Different neuronal Na+/K+‐ATPase isoforms are involved in diverse signaling pathways

Inhibition of rat neuronal Na⁺/K⁺‐ATPase α3 isoform at low (100 nM) ouabain concentration led to activation of MAP kinase cascade via PKC and PIP₃ kinase. In contrast to ouabain‐sensitive α3 isoform of Na⁺/K⁺‐ATPase, an ouabain‐resistant α1 isoform (inhibition with 1 mM of ouabain) of Na⁺/K⁺‐ATPase regulates MAP kinase via Src kinase dependent reactions. Using of Annexin V‐FITC apoptotic test to determine the cells with early apoptotic features allows to conclude that α3 isoform stimulates and α1 suppresses apoptotic process in cerebellum neurons. These data are the first demonstration showing participation of ouabain‐resistant (α1) and ouabain‐sensitive (α3) Na⁺/K⁺‐ATPase isoforms in diverse signaling pathways in neuronal cells. Copyright © 2010 John Wiley & Sons, Ltd.     

The program of friend cell erythroid differentiation: Early changes in Na+/K+ ATPase function

Treatment of Friend erythroleukemia cells with several different chemical agents causes an early decrease in the ⁸⁶Rb⁺ influx mediated by Na⁺/K⁺ adenosine triphosphatase (ATPase). These agents, which induced Friend cells to differentiate, include dimethylsulfoxide (DMSO), ouabain, hypoxanthine, and actinomycin D. The magnitude of the early decrease in ⁸⁶Rb⁺ influx correlates with the proportion of cells in cultures of inducible Friend cell clones which later go on to synthesize hemoglobin. Compounds which do not incude differentiation in these cells, such as xanthine, exogenous hematin, and erythropoietin, do not cause a change in ⁸⁶Rb⁺ influx. A change in the intracellular K⁺ ion concentration does not occur during induction by DMSO because, although there is a decrease in K⁺ content per cell soon after induction, there is a parallel decrease in cell volume. These results and previous observations from this laboratory are discussed in terms of the posible involvement of the Na⁺/K⁺ ATPase in Friend cell differentiation.     

Glutathione S‐transferase π complexes with and stimulates Na+,K+‐ATPase

Glutathione S‐transferase (GST) was found to complex with the Na⁺,K⁺‐ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α‐subunit of the Na⁺,K⁺‐ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺‐ATPase. Co‐immunoprecipitation experiments, using the anti‐α‐subunit antiserum to precipitate the GST‐Na⁺,K⁺‐ATPase complex and then using antibodies specific to an isoform of GST to identify the co‐precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺‐ATPase. GST stimulated the Na⁺,K⁺‐ATPase activity up to 1.4‐fold. The level of stimulation exhibited a saturable dose–response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺‐ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺‐ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺‐ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Temperature-specific inhibition of human red cell Na+/K+ ATPase by 2,450-MHz microwave radiation

The ATPase activity in human red blood cell membranes was investigated in vitro as a function of temperature and exposure to 2,450-MHz continuous wave microwave radiation to confirm and extend a report of Na+ transport inhibition under certain conditions of temperature and exposure. Assays were conducted spectrophotometrically during microwave exposure with a custom-made spectrophotometer-waveguide apparatus. Temperature profiles of total ATPase and Ca+2 ATPase (ouabain-inhibited) activity between 17 and 31 degrees C were graphed as an Arrhenius plot. Each data set was fitted to two straight lines which intersect between 23 and 24 degrees C. The difference between the total and Ca+2 ATPase activities, which represented the Na+/K+ ATPase activity, was also plotted and treated similarly to yield an intersection near 25 degrees C. Exposure of membrane suspensions to electromagnetic radiation, at a dose rate of 6 W/kg and at five temperatures between 23 and 27 degrees C, resulted in an activity change only for the Na+/K+ ATPase at 25 degrees C. The activity decreased by approximately 35% compared to sham-irradiated samples. A possible explanation for the unusual temperature/microwave interaction is proposed.     

