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Cytochrome P450 oxidoreductase participates in nitric oxide consumption by rat brain.

Hall CN, Keynes RG, Garthwaite J - Biochem. J. (2009)

Bottom Line: Purification of this activity yielded CYPOR (cytochrome P450 oxidoreductase).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.Cytochrome P450 inhibitors inhibited NO consumption by brain membranes, making these proteins likely candidates.

View Article: PubMed Central - PubMed

Affiliation: Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, UK. catherine.hall@ucl.ac.uk

ABSTRACT
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.

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CYPOR requires a partner to inactivate NO(A) Plateau NO concentrations following application of NADPH to 300 μMDETA/NO and purified CYPOR (100 m-units/ml) or brain membranes (2 mg ofprotein/ml)±DTPA and Trolox (both 100 μM). In the case of purified CYPOR, NADPHreduced the NO concentration (and therefore increased NO consumption) only when DTPA and Trolox werepresent, whereas DTPA and Trolox had no effect on NADPH-dependent NO consumption by brain membranes.n=4. (B) Example traces showing the NO profile when300 μM DETA/NO was added to 100 m-units/ml purified CYPOR. Trolox, but not DTPA (both100 μM), was required for NADPH-dependent NO consumption. (C) The effectof 100 μM NADPH addition to 300 μM DETA/NO±100 m-units/ml CYPORand NADPH plus different Trolox concentrations. A logistic fit to the data (continuous line) givesan EC50 for Trolox of 270 μM. n=4. (D) Thedecrease in NO concentration on the addition of NADPH to brain membranes (2 mg/ml) plus300 μM DETA/NO was reduced by the cytochrome P450 inhibitors clotrimazole (squares)and ketaconazole (circles). IC50 values were calculated from logistic fits to the data(solid lines). n=4.
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Figure 3: CYPOR requires a partner to inactivate NO(A) Plateau NO concentrations following application of NADPH to 300 μMDETA/NO and purified CYPOR (100 m-units/ml) or brain membranes (2 mg ofprotein/ml)±DTPA and Trolox (both 100 μM). In the case of purified CYPOR, NADPHreduced the NO concentration (and therefore increased NO consumption) only when DTPA and Trolox werepresent, whereas DTPA and Trolox had no effect on NADPH-dependent NO consumption by brain membranes.n=4. (B) Example traces showing the NO profile when300 μM DETA/NO was added to 100 m-units/ml purified CYPOR. Trolox, but not DTPA (both100 μM), was required for NADPH-dependent NO consumption. (C) The effectof 100 μM NADPH addition to 300 μM DETA/NO±100 m-units/ml CYPORand NADPH plus different Trolox concentrations. A logistic fit to the data (continuous line) givesan EC50 for Trolox of 270 μM. n=4. (D) Thedecrease in NO concentration on the addition of NADPH to brain membranes (2 mg/ml) plus300 μM DETA/NO was reduced by the cytochrome P450 inhibitors clotrimazole (squares)and ketaconazole (circles). IC50 values were calculated from logistic fits to the data(solid lines). n=4.

Mentions: As CYPOR seems to be involved in NO consumption by brain membranes, we tested whether purifiedCYPOR could also consume NO in an NADPH-dependent manner. In contrast with brain membranes, purifiedCYPOR did not consume NO upon addition of NADPH, unless Trolox and DTPA (100 μM) werepresent in the reaction mix (Figure 3A). Further study revealedthat the critical component was Trolox, which concentration-dependently enabled NO consumption bypurified CYPOR, whereas DTPA did not (Figures 3B and 3C). Other antioxidants, edaravone (500 μM) andphenothiazine (200 μM), could not sustain NO consumption by CYPOR (results not shown),indicating that this is a special property of Trolox rather than a general antioxidant effect.


Cytochrome P450 oxidoreductase participates in nitric oxide consumption by rat brain.

Hall CN, Keynes RG, Garthwaite J - Biochem. J. (2009)

CYPOR requires a partner to inactivate NO(A) Plateau NO concentrations following application of NADPH to 300 μMDETA/NO and purified CYPOR (100 m-units/ml) or brain membranes (2 mg ofprotein/ml)±DTPA and Trolox (both 100 μM). In the case of purified CYPOR, NADPHreduced the NO concentration (and therefore increased NO consumption) only when DTPA and Trolox werepresent, whereas DTPA and Trolox had no effect on NADPH-dependent NO consumption by brain membranes.n=4. (B) Example traces showing the NO profile when300 μM DETA/NO was added to 100 m-units/ml purified CYPOR. Trolox, but not DTPA (both100 μM), was required for NADPH-dependent NO consumption. (C) The effectof 100 μM NADPH addition to 300 μM DETA/NO±100 m-units/ml CYPORand NADPH plus different Trolox concentrations. A logistic fit to the data (continuous line) givesan EC50 for Trolox of 270 μM. n=4. (D) Thedecrease in NO concentration on the addition of NADPH to brain membranes (2 mg/ml) plus300 μM DETA/NO was reduced by the cytochrome P450 inhibitors clotrimazole (squares)and ketaconazole (circles). IC50 values were calculated from logistic fits to the data(solid lines). n=4.
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Related In: Results  -  Collection

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Figure 3: CYPOR requires a partner to inactivate NO(A) Plateau NO concentrations following application of NADPH to 300 μMDETA/NO and purified CYPOR (100 m-units/ml) or brain membranes (2 mg ofprotein/ml)±DTPA and Trolox (both 100 μM). In the case of purified CYPOR, NADPHreduced the NO concentration (and therefore increased NO consumption) only when DTPA and Trolox werepresent, whereas DTPA and Trolox had no effect on NADPH-dependent NO consumption by brain membranes.n=4. (B) Example traces showing the NO profile when300 μM DETA/NO was added to 100 m-units/ml purified CYPOR. Trolox, but not DTPA (both100 μM), was required for NADPH-dependent NO consumption. (C) The effectof 100 μM NADPH addition to 300 μM DETA/NO±100 m-units/ml CYPORand NADPH plus different Trolox concentrations. A logistic fit to the data (continuous line) givesan EC50 for Trolox of 270 μM. n=4. (D) Thedecrease in NO concentration on the addition of NADPH to brain membranes (2 mg/ml) plus300 μM DETA/NO was reduced by the cytochrome P450 inhibitors clotrimazole (squares)and ketaconazole (circles). IC50 values were calculated from logistic fits to the data(solid lines). n=4.
Mentions: As CYPOR seems to be involved in NO consumption by brain membranes, we tested whether purifiedCYPOR could also consume NO in an NADPH-dependent manner. In contrast with brain membranes, purifiedCYPOR did not consume NO upon addition of NADPH, unless Trolox and DTPA (100 μM) werepresent in the reaction mix (Figure 3A). Further study revealedthat the critical component was Trolox, which concentration-dependently enabled NO consumption bypurified CYPOR, whereas DTPA did not (Figures 3B and 3C). Other antioxidants, edaravone (500 μM) andphenothiazine (200 μM), could not sustain NO consumption by CYPOR (results not shown),indicating that this is a special property of Trolox rather than a general antioxidant effect.

Bottom Line: Purification of this activity yielded CYPOR (cytochrome P450 oxidoreductase).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.Cytochrome P450 inhibitors inhibited NO consumption by brain membranes, making these proteins likely candidates.

View Article: PubMed Central - PubMed

Affiliation: Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, UK. catherine.hall@ucl.ac.uk

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus