Ouabain-Induced Changes in the Expression of Voltage-Gated Potassium Channels in Epithelial Cells Depend on Cell–Cell Contacts

Abstract

Ouabain is a cardiac glycoside, initially isolated from plants, and currently thought to be a hormone since some mammals synthesize it endogenously. It has been shown that in epithelial cells, it induces changes in properties and components related to apical–basolateral polarity and cell–cell contacts. In this work, we used a whole-cell patch clamp to test whether ouabain affects the properties of the voltage-gated potassium currents (Ik) of epithelial cells (MDCK). We found that: (1) in cells arranged as mature monolayers, ouabain induced changes in the properties of Ik; (2) it also accelerated the recovery of Ik in cells previously trypsinized and re-seeded at confluence; (3) in cell–cell contact-lacking cells, ouabain did not produce a significant change; (4) Na+/K+ ATPase might be the receptor that mediates the effect of ouabain on Ik; (5) the ouabain-induced changes in Ik required the synthesis of new nucleotides and proteins, as well as Golgi processing and exocytosis, as evidenced by treatment with drugs inhibiting those processes; and (5) the signaling cascade included the participation of cSrC, PI3K, Erk1/2, NF-κB and β-catenin. These results reveal a new role for ouabain as a modulator of the expression of voltage-gated potassium channels, which require cells to be in contact with themselves.

Ouabain’s Influence on TRPV4 Channels of Epithelial Cells: An Exploration of TRPV4 Activity, Expression, and Signaling Pathways

“…Specifically, we have described that ouabain (1) boosts intercellular connections facilitated by tight junctions, as proven by an increase in transepithelial electrical resistance (TER), a simultaneous decrease in the movement of dextran between cells, and an increase in the expression of claudins 1, 2, and 4 [13]; (2) induces alterations in adherens junctions, resulting in heightened expression of E-cadherin, β-catenin, and γ-catenin [14]; (3) fosters the enhancement of gap junctional communication among cells forming fully developed epithelial monolayers [15] by modifying the distribution of Cx32 and Cx43 within the membrane [16]; and (4) expedites the recovery of the epithelial phenotype in cells seeded at confluence after trypsinization, as indicated by the presence of an apical cilium in MDCK cells [17]. Apart from its involvement in regulating intercellular contact components, we have found that ouabain also influences the expression of voltage-gated potassium channels, entailing Na + /K + -ATPase acting as a receptor and the engagement of multiple signaling pathways, including cSrc, PI3K, Erk1/2, NF-κB, and β-catenin [18].…”

Na+/K+-ATPase: More than an Electrogenic Pump

“…It has also been shown that in all of these effects induced by ouabain, NKA functions as a receptor, initiating various signaling pathways. For cell-cell induced effects, a signaling pathway involving SRC/RAS/RAF/MEK/ERK is implicated, while ion current en- Moreover, electrophysiological assays (whole-cell patch clamp) demonstrate that in MDCK cells organized as epithelial monolayers, nanomolar doses of ouabain induce the expression of ionic currents attributed to the presence of voltage-gated potassium channels (IKv) [243] alongside ionic currents associated with the presence of TRPV4 channels (ITRPV4) [244].…”

“…Moreover, electrophysiological assays (whole-cell patch clamp) demonstrate that in MDCK cells organized as epithelial monolayers, nanomolar doses of ouabain induce the expression of ionic currents attributed to the presence of voltage-gated potassium channels (IKv) [ 243 ] alongside ionic currents associated with the presence of TRPV4 channels (ITRPV4) [ 244 ].…”

“…Consequently, treatment with a concentration of 10 nanomolar ouabain has been observed to hasten ciliogenesis, a critical step in consolidating the epithelial phenotype [ 245 ]. Furthermore, cells exhibit a more rapid recovery of ionic current values, attributed to voltage-gated potassium channels [ 243 ] and TRPV4 channels [ 244 ].…”

“…Furthermore, treatment of mature monolayer MDCK cells with nanomolar doses of ouabain induces changes in various transcripts, including the transcript encoding myosin IXa, which regulates cell shape and collective epithelial cell migration by targeting RhoGAP activity to cell–cell junctions [ 243 ]. This finding implicates RhoA and its downstream effector ROCK in the signaling pathways through which ouabain influences cell-to-cell contacts in epithelial cells [ 244 ].…”