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1.
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.
Low concentrations (< 10?7 M) of ouabain stimulate the activity of Na+, K+-ATPase in whole homogenates of rat brain. The magnitude of this stimulation varies from 5 to 70%. The concentrations of ouabain which induces maximal stimulation is also highly variable and ranges between 10?9 to 10?7 M. The ouabain stimulation disappears following 1:50 dilution and 2 h preincubation or freezing and thawing of the membranes or their treatment with deoxycholate. “Aging” of a preparation of ATPase also results in loss of its ability to be stimulated by ouabain but ouabain inhibition is preserved. No stimulation of enzyme activity by ouabain is observed in rat brain microsomal fraction. The β-adrenergic blocker propranolol does not inhibit the ouabain induced stimulation of ATPase activity. It is suggested that the stimulation of Na+, K+-ATPase activity by low concentrations of cardiac glycosides if a result of either the displacement of an endogenous ouabain-like compound from the enzyme or an indirect effect by changing membrane surrounding environment of the Na+, K+-ATPase.  相似文献   

3.
We investigated modulation by ATP, Mg2+, Na+, K+ and NH4 + and inhibition by ouabain of (Na+,K+)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na+,K+)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (K M = 0.09 ± 0.01 mmol L−1) of the decapodid III (Na+,K+)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na+,K+)-ATPase activity by K+ also revealed a threefold greater affinity for K+ (K 0.5 = 0.91 ± 0.04 mmol L−1) in decapodid III than in other stages; NH4 + had no modulatory effect. The affinity for Na+ (K 0.5 = 13.2 ± 0.6 mmol L−1) of zoea I (Na+,K+)-ATPase was fourfold less than other stages. Modulation by Na+, Mg2+ and NH4 + obeyed cooperative kinetics, while K+ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg2+ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg2+-stimulated ATPases other than (Na+,K+)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na+-ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH4 +-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.  相似文献   

4.
The expression of Na+, K+-ATPase α3 subunit and synaptosomal membrane Na+, K+-ATPase activity were analyzed after administration of ouabain and endobain E, respectively commercial and endogenous Na+, K+-ATPase inhibitors. Wistar rats received intracerebroventricularly ouabain or endobain E dissolved in saline solution or Tris–HCl, respectively or the vehicles (controls). Two days later, animals were decapitated, cerebral cortex and hippocampus removed and crude and synaptosomal membrane fractions were isolated. Western blot analysis showed that Na+, K+-ATPase α3 subunit expression increased roughly 40% after administration of 10 or 100 nmoles ouabain in cerebral cortex but remained unaltered in hippocampus. After administration of 10 μl endobain E (1 μl = 28 mg tissue) Na+, K+-ATPase α3 subunit enhanced 130% in cerebral cortex and 103% in hippocampus. The activity of Na+, K+-ATPase in cortical synaptosomal membranes diminished or increased after administration of ouabain or endobain E, respectively. It is concluded that Na+, K+-ATPase inhibitors modify differentially the expression of Na+, K+-ATPase α3 subunit and enzyme activity, most likely involving compensatory mechanisms.  相似文献   

5.
Sporadic hemiplegic migraine type 2 (SHM2) and familial hemiplegic migraine type 2 (FHM2) are rare forms of hemiplegic migraine caused by mutations in the Na+,K+-ATPase α2 gene. Today, more than 70 different mutations have been linked to SHM2/FHM2, randomly dispersed over the gene. For many of these mutations, functional studies have not been performed. Here, we report the functional characterization of nine SHM2/FHM2 linked mutants that were produced in Spodoptera frugiperda (Sf)9 insect cells. We determined ouabain binding characteristics, apparent Na+ and K+ affinities, and maximum ATPase activity. Whereas membranes containing T345A, R834Q or R879W possessed ATPase activity significantly higher than control membranes, P796S, M829R, R834X, del 935–940 ins Ile, R937P and D999H membranes showed significant loss of ATPase activity compared to wild type enzyme. Further analysis revealed that T345A and R879W showed no changes for any of the parameters tested, whereas mutant R834Q possessed significantly decreased Na+ and increased K+ apparent affinities as well as decreased ATPase activity and ouabain binding. We hypothesize that the majority of the mutations studied here influence interdomain interactions by affecting formation of hydrogen bond networks or interference with the C-terminal ion pathway necessary for catalytic activity of Na+,K+-ATPase, resulting in decreased functionality of astrocytes at the synaptic cleft expressing these mutants.  相似文献   

