In vitro assay for the efficacy assessment of AAV vectors expressing microdystrophin.

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.

Effect of Dynamic and Static Load on the Concentration of Myokines in the Blood Plasma and Content of Sodium and Potassium in Mouse Skeletal Muscles.

Modulation of cytokine production by physical activity is of considerable interest, since it might be a promising strategy for correcting metabolic processes at both cellular and systemic levels. The content of IL-6, IL-8, and IL-15 in the plasma and the concentration of monovalent cations in the skeletal muscles of trained and untrained mice were studied at different periods after static and dynamic exercises. Dynamic loads caused an increase in the IL-6 content and decrease in the IL-15 content in the plasma of untrained mice, but produced no effect on the concentration of IL-8. In trained mice, the effect of a single load on the concentration of IL-6 and IL-15 in the plasma was enhanced, while the concentration of IL-8 decreased. Static loads produced a similar, but more pronounced effect on the plasma concentration of IL-6 and IL-15 compared the dynamic exercises; however, the concentration of IL-8 in response to the static exercise increased significantly. Prior training reinforced the described response for all the myokines studied. Dynamic load (swimming) increased the intracellular content of sodium but decreased the content of potassium in the mouse musculus soleus. Similar response was observed after the static load (grid hanging) in the musculus biceps; but no correlation of this response with the prior training was found. Possible mechanisms involved in the regulation of cytokine secretion after exercise are discussed, including triggering of gene transcription in response to changes in the [Na+]i/[K+]I ratio.

Depth of the Steroid Core Location Determines the Mode of Na,K-ATPase Inhibition by Cardiotonic Steroids.

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 -resistant (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).

Transcriptomic Changes in Endothelial Cells Triggered by Na,K-ATPase Inhibition: A Search for Upstream Na+i/K+i Sensitive Genes.

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.

Elevation of Intracellular Na+ Contributes to Expression of Early Response Genes Triggered by Endothelial Cell Shrinkage.

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.

Anti-fibrotic effects of tannic acid through regulation of a sustained TGF-beta receptor signaling.

BACKGROUND: Pulmonary fibrosis is a progressive disease characterized by structural distortion of the lungs. Transforming growth factor-beta (TGF-beta) is a key cytokine implicated in the pathogenesis of pulmonary fibrosis. TGF-beta-induced myofibroblast differentiation characterized by expression of smooth muscle alpha-actin and extracellular matrix proteins is a key process in pathogenesis of fibrotic disease. Tannic acid is a natural polyphenol with diverse applications. In this study, we investigated the effect of tannic acid on myofibroblast differentiation and pulmonary fibrosis in cultured cells and in bleomycin model of the disease. METHODS: Primary cultured human lung fibroblasts (HLF) were used. The relative levels of proteins were determined by Western blotting. HLF contraction was measured by traction microscopy. Bleomycin-induced pulmonary fibrosis in mice was used as the disease model. RESULTS: Tannic acid inhibited TGF-beta-induced expression of collagen-1 and smooth muscle alpha-actin (SMA) as well as force generation by HLF. Tannic acid did not affect initial phosphorylation of Smad2 in response to TGF-beta, but significantly inhibited sustained Smad2 phosphorylation, which we recently described to be critical for TGF-beta-induced myofibroblast differentiation. Accordingly, tannic acid inhibited Smad-dependent gene transcription in response to TGF-beta, as assessed using luciferase reporter for the activity of Smad-binding elements. Finally, in mouse model of bleomycin-induced pulmonary fibrosis, therapeutic application of tannic acid resulted in a significant reduction of lung fibrosis, decrease in collagen-1 content and of Smad2 phosphorylation in the lungs. CONCLUSIONS: This study demonstrates the anti-fibrotic effect of tannic acid in vitro and in vivo through a regulation of sustained Smad2 phosphorylation.

Control of lung myofibroblast transformation by monovalent ion transporters.