High salt‐treatment‐induced Na+ extrusion and low salt‐treatment‐induced Na+ accumulation in suspension‐cultured cells of the mangrove plant,Bruguiera sexangula

A suspension‐cultured cell strain of the mangrove plant (Bruguiera sexangula) was established from a callus culture and maintained in an amino acid medium in the absence of NaCl. NaCl non‐adapted cells were transferred to media containing 0–200 mm NaCl. The initial growth rate decreased gradually with increasing salt concentrations. However, at up to 150 mm NaCl, cell number growth at the highest point was almost the same as that at lower salt concentrations. Cells even continued to grow in the presence of 200 mm NaCl. Cells incubated in a medium containing 50 mm NaCl for 3 weeks accumulated Na⁺, while those incubated in 150 mm NaCl for 2 d showed only a transient increase in Na⁺ and Cl^– concentrations. In the latter treatment, the intracellular concentration of Na⁺ returned to the original low level within 2 weeks. It took a longer time for Cl^– to return to its original level. As a result, the Na⁺ and Cl^– concentrations in cells cultured with 50 mm NaCl were much larger than those in cells cultured with 150 mm NaCl. The intracellular distribution of ions after transfer to the medium containing 150 mm NaCl was analysed by isolating the vacuoles. Treatment with amiloride, an inhibitor of the Na⁺/H⁺ antiporter, suppressed the recovery of Na⁺ to the original level in the cells. Treatment with 150 mm NaCl for 3 d stimulated the activities of both the vanadate‐dependent H⁺‐ATPase and the Na⁺/H⁺ antiporter in the plasma membrane fraction.     

Na+,K+/H+ antiporters regulate the pH of endoplasmic reticulum and auxin‐mediated development

AtNHX5 and AtNHX6 are endosomal Na⁺,K⁺/H⁺ antiporters that are critical for growth and development in Arabidopsis, but the mechanism behind their action remains unknown. Here, we report that AtNHX5 and AtNHX6, functioning as H⁺ leak, control auxin homeostasis and auxin‐mediated development. We found that nhx5 nhx6 exhibited growth variations of auxin‐related defects. We further showed that nhx5 nhx6 was affected in auxin homeostasis. Genetic analysis showed that AtNHX5 and AtNHX6 were required for the function of the endoplasmic reticulum (ER)‐localized auxin transporter PIN5. Although AtNHX5 and AtNHX6 were colocalized with PIN5 at ER, they did not interact directly. Instead, the conserved acidic residues in AtNHX5 and AtNHX6, which are essential for exchange activity, were required for PIN5 function. AtNHX5 and AtNHX6 regulated the pH in ER. Overall, AtNHX5 and AtNHX6 may regulate auxin transport across the ER via the pH gradient created by their transport activity. H⁺‐leak pathway provides a fine‐tuning mechanism that controls cellular auxin fluxes.     

Hypoxia‐induced increase in intracellular calcium concentration in endothelial cells: Role of the Na+‐glucose cotransporter

Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet‐activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca²⁺]_i) which then activates the phospholipase A₂ (PLA₂). The link between the decrease in ATP and the increase in [Ca²⁺]_i was not known and is investigated in this work. We first showed that the presence of extracellular Na⁺ was necessary to observe the hypoxia‐induced increase in [Ca²⁺]_i and the activation of PLA₂. This increase was not due to the release of Ca²⁺ from intracellular stores, since thapsigargin did not inhibit this process. The Na⁺/Ca²⁺ exchanger was involved since dichlorobenzamil inhibited the [Ca²⁺]_i and the PLA₂ activation. The glycolysis was activated, but the intracellular pH (pH_i) in hypoxic cells did not differ from control cells. Finally, the hypoxia‐induced increase in [Ca²⁺]_i and PLA₂ activation were inhibited by phlorizin, an inhibitor of the Na⁺‐glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na⁺ through the activated Na⁺‐glucose cotransport followed by the activation of the Na⁺/Ca²⁺ exchanger, resulting in a net influx of Ca²⁺. J. Cell. Biochem. 84: 115–131, 2002. © 2001 Wiley‐Liss, Inc.     

