Cardiovascular Pharmacology Des-acyl ghrelin fragments evoke endothelium-dependent vasodilatation of rat mesenteric vascular bed via activation of potassium channels Banafsheh Moazed, Dale Quest, Venkat Gopalakrishnan ⁎ Department of Pharmacology and the Cardiovascular Research Group (CVRG), College of Medicine, University of Saskatchewan, Saskatoon, SK., Canada abstract article info Article history: Received 10 April 2008 Received in revised form 8 October 2008 Accepted 13 October 2008 Available online 21 October 2008 Keywords: Acetylcholine Calcium-activated potassium channel Des-acyl ghrelin Endothelium Ghrelin L-serine Mesenteric vascular bed Phenylephrine Vasodilatation The mechanisms that subserve ghrelin-evoked vasodilatation have not been elucidated in previous studies. Changes in perfusion pressure evoked by ghrelin and its N-terminal fragments were examined ex vivo in phenylephrine-constricted perfused mesenteric vascular beds of male Sprague Dawley rats maintained at a constant flow rate. Both ghrelin (maximum effect [E max ] 45%) and des-acyl ghrelin (E max 43%) evoked vasodilatation at concentrations between 10 pM and 1 nM, compared to acetylcholine (median effective concentration [EC 50 ] 3 nM; E max 93%). Those responses were abolished in endothelium-denuded preparations, and in endothelium-intact preparations exposed to either calcium-activated potassium channel, or a depolarizing stimulus, or in the presence of a combination of either apamin and 1,2- chlorophenyl diphenylmethyl-1 H-pyrazole (triarylmethane-34 [TRAM-34]), or ouabain and barium. ATP- activated potassium channel blockade, or a combination of nitric oxide synthase and cyclooxygenase inhibition had no effect. The classical growth hormone secretagogue antagonist, [D-Lys 3 ]-growth hormone- releasing peptide (10 nM), or several N-terminal fragments of des-acyl ghrelin, including the tripeptide glycine–serine–serine (G–S–S [1 nM]), showed endothelium-dependent vasodilatation like des-acyl ghrelin, while responses to glycine–serine or serine–serine were relatively lower. A higher concentration (100 μM) of L-serine, but not glycine, evoked vasodilatation of similar magnitude. The serine dense N-terminal sequence of des-acyl ghrelin mediates endothelium-dependent vasodilatation via activation of apamin+ TRAM-34 sensitive small- and intermediate-conductance calcium-activated potassium channels present on the mesenteric endothelium. Thus, the vasodilator response to ghrelins in the perfused rat mesenteric vascular bed is not mediated by the classical growth hormone secretagogue receptor type 1a. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Ghrelin, a 28 amino acid acylated peptide released from the stomach, is an endogenous ligand for growth hormone secretagogue receptor type 1a (Kojima et al., 1999; Kojima et al., 2001; Matsumoto et al., 2001; Nakazato et al., 2001; Tschop et al., 2000). The presence of an n-octanoyl group in the serine-3 position of ghrelin is essential for ghrelin to induce growth hormone release and enhance food intake (Kojima et al., 1999; Kojima et al., 2001). The des-octanoylated form of ghrelin (des-acyl ghrelin) abundantly present in circulation, is devoid of growth hormone secretagogue activity as it does not activate growth hormone secretago- gue receptor type 1a (Kojima et al., 1999; Kojima et al., 2001; Matsumoto et al., 2001; Nakazato et al., 2001; Tschop et al., 2000). A physiological role for des-acyl ghrelin is uncertain. Systemic infusion of ghrelin has been shown to reduce blood pressure and pulomonary hypertension in rat models and human volunteers (Nagaya et al., 2001a,b; Henriques-Coelho et al., 2004; Shinde et al., 2005). Initial studies assumed that the vasodilator effect of ghrelin is mediated via activation of growth hormone secretagogue receptor type 1a (Bedendi et al., 2001; Katugampola et al., 2002; Okumura et al., 2002; Wiley and Davenport, 2002; Wu et al., 2004). Ghrelin has been shown to induce coronary vasoconstriction in rats via increased calcium mobilization (Pemberton et al., 2004). This is consistent with reports that the somatotrophic and orexigenic actions of ghrelin are mediated by activation of growth hormone secretagogue receptor type 1a, which is linked to calcium mobilization (Kojima et al., 1999; Kojima et al., 2001). It was thus proposed that the vasodilator effect of ghrelin might be due to activation of a novel receptor on vascular smooth muscle cells (Katugampola et al., 2002; Nagaya et al., 2001a,b). Endothelial cell binding sites for ghrelin have been characterized (Baldanzi et al., 2002; Li et al., 2004; Rossi et al., 2007; Tesauro et al., 2005), but the mechanisms of ghrelin-induced vasodilatation have not been adequately characterized. Vasodilators such as acetylcholine and bradykinin evoke endothelium- dependent vasodilatation by recruiting nitric oxide, as well as nitric oxide-independent mediators such as prostacyclin and the putative endothelium-derived hyperpolarizing factor (Busse et al., 2002; Félétou and Vanhoutte, 2007). Previous studies have suggested that European Journal of Pharmacology 604 (2009) 79–86 ⁎ Corresponding author. Department of Pharmacology, College of Medicine, University of Saskatchewan 107, Wiggins Road, Saskatoon, SK., Canada, S7N 5E5. Tel.: +1 306 966 6293; fax: +1 306 966 6220. E-mail address: venkat.gopal@usask.ca (V. Gopalakrishnan). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.10.032 Contents lists available at ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar