Effect of a novel P2Y14 receptor antagonist on the porcine coronary artery.

ZSB Abbas1, N Dovlatova2, SC Fox2, WR Dunn1, V Ralevic1. 1School of Life Sciences, University of Nottingham, Nottingham, UK, 2Cardiovascular Medicine, School of Medical and Surgical Sciences, University of Nottingham, Nottingham, Nottingham, UK

Introduction:The P2Y14 receptor (P2Y14-R) is activated by sugar nucleotides and more selectively by MRS2690 [2,4]. We have previously shown that UDP-glucose and MRS2690 mediate contraction of porcine coronary arteries (PCA) [1]. The present study investigated the effects of related sugar-nucleotides on PCA and also the effect of a novel selective P2Y14-R antagonist, PPTN (4,7-disubstituted naphthoic acid derivative) [3,5], on responses to MRS2690. P2Y14-R coupling to the inhibition of cAMP was also investigated.

Methods: Pig hearts were obtained from a local abattoir and coronary segments were mounted for isometric tension in warmed (37°C) and gassed Krebs (95% O2, 5% CO2). Tissue viability was assessed using KCl (60mM). Vessels were pre-contracted with U46619, forskolin (an activator of cAMP) (0.1µM) was added to return the tone to baseline. Contractile response curves to MRS2690 (0.001-10µM), UDP-glucose, UDP-N-acetylglucosamine and UDP-glucuronic acid (0.1-1000µM) were constructed in isolated PCA segments. In some preparations the P2Y14 receptor antagonist PPTN (1µM) was added prior to addition of U46619 and forskolin and response curves to UDP-glucose and MRS2690 were constructed. Vasodilator stimulated phosphoprotein phosphorylation (VASP-P) (a cyclic AMP activated protein) was measured in vessels stimulated with forskolin (0.1µM) in the absence and presence of UDP-glucose and MRS2690 using flow cytometric bead assay.

Results: P2Y14-R agonists elicited concentration-dependent contractions in PCA. UDP-glucose, UDP-N-acetylglucosamine and UDP-glucuronic acid elicited contractions of equal potency, but MRS290 contraction was more potent. PPTN significantly antagonized the contractile responses to MRS2690 (P 0.0001, n=5-6). The concentrations of MRS2690 required to produce a contractile response of 0.2g showed that 1µM PPTN produced an approximately 20-fold shift (control, 1.4 x 10-7 M and 1µM PPTN 2.6 x 10-6 M) in the concentration response curve. UDP-glucose and MRS2690 significantly decreased forskolin induced VASP-P in PCA.

Conclusion: The greater potency of MRS2690 over UDP-glucose and related sugar nucleotides in eliciting contraction of the PCA is consistent with an involvement of the P2Y14-R. PPTN block of responses to MRS2690 is also consistent with an involvement of the P2Y14-R. Furthermore, UDP-glucose and MRS2690 decreased cAMP levels in PCA which is consistent with reports identifying the P2Y14 receptor as a Gi/o protein-coupled receptor.

References

1. Abbas, Z et al. (2011). Proc BPS http://www.pa2online.org/abstracts/vol9issue3abst051p.pdf

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