Fig 1.
Alveolar epithelial Na+ transport.
Na+ enters the apical membrane of alveolar epithelial cells through ENaC and is actively extruded by the Na,K-ATPase in the basolateral membrane compartment, whereas K+ ions are recycled by K+ channels. Thereby pulmonary fluid is absorbed as it passively follows the osmotic gradient generated by vectorial Na+ transport.
Table 1.
Primer sequences.
Fig 2.
Female FDLE cells display an increased Na+ transport.
(A) Ibase was the ISC after mounting the monolayers in the Ussing chambers, the amiloride-sensitive (Iamil) current represents the current reduction induced by amiloride (10 μM). Ibase and Iamil, but not the amiloride-insensitive ISC, were significantly higher in FDLE cells of female origin (n = 45/56; mean + SEM; ***p<0.001 by T-test). (B) Representative current tracing of FDLE monolayers. (C) Vbase and Vamil were significantly higher in FDLE cells of female origin (n = 45/56; mean + SEM; *p<0.05 by T-test).
Fig 3.
Female FDLE monolayers show a higher ENaC and Na,K-ATPase activity in permeabilized membrane measurements.
After the ISC reached a plateau (Ibase), amphotericin B was used to permeabilize either the basolateral (100 μM) or the apical (10 μM) membrane. At the maximal current increase either amiloride (10 μM) or ouabain (1 mM) were applied and amilmax (A) or ouabmax (C) determined. Ibase, amilmax and ouabmax were significantly higher in FDLE cells of female origin (A: n = 35/32; C: n = 53/51; mean + SEM, *p<0.05, **p<0.01 and ***p<0.001 by T-test). (B/D) Representative current tracings of permeabilized membrane measurements.
Fig 4.
mRNA expression of ENaC and Na,K-ATPase subunits is higher in female FDLE cells.
mRNA expression of (A) α-ENaC, β-ENaC and γ-ENaC and (B) Na,K-ATPase-α1, Na,K-ATPase-α2 and Na,K-ATPase-β1 was significantly higher in female compared with male FDLE cells after 4 days in culture (n = 7–12; mean + SEM; **p<0.01 and ***p<0.001 by T-test).
Fig 5.
mRNA expression of ER-β and PR-A/B is higher in female FDLE cells.
mRNA expression of ER-β and PR-A/B was significantly higher in female compared with male FDLE cells after 4 days in culture (n = 7–8; mean + SEM; *p<0.05 and **p<0.01 by T-test).
Fig 6.
mRNA expression of ENaC and Na,K-ATPase subunits, ER-β and PR-A/B is higher in non-cultured female FDLE cells.
mRNA expression determined directly after cell isolation was significantly higher in female compared with male fetuses for (A) α-ENaC, β-ENaC and γ-ENaC, (B) Na,K-ATPase-α1, Na,K-ATPase-α2 and Na,K-ATPase-β1, (C) ER-β and PR-A/B (n = 6–10; mean + SEM, **p<0.01 and ***p<0.001 by T-test).
Fig 7.
Inhibition of ER-β abolishes the sex differences in Na+ transport and female FDLE cells are more responsive to E2 stimulation.
(A) Ibase and Iamil were significantly higher in FDLE cells of female origin, while PHTPP abolished the sex difference (n = 23–28; mean + SEM; *p<0.05; **p<0.01 by T-test). (B) Vbase and Vamil were significantly higher in FDLE cells of female origin, while PHTPP abolished the sex difference (n = 23–28; mean + SEM; *p<0.05 by T-test). (C) PHTPP decreased Rte, but no Rte differences were observed between male and female FDLE cells. (D) PDGF-A mRNA expression in response to different E2 concentrations in male and female FDLE monolayers. Female cells are more responsive to E2 since PDGF-A mRNA expression was significantly increased in females, whereas PDGF-A mRNA expression was not affected by E2 in male FDLE cells. (♂: relative difference to male control; ♀: relative difference to female control; n = 6; mean + SEM; *p<0.05 by ANOVA with Dunnett’s post hoc test).
Fig 8.
FDLE cell number per fetus is higher and lung W/D weight ratio lower in female rat pups.
(A) FDLE cell numbers were significantly higher in females (n = 13 cell isolations; mean + SEM; *p<0,05 by T-test). (B) Lung W/D weight ratio was significantly lower in female fetal rats (n = 18/18; mean + SEM, *p<0.05 by T-test) and female newborn rats (n = 9/9; mean + SEM, **p<0.01 by T-test).