Yeast plasma membrane Ena1p ATPase alters alkali-cation homeostasis and confers increased salt tolerance in tobacco cultured cells

In plants, the plasma membrane Na(+)/H(+) antiporter is the only key enzyme that extrudes cytosolic Na(+) and contributes to salt tolerance. But in fungi, the plasma membrane Na(+)/H(+) antiporter and Na(+)-ATPase are known to be key enzymes for salt tolerance. Saccharomyces cerevisiae Ena1p ATPase encoded by the ENA1/PMR2A gene is primarily responsible for Na(+) and Li(+) efflux across the plasma membrane during salt stress and for K(+) efflux at high pH and high K(+). To test if the yeast ATPase would improve salt tolerance in plants, we expressed a triple hemagglutinin (HA)-tagged Ena1p (Ena1p-3HA) in cultured tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY2) cells. The Ena1p-3HA proteins were correctly localized to the plasma membrane of transgenic BY2 cells and conferred increased NaCl and LiCl tolerance to the cells. Under moderate salt stress conditions, the Ena1p-3HA-expressing BY2 clones accumulated lower levels of Na(+) and Li(+) than nonexpressing BY2 clones. Moreover, the Ena1p-3HA expressing BY2 clones accumulated lower levels of K(+) than nonexpressing cells under no-stress conditions. These results suggest that the yeast Ena1p can also function as an alkali-cation (Na(+), Li(+), and K(+)) ATPase and alter alkali-cation homeostasis in plant cells. We conclude that, even with K(+)-ATPase activity, Na(+)-ATPase activity of the yeast Ena1p confers increased salt tolerance to plant cells during salt stress.     

Stress response to high osmolarity in Trypanosoma cruzi epimastigotes

Trypanosoma cruzi undergoes differentiation in the rectum of triatomine, where increased osmolarity is caused mainly by elevated content of NaCl from urine. Early biochemical events in response to high osmolarity in this parasite have not been totally elucidated. In order to clarify the relationship between these events and developmental stages of T. cruzi, epimastigotes were subjected to hyperosmotic stress, which caused activation of Na(+)/H(+) exchanger from acidic vacuoles and accumulation of inositol trisphosphate (InsP(3)). Suppression of InsP(3) levels was observed in presence of intracellular Ca(2+) chelator or pre-treatment with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), which also inhibited the alkalinization of acidic vacuoles via a Na(+)/H(+) exchanger and the consequent increase in cytosolic calcium. These effects were activated and inhibited by PMA and Chelerythrine respectively, suggesting regulation by protein kinase C. The T. cruzi Na(+)/H(+) exchanger, TcNHE1, has 11 transmembrane domains and is localized in acidic vacuoles of epimastigotes. The analyzed biochemical changes were correlated with morphological changes, including an increase in the size of acidocalcisomes and subsequent differentiation to an intermediate form. Both processes were delayed when TcNHE1 was inhibited by EIPA, suggesting that these early biochemical events allow the parasite to adapt to conditions faced in the rectum of the insect vector.     

Plasma membrane depolarization without repolarization is an early molecular event in anti-Fas-induced apoptosis

The movement of intracellular monovalent cations has previously been shown to play a critical role in events leading to the characteristics associated with apoptosis. A loss of intracellular potassium and sodium occurs during apoptotic cell shrinkage establishing an intracellular environment favorable for nuclease activity and caspase activation. We have now investigated the potential movement of monovalent ions in Jurkat cells that occur prior to cell shrinkage following the induction of apoptosis. A rapid increase in intracellular sodium occurs early after apoptotic stimuli suggesting that the normal negative plasma membrane potential may change during cell death. We report here that diverse apoptotic stimuli caused a rapid cellular depolarization of Jurkat T-cells that occurs prior to and after cell shrinkage. In addition to the early increase in intracellular Na(+), (86)Rb(+) studies reveal a rapid inhibition of K(+) uptake in response to anti-Fas. These effects on Na(+) and K(+) ions were accounted for by the inactivation of the Na(+)/K(+)-ATPase protein and its activity. Furthermore, ouabain, a cardiac glycoside inhibitor of the Na(+)/K(+)-ATPase, potentiated anti-Fas-induced apoptosis. Finally, activation of an anti-apoptotic signal, i.e. protein kinase C, prevented both cellular depolarization in response to anti-Fas and all downstream characteristics associated with apoptosis. Thus cellular depolarization is an important early event in anti-Fas-induced apoptosis, and the inability of cells to repolarize via inhibition of the Na(+)/K(+)-ATPase is a likely regulatory component of the death process.     

