Characterization of adenosine receptors in rat brain by (-)[3H]N6-phenylisopropyladenosine.

Abstract

(−)N6-Phenylisopropyladenosine, a potent agonist in adenosine-responsive cellular systems, has been labeled with tritium to high specific activity (26 Ci/mmol) and used to identify adenosine binding sites in rat brain membranes. (−)[H3]N6-Phenylisopropyladenosine binding was studied by a vacuum filtration technique. The binding was rapid, rapidly reversible, dependent on pH and temperature and stereospecific since the (−)isomer of N6-phenylisopropyladenosine was 40-fold more potent than the (+)isomer in competition experiments. The stereospecific binding sites were saturable and bound 0.8 pmol of (−)N6-phenylisopropyladenosine per mg of membrane protein. The dissociation constant (KD) of (−)N6-phenylisopropyladenosine for these sites was 5–12 nM as determined independently by saturation and kinetic binding studies. Endogeneous ligands seem to occupy the binding sites since pretreatment with adenosine deaminase increased the specific binding. Adenosine and several adenosine derivatives were studied for their ability to compete with (−)[3H]N6-phenylisopropyladenosine binding. (−)N6-Phenylisopropyladenosine-5′-monophosphate, N6-phenyladenosine, N6-benzyladenosine, 2-chloroadenosine and adenosine were most potent in displacing the radioligand from its binding sites and the IC50-values ranged from 0.3–7 μM. Physiologically inactive compounds such as inosine, hypoxanthine, adenine and the ribose-modified analogues 2′-deoxyadenosine and 2′,5′-dideoxyadenosine did not substantially inhibit binding at concentrations up to 100 μM. The adenosine antagonists isobutylmethylxanthine (IC50 3.2 μM), theophylline (IC50 7.6 μM) and caffeine (IC50 99 μM) competed for the binding sites of (−)[3H]N6-phenylisopropyladenosine in a manner which parallels their known pharmacological activity whereas other phosphodiesterase inhibitors were ineffective. The (−)[3H]N6-phenylisopropyladenosine binding sites in rat brain membranes appear to be equivalent to adenosine receptor sites on the cell surface which have recently been classified as R-site adenosine receptors.