With an exception of few reports, the plasma concentration of ouabain and marinobufagenin, mostly studied cardiotonic steroids (CTS) assessed by immunoassay techniques, is less than 1 nM. During the last 3 decades, the implication of these endogenous CTS in the pathogenesis of hypertension and other volume-expanded disorders is widely disputed. The threshold for inhibition by CTS of human and rodent α1-Na,K-ATPase is ∼1 and 1000 nM, respectively, that rules out the functioning of endogenous CTS (ECTS) as natriuretic hormones and regulators of cell adhesion, cell-to-cell communication, gene transcription and translation, which are mediated by dissipation of the transmembrane gradients of monovalent cations. In several types of cells ouabain and marinobufagenin at concentrations corresponding to its plasma level activate Na,K-ATPase, decrease the [Na+]i/[K+]i-ratio and increase cell proliferation. Possible physiological significance and mechanism of non-canonical Na+i/K+i-dependent and Na+i/K+i-independent cell responses to CTS are discussed.

Ouabain is of cardiotonic steroids (CTS) family that is plant-derived compounds and is known for many years as therapeutic and cytotoxic agents. They are specific inhibitors of Na,K-ATPase, the enzyme, which pumps Na⁺ and K⁺ across plasma membrane of animal cells. Treatment of cells by CTS affects various cellular functions connected with the maintenance of the transmembrane gradient of Na⁺ and K⁺. Numerous studies demonstrated that binding of CTS to Na,K-ATPase not only suppresses its activity but also induces some signal pathways. This review is focused on different mechanisms of two ouabain effects: their ability (1) to protect rodent cells from apoptosis through the expression of [Na⁺]_i-sensitive genes and (2) to trigger death of non-rodents cells (so-called «oncosis»), possessing combined markers of «classic» necrosis and «classic» apoptosis. Detailed study of oncosis demonstrated that the elevation of the [Na⁺]_i/[K⁺]_i ratio is not a sufficient for its triggering. Non-rodent cell death is determined by the characteristic property of “sensitive” to ouabain α1-subunit of Na,K-ATPase. In this case, ouabain binding leads to enzyme conformational changes triggering the activation of p38 mitogen-activated protein kinases (MAPK) signaling. The survival of rodent cells with ouabain-«resistant» α1-subunit is connected with another conformational transition induced by ouabain binding that results in the activation of ERK 1/2 signaling pathway.

Cardiotonic steroids (CTSs) are specific inhibitors of Na,K-ATPase (NKA). They induce diverse physiological effects and were investigated as potential drugs in heart diseases, hypertension, neuroinflammation, antiviral and cancer therapy. Here, we compared the inhibition mode and binding of CTSs, such as ouabain, digoxin and marinobufagenin to NKA from pig and rat kidneys, containing CTSs-sensitive (α1S) and - r esistant (α1R) α1-subunit, respectively. Marinobufagenin in contrast to ouabain and digoxin interacted with α1S-NKA reversibly, and its binding constant was reduced due to the decrease in the deepening in the CTSs-binding site and a lower number of contacts between the site and the inhibitor. The formation of a hydrogen bond between Arg111 and Asp122 in α1R-NKA induced the reduction in CTSs’ steroid core deepening that led to the reversible inhibition of α1R-NKA by ouabain and digoxin and the absence of marinobufagenin’s effect on α1R-NKA activity. Our results elucidate that the difference in signaling, and cytotoxic effects of CTSs may be due to the distinction in the deepening of CTSs into the binding side that, in turn, is a result of a bent-in inhibitor steroid core (marinobufagenin in α1S-NKA) or the change of the width of CTSs-binding cavity (all CTSs in α1R-NKA).

