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Aims/hypothesis: The small, highly conserved vasoactive peptide urotensin II (UII) is upregulated in atherosclerosis. However, its effects in diabetes-associated atherosclerosis have not been assessed., Methods: Endothelial cells were grown in normal- and high-glucose (5 and 25 mmol/l) media with and without UII (10⁻⁸ mol/l) and/or the UII receptor antagonist, SB-657510 (10⁻⁸ mol/l). Apoe knockout (KO) mice with or without streptozotocin-induced diabetes were treated with or without SB-657510 (30 mg kg⁻¹ day⁻¹; n = 20 per group) and followed for 20 weeks. Carotid endarterectomy specimens from diabetic and non-diabetic humans were also evaluated., Results: In high (but not normal) glucose medium, UII significantly increased CCL2 (encodes macrophage chemoattractant protein 1 [MCP-1]) gene expression (human aortic endothelial cells) and increased monocyte adhesion (HUVECs). UII receptor antagonism in diabetic Apoe KO mice significantly attenuated diabetes-associated atherosclerosis and aortic staining for MCP-1, F4/80 (macrophage marker), cyclooxygenase-2, nitrotyrosine and UII. UII staining was significantly increased in carotid endarterectomies from diabetic compared with non-diabetic individuals, as was staining for MCP-1., Conclusions/interpretation: This is the first report to demonstrate that UII is increased in diabetes-associated atherosclerosis in humans and rodents. Diabetes-associated plaque development was attenuated by UII receptor antagonism in the experimental setting. Thus UII may represent a novel therapeutic target in the treatment of diabetes-associated atherosclerosis.

Background and Purpose: Recently identified antagonists of the urotensin-II (U-II) receptor (UT) are of limited utility for investigating the (patho)physiological role of U-II due to poor potency and limited selectivity and/or intrinsic activity., Experimental Approach: The pharmacological properties of two novel UT antagonists, GSK1440115 and GSK1562590, were compared using multiple bioassays., Key Results: GSK1440115 (pK(i)= 7.34-8.64 across species) and GSK1562590 (pK(i)= 9.14-9.66 across species) are high affinity ligands of mammalian recombinant (mouse, rat, cat, monkey, human) and native (SJRH30 cells) UT. Both compounds exhibited >100-fold selectivity for UT versus 87 distinct mammalian GPCR, enzyme, ion channel and neurotransmitter uptake targets. GSK1440115 showed competitive antagonism at UT in arteries from all species tested (pA(2)= 5.59-7.71). In contrast, GSK1562590 was an insurmountable UT antagonist in rat, cat and hUT transgenic mouse arteries (pK(b)= 8.93-10.12 across species), but a competitive antagonist in monkey arteries (pK(b)= 8.87-8.93). Likewise, GSK1562590 inhibited the hU-II-induced systemic pressor response in anaesthetized cats at a dose 10-fold lower than that of GSK1440115. The antagonistic effects of GSK1440115, but not GSK1562590, could be reversed by washout in rat isolated aorta. In ex vivo studies, GSK1562590 inhibited hU-II-induced contraction of rat aorta for at least 24 h following dosing. Dissociation of GSK1562590 binding was considerably slower at rat than monkey UT., Conclusions and Implications: Whereas both GSK1440115 and GSK1562590 represent high-affinity/selective UT antagonists suitable for assessing the (patho)physiological role of U-II, only GSK1562590 exhibited sustained UT residence time and improved preclinical efficacy in vivo.

Background and Purpose: The recent development of the UT ligand palosuran (1-[2-(4-benzyl-4-hydroxy-piperidin-1-yl)-ethyl]-3-(2-methyl-quinolin-4-yl)-urea sulphate salt) has led to the proposition that urotensin-II (U-II) plays a significant pathological role in acute and chronic renal injury in the rat., Experimental Approach: In the present study, the pharmacological properties of palosuran were investigated further using a series of radioligand binding and functional bioassays., Key Results: Palosuran functioned as a 'primate-selective' UT ligand in recombinant cell membranes (monkey and human UT K(i) values of 4 +/- 1 and 5 +/- 1 nM), lacking appreciable affinity at other mammalian UT isoforms (rodent and feline K(i) values >1 microM). Paradoxically, however, palosuran lost significant (10- to 54-fold) affinity for native and recombinant human UT when radioligand binding was performed in intact cells (K(i) values of 50 +/- 3 and 276 +/- 67 nM). In accordance, palosuran also exhibited diminished activity in hUT (human urotensin-II receptor)-CHO (Chinese hamster ovary) cells (IC50 323 +/- 67 nM) and isolated arteries (K(b)>10 microM in rat aorta; K(b)>8.5 microM in cat arteries; K(b)>1.6 microM in monkey arteries; K(b) 2.2 +/- 0.6 microM in hUT transgenic mouse aorta). Relative to recombinant binding K(i) values, palosuran was subjected to a 392- to 690-fold reduction in functional activity in monkey isolated arteries. Such phenomena were peculiar to palosuran and were not apparent with an alternative chemotype, SB-657510 (2-bromo-N-[4-chloro-3-((R)-1-methyl-pyrrolidin-3-yloxy)-phenyl]-4,5-dimethoxybenzenesulphonamide HCl)., Conclusions and Implications: Collectively, such findings suggest that caution should be taken when interpreting data generated using palosuran. The loss of UT affinity/activity observed in intact cells and tissues cf. membranes offers a potential explanation for the disappointing clinical efficacy reported with palosuran in diabetic nephropathy patients. As such, the (patho)physiological significance of U-II in diabetic renal dysfunction remains uncertain.

Background and Purpose: Atypical cannabinoids are thought to cause vasodilatation through an as-yet unidentified 'CBx' receptor. Recent reports suggest GPR55 is an atypical cannabinoid receptor, making it a candidate for the vasodilator 'CBx' receptor. The purpose of the present study was to test the hypothesis that human recombinant GPR55 is activated by atypical cannabinoids and mediates vasodilator responses to these agents., Experimental Approach: Human recombinant GPR55 was expressed in HEK293T cells and specific GTPgammaS activity was monitored as an index of receptor activation. In GPR55-deficient and wild-type littermate control mice, in vivo blood pressure measurement and isolated resistance artery myography were used to determine GPR55 dependence of atypical cannabinoid-induced haemodynamic and vasodilator responses., Key Results: Atypical cannabinoids O-1602 and abnormal cannabidiol both stimulated GPR55-dependent GTPgammaS activity (EC50 approximately 2 nM), whereas the CB1 and CB2-selective agonist WIN 55,212-2 showed no effect in GPR55-expressing HEK293T cell membranes. Baseline mean arterial pressure and heart rate were not different between WT and GPR55 KO mice. The blood pressure-lowering response to abnormal cannabidiol was not different between WT and KO mice (WT 20+/-2%, KO 26+/-5% change from baseline), nor was the vasodilator response to abnormal cannabidiol in isolated mesenteric arteries (IC50 approximately 3 micro M for WT and KO). The abnormal cannabidiol vasodilator response was antagonized equivalently by O-1918 in both strains., Conclusions: These results demonstrate that while GPR55 is activated by atypical cannabinoids, it does not appear to mediate the vasodilator effects of these agents.