Background At the time of birth, the major pathway regulating lung fluid absorption is transepithelial Na+ movement via epithelial sodium channels (ENaCs). The density of ENaC proteins is high in the apical membranes of the type II alveolar cells and its expression and function are regulated by many agents, including steroids and growth factors. The rat parotid gland cell line (PAR-C5) expresses all ENaC subunit proteins (α, β, andγ), but monolayers grown in media containing EGF do not demonstrate significant ENaC function.
Objective and Methods PAR-C5 cells were studied to examine the effect of EGF on ENaC function and trafficking. Cells were seeded on permeable filter supports in the presence or absence of EGF (20 ng/mL) and/or dexamethasone (0.1 μM). After the monolayers formed tight junctions as demonstrated by transepithelial resistances > 200 Ω.cm2 (mean resistance 513 Ω.cm2), permeable filters were mounted in Ussing chambers and Na+ transport was determined by the amiloride (10 μM)-sensitive component of the short-circuit current (Isc). α-ENaC protein content of similarly treated cells was examined by Western blot. α-ENaC localization in PAR-C5 cells was determined by confocal microscopy following staining with anti-α-ENaC antibody.
Results The percent change in amiloride-sensitive Isc was 41.8 ± 5.0% in the absence versus 8.5 ± 5.5% in the presence of EGF (p = .0131). Dexamethasone increased the percent change in amiloride-sensitive Isc to 67.7 ± 12.1% in the absence and 60.3 ± 5.7% in the presence of EGF (p = .234). Despite differences in ENaC function, the amount of α-ENaC protein expression was similar in the presence or absence of EGF. However, PAR-C5 cells grown in the presence of EGF demonstrated less α-ENaC localization in the membrane portion compared with those cells grown in the absence of EGF.
Conclusion These results suggest that EGF inhibits the intracellular trafficking of α-ENaC into the plasma membranes in the PAR-C5 cell line. Since animal in utero studies suggest that EGF supplementation may be used to reverse pulmonary hypoplasia, further investigation is needed to characterize the signaling mechanisms whereby EGF and other growth factors modulate intracellular ENaC trafficking in different cell types.
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