Characteristics of an adenosine A1 binding site in human placental membranes

Abstract

Binding sites were solubilized from human placental membrane using 1.5% sodium cholate and were assayed using polyethylene glycol precipitation. These soluble binding sites had properties of an adenosine A1 binding site. 2-[3H]Chloroadenosine and N-[3H]-ethylcarboxamidoadenosine (NECA) binding were time dependent and reversible. Scatchard plots indicate two classes of binding sites with Kd values of 6 and 357 n for 2-chloro[8-3H]adenosine and 0.1 and 26 n with [3H]NECA. The specificity of [3H]NECA binding was assessed by the ability of adenosine analogs to compete for binding sites. Using this approach the estimated IC50 values were 60 n for N6-((R)-1-methyl-2-phenylethyl)adenosine (R-PIA), 160 n for S-PIA, 80 n for NECA, and 20 n for 2-chloroadenosine. Binding of [3H]NECA to the soluble sites is inhibited to 48% of the control value by 100 [mu] guanylyl-5'-imidodiphosphate (Gpp(NH)p). The IC50 value for NECA binding to the soluble binding site was increased from 80 n to 1500 by Gpp(NH)p. There was a shift of binding affinity from a mixture of high and low affinity to only low affinity with 100 [mu] Gpp(NH)p. Despite these alterations a NECA prelabeled molecular species of 150 kDa did not decrease in molecular weight upon the addition of 100 [mu] Gpp(NH)p during high-performance liquid chromatography on a Superose 12 column. Other evidence to support the concept of preferential solubilization and assay of a small population of A1 binding sites was obtained. Following solubilization adenosine A2-like binding sites could be detected only in reconstituted vesicles. The existence of small amounts of A1 binding sites in intact human placental membranes was directly demonstrated using the A1 agonist ligand N6-[3H]cyclohexyladenosine and the A1 antagonist ligand 8-[3H]cyclopentyl-1,3-dipropylxanthine. JAR choriocarcinoma cells have "A2-like" membrane binding sites. In contrast to placental membranes, only A2-like binding sites could be solubilized from JAR choriocarcinoma cells. These observations indicate that human placental membranes contain adenosine A1 binding sites in addition to A2-like binding sites. These sites are guanine nucleotide sensitive, but do not shift to a lower molecular weight form upon assumption of a low affinity state.