Ouabain-sensitive ATPase Research Articles

Different antihypertensive and metabolic responses to rostafuroxin in undernourished and normonourished male rats: Outcomes on bodily Na+ handling.

Hypertension is a pandemic nowadays. We aimed to investigate whether chronic undernutrition modifies the response to the antihypertensive drug rostafuroxin in juvenile hypertensive rats. Chronic undernutrition was induced in male rats using a multideficient diet known as the Regional Basic Diet (RBD), mimicking alimentary habits in impoverished regions worldwide. Animals were given RBD-or a control/CTRL normal diet for rodents-from weaning to 90 days, and rostafuroxin (1 mg/kg body mass) was orally administered from day 60 onwards. For the last 2 days, the rats were hosted in metabolic cages to measure food/energy, water, Na+ ingestion, and urinary volume. Rostafuroxin increased food/energy/Na+ intake in CTRL and RBD rats but had opposite effects on Na+ balance (intake minus urinary excretion). The drug normalized the decreased plasma Na+ concentration in RBD rats, increased urinary volume in RBD but not in CTRL, and decreased and increased urinary Na+ concentration in the RBD and CTRL groups, respectively. Rostafuroxin decreased the ouabain-sensitive (Na+ +K+ )ATPase and increased the ouabain-resistant Na+ -ATPase from proximal tubule cells in both groups and normalized the systolic blood pressure in RBD without effect in CTRL rats. We conclude that chronic undernutrition modifies the response of blood pressure and metabolic responses to rostafuroxin.

Effects of in vivo exposures to nanoparticles (Al<sub>2</sub>O<sub>3</sub>, CuO, TiO<sub>2</sub>) on the activities of ATPases in the gill and muscle of freshwater mussel (Unio tigridis)

Mussels are effective bioindicator organisms for aquatic environments. Therefore, they were often used to determine the effects of various xenobiotics in the aquatic systems. There is no study to our knowledge on the in vivo effects of nanoparticles (NPs) on the activities of ATPases in freshwater mussels (Unio tigridis). This work demonstrates the effects of Al$_{2}$O$_{3}$, CuO, and TiO$_{2}$ NPs on Na- ATPase, Ca-ATPase, and Mg-ATPase activities in the gill and muscle of mussels following 14-day exposures to different concentrations of NPs (0, 1, 3, 9 mg/L). Mussels were fed with laboratory cultured algae (Chlorella vulgaris) during the exposures. There was no ouabainsensitive ATPase activity in the tissues of U. tigridis. The activities of Na-ATPase and Ca-ATPase in the gill decreased significantly (p < 0.05), while Mg-ATPase activity increased. However, the activities of all ATPases decreased significantly after NP exposures in the muscle. There were more significant alterations in the gill compared to the alterations in the muscle. The order of NP effects was determined as TiO$_{2}$ > CuO > Al$_{2}$O$_{3}$. There was no significant alteration in algae consumption between control and NP-exposed mussels. The present study reporting the first data on the effects of NPs in U. tigridis demonstrated the sensitivity of mussel physiology towards NP exposures, suggesting further studies to understand better the physiological response of mussels.

Characterization of ATPases in the gill of freshwater mussel (Unio tigridis) and effects of ionic and nanoparticle forms of aluminium and copper

Mussels are often used to monitor the aquatic systems for different ecological aims, as they are one of the best bioindicator animals. However, the characterization of biomarkers should be known before using them in environmental monitoring and toxicology studies. There is no study to our knowledge on the characterization of Na-ATPase and Ca-ATPase in tissues of freshwater mussel (Unio tigridis). Thus, this study was undertaken to characterize the optimum working conditions of Na-ATPase and Ca-ATPase in the gill of mussels, determining the highest levels of parameters (Na+, Mg2+, Ca2+, ATP, pH, temperature, enzyme amount, incubation time) to obtain maximum activity. The present study also aimed to investigate in vitro effects of ionic and nanoparticle (Al2O3, CuO) forms of aluminium and copper (0, 30, 90, 270 μg/L) on the activities of Na-ATPase and Ca-ATPase. Data showed that there was no ouabain-sensitive ATPase activity in the gill up to 10 mM ouabain concentrations. Na-ATPase and Ca-ATPase activities in the gill of control mussels were 5.124 ± 0.373 and 3.750 ± 0.211 μmol Pi/mg pro./h, respectively. Exposure to different concentrations of nanoparticles did not alter significantly (P > 0.05) the activities of Na-ATPase and Ca-ATPase in vitro, whereas the same concentrations of ionic aluminium and copper significantly decreased (P < 0.05) the enzyme activities. Data emphasized that there were different modes of action between ionic and nanoparticle forms of aluminium and copper. Data also suggested that in vivo studies should also be carried out to estimate better the effects of nanoparticle and ionic forms of metals on ATPases of U. tigridis.

