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

Abstract

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. DDT 1 MF-2 smooth muscle cells were incubated with 10 μM [ 3H]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 μM), indicating that the increased tritium release was primarily a purine nucleoside. HPLC verified this to be [ 3H]adenosine. The adenosine A 1 receptor selective agonist N 6-cyclohexyladenosine (CHA, 300 nM) increased the release of [ 3H]purine nucleoside induced by IAA treatment by 39% ( P < 0.05). This increase was blocked by the A 1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (10 μM). Treatment of cells with UTP (100 μM), histamine (100 μM), or phorbol-12-myristate-13-acetate (PMA, 10 μM) also increased [ 3H]purine nucleoside release. The protein kinase C inhibitor chelerythrine chloride (500 nM) inhibited the increase in [ 3H]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 1 receptor activation increased nucleoside efflux from metabolically stressed DDT 1 MF-2 cells by a PKC-dependent inhibition of adenosine kinase activity.