A glucocorticoid‐induced Na⁺ conductance in human airway epithelial cells identified by perforated patch recording

Abstract

The perforated patch recording technique was used to investigate the effects of dexamethasone (0.2 microm, 24-30 h), a synthetic glucocorticoid, on membrane conductance in the human airway epithelial cell line H441. Under zero current clamp conditions this hormone induced amiloride-sensitive depolarization of the membrane potential (V(m)). Lowering external Na(+) to 10 mm by replacing Na(+) with N-methyl-d-glucammonium (NMDG(+)) also hyperpolarized the dexamethasome-treated cells, whilst replacing Na(+) with Li(+) caused a small depolarization. Although V(m) was insensitive to amiloride in control cells, NMDG(+) substitution caused a small hyperpolarization and so an amiloride-insensitive cation conductance is present. Replacing Na(+) with Li(+) had no effect on V(m) in such cells. Voltage clamp studies of dexamethasone-treated cells showed that the amiloride-sensitive component of the membrane current reversed at a potential close to the Na(+) equilibrium potential (E(Na)), and replacing Na(+) with K(+) caused a leftward shift in reversal potential (V(Rev)) that correlated with the corresponding shift in E(Na). Lowering [Na(+)](o) to 10 mm, the concentration in the pipette solution, by substitution with NMDG(+) shifted V(Rev) to 0 mV, whilst replacing Na(+) with Li(+) caused a rightward shift. Exposing dexamethasone-treated cells to a cocktail of cAMP-activating compounds (20 min) caused a approximately 2-fold increase in amiloride-sensitive conductance that was associated with no discernible change in ionic selectivity and an 18 mV depolarization. Dexamethasone thus induces the expression of a selective Na(+) conductance with a substantial permeability to Li(+) that is subject to acute regulation via cAMP. These data thus suggest that selective Na(+) channels underlie cAMP-regulated Na(+) transport in airway epithelia.

The regulation of selective and nonselective Na+ conductances in H441 human airway epithelial cells

“…14). Subsequently, lowering [Na ϩ ] o to 10 mM (NMDG ϩ substitution), in the continued presence of 10 M amiloride, caused further hyperpolarization to Ϫ68.1 Ϯ 2.4 mV (P Ͻ 0.05), which also accords with the earlier data (14), and analysis of I Na(Ϫ82 mV) (see METHODS) indicated that amiloride caused 92.8% Ϯ 1.2% inhibition (P Ͻ 0.005) of I Na under these conditions (Fig. 9B).…”

“…Analysis of the present data showed that V m was normally Ϫ3.7 Ϯ 3.8 mV, although, as reported previously (14), there was variability between the values of V m measured in different cells (range: Ϫ20.7 to 20.2 mV), and analysis of the data from all experiments included in the present study in which membrane currents were recorded from such groups of cells showed that the mean value of V m was approximately Ϫ20 mV. This is very similar to the previously reported value (14). Under these conditions, amiloride (10 M, 30 -45 s) hyperpolarized V m to Ϫ56.7 Ϯ 1.5 mV (P Ͻ 0.005) and reduced the inward currents flowing at negative values of V Hold .…”

“…Standard, serial culture techniques were used to maintain H441 cells in RPMI 1640 culture medium supplemented with FBS (8.5%), newborn calf serum (8.5%), glutamine (2 mM), transferrin (5 g/ml), selenium (5 ng/ml), and an antibiotic/antimycotic mixture (Sigma, Poole, Dorset, United Kingdom). This differs from the medium used in our earlier studies (11,14,24,45,56) as it contains no insulin. For experiments, cells removed from culture flasks using trypsin/EDTA were plated onto glass coverslips and incubated (24 -48 h) in medium identical to that described above except that the serum components were replaced with FBS (8.5%) that had been dialyzed to remove hormones/growth factors.…”

“…The dexamethasone-induced depolarization thus reflects a rise in G Na (see also Ref. 14) with no change in G Cl . Figure 6A shows values of I Na derived from these data (see METHODS) and plotted against V Hold and also includes equivalent data from hormone-deprived cells (Fig.…”

“…These experiments showed that 20 -40% of the cells expressed sufficient GFP to be visible under epifluorescence. We often saw groups of 3-5 cells that included only a single transfected cell, and, since earlier work had indicated that such cells are electrically interconnected (14), we decided to record currents from single cells to be sure that data were derived from transfected cells. Experiments in which membrane currents were recorded from such GFP-expressing cells (identified by epifluorescence) showed that heterologous expression of this marker protein did not effect the electrical properties of hormone-deprived cells.…”