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1.
Using dialysed squid axons we have been able to control internal and external ionic compositions under conditions in which most of the Na+ efflux goes through the Na+ pump. We found that (i) internal K+ had a strong inhibitory effect on Na+ efflux; this effect was antagonized by ATP, with low affinity, and by internal Na+, (ii) a reduction in ATP levels from 3 mM to 50 μM greatly increased the apparent affinity for external K+, but reduced its effectiveness compared with other monovalent cations, as an activator of Na+ efflux, and (iii) the relative effectiveness of different K+ congeners as external activator of the Na+ efflux, though affected by the ATP concentration, was not affected by the Na+/+ ratio inside the cells. These results are consistent with the idea that the same conformation of the (Na+ + K+)-ATPase can be reached by interaction with external K+ after phosphorylation and with internal K+ before rephosphorylation. They also stress a nonphosphorylating regulatory role of ATP.  相似文献   

2.
Decreased cellular accumulation of cisplatin is a frequently observed mechanism of resistance to the drug. Beside passive diffusion, several cellular proteins using ATP hydrolysis as an energy source are assumed to be involved in cisplatin transport in and out of the cell. This investigation aimed at clarifying the contribution of intracellular ATP as an indicator of energy-dependent transport to cisplatin resistance using the A2780 human ovarian adenocarcinoma cell line and its cisplatin-resistant variant A2780cis. Depletion of intracellular ATP with oligomycin significantly decreased cellular platinum accumulation (measured by flameless atomic absorption spectrometry) in sensitive but not in resistant cells, and did not affect cisplatin efflux in both cell lines. Inhibition of Na+,K+-ATPase with ouabain reduced platinum accumulation in A2780 cells but to a lesser extent compared with oligomycin. Western blot analysis revealed lower expression of Na+,K+-ATPase α1 subunit in resistant cells compared with sensitive counterparts. The basal intracellular ATP level (determined using a bioluminescence-based assay) was significantly higher in A2780cis cells than in A2780 cells. Our results highlight the importance of ATP-dependent transport, among other processes mediated by Na+,K+-ATPase, for cisplatin influx in sensitive cells. Cellular platinum accumulation in resistant cells is reduced and less dependent on energy sources, which may partly result from Na+,K+-ATPase downregulation. Our data suggest the involvement of other ATP-dependent processes beside those regulated by Na+,K+-ATPase. Higher basal ATP level in cisplatin-resistant cells, which appears to be a consequence of enhanced mitochondrial ATP production, may represent a survival mechanism established during development of resistance.  相似文献   

3.
The partition of Li+, Br-, and I- across the membrane of the sartorius muscle of the toad Bufo marinus has been investigated both at the steady state and with kinetic methods. Li+ was found to have access to an amount of muscle water similar to that of Na+. Br- and I- could be regarded as being interchangeable with cellular Cl-. None of the foreign ions caused significant losses of cellular K+. Li+ efflux from the cell was slower in muscles which were equilibrated for long periods in Li+ than in short equilibrated muscles. Na+ efflux from Li+-treated muscles was similar in rate to normal controls, but the amount of Na+ in the slow fraction was increased by Li+. I- efflux was extremely rapid, and it was not possible to differentiate kinetically between intra- and extracellular material. These results have been found to be consistent with the hypothesis of a three phase system for muscle.  相似文献   

