Identification and impact of Kv7 channels in rat mesenteric artery endothelium

S. N. Baldwin¹, S. L. Sandow², J. B. Stott³, I. A. Greenwood³. ¹St George’s London, London, United Kingdom, ²University of the Sunshine Coast, Brisbane, Australia, ³Vascular Biology Research Centre, London, United Kingdom.

Introduction: Kv7 channels have been identified as key regulators of arterial diameter and receptor mediated dilatation in many arteries. Of the 5 isoforms Kv7.1, 7.4 and 7.5 are routinely identified in rodent and human arteries. Molecular and pharmacological interventions have implicated Kv7.4/7.5 as the main functional determinants. 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 relaxation.

Methods and materials: Male Wistar rats (175-225g) were killed by methods in accordance with UK Animals Scientific Procedures Act and Australian legislation. 2^nd-4^th 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 μM carbachol producing greater than 90% relaxation of arteries precontracted with 3 μM 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 (100 nM-10 μM) produced contraction-dependent relaxations that were reduced by incubation with the NO synthase inhibitor L-NAME (100 μM) and significantly impaired by a combination of apamin (10 nM) and TRAM-34 (1 μM). Incubation with the pan Kv7 blocker linopirdine (10 μM) reduced the relaxation produced by 300 nM carbachol from 57 ± 4 % to 25 ± 7 %(n=5). ML213 and acrylamide S1 are enhancers of Kv7.2-7.5, but not Kv7.1, and both agents produced concentration-dependent relaxation of precontracted mesenteric arteries. In arteries with intact endothelium the response of both Kv7 activators was enhanced compared to arteries without endothelium (1μM ML213 produced 60 ± 7 % relaxation and 19 ± 6 % relaxation in the presence and absence of endothelium, respectively n=6).

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; and the presence of endothelium enhanced responses to Kv7 activators.