UDP-sugars activate P2Y14 receptors to mediate vasoconstriction of the porcine coronary artery

Abstract

Aims: UDP-sugars can act as extracellular signalling molecules, but relatively little is known about their cardiovascular actions. The P2Y₁₄ receptor is a G_i/o-coupled receptor which is activated by UDP-glucose and related sugar nucleotides. In this study we sought to investigate whether P2Y₁₄ receptors are functionally expressed in the porcine coronary artery using a selective P2Y₁₄ receptor agonist, MRS2690, and a novel selective P2Y₁₄ receptor antagonist, PPTN (4,7-disubstituted naphthoic acid derivative).

Methods and results: Isometric tension recordings were used to evaluate the effects of UDP-sugars in porcine isolated coronary artery segments. The effects of the P2 receptor antagonists suramin and PPADS, the P2Y₁₄ receptor antagonist PPTN, and the P2Y₆ receptor antagonist MRS2578, were investigated. Measurement of vasodilator-stimulated phosphoprotein (VASP) phosphorylation using flow cytometry was used to assess changes in cAMP levels. UDP-glucose, UDP-glucuronic acid UDP-N-acetylglucosamine (P2Y₁₄ receptor agonists), elicited concentration-dependent contractions of the porcine coronary artery. MRS2690 was a more potent vasoconstrictor than the UDP-sugars. Concentration dependent contractile responses to MRS2690 and UDP-sugars were enhanced in the presence of forskolin (activator of cAMP), where the level of basal tone was maintained by addition of U46619, a thromboxane A₂ mimetic. Contractile responses to MRS2690 were blocked by PPTN, but not by MRS2578. Contractile responses to UDP-glucose were also attenuated by PPTN and suramin, but not by MRS2578. Forskolin-induced VASP-phosphorylation was reduced in porcine coronary arteries exposed to UDP-glucose and MRS2690, consistent with P2Y₁₄ receptor coupling to G_i/o proteins and inhibition of adenylyl cyclase activity.

Conclusions: Our data support a role of UDP-sugars as extracellular signalling molecules and show for the first time that they mediate contraction of porcine coronary arteries via P2Y₁₄ receptors.

Keywords: Coronary artery; MRS2690; P2Y(14) receptor; P2Y(6) receptor; Sugar nucleotides; UDP sugars; UDP-glucose.

Fig. 1

UDP-glucose and the selective P2Y₁₄ receptor agonist MRS2690 mediate contraction of porcine coronary arteries. Representative traces showing concentration-dependent contractile responses of porcine coronary arteries to UDP-glucose (a, c) and MRS2690 (b, d) at basal tone in the absence (Control) and presence of forskolin (0.1 μM) plus U46619. Lines on traces represent addition of cumulative concentrations of agonists. The mean data ± S.E.M. are of experiments from 6 to 12 different pigs (e, f). For UDP-glucose (e) the R_max was significantly different between the groups (P < 0.001, extra sum of squares F test) with no difference in logEC₅₀values. For MRS2690 (f) the curves were significantly different (P < 0.001, extra sum of squares F test).

Fig. 2

Forskolin plus U46619 do not augment contractions to acetylcholine and histamine in porcine coronary arteries. Cumulative concentration-response curves to acetylcholine (ACh) (a) and histamine (b), under control conditions and the effect of forskolin (0.1 μM) plus U46619. There was no significant difference in the R_max values for either compound, or in the logEC₅₀ values for ACh, but there was a small but significant difference in logEC₅₀ values for histamine, indicating a slightly reduced potency in the presence of forskolin plus U46619 (P < 0.01, extra sum of squares F-test). Data are mean ± S.E.M.

Fig. 3

UDP-sugars mediate contraction of porcine coronary arteries and the responses are augmented by forskolin. Cumulative concentration-response curves to P2Y₁₄ receptor agonists in the porcine coronary artery. UDP-glucose, UDP-glucuronic acid, UDP-N-acetylglucosamine and the P2Y₁₄ receptor agonist MRS2690 were investigated in isolated porcine coronary arteries under control conditions (a) and in arteries to which forskolin (0.1 μM) and U46619 had been added to elevate cAMP and maintain tone, respectively (b). Note the different y axes scales in panels a and b. There was a significant difference in the agonist logEC₅₀ values in the presence of forskolin plus U46619 (P < 0.001, extra sum of squares F test) (see also Table 1). Data are mean ± S.E.M.