Myofibroblast differentiation is a critical process in the pathogenesis of tissue fibrosis. We focus our mini-review on recent data showing an implication of monovalent ion transporters in fibroblast to myofibroblast transformation of human lung fibroblasts (HLF). In cultured HLF, cardiotonic steroids (CTS) known as potent inhibitors of Na+,K+-ATPase suppress myofibroblast differentiation in parallel with up- and down-regulated expression of cyclooxygenase-2 (COX-2) and TGF-ß receptor subunit TGFBR2, respectively. K+-free medium mimics antifibrotic action of CTS indicating a key role of elevated intracellular [Na+]i/[K+]i ratio. Augmented expression of COX-2 is abolished by inhibition of Na+/Ca2+ exchanger. Side-by-side with CTS acting via elevation of the [Na+]i/[K+]i ratio fibroblast to myofibroblast transformation is also suppressed by potent inhibitors of Ca2+-activated chloride channels tannic acid and K+,Cl- cotransporter DIOA. The relative impact of [Formula: see text] -mediated and -independent signaling triggered by elevated [Na+]i/[K+]i ratio and altered intracellular anion handling in transcriptomic changes involved in myofibroblast differentiation should be examined further.

Ubiquitous and cell type-specific transcriptomic changes triggered by dissipation of monovalent cation gradients in rodent cells: Physiological and pathophysiological implications.

Elevation of [Na+]i/[K+]i-ratio is considered as one of the major signals triggering transcriptomic changes in various cells types. In this study, we identified ubiquitous and cell type-specific [Formula: see text] -sensitive genes by comparative analysis of transcriptomic changes in ouabain-treated rat aorta smooth muscle cells and rat aorta endothelial cells (RASMC and RAEC, respectively), rat cerebellar granule cells (RCGC), and mouse C2C12 myoblasts. Exposure of the cells to ouabain increased intracellular Na+ content by ~14, 8, 7, and 6-fold and resulted in appearance of 7577, 2698, 2120, and 1146 differentially expressed transcripts in RAEC, RASMC, C2C12, and RCGC, respectively. Eighty-three genes were found as the intersection of the four sets of identified transcripts corresponding to each cell type and are classified as ubiquitous. Among the 10 top upregulated ubiquitous transcripts are the following: Dusp6, Plk3, Trib1, Ccl7, Mafk, Atf3, Ptgs2, Cxcl1, Spry4, and Coq10b. Unique transcripts whose expression is cell-specific include 4897, 1523, 789, and 494 transcripts for RAEC, RASMC, C2C12, and RCGC, respectively. The role of gene expression and signal pathways induced by dissipation of transmembrane gradient of monovalent cations in the development of various diseases is discussed with special attention to cardiovascular and pulmonary illnesses.

Search for Upstream Cell Volume Sensors: The Role of Plasma Membrane and Cytoplasmic Hydrogel.

The plasma membrane plays a prominent role in the regulation of cell volume by mediating selective transport of extra- and intracellular osmolytes. Recent studies show that upstream sensors of cell volume changes are mainly located within the cytoplasm that displays properties of a hydrogel and not in the plasma membrane. Cell volume changes occurring in anisosmotic medium as well as in isosmotic environment affect properties of cytoplasmic hydrogel that, in turn, trigger rapid regulatory volume increase and decrease (RVI and RVD). The downstream signaling pathways include reorganization of 2D cytoskeleton and altered composition of polyphosphoinositides located on the inner surface of the plasma membrane. In addition to its action on physico-chemical properties of cytoplasmic hydrogel, cell volume changes in anisosmotic conditions affect the ionic strength of the cytoplasm and the [Na+]i/[K+]i ratio. Elevated intracellular ionic strength evoked by long term exposure of cells to hypertonic environment resulted in the activation of TonEBP and augmented expression of genes controlling intracellular organic osmolyte levels. The role of Na+i/K+i -sensitive, Ca2+i -mediated and Ca2+i-independent mechanisms of excitation-transcription coupling in cell volume-adjustment remains unknown.

Electrical pulse stimulation decreases electrochemical Na+ and K+ gradients in C2C12 myotubes.

Electrical pulse stimulation (EPS)-treated cultured myotubes are widely employed as an in vitro model of muscle contraction. Here we examined time-dependent EPS action and dose-dependent ouabain action on [Na+]i and [K+]i in C2C12 myotubes. After 2 h of EPS (40 V, 1 Hz, 10 ms) [Na+]i increased by ∼150% whereas [K+]i declined by ∼20%. 3 µM ouabain had a negligible impact on [Na+]i and [K+]i in control cells but increased the [Na+]i/[K+]i ratio in EPS-treated myotubes by 85%. Thus, our results show for the first time that EPS results in dissipation of Na+ and K+ gradients in cultured myotubes and suggest that the augmented production of endogenous cardiotonic steroids may contribute to elevation of the [Na+]i/[K+]i ratio in exercising muscle.

Na⁺i,K⁺i-Dependent and -Independent Signaling Triggered by Cardiotonic Steroids: Facts and Artifacts.

