Ouabain exerts biphasic effects on connexin functionality and expression in vascular smooth muscle cells
Correction to: British Journal of Pharmacology (2003) 140, 1261–1271. 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)...
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Published in | British journal of pharmacology Vol. 141; no. 2; pp. 374 - 384 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
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Oxford, UK
Blackwell Publishing Ltd
01.01.2004
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Abstract | Correction to: British Journal of Pharmacology (2003) 140, 1261–1271. 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 (Cx) 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 Cx40 and Cx43 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 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 (2004) 141, 374–384. doi:10.1038/sj.bjp.0705671 |
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AbstractList | 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 (Cx) 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 Cx40 and Cx43 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 90 min following washout of the glycoside. 6. We conclude that ouabain exerts biphasic effects on 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. Correction to: British Journal of Pharmacology (2003) 140, 1261–1271. 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 (Cx) 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 Cx40 and Cx43 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 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 (2004) 141, 374–384. doi:10.1038/sj.bjp.0705671 Correction to : British Journal of Pharmacology (2003) 140 , 1261–1271. 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 m M ) 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 α 1 Na + /K + ‐ATPase subunit, which is ouabain resistant. By contrast, low concentrations of ouabain (1–10 μ M ) attenuated dye transfer in wild‐type COS‐1 and HeLa cells, whose endogenous α 1 subunits possess relatively high affinity for the glycoside (K i ∼0.3 vs ∼100 μ M ) 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 [Ca 2+ ] i or pH i could be identified following incubation with ouabain. In separate experiments, the effects of ouabain on real‐time trafficking of connexin (Cx) 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 Cx40 and Cx43 evaluated in A7r5 cells by immunochemical and Western blot analysis. Ouabain (1 m M ) 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 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 (2004) 141 , 374–384. doi: 10.1038/sj.bjp.0705671 Correction to : British Journal of Pharmacology (2003) 140 , 1261–1271. 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 m M ) 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 α 1 Na + /K + -ATPase subunit, which is ouabain resistant. By contrast, low concentrations of ouabain (1–10 μ M ) attenuated dye transfer in wild-type COS-1 and HeLa cells, whose endogenous α 1 subunits possess relatively high affinity for the glycoside (K i ∼0.3 vs ∼100 μ M ) 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 [Ca 2+ ] i or pH i could be identified following incubation with ouabain. In separate experiments, the effects of ouabain on real-time trafficking of connexin (Cx) 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 Cx40 and Cx43 evaluated in A7r5 cells by immunochemical and Western blot analysis. Ouabain (1 m M ) 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 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. |
Author | Martin, Patricia E M Errington, Rachael J Kristensen, Bo Hill, Nathan S Griffith, Tudor M |
AuthorAffiliation | 1 1 Department of Diagnostic Radiology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN 3 3 Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN 2 2 Biomembrane Center, The August Krogh Institute, Universitetsparken 13, Copenhagen, DK-2100, Denmark |
AuthorAffiliation_xml | – name: 1 1 Department of Diagnostic Radiology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN – name: 2 2 Biomembrane Center, The August Krogh Institute, Universitetsparken 13, Copenhagen, DK-2100, Denmark – name: 3 3 Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN |
Author_xml | – sequence: 1 givenname: Patricia E M surname: Martin fullname: Martin, Patricia E M – sequence: 2 givenname: Nathan S surname: Hill fullname: Hill, Nathan S – sequence: 3 givenname: Bo surname: Kristensen fullname: Kristensen, Bo – sequence: 4 givenname: Rachael J surname: Errington fullname: Errington, Rachael J – sequence: 5 givenname: Tudor M surname: Griffith fullname: Griffith, Tudor M |
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Notes | Current address: Department Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, U.K. |
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Snippet | Correction to: British Journal of Pharmacology (2003) 140, 1261–1271. doi:10.1038/sj.bjp.0705556
We have compared the effects of ouabain on the maintenance of... 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... Correction to : British Journal of Pharmacology (2003) 140 , 1261–1271. doi: 10.1038/sj.bjp.0705556 We have compared the effects of ouabain on the maintenance... |
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SubjectTerms | Animals Cell communication Chlorocebus aethiops Connexin 43 - biosynthesis Connexins - biosynthesis COS Cells Dose-Response Relationship, Drug EDHF Erratum Gap Junction alpha-5 Protein gap junctions Gene Expression Regulation - drug effects Gene Expression Regulation - physiology HeLa Cells Humans Muscle, Smooth, Vascular - drug effects Muscle, Smooth, Vascular - metabolism Ouabain - pharmacology Rats |
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Title | Ouabain exerts biphasic effects on connexin functionality and expression in vascular smooth muscle cells |
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