The experiments indicate that a major component of the vasod

The experiments indicate that a major component of the vasodilatation is due to antagonism of α 1-adrenoceptors. Evidence for α1-adrenoceptor antagonism is that vasodilatation was observed in 486 2 synthesis preconstricted with α1-adrenoceptor agonists, phenylephrine or methoxamine, but not in vessels preconstricted with endothelin-1 or the thromboxane A2 agonist U46619. Moreover, in rat mesenteric artery, ulimorelin shifted the concentration–response curve for phenylephrine to the right with a pA2 of 5.7. Performing these studies in mice lacking α1-adrenoceptors may assist in confirming these conclusions. A number of off-target effects for other ghrelin receptor ligands at known non-ghrelin receptors have been identified, including β3-adrenoceptor agonist activity by (4-[(aminocarbonyl)amino]-N-[4-(2-aminoethyl)phenyl]benzenesulfonamides (Pasternak et al., 2009) and muscarinic receptor agonist activity by spiro-azetidine–piperidine analogues (McClure et al., 2013). In our study, propranolol did not reduce the vasodilatator action of ulimorelin, excluding a role for β3-adrenoceptors. However, atropine did reduce the vasodilatator action of ulimorelin, suggesting it may in part be mediated via endothelial muscarinic receptors. Previously it has been reported that in rat mesenteric artery endothelial denudation reduced but did not abolish vasodilatation to ulimorelin (Callaghan et al., 2014). In addition to its vasodilatator effect, relatively high concentrations of ulimorelin caused constriction of rat and mouse saphenous and rat basilar artery. Constriction was observed in saphenous artery segments from Ghsr null mice suggesting this effect was not mediated by the ghrelin receptor. Similar concentrations of TZP102 also caused constriction of rat saphenous artery, but other ghrelin receptor agonists investigated had no constrictor effect in this vessel. This suggests that the macrocyclic molecules, ulimorelin and TZP102, may possess a structurally related molecular motif that is responsible for their contractile effects and that is absent in the other ghrelin receptor agonists investigated. Comprehensive structure–activity relationship investigations will be required to identify the particular motif responsible. The basilar artery was contracted using 60mM [K+] as there was no contraction to phenylephrine. This absence of α-adrenoceptor-mediated contractions in rat basilar arteries has been previously reported (Chang et al., 1988). Only a small vasodilatation of the basilar artery contracted with 60mM [K+] was observed following application of ulimorelin, making this vessel an ideal place to investigate the vasoconstriction. The reduced vasodilatation to ulimorelin observed in this artery adds further evidence to support the α-adrenoceptor antagonist effect of ulimorelin observed in both the saphenous and mesenteric arteries.
Conclusion Here, for the first time, the ghrelin receptor agonist ulimorelin has been shown to have two distinct, regionally selective, non-ghrelin receptor mediated actions on arteries in rodents, namely a reduction in α1-adrenoceptor-mediated constriction and, at a higher concentrations, a vasoconstrictor action. The vasodilatator mechanisms are unknown; however the evidence presented suggests inhibition of α1-adrenoceptors, and activation of muscarinic acetylcholine receptors, mediates this action of ulimorelin. The mechanism of constriction is currently unknown although opening of L-type calcium channels appears to be required. As the concentrations of ulimorelin producing these non-ghrelin receptor-mediated effects may be achieved in vivo, this may aid interpretation of studies of the effects of ulimorelin on blood pressure.
Acknowledgements John Broad was supported by the Outstanding Young Investigator Award of the British Pharmacological Society and the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists. Research support was provided by the National Health and Medical Research Council of Australia (Project Grant 1005811) and the Transport Accident Commission through the Institute for Safety Compensation and Recovery Research (Grant N-13-085).