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

Dayle Houston, Michihiro Ohno, Robert A. Nicholas, Kenneth A. Jacobson, T. Kendall Harden

Research output: Contribution to journalArticle

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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 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 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 [γ 32P]ATP to yield [ 32P]MRS2500. [ 32P]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 [ 32P]MRS2500 was saturable with a K D of 1.2 nM. Agonists and antagonists of the P2Y 1 receptor inhibited [ 32P]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 [ 32P]MRS2500 (K D=0.33 nM) 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 [ 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 P2Y 1 receptor radioligand and its utility for examining P2Y 1 receptor expression in native mammalian tissues.

LanguageEnglish (US)
Pages459-467
Number of pages9
JournalBritish Journal of Pharmacology
Volume147
Issue number5
DOIs
StatePublished - Mar 1 2006

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Binding Sites
Brain
Polynucleotide 5'-Hydroxyl-Kinase
Sf9 Cells
Membranes
G-Protein-Coupled Receptors
2'-deoxyadenosine
2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate
Cerebellum
Radioactivity
Insects
Nucleotides
Adenosine Triphosphate
Cell Membrane
Pharmacology
Lung
Liver
2-chloro-N6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate

Keywords

  • Competitive antagonist
  • MRS2279
  • MRS2500
  • P2Y receptor
  • Radioligand

ASJC Scopus subject areas

  • Pharmacology

Cite this

[ 32P]2-iodo-N 6-methyl-(N)-methanocarba-2′- deoxyadenosine-3′,5′-bisphosphate ([ 32P]MRS2500), a novel radioligand for quantification of native P2Y 1 receptors. / Houston, Dayle; Ohno, Michihiro; Nicholas, Robert A.; Jacobson, Kenneth A.; Harden, T. Kendall.

In: British Journal of Pharmacology, Vol. 147, No. 5, 01.03.2006, p. 459-467.

Research output: Contribution to journalArticle

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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 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 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 [γ 32P]ATP to yield [ 32P]MRS2500. [ 32P]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 [ 32P]MRS2500 was saturable with a K D of 1.2 nM. Agonists and antagonists of the P2Y 1 receptor inhibited [ 32P]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 [ 32P]MRS2500 (K D=0.33 nM) 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 [ 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 P2Y 1 receptor radioligand and its utility for examining P2Y 1 receptor expression in native mammalian tissues.",
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AU - Houston,Dayle

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AU - Nicholas,Robert A.

AU - Jacobson,Kenneth A.

AU - Harden,T. Kendall

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N2 - 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 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 [γ 32P]ATP to yield [ 32P]MRS2500. [ 32P]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 [ 32P]MRS2500 was saturable with a K D of 1.2 nM. Agonists and antagonists of the P2Y 1 receptor inhibited [ 32P]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 [ 32P]MRS2500 (K D=0.33 nM) 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 [ 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 P2Y 1 receptor radioligand and its utility for examining P2Y 1 receptor expression in native mammalian tissues.

AB - 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 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 [γ 32P]ATP to yield [ 32P]MRS2500. [ 32P]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 [ 32P]MRS2500 was saturable with a K D of 1.2 nM. Agonists and antagonists of the P2Y 1 receptor inhibited [ 32P]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 [ 32P]MRS2500 (K D=0.33 nM) 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 [ 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 P2Y 1 receptor radioligand and its utility for examining P2Y 1 receptor expression in native mammalian tissues.

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