We studied the effects of adenosine on injury caused by hypoxia and reoxygenation in LLC-PK1 cells. Lactate dehydrogenase and gamma-glutamyltranspeptidase were released from cells exposed to hypoxia for 6 hr and then reoxygenation for 1 hr. The addition of adenosine at 100 microM to the medium before hypoxia began significantly decreased enzyme leakage into medium during both hypoxia and reoxygenation. The adenosine A1-receptor agonist, R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA), at the concentration of 100 microM, did not affect enzyme release, but the adenosine A2-receptor agonist 2-p-[2-car-boxyethyl]phenethyl-amino-5'-N-ethylcarboxamido-adenosi ne hydrochloride (CGS 21680) at the concentration of 100 nM, suppressed the injury caused by hypoxia and reoxygenation. There were decreases in cAMP contents and ATP levels in LLC-PK1 cells injured by hypoxia and reoxygenation. Adenosine (100 microM) restored ATP levels in the cells during reoxygenation. With adenosine, the intracellular cAMP level was increased prominently during reoxygenation. These results suggest that adenosine protects LLC-PK1 cells from injury caused by hypoxia and reoxygenation by increasing the intracellular cAMP level via adenosine A2 receptor.
ABSTRACT-The aim of this study was to characterize injuries of LLC-PK1 and MDCK cells exposed to hypoxia and reoxygenation. Exposure of LLC-PK1 cells to hypoxia reduced the ATP contents and increased the leakage of lactate dehydrogenase (LDH), but MDCK cells had no such injuries. Hypoxia-reoxygenation of LLC-PK1 cells dramatically increased LDH leakage, which was suppressed by free radical scavengers, N N diphenyl p-phenylenediamine, superoxide dismutase and N. N dimethylthiourea. These results suggest that use of LLC-PK1 cells has advantages for the investigation of ischemia-reperfusion injury of the kidney as an in vitro model and that generation of oxygen radicals is involved in the cellular injury induced by hypoxia-reoxygenation.
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