Adeno-Associated Virus-Mediated Gene Therapy in the Mashlool, Atp1a3Mashl/+, Mouse Model of Alternating Hemiplegia of Childhood.

Alternating Hemiplegia of Childhood (AHC) is a devastating autosomal dominant disorder caused by ATP1A3 mutations, resulting in severe hemiplegia and dystonia spells, ataxia, debilitating disabilities, and premature death. Here, we determine the effects of delivering an extra copy of the normal gene in a mouse model carrying the most common mutation causing AHC in humans, the D801N mutation. We used an adeno-associated virus serotype 9 (AAV9) vector expressing the human ATP1A3 gene under the control of a human Synapsin promoter. We first demonstrated that intracerebroventricular (ICV) injection of this vector in wild-type mice on postnatal day 10 (P10) results in increases in ouabain-sensitive ATPase activity and in expression of reporter genes in targeted brain regions. We then tested this vector in mutant mice. Simultaneous intracisterna magna and bilateral ICV injections of this vector at P10 resulted, at P40, in reduction of inducible hemiplegia spells, improvement in balance beam test performance, and prolonged survival of treated mutant mice up to P70. Our study demonstrates, as a proof of concept, that gene therapy can induce favorable effects in a disease caused by a mutation of the gene of a protein that is, at the same time, an ATPase enzyme, a pump, and a signal transduction factor.

Intestinal response to ocean acidification in the European sea bass (Dicentrarchus labrax)

The intestine of marine fishes contributes to the ocean carbon cycle producing carbonate aggregates as part of the osmoregulatory process. Therefore, this study aimed to evaluate physiological adjustments of European sea bass (Dicentrarchus labrax) intestine to a higher pCO2 environment likely in the near future (~1700 μatm). At the whole-body level, hypercapnia for 5 weeks resulted in fish having a significantly diminished specific growth rate, condition factor and hepatosomatic index. An increase in plasma osmolality and HCO3− concentration was detected, paralleled by decreased metabolites concentrations. In the intestine, high seawater pCO2 was without effect on ouabain-sensitive ATPase activities, while Bafilomycin A1-sensitive ATPase activity significantly decreased in the anterior intestine. Anterior and mid intestine were mounted in Ussing chambers in order to measure bioelectrical parameters and bicarbonate secretion by pH-Stat ex-vivo. Hypercapnia induced a 2.3 and 2.8-fold increase in bicarbonate secretion rates in the anterior and mid intestine, respectively. In the intestinal fluid, HCO3− concentration increased 2.2-fold, and carbonate precipitates showed a 4.4-fold increase in response to hypercapnia, paralleled by a >3-fold increase of drinking and a >2-fold increase of intestinal volume at any given time. At the molecular level, hypercapnia elicited higher intestinal mRNA expression levels for atp6v1b (V-ATPase B subunit), slc4a4, slc26a3, and slc26a6, both in the anterior and mid intestine. As a whole, our results show that the intestine of sea bass responds to high seawater pCO2, a response that comes at a cost at the whole-body level with an impact in the fish specific growth rate, condition factor, and hepatosomatic index.

Hypercapnia Impairs Na,K-ATPase Function by Inducing Endoplasmic Reticulum Retention of the β-Subunit of the Enzyme in Alveolar Epithelial Cells.

