CHARACTERIZATION OF ADENOSINE-A1-RECEPTOR IN A CELL-LINE (28A) DERIVED FROM RABBIT COLLECTING TUBULE

We have previously reported that in several renal cell types, adenosine receptor agonists inhibit adenylyl cyclase and activate phospholipase C via a pertussis toxin-sensitive G protein. In the present study, in 28A cells, both of these adenosine receptor-mediated responses were inhibited by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a highly selective A1 adenosine receptor antagonist. The binding characteristics of the adenosine A1 receptor in the 28A renal cell line were studied using the radiolabeled antagonist [H-3]DPCPX to determine whether two separate binding sites could account for these responses. Saturation binding of [H-3]DPCPX to 28A cell membranes revealed a single class of A1 binding sites with an apparent K(d) value of 1.4 nM and maximal binding capacity of 64 fmol/mg protein. Competition experiments with a variety of adenosine agonists gave biphasic displacement curves with a pharmacological profile characteristic of A1 receptors. Comparison of [H-3]DPCPX competition binding data from 28A cell membranes with rabbit brain membranes, a tissue with well-characterized A1 receptors, reveals that the A1 receptor population in 28A cells has similar agonist, binding affinities to the receptor population in brain but has a considerably lower density. Addition of guanosine 5'-triphosphate (100-mu-M) to 28A cell membranes caused the competition curves to shift from biphasic to monophasic, indicating that the A1 receptors exist in two interconvertible affinity states because of their coupling to G proteins. In the absence of evidence for subpopulations of the A1 receptor, it appears that in 28A cells, a single A1 receptor population, as defined by ligand binding characteristics, couples via one or more pertussis toxin-sensitive guanine nucleotide binding proteins to two different biological signaling mechanisms.