Identification of a VxP Targeting Signal in the Flagellar Na+/K+‐ATPase

Na⁺/K⁺‐ATPase (NKA) participates in setting electrochemical gradients, cardiotonic steroid signaling and cellular adhesion. Distinct isoforms of NKA are found in different tissues and subcellular localization patterns. For example, NKA α1 is widely expressed, NKA α3 is enriched in neurons and NKA α4 is a testes‐specific isoform found in sperm flagella. In some tissues, ankyrin, a key component of the membrane cytoskeleton, can regulate the trafficking of NKA. In the retina, NKA and ankyrin‐B are expressed in multiple cell types and immunostaining for each is striking in the synaptic layers. Labeling for NKA is also prominent along the inner segment plasma membrane (ISPM) of photoreceptors. NKA co‐immunoprecipitates with ankyrin‐B, but on a subcellular level colocalization of these two proteins varies dependent on the cell type. We used transgenic Xenopus laevis tadpoles to evaluate the subcellular trafficking of NKA in photoreceptors. GFP‐NKA α3 and α1 are localized to the ISPM, but α4 is localized to outer segments (OSs). We identified a VxP motif responsible for the OS targeting by using a series of chimeric and mutant NKA constructs. This motif is similar to previously identified ciliary targeting motifs. Given the structural similarities between OSs and flagella, our findings shed light on the subcellular targeting of this testes‐specific NKA isoform.      

Decreased neuronal Na+, K+ -ATPase activity in Atp1a3 heterozygous mice increases susceptibility to depression-like endophenotypes by chronic variable stress

Unipolar depression and bipolar depression are prevalent and debilitating diseases in need of effective novel treatments. It is becoming increasingly evident that depressive disorders manifest from a combination of inherited susceptibility genes and environmental stress. Genetic mutations resulting in decreased neuronal Na(+) ,K(+) -ATPase (sodium-potassium adenosine triphosphatase) activity may put individuals at risk for depression given that decreased Na(+) ,K(+) -ATPase activity is observed in depressive disorders and animal models of depression. Here, we show that Na(+) ,K(+) -ATPase α3 heterozygous mice (Atp1a3(+/-) ), with 15% reduced neuronal Na(+) ,K(+) -ATPase activity, are vulnerable to develop increased depression-like endophenotypes in a chronic variable stress (CVS) paradigm compared to wild-type littermates (Atp1a3(+/+) ). In Atp1a3(+/+) mice CVS did not decrease Na(+) ,K(+) -ATPase activity, however led to despair-like behavior in the tail suspension test (TST), anhedonia in a sucrose preference test and a minimal decrease in sociability, whereas in Atp1a3(+/-) mice CVS decreased neuronal Na(+) ,K(+) -ATPase activity to 33% of wild-type levels, induced despair-like behavior in the TST, anhedonia in a sucrose preference test, anxiety in the elevated plus maze, a memory deficit in a novel object recognition task and sociability deficits in a social interaction test. We found that a mutation that decreases neuronal Na(+) ,K(+) -ATPase activity interacts with stress to exacerbate depression. Furthermore, we observed an interesting correlation between Na(+) ,K(+) -ATPase activity and mood that may relate to both unipolar depression and bipolar disorder. Pharmaceuticals that increase Na(+) ,K(+) -ATPase activity or block endogenous Na(+) , K(+) -ATPase inhibition may provide effective treatment for depressive disorders and preclude depression in susceptible individuals.     