4.
The addition of LiCl stimulated the (Na++K+)-dependent ATPase activity of a rat brain enzyme preparation. Stimulation was greatest in high Na+/low K+ media and at low Mg. ATP concentrations. Apparent affinities for Li+ were estimated at the α-sites (moderate-affinity sites for K+ demonstrable in terms of activation of the associated K+-dependent phosphatase reaction), at the β-sites (high-affinity sites for K+ demonstrable in terms of activation of the overall ATPase reaction), and at the Na+ sites for activation. The relative efficacy of Li+ was estimated in terms of the apparent maximal velocity of the phosphatase and ATPase reactions when Li+ was substituted for K+, and also in terms of the relative effect of Li+ on the apparent KM for Mg· ATP. With these data, and previously determined values for the apparent affinities of K+ and Na+ at these same sites, quantitative kinetic models for the stimulation were examined. A composite model is required in which Li+ stimulates by relieving inhibition due to K+ and Na+ (i) by competing with K+ for the α-sites on the enzyme through which K+ decreases the apparent affinity for Mg·ATP and (ii) by competing with Na+ at low-affinity inhibitory sites, which may represent the external sites at which Na+ is discharged by the membrane NA+/K+ pump that this enzyme represents. Both these sites of action for Li+ would thus lie, in vivo, on the cell exterior.  相似文献   

5.
Cation transport is thought to be an important process for ion homeostasis in plant cells. Here, we report that a soybean putative cation/proton antiporter GmCAX1 may be a mediator of this process. GmCAX1 is expressed in all tissues of the soybean plants but at a lower level in roots. Its expression was induced by PEG, ABA, Ca2+, Na+ and Li+ treatments. The GmCAX1-GFP fusion protein was mainly localized in plasma membrane of the transgenic Arabidopsis plant cells and onion epidermal cells. Transgenic Arabidopsis plants overexpressing GmCAX1 accumulated less Na+, K+, and Li+, and were more tolerant to elevated Li+ and Na+ levels during germination when compared with the controls. These results suggest that GmCAX1 may function as an antiporter for Na+, K+ and Li+. Modulation of this antiporter may be beneficial for regulation of ion homeostasis and thus plant salt tolerance.  相似文献   

6.
7.
Summary It is shown that the ouabain-resistant (OR) furosemide-sensitive K+(Rb+) transport system performs a net efflux of K+ in growing mouse 3T3 cells. This conclusion is based on the finding that under the same assay conditions the furosemidesensitive K+(Rb+) efflux was found to be two- to threefold higher than the ouabain-resistant furosemide-sensitive K+(Rb+) influx. The oubain-resistant furosemide-sensitive influxes of both22Na and86Rb appear to be Cl dependent, and the data are consistent with coupled unidirectional furosemide-sensitive influxes of Na+, K+ and Cl with a ratio of 1 1 2. However, the net efflux of K+ performed by this transport system cannot be coupled to a ouabain-resistant net efflux of Na+ since the unidirectional ouabain-resistant efflux of Na+ was found to be negligible under physiological conditions. This latter conclusion was based on the fact that practically all the Na+ efflux appears to be ouabainsensitive and sufficient to balance the Na+ influx under such steady-state conditions. Therefore, it is suggested that the ouabain-resistant furosemide-sensitive transport system in growing cells performs a facilitated diffusion of K+ and Na+, driven by their respective concentration gradients: a net K+ efflux and a net Na+ influx.  相似文献   

8.
In several tissues a coupling between glycolysis and (Na++K+)-ATPase has been observed. We report here studies on the coupling of glycolysis and (Na++K+)-ATPase in Rous-transformed hamster cells and Ehrlich ascites tumor cells. The rate of (Na++K+)-ATPase was estimated by the initial rate of ouabain-sensitive K+ influx after K+ reintroduction to K+-depleted cells. Experiments were performed with cells producing ATP via oxidative phosphorylation alone (i.e., lactate sole substrate), glycolysis alone (i.e., glucose as substrate in the absence of oxygen or with antimycin A), or glycolysis and oxidative phosphorylation (i.e., glucose as substrate in the presence of oxygen). The cells produced ATP at approximately the same rate under all of these conditions, but the initial rate of K+-influx was approx. 2-fold higher when AtP was produced from glycolysis. Changes in cell Na+ due to other transport processes related to glycolysis, such as Na+-H+ exchange, Na+-glucose cotransport, and K+-H+ exchange were ruled out as mediators of this effect on (Na++K+)-ATPase. These data suggest that glycolysis is more effective than oxidative phosphorylation in providing ATP to (Na++K+)-ATPase to these cultured cells.  相似文献   

