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

• Published in: British journal of pharmacology Vol. 140; no. 7; pp. 1261 - 1271
• Main Authors: Martin, Patricia E M, Hill, Nathan S, Kristensen, Bo, Errington, Rachael J, Griffith, Tudor M
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.12.2003; Nature Publishing
• Subjects: Animals; Aorta - cytology; Biological and medical sciences; Blotting, Western; Calcium - analysis; Cell communication; Cell Line; Cell Survival; Chlorocebus aethiops; Connexins - drug effects; Connexins - genetics; Connexins - metabolism; COS Cells; EDHF; Fluorescent Dyes; Fura-2; gap junctions; Gap Junctions - drug effects; Gap Junctions - metabolism; Glycosides - pharmacology; HeLa Cells; Humans; Hydrogen-Ion Concentration; Immunohistochemistry; Kinetics; Medical sciences; Muscle, Smooth, Vascular - cytology; Muscle, Smooth, Vascular - drug effects; Ouabain - pharmacology; Pharmacology. Drug treatments; Rats; Sodium-Potassium-Exchanging ATPase - metabolism; Connexin; gap junctions; Cardiotonic agent; Ouabain; Cell junction; Smooth muscle; Cell communication; Glycoside; EDHF; Gap junction
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0705556

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. Ouabain (1 mM) interrupted dye coupling between confluent A7r5 cells within ∼1 h, and high concentrations of ouabain were similarly required to reduce coupling between COS‐1 cells selected to express the rat α1 Na+/K+‐ATPase subunit, which is ouabain resistant. By contrast, low concentrations of ouabain (1–10 μM) attenuated dye transfer in wild‐type COS‐1 and HeLa cells, whose endogenous α1 subunits possess relatively high affinity for the glycoside (Ki∼0.3 vs ∼100 μM) 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. No consistent relationship between inhibition of intercellular dye transfer and secondary changes in [Ca2+]i or pHi could be identified following incubation with ouabain. 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. Ouabain (1 mM) depressed vesicular trafficking of Cx43‐GFP after ∼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 ∼90 min following washout of the glycoside. 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. British Journal of Pharmacology (2003) 140, 1261–1271. doi:10.1038/sj.bjp.0705556