Na⁺,K⁺-ATPase is the only known receptor of cardiotonic steroids (CTS) whose interaction with catalytic α-subunits leads to inhibition of this enzyme. As predicted, CTS affect numerous cellular functions related to the maintenance of the transmembrane gradient of monovalent cations, such as electrical membrane potential, cell volume, transepithelial movement of salt and osmotically-obliged water, symport of Na⁺ with inorganic phosphate, glucose, amino acids, nucleotides, etc. During the last two decades, it was shown that side-by-side with these canonical Na⁺i/K⁺i-dependent cellular responses, long-term exposure to CTS affects transcription, translation, tight junction, cell adhesion and exhibits tissue-specific impact on cell survival and death. It was also shown that CTS trigger diverse signaling cascades via conformational transitions of the Na⁺,K⁺-ATPase α-subunit that, in turn, results in the activation of membrane-associated non-receptor tyrosine kinase Src, phosphatidylinositol 3-kinase and the inositol 1,4,5-triphosphate receptor. These findings allowed researchers to propose that endogenous CTS might be considered as a novel class of steroid hormones. We focus our review on the analysis of the relative impact Na⁺i,K⁺i-mediated and -independent pathways in cellular responses evoked by CTS.

Role of cytoskeleton network in anisosmotic volume changes of intact and permeabilized A549 cells.

Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium. It was significantly slowed-down in both cell preparations after disruption of actin microfilaments by cytochalasin B, suggesting that rapid water release by intact cytoplasmic hydrogel contributes to hyperosmotic shrinkage. In hyposmotic swelling experiments, disruption of microtubules by vinblastine attenuated the maximal amplitude of swelling in intact cells and completely abolished it in permeabilized cells. The swelling of intact cells also triggered ~10-fold elevation of furosemide-resistant (86)Rb+ (K+) permeability and the regulatory volume decrease (RVD), both of which were abolished by Ba2+. Interestingly, RVD and K+ permeability remained unaffected in cytocholasin/vinblastine treated cells demonstrating that cytoskeleton disruption has no direct impact on Ba2+-sensitive K+-channels involved in RVD. Our results show, for the first time, that the cytoskeleton network contributes directly to passive cell volume adjustments in anisosmotic media via the modulation of the water retained by the cytoplasmic hydrogel.

Calcium is not required for triggering volume restoration in hypotonically challenged A549 epithelial cells.

Maintenance of cell volume is a fundamental housekeeping function in eukaryotic cells. Acute cell swelling activates a regulatory volume decrease (RVD) process with poorly defined volume sensing and intermediate signaling mechanisms. Here, we analyzed the putative role of Ca2+ signaling in RVD in single substrate-adherent human lung epithelial A549 cells. Acute cell swelling was induced by perfusion of the flow-through imaging chamber with 50 % hypotonic solution at a defined fluid turnover rate. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and cell volume were monitored simultaneously with ratiometric Fura-2 fluorescence and 3D reconstruction of stereoscopic single-cell images, respectively. Hypotonic challenge caused a progressive swelling peaking at ∼20 min and followed, during the next 20 min, by RVD of 60 ± 7 % of the peak volume increase. However, at the rate of swelling used in our experiments, these processes were not accompanied by a measurable increment of [Ca2+]i. Loading with intracellular Ca2+ chelator BAPTA slightly delayed peak of swelling but did not prevent RVD in 82 % of cells. Further, electrophysiology whole-cell patch-clamp experiments showed that BAPTA did not block activation of volume-regulated anion channel (VRAC) measured as swelling-induced outwardly rectifying 5-nitro-2-(3-phenylpropyl-amino) benzoic acid sensitive current. Together, our data suggest that intracellular Ca2+-mediated signaling is not essential for VRAC activation and subsequent volume restoration in A549 cells.

Regulation of myofibroblast differentiation by cardiac glycosides.

