Japanese Journal of Veterinary Research;Volume 48, Number 1

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Spin-trapping detection of superoxides in polymorphonuclear leukocytes stimulated with serum-opsonized zymosan

KUWABARA, Mikinori;TAKAHASHI, Tsuneo A.;NAGAHATA, Hajime;INANAMI, Osamu

Permalink : http://hdl.handle.net/2115/2791
JaLCDOI : 10.14943/jjvr.48.1.3
KEYWORDS : ESR-spin-trapping;hydroxyl radical;opsonized zymosan;polymorphonuclear leukocyte (PMN);superoxide anion

Abstract

To clarify where oxygen radicals are generated in polymorphonuclear leukocytes (PMNs) during phagocytosis, superoxides (O_2^-) from opsonized zymosan (OZ)-stimulated human PMNs were detected by the ESR and spin-trapping methods. PMNs were preactivated with OZ for the indicated periods of time at 37℃. Then a spin-trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), was added to them, and they were further incubated for 30sec for ESR observations. The ESR spectra consisted of two components due to the DMPO-OOH and DMPO-OH spin adducts. To clarify where these spin-adducts were present, cells were separated from extracellular fluid by brief centrifugation and resuspended in Hanks' balanced salt solution. ESR examination of two fractions showed that the DMPO-OOH adducts was present in the cell fraction, whereas the DMPO-OH adducts were present in the extracellular fluid. When DMSO was used as a scavenger of hydroxyl radicals (OH), DMPO-CH_3 adducts were observed in the fluid fraction but not in the cell fraction. Both spin adducts were completely abolished by Cu, Zn-SOD but not catalase. These results indicated that O_2^- were produced inside phagosomes of OZ-stimulated PMNs and OH were produced outside them by spontaneous decomposition of the DMPO-OOH adducts.

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