Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange

Abstract

Regional alveolar hypoxia causes local vasoconstriction in the lung, shifting blood flow from hypoxic to normoxic areas, thereby maintaining gas exchange. This mechanism is known as hypoxic pulmonary vasoconstriction (HPV). Disturbances in HPV can cause life-threatening hypoxemia whereas chronic hypoxia triggers lung vascular remodeling and pulmonary hypertension. The signaling cascade of this vitally important mechanism is still unresolved. Using transient receptor potential channel 6 (TRPC6)-deficient mice, we show that this channel is a key regulator of acute HPV as this regulatory mechanism was absent in TRPC6(-/-) mice whereas the pulmonary vasoconstrictor response to the thromboxane mimetic U46619 was unchanged. Accordingly, induction of regional hypoventilation resulted in severe arterial hypoxemia in TRPC6(-/-) but not in WT mice. This effect was mirrored by a lack of hypoxia-induced cation influx and currents in smooth-muscle cells from precapillary pulmonary arteries (PASMC) of TRPC6(-/-) mice. In both WT and TRPC6(-/-) PASMC hypoxia caused diacylglycerol (DAG) accumulation. DAG seems to exert its action via TRPC6, as DAG kinase inhibition provoked a cation influx only in WT but not in TRPC6(-/-) PASMC. Notably, chronic hypoxia-induced pulmonary hypertension was independent of TRPC6 activity. We conclude that TRPC6 plays a unique and indispensable role in acute hypoxic pulmonary vasoconstriction. Manipulation of TRPC6 function may thus offer a therapeutic strategy for the control of pulmonary hemodynamics and gas exchange.

Fig. 1.

Acute and prolonged HPV and arterial oxygenation in WT and TRPC6-deficient (TRPC6^−/−) mice. (A) Time course of hypoxic vasoconstriction in isolated, buffer-perfused, and ventilated mouse lungs during 160 min of hypoxic ventilation. Changes in pulmonary artery pressure (ΔPAP) are shown for lungs from WT (filled circles, n = 5) and TRPC6^−/− (open circles, n = 6) mice ventilated with a hypoxic gas of 1% O₂. Control lungs were ventilated normoxically [WT, black triangles, n = 6; TRPC6^−/−, white triangles, n = 5; ∗1, significant difference (P < 0.05) between WT and TRPC6^−/− mice after applying acute hypoxia; ∗2, significant differences (P < 0.05) between normoxic (WT and TRPC6^−/−) and hypoxic (WT and TRPC6^−/−) mice]. (B) Specificity of TRPC6 for the hypoxia-induced vasoconstrictor response. Lungs of WT (filled bars) and TRPC6^−/− (open bars) mice were challenged either with hypoxic ventilation (1% O₂, 10 min, acute hypoxia) or with injection of the thromboxane mimetic U46619 into the pulmonary artery (U46619). ΔPAP, maximum increase in pulmonary artery pressure. Data are from n = 5 mice per group; ∗, significant difference (P < 0.05) between WT and TRPC6^−/− mice. (C) Arterial oxygenation in anesthetized WT (filled circles) and TRPC6^−/− (open circles) mice after provocation of regional ventilatory failure. Mice were ventilated with room air and challenged with an airway fluid load of 25 μl of saline by tracheal administration at time point zero. Arterial oxygenation was measured during the following 10 min. ∗, significant difference (P < 0.05) between WT and TRPC6^−/− mice. Data are from n = 4 mice per group.

Fig. 2.

