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J. Biol. Chem., Vol. 281, Issue 18, 12546-12554, May 5, 2006

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Characterization of the Mechanism of Cytochrome P450 Reductase-Cytochrome P450-mediated Nitric Oxide and Nitrosothiol Generation from Organic Nitrates^*

From the Center for Biomedical EPR Spectroscopy and Imaging, Davis Heart and Lung Research Institute, and the Division of Cardiovascular Medicine, Department of Internal Medicine, Ohio State University College of Medicine, Columbus, Ohio 43210-1252

Mammalian cytochrome P450 reductase (CPR) and cytochrome P450 (CP) play important roles in organic nitrate bioactivation; however, the mechanism by which they convert organic nitrate to NO remains unknown. Questions remain regarding the initial precursor of NO that serves to link organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of CPR-CP-mediated organic nitrate bioactivation, EPR, chemiluminescence NO analyzer, NO electrode, and immunoassay studies were performed. With rat hepatic microsomes or purified CPR, the presence of NADPH triggered organic nitrate reduction to . The CPR flavin site inhibitor diphenyleneiodonium inhibited this generation, whereas the CP inhibitor clotrimazole did not. However, clotrimazole greatly inhibited -dependent NO generation. Therefore, CPR catalyzes organic nitrate reduction, producing nitrite, whereas CP can mediate further nitrite reduction to NO. Nitrite-dependent NO generation contributed <10% of the CPR-CP-mediated NO generation from organic nitrates; thus, is not the main precursor of NO. CPR-CP-mediated NO generation was largely thiol-dependent. Studies suggested that organic nitrite (R-O-NO) was produced from organic nitrate reduction by CPR. Further reaction of organic nitrite with free or microsome-associated thiols led to NO or nitrosothiol generation and thus stimulated the activation of sGC. Thus, organic nitrite is the initial product in the process of CRP-CP-mediated organic nitrate activation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation.

Received for publication, November 2, 2005 , and in revised form, March 2, 2006.

^* This work was supported by National Institutes of Health Grants HL63744, HL65608, and HL38324 (to J. L. Z.) and by Ohio Valley Affiliate of the American Heart Association Grant BGIA-0565249B (to H. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed: 110G Davis Heart and Lung Research Inst., 473 West 12th Ave., Columbus, OH 43210-1252. Tel.: 614-247-7857; Fax: 614-247-7845. E-mail: haitao.li{at}osumc.edu. ² To whom correspondence may be addressed: 110G Davis Heart and Lung Research Inst., 473 West 12th Ave., Columbus, OH 43210-1252. Tel.: 614-247-7857; Fax: 614-247-7845; E-mail: jay.zweier{at}osumc.edu.

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