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1.
Khoa T. D. Nguyen Jae-Won Shin Caroline Rae Ellas K. Nanitsos Gabriela B. Acosta David V. Pow Vlado Buljan Maxwell R. Bennett Paul L. Else Vladimir J. Balcar 《Neurochemical research》2009,34(10):1767-1774
The naturally occurring toxin rottlerin has been used by other laboratories as a specific inhibitor of protein kinase C-delta
(PKC-δ) to obtain evidence that the activity-dependent distribution of glutamate transporter GLAST is regulated by PKC-δ mediated
phosphorylation. Using immunofluorescence labelling for GLAST and deconvolution microscopy we have observed that d-aspartate-induced redistribution of GLAST towards the plasma membranes of cultured astrocytes was abolished by rottlerin.
In brain tissue in vitro, rottlerin reduced apparent activity of (Na+, K+)-dependent ATPase (Na+, K+-ATPase) and increased oxygen consumption in accordance with its known activity as an uncoupler of oxidative phosphorylation
(“metabolic poison”). Rottlerin also inhibited Na+, K+-ATPase in cultured astrocytes. As the glutamate transport critically depends on energy metabolism and on the activity of
Na+, K+-ATPase in particular, we suggest that the metabolic toxicity of rottlerin and/or the decreased activity of the Na+, K+-ATPase could explain both the glutamate transport inhibition and altered GLAST distribution caused by rottlerin even without
any involvement of PKC-δ-catalysed phosphorylation in the process. 相似文献
2.
Crush syndrome (CS) results from severe traumatic damage to the organism that is characterized by stress, acute homeostatic
failure of the tissues, and myoglobinuria with severe intoxication. This leads to an acute impairment of kidneys and heart.
The peripheral and central nervous systems are also the subject of significant changes in CS. Na+, K+-ATPase is a critical enzyme in neuron that is essential for the regulation of neuronal membrane potential, cell volume as
well as transmembrane fluxes of Ca++ and Excitatory Amino Acids. In the present study, Na+, K+-ATPase activity of rat brain regions [Olfactory lobes (OL), Cerebral cortex (CC), Cerebellum (CL), and Medulla oblongata
(MO)] during CS was investigated. Experimental model of CS in albino rats was induced by 2-h of compression followed by 2,
24, and 48-h of decompression of femoral muscle tissue. In this study, we have observed elevation in Na+, K+-ATPase activity above normal/control levels in all parts of brain (OL: 34.4%; CC: 1.0%; CL: 3.3% and MO: 45%) during 2-h
compression in comparison to controls. 相似文献
3.
Bersier MG Miksztowicz V Peña C Rodríguez de Lores Arnaiz G 《Neurochemical research》2005,30(4):479-486
The isolation of a soluble brain fraction which behaves as an endogenous ouabain-like substance, termed endobain E, has been described. Endobain E contains two Na+, K+-ATPase inhibitors, one of them identical to ascorbic acid. Neurotransmitter release in the presence of endobain E and ascorbic acid was studied in non-depolarizing (0 mM KCl) and depolarizing (40 mM KCl) conditions. Synaptosomes were isolated from cerebral cortex of male Wistar rats by differential centrifugation and Percoll gradient. Synaptosomes were preincubated in HEPES-saline buffer with 1 mM d-[3H]aspartate (15 min at 37°C), centrifuged, washed, incubated in the presence of additions (60 s at 37°C) and spun down; radioactivity in the supernatants was quantified. In the presence of 0.5–5.0 mM ascorbic acid, d-[3H]aspartate release was roughly 135–215% or 110–150%, with or without 40 mM KCl, respectively. The endogenous Na+, K+-ATPase inhibitor endobain E dose-dependently increased neurotransmitter release, with values even higher in the presence of KCl, reaching 11-times control values. In the absence of KCl, addition of 0.5–10.0 mM commercial ouabain enhanced roughly 100% d-[3H]aspartate release; with 40 mM KCl a trend to increase was recorded with the lowest ouabain concentrations to achieve statistically significant difference vs. KCl above 4 mM ouabain. Experiments were performed in the presence of glutamate receptor antagonists. It was observed that MPEP (selective for mGluR5 subtype), failed to decrease endobain E response but reduced 50–60% ouabain effect; LY-367385 (selective for mGluR1 subtype) and dizocilpine (for ionotropic NMDA glutamate receptor) did not reduce endobain E or ouabain effects. These findings lead to suggest that endobain E effect on release is independent of metabotropic or ionotropic glutamate receptors, whereas that of ouabain involves mGluR5 but not mGluR1 receptor subtype. Assays performed at different temperatures indicated that in endobain E effect both exocytosis and transporter reversion are involved. It is concluded that endobain E and ascorbic acid, one of its components, due to their ability to inhibit Na+, K+-ATPase, may well modulate neurotransmitter release at synapses. 相似文献
4.
