Solubilization of an Adenosine Uptake Site in Brain

Abstract

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [(3H]NBI) the solubilized site is characterized kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific and high affinity in nature. The Kd and Bmax [maximum binding capacity] of guinea pig extracts are 0.13 .+-. 0.02 nM and 133 .+-. 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep > hexobendine > dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding; caffeine is without effect. The solubilized adenosine uptake site has virtually identical properties to the native membrance site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine ([3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to 2 apparent populations of adenosine receptor, a high-affinity site with a Kd of 0.32 .+-. 0.06 nM and a Bmax of 105 .+-. 30 fmol/mg protein and a lower-affinity site with a Kd of 5.50 .+-. 0.52 nM and Bmax of 300 .+-. 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake ([3H]NBI binding) site. The adenosine uptake site can be solubilized and retains its binding and pharmacologic properties in the solubilized state.