Decrease in γ-Glutamyltransferase Activity in Rat Type II Cells Exposed in Vitro to Hyperoxia: Effects of the 21-Aminosteroid U-74389G

Abstract

Although the effect of hyperoxia on antioxidant enzymes is well known, the effect of subtoxic levels of hyperoxia on γ-glutamyltransferase (γ-GT), involved in the degradation and uptake of extracellular GSH for intracellular GSH synthesis, is unknown. The aim of the study was to investigate (1) the effects of in vitro hyperoxia on γ-GT activity of type II cells and (2) the effects of the lazaroid U-74389G and N-acetylcysteine (NAC) on the hyperoxia-induced changes in γ-GT and antioxidant enzyme activities. At 48 h after isolation, rat type II cells were exposed for 2 days to air, 60% O₂ or 85% O₂, with or without 30 μM U-74389G or 100 μM NAC. After the exposure, the cells were harvested and assayed for superoxide dismutase (SOD), glutathione peroxidase (GPx), γ-GT activity, and GSH levels. In another series of experiments 85% O₂-exposed cells, with or without U-74389G, were used for Northern blotting of γ-GT mRNA. Exposure to 60% O₂ decreased γ-GT and GSH by -47 and -34%, respectively, while SOD and GPx activities remained unchanged. After 85% O₂-exposure γ-GT decreased by -55%, SOD and GPx increased by +55 and +87%, respectively, while GSH decreased by -35%. NAC treatment decreased γ-GT activity by -42% in the air-exposed cells. After 60% _O2, U-74389G led to significantly higher γ-GT (+117%,) and GSH (+26%) while NAC only led to higher GSH (+28%) compared to the oxygen-exposed cells not treated with NAC or U-74389G. After 85% _O2, U-74389G increased γ-GT, SOD, and GSH by +72, +58, and +68%, respectively, while NAC only increased SOD (+49%) and GSH (+26%) compared to the oxygen-exposed cells not treated with NAC or U-74389G. The 85%, O₂ exposure, with or without U-74389G, had no effect on γ-GT mRNA levels. The results show that hyperoxia decreases rat type II cell γ-GT activity in vitro. This effect was not related to an altered regulation at mRNA level and it was not associated with the hyperoxia-induced decrease in intracellular GSH, since restoration of the GSH levels by NAC did not restore γ-GT activity. The lazaroid U-74389G with vitamin E-like properties effectively prevented the decrease in γ-GT and GSH, so that direct inactivation of the membrane-bound γ-GT by hyperoxia is the most likely mechanism.

Key Words:

• glutathione peroxidase
• lazaroids
• N-acetylcysteine
• reactive oxygen species
• superoxide dismutase