Ouabain Increases Gap Junctional Communication in Epithelial Cells

Abstract

Background/Aims: The finding that endogenous ouabain acts as a hormone prompted efforts to elucidate its physiological function. In previous studies, we have shown that 10 nM ouabain (i.e., a concentration within the physiological range) modulates cell-cell contacts such as tight junctions and apical/basolateral polarity. In this study, we examined whether 10 nM ouabain affects another important cell-cell feature: gap junction communication (GJC). Methods: We employed two different approaches: 1) analysis of the cell-to-cell diffusion of neurobiotin injected into a particular MDCK cell (epithelial cells from dog kidneys) in a confluent monolayer by counting the number of neighboring cells reached by the probe and 2) measurement of the electrical capacitance. Results: We found that 10 nM ouabain increase GJC by 475% within 1 hour. The Na+-K+-ATPase acts as a receptor of ouabain. In previous works we have shown that ouabain activates c-Src and ERK1/2 in 1 hour; in the present study we show that the inhibition of these proteins block the effect of ouabain on GJC. This increase in GJC does not require synthesis of new protein components, because the inhibitors cycloheximide and actinomycin D did not affect this phenomenon. Using silencing assays we also demonstrate that this ouabain-induced enhancement of GJC involves connexins 32 and 43. Conclusion: Ouabain 10 nM increases GJC in MDCK cells.

Ouabain Modulates the Distribution of Connexin 43 in Epithelial Cells

“…Our previous findings, that 10 nM ouabain triggers a quick enhancement of GJIC, plus the fact that this response is not impaired by inhibitors of RNA or protein synthesis [44] suggest that no synthesis of connexins are required to achieve this effect. To test this (A) Cell lysates were prepared from MDCK monolayers, either control or incubated with 10 nM ouabain for 1 hour, then equal amounts of total cell protein were subjected to SDS-PAGE and western blotting with antiCnx43 antibodies.…”

“…As part of our interest in the effect that ouabain, in a nanomolar range, has on epithelial (MDCK) cells, we evaluated in a previous work, how it influences gap junctional intercellular communication (GJIC) by dye transfer assays and by electrical capacitance measurements [44]. We found that it, triggers an enhanced GJIC response, that is significantly noticeable from 10 minutes after addition of ouabain to the bathing media and reaches a maximum at about one hour, followed by a slow decaying, then a sustained response that last, al least 24 hrs.…”

“…In a previous work we showed, by dye transfer assays and electrical capacitance measurements, that ouabain 10 nM enhances GJIC up to 475% within 1 hour in MDCK cells [44]. The fact that such response occurs quickly (after 10 minutes of addition of ouabain), and that it is not affected by cycloheximide or Actinomycin D suggest that ouabain does not induce the synthesis of new connexins in order to enhance GJIC but rather promotes the relocation of subunits already synthesized.…”

“…On the other hand, c-Src is a tyrosine kinase that is mutated in several cancers. We have previously found that ouabain mediates its effect on GJIC by a signal transduction cascade that includes c-Src and ERK1/2 [44]. To determine whether such signal transduction cascade is also involved in the relocation of Cnx43 induced by ouabain, we probed whether commercial inhibitors of c-SRC or ERK1/2 impairs any of the effects caused by ouabain, either the increase of Cnx43 protein density from Western blot assays of membrane extracts, or the increase of the density of clusters of cells observed in immunohistochemical assays.…”

Role of Myoendothelial Gap Junctions in the Regulation of Human Coronary Artery Smooth Muscle Cell Differentiation by Laminar Shear Stress

“…The expression of connexins can depend on the physiological state, including hibernation, arousal, level of hormone and mechanical environments [34][35][36]. Several studies demonstrated that Cx43 was increased in ECs cultured alone by high laminar shear stress (15 dyn/cm 2 ) compared to static conditions [19,37].…”