Figure 4. E2 increases Na⁺ absorption in CuFi-1 cells.

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).