Our previous investigations have established that Na+ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na+-pump, Na+-ATPase, is accompanied by H+ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na+-ATPase of T. viridis operates as an Na+/H+ exchanger is tested in the present work. The study of Na+ and H+ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO3– caused (i) an increase in ATP-driven Na+ uptake by the vesicles, (ii) an increase in (Na++ATP)-dependent vesicle lumen alkalization resulting from H+ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na+-ATPase. The (Na++ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K+ abolished at the vesicle membrane. The fact that the Na+-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na+-ATPase in driving H+ outside the vesicles. The findings allowed us to conclude that the Na+-ATPase of T. viridis directly performs an exchange of Na+ for H+. Since the Na+-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa+/nH+, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane 相似文献
5.
6.
This study addresses the mechanisms of oxygen-induced regulation of ion transport pathways in mouse erythrocyte, specifically focusing on the role of cellular redox state and ATP levels. Mouse erythrocytes possess Na+/K+ pump, K+-Cl– and Na+-K+-2Cl– cotransporters that have been shown to be potential targets of oxygen. The activity of neither cotransporter changed in response to hypoxia-reoxygenation. In contrast, the Na+/K+ pump responded to hypoxic treatment with reversible inhibition. Hypoxia-induced inhibition was abolished in Na+-loaded cells, revealing no effect of O2 on the maximal operation rate of the pump. Notably, the inhibitory effect of hypoxia was not followed by changes in cellular ATP levels. Hypoxic exposure did, however, lead to a rapid increase in cellular glutathione (GSH) levels. Decreasing GSH to normoxic levels under hypoxic conditions abolished hypoxia-induced inhibition of the pump. Furthermore, GSH added to the incubation medium was able to mimic hypoxia-induced inhibition. Taken together these data suggest a pivotal role of intracellular GSH in oxygen-induced modulation of the Na+/K+ pump activity. 相似文献
7.
A ouabain sensitive inward current occurs in Xenopus oocytes in Na+ and K+ -free solutions. Several laboratories have investigated the properties of this current and suggested that acidic extracellular pH (pHo) produces a conducting pathway through the Na+/K+ pump that is permeable to H+ and blocked by [Na+]o. An alternative suggestion is that the current is mediated by an electrogenic H+-ATPase. Here we investigate the effect of pHo and [Na+]o on both transient and steady-state ouabain-sensitive current. At alkaline or neutral pHo the relaxation rate of pre-steady-state current is an exponential function of voltage. Its U-shaped voltage dependence becomes apparent at acidic pHo, as predicted by a model in which protonation of the Na+/K+ pump reduces the energy barrier between the internal solution and the Na+ occluded state. The model also predicts that acidic pHo increases steady-state current leak through the pump. The apparent pK of the titratable group(s) is 6, suggesting that histidine is involved in induction of the conductance pathway. 22Na efflux experiments in squid giant axon and current measurements in oocytes at acidic pHo suggest that both Na+ and H+ are permeant. The acid-induced inward current is reduced by high [Na+]o, consistent with block by Na+. A least squares analysis predicts that H+ is four orders of magnitude more permeant than Na+, and that block occurs when 3 Na+ ions occupy a low affinity binding site (K
0.5=130±30 mM) with a dielectric coefficient of 0.23±0.03. These data support the conclusion that the ouabain-sensitive conducting pathway is a result of passive leak of both Na+ and H+ through the Na+/K+ pump. 相似文献
8.
R. V. Lun'kov I. M. Andreev N. A. Myasoedov G. F. Khailova E. B. Kurkova Yu. V. Balnokin 《Russian Journal of Plant Physiology》2005,52(5):635-644
A membrane fraction enriched in plasma membrane (PM) vesicles was isolated from the root cells of a salt-accumulating halophyte Suaeda altissima (L.) Pall. by means of centrifugation in discontinuous sucrose density gradient. The PM vesicles were capable of generating ΔpH at their membrane and the transmembrane electric potential difference (Δψ). These quantities were measured with optical probes, acridine orange and oxonol VI, sensitive to ΔpH and Δψ, respectively. The ATP-dependent generation of ΔpH was sensitive to vanadate, an inhibitor of P-type ATPases. The results contain evidence for the functioning of H+-ATPase in the PM of the root cells of S. altissima. The addition of Na+ and Li+ ions to the outer medium resulted in dissipation of ΔpH preformed by the H+-ATPase, which indicates the presence in PM of the functionally active Na+/H+ antiporter. The results are discussed with regard to involvement of the Na+/H+ antiporter and the PM H+-ATPase in loading Na+ ions into the xylem of S. altissima roots. 相似文献
9.
