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. 2017 Jan;56(1):99-108.
doi: 10.1165/rcmb.2016-0226OC.

The Cystic Fibrosis Transmembrane Conductance Regulator Potentiator Ivacaftor Augments Mucociliary Clearance Abrogating Cystic Fibrosis Transmembrane Conductance Regulator Inhibition by Cigarette Smoke (VSports注册入口)

S Vamsee Raju  1   2 , Vivian Y Lin  1 , Limbo Liu  3 , Carmel M McNicholas  2   4 , Suman Karki  1 , Peter A Sloane  1 , Liping Tang  1   2 , Patricia L Jackson  1   2 , Wei Wang  2   4 , Landon Wilson  5 , Kevin J Macon  6 , Marina Mazur  2 , John C Kappes  1   2 , Lawrence J DeLucas  2   6 , Stephen Barnes  5   7 , Kevin Kirk  2   4 , Guillermo J Tearney  3 , Steven M Rowe  1   2   4   8
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The Cystic Fibrosis Transmembrane Conductance Regulator Potentiator Ivacaftor Augments Mucociliary Clearance Abrogating Cystic Fibrosis Transmembrane Conductance Regulator Inhibition by Cigarette Smoke

"VSports手机版" S Vamsee Raju et al. Am J Respir Cell Mol Biol. 2017 Jan.

Abstract

Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction may contribute to chronic obstructive pulmonary disease pathogenesis and is a potential therapeutic target. We sought to determine the acute effects of cigarette smoke on ion transport and the mucociliary transport apparatus, their mechanistic basis, and whether deleterious effects could be reversed with the CFTR potentiator ivacaftor (VX-770). Primary human bronchial epithelial (HBE) cells and human bronchi were exposed to cigarette smoke extract (CSE) and/or ivacaftor. CFTR function and expression were measured in Ussing chambers and by surface biotinylation. CSE-derived acrolein modifications on CFTR were determined by mass spectroscopic analysis of purified protein, and the functional microanatomy of the airway epithelia was measured by 1-μm resolution optical coherence tomography. CSE reduced CFTR-dependent current in HBE cells (P < 0. 05) and human bronchi (P < 0. 05) within minutes of exposure. The mechanism involved CSE-induced reduction of CFTR gating, decreasing CFTR open-channel probability by approximately 75% immediately after exposure (P < 0. 05), whereas surface CFTR expression was partially reduced with chronic exposure, but was stable acutely. CSE treatment of purified CFTR resulted in acrolein modifications on lysine and cysteine residues that likely disrupt CFTR gating VSports手机版. In primary HBE cells, CSE reduced airway surface liquid depth (P < 0. 05) and ciliary beat frequency (P < 0. 05) within 60 minutes that was restored by coadministration with ivacaftor (P < 0. 005). Cigarette smoking transmits acute reductions in CFTR activity, adversely affecting the airway surface. These effects are reversible by a CFTR potentiator in vitro, representing a potential therapeutic strategy in patients with chronic obstructive pulmonary disease with chronic bronchitis. .

Keywords: cigarette smoke; cystic fibrosis transmembrane conductance regulator potentiator; ivacaftor; mucociliary transport; optical coherence tomography. V体育安卓版.

