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 of A2 receptors, activated adenosine transport. Km values for adenosine were 4.6 +/- 1.0 (n = 5) and 10.2 +/- 3.0 microM (n = 5) for controls and 100 nM NECA, respectively. The Vmax values were 66.7 +/- 23.5 and 170.2 +/- 30 pmol/10(6) cells/min for controls 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, respectively. 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.
In cultured chromaffin cells and plasma membrane vesicles from chromaffin tissue, the transport of D-[3H]adenosine followed Michaelis-Menten saturation kinetics, with Km values of 1.5 +/- 0.3 microM and 1.9 +/- 0.2 microM, respectively. The transport of the isomer, L-[3H]adenosine, showed sigmoidal kinetics in both preparations. In plasma membrane vesicles the S0.5 was 2.5 +/- 0.2 microM with a Hill coefficient of 2.8 and the Vmax value of 0.26 +/- 0.01 pmol s-1 (mg of protein)-1. In cultured chromaffin cells the kinetic parameters for L-[3H]adenosine were S0.5 = 6.2 +/- 0.2 microM and a Vmax 19.7 +/- 0.5 pmol/min per 10(6) cells, with a pronounced positive cooperativity. The Hill coefficient was 4.9. The transport of the L-isomer in cultured cells followed Michaelis-Menten kinetics at the lowest concentrations employed, below 2 microM. On the basis of these results, we propose a kinetic model whereby the adenosine transporter functions mnemonically.
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