Binding characteristics of an adenosine receptor in human placenta

The binding characteristics of human placental microsomes were evaluated for properties related to an adenosine receptor using 2-chloro[8-3H]adenosine as the ligand. Saturation of binding sites occurred with 0.75 pmol of ligand/mg of protein. Analysis of these data by Scatchard plot indicates a single class of binding sites with a Kd of 56 nM with 1.1 pmol of ligand bound/mg of protein. The specificity of 2-chloro[8-3H] adenosine binding was assessed by the ability of adenosine analogs to compete for binding sites. Using this approach, the Kd for 5'-N-ethylcarboxamideadenosine was estimated to be 0.3 microM and for N6-(L-2-phenylisopropyl)adenosine or N6-cyclohexyladenosine to be greater than 100 microM. Isobutylmethylxanthine and theophylline, receptor antagonists, have Kd values of 19 microM and 150 microM, respectively. Chloroadenosine binding to placental microsomes was time-dependent and reached equilibrium at approximately 20 min. The kob was 0.24 min-1 and the k1 was 0.71 X 10(8) min-1 M-1. Reversibility of chloroadenosine binding at equilibrium was completed at 5 min with a k2 value of 1.17 min-1. The Kd calculated from the reverse rate constant was 17 nM. Our observations suggest that 2-chloro[8-3H]adenosine identifies binding sites in human placenta with characteristics of an adenosine receptor. The binding properties conform to the previous description of a low affinity stimulatory receptor (Ra or A2 receptor).