ESR detection of intraphagosomal superoxide in polymorphonuclear leukocytes using 5-(diethoxyphosphoryl)-5-methyl-l-pyrroline-N-oxide.
We applied a spin trap, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), to detect O2*- generation during phagocytosis in human polymorphonuclear leukocytes (PMNs). PMNs were activated with serum-opsonized zymosan (sOZ) in the presence of DEPMPO. The ESR spectra mainly consisted of Cu,Zn-SOD-sensitive DEPMPO-OOH spin adducts. To clarify where these spin-adducts were present, cells after stimulation were separated from extracellular fluid by brief centrifugation and resuspended in Hanks' balanced salt solution. ESR examination showed that DEPMPO-OOH adducts were present in both fractions. When cells were stimulated by phorbol myristate acetate (PMA), the DEPMPO-OOH was detected in extracellular fluid but not in the cell fraction. Furthermore, DEPMPO-OOH adducts were quickly converted into ESR-silent compounds by addition of cell lysate of PMNs. These results indicate that DEPMPO is useful to detect O2*- of extracellular space including the intraphagosome but not that of intracellular space in sOZ-stimulated phagocytes.
Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. inanami@vetmed.hokudai.ac.jp
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Inanami
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Yamamori
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Takahashi
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Nagahata
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Kuwabara
D003497:Cyclic N-Oxides / Q000737:chemistry*; D004355:Drug Stability; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006801:Humans; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism*; D013113:Spin Labels; D013481:Superoxides / Q000032:analysis* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015054:Zymosan / Q000494:pharmacology
D003497:Cyclic N-Oxides; D013113:Spin Labels; D013481:Superoxides; C098505:5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide; D015054:Zymosan; D013755:Tetradecanoylphorbol Acetate