Alveolar edema, impaired alveolar fluid clearance, and elevated CO2 levels (hypercapnia) are hallmarks of the acute respiratory distress syndrome (ARDS). This study investigated how hypercapnia affects maturation of the Na,K-ATPase (NKA), a key membrane transporter, and a cell adhesion molecule involved in the resolution of alveolar edema in the endoplasmic reticulum (ER). Exposure of human alveolar epithelial cells to elevated CO2 concentrations caused a significant retention of NKA-β in the ER and, thus, decreased levels of the transporter in the Golgi apparatus. These effects were associated with a marked reduction of the plasma membrane (PM) abundance of the NKA-α/β complex as well as a decreased total and ouabain-sensitive ATPase activity. Furthermore, our study revealed that the ER-retained NKA-β subunits were only partially assembled with NKA α-subunits, which suggests that hypercapnia modifies the ER folding environment. Moreover, we observed that elevated CO2 levels decreased intracellular ATP production and increased ER protein and, particularly, NKA-β oxidation. Treatment with α-ketoglutaric acid (α-KG), which is a metabolite that has been shown to increase ATP levels and rescue mitochondrial function in hypercapnia-exposed cells, attenuated the deleterious effects of elevated CO2 concentrations and restored NKA PM abundance and function. Taken together, our findings provide new insights into the regulation of NKA in alveolar epithelial cells by elevated CO2 levels, which may lead to the development of new therapeutic approaches for patients with ARDS and hypercapnia.

Na+/K+‐ATPase α1 and Cardiac Resilience

Systemic levels of endogenous cardiotonic steroids (CTS) vary in response to physiological and pathophysiological stresses such as exercise, pregnancy, hypertension, and heart failure. Ample experimental evidence has shown that exposure to CTS modulates cardiac Na/K‐ATPase (NKA) α1 ion‐pumping and non‐ion‐pumping functions, which profoundly affects cardiac contractility and structure. However, the physiological role of CTS as a group of hormones with distinct but related modulatory effects on cardiovascular equipoise and dysfunction remains poorly understood. We used 2 genetically engineered mouse models to test the hypothesis that the endogenous CTS/NKA α1 interaction confers cardiac resilience during a sustained hemodynamic challenge.Wild type mice expressing the relatively CTS‐insensitive NKA α1 and the CTS‐sensitive NKA α2 (α1r/rα2s/s), mice expressing NKA α1 mutated to CTS‐sensitivity (α1s/sα2s/s), and mice with cardiomyocyte‐specific NKA α1 knockout (α1−/−α2s/s) were treated with a pressor dose of Angiotensin II (Ang II) of 1.5 mg.kg−1. d−1 for 14 days via osmotic minipumps to induce adverse cardiac remodeling.All mice were born with the expected Mendelian frequency and survived to adulthood. In the α1s/sα2s/s mouse, total cardiac expression of NKA α1 and α2 assessed by western blotting and ouabain‐sensitive ATPase activity were unchanged, and the expected increased affinity for the CTS ouabain was detected. NKA expression and activity in the α1−/−α2s/s heart was consistent with a nearly complete ablation of NKA α1, partially compensated by an upregulation of NKA α2. Ang II or vehicle treatment was administered to 3 month‐old males (n=6–8/group), and cardiac remodeling was assessed by gravimetry, histology, and qPCR. In contrast to wild‐type α1r/rα2s/s, the Ang II treatment did not induce any significant cardiac hypertrophy or fibrosis in α1s/sα2s/s hearts. Conversely, the Ang II treatment was lethal in α1−/−α2s/s mice, with a 70% mortality rate at 8 days. A shorter (7 days) Ang II treatment revealed massive cardiac fibrosis (50% increase compared to α1r/rα2s/s). Taken together, these results support a physiological role of endogenous CTS in cardiac resilience through an interaction with the signaling cardiac NKA α1 isoform.Support or Funding InformationN/AThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression of rat Na-K-ATPase α2 enables ion pumping but not ouabain-induced signaling in α1-deficient porcine renal epithelial cells.