9.
Effects of external ionic conditions ofD. discoideum cells were examined in relation to intracellular ionic concentrations, the activity of pyruvate kinase and the amount of ATP. Main components of metal cations in heat extracts of vegetative cells were K+, Na+, Mg2+ and Ca2+ whose concentrations in a cell were about 35.0, 3.6, 10.6 and 2.3 mM, respectively. External Na+ at the concentration more than 50 mM inhibited the formation of cell aggregates in the presence of 10?4M Ca2+. Such an inhibitory effect of Na+ was completely nullified by the addition of more than 10 mM K+. External Na+ caused a rapid decrease in intracellular K+, but an increase in intracellular Na+. Furthermore, it was found that the cells containing a high concentration of Na+ can develop normally in the presence of exogenous 10 mM K+, where intracellular K+ was maintaned at about 30 mM, irrespective of a high concentration of intracellular Na+ (about 30 mM). These suggest that the Na+-inhibition of the development is caused by a decrease in intracellular K+, but not by an increase in intracellular Na+. Pyruvate kinase extracted from the organism required K+ for its activation. The vegetative cells incubated in 50 mM Na+ contained only about 10 mM K+ which is insufficient for the enzyme activation. However, the amount of ATP in the cells containing less K+ was similar to that in those with much K+. These results are discussed in relation to the activity of glycolysis.  相似文献   

10.
The kinetic response of swine erythrocyte (Na + K)-ATPase to Na+ concentration was hyperbolic in low KCl (5–25 mm) but became increasingly sigmoidal (n = 2.2) as KCl was increased to 150 mm. The addition of 150 mm LiCl did not cause an increase in sigmoidicity although it decreased the apparent affinity for Na+. The dependence of ouabain-inhibited efflux of Na+ on internal Na+ concentration was measured in intact cells with intracellular cation concentrations altered by incubation in p-ehloromercuriphenyl sulfonate. The response to Na+ was sigmoidal (n = 2.2) in cells containing high K+ but hyperbolic in preparations in which most of the intracellular K+ was replaced by Li+, even in the presence of 150 mm external KCl. The data are consistent with a model in which internal K+ is an allosteric (feedback) inhibitor of Na+ efflux and there are three Na+ sites which interact cooperatively.  相似文献   

11.
Transport Pathways for Therapeutic Concentrations of Lithium in Rat Liver   总被引:1,自引:0,他引:1  
Although both amiloride- and phloretin-sensitive Na+/Li+ exchange activities have been reported in mammalian red blood cells, it is still unclear whether or not the two are mediated by the same pathway. Also, little is known about the relative contribution of these transport mechanisms to the entry of therapeutic concentrations of Li+ (0.2–2 mm) into cells other than erythrocytes. Here, we describe characteristics of these transport systems in rat isolated hepatocytes in suspension. Uptake of Li+ by hepatocytes, preloaded with Na+ and incubated in the presence of ouabain and bumetanide, comprised three components. (a) An amiloride-sensitive component, with apparent K m 1.2 mm Li+, V max 40 μmol · (kg dry wt · min)−1, showed increased activity at low intracellular pH. The relationship of this component to the concentration of intracellular H+ was curvilinear suggesting a modifier role of [H+] i . This system persisted in Na+-depleted cells, although with apparent K m 3.8 mm. (b) A phloretin-sensitive component, with K m 1.2 mm, V max 21 μmol · (kg · min)−1, was unaffected by pH but was inactive in Na+-depleted cells. Phloretin inhibited Li+ uptake and Na+ efflux in parallel. (c) A residual uptake increased linearly with the external Li+ concentration and represented an increasing proportion of the total uptake. The results strongly suggest that the amiloride-sensitive and the phloretin-sensitive Li+ uptake in rat liver are mediated by two separate pathways which can be distinguished by their sensitivity to inhibitors and intracellular [H+]. Received: 8 April 1999/Revised: 19 July 1999  相似文献   

