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1.
Reduction of external ferricyanide by the human erythrocyte is significantly stimulated by insulin and somatotrophin at concentrations above physiological levels. Basal (in absence of hormones) and hormone-stimulated activities are attenuated in the presence of glycolytic inhibitors iodoacetate and vanadate indicating the requirement of glycolytic substrates for the reduction process and for the activation of cellular metabolism in response to the hormones. Sulfhydryl reagents like N-ethylmaleimide also attenuate the basal and hormone-stimulated activities and this effect was rationalized on the basis of action at SH sites which trigger responses to hormones. Stimulation of ferricyanide reduction by insulin and somatotrophin may be also the result of Na+/H+ antiport activation which may be prevented by amiloride. This suggests that Na+/H+ antiport is part of the membrane transduction system for insulin and somatotrophin in the human erythrocyte. These observations are a contribution to the study of plasma membrane oxidoreductase systems involved in physiological and metabolic functions of the cell.  相似文献   

2.
Summary We have previously reported that ferricyanide reductase activity in human erythrocytes depended on glycolysis and could be modulated by several compounds including oxidants and hormones like insulin. Insulin could activate glycolysis, probably as a consequence of tyrosine phosphorylation of protein band 3, implicating phosphorylation reactions as an important signal for activation of the reductase by insulin. Reversible phosphorylation of cellular proteins is also believed to play a key role in the action of insulin. Cytosolic acid phosphatase activity has been found in human erythrocytes. To further extend initial reports, we studied the effect of modulators on the cytosolic erythrocyte acid phosphatase. Mild oxidants like ferricyanide (1 mM), vanadate (1 mM), Mn2+ (0.5 and 1 mM), and phenylarsine oxide (10 and 100 M) inhibited the phosphatase activity. Similarly, insulin at concentrations that stimulate ferricyanide reduction (500, 1000 IU/ml) inhibited the activity of the phosphatase enzyme. The overall results indicated that oxidants are able to inhibit the acid phosphatase and stimulate the redox enzyme. In addition, a significant negative correlation (r = –0.400; P = 0.006) was observed between phosphatase and reductase activities. The observations discussed here, together with previous ones, emphasize that a close association between reductase and phosphatase enzymes may exist and also suggest a role for redox reactions in tyrosine phosphorylation/dephosphorylation-mediated signal transduction pathways.  相似文献   

3.
In rat erythrocytes, the regulation of Na+/Mg2+ antiport by protein kinases (PKs), protein phosphatases (PPs), intracellular Mg2+, ATP and Cl was investigated. In untreated erythrocytes, Na+/Mg2+ antiport was slightly inhibited by the PK inhibitor staurosporine, slightly stimulated by the PP inhibitor calyculin A and strongly stimulated by vanadate. PMA stimulated Na+/Mg2+ antiport. This effect was completely inhibited by staurosporine and partially inhibited by the PKC inhibitors Ro-31-8425 and BIM I. Participation of other PKs such as PKA, the MAPK cascade, PTK, CK I, CK II, CAM II-K, PI 3-K, and MLCK was excluded by use of inhibitors. Na+/Mg2+ antiport in rat erythrocytes can thus be stimulated by PKCα.In non-Mg2+-loaded erythrocytes, ATP depletion reduced Mg2+ efflux and PMA stimulation in NaCl medium. A drastic activation of Na+/Mg2+ antiport was induced by Mg2+ loading which was not further stimulated by PMA. Staurosporine, Ro-31-8425, BIM I and calyculin A did not inhibit Na+/Mg2+ antiport of Mg2+-loaded cells. Obviously, at high [Mg2+]i Na+/Mg2+ antiport is maximally stimulated. PKCα or PPs are not involved in stimulation by intracellular Mg2+. ATP depletion of Mg2+-loaded erythrocytes reduced Mg2+ efflux and the affinity of Mg2+ binding sites of the Na+/Mg2+ antiporter to Mg2+. In non-Mg2+-loaded erythrocytes Na+/Mg2+ antiport essentially depends on Cl. Mg2+-loaded erythrocytes were less sensitive to the activation of Na+/Mg2+ antiport by [Cl]i.  相似文献   