TRPC expression analysis of, and cation influx in, PASMCs from WT and TRPC6^−/− mice. (A) Total RNA was prepared from primary cultured precapillary pulmonary artery SMCs of WT (n = 3 mice) and TRPC6-deficient (TRPC6^−/−, n = 3 mice) mice and reverse-transcribed. Products of the first strand synthesis were analyzed for the presence of amplification products obtained with primer pairs described in Materials and Methods. mRNAs coding for TRPCs and β-actin (as reference gene) were quantified with the aid of a light cycler. Values are presented as percentage of reference mRNA expression (β-actin mRNA expression). Except for TRPC6 expression (P < 0.05), no significant expression differences in WT and TRPC6^−/− cells were observed (P > 0.05). (B–D) Cation influx in PASMCs from WT and TRPC6^−/− mice. (B Left) Increase in [Ca²⁺]_i upon exposure to hypoxia. Primary cultured PASMC were loaded with fura-2 and analyzed by single-cell fluorescence imaging. A horizontal bar indicates hypoxic superfusion of the cells. ET-1 (4 nM) was added 5 min after starting the experiment and was present during the remainder of the experiment. Data are from n = 15 WT and n = 55 TRPC6^−/− cells from four mice each. (B Right) Maximal [Ca²⁺]_i in PASMC (n = 6 cells) from TRPC6-deficient mice infected with AAVs coding for TRPC6-EGFP (a fusion protein of TRPC6 and the EGFP) before (open bar) and after (filled bar) application of hypoxia. ET-1 (4 nM) was added 5 min after starting the experiment and was present during the remainder of the experiment. (C Left) Hypoxia-induced Mn²⁺ influx in TRPC6^−/− and WT PASMC. Mn²⁺ (0.3 mM) was added to the Ca²⁺-containing bath solution, and hypoxia was applied to PASMC from WT (n = 4 cells) and a TRPC6^−/− (TRPC6^−/−, n = 4 cells) mice as indicated by bars. ET-1 (4 nM) was added 5 min after starting the experiment and was present during the remainder of the experiment. Time courses of total fura-2 fluorescence at the isosbestic wavelength are shown. Total fura-2 fluorescence corrected for background signals was recorded by exciting fura-2 at the isosbestic wavelength (360 nm) and normalized to the initial values of each single cell. Dashed lines indicate the initial slopes, i.e., the rates of fura-2 quenching by Mn²⁺. (C Right) Summary of Mn²⁺ influx experiments. Differences in the linear range of the Mn²⁺ quench rate after Mn²⁺ addition and after application of hypoxic conditions were calculated for WT (filled bars, n = 12 cells) and TRPC6^−/− PASMC (open bars, n = 22 cells). ∗ significant difference (P < 0.05) between WT and TRPC6^−/− mice. (D) Effects of a DAG kinase inhibitor II (R59949) on [Ca²⁺]_i in WT versus TRPC6^−/− PASMC. ET-1 (4 nM) was added 1 min after starting the experiment and was present during the remainder of the experiment. The DAG kinase inhibitor R59949 induced a rise in [Ca²⁺]_i in WT cells (n = 15) but not in TRPC6^−/− cells (n = 75).

Fig. 3.

Hypoxia-induced activation of TRPC6-mediated cationic current. (A) Representative traces of whole-cell currents in normoxic and hypoxic PASMC from WT (Left) and TRPC6-deficient (TRPC6^−/−, Right) mice. (B and C) Summarized data of normalized currents elicited at a potential of −60 mV and +50 mV in normoxia (black bars) and after perfusion with hypoxic bath solution (gray bars) as well as after addition of the membrane-permeable analogue of diacylglycerol (OAG), during hypoxia (gray hatched bars; n = 7 for WT and n = 11 for TRPC6^−/− cells) or normoxia (black hatched bars; n = 4 each). Cells were primed with ET-1 2 min before treatment. Nifedipine was present throughout the experiments. ∗, P < 0.05 in comparison with normoxia.

Fig. 4.

Hypoxia-induced DAG accumulation in PASMC. (A) Confocal images before treatment by hypoxia (normoxia) and after treatment by hypoxia (hypoxia) or DAG kinase inhibitor (R59949) of TRPC6^−/− and WT PASMC infected with recombinant lentiviruses coding for a fluorescent DAG sensor consisting of the C1 domain of protein kinase C δ (PKCδ) (33) fused to the EGFP. Cells were primed with ET-1 1 min before treatment and were fixed by ice-cold acetone 5 min after treatment. Arrows indicate DAG accumulation at the plasma membrane. (B) Summarized data for the ratios of plasma membrane versus cytosolic fluorescence intensities before treatment (black bars) and after treatment of WT and TRPC6^−/− PASMC with hypoxia (gray bars) or DAG kinase inhibitor (gray hatched bars) (n = 37 cells for each group). Data were generated by calculating the fluorescence intensities in regions of interest defined over the plasma membrane and the cytosol of optical slices monitored by confocal microscopy.