Airway surface liquid depth measured in ex vivo fragments of pig and human trachea: dependence on Na+ and Cl- channel function - PubMed (original) (raw)

Airway surface liquid depth measured in ex vivo fragments of pig and human trachea: dependence on Na+ and Cl- channel function

Yuanlin Song et al. Am J Physiol Lung Cell Mol Physiol. 2009 Dec.

Abstract

The airway surface liquid (ASL) is the thin fluid layer lining the airways whose depth may be reduced in cystic fibrosis. Prior measurements of ASL depth have been made in airway epithelial cell cultures. Here, we established methodology to measure ASL depth to approximately 1-microm accuracy in ex vivo fragments of freshly obtained human and pig tracheas. Airway fragments were mounted in chambers designed for perfusion of the basal surface and observation of the apical, fluorescently stained ASL by scanning confocal microscopy using a high numerical aperture lens immersed in perfluorocarbon. Measurement accuracy was verified using standards of specified fluid thickness. ASL depth in well-differentiated primary cultures of human nasal respiratory epithelium was 8.0 +/- 0.5 microm (SE 10 cultures) under basal conditions, 8.4 +/- 0.4 microm following ENaC inhibition by amiloride, and 14.5 +/- 1.2 microm following CFTR stimulation by cAMP agonists. ASL depth in human trachea was 7.0 +/- 0.7 microm under basal conditions, 11.0 +/- 1.7 microm following amiloride, 17.0 +/- 3.4 microm following cAMP agonists, and 7.1 +/- 0.5 microm after CFTR inhibition. Similar results were found in pig trachea. This study provides the first direct measurements of ASL depth in intact human airways and indicates the involvement of ENaC sodium channels and CFTR chloride channels in determining ASL depth. We suggest that CF lung disease may be caused by the inability of CFTR-deficient airways to increase their ASL depth transiently following secretory stimuli that in non-CF airways produce transient increases in ASL depth.

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Figures

Fig. 1.

Fig. 1.

Methodology for measurement of airway surface liquid (ASL) thickness (ASL depth). A: schematic of perfusion chamber showing a cell layer/tissue fragment with mucosal surface facing upward. The mucosal ASL is covered with a high-boiling point perfluorocarbon and visualized from above using a high-numerical aperture immersion lens. The basal, downward-facing surface is exposed to perfusate. B: photograph of perfusion chamber (from above). C: thickness measurements in solution standards consisting of specified volumes of PBS containing rhodamine B-dextran between 2 cover glasses to give fluid thicknesses of 4, 5, 6, 10, and 20 μm. The 0-μm sample consists of a dried thin fluorescent film in place of the fluorescent solution. Left: representative x-z confocal reconstructions. Right: fluorescence intensity profiles in the _z_-direction, showing 10 profiles at different _x,y_-positions overlaid for each solution thickness. The horizontal dashed line drawn at 70% of maximum fluorescence was used as a threshold to define boundaries of the fluorescent solution layers. D: histograms of number vs. _z_-depth determined from data as in C. ASL depths were binned in 0.01-μm intervals for histogram construction.

Fig. 2.

Fig. 2.

ASL depth measurements in well-differentiated human airway epithelial cell cultures grown on porous supports. A: hematoxylin and eosin staining of non-CF and CF nasal respiratory cell cultures. B: Ussing chamber measurements of short-circuit current in response to ENaC inhibition (amiloride, 10 μM), CFTR activation (forskolin, 10 μM) and inhibition (CFTRinh-172, 10 μM), and calcium-activated chloride channel activation (UTP, 100 μM) and inhibition (CaCCinh-01, 30 μM). C, left: representative x-z confocal reconstructions shown together with _z_-fluorescence profiles for nasal respiratory cell cultures. The ASL was stained red with rhodamine B-dextran and surface epithelial cells green with Cell Track Green. Channel activators/inhibitors were present in culture medium for 4 h as indicated: 100 μM amiloride, 20 μM CFTRinh-172, 100 μM IBMX, 20 μM forskolin. Ten _z_-fluorescence profiles overlaid for each condition. Right: number vs. _z_-depth histograms. D, top: average ASL depths (means ± SE, n = 4–8 cultures/condition). *P < 0.05 between non-CF and CF cultures for basal ASL depth; #P < 0.02 for basal vs. forskolin/IBMX-treated cultures; **P < 0.05 for amiloride-treated non-CF vs. CF cultures. Bottom: time course of ASL depth in non-CF cultures following addition of IBMX and forskolin to the perfusate.

Fig. 3.

Fig. 3.

ASL depth measurements in pig trachea. A, left: hematoxylin/eosin staining of whole pig trachea. Right: staining of the posterior membranous region of trachea that is devoid of cartilaginous rings. B: short-circuit analysis. ENaC and CFTR modulators added where indicated: 100 μM amiloride, 20 μM forskolin, 100 μM IBMX, 20 μM MalH-2. C: photograph of perfusion chamber containing tracheal fragment surrounded by temperature controller. D: time course of ASL depth in pig trachea following addition of 20 μl of fluid on the mucosa (SE, n = 3). E, left: representative x-z confocal reconstructions and _z_-fluorescence profiles. Right: number vs. ASL depth histograms for measurements on different pig tracheas.

Fig. 4.

Fig. 4.

ASL depth measurements in human trachea. A: hematoxylin and eosin staining of human tracheal mucosa. B: short-circuit analysis showing effects of ENaC and CFTR modulators added where indicated: 100 μM amiloride, 20 μM forskolin, 100 μM IBMX, 20 μM CFTRinh-172. C, left: representative x-z confocal reconstructions and _z_-fluorescence profiles. Right: number vs. ASL depth histograms for measurements on different human tracheas.

Fig. 5.

Fig. 5.

Effect of Na+ and Cl− channel modulators on ASL depth in pig and human trachea. A and B: measurements made on pig (A) and human (B) tracheal fragments in which the mucosa was incubated at 37°C for 6 h without or with amiloride (100 μM), forskolin (20 μM), IBMX (100 μM), MalH-2 (20 μM), or CFTRinh-172 (20 μM). Left: representative x-z confocal fluorescence reconstructions. Right: number vs. ASL depth histograms. C and D: summary of ASL depths (means ± SE, n = 3–6) in pig trachea (C) and human trachea (D). *P < 0.05 compared with control.

Fig. 6.

Fig. 6.

Effect of amiloride and CFTR activators on steady-state ASL depth in pig trachea. A: the ASL was initially allowed to reach steady-state depth by 6-h incubation at 37°C in the absence of modulators. Amiloride or forskolin/IBMX were then applied as described in

methods

, and ASL depth was measured 2 h later (means ± SE, n = 4–5). Control indicates no modulator added. *P < 0.05 compared with control. B: control study showing amiloride inhibition of fluid absorption. Fluid was added onto the tracheal mucosa to give initial ASL depth ∼20 μm. The ASL was covered with perfluorocarbon, and ASL depth measured at 1 and 2 h. Where indicated, amiloride was added at time 0 to the ASL and serosal perfusate, as done in A (means ± SE, n = 3). *P < 0.05.

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