Influence of Cardiac Glycosides and Prostaglandins on the Physiology of Epithelial Cells

Epithelial cells play a major role in animal and human homeostasis because they selectively regulate the exchange of solutes between two given media, such as blood or urine. Cardiac glycosides (CG) are a group of highly toxic compounds whose best therapeutic known effect is on heart, although recent evidence has shown that it exerts a wide range of physiological effects on cells and tissues other than the heart. Prostaglandins, on the other hand, are a group of lipids that produce diverse physiological and pathological effects among which inflammation stands out. In this chapter, we describe that cardiac glycosides modulate key features of epithelial cell physiology, including cell-cell contact junctional complexes, cilliogenesis, and gap junction-mediated intercellular communication (GJIC) in epithelial cells. Prostaglandin PGE2 also modulates GJIC through a different signaling pathway. In addition, we describe that CG induce paracrine release of prostaglandin PGE2, which in turn modulates GJIC by itself

Ouabain Enhances Cell-Cell Adhesion Mediated by beta1 Subunits of the Na(+),K(+)-ATPase in CHO Fibroblasts

Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na(+),K(+)-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine beta1 subunit become adhesive, and those homotypic interactions amongst beta1 subunits of the Na(+),K(+)-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the alpha subunit of the Na(+),K(+)-ATPase, inhibits the pump activity and induces the detachment of epithelial cells when used at concentrations above 300 nM. At nanomolar non-inhibiting concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through the activation of signaling pathways associated with the alpha subunit. In this study, we investigated whether the adhesion between beta1 subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the beta1 subunit of the Na(+),K(+)-ATPase (CHO beta1), and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO beta1 cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the beta1 subunit of the Na(+),K(+)-ATPase at the cell membrane. We also examined the effect of ouabain on the activation of signaling pathways in CHO beta1 cells, and their subsequent effect on cell adhesion. We found that cSrc is activated by ouabain and, therefore, that it likely regulates the adhesive properties of CHO beta1 cells. Collectively, our findings suggest that the beta1 subunit adhesion is modulated by the expression levels of the Na(+),K(+)-ATPase at the plasma membrane, which is regulated by ouabain

Epithelial Na+,K+-ATPase — A Sticky Pump

Na+,K+-ATPase is an ATP-powered ion pump that establishes concentration gradients for Na+ and K+ ions across the plasma membrane in all animal cells by pumping Na+ from the cytoplasm and K+ from the extracellular medium. This heterodimeric enzyme, a member of P-type ATPases, is composed of a catalytic α-subunit with ten transmembrane domains and a heavily glycosylated auxiliary β-subunit. The Na+,K+-ATPase is specifically inhibited by cardiotonic steroids like ouabain, which bind to the enzyme’s α-subunit from the extracellular side and thereby block the ion pumping cycle. Na+,K+-ATPAse generates ion gradients that establishes the driving force for the transepithelial transport of several solutes and nutrients. The effectiveness of this vectorial transport motivated by Na+,K+-ATPase depends on the integrity of epithelial junctions that are essential for the maintenance of the polarized localization of membrane transporters, including the lateral sodium pump. This chapter reviews the facts showing that, in addition to pumping ions, the Na+,K+-ATPase located at the cell borders functions as a cell adhesion molecule and discusses the role of the Na+,K+-ATPase β-subunit in establishing and maintaining cell–cell interactions. Furthermore, Na+,K+-ATPase is a multifunctional protein that, in addition to pumping ions asymmetrically and participating in cell–cell contacts, acts as specific receptor for the hormone ouabain and transduces extracellular signals. Thus, when bearing in mind with transporting epithelia phenotype, the importance of modulation of cell contacts by Na+,K+-ATPase can hardly be underestimated

