KCNQ modulators reveal a key role for KCNQ potassium channels in regulating the tone of rat pulmonary artery smooth muscle

Abstract

Potassium channels are central to the regulation of pulmonary vascular tone. The smooth muscle cells of pulmonary artery display a background K(+) conductance with biophysical properties resembling those of KCNQ (K(V)7) potassium channels. Therefore, we investigated the expression and functional role of KCNQ channels in pulmonary artery. The effects of selective KCNQ channel modulators were investigated on K(+) current and membrane potential in isolated pulmonary artery smooth muscle cells (PASMCs), on the tension developed by intact pulmonary arteries, and on pulmonary arterial pressure in isolated perfused lungs and in vivo. The KCNQ channel blockers, linopirdine and XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone], inhibited the noninactivating background K(+) conductance in PASMCs and caused depolarization, vasoconstriction, and raised pulmonary arterial pressure without constricting several systemic arteries or raising systemic pressure. The KCNQ channel openers, retigabine and flupirtine, had the opposite effects. PASMCs were found to express KCNQ4 mRNA, at higher levels than mesenteric artery, along with smaller amounts of KCNQ1 and 5. It is concluded that KCNQ channels, most probably KCNQ4, make an important contribution to the regulation of pulmonary vascular tone, with a greater contribution in pulmonary compared with systemic vessels. The pulmonary vasoconstrictor effect of KCNQ blockers is a potentially serious side effect, but the pulmonary vasodilator effect of the openers may be useful in the treatment of pulmonary hypertension.

Fig. 1.

PA effects of KCNQ blockers. A, constriction evoked by linopirdine (10 μM) and XE991 (1 μM) in the arteries indicated. Measured as percentage of K⁺-induced constriction (n = 4–6). Inset, original traces showing responses to linopirdine, XE991, and 50 mM K⁺ (K) in pulmonary (left) and renal (right) arteries. Calibration bars, 1 mN vertical and 10 min horizontal. Bi, PA perfusion pressure in isolated, salt-perfused lungs before and during bolus injection of AT and infusion of linopirdine (10 μM). Bii, concentration dependence of linopirdine-induced PA pressure increase; control pressure (c) in the absence of drug plotted for comparison; n = 4. Ci, mean systemic and PA pressures recorded in vivo before and during intravenous linopirdine at doses shown. Cii, dose-dependent increase in PA but not systemic pressure by linopirdine (n = 5). *, p < 0.05; **, p < 0.01; ***, p < 0.001; unpaired Student's t test versus PA (A); analysis of variance followed by Tukey's comparison versus control (Bii and Cii).

Fig. 2.

Effect of KCNQ channel openers on rat PA. A, sustained constriction induced by 10 μM phenylephrine followed by relaxation upon cumulative application of retigabine at concentrations indicated. Calibration bars, 1 mN vertical and 10 min horizontal. B, concentration-response curves for retigabine-induced relaxation in the presence (+E) or absence (-E) of endothelium and flupirtine (n = 5). Relaxation measured as percentage of constrictor-induced pretone. C, constriction induced by 50 mM K⁺ followed by relaxation upon cumulative application of retigabine at concentrations indicated. Calibration as in A. D, histogram comparing mean relaxation amplitudes for flupirtine and retigabine (10 and 100 μM) when the preconstrictor agent was phenylephrine (10 μM), PGF_2α (3 μM), or 50 mM K⁺; n = 4–6. E, histogram comparing relaxation responses to KCNQ activators and levcromakalim (levcrom), all at 10 μM, in the absence and presence of 10 μM glibenclamide (n = 3). F, histogram comparing constrictor effects of linopirdine (10 μM) and XE991 (1 μM) in the presence and absence of retigabine at the concentrations indicated (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001, paired Student's t test versus control.

Fig. 3.

Effects of KCNQ channel blockers on membrane potential and I_KN. A, membrane potential traces show depolarization upon application of linopirdine (10 μM) or XE991 (5 μM). B, mean depolarizations induced by linopirdine and XE991 at the concentrations shown (n indicated above bars). C, I_KN under control conditions, in the presence of 10 μM linopirdine (left) or 5 μM XE991 (right) and after 15 to 20 min of washing. Inset, voltage protocol. D, histogram comparing linopirdine and XE991 effects on I_KN measured at 0 mV (paired data, n indicated above bars). P, control PSS application in place of drug. *, p < 0.05. E, mean amplitude of delayed rectifier K⁺ current, under control conditions, after 5 μM XE991 application, then washout (n = 5). Inset, voltage protocol and typical traces before (con) and during (XE) XE991 application and after drug washout, recorded with a step to +40 mV.

Fig. 4.

Effects of KCNQ channel openers on membrane potential and I_KN. A, membrane potential traces upon application of retigabine (10 μM) or flupirtine (10 μM). B, mean I_KN amplitude over a range of potentials, under control conditions (130 mM K⁺) and after application of 10 μM retigabine (n = 4). Inset, voltage protocol. *, p < 0.05, paired Student's t test versus control.

Fig. 5.

KCNQ transcripts in pulmonary artery. RT-PCR detection of KCNQ transcripts in whole-rat PA, heart, and brain (A) and isolated PASMCs (B). Each column represents a separate reaction with (+RT) or without (-RT) reverse transcriptase. M, size marker; B, water blank.

Fig. 6.

Expression of KCNQ. A, expression profile of KCNQ1, 4, and 5 channels in whole-rat PA and mesenteric artery (MA) measured with qRT-PCR and normalized to GAPDH (n = 3). *, p < 0.05, analysis of variance with Tukey's pair-wise comparison versus KCNQ4 in PA. B, KCNQ subunit expression in PA expressed relative to brain and heart (n = 3). C, fluorescence images of PASMCs labeled separately with three different anti-KCNQ4 antibodies, G14, S18, and N10. Staining was absent in control cells treated identically but without primary antibody (fluorescence and bright-field images of the same cell shown). Calibration bar, 20 μm.