We have found that cultured differentiated astrocytes pretreated with N⁶, 2^'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP), a permeable analogue of cAMP, incorporate thymidine, but not uridine, via nucleoside transporters including equilibrative nucleoside transporters (ENTs） into TCA insoluble fraction for repair on DNA injury in the presence of hydrogen peroxide (H₂O₂) at an early time, and these phenomena are specific in differentiated astrocytes, but not undifferentiated astrocytes and neurons.
We studied expression of ENTs in cultured astrocytes by RT-PCR, western blot analysis and immunocytochemistry. We could confirm ENT1, that is hypersensitive nucleoside transporter, and ENT2, that is low-sensitive nucleoside transporter in cultured astrocytes by RT-PCR and western blot analysis. This time, astrocytes ware double-stained by anti-GFAP antibody and anti-ENT3 antibody. We could confirm ENT3, that is assumed to be presented in lysosome, in cultured astrocytes co-stained by GFAP.
H₂O₂-induced thymidine incorporation into cultured astrocytes decreased by S-(4-Nitrobenzyl)-6-thioinosine (NBMPR), dilazep and dipyridamole, ENT inhibitors, at micromolar concentrations but not nanomolar concentrations, so thymidine was incorporated via ENT2, but not ENT1.
These results indicate that ENTs expressed in membrane and lysosome could relate with H₂O₂-induced thymidine incorporation and DNA repair in cultured astrocytes.