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 and function of ENTs and LIMPII (Lysosomal Integral Membrane Protein II). 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 and [³H]thymidine incorporation experiment. Astrocytes were 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. We found the expression of ENT3 and LIMPII by RT-PCR and western blot analysis, and the coexpression of ENT3 and LIMPII in cultured astrocytes by immunocytochemistry. We clarified that ENT3 is localized in astrocytic lysosome.
These results indicate that ENTs expressed and function in cell membrane and lysosome could relate with H₂O₂-induced thymidine incorporation and DNA repair and disassembly in cultured astrocytes.