12.
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   

13.
The membrane potential of the Ehrlich ascites tumor cell was shown to be influenced by its amino acid content and the activity of the Na+: K+ pump. The membrane potential (monitored by the fluorescent dye, 3,3′-dipropylthiodicarbocyanine iodide) varied with the size of the endogenous amino acid pool and with the concentration of accumulated 2-aminoisobutyrate. When cellular amino acid content was high, the cells were hyperpolarized; as the pool declined in size, the cells were depolarized. The hyperpolarization seen with cellular amino acid required cellular Na+ but not cellular ATP. Na+ efflux was more rapid from cells containing 2-aminoisobutyrate than from cells low in internal amino acids. These observations indicate that the hyperpolarization recorded in cells with high cellular amino acid content resulted from the electrogenic co-efflux of Na+ and amino acids.Cellular ATP levels were found to decline rapidly in the presence of the dye and hence the influence of the pump was seen only if glucose was added to the cells. When the cells contained normal Na+ (approx. 30 mM), the Na+: K+ pump was shown to have little effect on the membrane potential (the addition of ouabain had little effect on the potential). When cellular Na+ was raised to 60 mM, the activity of the pump changed the membrane potential from the range ?25 to ?30 mV to ?44 to ?63 mV. This hyperpolarization required external K+ and was inhibited by ouabain.  相似文献   

14.
A technique was developed which permitted the release of ATP from synaptosomes by elevated extracellular K+ or by veratridine to be directly and continuously monitored. The released ATP interacted with firefly luciferin and luciferase in the incubation medium to produce light which could be detected by a photomultiplier. The assay system was specific for ATP, in that similar concentrations of adenosine, AMP or ADP did not produce chemiluminescence. Moreover, the maximum peak of light emission correlated linearly with the concentrations of ATP present in the medium, so that semiquantitative estimates of ATP release could be made. Elevating the extracellular K+ concentration produced a graded release of ATP from synaptosomes. Rb+ also released ATP but Na+, Li+ and choline did not. The response to elevated K+ was not blocked by tetrodotoxin (TTX), indicating that this effect was not mediated by the opening of Na+-channels in synaptosomal membranes. Veratridine (50 μM) caused a graded release of ATP which was larger and more prolonged than that caused by elevated K+. The release of ATP by veratridine was blocked by TTX indicating that the opening of Na+-channels was involved. Neither veratridine nor elevated K+ released ATP from microsomal or mitochondrial fractions, showing that the release of ATP probably did not originate from microsomal, vesicular or mitochondrial contaminants of the synaptosomal preparation. Release of ATP by elevated K+ was diminished in a medium lacking CaCl+ or when EGTA was added to chelate Ca2+. In contrast, release by veratridine appeared to be augmented in Ca2+-free media or in the presence of EGTA. The K+-induced release of ATP, which is Ca2+ dependent, closely resembles the exocytotic release of putative neurotransmitters from presynaptic nerve-terminals. On the other hand, the apparent lack of a Ca2+ requirement for veratridine's action suggests that this process could originate from other sites, or involve mechanisms other than conventional neurotransmitter release processes.  相似文献   

