[ 32 P]2‐iodo‐ N 6 ‐methyl‐( N )‐methanocarba‐2′‐deoxyadenosine‐3′,5′‐bisphosphate ([ 32 P]MRS2500), a novel radioligand for quantification of native P2Y 1 receptors

Analysis of the P2Y family of nucleotide‐activated G‐protein‐coupled receptors has been compromised by the lack of selective high‐affinity, high‐specific‐radioactivity radioligands. We have pursued quantification of the P2Y 1 receptor through the development of a series of selective P2Y 1 receptor antagonists. Recently, we synthesized 2‐iodo‐ N 6 ‐methyl‐( N )‐methanocarba‐2′‐deoxyadenosine 3′,5′‐bisphosphate (MRS2500), a selective, competitive antagonist that exhibits a K i of 0.8 n M in competition‐binding assays with [ 3 H]MRS2279. A 3′‐monophosphate precursor molecule, MRS2608, was radiolabeled at the 5′ position with 32 P using polynucleotide kinase and [ γ 32 P]ATP to yield [ 32 P]MRS2500. [ 32 P]MRS2500 bound selectively to Sf9 insect cell membranes expressing the human P2Y 1 receptor (Sf9‐P2Y 1 ), but did not detectably bind membranes expressing other P2Y receptors. P2Y 1 receptor binding to [ 32 P]MRS2500 was saturable with a K D of 1.2 n M . Agonists and antagonists of the P2Y 1 receptor inhibited [ 32 P]MRS2500 binding in Sf9‐P2Y 1 membranes with values in agreement with those observed in functional assays of the P2Y 1 receptor. A high‐affinity binding site for [ 32 P]MRS2500 ( K D =0.33 n M ) was identified in rat brain, which exhibited the pharmacological selectivity of the P2Y 1 receptor. Distribution of this binding site varied among rat tissues, with the highest amount of binding appearing in lung, liver, and brain. Among brain regions, distribution of the [ 32 P]MRS2500 binding site varied by six‐fold, with the highest and lowest amounts of sites detected in cerebellum and cortex, respectively. Taken together, these data illustrate the synthesis and characterization of a novel P2Y 1 receptor radioligand and its utility for examining P2Y 1 receptor expression in native mammalian tissues.British Journal of Pharmacology (2006) 147 , 459–467. doi: 10.1038/sj.bjp.0706453