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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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NADPH-dependent NO consumption involves CYPOR(A) 300 μM DETA/NO was added to membranes from different cell types(as labelled) in cell incubation buffer, with DTPA but no Trolox. 1 Buffer alone; 2, T47D wild-typecells (0.05 mg of protein/ml); 3, white blood cells (2 mg of protein/ml); 4, platelets(2 mg of protein/ml); 5, crude brain (0.5 mg protein/ml); 6, partially purified brain(0.42 mg protein/m); 7, glia (1 mg of protein/ml); 8, T47D transfected cells(0.05 mg of protein/ml). Note that the experiment with T47D wild-type and transfected cellswas carried out on two separate occasions, so there are duplicates of the data from these celltypes. The change in NO level on subsequent addition of NADPH was plotted against CYPOR content.CYPOR content significantly correlates with NO consumption (Pearson's r=0.85, whichis significantly different from zero; P=0.002, n=3–6).(B) To assay cytochrome c reduction by CYPOR, absorbance at550 nm was followed with time. After incubation for 2 h on ice with buffer (closedcircles), absorbance increased linearly. The rate was only slightly slower when incubated with1 mg/ml IgG (open triangles), but was greatly reduced by incubation with 0.33 or1 mg/ml of a CYPOR antibody (filled triangles and open circles, respectively).(C) NO concentration reached after 100 μM DETA/NO addition to brainmembranes at 1 mg of protein/ml, following incubation for 2 h on ice with controlbuffer, 0.33 mg/ml of γ-IgG or 0.33 mg/ml of α-CYPOR, before (open bars)and after (solid bars) addition of 100 μM NADPH. The change in NO on NADPH additionwas significantly less following incubation with α-CYPOR (one-way ANOVA with Tukeypost-hoc tests; P<0.001 compared with buffer and IgG;n=4), but was not affected by incubation with γ-IgG(P>0.05 compared with buffer). (D) NO concentration reachedafter sequential additions of 100 μM DETA/NO, 100 μM NADPH and50 μM DPI to synaptosome membranes (1 mg protein/ml). DPI significantlyinhibits NADPH-dependent NO consumption (one-way ANOVA with Tukey post-hoc tests:NADPH compared with DPI, P<0.01; DPI compared with DETA/NO,P>0.05; n=6). DPI and α-CYPOR inhibit the NADPHeffect to the same extent (Student's t test: 100 μM DETA/NO plus100 μM NADPH plus 50 μM DPI compared with 100 μM DETA/NOplus 100 μM NADPH plus 0.33 mg/ml of α-CYPOR; P=0.41,n=4–6).
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Figure 2: NADPH-dependent NO consumption involves CYPOR(A) 300 μM DETA/NO was added to membranes from different cell types(as labelled) in cell incubation buffer, with DTPA but no Trolox. 1 Buffer alone; 2, T47D wild-typecells (0.05 mg of protein/ml); 3, white blood cells (2 mg of protein/ml); 4, platelets(2 mg of protein/ml); 5, crude brain (0.5 mg protein/ml); 6, partially purified brain(0.42 mg protein/m); 7, glia (1 mg of protein/ml); 8, T47D transfected cells(0.05 mg of protein/ml). Note that the experiment with T47D wild-type and transfected cellswas carried out on two separate occasions, so there are duplicates of the data from these celltypes. The change in NO level on subsequent addition of NADPH was plotted against CYPOR content.CYPOR content significantly correlates with NO consumption (Pearson's r=0.85, whichis significantly different from zero; P=0.002, n=3–6).(B) To assay cytochrome c reduction by CYPOR, absorbance at550 nm was followed with time. After incubation for 2 h on ice with buffer (closedcircles), absorbance increased linearly. The rate was only slightly slower when incubated with1 mg/ml IgG (open triangles), but was greatly reduced by incubation with 0.33 or1 mg/ml of a CYPOR antibody (filled triangles and open circles, respectively).(C) NO concentration reached after 100 μM DETA/NO addition to brainmembranes at 1 mg of protein/ml, following incubation for 2 h on ice with controlbuffer, 0.33 mg/ml of γ-IgG or 0.33 mg/ml of α-CYPOR, before (open bars)and after (solid bars) addition of 100 μM NADPH. The change in NO on NADPH additionwas significantly less following incubation with α-CYPOR (one-way ANOVA with Tukeypost-hoc tests; P<0.001 compared with buffer and IgG;n=4), but was not affected by incubation with γ-IgG(P>0.05 compared with buffer). (D) NO concentration reachedafter sequential additions of 100 μM DETA/NO, 100 μM NADPH and50 μM DPI to synaptosome membranes (1 mg protein/ml). DPI significantlyinhibits NADPH-dependent NO consumption (one-way ANOVA with Tukey post-hoc tests:NADPH compared with DPI, P<0.01; DPI compared with DETA/NO,P>0.05; n=6). DPI and α-CYPOR inhibit the NADPHeffect to the same extent (Student's t test: 100 μM DETA/NO plus100 μM NADPH plus 50 μM DPI compared with 100 μM DETA/NOplus 100 μM NADPH plus 0.33 mg/ml of α-CYPOR; P=0.41,n=4–6).

