Ouabain Modulates the Distribution of Connexin 43 in Epithelial Cells.
OBJECTIVE
The fact that ouabain has been identified as an endogenous substance, led us to inquire its physiological role in epithelial cells. Based on previous observations, we hypothesized that it influences processes related to cell contacts. Previously we have shown that nanomolar concentrations of ouabain up-regulate tight junctions, accelerate ciliogenesis, and increase gap junctional intercellular communication (GJIC). Given that silencing assays indicated that connexin 43 (Cnx43) is involved in the GJIC response, in the present work we study whether ouabain affects Cnx43 expression and distribution.


METHODS
We seeded confluent monolayers of epithelial renal MDCK cells and incubated them with 10 nM ouabain during 1 h. Then we measured, by densitometric analysis of Western blot assays, the amount of Cnx43 in cells and in fractions enriched of plasma membrane. We also studied its localization with immunofluorescence and confocal microscopy.


RESULTS
Cnx43 is remarkably displayed, outlining the borders of cells gathered in clusters, randomly scattered throughout the monolayer. Ouabain increases the density of such clusters, as well as the average number of cells per cluster, without inducing the synthesis of new Cnx43. It also promotes relocation towards the membrane, of subunits already available. The fact that such changes are inhibited by PP2 and PD98059 indicates that a signaling pathway, that includes c-Src and ERK1/2, is involved in this response.


CONCLUSIONS
Ouabain induces the translocation of Cnx43 from the cytoplasm to the plasma membrane. These findings support our hypothesis that one of the physiological roles of ouabain is the modulation of physiological processes that depend on cell to cell contacts.
Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies (Cinvestav), México D. F., México.
Arturo
A
Ponce
Isabel
I
Larre
Aida
A
Castillo
Catalina
C
Flores-Maldonado
Odette
O
Verdejo-Torres
Ruben Gerardo
RG
Contreras
Marcelino
M
Cereijido
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002450:Cell Communication / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D018031:Connexin 43 / Q000235:genetics* / Q000378:metabolism; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation; D061985:Madin Darby Canine Kidney Cells; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology*; D021381:Protein Transport; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction; D019108:Tight Junctions / Q000187:drug effects* / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
C412373:AG 1879; D018031:Connexin 43; D004791:Enzyme Inhibitors; D005419:Flavonoids; D011743:Pyrimidines; D010042:Ouabain; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one