Epithelial Na⁺ channel activity in human airway epithelial cells: the role of serum and glucocorticoid-inducible kinase 1

Abstract

Background and purpose: Glucocorticoids appear to control Na⁺ absorption in pulmonary epithelial cells via a mechanism dependent upon serum and glucocorticoid-inducible kinase 1 (SGK1), a kinase that allows control over the surface abundance of epithelial Na⁺ channel subunits (α-, β- and γ-ENaC). However, not all data support this model and the present study re-evaluates this hypothesis in order to clarify the mechanism that allows glucocorticoids to control ENaC activity.

Experimental approach: Electrophysiological studies explored the effects of agents that suppress SGK1 activity upon glucocorticoid-induced ENaC activity in H441 human airway epithelial cells, whilst analyses of extracted proteins explored the associated changes to the activities of endogenous protein kinase substrates and the overall/surface expression of ENaC subunits.

Key results: Although dexamethasone-induced (24 h) ENaC activity was dependent upon SGK1, prolonged exposure to this glucocorticoid did not cause sustained activation of this kinase and neither did it induce a coordinated increase in the surface abundance of α-, β- and γ-ENaC. Brief (3 h) exposure to dexamethasone, on the other hand, did not evoke Na⁺ current but did activate SGK1 and cause SGK1-dependent increases in the surface abundance of α-, β- and γ-ENaC.

Conclusions and implications: Although glucocorticoids activated SGK1 and increased the surface abundance of α-, β- and γ-ENaC, these responses were transient and could not account for the sustained activation of ENaC. The maintenance of ENaC activity did, however, depend upon SGK1 and this protein kinase must therefore play an important but permissive role in glucocorticoid-induced ENaC activation.

Figure 1

Prolonged (24 h) exposure to dexamethasone activates epithelial Na⁺ channels (ENaC). (A) Relationships (n= 12, C_m= 45.7 ± 5.2 pF; R_a= 22.2 ± 4.1 MΩ) between total membrane current (I_m) and holding potential (V_Hold) quantified in glucocorticoid-deprived cells during exposure to the standard extracellular solution (ECS) and after 20–30 s exposure to 10 µM amiloride (+Amil.). (B) Equivalent data from dexamethasone-treated (0.2 µM, ∼24 h) cells (n= 9, C_m= 47.6 ± 8.0 pF; R_a= 28.9 ± 3.1 MΩ). (C) Values of membrane potential (V_m) derived by analysis of the data in A and B (means ± SEM). ***P < 0.001, significant effects of amiloride, Student's paired t–test. †P < 0.05, significant effect of dexamethasone, one-way anova with Bonferroni post hoc test. (D). The amiloride-sensitive component of the total membrane current (I_Amil) was isolated by further analysis of the data in A (Gluc.-deprived) and B (Dex.-treated) and has been plotted against V_Hold.

Figure 2

Phosphorylation status of endogenous kinase substrates. Control (Cont) cells were maintained in dexamethasone-containing (0.2 µM) medium for 24 h before being used in experiments while inhibitor-treated cells were exposed to 0.5 µM PI103 (PI103, n= 11), 0.1 µM rapamycin (Rap., n= 8) or 10 µM GSK650394 (GSK, n= 4) for the final 3 h of this incubation period. Aliquots of extracted protein (40 µg) were then subject to Western analysis using antibodies against (A, B) Ser⁴⁷³-phosphorylated/total protein kinase B (P-PKB-Ser⁴⁷³/T-PKB); (C, D) Thr³⁰⁸-phosphorylated/total PKB (P-PKB-Thr³⁰⁸/T-PKB), and (E, F) Thr^346/356/366-phosphorylated/total n-myc downstream regulated gene 1 protein (P-NDRG1-Thr^346/356/366/T-NDRG1). A, C and E each show representative blots from single experiments while B, D and F present the pooled data obtained by densitometric analysis of the entire series of experiments (means ± SEM). ***P < 0.01; **P < 0.02, significantly different from control, Student's paired t-test.

Figure 3

Effects of TORIN1. Dexamethasone-treated (0.2 µM, 24 h) cells were either maintained under control conditions or exposed to TORIN1 (3–100 nM) for the final 3 h of this incubation period. Aliquots of protein extracted from control and TORIN1-treated cells (n= 5 for each) were then analysed by Western blotting using antibodies against Ser⁴⁷³-phosphorylated/total protein kinase B (P-PKB-Ser⁴⁷³/T-PKB, A, B), Thr³⁰⁸-phosphorylated/total PKB (P-PKB-Thr³⁰⁸/T-PKB, C, D) and Thr^346/356/366-phosphorylated/total protein encoded by n-myc downstream regulated gene 1 (P-NDRG1-Thr^346/356/366/T-NDRG1, E, F). A, C and E show representative blots from single experiments while B, D and F show pooled data derived from densitometric analysis of the entire series of experiments (mean ± SEM). ***P < 0.01, **P < 0.05, one-way anova with Bonferroni post hoc test.; †P < 0.05, Student's paired t–test; significant effects of TORIN1.

