Lack of specificity of [S-35]-ATP gamma S and [S-35]-ADP beta S as radioligands for ionotropic and metabotropic P2 receptor binding

Adenosine-5'-O-3-thio[S-35]triphosphate ([S-35]-ATP gamma S) has been repor ted to specifically bind several P2X receptor subtypes, including P2X(1), P 2X(2), P2X(3), and P2X(4). Similarly, adenosine-5'-O-2-thio[S-35]diphosphat e ([S-35]-ADP beta S) has been reported to label putative P2Y receptors. To address whether these radioligands selectively label P2 receptors, the fun ctional activity of various P2 ligands was compared with their ability to c ompete for [S-35]-ATP gamma S and [S-35]-ADP beta S binding to cell membran e preparations from rat brain, HEK293 cells, and to native and P2X(4) trans fected 1321N1 astrocytoma cells. [S-35]-ATP gamma S (0.2 nM) and [S-35]-ADP beta S (0.1 nM) displayed a high percentage of specific binding to membran es prepared from 1321N1 human astrocytoma cells, which were found to be dev oid of detectable P2X and P2Y Functional activity [S-35]-ATP gamma S and [S -35]-ADP beta S also exhibited equivalent high percentages of specific bind ing to HEK293 cell membranes, which endogenously express the P2Y(1) and P2Y (2) receptor subtypes, to 1321N1 cells stably transfected with the human P2 X(4) receptor, and to rat brain membranes, which have previously been shown to contain both P2X and P2Y receptor subtypes. The potency order of P2 ago nists to compete for radioligand binding to these cell membrane preparation s was significantly different from the functional rank order potencies dete rmined in HEK293 cells and 1321N1 cells expressing the P2X(4) receptor, as measured by cytosolic calcium flux. These data indicate that [S-35]-ATP gam ma S and [S-35]-ADP beta S appear to bind sites that do not correspond to k nown functional P2 receptor subtypes. The apparent lack of specificity of t hese radioligands for labeling P2 receptors is similar to that reported for other radiolabeled nucleotides and illustrates the need for caution in int erpreting the apparent pharmacology of native P2 receptors on the basis of binding data alone. (C) 1999 Wiley-Liss, Inc.