A new class of adenosine receptors in brain: Characterization by 2-chloro[ 3H]adenosine binding

Abstract

Micromolar concentrations of adenosine and its analogs have profound depressant effects on neuronal firing and synaptic transmission in many brain areas. Using the adenosine agonist 2-chloro[3H]-adenosine (Cl[ 3H]Ado), we have identified a distinct class of micromolar-affinity adenosine binding sites in rat forebrain membranes. Specific Cl[ 3H]Ado binding was reversible and saturable with an apparent K D of 9.1 μM and a B max of 61 pmoles/mg protein. The present studies were conducted using washed brain membrane fractions not treated with adenosine deaminase. Specific Cl[ 3H]Ado binding under these conditions was insensitive to (−)- N 6( R-phenylisopropyl) adenosine ((-)PIA) and treatment with 3 mM N-ethylmaleimide, unlike high-affinity A1 adenosine receptor binding. Treatment of membranes with adenosine deaminase revealed an additional population of binding sites sensitive to (-)PIA. Inhibition of Cl[ 3H]Ado binding by adenosine analogs exhibited an order of potency ClAdo > 5′- N-ethylcarboxamide adenosine (NECA) > (-)PIA which differs from that of both A1 and A2 adenosine receptors. The potent A1 and A2 receptor antagonist 8-phenyltheophylline had no significant effect on binding up to 10 μM. Specific binding, however, was inhibited by the adenosine antagonists 8( p-sulfophenyl)theophylline, isobutylmethylxanthine, theophylline, and caffeine. Micromolar Cl[ 3H]Ado binding was highly selective for adenosine agonists and antagonists. These results suggest that the micromolar-affinity Cl[ 3H]Ado binding sites may represent a novel central purinergic receptor, distinct from the A1 and A2 adenosine receptors involved in the regulation of adenylate cyclase.