Changes in intracellular sodium, chlorine, and potassium concentrations in staurosporine-induced apoptosis

Ion gradients across the plasma membrane, fundamentally K(+), play a pivotal role in the execution phase of apoptosis. However, little is known about other monovalent anions (Cl(-)) or cations (Na(+)) in apoptosis. In addition, the relationship between changes in total ion composition and morphological and biochemical events are poorly understood. We investigated simultaneous changes in sodium (Na), chlorine (Cl), and potassium (K) concentrations in stauroporine-induced apoptosis by quantitative electron probe X-ray microanalysis (EPXMA) in single cells. Apoptotic cells identified unequivocally from the presence of chromatin condensation in backscattered electron images were characterized by an increase in intracellular Na, a decrease in intracellular Cl and K concentrations, and a decrease in K/Na ratio. The ouabain-sensitive Rb-uptake assay demonstrated a net decrease in Na(+)/K(+)-ATPase activity, suggesting that increases in Na and decreases in K and the K/Na ratio in apoptotic cells were related with inhibition of the Na(+)/K(+)-ATPase pump. These changes in diffusible elements were associated with externalization of phosphatidyl serine and oligonucleosomal fragmentation of DNA. This alteration in ion homeostasis and morphological hallmarks of apoptosis occur in cells that have lost their inner mitochondrial transmembrane potential and before the plasma membrane becomes permeable.     

Phospholemman expression is high in the newborn rabbit heart and declines with postnatal maturation

Phospholemman (PLM) is a small sarcolemmal protein that modulates the activities of Na(+)/K(+)-ATPase and the Na(+)/Ca(2+) exchanger (NCX), thus contributing to the maintenance of intracellular Na(+) and Ca(2+) homeostasis. We characterized the expression and subcellular localization of PLM, NCX, and the Na(+)/K(+)-ATPase alpha1-subunit during perinatal development. Western blotting demonstrates that PLM (15kDa), NCX (120kDa), and Na(+)/K(+)-ATPase alpha-1 (approximately 100kDa) proteins are all more than 2-fold higher in ventricular membrane fractions from newborn rabbit hearts (1-4-day old) compared to adult hearts. Our immunocytochemistry data demonstrate that PLM, NCX, and Na(+)/K(+)-ATPase are all expressed at the sarcolemma of newborn ventricular myocytes. Taken together, our data indicate that PLM, NCX, and Na(+)/K(+)-ATPase alpha-1 proteins have similar developmental expression patterns in rabbit ventricular myocardium. Thus, PLM may have an important regulatory role in maintaining cardiac Na(+) and Ca(2+) homeostasis during perinatal maturation.     

Energetics of potassium ion transport in Aplysia gut

Basolateral membranes of Aplysia californica foregut epithelia contain an ATP-dependent Na(+)/K(+) transporter (Na(+)/K(+) pump or Na(+)/K (+) -ATPase). This Na(+)/K(+) pump accounts for both the intracellular Na(+) electrochemical potential (micro) being less than the extracelluar Na(+) micro and the intracellular K(+) micro being more than the extracellular K(+ ) micro. Also, K(+) channel activity resides in both luminal and basolateral membranes of the Aplysia foregut epithelial cells. Increased activity of the Na(+)/K(+) pump, coupled to luminal and basolateral membrane depolarization altered the K(+) transport energetics across the basolateral membrane to a greater extent than the alteration in K(+) transport energetics across the luminal membrane. These results suggest that K(+) transport, either into or out of the Aplysia foregut epithelial cells, is rate-limiting at the basolateral membrane.     

Agonist-dependent regulation of renal Na+,K+-ATPase activity is modulated by intracellular sodium concentration.

We tested the hypothesis that the level of intracellular sodium modulates the hormonal regulation of the Na(+),K(+)-ATPase activity in proximal tubule cells. By using digital imaging fluorescence microscopy of a sodium-sensitive dye, we determined that the sodium ionophore monensin induced a dose-specific increase of intracellular sodium. A correspondence between the elevation of intracellular sodium and the level of dopamine-induced inhibition of Na(+),K(+)-ATPase activity was determined. At basal intracellular sodium concentration, stimulation of cellular protein kinase C by phorbol 12-myristate 13-acetate (PMA) promoted a significant increase in Na(+),K(+)-ATPase activity; however, this activation was gradually reduced as the concentration of intracellular sodium was increased to become a significant inhibition at concentrations of intracellular sodium higher than 16 mm. Under these conditions, PMA and dopamine share the same signaling pathway to inhibit the Na(+),K(+)-ATPase. The effects of PMA and dopamine on the Na(+),K(+)-ATPase activity and the modulation of these effects by different intracellular sodium concentrations were not modified when extracellular and intracellular calcium were almost eliminated. These results suggest that the level of intracellular sodium modulates whether hormones stimulate, inhibit, or have no effect on the Na(+),K(+)-ATPase activity leading to a tight control of sodium reabsorption.     

