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 dissociation constant (Kd) calculated for chloroadenosine by Scatchard analysis of equilibrium data was 280 nmol/L, with a maximum binding (Bmax) of 1.6 pmol/mg protein, suggesting a single class of binding sites. The specificity of chloroadenosine binding was assessed by the ability of adenosine analogues to compete for binding sites. With this approach, the apparent Kd was estimated to be 0.74 mumol/L for 5'-N-ethylcarboxamideadenosine, 1 mumol/L for cyclohexyladenosine, and 13 mumol/L for N6-(L-2-phenylisopropyl)adenosine. Isobutylmethylxanthine and theophylline, receptor antagonists, had apparent Kd values of 84 mumol/L and 105 mumol/L, 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 t 1/2 of 10 minutes. The kob was 0.080/min, and the k1 was 7.5 X 10(4)/min/mol/L. Reversibility of chloroadenosine binding at equilibrium was completed at approximately 10 minutes, with a k2 value of 0.074/min. The Kd calculated from the rate constants was 990 nmol/L. Cyclohexyladenosine binding was concentration dependent. The Kd calculated for cyclohexyladenosine via Scatchard analysis of equilibrium data was 5 nmol/L with a Bmax of 0.35 pmol/mg protein. Cyclohexyladenosine binding was displaced by three known receptor agonists: N6-(L-2-phenylisopropyl)adenosine (Kd 4 nmol/L), 2-chloroadenosine (Kd 10 nmol/L) and 5'-N-ethylcarboxamideadenosine (Kd 6 nmol/L).(ABSTRACT TRUNCATED AT 250 WORDS)