Ulimorelin also known as TPZ is a macrocyclic molecule
Ulimorelin, also known as TPZ101, is a macrocyclic molecule that is a potent agonist of the ghrelin receptor (Hoveyda et al., 2011). Due to its ability to stimulate gastrointestinal motility, ulimorelin has been evaluated as a possible treatment in gastroparesis (Ejskjaer et al., 2010), ileus (Fraser et al., 2009) and constipation (Pustovit et al., 2014).
We previously showed, in rats, that ulimorelin caused a biphasic reduction in blood pressure with an initial rapid decrease (resistant to ghrelin receptor antagonists), followed by a slower decrease (Callaghan et al., 2014). Ulimorelin also relaxed rat mesenteric SB 203580 hydrochloride australia preconstricted with phenylephrine, an action not blocked by ghrelin receptor antagonists. Whether ulimorelin relaxes vessels constricted with other agents, or affects arteries in other vascular beds was not investigated, and mechanisms of action were also not investigated. In the current study, we investigated the mechanism(s) by which ulimorelin mediates its effects on vascular constriction, and investigated arteries supplying the viscera, limbs and central nervous system.
Materials and methods Male Sprague-Dawley rats, mice with knockout of the gene for the ghrelin receptor, and wild-type C57BL6 mice were used. The knockout was created by inserting a transcriptional blocking cassette into intron 1 of GHSR1a in C57BL6 mice, creating a GHSR1a-null allele (Zigman et al., 2005). All animal procedures were approved by the University of Melbourne Animal Experimentation Ethics Committee. The procedures abided by the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. 25 adult male Sprague-Dawley rats (300–400g) and 8 adult mice (30–40g) were killed by increasing atmospheric CO2, followed by exsanguination. The saphenous artery was dissected from its origin at the femoral artery to a point just above the knee joint. Second/third order mesenteric arteries supplying the distal 15cm of ileum were isolated from the mesentery. The whole of the basilar artery was isolated. After dissection, all vessels were maintained in physiological saline of the following composition (in mM): Na+, 150.6; K+, 4.7; Ca2+, 2; Mg2+, 1.2; Cl−, 144.1; H2PO42−, 1.3; HCO3−, 16.3; glucose, 7.8. This solution was gassed with 95% O2/5% CO2. The arteries were cut into 1.2–2mm length sections, and mounted isometrically onto stainless steel wires. The rat saphenous arteries were mounted onto 50μm diameter wires, whereas the rat mesenteric, basilar, and mouse saphenous arteries were mounted onto 40μm diameter wires. The basal conditions were normalised by gradually stretching the vessel in small steps until the effective transmural pressure calculated using Laplace׳s equation [transmural pressure=wall tension/(internal circumference/2π), where wall tension=force/2×vessel segment length] was 13.3mN/mm2 (100mmHg; see Mulvany and Halpern, 1977). The rat and mouse saphenous arteries were suspended at an effective transmural pressure of 100mmHg (Rummery et al., 2010). The rat mesenteric and basilar arteries were suspended at 90% of lumen circumference measured at an effective transmural pressure 100mmHg (see Mulvany and Halpern, 1977). Under these conditions the vessels are mounted at close to the peak of their length–tension relationship. The baths were filled with 6ml of physiological saline that was continuously bubbled with 95% O2/5% CO2 and heated to ~36.5°C. Following mounting, the arteries were allowed to recover for 30min. At the start of all experiments, the tissues were stimulated with three applications of phenylephrine (3 or 10µM) to confirm viability and when the contraction to the third application of phenylephrine had plateaued, carbachol (1µM) was applied to determine if the endothelium was intact. Different tissues were used to assess the effects of each concentration of ghrelin receptor agonist on contractions induced by phenylephrine, methoxamine, U46619, ET-1 and 60mM [K+]. The 60mM [K+] experiments were performed in the presence of the α-adrenoceptor agonists prazosin 100nM and idazoxan 1µM to prevent the actions of noradrenaline released from the perivascular sympathetic nerve terminals. The test agonists (ghrelin, desacyl ghrelin, ulimorelin, capromorelin, or AZP-351) were applied and left in contact with the tissue for at least 5min or until the relaxation produced had plateaued. When applied on their own, the concentration of ghrelin receptor agonists was increased cumulatively, with at least 8min between each increase in concentration. For cumulative concentration curves to phenylephrine, at least 4min was allowed to elapse between each addition of phenylephrine. When the effects of receptor antagonists, L-NAME or ion channel blockers were investigated, they were applied at least 30min prior to application of ulimorelin.