Reevaluation of the spin-trapped adduct formed from 5,5-dimethyl-1-pyrroline-1-oxide during the respiratory burst in neutrophils.
By employing EPR spectrometry with the aid of a spin-trapping agent, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), the generation of superoxide anion and hydroxyl radical was reevaluated during the respiratory burst of porcine and human neutrophils. Properly prepared resting neutrophils did not generate any spin-trapped radical, and, when the cells were stimulated with phorbol myristate acetate, only DMPO-OOH, the spin-trapped adduct of superoxide anion, was detected. No formation of DMPO-OH, the spin-trapped adduct of the hydroxyl radical, was observed. DMPO-OOH was also detected principally when the neutrophils were stimulated with opsonized zymosan, a particulate stimulus. In the latter case, however, the formation of DMPO-OOH ceased shortly after the addition of zymosan and subsequent production of DMPO-OH was observed. The production of DMPO-OH was found to be associated with cell injury. DMPO at the concentration usually used for the experiment (0.045-0.09 M) injured phagocytizing neutrophils, causing lysis of the cells. On the other hand, an addition of cell homogenate or glutathione-glutathione peroxidase system to the suspension of intact cells which were producing DMPO-OOH resulted in the formation of DMPO-OH. Thus, DMPO-OH was probably derived from DMPO-OOH by the action of enzymes and/or factor(s) which were released from the lysed cells.
Ueno
I
I
Institute of Medical Science, University of Tokyo.
Kohno
M
M
Mitsuta
K
K
Mizuta
Y
Y
Kanegasaki
S
S
D000818:Animals; D002374:Catalase / Q000378:metabolism; D003497:Cyclic N-Oxides / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D066298:In Vitro Techniques; D009504:Neutrophils / Q000378:metabolism*; D009895:Opsonin Proteins / Q000378:metabolism; D010101:Oxygen Consumption / Q000187:drug effects; D010587:Phagocytosis / Q000187:drug effects; D013113:Spin Labels; D013482:Superoxide Dismutase / Q000378:metabolism; D013552:Swine; D015054:Zymosan / Q000494:pharmacology
D003497:Cyclic N-Oxides; D009895:Opsonin Proteins; D013113:Spin Labels; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D015054:Zymosan; D002374:Catalase; D013482:Superoxide Dismutase