15.
The mechanism of volume regulation in hypotonic media was analysed in human peripheral blood mononuclear (PBM) cells. Electronic cell sizing showed that hypotonic swelling is followed by a regulatory volume decrease (RVD) phase. This was confirmed by both electron microscopy and by cellular water determinations. The rate of regulatory shrinking was proportional to the degree of hypotonicity in the 0.5–0.9 X isotonic range. Cell viability was only marginally affected in this range. The content of cellular K+ decreased during RVD, while Na+ content remained unchanged. Similarly, the efflux of 86Rb (used as a K+ analog) increased upon dilution, whereas 22Na efflux was not altered. 86Rb uptake was enhanced by hypotonic stress and both ouabain-sensitive and -insensitive components were affected. A ouabain-sensitive stimulation was also seen in Na+- free media. Ouabain partially inhibited RVD only if added to the cells hours before hypotonic challenge. A normal shrinking response was observed in K+-free media, and also in Na+-free media when Li+, choline+, or Tris+ were the substitutes. In high K+ or Rb+ hypotonic media shrinking was absent and a second swelling phase was observed. Cs+ displayed an intermediate behavior, with shrinking observed at lower dilutions and secondary swelling at higher ones. The direction and magnitude of the response also changed when the external K+ concentration was varied and, with 50 mM K+, no regulatory volume change occurred following hypotonic stress. These findings suggest that RVD occurs largely by a passive loss of cellular K+, resulting from a selective increase in permeability to this ion. In addition, the (Na-K) pump appears to be activated upon cell swelling by a mechanism other than Na+ entry into the cell, but this activation is not essential for RVD.  相似文献   

16.
Electrophoretic measurements on membrane coated particles were performed with a Zytopherometer. Tris-HCl buffer 0.2 M pH 7.0 at 37°C with addition of different combinations of Na+, K+, Mg2+ and ATP was used as test medium. The membranes were of two types, an untreated preparation with low NaK ATPase activity and a deoxycholate treated preparation with high NaK ATPase activity. There was no marked difference in reaction between the two types of membranes. To both types of membranes Mg2+ gave a strong positive and ATP a slight negative addition to the membrane charge. In the presence of ATP Na+ gave a higher charge contribution than did K+ or a combination of Na+ and K+. This implies that K+ gives a higher affinity for ATP than Na+ does and or that ATP mediates a higher affinity for Na+ than for K+.  相似文献   

17.
Summary As different structural states of the (Na+–K+)-ATPase (EC 3.6.1.3) may lead to a changed reactivity to antibodies, the influence of Na+, K+, Mg++, Pi and ATP on the reaction between highly purified (Na+–K+)-ATPase and antibodies directed against the membrane-bound enzyme was measured. The antigen antibody reaction was registered by measuring the antibody inhibition of (Na+–K+)-ATPase activity.In themembrane-bound but not in thesolubilized enzyme four different degrees of antibody inhibition were obtained at equilibrium of the antigen antibody reaction if different combinations of Na+, K+, Mg++ and ATP were present during the incubation with the antibodies. Corresponding to the different degrees of inhibition, different rates of enzyme inhibition were measured. (a) The smallest degree of enzyme inhibition was obtained when (i) only Mg++, (ii) Mg++ and Na+ or (iii) Mg++ and K+ were present during the antigen antibody reaction. (b) The enzyme activity was inhibited more strongly if Na+, Mg++ and ATP were present together. (c) It was inhibited even more if only (i) Na+, (ii) K+, (iii) ATP or both (iv) ATP and Na+, (v) ATP and K+, (vi) ATP and Mg++, or if (vii) no ATP and activating ions were present. (d) The highest degree of antibody inhibition was obtained if Mg++, ATP and K+ were present together.In the presence of Mg++ plus ADP and in the presence of Mg++ plus the ATP analog adenylyl (--methylene) diphosphonate, Na+ and K+ did not influence the degree of antibody inhibition as they did in the presence of Mg++ plus ATP. It was further found that the degree of antibody inhibition in the presence of Mg++, ATP and K+ was affected by the sequence in which K+ and ATP were added to the enzyme prior to the addition of the antibodies.It is suggested that by antibody inhibition different conformations of the (Na+–K+)-ATPase could be detected. These conformations may possibly not occur in the solubilized enzyme and therefore do not seem to be necessarily linked to the intermediary steps of the ATP hydrolysis of the enzyme. The structural changes which are induced by Na+ and K+ in the presence of Mg++ plus ATP are proposed to occur during the Na+–K+ transport.  相似文献   

