Alsaqati, Mouhamed
(2014)
Characterisation of the P2Y14 receptor in the pancreas: control of vascular tone and insulin secretion.
PhD thesis, University of Nottingham.
Abstract
The P2Y14 receptor is the most recently identified member of the P2Y family of receptors for adenine and uridine nucleotides and nucleotide sugars. It is activated by UDP, UDP-glucose and its analogues, as well as the synthetic analogue MRS2690, which exhibits greater potency and selectivity at the P2Y14 receptor. The principle aim of this study was to investigate the functional expression of the P2Y14 receptor in porcine pancreatic arteries, and the signalling pathways underlying the vasoconstriction evoked by P2Y14 receptor agonists, together with an examination of the effects of UDP-glucose and MRS2690 on insulin secretion from the rat INS-1 823/13 β-cell line.
Segments of porcine pancreatic arteries were prepared for isometric tension recordings in warmed oxygenated Krebs’-Henseleit buffer. Agonists were applied after preconstriction with U46619, a thromboxane A2 mimetic. ATP induced vasoconstriction followed by a vasorelaxation in pancreatic arteries; the contraction was blocked by NF449 (a P2X1 receptor selective antagonist), while the relaxation to ATP was blocked by an adenosine receptor antagonist. Neither the contraction, nor the relaxation to ATP were affected by removal of the endothelium. ADP evoked vasorelaxation, which was inhibited in the presence of SCH58261 (a selective adenosine A2A receptor antagonist). UTP-induced vasoconstriction was attenuated significantly in endothelium-denuded arteries. UDP, UDP-glucose and MRS2690 induced concentration-dependent contractions in porcine pancreatic arteries with a rank order of potency of MRS2690 (10-fold) > UDP-glucose = UDP. The contractions evoked by UDP-glucose and MRS2690 were significantly attenuated in the presence of PPTN (a selective P2Y14 receptor antagonist), indicating actions at P2Y14 receptors. The expression of P2Y14-like receptor was shown by immunohistochemical and contractile studies to be on the endothelium of the pancreatic arteries. UDP-glucose and MRS2690 inhibited forskolin-stimulated cAMP production. UDP-glucose and MRS2690 increased the level of MLC2 phosphorylation; this effect was blocked by PPTN, indicating the involvement of P2Y14 receptors. UDP-glucose increased the level of ERK1/2 phosphorylation. UDP-glucose and MRS2690 inhibited glucose-induced insulin release from the rat INS-1 823/13 β-cell line; this effect was blocked by PPTN, indicating actions through P2Y14 receptors. PPTN itself was able to elevate significantly basal insulin secretion from INS-1 823/13 β-cells, which may suggest a constitutive release of UDP-glucose from these cells.
These results suggest that, in porcine pancreatic arteries, ATP induces a vasoconstriction mediated by P2X1 receptors followed by a vasorelaxation evoked by adenosine receptors present on the vascular smooth muscle. ADP induced a relaxation mediated by adenosine A2A receptor. Moreover, my data indicate for the first time, an endothelium-dependent contraction evoked by UTP. A novel vasocontractile role of P2Y14 receptors in porcine pancreatic arteries was also documented. The contractile response was mediated largely by the endothelium. P2Y14-mediated contraction involves a cAMP-dependent mechanism, which is consistent with P2Y14 receptor coupling to Gi protein, and an elevation in phosphorylated MLC2 and ERK1/2. Activation of the P2Y14 receptor evoked a decrease in the level of insulin secreted from the rat pancreas. The current data have identified novel roles of the P2Y14 receptor as a mediator of pancreatic artery contractility and in regulation of insulin secretion. If its role within the vasculature is shown to be more widespread, the P2Y14 receptor may be a novel target for the treatment of cardiovascular disease.
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