Mentions: To prepare the suspension of mixed glia for studies of NO consumption, dishes were washed with∼100 ml of cell incubation buffer (20 mM Tris/HCl, 130 mM NaCl,5 mM KCl, 1.2 mM Na2HPO4 and 11 mM glucose, adjusted topH 7.45 at 37 °C) and incubated with 30 ml of 0.05% (w/v) trypsin,0.53 mM EDTA in HBSS (Hanks balanced salt solution) for 15 min at 37 °Cto dissociate the cells, which were washed and resuspended at 3×106 cells/ml inincubation buffer. Cell viability was verified at more than 95% based on Trypan Blue staining.


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

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

NADPH-dependent NO consumption involves CYPOR(A) 300 μM DETA/NO was added to membranes from different cell types(as labelled) in cell incubation buffer, with DTPA but no Trolox. 1 Buffer alone; 2, T47D wild-typecells (0.05 mg of protein/ml); 3, white blood cells (2 mg of protein/ml); 4, platelets(2 mg of protein/ml); 5, crude brain (0.5 mg protein/ml); 6, partially purified brain(0.42 mg protein/m); 7, glia (1 mg of protein/ml); 8, T47D transfected cells(0.05 mg of protein/ml). Note that the experiment with T47D wild-type and transfected cellswas carried out on two separate occasions, so there are duplicates of the data from these celltypes. The change in NO level on subsequent addition of NADPH was plotted against CYPOR content.CYPOR content significantly correlates with NO consumption (Pearson's r=0.85, whichis significantly different from zero; P=0.002, n=3–6).(B) To assay cytochrome c reduction by CYPOR, absorbance at550 nm was followed with time. After incubation for 2 h on ice with buffer (closedcircles), absorbance increased linearly. The rate was only slightly slower when incubated with1 mg/ml IgG (open triangles), but was greatly reduced by incubation with 0.33 or1 mg/ml of a CYPOR antibody (filled triangles and open circles, respectively).(C) NO concentration reached after 100 μM DETA/NO addition to brainmembranes at 1 mg of protein/ml, following incubation for 2 h on ice with controlbuffer, 0.33 mg/ml of γ-IgG or 0.33 mg/ml of α-CYPOR, before (open bars)and after (solid bars) addition of 100 μM NADPH. The change in NO on NADPH additionwas significantly less following incubation with α-CYPOR (one-way ANOVA with Tukeypost-hoc tests; P<0.001 compared with buffer and IgG;n=4), but was not affected by incubation with γ-IgG(P>0.05 compared with buffer). (D) NO concentration reachedafter sequential additions of 100 μM DETA/NO, 100 μM NADPH and50 μM DPI to synaptosome membranes (1 mg protein/ml). DPI significantlyinhibits NADPH-dependent NO consumption (one-way ANOVA with Tukey post-hoc tests:NADPH compared with DPI, P<0.01; DPI compared with DETA/NO,P>0.05; n=6). DPI and α-CYPOR inhibit the NADPHeffect to the same extent (Student's t test: 100 μM DETA/NO plus100 μM NADPH plus 50 μM DPI compared with 100 μM DETA/NOplus 100 μM NADPH plus 0.33 mg/ml of α-CYPOR; P=0.41,n=4–6).
