Characterization of nitrobenzylthioinosine binding to nucleoside transport sites selective for adenosine in rat brain.

Nucleoside transport sites in rat brain membrane preparations were labeled with [3H]nitrobenzylthioinosine ([3H] NBI), a potent inhibitor of nucleoside transport systems. The membranes contained a single class of very high affinity binding sites with KD and Bmax values of 0.06 nM and 147 fmol/mg of protein, respectively. The displacement of [3H]NBI binding by various nucleosides, adenosine receptor agonists and antagonists, and known nucleoside transport inhibitors was examined. The Ki values (micromolar concentration) of [3H]NBI displacement by the nucleosides tested were: adenosine, 3.0; inosine, 160; thymidine, 240; uridine, 390; guanosine, 460; and cytidine, 1000. These nucleosides displayed parallel displacement curves indicating their interaction with a common site labeled by [3H]NBI. The nucleobases, hypoxanthine and adenine, exhibited Ki values of 220 and 3640 microM, respectively. Adenosine receptor agonists exhibited moderate affinities for the [3H]NBI site, whereas the adenosine receptor antagonists, caffeine, theophylline, and enprofylline, were ineffective displacers. The Ki values for cyclohexyladenosine, (+)- and (-)-phenylisopropyladenosine, 2-chloroadenosine, and adenosine 5'-ethylcarboxamide were 0.8, 0.9, 2.6, 12, and 54 microM, respectively. These affinities and the rank order of potencies indicate that [3H]NBI does not label any known class of adenosine receptors (i.e., A1, A2, and P). The Ki values of other nucleoside transport inhibitors were: nitrobenzylthioguanosine, 0.05 nM; dipyridamole, 16 nM; papaverine, 3 microM; and 2'-deoxyadenosine, 22 microM. These results indicate that [3H]NBI binds to a nucleoside transporter in brain which specifically recognizes adenosine as its preferred endogenous substrate. This ligand may aid in the identification of CNS neural systems that selectively accumulate adenosine and thereby control "adenosinergic" function.