Modulation of sodium transport in alveolar epithelial cells by estradiol and progesterone

Abstract

The effects of estradiol (E2) and progesterone (P) on alveolar epithelial Na+ transport were studied in isolated alveolar epithelial cells from 18- to 19-d GA rat fetuses, grown to confluence in serum-free media supplemented with E2 (0-1 μM) and P (0-2.8 μM). Short-circuit currents (ISC) were measured, showing an increase by E2 and P in a dose-dependent manner. The Na,K-ATPase subunits -α1 and -β1 were detected by Western blotting, but total expression was not significantly altered. Furthermore, all three epithelial Na+ channel (ENaC) subunits -α, -β, and -γ were detected, with trends toward a higher expression in the presence of E2 and P. Real-time PCR revealed an increase of α- and β-ENaC expression but no alteration of γ-ENaC. In addition, the mRNA expression of cystic fibrosis transmembrane conductance regulator (CFTR) and Na,K-ATPase-β1 subunit were elevated in the presence of E2 and P. Single-channel patch clamp analysis demonstrated putative highly selective and nonselective cation channels in the analyzed cells, with a higher percentage of responsive patches under the influence of E2 and P. We conclude that E2 and P increased Na+ transport in alveolar epithelial cells by enhancing the expression and activity of ENaC and Na,K-ATPase.

Figure 1

Effect of E2 and P exposure during cell culture on I_SC in rat FDLE cells. Baseline was the I_SC after mounting the monolayers in the Ussing chambers, I_amil the current reduction by amiloride (10 μM), and I_ouab the ouabain- (1 mM) sensitive current. Media 1 to 6 refer to the concentrations of E2 and P in the culture medium as outlined in Table 1. Error bars represent SEM, *p < 0.05 by Dunnett's post hoc test. Medium 1 (▪), medium 2 (□), medium 3 (), medium 4 (⊠), medium 5 (), and medium 6 ().

Figure 2

(A) Effect of E2 and P exposure during cell culture on ouab_max in rat FDLE cells. Baseline was the I_SC after mounting the monolayers in the Ussing chambers, I_amph the current after adding 10 μM amphotericin B to the apical compartment, and ouab_max the current reduction caused by 1 mM ouabain. (B) Effect of E2 and P exposure during cell culture on amil_max in rat FDLE cells. The monolayers were subjected to a 145:5 apical to basolateral Na⁺ gradient, the basolateral membrane permeabilized by 100 μM amphotericin B, and 10 μM amiloride was given into the apical compartment at the maximum current increase. *p < 0.05 by Dunnett's post hoc test. Medium 1 (▪), medium 2 (□), medium 3 (), medium 4 (⊠), medium 5 (), and medium 6 ().

Figure 3

(A) Single-channel recordings of rat FDLE cells subjected to different concentrations of E2 and P. HSC represents a putative highly-selective cation channel of ENaC with a unitary conductance of ∼4 pS. NSC refers to a putative nonselective cation channel of ENaC with a unitary conductance of ∼23 pS. (B) Comparison of the percentage of responsive patches showing single-channel activity in cells incubated with different concentrations of E2 and P (n = 20 for medium 1, n = 19 for medium 5, and n = 11 for medium 6). *p < 0.05 by Fisher's exact test. Medium 1 (▪), medium 5 (), and medium 6 ().

Figure 4

The RT-PCR analysis of the mRNA expression level of the α-ENaC subunit (A), the β-ENaC subunit (B), and the γ-ENaC subunit (C) in rat FDLE cells subjected to different concentrations of E2 and P. *p < 0.05 by Dunnett's post hoc test. Medium 1 (▪), medium 4 (⊠), medium 5 (), and medium 6 ().

Figure 5

The RT-PCR analysis of the mRNA expression level of the Na,K-ATPase-β₁ subunit (A), CFTR (B), VEGF-A (C), and ER-β (D) in rat FDLE cells subjected to different concentrations of E2 and P. *p < 0.05 by Dunnett's post hoc test. Medium 1 (▪), medium 4 (⊠), medium 5 (), and medium 6 ()..

Figure 6

(A) Western blot of α-, β-, and γ-ENaC subunits in FDLE cells grown in the presence of different concentrations of E2 and P. The numbers on top refer to the E2 and P concentrations in the culture medium as outlined in Table 1. KNRK refers to a commercial rat kidney cell lysate, serving as a positive control. (B) Densitometric analysis of the Western blots of α-, β-, and γ-ENaC subunits. Values are shown as percentage of the density obtained from cells grown in medium 1 (all bars, n = 6). Medium 4 (⊠), medium 5 (), and medium 6 ().

Figure 7

(A) Western blot of Na,K-ATPase-α₁ and -β₁ subunits in FDLE cells grown in the presence of different concentrations of E2 and P. B is the positive control obtained from rat brain tissue. (B) Densitometric analysis of the Western blots of Na,K-ATPase-α₁ and -β₁ subunits. Values are shown as percentage of the density obtained from cells grown in medium 1 (n = 6 for α₁ and n = 10 for β₁). Medium 2 (□), medium 4 (⊠), and medium 6 ().