Schematic representation of the signaling pathways affected by low concentration of ouabain to regulate TJ protein expression and function. Ouabain (red silhouette) activates ERK1/2 via c-Src–dependent and -independent (green square) pathways, which in turn regulate the expression and localization of cln-1 and -4. The ouabain receptor is likely to be Na⁺,K⁺-ATPase located on the basolateral surface. Regulation of cln-1 depends on c-Src and ERK1/2. In turn, regulation of ionic permeability or TER depends on c-Src and partially on ERK1/2. The c-Src–independent activation of ERK1/2 leads to the regulation of cln-4 and nonionic permeability or J_DEX.

c-Src and ERK1/2 modulate the effect of ouabain on TER and J_DEX. (A) TER across monolayers of MDCK cells incubated with or without ouabain (n = 23), in the absence (columns 1 and 2) or presence of PP2 (n = 18, columns 3 and 4) or PD98059 (n = 17 to 18, columns 5 and 6). Values are mean ± SEM. (B) J_DEX was determined under analogous conditions. For control conditions or in the presence of ouabain, n = 14–16; for PP2, n = 8–9; for PD98059, n = 8–10. Values are mean ± SEM.

c-Src and ERK1/2 modulate the effect of ouabain on claudin expression. (A) Representative image of a Western blot of cln-1 in extracts from cells incubated with or without ouabain for 2 d in the presence or absence of PP2 or PD98059. This and the following images are composed by a set of upper bands corresponding to claudins, and lower ones to actin, which were obtained from the same membrane after stripping and blotting against this protein. (B) Statistical analysis of Western blots for cln-1. Values are mean ± SEM, n = 6 for each condition. (C) Western blot of cln-4 of cells incubated under control conditions or in the presence of ouabain for 3 d, with or without PP2 or PD98059. (D) Statistical analysis of Western blots for cln-4. Values are mean ± SEM. n = 8 for control condition (with or without ouabain); n = 8 for PP2; n = 6 for PD98059.

Ouabain at 10 nM stimulates tyrosine phosphorylation of c-Src and ERK1/2. (A) Ouabain was added from the apical (ap, second column) or basolateral (bl, third column) side of the monolayer, and TER was measured on the second day. Values are mean ± SEM, n = 3 for each time point at each condition. (B) Representative images of Western blots for c-Src phosphorylated at Y-418 (p-Y418) and total c-Src in the presence or absence of ouabain for 3 min. Values are mean ± SEM, n = 6 for each condition. This and the following images are composed by a set of upper bands corresponding to c-Src Y418, and lower ones to total c-Src, which were obtained from the same membrane after stripping and blotting against this protein. (C) Western blots and statistical analysis of ERK1/2 phosphorylation (pERK1/2) in the presence and absence of ouabain (n = 14) and in the presence of PP2 (n = 7) or PD98059 (n = 3). Values are mean ± SEM. This and following images are composed as explained in C, except that upper bands correspond to phosphorylated ERK1/2, and lower ones to total ERK1/2, which were obtained from the same membrane after stripping and blotting against this protein.

Ouabain at 10 nM increases TER and decreases nonionic permeability in MDCK cells without blocking ion pumping. (A) J_K is the influx of K⁺ as measured with ⁸⁶Rb in control cells (○). Cells were treated with 1 μM ouabain for 3 h added at day 1 (■) or 10 nM ouabain added at time 0 (●). Values are mean ± SEM, n = 6 for each time point at each condition. (B) Effect of 10 nM ouabain on K_c, the cellular content of K⁺. Values are mean ± SEM, n = 12 for each experimental point. (C) TER as measured in monolayers incubated in the absence or presence of ouabain (10 or 50 nM and 1 μM, ● and ■, respectively) for 3 d. Values are mean ± SEM. For values at time 0, n = 35; for the other experimental time points, n = 4 for each time point at each condition. (D) Unidirectional flux of 3 kDa dextran (J_DEX) through the monolayer under control conditions or upon treatment with 10 or 50 nM or 1 μM ouabain. Values are mean ± SEM, n = 9–17 for each time point.

Ouabain at 10 nM modifies the protein content and distribution of claudins. (A) Cell extracts from MDCK cells treated with or without ouabain for 1, 2, or 3 d were analyzed by Western blot. Values of the curves are mean ± SEM. For cln-1, n = 4, except for time = 0, where n = 3 for each condition and each point; for cln-2, n = 3–6; and for cln-4, n = 5, except for time = 0 for each time point at each condition. (Insets) Representative images of cln-1 and -2 as observed on the second day and cln-4 on the third day. This and the following images are composed by a set of upper bands corresponding to claudins, and lower ones to actin, which were obtained from the same membrane after stripping and blotting against this protein. (B) Expression of occludin over 3 d (blot image taken on the second day). Values are mean ± SEM, n = 4 for each time point at each condition. The representative image is a composite one, as in the case of claudins. (C) Representative images of claudins 1, 2, and 4, as well as (D) occludin, as observed by confocal microscopy on the second day after treatment with or without 10 nM ouabain.