T. V. Roslyakova E. M. Lazareva N. V. Kononenko A. V. Babakov 《Biochemistry. Biokhimii?a》2009,74(5):549-556
The gene HvNHX3 encoding a new isoform of vacuolar Na+/H+-antiporter was identified in barley. This gene is expressed in roots and leaves of barley seedlings, and it encodes a protein consisting of 541 amino acid residues with pre-dicted molecular weight 59.7 kDa. It was found that by its amino acid sequence HvNHX3 is closest to the Na+/H+-antiporter HbNHX1 of wild type from Hordeum brevisibulatum that grows on salt-marsh (solonchak) soils (95% homology). The expression of HvNHX3 during salt stress is increased several-fold in roots and leaves of barley seedlings. At the same time, the amount of HvNHX3 protein in roots does not change, but in leaves it increases significantly. It was shown using HvNHX3 immunolocalization in roots that this protein is present in all tissues, but in control plants it was clustered and in experimental plants after salt stress it was visualized as small granules. It has been proposed that HvNHX3 is converted into active form during declusterization. The conversion of HvNHX3 into its active form along with its quantitative increase in leaves during salt stress activates Na+/H+-exchange across the vacuolar membrane and Na+ release from cytoplasm, and, as a consequence, an increase of salt stress tolerance. 相似文献
10.
We investigated the effect of salinity on the relationship between Na+-K+-ATPase and sulfogalactosyl ceramide (SGC) in the basolateral membrane of rainbow trout (Oncorhynchus mykiss) gill epithelium. SGC has been implicated as a cofactor in Na+-K+-ATPase activity, especially in Na+-K+-ATPase rich tissues. However, whole-tissue studies have questioned this role in the fish gill. We re-examined SGC cofactor function from a gill basolateral membrane perspective. Nine SGC fatty acid species were quantified by tandem mass spectrometry (MS/MS) and related to Na+-K+-ATPase activity in trout acclimated to freshwater or brackish water (20 ppt). While Na+-K+-ATPase activity increased, the total concentration and relative proportion of SGC isoforms remained constant between salinities. However, we noted a negative correlation between SGC concentration and Na+-K+-ATPase activity in fish exposed to brackish water, whereas no correlation existed in fish acclimated to freshwater. Differential Na+-K+-ATPase/SGC sensitivity is discussed in relation to enzyme isoform switching, the SGC cofactor site model and saltwater adaptation.This revised version was published online in June 2005 with a corrected cover date. 相似文献
11.
12.
1. Patients affected by isovaleric acidemia (IVAcidemia) suffer from acute episodes of encephalopathy. However, the mechanisms
underlying the neuropathology of this disease are poorly known. The objective of the present study was to investigate the
in vitro effects of the metabolites that predominantly accumulate in IVAcidemia, namely isovaleric acid (IVA), 3-hydroxyisovaleric
acid (3-OHIVA) and isovalerylglycine (IVG), on important parameters of energy metabolism, such as 14CO2 production from acetate and the activities of the respiratory chain complexes I–IV, creatine kinase and Na+, K+-ATPase in synaptic plasma membranes from cerebral cortex homogenates of 30-day-old rats.
2. We observed that 3-OHIVA acid and IVG did not affect all the parameters analyzed. Similarly, 14CO2 production from acetate (Krebs cycle activity), the activities of creatine kinase, and of the respiratory chain complexes
was not modified by IVA. In contrast, IVA exposition to cortical homogenates provoked a marked inhibition of Na+, K+-ATPase activity. However, this activity was not changed when IVA was directly exposed to purified synaptic plasma membranes,
suggesting an indirect effect of this organic acid on the enzyme. Furthermore, pretreatment of cortical homogenates with α-tocopherol
and creatine totally prevented IVA-induced inhibition on Na+, K+-ATPase activity from synaptic plasma membranes, whereas glutathione (GSH) and the NO synthase inhibitor Nω-nitro-l-arginine methyl ester (L-NAME) did not alter this inhibition.
