Anoxia redistributes adenosine A_2A receptors in PC12 cells and increases receptor-mediated formation of cAMP

Abstract.

Undifferentiated and NGF-treated PC12 cells were subjected to anoxia for up to 24 h. The adenosine A_2A receptor antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo[1,5-c]pyrimidine (SCH 58261) decreased viability of undifferentiated, but not NGF-treated PC12 cells after 6 h of anoxia. Anoxia also transiently enhanced cAMP responses induced via activation of adenosine A_2A receptors in undifferentiated PC12 cells (20-fold decrease in the EC₅₀ value for the agonist 2-[p-(2-carbonylethyl) phenylethylamino]-5'-N-ethylcarboxamidoadenosine, CGS 21680). In NGF-treated PC12 cells, by contrast, anoxia decreased both the maximal response to and the potency of CGS 21680. In undifferentiated PC12 cells subjected to anoxia a very modest increase of A_2A receptor mRNA was detected in cells by Northern blotting, but no changes in the amount of the receptor protein could be seen by Western blotting. However, surface biotinylation of the cells followed by avidin pull-down showed that the A_2A receptor at the cell membrane was increased after anoxia. This was supported by immunolabelling of the A_2A receptors: much of the receptor protein was present in the cytoplasm of normoxic cells, but in cells subjected to anoxia the A_2A receptor immunolabelling at the cell membrane was more pronounced, indicating redistribution of the receptors from intracellular pools to the cell membrane during anoxia.