Characterization of adenosine receptors in brain using N6 cyclohexyl [3H]adenosine

The presence of distinct binding sites for adenosine in both the CNS and PNS has been proposed in numerous studies. The recent availability of stable adenosine analogues such as cyclohexyladenosine, 2-chloroadenosine and diethylphenylxanthine has made the characterization of such a receptor feasible. In the present report the binding of N6 cyclohexyl [3H]adenosine ([3H]CHA) to rat brain synaptosomal membranes is characterized. [3H]CHA binding is saturable and exhibits a biphasic kinetic saturation profile characteristic of 2 binding sites. The high affinity site has a Kd of 0.7 nM and the low affinity site 2.4 nM. The respective Bmax values are 230 and 120 fmol/mg protein in fat forebrain. The highest density of binding sites is found in the hippocampus and subcellular distribution studies indicate that the [3H]CHA site is predominantly synaptosomal. [3H]CHA binding is highly dependent in the presence of adenosine deaminase since only 30% of the binding capacity is observed in synaptosomal membranes not treated with this enzyme. Of the many cations and anions tested only copper and zinc have effects on [3H]CHA binding. Both metals are potent inhibitors of binding with copper having an IC50 of 30 microM and zinc 150 microM. Sulfhydryl reducing and alkylating agents also inhibit binding indicating that the binding site is a sulfhydryl-dependent protein.