Role of Kv7 channels in responses of the pulmonary circulation to hypoxia

Abstract

Hypoxic pulmonary vasoconstriction (HPV) is a beneficial mechanism that diverts blood from hypoxic alveoli to better ventilated areas of the lung, but breathing hypoxic air causes the pulmonary circulation to become hypertensive. Responses to airway hypoxia are associated with depolarization of smooth muscle cells in the pulmonary arteries and reduced activity of K(+) channels. As Kv7 channels have been proposed to play a key role in regulating the smooth muscle membrane potential, we investigated their involvement in the development of HPV and hypoxia-induced pulmonary hypertension. Vascular effects of the selective Kv7 blocker, linopirdine, and Kv7 activator, flupirtine, were investigated in isolated, saline-perfused lungs from rats maintained for 3-5 days in an isobaric hypoxic chamber (FiO2 = 0.1) or room air. Linopirdine increased vascular resistance in lungs from normoxic, but not hypoxic rats. This effect was associated with reduced mRNA expression of the Kv7.4 channel α-subunit in hypoxic arteries, whereas Kv7.1 and Kv7.5 were unaffected. Flupirtine had no effect in normoxic lungs but reduced vascular resistance in hypoxic lungs. Moreover, oral dosing with flupirtine (30 mg/kg/day) prevented short-term in vivo hypoxia from increasing pulmonary vascular resistance and sensitizing the arteries to acute hypoxia. These findings suggest a protective role for Kv7.4 channels in the pulmonary circulation, limiting its reactivity to pressor agents and preventing hypoxia-induced pulmonary hypertension. They also provide further support for the therapeutic potential of Kv7 activators in pulmonary vascular disease.

Keywords: KCNQ; Kv7 channels; P/Q relationship; flupirtine; hypoxic pulmonary vasoconstriction; isolated lungs.

Fig. 1.

Linopirdine primes hypoxic pulmonary vasoconstriction (HPV) in saline-perfused rat lungs. HPV response measured in unprimed lungs 15, 30, or 65 min after bolus injection of 230 μg linopirdine into the inflow cannula to give an effective concentration of ∼12 μM (gray, n = 6) and in time-matched controls (black, n = 5). *P < 0.05 vs. control.

Fig. 2.

Kv channel inhibition modulates pulmonary vascular responses to hypoxia and angiotensin II in primed lungs. Baseline perfusion pressure (A), angiotensin II-induced vasoconstriction (B), and HPV (C) measured in primed lungs before (control, black bars) and after exposure to 12 μM linopirdine (LNP) or 12 μM linopirdine plus 3 mM 4-aminopyridine (4-AP) (LNP + 4-AP) (gray bars). #P < 0.05 control vs. LNP or LNP + 4-AP, *P < 0.05 LNP vs. LNP + 4-AP; n = 6 for both group. NS, not significant.

Fig. 3.

Loss of response to linopirdine in chronic hypoxia. Pressure-flow (P/Q) plots measured in primed lungs from normoxic (A) and 3-day hypoxic (B) rats in control conditions and after exposure to linopirdine (10 μM). *P < 0.05 linopirdine vs. control; n = 6 for each group.

Fig. 4.

Flupirtine causes pulmonary vasodilatation in chronic hypoxia. Effect of flupirtine (20 μM) on P/Q plots measured in primed lungs from normoxic controls (A) and rats exposed for 5 days to hypoxia (B). *P < 0.05 flupirtine vs. control; n = 5 for each group.

Fig. 5.

Hypoxia downregulates Kv7.4 mRNA expression. A: fluorescence images of lung sections from normoxic rats showing autofluorescence (green) and labeling with an anti-Kv7.4 antibody (red) and the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI) (blue). Sections were treated identically, except for omission of the Kv7.4 antibody in the control. Calibration bars 100 μm. B: expression profile of KCNQ1, KCNQ4, and KCNQ5 subunit mRNAs in rat pulmonary artery from rats maintained in a normoxic (control) or hypoxic environment for 3 days. Detected with quantitative RT-PCR and normalized to the expression of GAPDH (n = 3). *P < 0.05 hypoxic vs. control. C: Western blots of pulmonary artery proteins from 5 separate normoxic (C1–C4) and hypoxic (H1–H4) rats and proteins from nontransfected HEK-293T cells (NT) and HEK-293T cells overexpressing Kv7.4 channels (T). Proteins were separated on a 10% SDS-PAGE and transferred to a PVDF membrane, which was cut between the 50 and 75 kDa markers and probed separately with antibodies against Kv7.4 and β-tubulin. Arrowheads indicate the positions of molecular weight markers (kDa). D: densitometric analysis of Western blots showing Kv7.4 expression normalized to β-tubulin in arteries from normoxic (control) and hypoxic rats, as well as rats administered flupirtine (F, 30 mg/kg/day) and exposed to hypoxia for 5 days (n = 4).