Novel vasocontractile role of the P2Y₁₄ receptor: characterization of its signalling in porcine isolated pancreatic arteries

Abstract

Background and purpose: The P2Y₁₄ receptor is the newest member of the P2Y receptor family; it is G(i/o) protein-coupled and is activated by UDP and selectively by UDP-glucose and MRS2690 (2-thiouridine-5'-diphosphoglucose) (7-10-fold more potent than UDP-glucose). This study investigated whether P2Y₁₄ receptors were functionally expressed in porcine isolated pancreatic arteries.

Experimental approach: Pancreatic arteries were prepared for isometric tension recording and UDP-glucose, UDP and MRS2690 were applied cumulatively after preconstriction with U46619, a TxA₂ mimetic. Levels of phosphorylated myosin light chain 2 (MLC2) were assessed with Western blotting. cAMP concentrations were assessed using a competitive enzyme immunoassay kit.

Key results: Concentration-dependent contractions with a rank order of potency of MRS2690 (10-fold) > UDP-glucose ≥ UDP were recorded. These contractions were reduced by PPTN {4-[4-(piperidin-4-yl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthoic acid}, a selective antagonist of P2Y₁₄ receptors, which did not affect responses to UTP. Contraction to UDP-glucose was not affected by MRS2578, a P2Y₆ receptor selective antagonist. Raising cAMP levels and forskolin, in the presence of U46619, enhanced contractions to UDP-glucose. In addition, UDP-glucose and MRS2690 inhibited forskolin-stimulated cAMP levels. Removal of the endothelium and inhibition of endothelium-derived contractile agents (TxA₂, PGF(2α) and endothelin-1) inhibited contractions to UDP glucose. Y-27632, nifedipine and thapsigargin also reduced contractions to the agonists. UDP-glucose and MRS2690 increased MLC2 phosphorylation, which was blocked by PPTN.

Conclusions and implications: P2Y₁₄ receptors play a novel vasocontractile role in porcine pancreatic arteries, mediating contraction via cAMP-dependent mechanisms, elevation of intracellular Ca²⁺ levels, activation of RhoA/ROCK signalling and MLC2, along with release of TxA₂, PGF(2α) and endothelin-1.

Keywords: MRS2690; P2Y14 receptor; UDP; UDP-glucose; endothelium; vasoconstriction.

Figure 1

(A) Concentration-dependent contractions evoked by UDP-glucose, UDP and MRS2690 in U46619-preconstricted porcine pancreatic arteries (**P < 0.01, two-way anova, MRS2690 response vs. UDP-glucose and UDP responses, F = 13.74, 16.03; n = 9–12). (B) Attenuation of UDP-glucose-induced contraction (the control) in the presence of UDP-glucose (100 μM) and UDP (100 μM) (added 10 min prior to U46619 addition). Both UDP-glucose and UDP significantly attenuated the contraction evoked by UDP-glucose (***P < 0.001, two-way anova, UDP-glucose contraction in the absence or presence of UDP-glucose or UDP, F = 63.11, 56.48; n = 10–13). Data are presented as mean ± SEM.

Figure 2

Effect of PPADS (10 μM) and suramin (100 μM) on responses to (A) UDP-glucose, (B) UDP and (C) MRS2690 in U46619-preconstricted porcine pancreatic arteries. (A) Suramin and PPADS enhanced the effects of UDP-glucose (***P < 0.001, two-way anova, UDP-glucose with suramin or PPADS vs. UDP-glucose alone, F = 19.85, 23.07; n = 6–10). (B) Suramin and PPADS enhanced the effects of UDP (***P < 0.001, two-way anova, UDP with suramin or PPADS vs. UDP alone, F = 16.83, 45.24; n = 8–16). (C) Suramin and PPADS had no effect on the contraction to MRS2690 (n = 5–9). Data are presented as mean ± SEM.

Figure 3

Effect of PPTN (1 μM), a P2Y₁₄ receptor antagonist, on responses to (A) UDP-glucose, (B) MRS2690 and (C) UTP in U46619-preconstricted porcine pancreatic arteries. (A) PPTN inhibited the effect of UDP-glucose (*P < 0.05, two-way anova, F = 6.56; n = 7). (B) PPTN inhibited the effect of MRS2690 (**P < 0.01, two-way anova, F = 12.85; n = 9). (C) PPTN had no effect on the response to UTP (n = 8–10). Data are presented as mean ± SEM. (D) Typical traces showing the effect of MRS2690 in the absence and presence of PPTN.