Fig. 4

Contraction to UDP-glucose in porcine coronary arteries is endothelium-independent and attenuated by suramin but augmented by PPADS. Concentration-response curves to UDP-glucose in endothelium-intact and endothelium-denuded arteries in the presence of forskolin plus U46619 (a). Effect of the P2 receptor antagonists suramin (b) and PPADS (c) on contractile responses to UDP-glucose in porcine coronary arteries in the presence of forskolin plus U46619. UDP-glucose R_max values were significantly different in the presence and absence of suramin (P < 0.01) and both R_max and logEC₅₀ values were significantly different in the presence and absence of PPADS (P < 0.01 and P < 0.001) (extra sum of squares F test). Data are mean ± S.E.M.

Fig. 5

Contractile responses to UDP-glucose and the selective P2Y₁₄ receptor agonist MRS2690 are attenuated by the selective P2Y₁₄ receptor antagonist PPTN. The effect of PPTN (1 μM) on contractile responses to UDP-glucose (a), MRS2690 (b), and UTP (c) in porcine coronary arteries in the presence of forskolin (0.1 μM) and U46619. Analysis of the curves showed that these were significantly different in the absence and presence of PPTN for UDP-glucose (P < 0.01) and MRS2690 (P < 0.001) (extra sum of squares F test). In contrast, there was no significant difference in the responses to UTP. Data are mean ± S.E.M.

Fig. 6

Contractile responses to UDP-glucose and MRS2690 are unaffected by the selective P2Y₆ receptor antagonist MRS2578. Effect of MRS2578, on concentration-response curves to (a) UDP-glucose and (b) MRS2690 in porcine coronary arteries. UDP-glucose and MRS260 caused concentration-dependent contractions in the presence of forskolin and U46619. Contractions to UDP-glucose were unaffected by MRS2578 or by prior incubation of the UDP-glucose solution and tissues with apyrase (5 U/ml, > 30 min) (a). Contractions to MRS2690 were also unaffected by MRS2578. Data are mean ± S.E.M.

Fig. 7

UDP-glucose-induced VASP-phosphorylation is blocked by the P2Y₁₄ receptor antagonist PPTN. Levels of VASP phosphorylation (VASP-P) in porcine coronary arteries at basal tone, in the absence (Control) and presence of forskolin and the effect of UDP-glucose (UDPG), MRS2690 (P2Y₁₄ receptor agonist) and PPTN (P2Y₁₄ receptor antagonist). (a) Levels of VASP-P in control conditions and in the presence of forskolin, forskolin + UDP-glucose (100 μM), and forskolin + MRS2690 (10 μM). (b) Effect of UDP-glucose (1 mM) and MRS2690 (10 μM) on forskolin-induced VASP phosphorylation in porcine coronary arteries in the absence and presence of PPTN (1 μM). (c) Effect of PPTN (1 μM) on forskolin-induced VASP-P. VASP-phosphorylation was measured using flow cytometry. *P < 0.05, **P < 0.01; ***P < 0.001, ANOVA with Tukey's post test. PPTN alone had no effect on VASP-P (Student's t-test). Data are mean ± S.E.M.

Fig. 8

Effect of platelets and the P2Y₁₄ receptor antagonist PPTN, on porcine coronary artery contractility. Platelets isolated from 7 healthy adult human donors were added to U46619-preconstricted porcine coronary arteries in the absence (a–c) and presence (d–g) of L-NAME (100 μM), a nitric oxide synthase inhibitor, in the absence and presence of PPTN (1 μM) (P2Y₁₄ receptor antagonist). In the absence of L-NAME the platelets elicited vasorelaxation. In the presence of L-NAME platelets elicited vasoconstriction. n indicates the number of different pigs from which coronary artery segments were obtained. *P < 0.05; **P < 0.01 for comparison of responses in the absence and presence of PPTN (two way ANOVA). Data are mean ± S.E.M.