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Effect of 5′‐(N‐Ethylcarboxamido)adenosine on Adenosine Transport in Cultured Chromaffin Cells

Abstract: Extracellular adenosine is transported into chromaffin cells by a high‐affinity transport system. The action of adenosine receptor ligands was studied in this cellular model 5‐(N‐Ethylcarboxamido)adenosine (NECA), an agonist receptors, activated adenosine transport. Km values for adenosine were 4.6 ± 1.0 (n = 5) and 10.2 ± 3.0 μM (n = 5) for controls and 100 nM NECA, respectively. The Vmax values were 66.7 ± 23.5 and 170.2 ± 30 pmol/106 cells/min for control and 100 nM NECA, respectively. The A1 agonist N6‐cyclohexyladenosine, the A1 antagonist 8‐cyclopentyl‐1, 3‐dipropylxanthine, and the A1‐A2 antagonist 1,3‐dipropyl‐8‐{4‐[(2‐aminoethyl)amino]‐carbonylmethyloxyphenyl}‐xanthine did not significantly modify the adenosine transport in this system. Binding studies done with [3H]dipyridamole, a nucleoside transporter ligand, did not show changes in either the number or affinity of transporter sites after NECA treatment. This ligand can enter cells and quantifies the total number of transporters. The binding studies with [3H]‐nitrobenzylthioinosine, which quantifies the plasma membrane transporters, showed a Bmax of 19,200 ± 800 and 23,200 ± 700 transporters/cell for controls and 100 nM NECA, repectively. No changes in the KD were obtained. The effects of NECA were not mediated through adenylate cyclase activation, because its action was not imitated by forskolin.