Na-K-ATPase is a fundamental component of ion transport. Four α isoforms of the Na-K-ATPase catalytic α subunit are expressed in human cells. The ubiquitous Na-K-ATPase α1 was recently discovered to also mediate signal transduction through Src kinase. In contrast, α2 expression is limited to a few cell types including myocytes, where it is coupled to the Na(+)/Ca(2+) exchanger. To test whether rat Na-K-ATPase α2 is capable of cellular signaling like its α1 counterpart in a recipient mammalian system, we used an α1 knockdown pig renal epithelial cell (PY-17) to create an α2-expressing cell line with no detectable level of α1 expression. These cells exhibited normal ouabain-sensitive ATPase, but failed to effectively regulate Src. In contrast to α1-expressing cells, ouabain did not stimulate Src kinase or downstream effectors such as ERK and Akt in α2 cells, although their signaling apparatus was intact as evidenced by EGF-mediated signal transduction. Additionally, α2 cells were unable to rescue caveolin-1. Unlike the NaKtide sequence derived from Na-K-ATPase α1, which downregulates basal Src activity, the corresponding α2 NaKtide was unable to inhibit Src in vitro. Finally, coimmunoprecipitation of cellular Src was diminished in α2 cells. These findings indicate that Na-K-ATPase α2 does not regulate Src and, therefore, may not serve the same role in signal transduction as α1. This further implies that the signaling mechanism of Na-K-ATPase is isoform specific, thereby supporting a model where α1 and α2 isoforms play distinct roles in mediating contraction and signaling in myocytes.

Heterogeneity of Signal Transduction by Na/K‐ATPase α‐Isoforms: Role of α2 Na/K‐ATPase

Na/K-ATPase α1 subunit is a ubiquitous transmembrane ion transporting enzyme. More and more studies provide evidences that α1 Na/K-ATPase mediates signal transduction through Src tyrosine kinases upon binding to ligands like cardiotonic steroid. Among four isoforms expressed in human, α2 Na/K-ATPase is predominantly expressed in cardiac myocytes and plays an important role in the regulation of muscle contraction. To understand whether α2 isoform participates in signal transduction, we generate a mammalian cell line expressing α2 isoform only (LX-α2-4), by transfecting α2 N/K-ATPase into α1 knockdown pig kidney cells (PY-17). LX-α2-4 cells exhibited normal ouabain-sensitive ATPase activity, but failed to effectively regulate Src and ERKs. Consistently, ouabain was unable to stimulate Src kinase and downstream effectors such as ERK and Akt in these cells. Additionally, α2 cells were unable to rescue the expression of caveolin. Furthermore, while α1 Na/K-ATPase is known to regulate Src via the NaKtide sequence, the corresponding α2 NaKtide was unable to inhibit Src in vitro. Finally, LX-α2-4 cells grew much slower than these of α1-expressing AAC-19 cells. In short, α2 Na/K-ATPase does not regulate Src as α1, implying that α1 and α2 isoforms play distinct roles in mediating contraction and signaling in myocytes. This suggests potential therapeutics utilities may be achieved through targeting different isoforms of Na/K-ATPase in various diseases. Figure 1. Possible signaling transduction mechanisms by isoforms of Na/K-ATPase

Nuclear Na,K-ATPase plays an active role in Nucleoplasmic Calcium Homeostasis*

Na(+)/K(+)-ATPase, an integral membrane protein, has been studied for over a half century with respect to its transporter function in the plasma membrane, where it expels three Na(+) ions from the cell in exchange for two K(+) ions. In this study, we demonstrate a functioning Na(+)/K(+)-ATPase within HEK293 cell nuclei. This subcellular localization was confirmed by western blotting, ouabain-sensitive ATPase activity of the nuclear membrane fraction, immunocytochemistry and delivery of fluorescently tagged Na(+)/K(+)-ATPase α- and β-subunits. In addition, we observed an overlap between nuclear Na(+)/K(+)-ATPase and Na/Ca-exchanger (NCX) when nuclei were immunostained with commercially available Na(+)/K(+)-ATPase and NCX antibodies, suggesting a concerted physiological coupling between these transporters. In keeping with this, we observed an ATP-dependent, strophanthidin-sensitive Na(+) flux into the nuclear envelope (NE) lumen loaded with the Na-sensitive dye, CoroNa-Green. Analogous experiments using Fluo-5N, a low affinity Ca(2+) indicator, demonstrated a similar ATP-dependent and strophanthidin-sensitive Ca(2+) flux into the NE lumen. Our results reveal an intracellular physiological role for the coordinated efforts of the Na(+)/K(+)-ATPase and NCX to actively remove Ca(2+) from the nucleoplasm into the NE lumen (i.e. the nucleoplasmic reticulum).

