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 EC(50) 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.
Arslan
Giulia
G
Department of Physiology and Pharmacology, Section of Molecular Neuropharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden, Giulia.Arslan@fyfa.ki.se
Kull
Björn
B
Fredholm
Bertil B
BB
D000818:Animals; D001692:Biological Transport; D019921:Biotinylation; D015152:Blotting, Northern; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D015687:Cell Hypoxia; D002470:Cell Survival / Q000187:drug effects; D000242:Cyclic AMP / Q000096:biosynthesis*; D008565:Membrane Proteins / Q000032:analysis; D016716:PC12 Cells; D058915:Purinergic P1 Receptor Antagonists; D011743:Pyrimidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D043705:Receptor, Adenosine A2A; D011956:Receptors, Cell Surface / Q000378:metabolism; D018047:Receptors, Purinergic P1 / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014230:Triazoles / Q000494:pharmacology
C098657:5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)pyrimidine; D008565:Membrane Proteins; D058915:Purinergic P1 Receptor Antagonists; D011743:Pyrimidines; D012333:RNA, Messenger; D043705:Receptor, Adenosine A2A; D011956:Receptors, Cell Surface; D018047:Receptors, Purinergic P1; D014230:Triazoles; D000242:Cyclic AMP