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An examination of deoxyadenosine 5'(alpha-thio)triphosphate as a ligand to define P2Y receptors and its selectivity as a low potency partial agonist of the P2Y1 receptor.
Br J Pharmacol 1997; 121(2):338-44BJ

Abstract

1. The functional activity of deoxyadenosine 5'(alpha-thio)triphosphate (dATP alpha S) was assessed at the cloned human P2Y1 receptor stably expressed in 1321N1 human astrocytoma cells and transiently expressed in Cos-7 cells. 2. Cells expressing the receptor responded to adenine nucleotides with an increase in [3H]-inositol phosphate accumulation. Half-maximal responses were obtained at approximately 30 nM for 2-methylthioadenosine-5'-triphosphate (2MeSATP), 300 nM for dATP alpha S, and 1000 nM for adenosine 5'-triphosphate (ATP). dATP alpha S produced a maximal response that was only 37 +/- 4% of that produced by ATP or 2MeSATP. dATP alpha S also competitively antagonized the phospholipase C response to 2MeSATP with a KB of 644 +/- 14 nM. Thus dATP alpha S acts as a low potency partial agonist at P2Y1 receptors. 3. The selectivity of dATP alpha S for P2Y1 receptors was determined by examining its capacity to activate P2Y2, P2Y4 and P2Y6 receptors also stably expressed in 1321N1 cells. Although dATP alpha S was a partial agonist at P2Y1 receptors it was a full agonist at P2Y2 receptors, albeit with a potency that was two orders of magnitude lower than at P2Y1 receptors. No agonist or antagonist activity was observed at P2Y4 and P2Y6 receptors. 4. Although [35S]-dATP alpha S bound to a relatively high density (ca 10 pmol mg-1 protein) of binding sites in membranes from 1321N1 or Cos-7 cells expressing the P2Y1 receptor, no difference in the total density of sites was observed between membranes from wild-type, empty vector-transfected, or P2Y1 receptor-expressing cells. Moreover, adenine nucleotide analogues inhibited [35S]-dATP alpha S binding with an order of potency that differed markedly from that for the accumulation of inositol phosphates in intact transfected P2Y1 receptor-expressing cells. Saturation binding experiments demonstrated multiple affinity states for [35S]-dATP alpha S binding in wild-type Cos-7 cell membranes. These data from 1321N1 and Cos-7 cells suggest that cellular membranes exhibit a large number of high affinity binding sites for [35S]-dATP alpha S that are not related to P2Y receptor subtypes.

Authors+Show Affiliations

Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599-7365, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

9154346

Citation

Schachter, J B., and T K. Harden. "An Examination of Deoxyadenosine 5'(alpha-thio)triphosphate as a Ligand to Define P2Y Receptors and Its Selectivity as a Low Potency Partial Agonist of the P2Y1 Receptor." British Journal of Pharmacology, vol. 121, no. 2, 1997, pp. 338-44.
Schachter JB, Harden TK. An examination of deoxyadenosine 5'(alpha-thio)triphosphate as a ligand to define P2Y receptors and its selectivity as a low potency partial agonist of the P2Y1 receptor. Br J Pharmacol. 1997;121(2):338-44.
Schachter, J. B., & Harden, T. K. (1997). An examination of deoxyadenosine 5'(alpha-thio)triphosphate as a ligand to define P2Y receptors and its selectivity as a low potency partial agonist of the P2Y1 receptor. British Journal of Pharmacology, 121(2), pp. 338-44.
Schachter JB, Harden TK. An Examination of Deoxyadenosine 5'(alpha-thio)triphosphate as a Ligand to Define P2Y Receptors and Its Selectivity as a Low Potency Partial Agonist of the P2Y1 Receptor. Br J Pharmacol. 1997;121(2):338-44. PubMed PMID: 9154346.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - An examination of deoxyadenosine 5'(alpha-thio)triphosphate as a ligand to define P2Y receptors and its selectivity as a low potency partial agonist of the P2Y1 receptor. AU - Schachter,J B, AU - Harden,T K, PY - 1997/5/1/pubmed PY - 1997/5/1/medline PY - 1997/5/1/entrez SP - 338 EP - 44 JF - British journal of pharmacology JO - Br. J. Pharmacol. VL - 121 IS - 2 N2 - 1. The functional activity of deoxyadenosine 5'(alpha-thio)triphosphate (dATP alpha S) was assessed at the cloned human P2Y1 receptor stably expressed in 1321N1 human astrocytoma cells and transiently expressed in Cos-7 cells. 2. Cells expressing the receptor responded to adenine nucleotides with an increase in [3H]-inositol phosphate accumulation. Half-maximal responses were obtained at approximately 30 nM for 2-methylthioadenosine-5'-triphosphate (2MeSATP), 300 nM for dATP alpha S, and 1000 nM for adenosine 5'-triphosphate (ATP). dATP alpha S produced a maximal response that was only 37 +/- 4% of that produced by ATP or 2MeSATP. dATP alpha S also competitively antagonized the phospholipase C response to 2MeSATP with a KB of 644 +/- 14 nM. Thus dATP alpha S acts as a low potency partial agonist at P2Y1 receptors. 3. The selectivity of dATP alpha S for P2Y1 receptors was determined by examining its capacity to activate P2Y2, P2Y4 and P2Y6 receptors also stably expressed in 1321N1 cells. Although dATP alpha S was a partial agonist at P2Y1 receptors it was a full agonist at P2Y2 receptors, albeit with a potency that was two orders of magnitude lower than at P2Y1 receptors. No agonist or antagonist activity was observed at P2Y4 and P2Y6 receptors. 4. Although [35S]-dATP alpha S bound to a relatively high density (ca 10 pmol mg-1 protein) of binding sites in membranes from 1321N1 or Cos-7 cells expressing the P2Y1 receptor, no difference in the total density of sites was observed between membranes from wild-type, empty vector-transfected, or P2Y1 receptor-expressing cells. Moreover, adenine nucleotide analogues inhibited [35S]-dATP alpha S binding with an order of potency that differed markedly from that for the accumulation of inositol phosphates in intact transfected P2Y1 receptor-expressing cells. Saturation binding experiments demonstrated multiple affinity states for [35S]-dATP alpha S binding in wild-type Cos-7 cell membranes. These data from 1321N1 and Cos-7 cells suggest that cellular membranes exhibit a large number of high affinity binding sites for [35S]-dATP alpha S that are not related to P2Y receptor subtypes. SN - 0007-1188 UR - https://www.unboundmedicine.com/medline/citation/9154346/An_examination_of_deoxyadenosine_5'_alpha_thio_triphosphate_as_a_ligand_to_define_P2Y_receptors_and_its_selectivity_as_a_low_potency_partial_agonist_of_the_P2Y1_receptor_ L2 - https://doi.org/10.1038/sj.bjp.0701136 DB - PRIME DP - Unbound Medicine ER -