3. These data indicate that peroxide radicals were probably involved in this inhibitory effect. Since Na+, K+-ATPase is a critical enzyme for normal brain development and functioning and necessary to maintain neuronal excitability,
it is presumed that the inhibitory effect of IVA on this activity may be involved in the pathophysiology of the neurological
dysfunction of isovaleric acidemic patients. 相似文献
13.
Previous studies showed that endobain E, an endogenous Na+, K+-ATPase inhibitor, decreases dizocilpine binding to NMDA receptor in isolated membranes. The effect of endobain E on expression
of NMDA receptor subunits in membranes of rat cerebral cortex and hippocampus was analyzed by Western blot. Two days after
administration of 10 μl endobain E (1 μl = 29 mg fresh tissue) NR1 subunit expression enhanced 5-fold and 2.5-fold in cerebral
cortex and hippocampus, respectively. NR2A subunit expression increased 2-fold in cerebral cortex and 1.5-fold in hippocampus.
The level of NR2B subunit raised 3-fold in cerebral cortex but remained unaltered in hippocampus. NR2C subunit expression
was unaffected in either area. NR2D subunit enhanced 1.6 and 2.1-fold for cerebral cortex and hippocampus, respectively. Results
indicate that endogenous Na+, K+-ATPase inhibitor endobain E differentially modifies the expression of NMDA receptor subunits. 相似文献
14.
Bettahi I Dasgupta G Renaudet O Chentoufi AA Zhang X Carpenter D Yoon S Dumy P BenMohamed L 《Cancer immunology, immunotherapy : CII》2009,58(2):187-200
Molecularly defined synthetic vaccines capable of inducing both antibodies and cellular anti-tumor immune responses, in a
manner compatible with human delivery, are limited. Few molecules achieve this target without utilizing external immuno-adjuvants.
In this study, we explored a self-adjuvanting glyco-lipopeptide (GLP) as a platform for cancer vaccines using as a model MO5,
an OVA-expressing mouse B16 melanoma. A prototype B and T cell epitope-based GLP molecule was constructed by synthesizing
a chimeric peptide made of a CD8+ T cell epitope, from ovalbumin (OVA257–264) and an universal CD4+ T helper (Th) epitope (PADRE). The resulting CTL–Th peptide backbones was coupled to a carbohydrate B cell epitope based
on a regioselectively addressable functionalized templates (RAFT), made of four α-GalNAc molecules at C-terminal. The N terminus
of the resulting glycopeptides (GP) was then linked to a palmitic acid moiety (PAM), obviating the need for potentially toxic
external immuno-adjuvants. The final prototype OVA-GLP molecule, delivered in adjuvant-free PBS, in mice induced: (1) robust
RAFT-specific IgG/IgM that recognized tumor cell lines; (2) local and systemic OVA257–264-specific IFN-γ producing CD8+ T cells; (3) PADRE-specific CD4+ T cells; (4) OVA-GLP vaccination elicited a reduction of tumor size in mice inoculated with syngeneic murine MO5 carcinoma
cells and a protection from lethal carcinoma cell challenge; (5) finally, OVA-GLP immunization significantly inhibited the
growth of pre-established MO5 tumors. Our results suggest self-adjuvanting glyco-lipopeptide molecules as a platform for B
Cell, CD4+, and CD8+ T cell epitopes-based immunotherapeutic cancer vaccines.
Both I. Bettahi and G. Dasgupta have contributed equally to this work. 相似文献
15.
Hui Chen Rui An Jiang-Hua Tang Xiang-Huan Cui Fu-Shun Hao Jia Chen Xue-Chen Wang 《Molecular breeding : new strategies in plant improvement》2007,19(3):215-225
To develop a salt-tolerant upland rice cultivar (Oryza sativa L.), OsNHX1, a vacuolar-type Na+/H+ antiporter gene from rice was transferred into the genome of an upland rice cultivar (IRAT109), using an Agrobacterium-mediated method. Seven independent transgenic calli lines were identified by polymerase chain reaction (PCR) analysis. These
35S::OsNHX1 transgenic plants displayed a little accelerated growth during seedling stage but showed delayed flowering time and a slight
growth retardation phenotype during late vegetative stage, suggesting that the OsNHX1 has a novel function in plant development. Northern and western blot analyses showed that the expression levels of OsNHX1 mRNA and protein in the leaves of three independent transgenic plant lines were significantly higher than in the leaves of
wild type (WT) plants. T2 generation plants exhibited increased salt tolerance, showing delayed appearance and development of damage or death caused
by salt stress, as well as improved recovery upon removal from this condition. Several physiological traits, such as increased
Na+ content, and decreased osmotic potential in transgenic plants grown in high saline concentrations, further indicated that
the transgenic plants had enhanced salt tolerance. Our results suggest the potential use of these transgenic plants for further
agricultural applications in saline soil. 相似文献
16.
Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca+2 mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na+/H+ exchanger, leading to reduced intracellular Ca+2 mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction. 相似文献
17.
Four Na+/H+ antiporters, Mrp, TetA(L), NhaC, and MleN have so far been described in Bacillus subtilis 168. We identified an additional Na+/H+ antiporter, YvgP, from B. subtilis that exhibits homology to the cation: proton antiporter-1 (CPA-1) family. The yvgP-dependent complementation observed in a Na+(Ca2+)/H+ antiporter-defective Escherichia coli mutant (KNabc) suggested that YvgP effluxed Na+ and Li+. In addition, effects of yvgP expression on a K+ uptake-defective mutant of E. coli indicated that YvgP also supported K+ efflux. In a fluorescence-based assay of everted membrane vesicles prepared from E. coli KNabc transformants, YvgP-dependent Na+ (K+, Li+, Rb+)/H+ antiport activity was demonstrated. Na+ (K+, Li+)/H+ activity was higher at pH 8.5 than at pH 7.5. Mg2+, Ca2+ and Mn2+ did not serve as substrates but they inhibited YvgP antiport activities. Studies of yvgP expression in B. subtilis, using a reporter gene fusion, showed a significant constitutive level of expression that was highest in stationary phase,
increasing as stationary phase progressed. In addition, the expression level was significantly increased in the presence of
added K+ and Na+. 相似文献
18.
Makoto Fujisawa Yuko Wada Takahiro Tsuchiya Masahiro Ito 《Archives of microbiology》2009,191(8):649-657
19.
The NADH dehydrogenase I from Escherichia coli is a bacterial homolog of the mitochondrial complex I which translocates Na+ rather than H+. To elucidate the mechanism of Na+ transport, the C-terminally truncated NuoL subunit (NuoLN) which is related to Na+/H+ antiporters was expressed as a protein A fusion protein (ProtA–NuoLN) in the yeast Saccharomyces cerevisiae which lacks an endogenous complex I. The fusion protein inserted into membranes from the endoplasmatic reticulum (ER), as
confirmed by differential centrifugation and Western analysis. Membrane vesicles containing ProtA–NuoLN catalyzed the uptake of Na+ and K+ at rates which were significantly higher than uptake by the control vesicles under identical conditions, demonstrating that
ProtA–NuoLN translocated Na+ and K+ independently from other complex I subunits. Na+ transport by ProtA–NuoLN was inhibited by EIPA (5-(N-ethyl-N-isopropyl)-amiloride) which specifically reacts with Na+/H+ antiporters. The cation selectivity and function of the NuoL subunit as a transporter module of the NADH dehydrogenase complex
is discussed.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
20.
Ryuichi Takahashi Takayoshi Nishio Nobumasa Ichizen Tetsuo Takano 《Acta Physiologiae Plantarum》2007,29(5):431-438
Reed plants (Phragmites australis Trinius) grow not only in fresh and brackish water areas but also in arid and high salinity regions. Reed plants obtained
from a riverside (Utsunomiya) were damaged by 257 mM NaCl, whereas desert plants (Nanpi) were not. When the plants were grown
under salt stress, the shoots of the Utsunomiya plants contained high levels of sodium and low levels of potassium, whereas
the upper part of the Nanpi plants contained low levels of sodium and high levels of potassium. One month salt stress did
not affect potassium contents in either Utsunomiya or Nanpi plants, but it did dramatically increase sodium contents only
in the Utsunomiya plants. The ratio of K+ to Na+ was maintained at a high level in the upper parts of the Nanpi plants, whereas the ratio markedly decreased in the Utsunomiya
plants in the presence of NaCl. Accumulation of Na+ in the roots and Na+ efflux from the roots were greater in the Nanpi plants than in the Utsunomiya plants. These results suggest that the salt
tolerance mechanisms of Nanpi reed plants include an improved ability to take up K+ to prevent an influx of Na+ and an improved ability to exclude Na+ from the roots. 相似文献