Activation of P2Y2 purinoceptor inhibits the activity of the Na+/K+-ATPase in HeLa cells

The role of ATP on regulation of the Na(+)/K(+)-ATPase activity in the human cancerous HeLa cells was investigated. HeLa cells stimulated with increasing ATP concentrations showed a dose-dependent inhibition of the Na(+)/K(+)-ATPase activity. These effects were also obtained by UTP. ATP and UTP provoked a rise in intracellular calcium concentration ([Ca(2+)](i)) persisting for at least 4 min. The inhibitor of phospholipase C, U73122, blocked the elevation of [Ca(2+)](i) provoked by ATP/UTP. The expression of mRNA for P2Y2 and P2Y6 receptors was demonstrated by RT-PCR. ATP/UTP activated PKC-alpha, -betaI and -epsilon isoforms, but not PKC-delta and -zeta. The inhibition of the Na(+)/K(+)-ATPase activity by ATP/UTP was blocked by G?6976, a specific inhibitor of the calcium-dependent PKCs. In conclusion, our results suggest that ATP/UTP modulate Na(+)/K(+)-ATPase activity in HeLa cells through the P2Y2 purinoceptor via calcium mobilisation and activation of calcium-dependent PKCs.     

A hybrid between Na+,K+-ATPase and H+,K+-ATPase is sensitive to palytoxin, ouabain, and SCH 28080

Na(+),K(+)-ATPase is inhibited by cardiac glycosides such as ouabain, and palytoxin, which do not inhibit gastric H(+),K(+)-ATPase. Gastric H(+),K(+)-ATPase is inhibited by SCH28080, which has no effect on Na(+),K(+)-ATPase. The goal of the current study was to identify amino acid sequences of the gastric proton-potassium pump that are involved in recognition of the pump-specific inhibitor SCH 28080. A chimeric polypeptide consisting of the rat sodium pump alpha3 subunit with the peptide Gln(905)-Val(930) of the gastric proton pump alpha subunit substituted in place of the original Asn(886)-Ala(911) sequence was expressed together with the gastric beta subunit in the yeast Saccharomyces cerevisiae. Yeast cells that express this subunit combination are sensitive to palytoxin, which interacts specifically with the sodium pump, and lose intracellular K(+) ions. The palytoxin-induced K(+) efflux is inhibited by the sodium pump-specific inhibitor ouabain and also by the gastric proton pump-specific inhibitor SCH 28080. The IC(50) for SCH 28080 inhibition of palytoxin-induced K(+) efflux is 14.3 +/- 2.4 microm, which is similar to the K(i) for SCH 28080 inhibition of ATP hydrolysis by the gastric H(+),K(+)-ATPase. In contrast, palytoxin-induced K(+) efflux from cells expressing either the native alpha3 and beta1 subunits of the sodium pump or the alpha3 subunit of the sodium pump together with the beta subunit of the gastric proton pump is inhibited by ouabain but not by SCH 28080. The acquisition of SCH 28080 sensitivity by the chimera indicates that the Gln(905)-Val(930) peptide of the gastric proton pump is likely to be involved in the interactions of the gastric proton-potassium pump with SCH 28080.     

Gramicidin A directly inhibits mammalian Na+/K+-ATPase

The linear pentadecapeptide gramicidin A forms an ion channel in the lipid bilayer to selectively transport monovalent cations. Nevertheless, we have surprisingly found that gramicidin A directly inhibits mammalian Na(+)/K(+)-ATPase. Gramicidin A inhibited ATP hydrolysis by Na(+)/K(+)-ATPase from porcine cerebral cortex at the IC(50) value of 8.1 microM, while gramicidin S was approximately fivefold less active. The synthetic gramicidin A analog lacking N-terminal formylation and C-terminal ethanolamine exhibited a weaker inhibitory effect on the ATP-hydrolyzing activity of Na(+)/K(+)-ATPase than gramicidin A, indicating that these end modifications are necessary for gramicidin A to inhibit Na(+)/K(+)-ATPase activity. Moreover, Lineweaver-Burk analysis showed that gramicidin A exhibits a mixed type of inhibition. In addition to the most well-studied ionophore activity, our present study has disclosed a novel biological function of gramicidin A as a direct inhibitor of mammalian Na(+)/K(+)-ATPase activity.     