Cellular localization of the K+‐dependent Na+–Ca2+ exchanger NCKX5 and the role of the cytoplasmic loop in its distribution in pigmented cells

NCKX5 is a bidirectional K⁺‐dependent Na⁺–Ca²⁺ exchanger, which belongs to the SLC24A gene family. In particular, the A111T mutation of NCKX5 has been associated with reduced pigmentation in European populations. In contrast to other NCKX isoforms, which function in the plasma membrane (PM), NCKX5 has been shown to localize either in the trans‐Golgi network (TGN) or in melanosomes. Moreover, sequences responsible for retaining its intracellular localization are unknown. This study addresses two major questions: (i) clarification of intracellular location of NCKX5 and (ii) identification of sequences that retain NCKX5 inside the cell. We designed a set of cDNA constructs representing NCKX5 loop deletion mutants and NCKX2–NCKX5 chimeras to address these two questions after expression in pigmented MNT1 cells. Our results show that NCKX5 is not a PM resident and is exclusively located in the TGN. Moreover, the large cytoplasmic loop is the determinant for retaining NCKX5 in the TGN.     

Comparative genetics of Na+/K+‐ATPase in monarch butterfly populations with varying host plant toxicity

Herbivores have evolved numerous behavioural and physiological adaptations to host plants; however, molecular adaptations are still poorly understood. One well‐studied case comprises the specialist insects that feed on cardenolide‐containing plants. Here, convergent molecular evolution in the Na⁺/K⁺‐ATPase results in a reduced sensitivity to cardenolides across four insect orders. Because different plant species and genotypes differ in toxicity, Na⁺/K⁺‐ATPase may be under differential selection from geographically varying host plants. We examined the α subunit of Na⁺/K⁺‐ATPase in monarch butterflies (Danaus plexippus) from six worldwide populations to test whether differences in their host plant chemistry result in local adaptation at the molecular level. Although our study revealed multiple synonymous changes, we did not find these to be population‐specific, nor did we identify nonsynonymous changes. Additionally, we compared the amino acid sequence of this subunit across 19 species. We identified two novel changes at sites 836 (K836N) and 840 (E840R) in the αM7‐αM8 regions in the genus Danaus. Although previous studies focused on the first two trans‐membrane domains, C‐terminal domains may also interact with cardenolides. These results reveal a lack of molecular evolution of Na⁺/K⁺‐ATPase at the population level, and call for additional attention regarding the C‐terminal regions of this important enzyme.     

Differences in epithelial morphology correlate to Na+‐transport: A study of the proximal,mid, and distal regions of the coprodeum from hens on high and low NaCl diet

A study was performed to correlate regional morphology and amiloride inhibitable Na⁺‐transport in the coprodeal epithelium in hens, Gallus domesticus, on low‐NaCl diet and in controls. Proximal (close to colon), mid and distal (close to urodeum) regions were examined using light microscopy, transmission‐ and scanning electron microscopy. Na⁺‐transport was measured electrophysiologically in Ussing‐chambers in the proximal and distal regions. The epithelium, simple and columnar, is composed of absorptive intestinal epithelial cells, goblet cells, brush cells, migrating lymphoid cells, and entero‐endocrine cells. Brush cells, identified in avians for the first time, occur in highest number in the proximal part of the coprodeum in low‐NaCl hens. Na⁺‐transport is high in the low‐NaCl hens, ranging from 347μA/cm² (proximal) to 187μA/cm² (distal). In control hens, which correspond to hens on high‐NaCl diet, it is low in all regions (0–4 μA/cm²). Absorptive intestinal epithelial cells as well as brush cells adapt to variations in transepithelial Na⁺‐transport by regulating height and packing density of their microvilli, number, size, and localization of apical vesicles, and the width of the intercellular space. Regional differences in the epithelial cell composition and ultrastructure are closely correlated to transepithelial Na⁺‐transport but only in low‐NaCl hens, as controls do not show these variations. J. Morphol. 239:75–86, 1999. © 1999 Wiley‐Liss, Inc.