Bradykinin activates calcium-dependent potassium channel of small conductance in cultured human airway smooth muscle cells Bo Liu, Anette Freyer & Ian P Hall. Department of Therapeutics and Molecular Medicine, University Hospital of Nottingham, UK.	Print Abstract Search PubMed for: Liu B Freyer A Hall IP

Airway smooth muscle potassium channels (K channels) play an important role in the regulation of bronchial tone and response to bronchodilatary therapy. Human airway smooth muscle cells (HASM) express voltage and calcium-dependent K channels (Snetkov, 1996). Bradykinin (a potent broncho-constrictor) causes hyper-polarisation and increase of inward currents in ASM in vitro (Tomasic, 1992); however, the effect of bradykinin on potassium currents (Ik) in cultured HASM is largely unknown. The purpose of the present study was to characterise the K currents induced by bradykinin in cultured HASM.

HASM were isolated from proximal bronchi of patients undergoing pneumonectomy or lobectomy following informed written patient consent and local ethical committee approval. Airway smooth muscle was dissected from the bronchus, enzymatically dispersed with collagenase then cultured in DMEM supplemented with 10% FCS and 2 mM glutamine. Whole-cell voltage-clamp recordings were made from trypsin-dispersed HASM (passage 1 to 6) held at holding potentials of -40mV and depolarised from -100 to +100 mV in 10mV increments of 400ms duration. The internal electrode solution consisted of (mM) 140 KCl, 1 MgCl₂, 1 or 0.6 CaCl₂, 1 EGTA, and 10 HEPES, pH 7.2 (free intracellular Ca²⁺ of 793 nM or 133 nM) and the bath solution (mM) of 135 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES, pH 7.2. Current amplitudes were normalised to cell capacitance and plotted as a function of test potentials. Resting membrane potentials were recorded under current-clamp. All pulse protocols and data acquisition were performed with an EPC-9 amplifier con-trolled by Pulse/Pulsefit (V8.53; HEKA, Germany).Results are expressed as mean ± SE of n observations, and were compared using Students unpaired t test.

The resting membrane potentials of HASM was -33.69 ± 2.89 mV) and the membrane capacitance 140.08±6.02 pF (both n=29). Step depolarisations of HASM from -100mV to +100mV led to the activation of slowly activating outward currents. Pharmacological dissection of the currents with 1, 10 and 20 mM TEA, reduced mean outward currents at +100 mV by 25.21%±1.63 (p=0.125), 56.19%±4.55 (p=0.033) and 63.43%±2.92 (p=0.015, n=12, respectively). Neither iberiotoxin (100nM) nor apamin (100nM) had any obvious effect on the basal Ik. Application of bradykinin caused hyper-polarisation and increased Ik in a dose-dependent manner (10µM maximally increased current 3.9±0.37 fold cf. baseline, EC50=0.33 µM, n=14). Pre-incubation with apamin abrogated this bradykinin-induced increase, though iberiotoxin did not alter the current significantly. Interestingly, neither nifedipine (1µM) nor verapamil (1µM) had any effect on the increased Ik induced by bradykinin.

Bradykinin activates the calcium-dependent K channel of small conductance (SK), rather than that of large conductance (BK), via a L-type calcium channel-independent pathway in cultured human airway smooth muscle cells.

Snetkov VA, et al. (1996), Exp Physiol. 81, 5:791-804.
Tomasic M, et al. (1992), Am J Physiol 263,(1Pt1):C106.

This study is funded by the National Asthma Campaign.