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Absence of proton channels in COS-7 cells expressing functional NADPH oxidase components.

Morgan D, Cherny VV, Price MO, Dinauer MC, DeCoursey TE - J. Gen. Physiol. (2002)

Bottom Line: Biol.PMA or AA did not elicit detectable H(+) current in COS(WT) or COS(phox) cells.Therefore, gp91(phox) does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Physiology, Rush Presbyterian St. Luke's Medical Center, 1750 W Harrison, Chicago, IL 60612, USA.

ABSTRACT
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O(2)(-)) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H(+) efflux was thought to be contained within the gp91(phox) subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063-36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COS(phox)). The 7D5 antibody, which detects an extracellular epitope of the gp91(phox) protein, labeled 96-98% of COS(phox) cells. NADPH oxidase was functional because COS(phox) (but not COS(WT)) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COS(WT)) or COS(phox) cells studied at pH(o) 7.0 and pH(i) 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H(+) current in COS(WT) or COS(phox) cells. Therefore, gp91(phox) does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.

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Superoxide production by COSphox cells. (A) The average time course of PMA-stimulated superoxide anion production detected as the reduction of cytochrome c is plotted. Cells (106 cells/ml) were incubated at 37°C in HBSS before the addition of PMA (concentrations of PMA are in nM). Control cells were not stimulated. Data are mean values from four experiments with the error bars removed for clarity. (B) The average maximum rate of O2− production (± SE, n = 4) determined in each experiment in A. Data were collected in 2-min intervals and the rate is given per minute. DPI (6 μM) and 100 μg/ml SOD were added simultaneously with PMA to establish that the reduction of cytochrome c was due to NADPH oxidase and O2−, respectively.
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fig6: Superoxide production by COSphox cells. (A) The average time course of PMA-stimulated superoxide anion production detected as the reduction of cytochrome c is plotted. Cells (106 cells/ml) were incubated at 37°C in HBSS before the addition of PMA (concentrations of PMA are in nM). Control cells were not stimulated. Data are mean values from four experiments with the error bars removed for clarity. (B) The average maximum rate of O2− production (± SE, n = 4) determined in each experiment in A. Data were collected in 2-min intervals and the rate is given per minute. DPI (6 μM) and 100 μg/ml SOD were added simultaneously with PMA to establish that the reduction of cytochrome c was due to NADPH oxidase and O2−, respectively.

Mentions: Superoxide anion (O2−) production by COSphox cells was assessed by their ability to reduce ferricytochrome c. Fig. 6 A shows the average time courses of cumulative O2− release by four batches of COSphox cells challenged with different concentrations of PMA. O2− production above control levels was seen at 6 nM PMA. PMA at 60 and 300 nM, respectively, produced similar levels of O2−. The O2− release time course was characterized by a delay of several minutes after the addition of PMA, followed by a rapid increase between 5 and 20 min, after which the rate of O2− production diminished. Fig. 6 B summarizes the average maximal rate of O2− production in these experiments. The highest maximal rate, observed at 300 nM PMA, was 26 ± 6 nmol min−1 (107 cells)−1. The response was almost completely inhibited by 100 μg/ml superoxide dismutase (SOD), showing that ferricytochrome c reduction was due to the release of O2− and not peroxides or other oxidizing agents. Diphenylene iodonium (DPI) at 6 μM also inhibited O2− production, implicating a flavoenzyme (O'Donnell et al., 1994), most likely NADPH oxidase (Cross and Jones, 1986). As reported previously (Price et al., 2002), COSWT cells studied under identical conditions did not produce detectable O2− (unpublished data).


Absence of proton channels in COS-7 cells expressing functional NADPH oxidase components.

Morgan D, Cherny VV, Price MO, Dinauer MC, DeCoursey TE - J. Gen. Physiol. (2002)

Superoxide production by COSphox cells. (A) The average time course of PMA-stimulated superoxide anion production detected as the reduction of cytochrome c is plotted. Cells (106 cells/ml) were incubated at 37°C in HBSS before the addition of PMA (concentrations of PMA are in nM). Control cells were not stimulated. Data are mean values from four experiments with the error bars removed for clarity. (B) The average maximum rate of O2− production (± SE, n = 4) determined in each experiment in A. Data were collected in 2-min intervals and the rate is given per minute. DPI (6 μM) and 100 μg/ml SOD were added simultaneously with PMA to establish that the reduction of cytochrome c was due to NADPH oxidase and O2−, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233867&req=5

fig6: Superoxide production by COSphox cells. (A) The average time course of PMA-stimulated superoxide anion production detected as the reduction of cytochrome c is plotted. Cells (106 cells/ml) were incubated at 37°C in HBSS before the addition of PMA (concentrations of PMA are in nM). Control cells were not stimulated. Data are mean values from four experiments with the error bars removed for clarity. (B) The average maximum rate of O2− production (± SE, n = 4) determined in each experiment in A. Data were collected in 2-min intervals and the rate is given per minute. DPI (6 μM) and 100 μg/ml SOD were added simultaneously with PMA to establish that the reduction of cytochrome c was due to NADPH oxidase and O2−, respectively.
Mentions: Superoxide anion (O2−) production by COSphox cells was assessed by their ability to reduce ferricytochrome c. Fig. 6 A shows the average time courses of cumulative O2− release by four batches of COSphox cells challenged with different concentrations of PMA. O2− production above control levels was seen at 6 nM PMA. PMA at 60 and 300 nM, respectively, produced similar levels of O2−. The O2− release time course was characterized by a delay of several minutes after the addition of PMA, followed by a rapid increase between 5 and 20 min, after which the rate of O2− production diminished. Fig. 6 B summarizes the average maximal rate of O2− production in these experiments. The highest maximal rate, observed at 300 nM PMA, was 26 ± 6 nmol min−1 (107 cells)−1. The response was almost completely inhibited by 100 μg/ml superoxide dismutase (SOD), showing that ferricytochrome c reduction was due to the release of O2− and not peroxides or other oxidizing agents. Diphenylene iodonium (DPI) at 6 μM also inhibited O2− production, implicating a flavoenzyme (O'Donnell et al., 1994), most likely NADPH oxidase (Cross and Jones, 1986). As reported previously (Price et al., 2002), COSWT cells studied under identical conditions did not produce detectable O2− (unpublished data).

Bottom Line: Biol.PMA or AA did not elicit detectable H(+) current in COS(WT) or COS(phox) cells.Therefore, gp91(phox) does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Physiology, Rush Presbyterian St. Luke's Medical Center, 1750 W Harrison, Chicago, IL 60612, USA.

ABSTRACT
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O(2)(-)) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H(+) efflux was thought to be contained within the gp91(phox) subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063-36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COS(phox)). The 7D5 antibody, which detects an extracellular epitope of the gp91(phox) protein, labeled 96-98% of COS(phox) cells. NADPH oxidase was functional because COS(phox) (but not COS(WT)) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COS(WT)) or COS(phox) cells studied at pH(o) 7.0 and pH(i) 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H(+) current in COS(WT) or COS(phox) cells. Therefore, gp91(phox) does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.

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