundan t expression of CYP2J3 protein in heart and liver, Immunohistochemical staining of formalin-fixed paraffin-embedded rat heart tissue section s using the anti-CYP2J2 IgG and avidin-biotin-peroxidase detection loc alized expression of CYP2J3 primarily to atrial and ventricular myocyt es, 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% o f base-line contractile function, The addition of 5 mu M 11,12-epoxyei cosatrienoic acid to the perfusate prior to global ischemia resulted i n a significant 1.6 fold improvement in recovery of cardiac contractil ity (69 +/- 5% of base line, p = 0.01 versus vehicle alone). Important ly, neither 14,15-epoxyeicosatrienoic acid nor 19-hydroxyeicosatetraen oic acid significantly improved functional recovery following global i schemia, demonstrating the specificity of the biological effect for th e 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 m yocytes and (b) 11,12-epoxyeicosatrienoic acid may play an important f unctional role in the response of the heart to ischemia.