32p]2-iodo-n6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate ([32p]mrs2500), a Novel Radioligand for Quantification of Native P2y1 Receptors.
From: Department of Pharmacology, University of North Carolina School of Medicine, CB# 7365 Chapel Hill, NC 27599, USA. dhouston@med.unc.edu
British journal of pharmacology
- Publish Date: Mar 2006
- ISSN: 0007-1188
- Volume: 147
- Issue: 5
- Pages: 459-67
- Medium: Print
- Language: English
- Citation (JAMA): Houston Dayle, Ohno Michihiro, Nicholas Robert A, et al. 32p]2-iodo-n6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate ([32p]mrs2500), a Novel Radioligand for Quantification of Native P2y1 Receptors.. Br. J. Pharmacol. Mar 2006;147:459-67
Abstract
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 P2Y1 receptor through the development of a series of selective P2Y1 receptor antagonists. Recently, we synthesized 2-iodo-N6-methyl-(N)-methanocarba-2’-deoxyadenosine 3’,5’-bisphosphate (MRS2500), a selective, competitive antagonist that exhibits a Ki of 0.8 nM in competition-binding assays with [3H]MRS2279. A 3’-monophosphate precursor molecule, MRS2608, was radiolabeled at the 5’ position with 32P using polynucleotide kinase and [gamma32P]ATP to yield [32P]MRS2500. [32P]MRS2500 bound selectively to Sf9 insect cell membranes expressing the human P2Y1 receptor (Sf9-P2Y1), but did not detectably bind membranes expressing other P2Y receptors. P2Y1 receptor binding to [32P]MRS2500 was saturable with a KD of 1.2 nM. Agonists and antagonists of the P2Y1 receptor inhibited [32P]MRS2500 binding in Sf9-P2Y1 membranes with values in agreement with those observed in functional assays of the P2Y1 receptor. A high-affinity binding site for [32P]MRS2500 (KD=0.33 nM) was identified in rat brain, which exhibited the pharmacological selectivity of the P2Y1 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 [32P]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 P2Y1 receptor radioligand and its utility for examining P2Y1 receptor expression in native mammalian tissues.
Mesh Headings (Keywords): Adenosine Diphosphate, Animals, Brain, Deoxyadenine Nucleotides, Male, Phosphorus Radioisotopes, Radioligand Assay, Rats, Rats, Sprague-Dawley, Receptors, Purinergic P2
Check for Full Text / PubMed Unique Identifier (PMID): 16299552
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