6.
Distal colon absorbs K+ through a Na+-independent, ouabain-sensitive H+/K+-exchange, associated to an apical ouabain-sensitive H+/K+-ATPase. Expression of HKα2, gene associated with this ATPase, induces K+-transport mechanisms, whose ouabain susceptibility is inconsistent. Both ouabain-sensitive and ouabain-insensitive K+-ATPase activities have been described in colonocytes. However, native H+/K+-ATPases have not been identified as unique biochemical entities. Herein, a procedure to purify ouabain-sensitive H+/K+-ATPase from guinea-pig distal colon is described. H+/K+-ATPase is Mg2+-dependent and activated by K+, Cs+ and NH4+ but not by Na+ or Li+, independently of K+-accompanying anion. H+/K+-ATPase was inhibited by ouabain and vanadate but insensitive to SCH-28080 and bafilomycin-A. Enzyme was phosphorylated from [32P]-γ-ATP, forming an acyl-phosphate bond, in an Mg2+-dependent, vanadate-sensitive process. K+ inhibited phosphorylation, effect blocked by ouabain. H+/K+-ATPase is an α/β-heterodimer, whose subunits, identified by Tandem-mass spectrometry, seems to correspond to HKα2 and Na+/K+-ATPase β1-subunit, respectively. Thus, colonic ouabain-sensitive H+/K+-ATPase is a distinctive P-type ATPase.  相似文献   

7.
Binding to Na+,K+-ATPase, cardiotonic steroids (CTS) activate intracellular signaling cascades that affect gene expression and regulation of proliferation and apoptosis in cells. Ouabain is the main CTS used for studying these processes. The effects of other CTS on nervous tissue are practically uncharacterized. Previously, we have shown that ouabain affects the activation of mitogen-activated protein kinases (MAP kinases) ERK1/2, p38, and JNK. In this study, we compared the effects of digoxin and bufalin, which belong to different subclasses of CTS, on primary culture of rat cortical cells. We found that CTS toxicity is not directly related to the degree of Na+,K+-ATPase inhibition, and that bufalin and digoxin, like ouabain, are capable of activating ERK1/2 and p38, but with different concentration and time profiles. Unlike bufalin and ouabain, digoxin did not decrease JNK activation after long-term incubation. We concluded that the toxic effect of CTS in concentrations that inhibit less than 80% of Na+,K+-ATPase activity is related to ERK1/2 activation as well as the complex profile of MAP kinase activation. A direct correlation between Na+,K+-ATPase inhibition and the degree of MAP kinase activation is only observed for ERK1/2. The different action of the three CTS on JNK and p38 activation may indicate that it is associated with intracellular signaling cascades triggered by protein–protein interactions between Na+,K+-ATPase and various partner proteins. Activation of MAP kinase pathways by these CTS occurs at concentrations that inhibit Na+,K+-ATPase containing the α1 subunit, suggesting that these signaling cascades are realized via α1. The results show that the signaling processes in neurons caused by CTS can differ not only because of different inhibitory constants for Na+,K+-ATPase.  相似文献   

8.
Low concentrations of cardiac glycosides including ouabain, digoxin, and digitoxin block cancer cell growth without affecting Na+,K+-ATPase activity, but the mechanism underlying this anti-cancer effect is not fully understood. Volume-regulated anion channel (VRAC) plays an important role in cell death signaling pathway in addition to its fundamental role in the cell volume maintenance. Here, we report cardiac glycosides-induced signaling pathway mediated by the crosstalk between Na+,K+-ATPase and VRAC in human cancer cells. Submicromolar concentrations of ouabain enhanced VRAC currents concomitantly with a deceleration of cancer cell proliferation. The effects of ouabain were abrogated by a specific inhibitor of VRAC (DCPIB) and knockdown of an essential component of VRAC (LRRC8A), and they were also attenuated by the disruption of membrane microdomains or the inhibition of NADPH oxidase. Digoxin and digitoxin also showed anti-proliferative effects in cancer cells at their therapeutic concentration ranges, and these effects were blocked by DCPIB. In membrane microdomains of cancer cells, LRRC8A was found to be co-immunoprecipitated with Na+,K+-ATPase α1-isoform. These ouabain-induced effects were not observed in non-cancer cells. Therefore, cardiac glycosides were considered to interact with Na+,K+-ATPase to stimulate the production of reactive oxygen species, and they also apparently activated VRAC within membrane microdomains, thus producing anti-proliferative effects.  相似文献   

