Osmotic water permeabilities of cultured, well-differentiated normal and cystic fibrosis airway epithelia

Abstract

Current hypotheses describing the function of normal airway surface liquid (ASL) in lung defense are divergent. One theory predicts that normal airways regulate ASL volume by modulating the flow of isosmotic fluid across the epithelium, whereas an alternative theory predicts that ASL is normally hyposmotic. These hypotheses predict different values for the osmotic water permeability (P(f)) of airway epithelia. We measured P(f) of cultures of normal and cystic fibrosis (CF) airway epithelia that, like the native tissue, contain columnar cells facing the lumen and basal cells that face a basement membrane. Xz laser scanning confocal microscopy recorded changes in epithelial height and transepithelial volume flow in response to anisosmotic challenges. With luminal hyperosmotic challenges, transepithelial and apical membrane P(f) are relatively high for both normal and CF airway epithelia, consistent with an isosmotic ASL. Simultaneous measurements of epithelial cell volume and transepithelial water flow revealed that airway columnar epithelial cells behave as osmometers whose volume is controlled by luminal osmolality. Basal cell volume did not change in these experiments. When the serosal side of the epithelium was challenged with hyperosmotic solutions, the basal cells shrank, whereas the lumen-facing columnar cells did not. We conclude that (a) normal and CF airway epithelia have relatively high water permeabilities, consistent with the isosmotic ASL theory, and the capacity to restore water on airway surfaces lost by evaporation, and (b) the columnar cell basolateral membrane and tight junctions limit transepithelial water flow in this tissue.

Figure 1

Fluorescence and height measurements by xz laser scanning confocal microscopy. (a) Relationship between Texas Red-dextran concentration and fluorescence. (b) Relationship between serosal bath volume and height. T-Col = Transwell-Col substratum, CSBndry = coverslip-serosal bath interface.

Figure 2

Responses of HBE cell cultures to luminal hypertonicity. (a) Highly differentiated HBE cell culture, typical of those used in study. (b) Xz scanning laser confocal images of an HBE passage 1 cell culture before and 60 seconds after elevation of luminal osmolality to 450 mosM with NaCl. (c) Time course of changes in serosal bath Texas Red-dextran fluorescence and height after luminal hyperosmotic challenge.

Figure 3

Osmotic permeabilities of HBE cultures. (a) Transepithelial osmotic permeability (P_f^T) for MDCK (n = 5) and for HBE (passage 1; n = 8) cell cultures challenged luminally with 150 mosM NaCl, as determined from changes in TR-dextran fluorescence of the serosal bath. (b) Transapical osmotic permeability (P_f^A) for MDCK (n = 5) and for HBE (passage 1; n = 8) cell cultures, as determined from changes in cell height. In both panels, data are expressed as mean ± SE, and for n HBE cell cultures, without and with luminal 1 mM HgCl₂. ^AP < 0.05, paired t test.

Figure 4

Changes in HBE culture height (filled circles) and serosal volume (open circles) measured simultaneously during luminally directed osmotic water flow. Time courses of changes in epithelial height and serosal bath TR-dextran fluorescence for HBE cultures challenged luminally with 150 mosM raffinose. The data are expressed as the mean ± SE (n = 6).

Figure 5

Hyperosmotic exposure of the basolateral membrane. HBE cultures were exposed serosally to TBR + 150 mM raffinose, by placing the T-Col on a droplet of the hyperosmotic medium positioned on the coverslip in the middle of the chamber and then focusing quickly on the epithelium with the confocal microscope in XZ laser scanning mode. The lumen-facing columnar and basal/intermediate cells in the cultures were monitored subsequently for changes in height from images collected at 7-second intervals. The initial image was collected 1–3 seconds from the time of exposure of the culture to the hyperosmotic medium. Within each culture, the heights of three cells of each type were determined in each image series over time, and these data are presented as the mean ± SE (n = 4).