4.
Internal pH (pH i ) is in Vero cells regulated mainly by three antiports. Na+/H+ antiport and Na+-dependent Cl+/HCO 3 + antiport increase pH i in acidified cells, and Na+-independent Cl+/HCO 3 + antiport lowers pH i in cells after alkalinization. The activities of the antiporters were altered in cells after exposure to 41–45°C. Under such conditions the Na+/H+ antiport and the Na+-dependent Cl+/HCO 3 + antiport were both stimulated, whereas the Na+-independent Cl+/HCO 3 + antiport was inhibited in such a way that a higher pH value was required to activate it. This alteration was also induced by some other forms of cellular stress, but did most likely not involve stress proteins as protein synthesis was not required. The possibility of regulation by alteration in protein phosphorylation is discussed.We are grateful to Mrs. Jorunn Jacobsen for her skillful handling of the cell cultures. This work was supported by the Norwegian Research Council for Science and Humanities, the Norwegian Cancer Society and the Lærdal Foundation.  相似文献   

5.
The apparent volume of neutrophils, as measured electronically with the Coulter counter, has been reported to increase upon treatment with chemotactic factors. The occurrence of a volume change was confirmed by forward angle light scattering and by isotopic measurements of intracellular water space in cells treated with 12-O-tetradecanoylphorbol 13, acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP). Cell swelling was associated with an increase in the osmotic content of the cells, determined from Boyle-van't Hoff plots, and with an increase in Na+ content, measured by flame photometry. The volume change was inhibited by replacement of extracellular Na+ with K+ or N-methyl-D-glucamine+, or by addition of amiloride. Swelling was also inhibited by the 5-N-substituted analogs of amiloride, which are potent specific inhibitors of the Na+/H+ antiport. This pathway is activated in neutrophils by both TPA and FMLP. Activation of Na+/H+ exchange, determined as a Na+-dependent and amiloride-sensitive cytoplasmic alkalinization, was also found when neutrophils were treated with hypertonic solutions. The hypertonic activation of the antiport was similarly followed by cell swelling, detectable by electronic sizing. The results indicate that activation of Na+/H+ exchange can lead to significant cell swelling in neutrophils.  相似文献   

6.
Polypeptide growth factors (mitogens) which stimulate proliferation of fibroblasts and epithelial cells also rapidly activate transmembrane ion transport systems. The mitogen-induced Na+ influx is due to the activation of a Na+/H+ antiport. Recent methodological developments allow, for the first time, precise measurements of the factors that control activation of the Na+/H+ antiport by these agents, and provide the tools needed to assess the physiological significance of this exchange mechanism.  相似文献   

7.
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   

8.
Changes of sodium ionic concentration of human erythrocytes applied to pulsed electrical field (PEF) were studied by using shift reagent and NMR spectroscopy. The results show that the concentration of intraceUular Na+ increases with the increasing intensity of PEF when the erythrocytes are applied to PEF with higher intensities. The relationship between intracellular Na+ concentrations and the intensities of PEF does not follow linear or exponential behavior. As the intensities increase, the intracellular Na+ concentrations increase even faster by an exponential curve. However under effects of PEF at lower intensities, intracellular Na+ concentration decreases. Ouabain can inhibit the decrease of intracellular Na+ concentration, and the inhibition increases with the increasing concentration of ouabain, suggesting that Na+, K+-ATPase on cell membrane can be activated by PEF at lower intensities. Direct measurement of activities of the enzyme by using Malachite green method has confirmed this observation. Cell permeabilities to ions, activation of enzymes by electrical fields and transmission of physical signals like PEF across cell membranes are discussed.  相似文献   

