Abstract
Cardiac glycosides are a group of compounds widely known for their action in cardiac tissue, some of which have been found to be endogenously produced (ECG). We have previously studied the effect of ouabain, an endogenous cardiac glycoside, on the physiology of epithelial cells, and we have shown that in concentrations in the nanomolar range, it affects key properties of epithelial cells, such as tight junction, apical basolateral polarization, gap junctional intercellular communication (GJIC), and adherent junctions. In this work, we study the influence of digoxin and marinobufagenin, two other endogenously expressed cardiac glycosides, on GJIC as well as the degree of transepithelial tightness due to tight junction integrity (TJ). We evaluated GJIC by dye transfer assays and tight junction integrity by transepithelial electrical resistance (TER) measurements, as well as immunohistochemistry and western blot assays of expression of claudins 2 and 4. We found that both digoxin and marinobufagenin improve GJIC and significantly enhance the tightness of the tight junctions, as evaluated from TER measurements. Immunofluorescence assays show that both compounds promote enhanced basolateral localization of claudin-4 but not claudin 2, while densitometric analysis of western blot assays indicate a significantly increased expression of claudin 4. These changes, induced by digoxin and marinobufagenin on GJIC and TER, were not observed on MDCK-R, a modified MDCK cell line that has a genetically induced insensitive α1 subunit, indicating that Na-K-ATPase acts as a receptor mediating the actions of both ECG. Plus, the fact that the effect of both cardiac glycosides was suppressed by incubation with PP2, an inhibitor of c-Src kinase, PD98059, an inhibitor of mitogen extracellular kinase-1 and Y-27632, a selective inhibitor of ROCK, and a Rho-associated protein kinase, indicate altogether that the signaling pathways involved include c-Src and ERK1/2, as well as Rho-ROCK. These results widen and strengthen our general hypothesis that a very important physiological role of ECG is the control of the epithelial phenotype and the regulation of cell-cell contacts.
Highlights
Cardiac steroids (CS) are a group of diverse compounds derived from plant and animal sources, with similar chemical properties, that have long been used to increase cardiac contractile force in patients with congestive heart failure and cardiac arrhythmias. ey all contain a structure consisting of 17 carbon atoms arranged in 4 cyclic carbon rings, known as sterane moiety, and an unsaturated lactone ring at the C-17 position
It indicates there exists a subpopulation of Na-KATPase, located in caveolae that does not function as a pump, but rather as a receptor that upon binding of cardiac glycosides activates one or more signaling pathways to produce a variety of changes on the physiology or even the genetic expression of cells [19, 20]. e binding of cardiac glycosides to Na-K-ATPase activates the Src/epidermal growth factor receptor complex to initiate multiple signal pathways, which include PLC/IP3/CICR, PI3K, reactive oxygen species (ROS), PLC/DG/PKC/Raf/MEK/ERK1/2, and Ras/Raf/MEK/ERK1/2 pathways [21]
Multiple repeats were made, and each repeat consisted of the injection of Lucifer yellow into a single cell, which was able to diffuse to the neighboring cells if there was Gap Junctional Intercellular Communication (GJIC). en, the average number of cells stained per trial was considered as a criterion to estimate the degree of GJIC. erefore, for each experimental treatment, we compared the average number of stained cells versus the corresponding value obtained from untreated cells
Summary
Cardiac steroids (CS) are a group of diverse compounds derived from plant and animal sources, with similar chemical properties, that have long been used to increase cardiac contractile force in patients with congestive heart failure and cardiac arrhythmias. ey all contain a structure consisting of 17 carbon atoms arranged in 4 cyclic carbon rings, known as sterane moiety, and an unsaturated lactone ring at the C-17 position. A second hypothesis, about the way that cardiac glycosides interact with Na-K-ATPase, has been described more recently It indicates there exists a subpopulation of Na-KATPase, located in caveolae that does not function as a pump, but rather as a receptor that upon binding of cardiac glycosides activates one or more signaling pathways to produce a variety of changes on the physiology or even the genetic expression of cells [19, 20]. A second fact that has given renewed interest to the study of cardiac glycosides is the finding that some of these compounds are produced endogenously by some mammalian species, including humans. In addition to demonstrating the influence of ouabain in the processes already described, we have shown that Na-K-ATPase is the primary receptor that mediates a signaling cascade involving c-Src and ERK1/2 [42]. Like with ouabain, Na-K-ATPase is the primary receptor that mediates such responses and whether c-Src, ERK1/2, and Rho/ROCK participate in the signaling pathways involved
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