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Copyright 2004 The British Pharmacological Society

010P University of Bath
Summer Meeting July 2004

Pharmacological evidence for a key role of voltage-gated K+ channels in the function of rat aortic smooth muscle cells

Sergey V. Smirnov, Paolo Tammaro, Amy L. Smith & Simon R. Hutchings, Department of Pharmacy and Pharmacology, University of Bath, Bath,
BA2 7AY, U.K

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Smith K
Haynes A
Chapman H
Smart D
Arch JRS

Potassium (K+) channels play an important role in the regulation of excitation-contraction coupling in vascular smooth muscle cells (VSMCs). Two major types of K+ channel currents, large conductance Ca2+-activated (BKCa) and voltage-dependent (IKv), are ubiquitously expressed in VSMCs. While IKv is mainly activated by membrane depolarisation, BKCa currents are also stimulated by an increase in intracellular Ca2+ concentration ([Ca2+]i) occurring in the presence of vasoconstrictors. The main aim of this study was to investigate a relative contribution of IKv and BKCa to the whole cell currents in rat aortic myocytes (RAMs) under increased [Ca2+]i and to phenylephrine-induced contraction of rat aortic rings using selective K+ channel pharmacology.

Thoracic aortas were isolated from male Wistar rats (225-300 g) humanely killed. Isometric tension recordings were performed from endothelium-denuded aortic rings (~ 3 mm in length) bathed in Krebs solution of the following composition (mM): 118 NaCl, 25 NaHCO3, 4.9 KCl, 1.2 KH2SO4, 2.5 CaCl2, 1.2 MgSO4, 11.7 glucose. Single RAMs were isolated using papain and collagenase (1 mg/ml each). K+currents were recorded with patch clamp technique at room temperature. Pipette solution contained (mM): 110 KCl, 10 NaCl, 5 MgCl2, 10 HEPES, 10 EGTA and 5 mM CaCl2 (calculated free [Ca2+]=200 nM)and 7 mM CaCl 2 was used to achieve 444 nM [Ca2+]. Perforated-patch recordings were performed using 100 µg/ml amphotericin B added to the pipette solution.

BKCa and IKv in the whole cell current were separated using 1 µM paxilline, a selective BKCa inhibitor. Electrophysiological analysis revealed that IKv activated at -40 mV, while BKCa was seen positive to -20 mV in all three conditions. Voltage-dependent characteristics, but not maximal conductance, of IKv was significantly altered in increased [Ca2+]i. The most significant differences were observed in the IKv steady-state activation in [Ca2+]i =444 nM (a significant shift by ~8 mV, <0.011P<0.037, unpaired t test). Cell dialysis with elevated [Ca2+]i also shifted the IKv availability by ~10 mV (0.008<P<0.012, compared to perforate patch recordings). 1 µM correolide (a Kv 1 blocker) did not inhibit the IKv. However, the IKv was blocked by tetra ethyl ammonium TEA (IC 50=3.1 ± 0.6 mM, n=5) and by millimolar concentrations of 4-aminopyridine (4-AP). In non-stimulated aortic rings 1-5 mM TEA and 4-AP (inhibitors of IKv), but not paxilline (1 µM), caused contraction. Phenylephrine (15-40 nM) induced sustained tension with superimposed slow oscillatory waves (OWs) of contraction. OWs were blocked by diltiazem and ryanodine, suggesting the involvement of L-type Ca2+ channels and ryanodine-sensitive Ca2+ stores in this process. TEA and 4-AP (which block I Kv in RAMs), but not IbTX and paxilline (BKCa inhibitors) nor correolide, increased the duration and amplitude of OWs. Our findings suggest that IKv, and not BKCa, plays an important role in the regulation of excitability of the rat aorta.