At birth, the alveolar epithelium functionally switches from fluid secretion to absorption, a process that is critical to initiate gas exchange in the newborn. The levels of various hormones, including catecholamines, thyroid hormones, mineralcorticoids and glucocorticoids, increase late in gestation potentially augmenting the expression of proteins involved in the Na⁺ transport pathway. The epithelial Na⁺ channel α-ENaC constitutes a rate-limiting step for Na⁺ transport across airway epithelia and is a principle target for hormonal regulation. In this study we investigated the role of glucocorticoids as physiological regulators of amiloride-sensitive Na⁺ transport utilising the rat fetal distal lung (FDLE) cell as an alveolar cell model. However, the majority of data published so far have been collected from rat FDLE cells cultured in serum containing media. Since serum itself contains a variety of factors known to influence cell growth, care was taken to avoid supplements containing hormones or growth factors. The initial aim of this study was to develop a fully defined culture medium, which supports the growth of FDLE cells in the absence of serum.

FDLE cells were isolated from fetuses (gestational age 19 days) that were removed from anaesthetised rats (3 % halothane, killed before regaining consciousness) following a method that has been previously described (Ramminger et al. 2000). Cells were plated onto Transwell-col membranes in a minimally defined serum-free media (MDSF) consisting of DMEM and Ham’s F12 nutrient mixture in a 1:1 ratio that was supplemented with 1.25 mg ml^-1 BSA, 2.0 mM glutamine and antibiotic. Once seeded, cells were incubated at either the fetal P_O2 (23 mmHg) or the adult alveolar P_O2 (100 mmHg) where they formed resistive monolayers within 48 h.

Monolayers that had been maintained at the adult alveolar P_O2 of 100 mmHg (48 h) exhibited an amiloride-sensitive short-circuit current (I_SC) that was ~2.8 fold greater (P < 0.02, Student’s t test) than that of cells maintained at the fetal P_O2 of 23 mmHg (23 mmHg: n = 10, 3.3 ± 0.5 µA cm^-2; 100 mmHg: n = 10, 7.8 ± 0.6 µA cm^-2; mean ± S.E.M.). Interestingly, FDLE cells that were maintained in the absence of any hormones or growth factors displayed amiloride-sensitive I_SC that was sensitive to P_O2. Despite this, the cells maintained in the serum-free media were essentially insensitive to isoprenaline, an agent known to stimulate Na⁺ transport in this cell type (Ramminger et al. 2000). Treatment with the glucocorticoid dexamethasone (dex, 200 nM, 24 h) increased the amiloride-sensitive I_SC (n = 10) in cells maintained at both fetal (P < 0.01) and adult alveolar (P < 0.01) P_O2 levels, although the evoked rise in I_SC was greater in the cells grown at fetal P_O2 (23 mmHg: control I_SC 2.6 ± 0.5 µA cm^-2, dex I_SC 4.2 ± 0.7 µA cm^-2; 100 mmHg: control I_SC 7.6 ± 0.8 µA cm^-2, dex I_SC 8.9 ± 0.2 µA cm^-2). Dexamethasone, however, failed to restore sensitivity (n = 6) to the β-adrenoceptor agonist isoprenaline. Therefore in rat FDLE cells, the P_O2-evoked rise in amiloride-sensitive I_SC is not dependent upon the presence of hormones or growth factors, whereas sensitivity to isoprenaline requires the presence of an unknown factor other than dexamethasone.

The authors thank The Wellcome Trust for their financial support.

Ramminger, S.J., Baines, D.L., Olver, R.E. & Wilson, S.M. (2000). J. Physiol. 524, 539-547. abstract