Kinetic and inhibitor specificity of adenosine transport in guinea pig cerebral cortical synaptosomes: evidence for two nucleoside transporters.

The transport of [(3)H]adenosine at 22 degrees C was investigated in guinea pig cerebral cortical synaptosomes using an inhibitor-stop filtration method. Under these conditions adenosine was not significantly metabolized during the incubation period used to determine the initial rates of adenosine transport. The dose response curves for the inhibition of adenosine transport by nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole were biphasic-approx. 50-60% of the transport activity was inhibited with IC(50) values of 0.7, 1 and 9 nM respectively, but the remaining activity was insensitive to concentrations as high as 1 ? M. Adenosine influx by both components was saturable (K(m) values of 17 +/- 3 and 68 +/- 8 ? M; V(max) values of 2.8 +/- 0.3 and 6.1 +/- 0.4 pmol/mg protein per s for NBMPR-sensitive and -insensitive components, respectively), and inhibited by other nucleosides and benzodiazepines. The two transport components also differed in their sensitivity to inhibition by other nucleosides and benzodiazepines indicating that the NBMPR-sensitive component of nucleoside transport in guinea pig synaptosomes exhibits a higher affinity than the NBMPR-insensitive component. However, both components have a broad specificity. Inhibition of adenosine transport by NBMPR was associated with high affinity binding of NBMPR to the synaptosomes (K(d) 88 +/- 6 pM). Binding of NBMPR to these sites was blocked by dilazep and dipyridamole with K(1) values similar to those measured for inhibiting NBMPR-sensitive adenosine influx. These results, together with previous findings using NBMPR and dipyridamole as ligand probes, suggest that there are two components of nucleoside transport in mammalian cerebral cortical synaptosomes that differ in their sensitivity to inhibition by NBMPR and other transport inhibitors.