Figure 4

Dexamethasone-induced Na⁺ currents – effects of kinase inhibitors. Total membrane currents (I_m) were recorded both under standard conditions and after 10–30 s exposure to 10 µM amiloride. The amiloride-sensitive component of the total membrane current (I_Amil) was then isolated by digitally subtracting the current that persisted in the presence of amiloride from the corresponding record of control current, and plotted (mean ± SEM) against V_Hold. (A) Data from cells exposed to 0.2 µM dexamethasone for ∼24 h under control conditions, and from cells that had also been exposed to 0.5 µM PI103 for the final 3–4 h of this incubation period (control: n= 5, C_m= 55.3 ± 10.8 pF; R_a= 27.5 ± 11.9 MΩ; PI103-treated: n= 6, C_m= 46.2 ± 9.3 pF; R_a= 32.8 ± 8.4 MΩ). (B) Data from experiments that used an identical protocol to explore the effects of 0.1 µM rapamycin (3–4 h) upon I_Amil in dexamethasone-treated (0.2 µM, ∼24 h) cells (control: n= 4, C_m= 67.4 ± 13.4 pF; R_a= 27.8 ± 10.8 MΩ; rapamycin-treated: n = 5, C_m= 79.3 ± 19.3 pF; R_a= 30.7 ± 4.1 MΩ). (C) Analogous data showing the effects of 0.1 µM TORIN1 (control: n= 4, C_m= 39.9 ± 10.1 pF; R_a= 20.1 ± 3.9 MΩ; TORIN1-treated: n= 4, C_m= 40.5 ± 5.8 pF; R_a= 33.1 ± 6.9 MΩ). (D) Analogous data showing the effects of 10 µM GSK650394 (control: n= 5, C_m= 69.7 ± 11.7 pF; R_a= 39.4 ± 10.1 MΩ; GSK650394-treated: n= 4, C_m= 35.4 ± 2.9 pF; R_a= 39.8 ± 11.8 MΩ). (E) Pooled data showing the amiloride-sensitive currents flowing at −82 mV (I_{Amil (−82 mV)}, mean ± SEM) quantified in control (i.e. 0.2 µM dexamethasone, ∼24 h) and inhibitor-treated (3–4 h) cells. ***P < 0.001, significantly different from appropriate control, Student's t-test.

Figure 5

Effects of prolonged (∼24 h) exposure to 0.2 µM dexamethasone upon the expression of epithelial Na⁺ channel α, β and γ subunits (α-, β- and γ-ENaC). (A) Western blots showing the abundance of α-ENaC in aliquots of total (40 µg) and surface-exposed (purified from 500 µg of total protein) protein derived from glucocorticoid-deprived and dexamethasone-treated (0.2 µM, ∼24 h) cells. Analogous data for β- (B), γ-ENaC (C) and β-actin (D) are also presented. (E–G) Densitometric analyses showing the pooled results from five independent experiments. *P < 0.05; **P < 0.001; ***P < 0.005, significant effects of dexamethasone, Student's paired t-test.

Figure 6

Effects of P1103 upon α-, β- and γ-ENaC expression in dexamethasone-treated cells. Total and surface-exposed proteins were extracted from dexamethasone-treated (0.2 µM, ∼24 h) cells that had either been maintained under control conditions or exposed to 0.5 µM PI103 for the final 3 h of the incubation period. The surface-exposed proteins purified from 500 µg aliquots of total protein were fractionated (SDS-PAGE) in parallel with 40 µg aliquots of total protein, and subject to Western analysis using antibodies against α-ENaC, β-ENaC, γ-ENaC and β-actin. (A) Western blot showing data from an individual experiment; bars to the right indicate the approximate position of the appropriate molecular weight markers. (B) Densitometric analysis showing the pooled data from four independent experiments (mean ± SEM). *P < 0.05, significant effects of PI103, Student's paired t-test.

Figure 7

Effects of rapamycin upon α-, β- and γ-ENaC expression in dexamethasone-treated (0.2 µM, ∼24 h) cells. Total and surface-exposed proteins were extracted from dexamethasone-treated (0.2 µM, ∼24 h) control cells and from dexamethasone-treated cells that had been exposed to 0.1 µM rapamycin for the final 3 h of the incubation period, and analysed as described in Figure 5. (A) Western blot showing data from an individual experiment; bars to the right indicate the approximate position of the appropriate molecular weight markers. (B) Densitometric analysis showing the pooled data from four independent experiments (mean ± SEM). *P < 0.05, significant effects of rapamycin, Student's paired t-test.

