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 (BK_Ca) and voltage-dependent (I_Kv), are ubiquitously expressed in VSMCs. While I_Kv is mainly activated by membrane depolarisation, BK_Ca 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 I_Kv and BK_Ca 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.

BK_Ca and I_Kv in the whole cell current were separated using 1 µM paxilline, a selective BK_Ca inhibitor. Electrophysiological analysis revealed that I_Kv activated at -40 mV, while BK_Ca was seen positive to -20 mV in all three conditions. Voltage-dependent characteristics, but not maximal conductance, of I_Kv was significantly altered in increased [Ca²⁺]_i. The most significant differences were observed in the I_Kv 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 I_Kv availability by ~10 mV (0.008<P<0.012, compared to perforate patch recordings). 1 µM correolide (a K_v 1 blocker) did not inhibit the I_Kv. However, the I_Kv 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 I_Kv), 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 (BK_Ca inhibitors) nor correolide, increased the duration and amplitude of OWs. Our findings suggest that I_Kv, and not BK_Ca, plays an important role in the regulation of excitability of the rat aorta.