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Figure 2: NADPH-dependent NO consumption involves CYPOR(A) 300 μM DETA/NO was added to membranes from different cell types(as labelled) in cell incubation buffer, with DTPA but no Trolox. 1 Buffer alone; 2, T47D wild-typecells (0.05 mg of protein/ml); 3, white blood cells (2 mg of protein/ml); 4, platelets(2 mg of protein/ml); 5, crude brain (0.5 mg protein/ml); 6, partially purified brain(0.42 mg protein/m); 7, glia (1 mg of protein/ml); 8, T47D transfected cells(0.05 mg of protein/ml). Note that the experiment with T47D wild-type and transfected cellswas carried out on two separate occasions, so there are duplicates of the data from these celltypes. The change in NO level on subsequent addition of NADPH was plotted against CYPOR content.CYPOR content significantly correlates with NO consumption (Pearson's r=0.85, whichis significantly different from zero; P=0.002, n=3–6).(B) To assay cytochrome c reduction by CYPOR, absorbance at550 nm was followed with time. After incubation for 2 h on ice with buffer (closedcircles), absorbance increased linearly. The rate was only slightly slower when incubated with1 mg/ml IgG (open triangles), but was greatly reduced by incubation with 0.33 or1 mg/ml of a CYPOR antibody (filled triangles and open circles, respectively).(C) NO concentration reached after 100 μM DETA/NO addition to brainmembranes at 1 mg of protein/ml, following incubation for 2 h on ice with controlbuffer, 0.33 mg/ml of γ-IgG or 0.33 mg/ml of α-CYPOR, before (open bars)and after (solid bars) addition of 100 μM NADPH. The change in NO on NADPH additionwas significantly less following incubation with α-CYPOR (one-way ANOVA with Tukeypost-hoc tests; P<0.001 compared with buffer and IgG;n=4), but was not affected by incubation with γ-IgG(P>0.05 compared with buffer). (D) NO concentration reachedafter sequential additions of 100 μM DETA/NO, 100 μM NADPH and50 μM DPI to synaptosome membranes (1 mg protein/ml). DPI significantlyinhibits NADPH-dependent NO consumption (one-way ANOVA with Tukey post-hoc tests:NADPH compared with DPI, P<0.01; DPI compared with DETA/NO,P>0.05; n=6). DPI and α-CYPOR inhibit the NADPHeffect to the same extent (Student's t test: 100 μM DETA/NO plus100 μM NADPH plus 50 μM DPI compared with 100 μM DETA/NOplus 100 μM NADPH plus 0.33 mg/ml of α-CYPOR; P=0.41,n=4–6).
Mentions: To prepare the suspension of mixed glia for studies of NO consumption, dishes were washed with∼100 ml of cell incubation buffer (20 mM Tris/HCl, 130 mM NaCl,5 mM KCl, 1.2 mM Na2HPO4 and 11 mM glucose, adjusted topH 7.45 at 37 °C) and incubated with 30 ml of 0.05% (w/v) trypsin,0.53 mM EDTA in HBSS (Hanks balanced salt solution) for 15 min at 37 °Cto dissociate the cells, which were washed and resuspended at 3×106 cells/ml inincubation buffer. Cell viability was verified at more than 95% based on Trypan Blue staining.

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