9.
Mechanisms underlying the tissue-specific impact of cardiotonic steroids (CTS) on cell survival and death remain poorly understood. This study examines the role of Na+,K+-ATPase α subunits in death of Madin-Darby canine kidney (MDCK) cells evoked by 24-h exposure to ouabain. MDCK cells expressing a variant of the α1 isoform, CTS-sensitive α1S, were stably transfected with a cDNA encoding CTS-resistant α1R-Na+,K+-ATPase, whose expression was confirmed by RT–PCR. In mock-transfected and α1R-cells, maximal inhibition of 86Rb influx was observed at 10 and 1000 μM ouabain, respectively, thus confirming high abundance of α1R-Na+,K+-ATPase in these cells. Six-hour treatment of α1R-cells with 1000 μM ouabain led to the same elevation of the [Na+]i/[K+]i ratio that was detected in mock-transfected cells treated with 3 μM ouabain. However, in contrast to the massive death of mock-transfected cells exposed to 3 μM ouabain, α1R-cells survived after 24-h incubation with 1000 μM ouabain. Inversion of the [Na+]i/[K+]i ratio evoked by Na+,K+-ATPase inhibition in K+-free medium did not affect survival of α1R-cells but increased their sensitivity to ouabain. Our results show that the α1R subunit rescues MDCK cells from the cytotoxic action of CTS independently of inhibition of Na+,K+-ATPase-mediated Na+ and K+ fluxes and inversion of the [Na+]i/[K+]i ratio.  相似文献   

10.
In addition to performing its essential transport function, the sodium pump also activates multiple cell signaling pathways in response to digitalis drugs such as ouabain. Based mainly on cell-free studies with mixtures of purified Src kinase and Na+/K+-ATPase, a well-advocated hypothesis on how ouabain initiates the activation of signaling pathways is that there is a preexisting physiological complex of inactive Src bound to the α-subunit of Na+/K+-ATPase, and that ouabain binding to this subunit disrupts the bound Src and activates it. Because of the published disagreements of the results of such cell-free experiments of two other laboratories, our aim was to attempt the resolution of these discrepancies. We reexamined the effects of ouabain, vanadate, and oligomycin on mixtures of Src, Na+/K+-ATPase, Mg2+, and ATP as specified in prior studies; and assayed for Src-418 autophosphorylation as the measure of Src activation. In contrast to the findings of the proponents of the above hypothesis, our results showed similar effects of the three inhibitors of Na+/K+-ATPase; indicating that Src activation in such experiments is primarily due to the ATP-sparing effect of the ATPase inhibitor on the mixture of two enzymes competing for ATP. We conclude that there is no solid evidence for direct molecular interaction of Src with Na+/K+-ATPase under physiological conditions.  相似文献   

11.
Internalization of the Na+/K+-ATPase (the Na+ pump) has been studied in the human lung carcinoma cell line H1299 that expresses YFP-tagged α1 from its normal genomic localization. Both real-time imaging and surface biotinylation have demonstrated internalization of α1 induced by ≥100 nm ouabain which occurs in a time scale of hours. Unlike previous studies in other systems, the ouabain-induced internalization was insensitive to Src or PI3K inhibitors. Accumulation of α1 in the cells could be augmented by inhibition of lysosomal degradation but not by proteosomal inhibitors. In agreement, the internalized α1 could be colocalized with the lysosomal marker LAMP1 but not with Golgi or nuclear markers. In principle, internalization could be triggered by a conformational change of the ouabain-bound Na+/K+-ATPase molecule or more generally by the disruption of cation homeostasis (Na+, K+, Ca2+) due to the partial inhibition of active Na+ and K+ transport. Overexpression of ouabain-insensitive rat α1 failed to inhibit internalization of human α1 expressed in the same cells. In addition, incubating cells in a K+-free medium did not induce internalization of the pump or affect the response to ouabain. Thus, internalization is not the result of changes in the cellular cation balance but is likely to be triggered by a conformational change of the protein itself. In physiological conditions, internalization may serve to eliminate pumps that have been blocked by endogenous ouabain or other cardiac glycosides. This mechanism may be required due to the very slow dissociation of the ouabain·Na+/K+-ATPase complex.  相似文献   