Myofibroblast differentiation is a key process in pathogenesis of fibrotic diseases. Cardiac glycosides (ouabain, digoxin) inhibit Na(+)-K(+)-ATPase, resulting in increased intracellular [Na(+)]-to-[K(+)] ratio in cells. Microarray analysis suggested that increased intracellular [Na(+)]/[K(+)] ratio may promote the expression of cyclooxygenase-2 (COX-2), a critical enzyme in the synthesis of prostaglandins. Given antifibrotic effects of prostaglandins through activation of protein kinase A (PKA), we examined if cardiac glycosides stimulate COX-2 expression in human lung fibroblasts and how they affect myofibroblast differentiation. Ouabain stimulated a profound COX-2 expression and a sustained PKA activation, which was blocked by COX-2 inhibitor or by COX-2 knockdown. Ouabain-induced COX-2 expression and PKA activation were abolished by the inhibitor of the Na(+)/Ca(2+) exchanger, KB-R4943. Ouabain inhibited transforming growth factor-ß (TGF-ß)-induced Rho activation, stress fiber formation, serum response factor activation, and the expression of smooth muscle α-actin, collagen-1, and fibronectin. These effects were recapitulated by an increase in intracellular [Na(+)]/[K(+)] ratio through the treatment of cells with K(+)-free medium or with digoxin. Although inhibition of COX-2 or of the Na(+)/Ca(2+) exchanger blocked ouabain-induced PKA activation, this failed to reverse the inhibition of TGF-ß-induced Rho activation or myofibroblast differentiation by ouabain. Together, these data demonstrate that ouabain, through the increase in intracellular [Na(+)]/[K(+)] ratio, drives the induction of COX-2 expression and PKA activation, which is accompanied by a decreased Rho activation and myofibroblast differentiation in response to TGF-ß. However, COX-2 expression and PKA activation are not sufficient for inhibition of the fibrotic effects of TGF-ß by ouabain, suggesting that additional mechanisms must exist.

Deoxygenation Affects Composition of Membrane-Bound Proteins in Human Erythrocytes.

BACKGROUND/AIMS: ATP release from erythrocyte plays a key role in hypoxia-induced elevation of blood flow in systematic circulation. We have previously shown that hemolysis contributes to erythrocyte ATP release triggered by several stimuli, including hypoxia, but the molecular mechanisms of hypoxia-increased membrane fragility remain unknown. METHODS: In this study, we compared the action of hypoxia on hemolysis, ATP release and the composition of membrane-bound proteins in human erythrocytes. RESULTS: Twenty minutes incubation of human erythrocytes in the oxygen-free environment increased the content of extracellular hemoglobin by ∼1.5 fold. Paired measurements of hemoglobin and ATP content in the same samples, showed a positive correlation between hemolysis and ATP release. Comparative analysis of SDS-PAGE electrophoresis of erythrocyte ghosts obtained under control and deoxygenated conditions revealed a ∼2-fold elevation of the content of membrane-bound protein with Mr of ∼60 kDa. CONCLUSION: Deoxygenation of human erythrocytes affects composition of membrane-bound proteins. Additional experiments should be performed to identify the molecular origin of 60 kDa protein and its role in the attenuation of erythrocyte integrity and ATP release in hypoxic conditions.

Hemolysis is a primary ATP-release mechanism in human erythrocytes.

The hypothesis that regulated ATP release from red blood cells (RBCs) contributes to nitric oxide-dependent control of local blood flow has sparked much interest in underlying release mechanisms. Several stimuli, including shear stress and hypoxia, have been found to induce significant RBC ATP release attributed to activation of ATP-conducting channels. In the present study, we first evaluated different experimental approaches investigating stimulated RBC ATP release and quantifying hemolysis. We then measured ATP and free hemoglobin in each and every RBC supernatant sample to directly assess the contribution of hemolysis to ATP release. Hypotonic shock, shear stress, and hypoxia, but not cyclic adenosine monophosphate agonists, significantly enhanced ATP release. It tightly correlated, however, with free hemoglobin in RBC supernatants, indicating that lysis was responsible for most, if not all, ATP release. Luminescence ATP imaging combined with simultaneous infrared cell imaging showed that ATP was released exclusively from lysing cells with no contribution from intact cells. In summary, with all stimuli tested, we found no evidence of regulated ATP release from intact RBCs other than by cell lysis. Such a release mechanism might be physiologically relevant in vivo, eg, during exercise and hypoxia where intravascular hemolysis, predominantly of senescent cells, is augmented.

Antifibrotic effects of noscapine through activation of prostaglandin E2 receptors and protein kinase A.

