Characterization of ATP and P2 agonists binding to the cardiac plasma membrane P1,P4-diadenosine 5′-tetraphosphate receptor

Abstract

P1,P4-Diadenosine 5'-tetraphosphate (Ap4A) acts as an extracellular modulator through its interaction with purinoceptors. Our laboratory has demonstrated the presence of an Ap4A receptor in cardiac tissue [1,2]. Due to the rapid hydrolysis of ATP by cardiac membranes the relationship of ATP and Ap4A binding to purinoceptors on cardiac membranes has not been characterized. In this communication we used two approaches to determine the relationship of ATP to the Ap4A receptor. Radioligand binding carried out with [alpha-32P]Ap4A and adenosine 5'-O-¿3-thiotriphosphate¿ ([gamma-35S]ATPgammaS) demonstrates the presence of a single high affinity binding site for Ap4A and the presence of two binding sites for ATPgammaS. The second approach utilized immunoaffinity purified Ap4A receptor that was shown to be free of ATPase and Ap4Aase activities. Non-radiolabeled Ap4A and ATPgammaS effectively inhibited photocrosslinking of [alpha-32P]8-N3Ap4A to the receptor polypeptide while ATP was a much less effective inhibitor. Furthermore, on plasma membranes [alpha-32P]8-N3Ap4A photocrosslinked to only a 50 kDa polypeptide. These data are consistent with Ap4A interacting with a homogeneous population of receptors on cardiac plasma membranes but with ATP having a low affinity for the receptor.

Adenosine 5′-Triphosphate: a P2-Purinergic Agonist in the Myocardium

“…It also activates them as well as p 38 MAPK in rat adult cardiomyocytes (5). Activation of the G protein-coupled P2Y 2 receptors by ATP and UTP stimulates p 42 and p 44 MAPK in endothelial cells (367) and in PC12 cells (444). In PC12 cells also, the activation of the Ca 2ϩ -dependent tyrosine kinase PYK 2 after ATP stimulation of P2X 2 receptors leads to MAPK activation (461).…”

“…It is worth noting that a significant ATP catabolism occurs even on isolated cardiomyocytes. Thus ATP is rapidly hydrolyzed to 65 and 33% after 3 and 15 min, respectively, by plasma membrane at 20°C (42).…”

“…However, much caution should be taken during these binding studies, not only because it would be difficult to distinguish sites with relatively similar binding affinities but mostly because secured experimental conditions are difficult to settle. Optimal binding conditions to obtain reliable equilibrium parameters require a 15-min incubation, a time during which Ͼ75% of the radioligand must remain intact, a situation not easily reached in view of the high ecto-ATPase activies (42).…”

“…To date, in cardiac tissues, only the presence of Ap 4 A receptor has been reported (202,203,504), with at least 77% of the active receptors on the plasma membranes (502). Recent work demonstrates photocross-linking of an Ap 4 A derivative with a 50-kDa polypeptide, suggesting a homogeneous population of receptors (42). At the highaffinity binding site, the apparent K d values for Ap 4 A and ATP␥S are 0.08 and 0.04 M, respectively; there is also a low-affinity site for ATP␥S with a K d at 1.0 M. The P2X agonist ␣,␤-met-ATP did not compete effectively with these two agonists and indicates that the Ap 4 A receptor is a P2Y receptor in cardiac plasma membranes.…”

“…Moreover, this field is characterized by the paucity of receptor subtype-selective agonists and antagonists that have their own propensity to undergo rapid hydrolysis and interconversion just like the natural agonists. It is also a problem that commercially available compounds might already contain breakdown products that act as effective agonists (42,198,342,513), a point already considered in 1934 by Gillepsie (180).…”