Stimulation of nucleoside efflux and inhibition of adenosine kinase by A1 adenosine receptor activation

Adenosine is produced intracellularly during conditions of metabolic stress and is an endogenous agonist for four subtypes of G-protein linked receptors, Nucleoside transporters are membrane-bound carrier proteins that transfer adenosine, and other nucleosides, across biological membranes. We investigated whether adenosine receptor activation could modulate transporter-mediated adenosine efflux from metabolically stressed cells. DDT1 MF-2 smooth muscle cells were incubated With 10 mu M [H-3]adenine to label adenine nucleotide pools. Metabolic stress with the glycolytic inhibitor iodoacetic acid (IAA, 5 mM) increased tritium release by 63% (P < 0.01), relative to cells treated with buffer alone. The IAA-induced increase was blocked by the nucleoside transport inhibitor nitrobenzylthioinosine (1 mu M), indicating that the increased tritium release was primarily a purine nucleoside. HPLC verified this to be [H-3]adenosine. The adenosine Al receptor selective agonist N-6-cyclohexyladenosine (CHA, 300 nM) increased the release of [H-3]purine nucleoside induced by IAA treatment by 39% (P < 0.05). This increase was blocked bl, the A, receptor antagonist 8-cyclopentyl-1,3-diptopylxanthine (10 mu M) Treatment of cells with UTP (100 mu M), histamine (100 mu M), or phorbol-12-myristate-13-acetate (PMA, 10 mu M) also increased [H-3]purine nucleoside release. The protein kinase C inhibitor chelerythrine chloride (500 nM) inhibited the increase in [H-3]purine nucleoside efflux induced by CHA or PMA treatment. The adenosine kinase activity of cells treated with CHA or PMA was found to be decreased significantly compared with buffer-treated. cells. These data indicated that adenosine A, receptor activation increased nucleoside efflux from metabolically stressed DD-T-1 MF-2 cells by a PKC-dependent inhibition of adenosine kinase activity. (C) 2000 Elsevier Science Inc.