Aim: To define the role of enzymes involved in glutathione metabolism in 6-hydroxydopamine (6-OHDA)-induced glutathione alteration in primary cultured astrocytes.
Methods: Total glutathione (GSx) levels were determined using the modified enzymatic microtiter plate assay. The mRNA levels of gamma-glutamylcysteine synthetase (gammaGCS), gamma-glutamyltransferase (gammaGT), glutathione peroxidase (GPx), GR(glutathione reductase), and glutathione transferases (GST) were determined using RT-PCR. gammaGT activity was determined using gammaGT assay kits.
Results: In primary cultured astrocytes, 6-OHDA induced a significant elevation of cellular GSx levels after treatment for 24 h. However, the GSx levels decreased after 24 h and the values were even lower than the value in the control group without 6-OHDA at 48 h. RT-PCR data showed that the mRNA levels of gammaGCS, the rate-limiting enzyme of gamma-L-glutamyl-L-cysteinylglycine (GSH) synthesis, were increased by 6-OHDA after treatment for 24 h and 48 h; the mRNA levels of GPx, GR, and GST did not alter in 6-OHDA-treated astrocytes after treatment for 24 h and 48 h; and 6-OHDA increased the mRNA levels and the activity of gammaGT after treatment for 48 h, which induced a decrease in GSx levels, despite the up-regulation of gammaGCS after exposure to 6-OHDA for 48 h.
Conclusion: The change in gammaGCS correlated with the increase in GSH levels induced by 6-OHDA after treatment for 24 h. GSx levels decreased because of increased gammaGT mRNA levels and gammaGT activity induced by 6-OHDA after treatment for 48 h.