Figure 4

Effect of removal of the endothelium on responses to (A) UDP-glucose, (B) UDP and (C) MRS2690 in U46619-preconstricted porcine pancreatic arteries. The removal of endothelium reduced the contractions evoked by (A) UDP-glucose, (B) UDP and (C) MRS2690 (**P < 0.01, ***P < 0.001, two-way anova, F = 8.15, 51.24, 9.48; n = 4–15). Data are presented as mean ± SEM.

Figure 5

Effect of DUP 697 (3 μM) on responses to (A) UDP-glucose, (B) UDP, (C) MRS2690 in U46619-preconstricted porcine pancreatic arteries. DUP 697 inhibited the contractions evoked by (A) UDP-glucose, (B) UDP and (C) MRS2690 (** P < 0.01, *** P < 0.001, two-way anova, F = 7.85, 35.31, 4.95; n = 4–15). Data are presented as mean ± SEM.

Figure 6

The contraction evoked by UDP-glucose in the presence of (A) NDGA (10 μM), (B) zafirlukast (10 μM), (C) BQ123 (1 μM) in U46619-preconstricted porcine pancreatic arteries, and (D) L-655,240 (1 μM) in endothelin-1-preconstricted porcine pancreatic arteries. (A, B) NDGA and zafirlukast did not alter the response to UDP-glucose (n = 7–10). (C, D) BQ-123 and L-655,240 inhibited the response evoked by UDP-glucose (*P < 0.05, ***P < 0.001, two-way anova, F = 4.97, 19.03; n = 9–14). Data are presented as mean ± SEM.

Figure 7

Effect of (A) nifedipine (1 μM) and (B) thapsigargin (100 nM) on the response to UDP-glucose in U46619-preconstricted porcine pancreatic arteries. Both inhibitors, nifedipine and thapsigargin, inhibited the contraction evoked by UDP-glucose (*P < 0.05, ***P < 0.001, two-way anova, F = 32.5, 5.84; n = 12–15). Data are presented as mean ± SEM. (C) Typical traces showing the responses of UDP-glucose in the absence and presence of nifedipine.

Figure 8

Effect of Y-27632 (5 μM), a selective inhibitor of the Rho-associated protein kinase, on responses to (A) UDP and (B) MRS2690 in U46619-preconstricted porcine pancreatic arteries. Y-27632 reduced the contraction to (A) UDP and (B) MRS2690 (***P < 0.001, two-way anova, F = 53.07, 10.32; n = 9–15). Data are presented as mean ± SEM. (C) MLC2 phosphorylation induced by 100 μM UDP-glucose in porcine pancreatic arteries. A time course (above) and a representative blot (below) of phospho- (green) and total- (red) MLC2 (*P < 0.05, one-way anova, n = 4).

Figure 9

PPTN, a selective high-affinity antagonist of P2Y₁₄ receptor, abolished the ability of UDP-glucose and MRS2690 to elevate the level of MLC phosphorylation (*P < 0.05, Student's unpaired t-test, n = 3). Data are presented as mean ± SEM. Representative immunoblots of phospho- (green) and total- (red) MLC2 from three separate experiments in the absence or presence of PPTN.

Effect of preconstriction with U46619 followed by relaxation with forskolin (1 μM) on the responses to (A) UDP-glucose and (B) UTP in porcine pancreatic arteries. (A) Exposing the tissues to U46619 followed by forskolin significantly enhanced the contraction evoked by UDP-glucose (***P < 0.001, two-way anova, F = 54.34; n = 8–13). Data are presented as mean ± SEM. (B) Exposing the tissues to U66619 followed by forskolin failed to affect the response to UTP. (C) Typical traces showing the effect of UDP-glucose on basal tone (inset) and after the tissues were preconstricted with U46619 and then relaxed with forskolin (main).

Figure 11

Effect of UTP (1 mM), UDP-glucose 1 (mM) and MRS2690 (10 μM) on the cAMP concentrations in porcine pancreatic arteries exposed to U46619 followed by forskolin. UTP had no significant effect on cAMP level, while UDP-glucose and MRS2690 significantly reduced the cAMP level (*P < 0.05, Student's unpaired t-test, the response to UTP or UDP-glucose or MRS2690 vs. their respective controls, n = 4). Basal cAMP level represents the level of cAMP in the absence of forskolin, which was also significantly different from that in the presence of forskolin (*P < 0.05, Student's unpaired t-test, n = 4). Data are presented as mean ± SEM.