Quantitative [3H]dipyridamole autoradiography: evidence for adenosine transporter heterogeneity in guinea pig brain.

[3H] Dipyridamole binding in guinea pig brain slices has been characterized. Binding of [3H] dipyridamole to guinea pig forebrain slices was found to be rapid, reversible and saturable. Saturation experiments revealed a class of high affinity binding sites with a Bmax value of 592 +/- 118 fmol/mg protein and Kd value of 10.8 nM +/- 2.1 nM in the analysed concentration range. In competition experiments, the adenosine transport inhibitors hexobendine and dipyridamole itself were the most potent displacers (inhibition constants of 4.6 nM +/- 1 nM and 11.5 nM +/- 3 nM) with "pseudo-Hill" coefficients close to 1. Competition curves with nitrobenzylthioinosine, another adenosine transport inhibitor, however, showed a biphasic profile with a "pseudo-Hill" coefficient of 0.33 +/- 0.04. Just 42% +/- 4% of [3H] dipyridamole binding were inhibited by nanomolar concentrations of nitrobenzylthionosine and only micromolar concentrations displaced the remainder. Subsequent quantitative autoradiography demonstrated regional differences in the inhibition of [3H] dipyridamole binding by submicromolar concentrations of nitrobenzylthioinosine. While in cortical areas of cerebrum and cerebellum 500 nM nitrobenzylthioinosine displaced binding of [3H] dipyridamole to only about one-third of its sites (in the Purkinje cell layer less than 10%), it showed similar potency as dipyridamole in various areas of the brainstem and hypothalamus. This biphasic and regionally heterogenous interaction of nitrobenzylthioinosine with [3H] dipyridamole binding sites in guinea pig brain slices strongly suggests heterogeneity of adenosine transporters.