The effects of P2X receptor agonists on renal sodium and water excretion in anaesthetized rats

To investigate in vivo effects of P2X receptor activation on sodium and water excretion in urine. The clearance experiments were carried out in anaesthetized rats during intravenous infusion (2 μmol kg(-1) + 20 nmol (kg min)(-1) , v = 40 μL min(-1)) of P2X receptors agonists: α,β-methylene ATP (α,β-meATP) and β,γ-methylene ATP (β,γ-meATP). Cortical blood flow (CBF) was estimated by laser Doppler flux during intrarenal artery infusion of β,γ-meATP (20 nmol (kg min)(-1) , v = 2 μL min(-1)). Influence of α,β-meATP and β,γ-meATP on the activity of Na-K-ATPase was investigated in isolated proximal tubules. Intravenous infusion of β,γ-meATP resulted in a marked, progressively increasing diuresis and this effect was accompanied by a progressive increase in the sodium excretion rate. The glomerular filtration rate was unaffected. The effects of β,γ-meATP were abolished by P2 receptor antagonist PPADS (70 nmol (kg min)(-1)). CBF increased by 16 ± 2% during renal artery infusion of β,γ-meATP. Furthermore, α,β-meATP and β,γ-meATP increased 1.5-fold lithium clearance (C(Li)). Sodium excretion, expressed as a fraction of the distal delivery (C(Na) C(Li) (-1)), increased 1.5-fold during infusion of α,β-meATP or β,γ-meATP. Both agonists at 10(-6) (M) produced a statistical significant decrement in the ouabain-sensitive ATPase activity about 16-20% and these effects were blocked in the presence of PPADS. Activation of P2X receptors increased renal sodium and water excretion. Mechanistically, P2X agonists increased renal perfusion and inhibited sodium reabsorption via an Na-K-ATPase-dependent mechanism.

A novel physiological role for Na, K‐ATPase: Maintenance of nucleoplasmic calcium

Na, K‐ATPase (NKA) is the integral membrane protein responsible for establishing and maintaining the Na+ electrochemical gradient across the plasma membrane of animal cells by actively exporting 3Na+ in exchange for 2K+. In this report, we present evidence suggesting that NKA builds a maintains a Na+ gradient across the nuclear envelope (NE) membrane, extending our earlier observation that NKA functions within the Endoplasmic Reticulum (Gatto et al., AJP‐cell phys., 281, C982‐C992, 2001). Immunocytochemistry of isolated nuclei from HEK‐293 cells with commercially available alpha and beta antibodies revealed that NKA was located on the nuclear envelope. In addition, membrane preparations from isolated nuclei had ouabain‐sensitive ATPase activity. Moreover, immunostaining of nuclei with antibodies against the Na/Ca‐exchanger (NCX) and NKA reveals an overlap between localization of nuclear NKA and NCX (Na/Ca‐exchanger). Given that NCX is driven by a Na+ gradient, we propose that the intracellular NKA function is utilized by NCX to extrude Ca2+ from the nucleus. We measured NKA function in isolated HEK cell nuclei via CoroNa green fluorescence. We observed an ATP‐dependent, strophanthidin‐sensitive Na accumulation in the nuclear envelope loaded with the Na‐sensitive dye. Analogous experiments are underway utilizing Ca‐sensitive probes. This work is supported by NIH grant DK083859.