12.
Summary The sulfatide content, phospholipid concentration, and (Na++K+)-ATPase activity from skin and gills of different stages of larval development ofCalyptocephalella caudiverbera (a Chilean frog) were analyzed. Additionally, the short-circuit current in skin was studied. When skin and gills, depending on the stage of larval development, present (Na++K+)-ATPase activity, they have a high ratio of sulfatide to amount of membrane and the phosphatidylserine concentration remains unchanged. Sulfatide content and (Na++K+)-ATPase activity in skin are in direct relationship with the level of sodium flux present during development. The specific enzymatic hydrolysis of sulfatide with partially purified arylsulfatase of pig kidney inhibits 100% of the ouabain-sensitive (Na++K+)-ATPase. The ouabain-insensitive ATPase remains virtually unchanged with the treatment, even with a high concentration of arylsulfatase or with ouabain present in the medium. These experiments strongly suggest a role of sulfatides in the (Na++K+)-ATPase activity and, as a consequence, in sodium ion transport.  相似文献   

13.
《Insect Biochemistry》1991,21(7):749-758
The present study confirms previous reports of the presence of (Na+ + K+)-ATPase and anion-stimulated ATPase activity in Malpighian tubules of Locusta. In addition, the presence of a K+-stimulated, ouabain-insensitive ATPase activity has been identified in microsomal fractions. Differential and sucrose density-gradient centrifugation of homogenates has been used to separate membrane fractions which are rich in mitochondria, apical membranes and basolateral membranes; as indicated by the presence of succinate dehydrogenase and the presence or absence of non-specific alkaline phosphatase activity, respectively. Relatively high specific (Na+ + K+)-ATPase activity was associated with the basolateral membrane-rich fractions with only low levels of this activity being associated with the apical membrane-rich preparation. K+-stimulated ATPase activity was also associated, predominantly, with the basolateral membrane-rich fractions. However, comparison of the distribution of this activity with that of the (Na+ + K+)-ATPase suggests that the two enzymes did not co-separate. The possibility that the K+-stimulated ATPase was not associated with the basolateral plasma membrane is discussed.Anion-stimulated ATPase activity was found in the apical and basolateral membrane-rich fractions and in the fraction contaning mainly mitochondria. Nevertheless, the fact that this bicarbonate-stimulated activity did not co-separate with succinate dehydrogenase activity suggests that it was not exclusively mitochondrial in origin. These results are consistent with physiological studies indicating a basolateral (Na+ + K+)-ATPase but do not support the K+-stimulated ATPase as a candidate for the apical electrogenic pump. The possible role of the bicarbonate-stimulated ATPase activity in ion transport across both the basolateral and apical cell membranes is discussed.  相似文献   

14.
Sodium plays a major role in different astrocytic functions, including maintenance of ion homeostasis and uptake of neurotransmitters and metabolites, which are mediated by different Na+-coupled transporters. In the current study, the role of an electrogenic sodium-bicarbonate cotransporter (NBCe1), a sodium-potassium-chloride transporter 1 (NKCC1) and sodium-potassium ATPase (Na+-K+-ATPase) for the maintenance of [Na+]i was investigated in cultured astrocytes of wild-type (WT) and of NBCe1-deficient (NBCe1-KO) mice using the Na+-sensitive dye, asante sodium green-2. Our results suggest that cytosolic Na+ was higher in the presence of CO2/HCO3 (15 mM) than CO2/HCO3-free, HEPES-buffered solution in WT, but not in NBCe1-KO astrocytes (12 mM). Surprisingly, there was a strong dependence of cytosolic [Na+] on the extracellular [HCO3] attributable to NBCe1 activity. Pharmacological blockage of NKCC1 with bumetanide led to a robust drop in cytosolic Na+ in both WT and NBCe1-KO astrocytes by up to 6 mM. There was a strong dependence of the cytosolic [Na+] on the extracellular [K+]. Inhibition of the Na+-K+-ATPase led to larger increase in cytosolic Na+, both in the absence of K+ as compared with the presence of ouabain and in NBCe1-KO astrocytes as compared with WT astrocytes. Our results show that cytosolic Na+ in mouse cortical astrocytes can vary considerably and depends greatly on the concentrations of HCO3 and K+, attributable to the activity of the Na+-K+-ATPase, of NBCe1 and NKCC1.  相似文献   