Cnh1 Na(+) /H(+) antiporter and Ena1 Na(+) -ATPase play different roles in cation homeostasis and cell physiology of Candida glabrata

Yeasts tightly regulate their intracellular concentrations of alkali metal cations. In Saccharomyces cerevisiae, the Nha1 Na(+) /H(+) -antiporter and Ena1 Na(+) -ATPase, mediate the efflux of toxic sodium and surplus potassium. We report the characterization of Candida glabrata CgCnh1 and CgEna1 homologues. Their substrate specificity and transport properties were compared upon expression in S. cerevisiae, and their function characterized directly in C. glabrata. The CgCnh1 antiporter and the CgEna1 ATPase transport both potassium and sodium when expressed in S. cerevisiae. CgEna1p fully complements the lack of S. cerevisiae own Na(+) -ATPases but the activity of the CgCnh1 antiporter is lower than that of ScNha1p. Candida glabrata deletion mutants and analyses of their phenotypes revealed that though both transporters have a broad substrate specificity, their function in C. glabrata cells is not the same. Their differing physiological roles are also reflected in their regulation of expression, CgENA1 is highly upregulated by an increased osmotic pressure or sodium concentration, whereas CgCNH1 is expressed constitutively. The Cnh1 antiporter is involved in the regulation of potassium content and the Ena1 ATPase in sodium detoxification of C. glabrata cells. This situation differs from S. cerevisiae, where the Nha1 antiporter and Ena ATPases both participate together in Na(+) detoxification and in the regulation of K(+) homeostasis.     

Naloxone and ouabain in ultralow concentrations restore Na+/K+-ATPase and cytoskeleton in lipopolysaccharide-treated astrocytes

Astrocytes respond to inflammatory stimuli and may be important modulators of the inflammatory response in the nervous system. This study aimed first to assess how astrocytes in primary culture behave in response to inflammatory stimuli concerning intracellular Ca(2+) responses, expression of Toll-like receptor 4 (TLR4), Na(+)/K(+)-ATPase, actin filament organization, and expression of cytokines. In a cell culture model with lipopolysaccharide (LPS), astrocyte response was assessed first in the acute phase and then after incubation with LPS for 1-48 h. The concentration curve for LPS-stimulated Ca(2+) responses was bell-shaped, and the astrocytes expressed TLR4, which detects LPS and evokes intracellular Ca(2+) transients. After a long incubation with LPS, TLR4 was up-regulated, LPS-evoked Ca(2+) transients were expressed as oscillations, Na(+)/K(+)-ATPase was down-regulated, and the actin filaments were disorganized. Interleukin-1β (IL-1β) release was increased after 24 h in LPS. A second aim was to try to restore the LPS-induced changes in astrocytes with substances that may have dose-dependent anti-inflammatory properties. Naloxone and ouabain were tested separately in ultralow or high concentrations. Both substances evoked intracellular Ca(2+) transients for all of the concentrations from 10(-15) up to 10(-4) M. Neither substance blocked the TLR4-evoked Ca(2+) responses. Naloxone and ouabain prevented the LPS-induced down-regulation of Na(+)/K(+)-ATPase and restored the actin filaments. Ouabain, in addition, reduced the IL-1β release from reactive astrocytes. Notably, ultralow concentrations (10(-12) M) of naloxone and ouabain showed these qualities. Ouabain seems to be more potent in these effects of the two tested substances.     

Ouabain assembles signaling cascades through the caveolar Na+/K+-ATPase

Based on the observation that the Na(+)/K(+)-ATPase alpha subunit contains two conserved caveolin-binding motifs, we hypothesized that clustering of the Na(+)/K(+)-ATPase and its partners in caveolae facilitates ouabain-activated signal transduction. Glutathione S-transferase pull-down assay showed that the Na(+)/K(+)-ATPase bound to the N terminus of caveolin-1. Significantly, ouabain regulated the interaction in a time- and dose-dependent manner and stimulated tyrosine phosphorylation of caveolin-1 in LLC-PK1 cells. When added to the isolated membrane fractions, ouabain increased tyrosine phosphorylation of proteins from the isolated caveolae but not other membrane fractions. Consistently, ouabain induced the formation of a Na(+)/K(+)-ATPase-Src-caveolin complex in the isolated caveolae preparations as it did in live cells. Finally, depletion of either cholesterol by methyl beta-cyclodextrin or caveolin-1 by siRNA significantly reduced the caveolar Na(+)/K(+)-ATPase and Src. Concomitantly, cholesterol depletion abolished ouabain-induced recruitment of Src to the Na(+)/K(+)-ATPase signaling complex. Like depletion of caveolin-1, it also blocked the effect of ouabain on ERKs, which was restored after cholesterol repletion. Clearly, the caveolar Na(+)/K(+)-ATPase represents the signaling pool of the pump that interacts with Src and transmits the ouabain signals.     

