Characterisation of P2Y₂ receptors in human vascular endothelial cells using AR‐C118925XX, a competitive and selective P2Y₂ antagonist

Abstract

Background and Purpose There is a lack of potent, selective antagonists at most subtypes of P2Y receptor. The aims of this study were to characterise the pharmacological properties of the proposed P2Y2 receptor antagonist, AR‐C118925XX, and then to use it to determine the role of P2Y2 receptors in the action of the P2Y2 agonist, UTP, in human vascular endothelial cells. Experimental Approach Cell lines expressing native or recombinant P2Y receptors were superfused constantly, and agonist‐induced changes in intracellular Ca2+ levels monitored using the Ca2+‐sensitive fluorescent indicator, Cal‐520. This set‐up enabled full agonist concentration–response curves to be constructed on a single population of cells. Key Results UTP evoked a concentration‐dependent rise in intracellular Ca2+ in 1321N1‐hP2Y2 cells. AR‐C118925XX (10 nM to 1 μM) had no effect per se on intracellular Ca2+ but shifted the UTP concentration–response curve progressively rightwards, with no change in maximum. The inhibition was fully reversible on washout. AR‐C118925XX (1 μM) had no effect at native or recombinant hP2Y1, hP2Y4, rP2Y6, or hP2Y11 receptors. Finally, in EAhy926 immortalised human vascular endothelial cells, AR‐C118925XX (30 nM) shifted the UTP concentration–response curve rightwards, with no decrease in maximum. Conclusions and Implications AR‐C118925XX is a potent, selective and reversible, competitive P2Y2 receptor antagonist, which inhibited responses mediated by endogenous P2Y2 receptors in human vascular endothelial cells. As the only P2Y2‐selective antagonist currently available, it will greatly enhance our ability to identify the functions of native P2Y2 receptors and their contribution to disease and dysfunction.

<p>P₂Y₂ Receptors Mediate Masseter Muscle Mechanical Hypersensitivity in Rats</p>

“…In accordance with recent studies, AR-C118925 was chosen for blockage of the upregulated P 2 Y 2 Rs. 8,18,19,36 The results of the current study showed attenuation of bilateral hypersensitivity after AR-C118925 was administered ipsilateral to the inflammation. This suggests that the analgesic effect of AR-C118925 on ipsilateral MM hypersensitivity may be mediated by its effect on P 2 Y 2 Rs.…”

“…18 Muoboghare et al have suggested that UTP acts via P 2 Y 2 Rs to mobilise Ca 2+ in human endothelial cells. 19 Recent studies have investigated the role of P 2 Y 2 Rs in pain mechanisms, but their exact function remains unclear, especially in the fields of orofacial pain and TMD. P 2 Y 2 Rs (along with P 2 Y 1 receptors) are the major P 2 YRs expressed in small-diameter sensory neurons.…”

P2Y1‐like nucleotide receptors—Structures, molecular modeling, mutagenesis, and oligomerization

“…In addition to investigation of agonists, a selected set of anthraquinone‐(AQ‐) derived antagonists ( 2.11 – 2.13 ) and the thiouracil derivative AR‐C118925 ( 2.16 ), a P2Y 2 R‐selective competitive antagonist, were tested on all of those mutants, that could be activated by UTP . A trend toward increased potency of 2.11 – 2.13 was observed for the mutants Y114A, Y114F, R194H, F261A, Y268F, and Y288F, indicating an orthosteric binding mode of these AQ derivatives.…”

That was then, this is now: the development of our knowledge and understanding of P2 receptor subtypes

“…The P2Y 2 R agonist, Diquafasol (UP4U, INS365), stimulates secretion of water and mucin by conjunctival epithelial and goblet cells in the eye and is approved for treatment of dry eye syndrome in South Korea and Japan. AR-C118925XX is the first potent (pA 2 = 8.43) [ 98 ] and highly selective competitive P2Y 2 R antagonist to become commercially available [ 99 ], and as such, it is likely to be used widely experimentally. Other potent competitive antagonists that display a degree of subtype selectivity include NF340 at P2Y 11 R (pA 2 = 8.02) [ 100 ], MRS2211 at P2Y 13 R (pA 2 = 6.3) [ 101 ] and PPTN at P2Y 14 R (pK i = 10.1) [ 102 ].…”