Lack of specificity of [35S]‐ATPγS and [35S]‐ADPβS as radioligands for ionotropic and metabotropic P2 receptor binding

Adenosine‐5′‐O‐3‐thio³⁵S]triphosphate (³⁵S]‐ATPγS) has been reported to specifically bind several P2X receptor subtypes, including P2X₁, P2X₂, P2X₃, and P2X₄. Similarly, adenosine‐5′‐O‐2‐thio³⁵S]diphosphate (³⁵S]‐ADPβS) has been reported to label putative P2Y receptors. To address whether these radioligands selectively label P2 receptors, the functional activity of various P2 ligands was compared with their ability to compete for ³⁵S]‐ATPγS and ³⁵S]‐ADPβS binding to cell membrane preparations from rat brain, HEK293 cells, and to native and P2X₄ transfected 1321N1 astrocytoma cells. ³⁵S]‐ATPγS (0.2 nM) and ³⁵S]‐ADPβS (0.1 nM) displayed a high percentage of specific binding to membranes prepared from 1321N1 human astrocytoma cells, which were found to be devoid of detectable P2X and P2Y functional activity. ³⁵S]‐ATPγS and ³⁵S]‐ADPβS also exhibited equivalent high percentages of specific binding to HEK293 cell membranes, which endogenously express the P2Y₁ and P2Y₂ receptor subtypes, to 1321N1 cells stably transfected with the human P2X₄ receptor, and to rat brain membranes, which have previously been shown to contain both P2X and P2Y receptor subtypes. The potency order of P2 agonists to compete for radioligand binding to these cell membrane preparations was significantly different from the functional rank order potencies determined in HEK293 cells and 1321N1 cells expressing the P2X₄ receptor, as measured by cytosolic calcium flux. These data indicate that ³⁵S]‐ATPγS and ³⁵S]‐ADPβS appear to bind sites that do not correspond to known functional P2 receptor subtypes. The apparent lack of specificity of these radioligands for labeling P2 receptors is similar to that reported for other radiolabeled nucleotides and illustrates the need for caution in interpreting the apparent pharmacology of native P2 receptors on the basis of binding data alone. Drug Dev. Res. 48:84–93, 1999. © 1999 Wiley‐Liss, Inc.