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

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概要

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• 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.

• 北海道大学の論文
• 2000-05-31

著者

• NAGAHATA Hajime

  Department of Animal Health, School of Veterinary Medicine, Rakuno Gakuen Univeristy

• TAKAHASHI Tsuneo

  Department of Cell Processing, The Institute of Medical Science, The University of Tokyo

• Takahashi T

  Cell Processing Department The Institute Of Medical Science University Of Tokyo

• KUWABARA MIKINORI

  Laboratory of Radiation Biology, Graduate School of Veterinary Medicine

• INANAMI Osamu

  Laboratory of Radiation Biology, Department of Environmental Veterinary Sciences, Graduate School of

• Kuwabara Mikinori

  Laboratory Of Radiation Biology Graduate School Of Veterinary Medicine

• Kuwabara Mikinori

  Laboratory Of Radiation Biology Department Of Environmental Veterinary Sciences Graduate School Of V

• Inanami Osamu

  Laboratory Of Radiation Biology Department Of Environmental Veterinary Medical Sciences Graduate Sch

• Kuwabara M

  Hokkaido Univ. Sapporo Jpn

• Noda Hiroshi

  Department Of Animal Health Faculty Of Veterinary Medicine Rakuno Gakuen University

• Inanami Osamu

  Lab. Radiat. Biol. Grad. Sch. Vet. Med. Hokkaido Univ.

• Tanabe Tsuyoshi

  Department Of Hematology/oncology The Institute Of Medical Science The University Of Tokyo

• Nagahata H

  Department Of Animal Health School Of Veterinary Medicine Rakuno Gakuen University

• Nagahata Hajime

  Department Of Animal Health School Of Veterinary Medicine Rakuno Gakuen Univeristy

• Takahashi Tsuneo

  Department Of Biochemistry And Molecular Biology Nihon University School Of Dentistry At Matsudo

• Takahashi Tsuneo

  Department Of Advanced Nuclear Heat Technology Japan Atomic Energy Research Institute

• Takahashi Tsuneo

  Cell Processing Department The Institute Of Medical Science University Of Tokyo

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