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Updated: Aug 17, 2026
Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases
Published on: February 24, 2018
The inhibition by diphenyleneiodonium and its analogues of superoxide generation by macrophages
Abstract:
Peritoneal macrophages were elicited in rats by using casein as a stimulus; when stimulated with phorbol 12-myristate 13-acetate (PMA) they produced O2.-. Nearly 60% of the total cytochrome b had a low Em,7.0 of -247 mV, typical of the cytochrome b component found in the NADPH-dependent O2(.-)-generating oxidase of neutrophils. The rate of O2.- generation by macrophages was 1.23 mol of O2.-/s per mol of cytochrome b. Treatment of intact macrophages with diphenyleniodonium (DPI) at 0.9 microM caused 50% inhibition of PMA-induced O2.- generation, with little effect on mitochondrial respiratory activity; KCN inhibited respiratory activity without affecting PMA-induced O2.- generation. A similar specificity of inhibition was found for di-2-thienyliodonium (50% inhibition of O2.- generation at 0.5 microM) and, at higher concentrations, for diphenyl iodonium. When macrophage suspensions were incubated with [125I]DPI followed by autoradiography of SDS/polyacrylamide-gel-electrophoresis-separated polypeptides, radioactivity was most strongly associated with a band of Mr 45,000, similar to that found in neutrophils [Cross & Jones (1986) Biochem. J. 237, 111-116]. The O2(.-)-generating oxidase of macrophages appears to have components in common with the NADPH oxidase of neutrophils, despite differences in activity. Its sensitivity to DPI suggests that selective prevention of radical generation by macrophages in vivo is possible.
Insights
Peritoneal macrophages generate superoxide (O2.-) via an NADPH oxidase similar to neutrophils. This radical generation is sensitive to diphenyleniodonium (DPI), suggesting potential for in vivo inhibition.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Peritoneal macrophages are key immune cells involved in host defense.
- Superoxide (O2.-) generation is a critical function of phagocytic cells.
- The NADPH oxidase enzyme complex is responsible for superoxide production in neutrophils.
Purpose of the Study:
- To investigate the characteristics of superoxide generation in rat peritoneal macrophages.
- To compare the O2.- generating oxidase in macrophages with that of neutrophils.
- To assess the potential for selective inhibition of macrophage O2.- production.
Main Methods:
- Elicitation of peritoneal macrophages using casein.
- Stimulation of macrophages with phorbol 12-myristate 13-acetate (PMA).
- Measurement of O2.- generation and cytochrome b content.
- Inhibition studies using diphenyleniodonium (DPI), di-2-thienyliodonium, and KCN.
- Autoradiography of SDS/polyacrylamide-gel-electrophoresis separated polypeptides after [125I]DPI incubation.
Main Results:
- PMA-stimulated macrophages produced O2.- with a rate of 1.23 mol O2.-/s per mol of cytochrome b.
- A significant portion of macrophage cytochrome b exhibited characteristics similar to that in neutrophil NADPH oxidase.
- Diphenyleniodonium (DPI) inhibited PMA-induced O2.- generation by 50% at 0.9 microM without affecting mitochondrial respiration.
- Radioactivity from [125I]DPI was associated with a 45,000 Mr polypeptide, similar to neutrophils.
- KCN inhibited respiration but not O2.- generation, indicating distinct pathways.
Conclusions:
- Rat peritoneal macrophages possess an O2.- generating oxidase with components common to the neutrophil NADPH oxidase.
- The sensitivity of macrophage O2.- production to DPI suggests a potential therapeutic target for selective inhibition in vivo.
- Further research into the specific components and regulation of macrophage NADPH oxidase is warranted.
