J Korean Med Sci.  2011 Mar;26(3):317-324. 10.3346/jkms.2011.26.3.317.

Suppression of CFTR-mediated Cl- Secretion of Airway Epithelium in Vitamin C-deficient Mice

• Kim Y
• Kim H
• Yoo HY
• Kang JS
• Kim SJ
• Kim JK
• Cho HS

• KMID: 2157866
• DOI: http://doi.org/10.3346/jkms.2011.26.3.317

Abstract

Hyperoxic ventilation induces detrimental effects on the respiratory system, and ambient oxygen may be harmful unless compensated by physiological anti-oxidants, such as vitamin C. Here we investigate the changes in electrolyte transport of airway epithelium in mice exposed to normobaric hyperoxia and in gulonolacton oxidase knock-out (gulo[-/-]) mice without vitamin C (Vit-C) supplementation. Short-circuit current (Isc) of tracheal epithelium was measured using Ussing chamber technique. After confirming amiloride-sensitive Na+ absorption (DeltaIsc,amil), cAMP-dependent Cl- secretion (DeltaIsc,forsk) was induced by forskolin. To evaluate Ca2+-dependent Cl- secretion, ATP was applied to the luminal side (DeltaIsc,ATP). In mice exposed to 98% PO2 for 36 hr, DeltaIsc,forsk decreased, DeltaIsc,amil and DeltaIsc,ATP was not affected. In gulo(-/-) mice, both DeltaIsc,forsk and DeltaIsc,ATP decreased from three weeks after Vit-C deprivation, while both were unchanged with Vit-C supplementation. At the fourth week, tissue resistance and all electrolyte transport activities were decreased. An immunofluorescence study showed that the expression of cystic fibrosis conductance regulator (CFTR) was decreased in gulo(-/-) mice, whereas the expression of KCNQ1 K+ channel was preserved. Taken together, the CFTR-mediated Cl- secretion of airway epithelium is susceptible to oxidative stress, which suggests that supplementation of the antioxidant might be beneficial for the maintenance of airway surface liquid.

Keyword

Hyperoxia; Airway Epithelium; Cystic Fibrosis Transmembrane Conductance Regulator; Electrolyte Transport; Ascorbic Acid

MeSH Terms

Animals
Ascorbic Acid Deficiency/*metabolism
Biological Transport/drug effects
Chlorides/*metabolism
Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors/drug
Forskolin/pharmacology
Hyperbaric Oxygenation
Hyperoxia/*physiopathology
Ion Transport/drug effects
Mice
Mice, Inbred C57BL
Mice, Inbred ICR
Mice, Knockout/metabolism
Mice, Transgenic
Microscopy, Fluorescence
Oxidative Stress
Oxygen/adverse effects/pharmacology
Potassium Channels/metabolism
Respiratory Mucosa/drug effects/*metabolism/secretion
Sodium
Sugar Acids/metabolism

Figure

• 

  Fig. 1 Ussing chamber experiments in tracheal epithelia obtained from control and hyperoxia-exposed mice. (A) Original recordings of the transepithelial voltage (Vte). The upper border of the trace is Vte, the downward deflection (ΔVte) is the response to current injections from which the transepithelial resistance (Rte) and the equivalent short-circuit current (Isc) were calculated. The bars below indicate the luminal (lu) or basolateral (bl) application of amiloride (10 µM), forskolin (2 µM)/IBMX (100 µM), chromanol 293B (293B 10 µM) or ATP (50 µM). (B) Summary of Isc measured in the initial control and during each phase of drug application as demonstrated above. Open and closed bar graphs indicate results from normoxia (21% PO2) and hyperoxia (98% PO2, 36 hr)-exposed mice, respectively. (C-E) Summaries of the changes in Isc (ΔIsc) caused by forskolin/IBMX (C), amiloride (D), and ATP (E), as demonstrated in the above trace. Data from the groups of mice exposed to different levels of oxygen tension (21%, 80%, 90%, and 98% PO2) are compared. Numbers of tested tissues are directly indicated in the figure. The asterisks indicate statistical significance (P < 0.05, paired t-test).

• 

  Fig. 2 Ussing chamber experiments in tracheal epithelia obtained from control and vitamin C-deficient mice. (A) Original recordings of the transepithelial voltages (Vte) in control (WT [C57BL/6], upper trace) and gulo(-/-) mice with no vitamin C supplementation for three weeks (K/O-3wk, lower trace). The bars below indicate the luminal (lu) or basolateral (bl) application of amiloride (10 µM), forskolin (2 µM)/IBMX (100 µM), chromanol 293B (293B 10 µM) or ATP (50 µM). (B) Summary of the Isc measured in the initial controls and during each phase of drug application, as demonstrated above. Open and closed bar graphs indicate results from WT (C57BL/6) and K/O-3wk, respectively. (C-E) Summaries of the changes in Isc (ΔIsc) caused by forskolin/IBMX (C), amiloride (D), and ATP (E), as demonstrated in the above trace. Data from the groups of WT and gulo(-/-) mice with or without vitamin C supplementation. The vitamin C deprivation period was varied from one to four weeks. Numbers of tested tissues are directly indicated in the figure. The asterisks indicate statistical significance (P < 0.05, paired t-test). (F) Summaries of tissue resistance (Rte) measured in each group.

• 

  Fig. 3 Decreased expression of CFTR in the tracheal epithelia from vitamin C-deprived mice. Using confocal microscopy, the expression of CFTR (red) was examined with immunostaining in the tracheal epithelia of wild type (A), vitamin C-supplemented gulo(-/-) (B), and vitamin C-deprived gulo(-/-) mice (C-F). Nuclei were counterstained with DAPI (blue). The expressions of CFTR in luminal membranes decreased in K/O-2wk (D) and almost disappeared in K/O-3wk mice (E). Note that the columnar shape of the epithelium changes to cube-like or flattened in K/O-4wk (F). Scale bar, 50 µm.

• 

  Fig. 4 Persistent expression of KCNQ1 in the tracheal epithelia from vitamin C-deprived mice. Immunofluorescence microcopy was used to compare the expressions of KCNQ1 (green) in WT (A) and gulo(-/-) mice in which vitamin C was either supplemented (B) or not for 1, 2, 3, and 4 weeks (C-F). Nuclei were counterstained with DAPI (blue). Scale bar, 50 µm.

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