9.
Intraerythrocytic malaria parasites produce vast amounts of lactic acid through glycolysis. While the egress of lactate is very rapid, the mode of extrusion of H+ is not known. The possible involvement of a Na+/H+ antiport in the extrusion of protons across the plasma membrane of Plasmodium falciparum has been investigated by using the fluorescent pH probe 6-carboxyfluorescein. The resting cytosolic pH was 7.27 ± 0.1 in ring stage parasites and 7.31 ± 0.12 in trophozoites. Spontaneous acidification of parasite cytosol was observed in Na+-medium and realkalinization occurred upon addition of Na+ to the medium in a concentration-dependent manner, with no apparent saturation. The rate of H+-at the ring stage was higher than that at the trophozoite stage due to the larger surface/volume ratio of the young parasite stage. Na+-H+-was: 1) inhibited by the Na+/H+ inhibitors amiloride and 5-(N-ethyl-isopropyl) amiloride (EIPA), though at relatively high concentrations; 2) augmented with rising pH6 (pHi = 6.2 [Na+]o = 30 mM); and 3) decreased with increasing pHi (pHo = 7.4; [Na+]o = 30 mM). The pHi and the pHo dependencies of H+-were almost identical at all parasite stages. Only at pHi > 7.6 efflux was totally obliterated. The target of this inhibitory effect is probably other than the antiport. Results indicate that H+-is mediated by a Na+/H+ antiport which is regulated by host and parasite pH and by the host cytosol sodium concentration. The proton transport capacity of the antiport can easily cope with all the protons of lactic acid produced by parasite's glycolysis. © 1993 Wiley-Liss, Inc.  相似文献   

10.
Summary Ehrlich ascites tumor cells contain a Na+ uptake system, which is activated by internal protons and is inhibited by amiloride with an IC50 of 25 m and by dimethylamiloride with an IC50 of 0.6 m at 1mm external Na+. Decrease of external Na+ or addition of amiloride is followed by a decrease of internal pH. Taken together, these findings suggest the presence of an operative Na+/H+ antiport system, which is involved in the regulation of internal pH. We cannot find a significant contribution of a proton pump activated by glycolysis to the pH gradient. At an external pH between 7.0 and 7.6, quiescent cells are more alkaline than exponentially growing cells (0.1 to 0.17 units). Accordingly, an increase of the affinity of the Na+/H+ antiport for internal protons in quiescent cells is demonstrated by the following findings: 1. The internal pH, at which the half-maximal activation of the amiloride-sensitive Na+ uptake occurs, is shifted from 6.85 to 7.1 at 1mm external Na+. 2. The threshold value of external pH, below which a pronounced effect of amiloride on steadystate internal pH is observed, is shifted from 7.0 in growing to 7.5 in quiescent cells at physiological Na+ concentrations. Therefore, we conclude that quiescent Ehrlich ascites tumor cells raise their internal pH by increasing the affinity of their Na+/H+ antiporter to internal protons. The Na+/H+ antiport cannot be activated further by addition of serum growth factors to quiescent cells. All experiments were performed at bicarbonate concentrations in the medium which do not exceed 0.5mm. The data are discussed in view of existing models of mitogenic activity of transitory pH changes.  相似文献   

11.
Cell membrane transport of K+ stimulates the rate of glycolysis in Ehrlich ascites tumor cells. A study of the characteristics of this relationship indicates that the stimulation occurs under anaerobic as well as under aerobic conditions. The data suggest that glycolysis is stimulated by a K+ transport mechanism that is coupled to Na+ transport because the effect is blunted or abolished when the principal intracellular ion is lithium or choline. This stimulus to glycolysis is blocked by ouabain and ethacrynic acid, agents that have been shown to inhibit monovalent cation transport in erythrocytes. In contrast to the action of ouabain, glycolysis is inhibited by ethacrynic acid in Ehrlich ascites tumor cells in the absence of cell membrane K+ transport. In studies with ghost-free hemolysates of human erythrocytes and with cytosol prepared from Ehrlich ascites tumor cells, ethacrynic acid significantly blocks lactate formation from fructose diphosphate demonstrating the direct inhibitory effect of this agent on one or more enzymes of the Embden-Meyerhof pathway. Since ethacrynic acid has no influence on lactate formation in intact erythrocytes utilizing an endogenous substrate, the presumptive site of inhibition is proximal to the 3-phosphoglycerate level.  相似文献   