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

The sodium pump, or Na+/K+-ATPase (NKA), is an essential enzyme found in the plasma membrane of all animal cells. Its primary role is to transport sodium (Na+) and potassium (K+) ions across the cell membrane, using energy from ATP hydrolysis. This transport creates and maintains an electrochemical gradient, which is crucial for various cellular processes, including cell volume regulation, electrical excitability, and secondary active transport. Although the role of NKA as a pump was discovered and demonstrated several decades ago, it remains the subject of intense research. Current studies aim to delve deeper into several aspects of this molecular entity, such as describing its structure and mode of operation in atomic detail, understanding its molecular and functional diversity, and examining the consequences of its malfunction due to structural alterations. Additionally, researchers are investigating the effects of various substances that amplify or decrease its pumping activity. Beyond its role as a pump, growing evidence indicates that in various cell types, NKA also functions as a receptor for cardiac glycosides like ouabain. This receptor activity triggers the activation of various signaling pathways, producing significant morphological and physiological effects. In this report, we present the results of a comprehensive review of the most outstanding studies of the past five years. We highlight the progress made regarding this new concept of NKA and the various cardiac glycosides that influence it. Furthermore, we emphasize NKA’s role in epithelial physiology, particularly its function as a receptor for cardiac glycosides that trigger intracellular signals regulating cell–cell contacts, proliferation, differentiation, and adhesion. We also analyze the role of NKA β-subunits as cell adhesion molecules in glia and epithelial cells

Ouabain Enhances Cell-Cell Adhesion Mediated by β₁ Subunits of the Na⁺,K⁺-ATPase in CHO Fibroblasts

Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na+,K+-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine β1 subunit become adhesive, and those homotypic interactions amongst β1 subunits of the Na+,K+-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the α subunit of the Na+,K+-ATPase, inhibits the pump activity and induces the detachment of epithelial cells when used at concentrations above 300 nM. At nanomolar non-inhibiting concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through the activation of signaling pathways associated with the α subunit. In this study, we investigated whether the adhesion between β1 subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the β1 subunit of the Na+,K+-ATPase (CHO β1), and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO β1 cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the β1 subunit of the Na+,K+-ATPase at the cell membrane. We also examined the effect of ouabain on the activation of signaling pathways in CHO β1 cells, and their subsequent effect on cell adhesion. We found that cSrc is activated by ouabain and, therefore, that it likely regulates the adhesive properties of CHO β1 cells. Collectively, our findings suggest that the β1 subunit adhesion is modulated by the expression levels of the Na+,K+-ATPase at the plasma membrane, which is regulated by ouabain

The β2-Subunit (AMOG) of Human Na+, K+-ATPase Is a Homophilic Adhesion Molecule

The β2 subunit of Na+, K+-ATPase was originally identified as the adhesion molecule on glia (AMOG) that mediates the adhesion of astrocytes to neurons in the central nervous system and that is implicated in the regulation of neurite outgrowth and neuronal migration. While β1 isoform have been shown to trans-interact in a species-specific mode with the β1 subunit on the epithelial neighboring cell, the β2 subunit has been shown to act as a recognition molecule on the glia. Nevertheless, none of the works have identified the binding partner of β2 or described its adhesion mechanism. Until now, the interactions pronounced for β2/AMOG are heterophilic cis-interactions. In the present report we designed experiments that would clarify whether β2 is a cell–cell homophilic adhesion molecule. For this purpose, we performed protein docking analysis, cell–cell aggregation, and protein–protein interaction assays. We observed that the glycosylated extracellular domain of β2/AMOG can make an energetically stable trans-interacting dimer. We show that CHO (Chinese Hamster Ovary) fibroblasts transfected with the human β2 subunit become more adhesive and make large aggregates. The treatment with Tunicamycin in vivo reduced cell aggregation, suggesting the participation of N-glycans in that process. Protein–protein interaction assay in vivo with MDCK (Madin-Darby canine kidney) or CHO cells expressing a recombinant β2 subunit show that the β2 subunits on the cell surface of the transfected cell lines interact with each other. Overall, our results suggest that the human β2 subunit can form trans-dimers between neighboring cells when expressed in non-astrocytic cells, such as fibroblasts (CHO) and epithelial cells (MDCK)