Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC346
Kv7.4 channels contribute to β-adrenoceptor regulation of rat renal artery
F. Zunke1, P. S. Chadha1, I. A. Greenwood1
1. Division of Biomedical Sciences, St George's University of London, London, United Kingdom.
KCNQ-encoded voltage-dependent potassium channels (Kv7) have been identified in vascular smooth muscle cells and shown to regulate vascular tone (Greenwood and Ohya, 2009). Kv7.4 channels have been identified as one of the main functional contributors in vascular tissue. Although modulation of these channels with selective activators and blockers causes profound changes in vascular tone, it is not known to what extent Kv7.4 channels contribute to endogenous dilator mechanisms. The present study utilises Kv7 modulators and a selective Kv7.4 knockdown protocol to characterise the impact of these channels in β-adrenoceptor-mediated relaxation of rat renal artery. Renal arteries were isolated from male Wistar rats (200-225 g). Isometric tension recordings were performed on a myograph. In some vessels, Kv7.4 channel knockdown was achieved by transfecting with specific KCNQ4 siRNA using a reverse permeabilisation protocol. Control vessels were transfected with non-specific scrambled siRNA. Subsequent experiments were conducted following 72 hrs incubation. Kv7 channel blocker (linopirdine) and activator (S-1) were used to assess Kv7 channel functionality. Furthermore, responses to isoprenaline (β-adrenoceptor agonist) and forskolin (adenylyl cyclase activator) were determined in arteries preconstricted with 3 μM Methoxamine, in the presence and absence of various K+ channel blockers. KCNQ gene expression was determined using endpoint and quantitative PCR. Kv7.4 channel expression was assessed by Western blotting. Values are presented as mean ± s.e.m., compared by two-tailed Student’s t-test. Amplicons of KCNQ1, KCNQ3, KCNQ4 and KCNQ5 mRNA as well as Kv7.4 protein were detected in rat renal arteries. Application of the Kv7 activator S-1 caused concentration-dependent relaxation in renal arteries, which was inhibited in the presence of linopirdine. In preconstricted arteries, isoprenaline-induced relaxation was inhibited in the presence of 10 µM linopirdine (Emax, 39.3±15.5 vs. 103.6±4.2 vehicle control; n=5-6; p<0.01). The Kv channel blocker 4-aminopyridine (1 mM) or KATP blocker glibenclamide (10 µM) did not inhibit isoprenaline responses. Relaxation caused by forskolin was also attenuated in the presence of linopirdine (pEC50, 6.3±0.1 vs. 7.2±0.1 vehicle control; n=4; p<0.01). In arteries transfected with KCNQ4 siRNA, S-1 relaxation was attenuated (1 μM S-1: 17.2% ± 5.8 vs. 43.5% ± 9.2 in control vessels; n=7; p<0.05). Moreover, in these arteries, relaxation in response to isoprenaline was significantly attenuated compared to control vessels (pEC50, 7.1±0.1 vs. 8.2±0.1 scrambled siRNA control; n=7; p<0.0001). These results show that Kv7.4 channels appear to be functional contributors to β-adrenoceptor-mediated relaxation.
Where applicable, experiments conform with Society ethical requirements