15.
De novo mutations in ATP1A3, the gene encoding the α3-subunit of Na+,K+-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N, D801N, E815K, and G947R) associated with AHC. Wild type and mutant Na+,K+-ATPases were expressed in Sf9 insect cells using the baculovirus expression system. Ouabain binding, ATPase activity, and phosphorylation were absent in mutants I274N, E815K and G947R. Mutants S137Y and D801N were able to bind ouabain, although these mutants lacked ATPase activity, phosphorylation, and the K+/ouabain antagonism indicative of modifications in the cation binding site. Mutant D220N showed similar ouabain binding, ATPase activity, and phosphorylation to wild type Na+,K+-ATPase. Functional impairment of Na+,K+-ATPase in mutants S137Y, I274N, D801N, E815K, and G947R might explain why patients having these mutations suffer from AHC. Moreover, mutant D801N is able to bind ouabain, whereas mutant E815K shows a complete loss of function, possibly explaining the different phenotypes for these mutations.  相似文献   

16.
Abstract: The activities of certain properties of sodium, potassium-activated adenosine triphosphatase (Na +, K+- ATPase; EC 3.6.1.3) were examined in cultures and peri- karya fractions enriched in rat cerebellar nerve cells or astrocytes, in comparison with preparations from whole immature and adult rat cerebellum and derived synapto- somal fractions, as well as nonneural tissue such as the kidney. The specific activity of Na +, K+-ATPase was markedly higher in the freshly isolated astrocytes than in the nerve cells (3–15-fold greater depending on neuronal cell type). In contrast, the specific activity of the enzyme was about twice as high in the primary neuronal as in the a'strocytic cultures after 14 days in vitro. In membrane preparations from the whole cerebellum, synaptosomal fractions, and total perikarya suspensions the inhibition of enzyme activity by ouabain indicated complex kinetics, which were consistent with the presence of two forms of the Na +, K+-ATPase (apparent Aj values of about 10–7M and 10–4-10–5M, respectively), the high- affinity form accounting for 60–75% of the total activity. The interaction of the enzyme with ouabain was apparently similar in perikarya preparations of granule neurones, Purkinje cells, and astrocytes. Differences were, however, observed in the properties of the Na +,K + - ATPase of cultured neurones and astrocytes. The latter contained predominantly, but not exclusively, an Na+,K+-ATPase with low affinity for ouabain (73% of the total) that is similar to the single enzyme form in the kidney. This form constituted a significantly smaller proportion of the Na +, K+-ATPase in the cultured neuronal preparations (55%). It would appear, therefore, that in membrane fractions from preparations enriched in different separated and cultured neural cell types both the high- and the low-affinity forms of the enzyme, in terms of interaction with ouabain, are expressed. Depending on the class of cells these enzyme forms constituted a different proportion of the total activity, but both forms seemed to be present in every type of cell examined, even after taking into acc.ount the contribution in the enriched preparations of the contaminating cell types. In contrast with the results on the Na+, K+-ATPase activity determined under optimal conditions in preparations derived from disrupted cells, differences could not be detected between the cultured cell types when the effect of ouabain on the uptake of 86Rb into “live cells” was estimated as a measure of in situ ion pump activity. Besides the interaction with ouabain, the K+ dependence of the Na+, K+-ATPase activity was also investigated in crude particulate preparations from cultured cerebellar neurones and astrocytes. Differences were observed as nearly maximal enzyme activity was obtained in the as- trocyte preparations at 1 mM KCl, when only about one- third of the maximal activity was displayed by the cultured nerve cells.  相似文献   

