CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction

Abstract

Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca(2+) mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca(2+) mobilization. Ca(2+) mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction.

Keywords: ceramide; neutral sphingomyelinase; pulmonary hypertension; transient receptor potential canonical 6.

Fig. 1.

CFTR is required for HPV and its role in lung vasoconstriction does not relate to CFTR’s function as a Cl⁻ channel. (A) Hypoxia (1% O₂)-induced increase in pulmonary artery pressure (ΔPpa) in isolated mouse lungs was attenuated by CFTR inhibition (CFTRinh-172; 10 µmol/L) and in lungs of CFTR-deficient (CFTR^−/−) mice. (B) CFTR^−/− mice developed more profound hypoxemia in response to partial airway occlusion by tracheal instillation of 25 μL saline compared with wild-type (WT) mice. (C) CFTR inhibition (CFTRinh-172; 10 µmol/L) or deficiency did not alter the increase in mean Ppa (ΔPpa) in response to angiotensin II (1 µg bolus). (D) Cl⁻ free perfusate did not affect the hypoxia-induced increase in Ppa. Representative immunoblots and quantitative data (normalized first to tubulin as corresponding loading control, and then to normoxia as baseline) show differential expression of CFTR in PASMCs during 2, 4, or 24 h of acute hypoxia (1% O₂) (E) or pulmonary arteries isolated from WT mice exposed to 5 wk of normoxia or hypoxia [10% (vol/vol) O₂] (F). Values are given as mean and SEM; n = 6, 5, 5, 4–5, 8, and 4 per group for A–F, respectively. *P ≤ 0.05, **P ≤ 0.01 vs. control group.

Fig. 2.

CFTR-deficient mice are partially protected from hypoxic PH. Cftr^tm1Unc Tg(FABPCFTR)1Jaw/J mice lacking pulmonary CFTR (CFTR^−/−) and corresponding wild types (CFTR^+/+) were housed under normoxic (21% O₂) or hypoxic (10% O₂) conditions for 5 wk. Hypoxia-induced increase in RVSP (A) and pulmonary arterial remodeling (B–D) were attenuated in mice lacking pulmonary CFTR [representative images of HE-stained lung section shown in B; quantitative analyses of medial wall thickness in arterioles of different caliber given in C, and fraction of nonmuscularized (N), partially muscularized (P), or fully muscularized (M) small pulmonary arteries in D]. Values are given as mean and SEM (A, C, and D); n = 8–15 or 7–11 per group for A or C/D, respectively. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs. indicated group for A and C; *P ≤ 0.05 vs. corresponding normoxia and #P ≤ 0.05 vs. corresponding CFTR^+/+ for D. (Scale bar: 50 µm.)

Fig. 3.

CFTR is required for hypoxia-induced Ca²⁺ signaling. (A) Representative images of the [Ca²⁺]_i response to hypoxia in human PASMCs show Fura-2–loaded PASMC color-coded for [Ca²⁺]_i at normoxia (pO₂ ∼ 150 mmHg; Left) or after 5 min of hypoxia (pO₂ ∼ 10 mmHg; Right) in the presence or absence of the CFTR inhibitor CFTRinh-172 (10 µmol/L). (Scale bar: 25 µm.) (B) Group data show the hypoxia-induced increase in human PASMC [Ca²⁺]_i and its inhibition by CFTRinh-172. (C) Group data show the hypoxia-induced [Ca²⁺]_i increase in murine PASMCs isolated from CFTR-deficient (CFTR^−/−) or corresponding wild-type (CFTR^+/+) mice. Group data show the effect of CFTRinh-172 (10 µmol/L) on the hypoxia- (D) or sphingosine-1-phosphate (E; 10 µmol/L)–induced [Ca²⁺]_i increase (ΔSMC [Ca²⁺]_i) in human PASMCs or CASMCs, respectively. Values are given as mean and SEM; n = 5, 3, 5, and 5 per group for B–E, respectively. *P ≤ 0.05, ***P ≤ 0.001 vs. indicated group.

Fig. 4.

CFTR is required for hypoxia-induced TRPC6 translocation. (A) The TRPC6 inhibitor LA (5 μM) attenuated the hypoxia-induced increase in ΔPpa in isolated mouse lungs. (B) TRPC6 translocation to caveolae in response to 15 min of hypoxia (1% O₂) was evident as TRPC6 abundance in membrane fractions 4–6 and was blocked by pretreatment with CFTRinh-172 (10 µmol/L). Values are given as mean and SEM; n = 5 or 3 per group for A and B, respectively. *P ≤ 0.05, **P ≤ 0.01 vs. indicated group. (C) PASMCs were exposed to either normoxia or hypoxia (1% O₂) for 15 min in the presence or absence of CFTRinh-172 (10 µmol/L); cell homogenates were immunoprecipitated (IP) for CFTR and precipitates were blotted for both CFTR and TRPC6. Co-IPs show hypoxia-induced complex formation between TRPC6 and CFTR that was blocked by CFTR inhibition.

