| Co-Investigator(Kenkyū-buntansha) |
INUI Makoto Osaka University, School of Medicine, Assistant Professor, 医学部, 助手 (70223237)
TADA Michihiko Osaka University, School of Medicine, Professor, 医学部, 教授 (90093434)
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| Research Abstract |
Activated neurtophils play an important role in myocardial injury during ischemia followed by reperfusion. To clarify the mechanism of the activation of neutrophils in the reperfused microvasculature, we co-incubated canine heart capillary endothelial cells and isolated neutrophils under hypoxia-reoxygenation. The extent of neutrophil adhesion was evaluated by counting of cells which were adhered to the endothelial cell monolayer. Production of an arachidonate lipoxygenase metabolite, hydroxyeicosa-tetraenoic acid (HETE), was assayed by HPLC. Free radical generation was assayed by luminol-enhanced chemiluminescence and 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) spin trapping of electron spin resonance (ESR) spectroscopy. After reoxygenation, serial changes of free radical generation revealed two peaks of increase ; the first was at 1 hour and the second was at 3 hours after reoxygenation. The first peak was diminished by allopurinol, whereas the second was diminished by nordihydroguaiare
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tic acid. Production of 15-, 5-, and 12-HETEs were augmented in endothelial cells after reoxygenation. Adhesion of neutrophils co-incubated with reoxygenated endothelial cells was also enhanced compared with that of neutrophils co-incubated with control endothelial cells. Adhesion of neutrophils was associated with the production of HETEs and free radicals at 3 hours after reoxygenation.
To elucidate whether these activated neutrophils produce myocardial cell injury, we co-cultured endothelial cells and myocardial cells in double chamber (myocytes on dish bottom ; lower chamber, endothelial cells on collagen sheet ; upper chamber) under hypoxia-reoxygenation. After reoxygenation, neutrophils were added to these cells and serial changes of free radical production was assayed by ESR and cell injury was determined by morphological change. Under mono-culture of endothelial cells, DMPO-OH signal was detected mainly at the late phase of reoxygenation, while DMPO-OOH signal was detected at the early phase of reoxygenation. Under the co-culture condition, DMPO-OOH signal was markedly enhanced in the upper chamber, compared with the sum of that generated in mono-culture. DMPO-OH signal was also enhanced after the late phase of reoxygenation. Injury of myocardial cells and endothelial cells was augmented in a close relation with the enhancement of free radical generation. These results suggested that reoxygenation injury of endothelial cells induced neutrophil activation and augmentation of free radical production, resulting in the enhancement of myocardial cell injury. Less
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