Selectivity of externally facing ion-binding sites in the Na/K pump to alkali metals and organic cations

The Na/K pump is a P-type ATPase that exchanges three intracellular Na(+) ions for two extracellular K(+) ions through the plasmalemma of nearly all animal cells. The mechanisms involved in cation selection by the pump's ion-binding sites (site I and site II bind either Na(+) or K(+); site III binds only Na(+)) are poorly understood. We studied cation selectivity by outward-facing sites (high K(+) affinity) of Na/K pumps expressed in Xenopus oocytes, under voltage clamp. Guanidinium(+), methylguanidinium(+), and aminoguanidinium(+) produced two phenomena possibly reflecting actions at site III: (i) voltage-dependent inhibition (VDI) of outwardly directed pump current at saturating K(+), and (ii) induction of pump-mediated, guanidinium-derivative-carried inward current at negative potentials without Na(+) and K(+). In contrast, formamidinium(+) and acetamidinium(+) induced K(+)-like outward currents. Measurement of ouabain-sensitive ATPase activity and radiolabeled cation uptake confirmed that these cations are external K(+) congeners. Molecular dynamics simulations indicate that bound organic cations induce minor distortion of the binding sites. Among tested metals, only Li(+) induced Na(+)-like VDI, whereas all metals tested except Na(+) induced K(+)-like outward currents. Pump-mediated K(+)-like organic cation transport challenges the concept of rigid structural models in which ion specificity at site I and site II arises from a precise and unique arrangement of coordinating ligands. Furthermore, actions by guanidinium(+) derivatives suggest that Na(+) binds to site III in a hydrated form and that the inward current observed without external Na(+) and K(+) represents cation transport when normal occlusion at sites I and II is impaired. These results provide insights on external ion selectivity at the three binding sites.

Effect of alcohol and kolanut interaction on brain sodium pump activity in wistar albino rats.

Effect of alcohol-kolanut interaction on sodium pump activity in wistar albino rats was studied. Thirty wistar albino rats were divided into six groups of five (5) rats per group and used for the study. The control group (1) received via oral route a placebo (4 ml of distilled water). Groups 2 to 6 were treated for a period of 21 days, with (10% v/v) of alcohol (group 2), 50mg/kg body weight of kolanut (group 3), 50 mg/kg body weight of caffeine (group 4), 4 ml of 10% v/v of alcohol and 50 mg/kg body weight kolanut (group 5), 4 ml of 10% v/v of alcohol and 50 mg/kg body weight of caffeine in 4.0 ml of the vehicle via gastric intubation respectively. A day after the final exposure, the brain of each rat was harvested and processed to examine several biochemical parameters, i.e., total ATpase, ouabain-insensitive ATpase, ouabain sensitive ATpase (Na(+)-K(+)ATPase), non-enzymatic breakdown of ATP and inorganic phosphate (Pi) released. The results showed that the essential enzyme of the brain responsible for neuronal function, Na(+)-K(+)ATPase, was inhibited by alcohol-kolanut co-administration relative to control, resulting in a decrease in Na(+)-K(+)ATPase activity, ATP production, ion transport and action potential, leading to loss of neuronal activities.

Interaction of alcohol and kola nut on brain sodium pump activity in Wistar rats

The effect of interaction of alcohol and kola nut on sodium pump activity was studied in Wistar rats. Thirty Wistar rats were divided into six groups of five rats per group. Control received a placebo (4 mL of distilled water). Groups 2–6 were treated for a period of 21 days with 10% (v/v) alcohol, 50 mg caffeine/kg, 50 mg kola nut/kg, or a combination of 10% (v/v) alcohol + 50 mg kola nut/kg, or 10% (v/v) alcohol + 50 mg caffeine/kg, respectively. One day after the final exposure, brains were harvested and several biochemical parameters examined including activities of total ATPase, ouabain-insensitive ATPase, ouabain sensitive ATPase (Na+–K+-ATPase) and levels of nonenzymatic breakdown of ATP and inorganic phosphate (Pi) released. Results showed that the essential enzyme of the brain responsible for neuronal function, Na+–K+-ATPase, was inhibited by alcohol–kola nut co-administration relative to control, resulting in a decreased ATP production, ion transport and action potential, leading to loss of neuronal activities.

