Quantitation of the P2Y(1) receptor with a high affinity radiolabeled antagonist.
2-Chloro-N(6)-methyl-(N )-methanocarba-2'-deoxyadenosine-3',5'- bisphosphate (MRS2279) was developed previously as a selective high-affinity, non-nucleotide P2Y(1) receptor (P2Y1-R) antagonist (J Med Chem 43:829-842, 2002; Br J Pharmacol 135:2004-2010, 2002). We have taken advantage of the N(6)-methyl substitution in the adenine base to incorporate [(3)H]methylamine into the synthesis of [(3)H]MRS2279 to high (89 Ci/mmol) specific radioactivity and have used this molecule as a radioligand for the P2Y1-R. [(3)H]MRS2279 bound to membranes from Sf9 insect cells expressing recombinant human P2Y1-R but not to membranes from wild-type Sf9 cells or Sf9 cells expressing high levels of recombinant P2Y(2) or P2Y(12) receptors. Equilibrium binding of [(3)H]MRS2279 to P2Y1-R expressed in Sf9 membranes was with a high affinity (K(d) = 8 nM) essentially identical to the apparent affinity of MRS2279 determined previously in studies of P2Y1-R-promoted inositol phosphate accumulation or platelet aggregation. A kinetically derived K(d) calculated from independent determinations of the rate constants of association (7.15 x 10(7) M(-1) min(-1)) and dissociation (0.72 min(-1)) of [(3)H]MRS2279 also was in good agreement with the K(d) derived from equilibrium binding studies. Competition binding assays with [(3)H]MRS2279 and P2Y1-R expressing Sf9 cell membranes revealed K(i) values for the P2Y1-R antagonists MRS2279 (K(i) = 13 nM), N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179; K(i) = 84 nM), adenosine-3', 5'-bisphosphate (K(i)=900 nM), and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (K(i) = 6 microM) that were in good agreement with antagonist activities of these molecules previously determined at the P2Y1-R in intact tissues. Moreover, [(3)H]MRS2279 also bound with high affinity (K(d) = 4-8 nM) to Chinese hamster ovary (CHO) or 1321N1 human astrocytoma cells stably expressing the human P2Y1-R, but specific binding was not observed in wild-type CHO or 1321N1 cells. [(3)H]MRS2279 bound with high affinity (K(d) = 16 nM) to a binding site on out-dated human platelets (5-35 receptors/platelet) and rat brain membranes (210 fmol/mg protein) that fit the expected drug selectivity of a P2Y1-R. Taken together, these results indicate that [(3)H]MRS2279 is the first broadly applicable antagonist radioligand for a P2Y receptor.
Waldo
Gary L
GL
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
Corbitt
James
J
Boyer
José L
JL
Ravi
Gnana
G
Kim
Hak Sung
HS
Ji
Xiao-Duo
XD
Lacy
James
J
Jacobson
Kenneth A
KA
Harden
T Kendall
TK
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D007313:Insecta / Q000166:cytology; D058919:Purinergic P2 Receptor Antagonists; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000032:analysis*; D058472:Receptors, Purinergic P2Y1; D014316:Tritium
C465253:2-chloro-N6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate; C546382:P2RY1 protein, human; C546384:P2ry1 protein, rat; D058919:Purinergic P2 Receptor Antagonists; D019275:Radiopharmaceuticals; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D014316:Tritium; D000244:Adenosine Diphosphate