Limits...
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.

Show MeSH

Related in: MedlinePlus

NO inactivation by glia requires NADPH and is dependent on a membrane protein(A) Typical NO profiles on addition of 100 μM DETA/NO to buffer aloneor 1 mg/ml cultured cerebellar glia ±0.5 μM DPI. (B)Representative traces of NO accumulation following sequential applications of 100 μMDETA/NO (at t=0) and 100 μM NADPH (open arrows) to buffer, gliallysate and, following centrifugation at 53000 rev./min using a TLA-100.2 rotor for1 h, cytosolic supernatant plus membrane pellet, the membrane pellet and supernatant alone.Lysed cells only inactivate NO after addition of NADPH. This activity remains in the pellet afterhigh-speed centrifugation. (C) Summary of results from (B). Buffer andcytosol are not significantly different from each other, but were different from the membranepellet, resuspended lysate and control lysate (repeated measures using ANOVA with Tukey posthoc tests, P<0.001; n=4). (D) Summaryof results when 100 μM DETA/NO and 100 μM NADPH were added to membranesfrom glia, synaptosomes and whole brain (all at 1 mg/ml). Results are normalized to the NOconcentration reached on each experimental day when 100 μM DETA/NO was added to bufferalone (n=4). (E) NO levels following addition of 100 μMDETA/NO and 100 μM NADPH to synaptosome and glial membranes at 1 mg ofprotein/ml before and after incubation at 37 °C for30 min±0.5 mg/ml proteinase K. NADPH decreased NO levels in controls but not inproteinase K-treated membranes (P=0.20; glial membranes: n=8;synaptosome membranes n=3). The incubation at 37 °C has caused somedecrease in the activity of the controls compared with that observed in (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2662488&req=5

Figure 1: NO inactivation by glia requires NADPH and is dependent on a membrane protein(A) Typical NO profiles on addition of 100 μM DETA/NO to buffer aloneor 1 mg/ml cultured cerebellar glia ±0.5 μM DPI. (B)Representative traces of NO accumulation following sequential applications of 100 μMDETA/NO (at t=0) and 100 μM NADPH (open arrows) to buffer, gliallysate and, following centrifugation at 53000 rev./min using a TLA-100.2 rotor for1 h, cytosolic supernatant plus membrane pellet, the membrane pellet and supernatant alone.Lysed cells only inactivate NO after addition of NADPH. This activity remains in the pellet afterhigh-speed centrifugation. (C) Summary of results from (B). Buffer andcytosol are not significantly different from each other, but were different from the membranepellet, resuspended lysate and control lysate (repeated measures using ANOVA with Tukey posthoc tests, P<0.001; n=4). (D) Summaryof results when 100 μM DETA/NO and 100 μM NADPH were added to membranesfrom glia, synaptosomes and whole brain (all at 1 mg/ml). Results are normalized to the NOconcentration reached on each experimental day when 100 μM DETA/NO was added to bufferalone (n=4). (E) NO levels following addition of 100 μMDETA/NO and 100 μM NADPH to synaptosome and glial membranes at 1 mg ofprotein/ml before and after incubation at 37 °C for30 min±0.5 mg/ml proteinase K. NADPH decreased NO levels in controls but not inproteinase K-treated membranes (P=0.20; glial membranes: n=8;synaptosome membranes n=3). The incubation at 37 °C has caused somedecrease in the activity of the controls compared with that observed in (C).

Mentions: The NO-consuming activity of brain tissue was assessed by comparing the NO concentration profilein control buffer and various preparations of brain tissue on application of the NO donor DETA/NO.Since this compound releases NO with a half-life of 20.5 h, the rate of release isessentially constant over the time-course of these experiments. The NO concentration initially risesbut reaches a steady concentration when the rate of release equals the rate of breakdown. In controlbuffer, NO is consumed by reaction with O2 (autoxidation; [22]), but in the presence of glia cultured from the cerebellum, NO reached a lowersteady-state concentration, signifying faster consumption (Figure1A; [18]).


