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Aim:To define the role of enzymes involved in glutathione metabolism in 6-hydroxydopamine(6-OHDA)-induced glutathione alteration in primary culturedastrocytes.Methods:Total glutathione(GSx)levels were determined using themodified enzymatic microtiter plate assay.The mRNA levels of γ-glutamylcysteinesynthetase(γGCS),γ-glutamyltransferase(γGT),glutathione peroxidase(GPx),GR(glutathione reductase),and glutathione transferases(GST)were determined us-ing RT-PCR.γGT activity was determined using 7GT assay kits.Results:Inprimary cultured astrocytes,6-OHDA induced a significant elevation of cellularGSx levels after treatment for 24 h.However,the GSx levels decreased after 24 hand 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 γGCS,the rate-limiting enzyme of γ-L-glutamyl-L-cysteinylglycine(GSH)synthesis,were increasedby 6-OHDA after treatment for 24 h and 48 h;the mRNA levels of GPx,GR,and GSTdid 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 TGT after treatment for 48 h,which induced a decrease in GSx levels,despite the up-regulation of γCS afterexposure to 6-OHDA for 48 h.Conclusion:The change in γGCS correlated withthe increase in GSH levels induced by 6-OHDA after treatment for 24 h.GSx levelsdecreased because of increased γGT mRNA levels and γGT activity induced by 6-OHDA after treatment for 48 h.
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 themodified enzymatic microtiter plate assay. The mRNA levels of γGlutamylcysteinesynthetase (γGCS), glutathione peroxidase (GPx), GR (glutathione reductase), and glutathione transferases (GST) were determined using RT-PCR. γGT activity was determined using 7GT assay kits. Results: Inprimary cultured astrocytes, 6-OHDA induced a significant elevation of cellularGSx levels after treatment for 24 h.However, the GSx levels decreased after 24 hand 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 γGCS, the rate-limiting enzyme of γ-L-glutamyl-L-cysteinylglycine (GSH) synthesis, were increased by 6-OHDA after treatment for 24 h and 48 h; of GPx, GR, and GSTdid 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 TGT after treatment for 48 h, which induced a decrease in GSx levels, despite the up-regulation of γCS afterexposure to 6-OHDA for 48 h.Conclusion: The change in γ GSS correlated with the increase in GSH levels induced by 6-OHDA after treatment for 24 h.GSx levels decreased due to increased γ GT mRNA levels and γ GT activity induced by 6-OHDA after treatment for 48 h.