TY - JOUR
T1 - Measurement and characterization of superoxide generation in microglial cells
T2 - Evidence for an NADPH oxidase-dependent pathway
AU - Sankarapandi, Sornampillai
AU - Zweier, Jay L.
AU - Mukherjee, Goutam
AU - Quinn, Mark T.
AU - Huso, David L.
N1 - Funding Information:
1This work was supported by grants from the National Institutes of Health (NIH), HL-38324 and HL-52315, and a Grant-in-Aid from the American Heart Association (AHA) (J.L.Z.), Grants NS35693, RR00080 (D.L.H.) and NIH Grant RO1 AR42426, USDA Grant NRICGP 9502274, Arthritis Foundation Biomedical Science Grant, and an Established Investigator Award from AHA (M.T.Q.).
PY - 1998/5/15
Y1 - 1998/5/15
N2 - While oxygen free radicals are important mediators of brain injury, questions remain regarding which cell types and enzyme pathways trigger this radical generation. Microglial cells have been hypothesized to be an important source of radical generation; however, the magnitude, kinetics, and mechanism of this process are unknown. Oxygen radical generation by stimulated primary microglia was directly measured and characterized by electron paramagnetic resonance spin trapping. Microglia, when stimulated by phorbol ester or opsonified zymosan, gave rise to EPR spectra characteristic of superoxide. Experiments performed in the presence of superoxide dismutase, catalase, deferoxamine, and dimethyl sulfoxide excluded generation of hydroxyl radicals in significant amounts. Microglial superoxide generation was blocked by the NADPH oxidase inhibitor diphenylene iodonium in a manner similar to that seen in neutrophils, suggesting that a neutrophil like NADPH oxidase was the source of superoxide production. However, microglia produced 20 to 40 times less superoxide compared to a similar number of neutrophils during the first 30 min following stimulation, indicating a marked difference in the regulation of NADPH oxidase activation. Western blots of microglia lysates demonstrated that both large (gp91-phox) and small (p22-phox) NADPH oxidase subunits are expressed in both unstimulated and stimulated microglia. Indirect immunofluorescence demonstrated localization at the membrane surfaces of activated cells. Thus, microglial cells generate superoxide via a neutrophil-like NADPH oxidase but exhibit distinctly different time course and magnitude of activation than that seen in neutrophils.
AB - While oxygen free radicals are important mediators of brain injury, questions remain regarding which cell types and enzyme pathways trigger this radical generation. Microglial cells have been hypothesized to be an important source of radical generation; however, the magnitude, kinetics, and mechanism of this process are unknown. Oxygen radical generation by stimulated primary microglia was directly measured and characterized by electron paramagnetic resonance spin trapping. Microglia, when stimulated by phorbol ester or opsonified zymosan, gave rise to EPR spectra characteristic of superoxide. Experiments performed in the presence of superoxide dismutase, catalase, deferoxamine, and dimethyl sulfoxide excluded generation of hydroxyl radicals in significant amounts. Microglial superoxide generation was blocked by the NADPH oxidase inhibitor diphenylene iodonium in a manner similar to that seen in neutrophils, suggesting that a neutrophil like NADPH oxidase was the source of superoxide production. However, microglia produced 20 to 40 times less superoxide compared to a similar number of neutrophils during the first 30 min following stimulation, indicating a marked difference in the regulation of NADPH oxidase activation. Western blots of microglia lysates demonstrated that both large (gp91-phox) and small (p22-phox) NADPH oxidase subunits are expressed in both unstimulated and stimulated microglia. Indirect immunofluorescence demonstrated localization at the membrane surfaces of activated cells. Thus, microglial cells generate superoxide via a neutrophil-like NADPH oxidase but exhibit distinctly different time course and magnitude of activation than that seen in neutrophils.
KW - Electron paramagnetic resonance
KW - Microglia
KW - NADPH oxidase
KW - Spin trapping
KW - Superoxide
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U2 - 10.1006/abbi.1998.0658
DO - 10.1006/abbi.1998.0658
M3 - Article
C2 - 9606965
AN - SCOPUS:0032524692
SN - 0003-9861
VL - 353
SP - 312
EP - 321
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -