Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA314

Poster Communications

Identification of Kv7 channels in rat mesenteric artery endothelium and role in endothelial derived relaxations.

S. Baldwin1, S. Sandow2, J. Stott1, I. A. Greenwood1

1. Institute of Molecular & Clinical Sciences, St George's London, London, United Kingdom. 2. University of the Sunshine Coast, Brisbane, Queensland, Australia.

Introduction: Kv7 channels have been identified as key regulators of arterial diameter and receptor mediated dilatation in many arteries (1). However, no study has considered expression of Kv7 channels in the endothelium and the possible impact of channels localised therein. Objective. The goal of the study was to; ascertain whether Kv7 were present in the endothelium of rat mesenteric arteries and to determine if Kv7 blockers modulate endothelium-dependent relaxations. Methods and materials. Male Wistar rats (175-225g) were killed by methods in accordance with UK Animals Scientific Procedures Act and Australian legislation. 2nd-4th order mesenteric arteries were dissected, cleaned of fat and then either mounted in a wire myograph (DMT, Aarhus) for isometric tension recording or perfusion fixed for intact artery immunocytochemistry using Kv7.4 and 7.5 primary (Abcam, ab65797, lot GR94754; Millipore; ABN1372, lot Q2476155, respectively) and Alexa 594 secondary antibody. In myograph studies endothelial integrity was tested with the addition of 10 uM carbachol producing greater than 90% relaxation of arteries precontracted with 3 uM methoxamine or 1 µM U46619. Where necessary endothelium was removed by gentle mechanical abrasion. Results. Confocal imaging (n=3-5) showed Kv7.4 as low diffuse in endothelium and absence at internal elastic lamina (IEL) holes; with 7.5 being diffuse and punctate in endothelium and at a proportion of IEL holes. In isometric tension studies carbachol mediated relaxations were reduced by incubation with either the NO synthase inhibitor L-NAME (100 mM) or prostacyclin IP receptor antagonist CAY-10471 (10 µM) and impaired markedly by apamin (10 nM) and TRAM-34 (1 uM) combination. Incubation with the pan Kv7 blocker linopirdine (10 mM) reduced the relaxation produced by 300 nM carbachol from 45.9 ± 4 % to 75.1 ± 7 % (n=6). Incubation with Kv7.1 specific antagonist HMR 1556 attenuated relaxation produced by 300nM CCh from 45.9 ± 4 % to 66.4 ± 7 % (n=7). A response that could not be emulated by the use of non-specific potassium channel inhibitors tetraethyl ammonium (1 mM) and 4-amminopyridine (1 mM). Illoprost mediated relaxations were abrogated by CAY-10471 and attenuated by linopirdine (10 µM, (n=5). Relaxations produced by the Kv7.2-7.5 enhancers ML213 and acrylamide S1, but not Kv7.1, were enhanced in arteries with intact endothelium compared to arteries without endothelium. Conclusions. This study has revealed that Kv7.4/5 are present in the endothelium of rat mesenteric arteries, as well as in the smooth muscle. Moreover, we show that Kv7 isoforms contribute to endothelial derived receptor mediated vasorelaxant responses. This study also shows the presence of endothelium enhanced responses only to Kv7.2-5 activators in rat mesenteric arteries.

Where applicable, experiments conform with Society ethical requirements