17.
Effects of long-term, subtotal inhibition of Na+-K+ transport, either by growth of cells in sublethal concentrations of ouabain or in low-K+ medium, are described for HeLa cells. After prolonged growth in 2 × 10?8 M ouabain, the total number of ouabain molecules bound per cell increases by as much as a factor of three, mostly due to internalization of the drug. There is only about a 20% increase in ouabain-binding sites on the plasma membrane, representing amodest induction of Na+, K+-ATPase. In contrast, after long-term growth in low K+ there can be a twofold or greater increase in ouabain binding per cell, and in this case the additional sites are located in the plasma membrane. The increase is reversible. To assess the corresponding transport changes, we have separately estimated the contributions of increased intracellular [Na+] and of transport capacity (number of transport sites) to transport regulation. During both induction and reversal, short-term regulation is achieved primarily by changes in [Na+]i. More slowly, long-term regulation is achieved by changes in the number of functional transporters in the plasma membrane as assessed by ouabain binding, Vmax for transport, and specific phosphorylation. Parallel exposure of cryptic Na+, K+-ATPase activity with sodium dodecyl sulfate in the plasma membranes of both induced and control cells showed that the induction cannot be accounted for by an exposure of preexisting Na+, K+-ATPase in the plasma membrane. Analysis of the kinetics of reversal indicates that it may be due to a post-translational event.  相似文献   

18.
Recent studies demonstrate that cytotoxic actions of ouabain and other cardiotonic steroids (CTS) on renal epithelial cells (REC) are triggered by their interaction with the Na+,K+-ATPase α-subunit but not the result of inhibition of Na+,K+-ATPase-mediated ion fluxes and inversion of the [Na+]i/[K+]i ratio. This study examined the role of mitogen-activated protein kinases (MAPK) in the death of ouabain-treated REC. Exposure of C7-MDCK cells that resembled principal cells from canine kidney to 3 μM ouabain led to phosphorylation of p38 without significant impact on phosphorylation of ERK and JNK MAPK. Maximal increment of p38 phosphorylation was observed at 4 h followed by cell death at 12 h of ouabain addition. In contrast to ouabain, neither cell death nor p38 MAPK phosphorylation were affected by elevation of the [Na+]i/[K+]i ratio triggered by Na+,K+-ATPase inhibition in K+-free medium. p38 phosphorylation was noted in all other cell types exhibiting death in the presence of ouabain, such as intercalated cells from canine kidney and human colon rectal carcinoma cells. We did not observe any action of ouabain on p38 phosphorylation in ouabain-resistant smooth muscle cells from rat aorta and endothelial cells from human umbilical vein. Both p38 phosphorylation and death of ouabain-treated C7-MDCK cells were suppressed by p38 inhibitor SB 202190 but were resistant to its inactive analogue SB 202474. Our results demonstrate that death of CTS-treated REC is triggered by Nai+,Ki+—independent activation of p38 MAPK.  相似文献   

19.
Cardiac glycosides are inhibitors of Na+,K+-ATPase, and K+-phosphatase activities of the transport enzyme. Previous studies have shown that when the sensitivities of these two activities to ouabain are compared by the addition of varying concentrations of the drug to the assay media, the K+-phosphatase is significantly less sensitive than Na+,K+-ATPase. This work was done to seek an explanation for this phenomenon. 3-O-Methyl-fluorescein phosphate was used as substrate for the continuous fluorimetric assay of K+-phosphatase obtained from human red cells. When ouabain was added to the assay medium, a time-dependent inhibition of K+-phosphatase was observed. The rate of inhibition was also influenced by the order of additions of K+ and ouabain. In view of these results, several enzyme samples exposed to ouabain for varying lengths of time were prepared, and their Na+,K+-ATPase and K+-phosphatase activities were then determined. A good correlation between the extent of inhibition of the two activities was obtained. These results prove that the previously observed discrepancies between the sensitivities of Na+,K+-ATPase and K+-phosphatase to ouabain are due to the different kinetics of drug interaction with the enzyme under the different conditions of the two assays and that once a certain level of ouabain binding to the enzyme is achieved, both activities are equally inhibited.  相似文献   

20.
Liposomes containing either purified or microsomal (Na+,K+)-ATPase preparations from lamb kidney medulla catalyzed ATP-dependent transport of Na+ and K+ with a ratio of approximately 3Na+ to 2K+, which was inhibited by ouabain. Similar results were obtained with liposomes containing a partially purified (Na+,K+-ATPase from cardiac muscle. This contrasts with an earlier report by Goldin and Tong (J. Biol. Chem. 249, 5907–5915, 1974), in which liposomes containing purified dog kidney (Na+,K+)-ATPase did not transport K+ but catalyzed ATP-dependent symport of Na+ and Cl?. When purified by our procedure, dog kidney (Na+,K+)-ATPase showed some ability to transport K+ but the ratio of Na+ : K+ was 5 : 1.  相似文献   

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