Characteristics of high affinity and low affinity adenosine binding sites in human cerebral cortex

The binding characteristics of human brain cortical membrane fractions were evaluated to test the hypothesis that there are A1 and A2 adenosine binding sites. The ligands used were 2-chloro[8-3H]adenosine and N6-[adenine-2,8-3H]cyclohexyladenosine. Binding of chloroadenosine to human brain cortical membranes was time dependent, reversible and concentration dependent. The Kd calculated for chloroadenosine by Scatchard analysis of equilibrium data was 280 nM, with a Bmax of 1.6 pmoles/mg protein, suggesting a single class of binding sites. The specificity of chloroadenosine binding was assessed by the ability of adenosine analogs to compete for binding sites. Using this approach, the apparent Kd was estimated to be 0.74 microM for 5'-N-ethyl-carboxamideadenosine, 1 microM cyclohexyladenosine, and 13 microM for N6-(L-2-phenylisopropyl)adenosine. Isobutylmethylxanthine and theophylline, receptor antagonists, had apparent Kd values of 84 microM and 105 microM, respectively. Hill slope factors ranged from 0.3 to 0.6. Chloroadenosine binding to human brain cortical membranes approached equilibrium at 90 minutes, with a T1/2 of 10 minutes. The kob was 0.080 min-1 and the k1 was 7.5 X 10(4) min-1 M-1. Reversibility of chloroadenosine binding at equilibrium was completed at approximately 10 minutes with a k2 value of 0.074 min-1. The Kd calculated from the rate constants was 990 nM. Cyclohexyladenosine binding was concentration dependent. The Kd calculated for cyclohexyladenosine via Scatchard analysis of equilibrium data was 5 nM with a Bmax of 0.35 pmoles/mg protein. Cyclohexyladenosine binding was displaced by 3 known receptor agonists: N6-(L-2-phenyliso propyl)adenosine (Kd 4 nM), 2-chloroadenosine (Kd 10 nM) and 5H-N-ethyl-carboxamideadenosine (Kd 6 nM). The apparent Kd values for the agonists were 1 to 3 orders of magnitude lower with this ligand as compared to radioactive chloroadenosine. Binding was also displaced by 2 known antagonists, isobutylmethylxanthine and theophylline, with apparent Kd values of 4 microM and 8 microM, respectively. Hill slope factors ranged from 0.5 to 0.8. Our data support the existence of two adenosine binding sites in human cortex compatible with the low affinity (A2) and high affinity (A1) adenosine receptors.