Concentration-dependence of 17β-estradiol effects on ASL height in NuLi-1 and CuFi-1 epithelial monolayers and the effect of 1 nM E2 on ASL height of primary CF bronchial epithelial cells.

<p>Epithelial cells were stained with Calcein Green and the ASL stained using dextran conjugated Texas Red™ fluorochrome 24 h before estrogen treatment. Panel A shows representative z-plane sections of NuLi-1 (left) and CuFi-1 (right) cells under basal conditions (top) and after 30 mins treatment with 1 nM E2 (bottom). Mean changes in ASL height in control conditions or following treatment with different concentrations of estrogen are shown in panel B for NuLi-1 (solid line) and CuFi-1 (dashed line) cells (n≥4, Error bars reflect standard error of the mean, ANOVA, ** p<0.01, *** p<0.001, compared to control conditions; δ p<0.05, δδ p<0.01, δδδ p<0.001, difference between NuLi-1 and CuFi-1 cells at the indicated estrogen concentration, ns: non significant). Representative z-plane sections of CF primary bronchial epithelial cells under basal conditions (left) and after 30 mins treatment with 1 nM E2 (right) (panel C) and mean changes in ASL height in primary bronchial epithelial cells of 3 CF female children (panel D) (n = 3, paired t-test, ** p<0.01)</p

E2 increases Na⁺ absorption in CuFi-1 cells.

<p>CuFi-1 cells were cultured on collagen-coated culture inserts. The monolayers were allowed to reach a transepithelial resistance of 1,000 Ω.cm⁻² or above prior the experiment. A. In the Ussing chambers, the cells were allowed to stabilize and were then treated basolaterally with E2. The changes in short-circuit current were measured in response to amiloride (10 µM), ATP (100 µM), forskolin (10 µM) and bumetanide (10 µM). Typical traces of Isc in vehicle and E2 treated CuFi-1 cells are shown. B. ASLh was measured in CuFi-1 cells in response to estrogen and NKCC1 inhibition. CuFi-1 monolayers were treated with E2 (1 nM), Bumetanide (Bum, 10 µM) or pretreated with estrogen before bumetanide was added (E2 + Bum) (n = 5, error bars reflect standard error of the mean, ANOVA, * p<0.05, ** p<0.01, ns: non significant). C. Amiloride-sensitive current in CuFi-1 cells treated with E2 or vehicle. Amiloride was added after 30 minutes (n = 6, Error bars reflect standard error of the mean, Student's t test, * p<0.05). D. ASLh measurement in CuFi-1 cells after treatment with E2 (30 min, 1 nM), Amiloride (Amil, 20 min, 300 µM) or pretreated with E2 and then treated with Amiloride (E2 + Amil) (n = 7, Error bars reflect standard error of the mean, ANOVA, * p<0.05, *** p<0.001). F. Ouabain–sensitive current in CuFi-1 cells mounted in Ussing chambers and bathed in modified low Na⁺ Krebs buffer in which NaCl was replaced by NMDG-Cl⁻. After the current stabilised, the apical membrane was permeabilized and the cells were treated with E2 or with vehicle and Ouabain (100 mM) was added to the basolateral chamber after 30 minutes (n = 4, Error bars reflect standard error of the mean, Student's t test, * p<0.05).</p

E2 inhibits K⁺ recycling in normal and CF bronchial cell lines.

<p>A. NuLi-1 (grey bars) and CuFi-1 (white bars) epithelial monolayers were stained with Calcein Green and ASL was stained with dextran conjugated to Texas Red^TM fluorochrome and ASL height was measured after treatment with E2 (30 min, 1 nM), and a cocktail of BaCl₂ and TEA to inhibit potassium channels (B/T 30 min, BaCl₂ 10 mM, TEA 1 mM) or pretreated with E2 and then treated with the K⁺ channels cocktail inhibitor (E2 + B/T) (n = 5, Error bars reflect standard error of the mean, ANOVA, * p<0.05, ** p<0.01). B. NuLi-1 (grey bars) and CuFi-1 (white bars) epithelial monolayers were treated with E2 (30 min, 1 nM), and the KCNQ1 channel specific inhibitor HMR1556 (HMR, 30 min, 1 µM) or pretreated with E2 and then treated with HMR1556 (E2 + HMR) (n≥3, Error bars reflect standard error of the mean, ANOVA, ** p<0.01).</p

Effect of the Estrogen Dendrimer Conjugate on ASL height.