Activity of BK(Ca) channel is modulated by membrane cholesterol content and association with Na+/K+-ATPase in human melanoma IGR39 cells

Interaction of large conductance Ca(2+)- and voltage-activated K(+) (BK(Ca)) channels with Na(+)/K(+)-ATPase, caveolin-1, and cholesterol was studied in human melanoma IGR39 cells. Functional BK(Ca) channels were enriched in caveolin-rich and detergent-resistant membranes, i.e. rafts, and blocking of the channels by a specific BK(Ca) blocker paxilline reduced proliferation of the cells. Disruption of rafts by selective depletion of cholesterol released BK(Ca) channels from these domains with a consequent increase in their activity. Consistently, cholesterol enrichment of the cells increased the proportion of BK(Ca) channels in rafts and decreased their activity. Immunocytochemical analysis showed that BK(Ca) channels co-localize with Na(+)/K(+)-ATPase in a cholesterol-dependent manner, thus suggesting their co-presence in rafts. Supporting this, ouabain, a specific blocker of Na(+)/K(+)-ATPase, inhibited BK(Ca) whole-cell current markedly in control cells but not in cholesterol-depleted ones. This inhibition required the presence of external Na(+). Collectively, these data indicate that the presence of Na(+)/K(+)-ATPase in rafts is essential for efficient functioning of BK(Ca) channels, presumably because the pump maintains a low intracellular Na(+) proximal to the BK(Ca) channel. In conclusion, cholesterol could play an important role in cellular ion homeostasis and thus modulate many cellular functions and cell proliferation.     

Insect Na(+)/K(+)-ATPase

Na(+)/K(+)-ATPase (sodium/potassium pump) is a P-type ion-motive ATPase found in the plasma membranes of animal cels. In vertebrates, the functions of this enzyme in nerves, heart and kidney are well characterized and characteristics a defined by different isoforms. In contrast, despite different tissue distributions, insects possess a single isoform of the alpha-subunit. A comparison of insect and vertebrate Na(+)/K(+)-ATPases reveals that although the mode of action and structure are very highly conserved, the specific roles of the enzyme in most tissues varies. However, the enzyme is essential for the function of nerve cells, and in this respect Na(+)/K(+)-ATPase appears to be fundamental in metazoan evolution.     

Comparative properties of caveolar and noncaveolar preparations of kidney Na+/K+-ATPase

To evaluate previously proposed functions of renal caveolar Na(+)/K(+)-ATPase, we modified the standard procedures for the preparation of the purified membrane-bound kidney enzyme, separated the caveolar and noncaveolar pools, and compared their properties. While the subunits of Na(+)/K(+)-ATPase (α,β,γ) constituted most of the protein content of the noncaveolar pool, the caveolar pool also contained caveolins and major caveolar proteins annexin-2 tetramer and E-cadherin. Ouabain-sensitive Na(+)/K(+)-ATPase activities of the two pools had similar properties and equal molar activities, indicating that the caveolar enzyme retains its ion transport function and does not contain nonpumping enzyme. As minor constituents, both caveolar and noncaveolar pools also contained Src, EGFR, PI3K, and several other proteins known to be involved in stimulous-induced signaling by Na(+)/K(+)-ATPase, indicating that signaling function is not limited to the caveolar pool. Endogenous Src was active in both pools but was not further activated by ouabain, calling into question direct interaction of Src with native Na(+)/K(+)-ATPase. Chemical cross-linking, co-immunoprecipitation, and immunodetection studies showed that in the caveolar pool, caveolin-1 oligomers, annexin-2 tetramers, and oligomers of the α,β,γ-protomers of Na(+)/K(+)-ATPase form a large multiprotein complex. In conjunction with known roles of E-cadherin and the β-subunit of Na(+)/K(+)-ATPase in cell adhesion and noted intercellular β,β-contacts within the structure of Na(+)/K(+)-ATPase, our findings suggest that interacting caveolar Na(+)/K(+)-ATPases located at renal adherens junctions maintain contact of two adjacent cells, conduct essential ion pumping, and are capable of locus-specific signaling in junctional cells.