Myofibroblast differentiation is a key process in the pathogenesis of fibrotic disease. We have shown previously that differentiation of myofibroblasts is regulated by microtubule polymerization state. In this work, we examined the potential antifibrotic effects of the antitussive drug, noscapine, recently found to bind microtubules and affect microtubule dynamics. Noscapine inhibited TGF-ß-induced differentiation of cultured human lung fibroblasts (HLFs). Therapeutic noscapine treatment resulted in a significant attenuation of pulmonary fibrosis in the bleomycin model of the disease. Noscapine did not affect gross microtubule content in HLFs, but inhibited TGF-ß-induced stress fiber formation and activation of serum response factor without affecting Smad signaling. Furthermore, noscapine stimulated a rapid and profound activation of protein kinase A (PKA), which mediated the antifibrotic effect of noscapine in HLFs, as assessed with the PKA inhibitor, PKI. In contrast, noscapine did not activate PKA in human bronchial or alveolar epithelial cells. Finally, activation of PKA and the antifibrotic effect of noscapine in HLFs were blocked by the EP2 prostaglandin E2 receptor antagonist, PF-04418948, but not by the antagonists of EP4, prostaglandin D2, or prostacyclin receptors. Together, we demonstrate for the first time the antifibrotic effect of noscapine in vitro and in vivo, and we describe a novel mechanism of noscapine action through EP2 prostaglandin E2 receptor-mediated activation of PKA in pulmonary fibroblasts.

Salt and gene expression: evidence for [Na+]i/[K+]i-mediated signaling pathways.

Our review focuses on the recent data showing that gene transcription and translation are under the control of signaling pathways triggered by modulation of the intracellular sodium/potassium ratio ([Na+]i/[K+]i). Side-by-side with sensing of osmolality elevation by tonicity enhancer-binding protein (TonEBP, NFAT5), [Na+]i/[K+]i-mediated excitation-transcription coupling may contribute to the transcriptomic changes evoked by high salt consumption. This novel mechanism includes the sensing of heightened Na+ concentration in the plasma, interstitial, and cerebrospinal fluids via augmented Na+ influx in the endothelium, immune system cells, and the subfornical organ, respectively. In these cells, [Na+]i/[K+]i ratio elevation, triggered by augmented Na+ influx, is further potentiated by increased production of endogenous Na+,K+-ATPase inhibitors documented in salt-sensitive hypertension.

Salt and osmosensing: role of cytoplasmic hydrogel.

Osmotic perturbations, occurring frequently under physiological and pathological conditions, alter cell size/volume and function. To protect cellular homeostasis, cell osmo- and volume-sensing mechanisms activate volume compensatory processes. The plasma membrane plays a prominent role in cell volume regulation by mediating the selective transport of extra- and intracellular osmolytes. The function of the membrane-enclosed cytoplasm in osmosensing and cell volume homeostasis is much less appreciated. We present current concepts and discuss evidence of cell volume sensors with emphasis on the hydrogel nature of the mammalian cytoplasm and its intrinsic osmosensitivity.

Critical role of the α1-Na(+), K(+)-ATPase subunit in insensitivity of rodent cells to cytotoxic action of ouabain.

In rodents, ubiquitous α1-Na(+), K(+)-ATPase is inhibited by ouabain and other cardiotonic steroids (CTS) at ~10(3)-fold higher concentrations than those effective in other mammals. To examine the specific roles of the CTS-sensitive α1S- and CTS-resistant α1R-Na(+), K(+)-ATPase isoforms, we compared the effects of ouabain on intracellular Na(+) and K(+) content, cell survival, and mitogen-activated protein kinases (MAPK) in human and rat vascular smooth muscle cells (HASMC and RASMC), human and rat endothelial cells (HUVEC and RAEC), and human and rat brain astrocytes. 6-h exposure of HASMC and HUVEC to 3 µM ouabain dramatically increased the intracellular [Na(+)]/[K(+)] ratio to the same extend as in RASMC and RAEC treated with 3000 µM ouabain. In 24, 3 µM ouabain triggered the death of all types of human cells used in this study. Unlike human cells, we did not detect any effect of 3000-5000 µM ouabain on the survival of rat cells, or smooth muscle cells from mouse aorta (MASMC). Unlike in the wild-type α1(R/R) mouse, ouabain triggered death of MASMC from α1(S/S) mouse expressing human α1-Na(+), K(+)-ATPase. Furthermore, transfection of HUVEC with rat α1R-Na(+), K(+)-ATPase protected them from the ouabain-induced death. In HUVEC, ouabain led to phosphorylation of p38 MAPK, whereas in RAEC it stimulated phosphorylation of ERK1/2. Overall, our results, demonstrate that the drastic differences in cytotoxic action of ouabain on human and rodent cells are caused by unique features of α1S/α1R-Na(+), K(+)-ATPase, rather than by any downstream CTS-sensitive/resistant components of the cell death machinery.