Pharmacological Characterization of Rapidly Accumulated Adenosine by Dissociated Brain Cells from Adult Rat

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Abstract
Mechanically dissociated brain cells from adult rats were used to study biochemically and pharmacologically their capacity to accumulate rapidly [3H]adenosine. The assay, which used an inhibitor‐stop method to prevent further uptake into cells, was characterized with respect to protein and optimal substrate concentrations, and incubation times that ranged from 5 to 180 s. The accumulation of [3H]adenosine using 15‐s incubation periods, conditions under which < 10% of accumulated [3H]adenosine was metabolized, was best described kinetically by a two‐component system with Km and Vmax values for the high‐affinity component of 0.8 μM and 6.2 pmol/mg protein/15 s and for the low‐affinity component 259 μM and 2,217 pmol/mg protein/15 s, respectively. The potencies with which nucleosides, adenosine deaminase resistant adenosine receptor agonists, and nucleoside uptake inhibitors competed for these uptake components were determined. Of the nucleosides examined, adenosine was the „preferred” substrate for the uptake site. The Ki value of adenosine for the high‐affinity component was 10.7 μM. Inosine and uridine competed for a single lower affinity uptake system: Ki values were 142 and 696 μM, respectively. Nucleoside uptake inhibitors—nitrobenzylthioinosine, dipyridamole. and dilazep—were the most potent inhibitors of [3H]adenosine accumulation tested: the Ki values for the high‐affinity system were 0.11, 1.3, and 570 nM, respectively. The adenosine analogs S‐phenylisopropyladenosine, R‐phenylisopropyladenosine, and cyclohexyladenosine inhibited the high‐affinity component with Ki values of 2.3, 9.3, and 14.5 μM, respectively. N‐Ethylcarboxamidoadenosine competed for a single lower affinity uptake system; Ki, 292 μM. These results indicate that dissociated brain cells avidly take up [3H]adenosine and that the pharmacological profile for nucleoside uptake is similar to that for [3H]nitrobenzylthioinosine binding to putative nucleoside transport sites when uptake studies are conducted using brief incubation intervals.