18.
Capsazepine (CPZ) inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity. In this study we have investigated the functional effects of CPZ on Na+,K+-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na+,K+-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na+,K+-ATPase current in the presence of extracellular K+. In contrast, CPZ stimulated pump current in the absence of extracellular K+. Similar conclusions were attained using HEK293 cells loaded with the Na+ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na+,K+-ATPase indicated that CPZ stabilizes ion interaction with the K+ sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na+ congener at the Na+ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na+,K+-ATPase. CPZ increased oxonol VI fluorescence in the absence of K+, reflecting increased Na+ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K+, likely indicating opening of an intracellular pathway selective for K+. As revealed by the recent crystal structure of the E1.AlF4 -.ADP.3Na+ form of the pig kidney Na+,K+-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K+ sites in Na+,K+-ATPase.  相似文献   

19.
To investigate the involvement of K+ efflux in apoptotic cell shrinkage, we monitored efflux of the K+ congener,86 Rb+, and cell volume during CD95-mediated apoptosis in Jurkat cells. An anti-CD95 antibody caused apoptosis associated with intracellular GSH depletion, a significant increase in 86Rb+ efflux, and a decrease in cell volume compared with control cells. Preincubating Jurkat cells with Val-Ala-Asp-chloromethylketone (VAD-cmk), an inhibitor of caspase proteases, prevented the observed 86Rb+ efflux and cell shrinkage induced by the anti- CD95 antibody. A wide range of inhibitors against most types of K+ channels could not inhibit CD95-mediated efflux of86 Rb+, however, the uptake of86 Rb+ by Jurkat cells was severely compromised when treated with anti-CD95 antibody. Uptake of86 Rb+ in Jurkat cells was sensitive to ouabain (a specific Na+/K+-ATPase inhibitor), demonstrating Na+/K+-ATPase dependent K+ uptake. Ouabain induced significant86 Rb+ efflux in untreated cells, as well as it seemed to compete with86 Rb+ efflux induced by the anti-CD95 antibody, supporting a role for Na+/K+-ATPase in the CD95-mediated86 Rb+ efflux. Ouabain treatment of Jurkat cells did not cause a reduction in cell volume, although together with the anti-CD95 antibody, ouabain potentiated CD95-mediated cell shrinkage. This suggests that the observed inhibition of Na++/K+-ATPase during apoptosis may also facilitate apoptotic cell shrinkage.  相似文献   

20.
In intact mitochondria supplemented with succinate or -hydroxybutyrate, the rates of oxygen consumption induced by beauvericin followed the ionic selectivity pattern: Na+>Rb+, Cs+, K+, Li+.When the respiratory substrate is glutamate plus malate in the absence of phosphate, the selectivity pattern is: K+>Rb+>Cs+>Li+>Na+.When the media are supplemented with phosphate, the Na+/K+ discrimination of beauvericin is considerably modified with all the respiratory substrates, being K+>Na+ with succinate and Na+>K+ with glutamate plus malate, whereas no significant ionic selectivity differences were obtained with -hydroxybutyrate.The respiratory control induced by oligomycin in submitochondrial particles is released by beauvericin only in the presence of a nigericin-like carboxylic antibiotic and an alkali metal cation, being far more effective in K+ than in Na+.This selectivity is maintained regardless of whether NADH or succinate is used as a respiratory substrate.Release of respiratory control can also be obtained with a combination of beauvericin and NH4Cl.This information indicates that the ionic selectivity pattern obtained with beauvericin in mitochondrial membranes is an intrinsic property of the antibiotic which, however, can be significantly modified by factors such as the nature of the translocatable substrate anion or other anionic species, as well as the possible operation of a Na+/H+ antiporter existent in the membrane.  相似文献   

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