Fig. 5.

Neutral sphingomyelinase (nSMase)-dependent pulmonary vasoconstriction and TRPC6 recruitment to caveolae require CFTR. (A) nSMase inhibitor GW4869 but not acid sphingomyelinase (aSMase) inhibitor ARC39 (each 10 µmol/L) blocked the hypoxia (1% O₂)-induced increase in ΔPpa in isolated mouse lungs. (B) GW4869 (10 µmol/L) blocked the hypoxia-induced abundance of TRPC6 in caveolar membrane fractions of PASMCs. (C) CFTR and TRPC6 are required for nSMase-induced pulmonary vasoconstriction: Exogenous nSMase (100 U/L) caused an increase in Ppa that was blocked by CFTRinh-172 (10 µmol/L) or the TRPC6 inhibitor LA (5 µmol/L). (D) Exogenous nSMase (100 U/L) caused translocation of TRPC6 to caveolar membrane fractions of PASMCs that was blocked by CFTRinh-172 (10 µmol/L). Values are given as mean and SEM; n = 5, 3, 5–10, or 4 per group for A–D, respectively. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs. indicated group.

Fig. 6.

Sphingosine-1-phosphate (S1P) signaling is required for hypoxia- and nSMase-induced pulmonary vasoconstriction, but not for TRPC6 translocation. (A) The hypoxia-induced increase in ΔPpa in isolated mouse lungs was attenuated by the sphingosine kinase (SphK) inhibitor SKI II (5 µmol/L) or the S1P receptor 2/4 (S1P_2/4) antagonist JTE-013 (10 µmol/L). (B) nSMase (100 U/L)-induced increase in Ppa was also attenuated by SKI II and JTE-013. (C) Hypoxia-induced increase in Ppa was attenuated in lungs of SphK1-deficient mice, but not in lungs of S1P₂- or S1P₄-deficient mice. (D) S1P (10 µmol/L) alone did not induce TRPC6 translocation to caveolar membrane fractions in PASMCs. (E) nSMase (100 U/L)-induced TRPC6 translocation to caveolar membrane fractions in PASMCs was not attenuated by SphK inhibition (5 µmol/L). Values are given as mean and SEM; n = 5, 5, 7–10, 3, or 4 per group for A–E, respectively. *P ≤ 0.05, **P ≤ 0.01 vs. indicated group.

Fig. 7.

Downstream signaling pathways of nSMase and S1P in isolated mouse lungs. (A) nSMase-induced pulmonary vasoconstriction requires PLC: The PLC inhibitor U73122, but not its inactive analog U73343 (each 10 µmol/L), attenuated the nSMase (100 U/L)-induced increase in ΔPpa. (B) S1P (10 µmol/L)-induced pulmonary vasoconstriction was not blocked by the TRPC6 inhibitor LA (5 µmol/L) or the PLC inhibitor U73122 (10 µmol/L), but inhibited by the RhoK inhibitor Y27632 (10 µmol/L). (C and D) Following nSMase-induced TRPC6 translocation, S1P induced an amplified vasoconstrictive response through activation of PLC, TRPC6, and RhoK: In the presence of the SphK inhibitor SKI II (5 µmol/L), the lung vasomotor response to exogenous nSMase (100 U/L) was largely blunted, yet the vasoconstrictive effect of S1P (10 µmol/L) was amplified in the presence of nSMase and SKI II (C). The resulting synergistic response was sensitive to inhibition of TRPC6 by LA (5 µmol/L), PLC by U73122 yet not U73343 (both 10 µmol/L), and RhoK by Y27632 (10 µmol/L) (D), with combined inhibition (combi) of all three pathways showing an additive effect. Values are given as mean and SEM; n = 5 per group. *P ≤ 0.05, **P ≤ 0.01 vs. indicated group.

Fig. 8.

Proposed concept for the role of CFTR and sphingolipids in HPV. Hypoxia activates neutral sphingomyelinase, resulting in the formation of ceramide, which causes recruitment of TRPC6 to caveolar membranes in a CFTR-dependent manner that involves the formation of a CFTR/TRPC6 protein complex. Concomitant conversion of ceramide to S1P via ceramidase and SphK1 stimulates S1P₂ and S1P₄ receptors, thereby triggering TRPC6-mediated Ca²⁺ influx via PLC-dependent diacylglyercol (DAG) synthesis, and, in parallel, Ca²⁺ sensitization via RhoK, ultimately leading to PASMC contraction.