Beta-amyloid (Aβ) has a dual role, both as an important factor in the pathology of Alzheimer’s disease and as a regulator in brain physiology. The inhibitory effect of Aβ₄₂ oligomers on Na,K-ATPase contributes to neuronal dysfunction in Alzheimer’s disease. Still, the physiological role of the monomeric form of Aβ₄₂ interaction with Na,K-ATPase remains unclear. We report that Na,K-ATPase serves as a receptor for Aβ₄₂ monomer, triggering Src kinase activation. The co-localization of Aβ₄₂ with α1- and β1-subunits of Na,K-ATPase, and Na,K-ATPase with Src kinase in SH-SY5Y neuroblastoma cells, was observed. Treatment of cells with 100 nM Aβ₄₂ causes Src kinase activation, but does not alter Na,K-ATPase transport activity. The interaction of Aβ₄₂ with α1β1 Na,K-ATPase isozyme leads to activation of Src kinase associated with the enzyme. Notably, prevention of Na,K-ATPase:Src kinase interaction by a specific inhibitor pNaKtide disrupts the Aβ-induced Src kinase activation. Stimulatory effect of Aβ₄₂ on Src kinase was lost under hypoxic conditions, which was similar to the effect of specific Na,K-ATPase ligands, the cardiotonic steroids. Our findings identify Na,K-ATPase as a Aβ₄₂ receptor, thus opening a prospect on exploring the physiological and pathological Src kinase activation caused by Aβ₄₂ in the nervous system.

The Alzheimer’s disease (AD)-associated breakdown of the blood–brain barrier (BBB) promotes the accumulation of beta-amyloid peptide (Aβ) in the brain as the BBB cells provide Aβ transport from the brain parenchyma to the blood, and vice versa. The breakdown of the BBB during AD may be caused by the emergence of blood-borne Aβ pathogenic forms, such as structurally and chemically modified Aβ species; their effect on the BBB cells has not yet been studied. Here, we report that the effects of Aβ₄₂, Aβ₄₂, containing isomerized Asp7 residue (iso-Aβ₄₂) or phosphorylated Ser8 residue (p-Aβ₄₂) on the mitochondrial potential and respiration are closely related to the redox status changes in the mouse brain endothelial cells bEnd.3. Aβ₄₂ and iso-Aβ₄₂ cause a significant increase in nitric oxide, reactive oxygen species, glutathione, cytosolic calcium and the mitochondrial potential after 4 h of incubation. P-Aβ₄₂ either does not affect or its effect develops after 24 h of incubation. Aβ₄₂ and iso-Aβ₄₂ activate mitochondrial respiration compared to p-Aβ₄₂. The isomerized form promotes a greater cytotoxicity and mitochondrial dysfunction, causing maximum oxidative stress. Thus, Aβ₄₂, p-Aβ₄₂ and iso-Aβ₄₂ isoforms differently affect the BBBs’ cell redox parameters, significantly modulating the functioning of the mitochondria. The changes in the level of modified Aβ forms can contribute to the BBBs’ breakdown during AD.

Stimulus-dependent elevation of intracellular Ca²⁺ affects gene expression via well-documented calmodulin-mediated signaling pathways. Recently, we found that the addition of extra- and intracellular Ca²⁺ chelators increased, rather than decreased, the number of genes expressed, and that this is affected by the elevation of [Na⁺]_i/[K⁺]_i-ratio. This assumes the existence of a novel Na⁺_i/K⁺_i-mediated Ca²⁺_i-independent mechanism of excitation-transcription coupling. To identify upstream Na⁺_i/K⁺_i-sensitive genes, we examined the kinetics of transcriptomic changes in human umbilical vein endothelial cells (HUVEC) subjected to Na,K-ATPase inhibition by ouabain or K⁺-free medium. According to our data, microRNAs, transcription factors, and proteins involved in immune response and inflammation might be considered as key components of Na⁺_i/K⁺_i-mediated excitation-transcription coupling. Special attention was focused on the FOS gene and the possible mechanism of transcription regulation via G-quadruplexes, non-canonical secondary structures of nucleic acids, whose stability depends on [Na⁺]_i/[K⁺]_i-ratio. Verification of the [Na⁺]_i/[K⁺]_i-sensitive transcription regulation mechanism should be continued in forthcoming studies.