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Figures

Figure 1.
Figure 1.
Cigarette smoke extract (CSE) acutely reduces cystic fibrosis (CF) transmembrane conductance regulator (CFTR)–dependent short-circuit current (ISC). (A) Representative ISC tracing of non-CF primary human bronchial epithelial (HBE) monolayers sequentially treated with amiloride (100 μM), chloride-free amiloride (low Cl−), forskolin (20 μM), CSE (2%), or DMSO vehicle control, and CFTRInh-172 (10 μM). (B) Summary of forskolin-stimulated ISC shown in A. ****P < 0.0001, n = 4/condition. (C) Change in ISC when CSE (2%) was added to non-CF HBE cultures before amiloride or forskolin addition. *P < 0.05, n = 3–4/condition. (D) Representative ISC tracing of human tracheal explant from a normal donor, sequentially stimulated with amiloride (100 μM), forskolin (100 nM), CSE (2%), or DMSO vehicle control, followed by CFTRInh-172 (10 μM). (E) Summary data of that shown in D. ***P < 0.001, n = 4/condition.
Figure 2.
Figure 2.
CSE reduces surface CFTR expression at 24-hours, but not 20-minute, exposure. (A) Top: representative Western blot of primary HBE cells exposed to CSE (2%) for 20 minutes or 24 hours to the apical compartment as compared with DMSO vehicle control. CFTR C band indicated by arrow. Middle: surface CFTR expression quantified by biotinylation followed by Western blot. Surface CFTR indicated by arrow. Bottom: tubulin loading control. (B) Quantitation of total CFTR Western blot, as expressed as CFTR:tubulin ratio in comparison to control. *P < 0.05, n = 4 blots/condition. (C) Surface CFTR:tubulin expression ratio. **P < 0.01, n = 4 blots/condition. ns, not significant.
Figure 3.
Figure 3.
CSE acutely reduces CFTR-dependent channel opening. (A) Representative recording from macropatch of HEK293 cells expressing wild-type CFTR then exposed to low-dose protein kinase A (PKA) inhibitor (PKI; 40 U/ml) to ascertain phosphorylation status, then CSE (1%). CFTRInh-172 (10 μM) was added to confirm CFTR dependence. Holding potential was −50 mV. (B) Summary data of that shown in A. *P < 0.05, n = 4–6/condition. (C) Unitary conductance tracing of inside-out patch derived from wild-type CFTR expressing HEK293 cells showing significant and immediate inhibition of CFTR channel opening upon addition of CSE (1%). Holding potential was −50 mV. Experiment performed in the presence of 1 mM MgATP plus 40 U/ml of the catalytic subunit of PKA. *P < 0.05, n = 4. Dotted lines represent zero current level. (D) Changes in the probability of opening (Po) for CFTR channels observed in C are summarized following addition of vehicle, CSE (1%) and ivacaftor (VX-770).
Figure 4.
Figure 4.
Covalent modification of CFTR by cigarette smoke and acrolein. Purified recombinant CFTR protein was treated with 1 μM acrolein or 1% CSE for 30 minutes at 37°C, resolved on SDS-PAGE gel, trypsin digested, and analyzed by nano–liquid chromatography tandem mass spectrometry. Spectra are shown for acrolein modifications of cysteine by Schiff’s base addition (+38 addition [A]) and for a Michael addition on a lysine (+56 addition [B]) that resulted from treatment of CFTR with both acrolein and CSE addition, but did not occur after treatment with vehicle control. The masses of the theoretical fragment ions representing both kinds of acrolein modifications are listed in a table inset within the spectra. (C) Summary of specific CFTR amino acids modified by acrolein and CSE (highlighted in red with their molecular weight within the native CFTR sequence) within eight CFTR peptide fragments detected by matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis. (D) 16HBE cells acutely treated with acrolein (5 ppm, 10 min) as compared with cells exposed to vehicle control were analyzed for changes in total protein (bottom) and for fraction of CFTR proteins that were modified by acrolein using biotin hydrazide directed toward acrolein-modified CFTR residues (top). Blots are representative of three separate studies.
Figure 4.
Figure 4.
Covalent modification of CFTR by cigarette smoke and acrolein. Purified recombinant CFTR protein was treated with 1 μM acrolein or 1% CSE for 30 minutes at 37°C, resolved on SDS-PAGE gel, trypsin digested, and analyzed by nano–liquid chromatography tandem mass spectrometry. Spectra are shown for acrolein modifications of cysteine by Schiff’s base addition (+38 addition [A]) and for a Michael addition on a lysine (+56 addition [B]) that resulted from treatment of CFTR with both acrolein and CSE addition, but did not occur after treatment with vehicle control. The masses of the theoretical fragment ions representing both kinds of acrolein modifications are listed in a table inset within the spectra. (C) Summary of specific CFTR amino acids modified by acrolein and CSE (highlighted in red with their molecular weight within the native CFTR sequence) within eight CFTR peptide fragments detected by matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis. (D) 16HBE cells acutely treated with acrolein (5 ppm, 10 min) as compared with cells exposed to vehicle control were analyzed for changes in total protein (bottom) and for fraction of CFTR proteins that were modified by acrolein using biotin hydrazide directed toward acrolein-modified CFTR residues (top). Blots are representative of three separate studies.
Figure 5.
Figure 5.
The 1-μm resolution optical coherence tomography (μOCT) reveals significant changes in airway surface liquid (ASL) depth and ciliary beat frequency (CBF) after acute CSE exposure and/or ivacaftor (VX-770) treatment of HBE cells expressing wild-type CFTR. (A) Representative μOCT images of each experimental group at baseline, 20 minutes, and 1 hour posttreatment indicating the surface microanatomy (red lines, ASL; scale bars, 10 μm). (B) After 20 minutes of exposure, CSE-treated cells exhibit diminished ASL when compared with baseline (aP < 0.05) and to air control cells at the same time point (bP < 0.05); 1 hour of ivacaftor exposure markedly augmented ASL depth, as compared with that at baseline (cP < 0.05) and to DMSO control at 1 hour (dP < 0.05). Similarly, at 1 hour, cells treated with both CSE and ivacaftor display significantly higher ASL levels than those exposed only to CSE (eP < 0.005). (C) CSE exposure significantly reduced CBF at 20 minutes when compared with baseline (aP < 0.0005) and air control at the same time point (bP < 0.005). n = 4–6 and repeated twice using cells collected from two separate donors.
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