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

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

NO inactivation by glia requires NADPH and is dependent on a membrane protein(A) Typical NO profiles on addition of 100 μM DETA/NO to buffer aloneor 1 mg/ml cultured cerebellar glia ±0.5 μM DPI. (B)Representative traces of NO accumulation following sequential applications of 100 μMDETA/NO (at t=0) and 100 μM NADPH (open arrows) to buffer, gliallysate and, following centrifugation at 53000 rev./min using a TLA-100.2 rotor for1 h, cytosolic supernatant plus membrane pellet, the membrane pellet and supernatant alone.Lysed cells only inactivate NO after addition of NADPH. This activity remains in the pellet afterhigh-speed centrifugation. (C) Summary of results from (B). Buffer andcytosol are not significantly different from each other, but were different from the membranepellet, resuspended lysate and control lysate (repeated measures using ANOVA with Tukey posthoc tests, P<0.001; n=4). (D) Summaryof results when 100 μM DETA/NO and 100 μM NADPH were added to membranesfrom glia, synaptosomes and whole brain (all at 1 mg/ml). Results are normalized to the NOconcentration reached on each experimental day when 100 μM DETA/NO was added to bufferalone (n=4). (E) NO levels following addition of 100 μMDETA/NO and 100 μM NADPH to synaptosome and glial membranes at 1 mg ofprotein/ml before and after incubation at 37 °C for30 min±0.5 mg/ml proteinase K. NADPH decreased NO levels in controls but not inproteinase K-treated membranes (P=0.20; glial membranes: n=8;synaptosome membranes n=3). The incubation at 37 °C has caused somedecrease in the activity of the controls compared with that observed in (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2662488&req=5

Figure 1: NO inactivation by glia requires NADPH and is dependent on a membrane protein(A) Typical NO profiles on addition of 100 μM DETA/NO to buffer aloneor 1 mg/ml cultured cerebellar glia ±0.5 μM DPI. (B)Representative traces of NO accumulation following sequential applications of 100 μMDETA/NO (at t=0) and 100 μM NADPH (open arrows) to buffer, gliallysate and, following centrifugation at 53000 rev./min using a TLA-100.2 rotor for1 h, cytosolic supernatant plus membrane pellet, the membrane pellet and supernatant alone.Lysed cells only inactivate NO after addition of NADPH. This activity remains in the pellet afterhigh-speed centrifugation. (C) Summary of results from (B). Buffer andcytosol are not significantly different from each other, but were different from the membranepellet, resuspended lysate and control lysate (repeated measures using ANOVA with Tukey posthoc tests, P<0.001; n=4). (D) Summaryof results when 100 μM DETA/NO and 100 μM NADPH were added to membranesfrom glia, synaptosomes and whole brain (all at 1 mg/ml). Results are normalized to the NOconcentration reached on each experimental day when 100 μM DETA/NO was added to bufferalone (n=4). (E) NO levels following addition of 100 μMDETA/NO and 100 μM NADPH to synaptosome and glial membranes at 1 mg ofprotein/ml before and after incubation at 37 °C for30 min±0.5 mg/ml proteinase K. NADPH decreased NO levels in controls but not inproteinase K-treated membranes (P=0.20; glial membranes: n=8;synaptosome membranes n=3). The incubation at 37 °C has caused somedecrease in the activity of the controls compared with that observed in (C).
Mentions: The NO-consuming activity of brain tissue was assessed by comparing the NO concentration profilein control buffer and various preparations of brain tissue on application of the NO donor DETA/NO.Since this compound releases NO with a half-life of 20.5 h, the rate of release isessentially constant over the time-course of these experiments. The NO concentration initially risesbut reaches a steady concentration when the rate of release equals the rate of breakdown. In controlbuffer, NO is consumed by reaction with O2 (autoxidation; [22]), but in the presence of glia cultured from the cerebellum, NO reached a lowersteady-state concentration, signifying faster consumption (Figure1A; [18]).

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