Stimulation of nucleoside efflux and inhibition of adenosine kinase by A(1) adenosine receptor activation

Adenosine is produced intracellularly during conditions of metabolic stress and is an endogenous agonist for four subtypes of G-protein linked recepto rs, Nucleoside transporters are membrane-bound carrier proteins that transf er adenosine, and other nucleosides, across biological membranes. We invest igated whether adenosine receptor activation could modulate transporter-med iated adenosine efflux from metabolically stressed cells. DDT1 MF-2 smooth muscle cells were incubated With 10 mu M [H-3]adenine to label adenine nucl eotide pools. Metabolic stress with the glycolytic inhibitor iodoacetic aci d (IAA, 5 mM) increased tritium release by 63% (P < 0.01), relative to cell s treated with buffer alone. The IAA-induced increase was blocked by the nu cleoside transport inhibitor nitrobenzylthioinosine (1 mu M), indicating th at the increased tritium release was primarily a purine nucleoside. HPLC ve rified this to be [H-3]adenosine. The adenosine Al receptor selective agoni st N-6-cyclohexyladenosine (CHA, 300 nM) increased the release of [H-3]puri ne 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 n ucleoside release. The protein kinase C inhibitor chelerythrine chloride (5 00 nM) inhibited the increase in [H-3]purine nucleoside efflux induced by C HA or PMA treatment. The adenosine kinase activity of cells treated with CH A or PMA was found to be decreased significantly compared with buffer-treat ed. cells. These data indicated that adenosine A, receptor activation incre ased nucleoside efflux from metabolically stressed DD-T-1 MF-2 cells by a P KC-dependent inhibition of adenosine kinase activity. (C) 2000 Elsevier Sci ence Inc.