Cytochrome P450 oxidoreductase participates in nitric oxide consumption by rat brain.
In low nanomolar concentrations, NO (nitric oxide) functions as a transmitter in brain and other tissues, whereas near-micromolar NO concentrations are associated with toxicity and cell death. Control of the NO concentration, therefore, is critical for proper brain function, but, although its synthesis pathway is well-characterized, the major route of breakdown of NO in brain is unclear. Previous observations indicate that brain cells actively consume NO at a high rate. The mechanism of this consumption was pursued in the present study. NO consumption by a preparation of central glial cells was abolished by cell lysis and recovered by addition of NADPH. NADPH-dependent consumption of NO localized to cell membranes and was inhibited by proteinase K, indicating the involvement of a membrane-bound protein. Purification of this activity yielded CYPOR (cytochrome P450 oxidoreductase). Antibodies against CYPOR inhibited NO consumption by brain membranes and the amount of CYPOR in several cell types correlated with their rate of NO consumption. NO was also consumed by purified CYPOR but this activity was found to depend on the presence of the vitamin E analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), included in the buffer as a precaution against inadvertent NO consumption by lipid peroxidation. In contrast, NO consumption by brain membranes was independent of Trolox. Hence, it appears that, during the purification process, CYPOR becomes separated from a partner needed for NO consumption. Cytochrome P450 inhibitors inhibited NO consumption by brain membranes, making these proteins likely candidates.
Hall
Catherine N
CN
Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, UK. catherine.hall@ucl.ac.uk
Keynes
Robert G
RG
Garthwaite
John
J
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D045744:Cell Line, Tumor; D002839:Chromans / Q000494:pharmacology; D003022:Clotrimazole / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005420:Flavoproteins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D009251:NADPH-Ferrihemoprotein Reductase / Q000276:immunology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D000906:Antibodies; D002839:Chromans; D005420:Flavoproteins; D009569:Nitric Oxide; D009251:NADPH-Ferrihemoprotein Reductase; D003022:Clotrimazole; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid