Background/Aims: Cultured skeletal muscle cells subjected to electrical pulse stimulation (EPS) are widely employed as an in vitro model of exercising skeletal muscle. Numerous studies demonstrated that sustained excitation of skeletal muscle results in the dissipation of the transmembrane gradient of monovalent cations. During exercises the impending loss of excitability has to be counterbalanced by rapid restoration of the Na+i/K+i ratio. To understand mechanisms of the maintenance of muscle contractility, it is necessary to know which transporters are participated in the dissipation of Na+i and K+i gradients and how to activate Na,K-ATPase for its regeneration. This study was aimed at the identification of ion transporters involved in the dissipation of the transmembrane gradients of Na+ and K+induced by EPS, and Na,K-ATPase isoforms involved in its restoration. Methods: The differentiated C2C12 myotubes were subjected to electrical pulse stimulation in the presence or absence of different ion transport systems inhibitors followed by measurement of intracellular monovalent cations by flame atomic absorption spectrometry. Results: Electrical pulse stimulation of C2C12 myotubes results in the dissipation of Na+i/K+i gradient, which is maintained by α2-Na,K-ATPase. Na-K-2Cl cotransporter (NKCC), voltage-gated Na+ (Nav), and large conductance Ca2+-activated K+ channels (BKCa), and Na/H exchanger (NHE) are involved in the dissipation of this gradient. Suppression of calmodulin-dependent protein kinase II (CaMKII) increases Na+ efflux in EPS-treated C2C12 myotubes. Conclusion: NKCC, Nav, BKCa, and NHE are involved in the dissipation of Na+i/K+i gradient in EPS-treated C2C12 myotubes.

The review presents the mechanisms of participation of ions (Na + , K + , and Ca 2+ ) in the processes of synaptic plasticity in the postsynaptic neuron during long-term potentiation and long-term depression. It is assumed that the main participants are AMPA and NMDA receptors, voltage-dependent Na + , K + , Ca 2+ channels, Ca 2+ and Na + -activated K + channels, ATP-sensitive K + channels, and Ca 2+ channels of the endoplasmic reticulum. The review provides their molecular characteristics and discusses their role in long-term potentiation and long-term depression. The significance of changes in the intracellular ratio [Na + ] i /[K + ] i and Ca 2+ -dependent mechanism are considered for the first time from the signal formation to the level of gene expression. We believe that additional research is needed to identify a subset of neuronal genes whose differential expression contributes to synaptic plasticity, which is implemented with the participation of [Na + ] i /[K + ] i -sensitive Ca 2+ -independent “excitation–transcription coupling” mechanism.

High-salt consumption contributes to the development of hypertension and is considered an independent risk factor for vascular remodelling, cardiac hypertrophy and stroke incidence. Alterations in NO production, inflammation and endothelial cell stiffening are considered now as plausible mediators of cardiovascular dysfunction. We studied early responses of endothelial cells (HUVEC) caused by a moderate increase in extracellular sodium concentration. Exposure of HUVEC to elevated sodium within the physiological range up to 24 h is accompanied by changes in monovalent cations fluxes and Na,K-ATPase activation, and, in turn, results in a significant decrease in the content of PTGS2, IL6 and IL1LR1 mRNAs. The expression of NOS3 and FOS genes, as well as the abundance of cytosolic and nuclear NFAT5 protein, remained unchanged. We assessed the mechanical properties of endothelial cells by estimating Young's modulus and equivalent elastic constant using atomic force and interference microscopy, respectively. These parameters were unaffected by elevated-salt exposure for 24 h. The data obtained suggest that even small and short-term elevations of extracellular sodium concentration affect the expression of genes involved in the control of endothelial function through the Na+i/K+i-dependent mechanism(s).

It was found that a decrease in the concentration of extracellular Ca2+ ([Ca2+]out) caused an increase in the surface charge of the erythrocyte membrane, which was accompanied by a change in the conformation of heme and globin, as well as in a redistribution of hemoglobin in the cell (a decrease in the optical path difference (OPD) of the cytoplasm and the content of the membrane-associated hemoglobin). It was also found that when [Ca2+]out was decreased from 10–3 to 10–6 M, the morphology of the erythrocyte practically did not change, the ζ-potential increased from –15.4 ± 0.2 to –14.5 ± 0.3 mV (at [Ca2+]out decrease from 10–3 to 5 × 10–4 M), and the OPD of the cell cytoplasm decreased. A decrease in [Ca2+]out to 10–6 M changed the conformation of heme, as well as the distribution of hemoglobin in the erythrocyte, as assessed by a decrease in the symmetric vibrations of the side CH groups of pyrrole semirings and an increase in the vibrations of the vinyl groups of hemoporphyrin as well as the affinity of heme to ligands. At the same time, the probability of finding heme in a flat conformation did not change, while the rigidity of its protein surrounding decreased. When [Ca2+]out was decreased from 10–3 to 10–4 M, a change in the globin conformation was observed as a decrease in the ordering of the CH groups of globin amino acids, an increase in the globin packing density, and the polarity of the globin surrounding. When erythrocytes were incubated in a medium containing [Ca2+]out = 10–6 M, the maximum intensity of the Raman scattering of erythrocyte hemoglobin (Raman spectrum bands at 1375 and 1580 cm–1) was detected in the center of the cell; it was 2.6 times less than that of erythrocytes incubated in a medium containing [Ca2+]out = 10–3 M.

The affinity of rodent Na+,K+-ATPase α1-subunit to cardiotonic steroids (CTS) is known to be approximately 1000-fold less than the affinity of Na+,K+-ATPase α1-subunit from other mammals. The CTS-resistant isoform of Na+,K+-ATPase α1-subunit (α1R) is expressed in rodent cells, in contrast to the CTS-sensitive isoform of the α1-subunit (α1S), which is expressed in cells of other mammals. Earlier we have established that incubation with ouabain in concentrations that completely suppressed the activity of Na+,K+-ATPase α1-isoform (α1S-Na+,K+-ATPase) led to a death of human endothelial and smooth muscle cells but did not affect the survival of rat cells expressing α1R-Na+,K+-ATPase. Conformational transitions that are induced by CTS binding to Na+,K+-ATPase play a key role in the process of cell survival. To reveal differences in the CTS-induced conformational changes of α1R- and α1S-Na+,K+-ATPase isoforms, we used three different CTS (two cardenelids, ouabain and digoxin, and one bufadienolid, marinobufagenin) and analyzed the trypsinolysis products of α1-subunits in two main conformations of Na+,K+-ATPase (E1 and E2-P). The treatment of the pig kidney α1S-Na,K-ATPase in E1 conformation by trypsin results in a significant decrease of the amount of α1S-subunit and in the appearance of protein fragments with molecular masses of about 40, 35, 23, and 19 kDa. Preincubation of Na+,K+-ATPase in E1 conformation with ouabain or with digoxin (1 mM) leads to a decrease of the amount of α1S-subunit, increase of the amount of polypeptide fragment with molecular mass of about 40 kDa, and a significant rise of the amount of fragment with molecular mass of about 35.5 kDa, which was not found after the preincubation of the Na+,K+-ATPase in E1 conformation with marinobufagenin (1 mM). In the absence of CTS the trypsinolysis of α1S-Na+,K+-ATPase in E2-P conformation results in a decrease of the amount of α1S-subunit and an increase of the amount of proteolytic products with molecular mass of about 40 and 35.5 kDa. Preincubation of the Na+,K+-ATPase in E2-P conformation in the presence of any of the CTS studied induces the appearance of big amount of an additional peptide fragment with molecular mass of about 45 kDa. Preincubation of α1R-Na+,K+-ATPase from rat kidney with any of the CTS does not change the composition of proteolytic products and their molecular masses in either E1 or E2-P conformation. The results suggest that the structure of the CTS-binding site and a conformational response of α1-Na+,K+-ATPase to binding of CTS is mainly determined by the primary structure of the CTS-resistant and CTS-sensitive α1-subunits of the Na+,K+-AТРase.

Abstract—Using Raman spectroscopy (RS) approach in a spectral range of 1000–3000 cm–1 were used to study the conformational and structural changes that arise in the heme group and globin moiety of hemoglobin in human red blood cells at various temperatures and oxygen contents. In hypoxia, the hemoglobin conformation was shown to change as a result of the increasing contribution of hematoporphyrin pyrrole rings and vibrational motions of vinyl groups. Modifications were additionally detected in the contributions of symmetric and asymmetric vibrations of the CH2 and CH3 radicals of histidine (2850, 2860, and 2900 cm–1) and lysine (2880 and 2860 cm–1) residues. The mechanisms of oxygen binding are discussed for hemoglobin located in the submembrane region and cytoplasm of the cell.

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.

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.

AAV-delivered microdystrophin genes hold great promise for Duchenne muscular dystrophy (DMD) treatment. It is anticipated that the optimization of engineered dystrophin genes will be required to increase the efficacy and reduce the immunogenicity of transgenic proteins. An in vitro system is required for the efficacy testing of genetically engineered dystrophin genes. We report here on the proof of concept for an in vitro assay based on the assessment of sarcolemma damage after repetitively applied electrical stimuli. The primary cell culture of myoblasts was established from wild-type C57BL/10ScSnJ and dystrophin-deficient mdx mice. The preparation parameters and the differentiation of contractile myotubes were optimized. DAPI and TO-PRO-3 dyes were used to assess myotubular membrane permeability in response to electrical pulse stimulation (EPS). Myotubes derived from mdx mice exhibited a greater increase in membrane damage, as assessed by TO-PRO-3-measured permeability after EPS, than was exhibited by the healthy control myotubes. AAV-DJ particles carrying the microdystrophin gene were used to transduce mdx-derived differentiated myotubes. Microdystrophin delivery ameliorated the disease phenotype and reduced the EPS-induced membrane damage to a level comparable to that of the healthy controls. Thus, the in vitro system was shown to be capable of supporting studies on DMD gene therapy.

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.

Long-term study on the identification of Na,K-ATPase endogenous inhibitors in mammalian tissues has resulted in the discovery of ouabain, marinobufagenin (MBG), and other cardiotonic steroids (CTS) in the blood plasma. Production of ouabain and MBG is increased in essential hypertension and other diseases associated with hypervolemia. Here, we compared the effects of ouabain and MBG on the Na,K-ATPase activity (measured as the transport of Na+, K+, and Rb+ ions) and proliferation and death of human renal epithelial cells (HRECs) and human umbilical vein endothelial cells (HUVEC) expressing α1-Na,K-ATPase. Ouabain concentration that provided the half-maximal inhibition of the Rb+ influx (IC50) into HRECs and HUVECs was 0.07 μM. In both types of cells, the IC50 values for MBG were 10 times higher than for ouabain. Incubation of HREC and HUVEC with 0.001-0.01 μM ouabain for 30 h resulted in 40% increase in the [3H]thymidine incorporation into DNA; further elevation of ouabain concentration to 0.1 μM completely suppressed DNA synthesis. MBG at the concentration of 0.1 μM activated DNA synthesis by 25% in HRECs, but not in HUVECs; 1 μM MBG completely inhibited DNA synthesis in HRECs and by 50% in HUVECs. In contrast to HRECs, incubation of HUVECs in the serum-free medium induced apoptosis, which was almost completely suppressed by ouabain and MBG at the concentrations of 0.1 and 3 μM, respectively. Based on these data, we can conclude that (i) the effect of MBG at the concentrations detected in the blood plasma (<0.01 μM) on HRECs and HUVECs was not due to the changes in the [Na+]i/[K+]i ratio; (ii) the effect of physiological concentrations of ouabain on these cells might be mediated by the activation of Na,K-ATPase, leading to cell proliferation.

BACKGROUND/AIMS:Prolonged hyperosmotic shrinkage evokes expression of osmoprotective genes via nuclear factor NFAT5-mediated pathway and activates Na+ influx via hypertonicity-induced cation channels (HICC). In human umbilical vein endothelial cells (HUVEC) elevation of intracellular sodium concentration ([Na+]i) triggers transcription of dozens of early response genes (ERG). This study examined the role of monovalent cations in the expression of Na+i-sensitive ERGs in iso- and hyperosmotically shrunken HUVEC.METHODS:Cell volume was measured by 3D reconstruction of cell shape and as 14C-urea available space. Intracellular Na+ and K+ content was measured by flame atomic absorption spectrometry. ERG transcription was estimated by RT-PCR.RESULTS:Elevation of medium osmolality by 150 mM mannitol or cell transfer from hypo- to isosmotic medium decreased cell volume by 40-50%. Hyperosmotic medium increased [Na+]i by 2-fold whereas isosmotic shrinkage had no impact on this parameter. Hyperosmotic but not isosmotic shrinkage increased up-to 5-fold the content of EGR1, FOS, ATF3, ZFP36 and JUN mRNAs. Expression of these ERGs triggered by hyperosmotic shrinkage and Na+,K+-ATPase inhibition by 0.1 µM ouabain exhibited positive correlation (R2=0.9383, p=0.0005). Isosmotic substitution of NaCl by N-methyl-D-glucamine abolished an increment of [Na+]i and ERG expression triggered by mannitol addition.CONCLUSION:Augmented expression of ERGs in hyperosmotically shrunken HUVEC is mediated by elevation of [Na+]i.

The review summarizes the history of the discovery in the mid-70s of the impaired ion transport across the plasma membrane of cells during primary arterial hypertension. A half-century's history of studies on the molecular nature of the ionic transporters underlying these disorders and the mechanisms mediated by them leading to the development of hypertension and complications caused by a long-term increase in blood pressure is analyzed.