Entamoeba histolytica: Ouabain-insensitive Na +-ATPase activity

Our aim was to determine the presence of sodium pumps in Entamoeba histolytica. It is shown through the measurement of ouabain-sensitive ATPase activity and immunoblotting that E. histolytica does not express (Na ++K +)ATPase. On the other hand, we observed a Na +-ATPase with the following characteristics: (1) stimulated by Na + or K +, but these effects are not addictive; (2) the apparent affinity is similar for Na + and K + ( K 0.5 = 13.3 ± 3.7 and 15.4 ± 3.1 mM, respectively), as well as the V max (24.9 ± 1.5 or 27.5 ± 1.6 nmol Pi mg −1 min −1, respectively); (3) insensitive up to 2 mM ouabain; and (4) inhibited by furosemide with an IC 50 of 0.12 ± 0.004 mM. Furthermore, this enzyme forms a Na +- or K +-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate phosphorylated intermediate.

Lys691 and Asp714 of the Na+/K+-ATPase α Subunit Are Essential for Phosphorylation, Dephosphorylation, and Enzyme Turnover

P-type ATPases such as the sodium pump appear to be members of a superfamily of hydrolases structurally typified by the L-2-haloacid dehalogenases. In the dehalogenase L-DEX-ps, Lys151 serves to stabilize the excess negative charge in the substrate/reaction intermediates and Asp180 coordinates a water molecule that is directly involved in ester intermediate hydrolysis. To investigate the importance of the corresponding Lys691 and Asp714 of the sodium pump alpha subunit, sodium pump mutants were expressed in yeast and analyzed for their properties. Lys691Ala, Lys691Asp, Asp714Ala, and Asp714Arg mutants were inactive, not only with respect to ATPase activity but also to interaction with the highly sodium pump-specific inhibitors ouabain or palytoxin (PTX). In contrast, conservative mutants Lys691Arg and Asp714Glu retained some of the partial activities of the wild-type enzyme, although they completely failed to display any ATPase activity. Yeast cells expressing Lys691Arg and Asp714Glu mutants are sensitive to the sodium pump-specific inhibitor PTX and lose intracellular K+. Their sensitivity to PTX, with EC50 values of 118 +/- 24 and 76.5 +/- 3.6 nM, respectively, was clearly reduced by almost 7- or 4-fold below that of the native sodium pump (17.8 +/- 2.7 nM). Ouabain was recognized under these conditions with low affinity by the mutants and inhibited the PTX-induced K+ efflux from the yeast cells. The EC50 for the ouabain effect was 183 +/- 20 microM for Lys691Arg and 2.3 +/- 0.08 mM for the Asp714Glu mutant. The corresponding value obtained with cells expressing the native sodium pump was 69 +/- 18 microM. In the presence of Pi and Mg2+, none of the mutant sodium pumps were able to bind ouabain. When Mg2+ was omitted, however, both Lys691Asp and Asp714Glu mutants displayed ouabain binding that was reduced by Mg2+ with an EC50 of 0.76 +/- 0.11 and 2.3 +/- 0.2 mM, respectively. In the absence of Mg2+, ouabain binding was also reduced by K+. The EC50 values were 1.33 +/- 0.23 mM for the wild-type enzyme, 0.93 +/- 0.2 mM for the Lys691Arg mutant, and 1.02 +/- 0.24 mM for the Asp714Glu enzyme. None of the neutral or nonconservative mutants displayed any ouabain-sensitive ATPase activity. Ouabain-sensitive phosphatase activity, however, was present in membranes containing either the wild-type (1105 +/- 100 micromol of p-nitrophenol phosphate hydrolyzed min(-1) mg of protein(-1)) or the Asp714Glu mutant (575 +/- 75 micromol min(-1) mg(-1)) sodium pump. Some phosphatase activity was also associated with the Lys691Arg mutant (195 +/- 63 micromol min(-1) mg(-1)). The results are consistent with Lys691 and Asp714 being essential for the phosphorylation/dephosphorylation process that allows the sodium pump to accomplish the catalytic cycle.