Figure 8

Effects of TORIN1 upon α-, β- and γ-ENaC expression in dexamethasone-treated (0.2 µM, ∼24 h) cells. Total and surface-exposed proteins were from dexamethasone-treated (0.2 µM, ∼24 h) control cells and from cells exposed to 0.1 µM TORIN1 for the final 3 h of the incubation period were analysed as described in Figure 5 (A) Western blot showing the results of an individual experiment; bars to the right indicate the approximate position of the appropriate molecular weight markers. (B) Densitometric analysis showing the pooled data from four independent experiments (mean ± sSEM). *P < 0.05, significant effects of TORIN1, Student's paired t-test.

Figure 9

Effects of GSK650394 upon α-, β- and γ-ENaC expression in dexamethasone-treated (0.2 µM, ∼24 h) cells. Total and surface-exposed proteins were from dexamethasone-treated (0.2 µM, ∼24 h) control cells and from cells exposed to 10 µM GSK650394 for the final 3 h of the incubation period were analysed as described in Figure 5. (A) Western blot showing the results of an individual experiment; bars to the right indicate the approximate position of the appropriate molecular weight markers. (B) Densitometric analysis showing the pooled data from four independent experiments (mean ± SEM). *P < 0.05, significant effects of GSK650394, Student's paired t-test.

Figure 10

Dexamethasone causes only a transient increase in SGK1 activity. (A) Typical Western blots showing the effects of dexamethasone (0.2 µM, 0–6 h) upon the abundance of Thr^346/356/366-phosphorylated (upper panel) and total (lower panel) n-myc downstream regulated gene 1 protein (NDRG1). (B) Densitometric analysis showing the pooled data from six such experiments. (C) Typical blots showing the results of experiments that used the same protocol to explore the effects of dexamethasone (0.2 µM, 0–6 h) upon the abundance of Ser⁴⁷³-phosphorylated (upper panel) and total (lower panel) PKB. (D) Densitometric analysis showing the pooled data from six such experiments. (E) Typical Western blots showing the effects of short (3h) and long-term (∼24 h) dexamethasone exposure (0.2 µM) upon the abundance of Thr^346/356/366-phosphorylated and total NDRG1. (F) Densitometric analysis showing the pooled data from five such experiments. (G) Typical Western blots showing the effects of short- (3 h) and long-term (∼24 h) dexamethasone stimulation (0.2 µM) upon the abundance of Ser⁴⁷³-phosphorylated (upper panel) and total PKB (lower panel).; essentially identical data were obtained in four independent experiments. (H) Densitometric analysis showing the pooled data from five such experiments. All data are mean ± SEM. †P < 0.001, one-way anova with Bonferroni post hoc test; ***P < 0.001, Student's paired t-test, significant effects of dexamethasone.

Figure 11

Brief (3 h) stimulation with dexamethasone increases the surface abundance of epithelial Na⁺ channel subunits (α-, β- and γ-ENaC). (A) Typical Western blots showing the effects of dexamethasone (0.2 µM, 3 h) upon the surface abundance of epithelial Na⁺ channel α, β and γ subunits (α-, β- and γ-ENaC) both under control conditions and in the presence of 10 µM GSK650394 (GSK). (B) Densitometric analysis of the data (mean ± SEM) from four independent experiments. ***P < 0.001, significant differences between glucocorticoid-deprived and dexamethasone-stimulated cells,; †P < 0.05 significant effects of GSK650394, one-way anova with Bonferroni post hoc test.

Figure 12

Effects of brief (∼3 h) and prolonged (∼24 h) stimulation of dexamethasone upon epithelial Na⁺ channel activity. (A) Relationships between amiloride-sensitive membrane current (I_Amil, mean ± SEM) and holding potential (V_Hold) quantified in glucocorticoid-deprived cells (Dex.-free, n= 3, C_m= 47.7 ± 7.2 pF; R_a= 30.7 ± 9.1 MΩ) and in cells that had been exposed to 0.2 µM dexamethasone for ∼3h (∼3 h Dex., n= 5, C_m= 33.6 ± 2.3 pF; R_a= 30.7 ± 4.8 MΩ) or ∼24 h (∼24 h Dex., n= 5,C_m= 48.0 ± 3.0pF;R_a= 19.3 ± 2.1 MΩ). (B) Values of I_{Amil (−82 mV)} derived by further analysis of these data. †P < 0.05, significantly different from corresponding values in glucocorticoid-free and in ∼3 h Dex.-treated cells, one-way anova with Bonferroni post hoc test.