Ouabain exerts biphasic effects on connexin functionality and expression in vascular smooth muscle cells

Abstract

1. We have compared the effects of ouabain on the maintenance of gap junctional communication in rat aortic A7r5 smooth muscle cells, monkey COS-1 fibroblasts and human HeLa epithelial cells. 2. Ouabain (1 mM) interrupted dye coupling between confluent A7r5 cells within approximately 1 h, and high concentrations of ouabain were similarly required to reduce coupling between COS-1 cells selected to express the rat alpha1 Na+/K+-ATPase subunit, which is ouabain resistant. By contrast, low concentrations of ouabain (1-10 microM) attenuated dye transfer in wild-type COS-1 and HeLa cells, whose endogenous alpha1 subunits possess relatively high affinity for the glycoside (Ki approximately 0.3 vs approximately 100 microM) Ouabain-induced reductions in dye transfer therefore correlated with the ability of the glycoside to bind to the Na+/K+-ATPase isoenzymes expressed in these different cell lines. 3. No consistent relationship between inhibition of intercellular dye transfer and secondary changes in [Ca2+]i or pHi could be identified following incubation with ouabain. 4. In separate experiments, the effects of ouabain on real-time trafficking of connexin protein were monitored by time-lapse microscopy of A7r5 cells transfected to express a fluorescent Cx43-green fluorescent protein (GFP) and the ability of the glycoside to modulate endogenous expression of connexins (Cx) 40 and 43 evaluated in A7r5 cells by immunochemical and Western blot analysis. 5. Ouabain (1 mM) depressed vesicular trafficking of Cx43-GFP after approximately 1 h, and caused a time-dependent loss of endogenous Cx40 and Cx43 protein that was first evident at 2 h and almost complete after 4 h. These effects of ouabain on Cx expression were reversed approximately 90 min following washout of the glycoside. 6. We conclude that ouabain exerts biphasic effects on the intercellular communication that involve an initial decrease in gap junctional permeability followed by a global reduction in the expression of Cx protein. Further studies are necessary to establish to what extent these actions of ouabain reflect inversion of the normal [Na+]i/[K+]i ratio and/or conversion of the Na+/K+-ATPase into a general signal transducer that regulates downstream protein synthesis.

Figure 1

Inhibitory effects of ouabain on dye coupling in A7r5 cells, HeLa cells expressing Cx43-GFP, wild-type COS-1 cells and ouabain-resistant COS-1 cells. Results are expressed as the percentage of cells transferring Lucifer yellow to 0–4, 5–10 and >10 neighbours (for A7r5 and COS cultures) and to 0–2, 3–4 and >5 neighbours in HeLa cultures (because of lower dye coupling). Experiments were performed in triplicate with >50 injections per plate, and this protocol repeated three times. Data are shown as mean± s.e.m. Asterisks indicate significant effects of ouabain on the highest level of coupling compared to control *P<0.05, ** P<0.005.

Figure 2

Effects of ouabain on intracellular [Ca²⁺] and pH. (a) Fura 2 fluorescence before and during exposure of HeLa, wtCOS, oubRCOS and A7r5 cells to 100 μM ouabain. A7r5 cells were also treated with 1 mM ouabain. (b) Fura 2 fluorescence in wtCOS and oubRCOS cells treated with 80 μM ATP. (c) BCECF fluorescence in wtCOS, oubRCOS and A7r5 cells under control conditions and during exposure to 1 mM ouabain. (d) BCECF fluorescence in wtCOS, oubRCOS and A7r5 cells treated with 10 mM NH₄Cl for 5 min followed by washout and a further 5 min monitoring. Results are expressed as the 340 : 380 nm ratio for Ca²⁺ measurements and 480 : 440 nm ratios for pH measurements. In all, 10 cells were analysed per field of view and results averaged (n=3 cultures for each experimental group).

Figure 3

(a) Integrity of gap junction plaques in A7r5 cells fixed and stained for Cx43 following incubation with 100 μM–1 mM ouabain for 1–4 h as indicated. Bar=10 μm; arrows indicate plaques at points of cell–cell contact. (b) Plaque integrity quantified by analysis of Cx43 fluorescence at the plasma membrane following the various treatments subtracted from background fluorescence. Results are given as mean relative fluorescence ±s.e.m. Asterisk indicates a significant difference from control (n=3, P<0.01).

Figure 4

Western blot analysis of Cx43 and Cx40 expression profiles in A7r5 cell lysates following treatment with 100 μM or 1 mM ouabain for 1–4 h and recovery of expression after 2 h treatment. Equivalent amounts of protein (50 μg) were loaded onto the gels and detected using a primary monoclonal antibody to α-tubulin as internal standard (top panels) and a polyclonal antibody against Cx43 or Cx40 (middle panels) as indicated. (i–iii): Densitometric analysis of the mean Cx expression ±s.e.m. Asterisk indicates a significant difference from control (n=3, P<0.05). Each blot was standardized as a percentage of the control signal.

Figure 5

(a) Cx43-GFP trafficking events in A7r5 cells in the presence of external Ca²⁺ (1.3 mM). Images were recorded every 15 s over a 15 min time period. To analyse the data, time points 1 (15 s, t1), 30 (7.5 min, t2) and 60 (15 min, t3) were assigned the colours red (i), green (ii) and blue (iii), respectively. Merging these three images allows the assessment of vesicular trafficking and movement of the cell (b) under control conditions or (c) following treatment for 45 min with 1 mM ouabain. (d) Scatter plots of the extent of colocalization of the red and blue channels in the absence (i) and presence (ii) of ouabain, where the line of identity is indicated. (e) Cx43-GFP trafficking events in A7r5 cells in the vicinity of the plasma membrane observed in Ca²⁺-free medium: (i) merged image for time 1–15 min, (ii) 20–35 min and (iii) 45–60 min. These findings indicate that imaging of the same cell for prolonged periods did not result in significant photobleaching and that intracellular trafficking was maintained, thus excluding secondary effects from laser damage and focus movement. Bar=10 μm. (f) Effects of 1 mM ouabain on Cx43-GFP trafficking in Ca²⁺-free medium, with merged images illustrating trafficking (i) before treatment, (ii) 35–45 min following addition of ouabain and (iii) 60–75 min following addition of ouabain. Bar=5 μm.