<p>NuLi-1 and CuFi-1 epithelial monolayers were treated with 1 nM E2, or EDC at concentrations providing 1 nM estrogen equivalents, or empty dendrimer at the concentration that matched that in EDC at a 1 nM estrogen equivalent. Panel A shows typical z-plane sections in control conditions or after treatment with E2, EDC or the empty dendrimer (D). The concentrations given for the EDC and the empty dendrimer are in equivalent E2 concentrations. Panel B shows the mean changes in ASL height in control conditions or following exposure to E2, EDC or the empty dendrimer (n≥4, Error bars reflect standard error of the mean, ANOVA, * p<0.05, ** p<0.01).</p

E2 inhibits Cl⁻ secretion in NuLi-1 cells.

<p>NuLi-1 cells were cultured on collagen-coated culture inserts. The monolayers were then allowed to reach a transepithelial resistance of 1,000 Ω.cm⁻² or above prior the experiment. A. When mounted in the Ussing chambers, the cells were allowed to stabilize and were then treated basolaterally with E2. The changes in short-circuit current were then measured in response to amiloride (10 µM), ATP (100 µM), forskolin (10 µM) and bumetanide (10 µM). Typical traces of Isc in control and E2 treated NuLi-1 cells are shown. B. Summary of the amiloride short circuit current in control and E2 treated cells. Panel C shows ASLh measurement in NuLi-1 in response to estrogen and ENaC inhibition. NuLi-1 monolayers were treated with E2 (1 nM), Amiloride (Am, 300 µM) or pretreated with estrogen and then treated with amiloride (E2 + Am) (n≥3, error bars reflect standard error of the mean, ANOVA, ** p<0.01, *** p<0.001). Panels D and F show ASLh measurement in NuLi-1 epithelial cells after treatment with (D) E2 (30 min, 1 nM), Bumetanide (Bum, 30 min, 10 µM) or pretreated with E2 and then treated with Bumetanide (E2 + Bum) or (F) E2 (30 min, 1 nM), Forskolin (FSK, 30 min, 10 µM) or pretreated with E2 and then treated with Forskolin (E2 + FSK) (n≥5, Error bars reflect standard error of the mean, ANOVA, * p<0.05, ** p<0.01, *** p<0.001). Panel E shows bumetanide-sensitive current in NuLi-1 cells treated with E2 or with vehicle. Bumetanide was added after 30 minutes (n≥5, Error bars reflect standard error of the mean, Student's t test, * p<0.05).</p

E2-induced PKCδ activation is responsible for the ASL height decrease in CuFi-1 cells.

<p>NuLi-1 (grey bars) and CuFi-1 (white bars) cells were treated basolaterally with E2 (1 nM) for 30 mins. After hormone treatment, cells were washed and total protein extracts were prepared in order to measure the level of phosphorylation of PKCα/βII and PKCζ/λ by immunoblot. Representative Western blots are shown in panel A and densitometric quantitation of phospho-PKCα/βII / β-actin and phospho-PKCζ/λ / β-actin are shown in panel B (n = 4, Error bars reflect standard error of the mean, Student’s t-test). B. NuLi-1 and CuFi-1 cells were treated basolaterally with E2 (1 nM) for the indicated times. After treatment, cells were washed and total protein extracts were prepared in order to measure the level of phosphorylation of PKCδ/θ. Representative images are shown in panel C and mean phosphorylation state of PKCδ/θ changes in control conditions, or following treatment with estrogen at different time points, are shown in panel D (n≥4, Error bars reflect standard error of the mean, ANOVA, * p<0.05). E. ASL height was measured in CuFi-1 (white bars) and NuLi-1 (grey bars) monolayers in response to estrogen and PKCδ inhibition. NuLi-1 and CuFi-1 monolayers were treated with E2 (1 nM) or pretreated with rottlerin (5 µM) and then treated with E2 (n = 5, Error bars reflect standard error of the mean, ANOVA, * p<0.05).</p