Single dexamethasone injection increases alveolar fluid clearance in adult rats.

Epithelial Na+ channels and Na+/K+-adenosine triphosphatase (ATPase) in alveolar epithelium have a very important role in the absorption of excessive fluid from the alveolar space. We examined whether single dexamethasone injection at therapeutic doses would modulate lung epithelial Na+ channels and Na+/K+-ATPase and increase alveolar fluid clearance in adult rats. Controlled laboratory study. University research laboratory. Adult male Sprague-Dawley rats (n = 138). Rats were intraperitoneally injected with dexamethasone at a dose ranging from 0.02 to 2.0 mg/kg, and allowed free access to food and water. Alveolar fluid clearance was determined by measuring the increase in albumin concentration in the lung instillate solution. We discovered a significant increase in alveolar fluid clearance at 48 and 72 hrs after dexamethasone treatment. The effect of dexamethasone was dose dependent. In addition, increased alveolar fluid clearance was associated with a faster recover from hypoxemia, which was induced by filling the alveolar space with instillate solution. The dexamethasone-induced increase in alveolar fluid clearance was inhibited by amiloride and ouabain. Quantitative reverse transcriptase-polymerase chain reaction showed that dexamethasone treatment increased lung beta-epithelial Na+ channel mRNA levels. The expression of gamma-epithelial Na+ channel mRNA was also increased slightly. In contrast, alpha-epithelial Na+ channel mRNA levels did not differ from control levels. There was no change in alpha1- or beta1-Na+/K+-ATPase mRNA levels over 72 hrs after dexamethasone treatment. However, we found that lung Na+/K+-ATPase hydrolytic activity, determined by monitoring the ouabain-sensitive ATPase hydrolysis, was increased at 48 and 72 hrs after dexamethasone treatment. Single dexamethasone injection at therapeutic doses is capable of modulating lung epithelial Na+ channels and Na+/K+-ATPase and increase alveolar fluid clearance, thereby accelerating recovery from pulmonary edema.

Protective role of L-methionine against free radical damage of rat brain synaptosomes.

Incubation of rat brain synaptosomal/mitochondrial fraction with tert-butylhydroperoxide resulted in accumulation of the lipid peroxidation product, conjugated dienes, damage of the synaptosomal membrane as evidenced by leakage of lactate dehydrogenase, and decrease of the total content of glutathione and of the GSH/GSSG ratio. This treatment also produced a considerable decrease of the ouabain-sensitive ATPase activity and a much smaller diminution of the activities of glutathione reductase and glutathione transferase. Preincubation of the synaptosomal/mitochondrial fraction with 0.5 or 1.0 mM L-methionine significantly protected against lipid peroxidation, membrane damage and changes in the glutathione system produced by low (1 mM) concentrations of tert-butylhydroperoxide and completely prevented inactivation of ouabain-sensitive ATPase, glutathione reductase and glutathione transferase by such treatment. The importance of L-methionine in antioxidant protection is discussed.

Protective role of L-methionine against free radical damage of rat brain synaptosomes.

Incubation of rat brain synaptosomal/mitochondrial fraction with tert-butylhydroperoxide resulted in accumulation of the lipid peroxidation product, conjugated dienes, damage of the synaptosomal membrane as evidenced by leakage of lactate dehydrogenase, and decrease of the total content of glutathione and of the GSH/GSSG ratio. This treatment also produced a considerable decrease of the ouabain-sensitive ATPase activity and a much smaller diminution of the activities of glutathione reductase and glutathione transferase. Preincubation of the synaptosomal/mitochondrial fraction with 0.5 or 1.0 mM L-methionine significantly protected against lipid peroxidation, membrane damage and changes in the glutathione system produced by low (1 mM) concentrations of tert-butylhydroperoxide and completely prevented inactivation of ouabain-sensitive ATPase, glutathione reductase and glutathione transferase by such treatment. The importance of L-methionine in antioxidant protection is discussed.