12.
Nature of the light-induced h efflux and na uptake in cyanobacteria   总被引:3,自引:0,他引:3       下载免费PDF全文
We investigated the nature of the light-induced, sodium-dependent acidification of the medium and the uptake of sodium by Synechococcus. The rate of acidification (net H+ efflux) was strongly and specifically stimulated by sodium. The rates of acidification and sodium uptake were strongly affected by the pH of the medium; the optimal pH for both processes being in the alkaline pH range. Net proton efflux was severely inhibited by inhibitors of adenosine triphosphatase activity, energy transfer, and photosynthetic electron transport, but was not affected by the presence of inorganic carbon (Ci). Light and Ci stimulated the uptake of sodium, but the stimulation by Ci was observed only when Ci was present at the time sodium was provided. Amiloride, a potent inhibitor of Na+/H+ antiport and Na+ channels, stimulated the rate of acidification but inhibited the rate of sodium uptake. It is suggested that acidification might stem from the activity of a light dependent proton excreting adenosine triphosphatase, while sodium transport seems to be mediated by both Na+/H+ antiport and Na+ uniport.  相似文献   

13.
Changes of sodium ionic concentration of human erythrocytes applied to pulsed electrical field (PEF) were studied by using shift reagent and NMR spectroscopy. The results show that the concentration of intracellular Na+ increases with the increasing intensity of PEF when the erythrocytes are applied to PEF with higher intensities. The relationship between intracellular Na concentrations and the intensities of PEF does not follow linear or exponen-tial behavior. As the intensities increase, the intracellular Na+concentrations increase even faster by an exponential curve. However under effects of PEF at lower intensities, intracellular Na+ concentration decreases. Ouabain can in-hibit the decrease of intracellular Na concentration, and the inhibition increases with the increasing concentration of ouabain, suggesting that Na+ , K+ -ATPase on cell membrane can be activated by PEF at lower intensities. Direct measurement of activities of the enzyme by using Malachite green method has confirmed this observation. Cell perme-abilities to ions, activation of enzymes by electrical fields and transmission of physical signals like PEF across cell mem-branes are discussed.  相似文献   

14.
The growth and methane formation ofMethanospirillum hungatei were inhibited by an inhibitor of Na+/H+ antiport amiloride. After addition of NaCl or LiCl, when the cells had a lower intracellular pH and were deenergized, they extruded protons into the external medium. The acidification of the external medium was stimulated by protonophores and inhibited by amiloride. These findings suggest the existence of an Na+/H+ antiport in the cytoplasmic membrane ofM. hungatei and its role in the energetics of methanogenic bacteria.  相似文献   

15.
Activation of Na+-H+ exchange in rat thymocytes was found to be followed by an increase in free cytoplasmic Ca2+ concentration ([Ca2+]i). We determined whether the change in [Ca2+]i was secondary to the uptake of Na+, or to the cytoplasmic alkalinization that result from activation of the antiport. Increasing intracellular [Na+] by treating the cells with ouabain or gramicidin failed to affect [Ca2+]i. In contrast, procedures that increased the cytoplasmic pH, such as addition of monensin or NH3, significantly elevated [Ca2+]i. These results suggest an important role of cytoplasmic pH in the control of [Ca2+]i in lymphocytes.  相似文献   

16.
The signal transduction pathway for insulin-mediated activation of sarcolemmal Na+–Ca2+ exchange was examined. Insulin stimulated Na+–Ca2+ exchanger activity in a dose-dependent manner, with the EC50 being about 0.7 U/l. The insulin effect was blocked by the protein kinase inhibitor, staurosporine, indicating possible involvement of a protein kinase in insulin action. Also, the relationship between the insulin effect and activation of a G protein, was examined by testing the effects of 5 guanylyl imidodiphosphate (Gpp(NH))p) on Na+–Ca2+ exchange in, the presence and absence of insulin. When exchanger activity was assayed at a calcium concentration of 40 M, insulin alone had no effect whereas ATP and Gpp(NH)p increased exchanger activity. However, insulin responsiveness was restored in vesicles preloaded with either ATP or Gpp(NH)p, suggesting that insulin may act through a combination of G protein coupling and protein phosphorylation to enhance Na+–Ca2+ exchanger activity. We conclude that calcium overload in the diabetic heart may involve a defect in acute activation of the exchanger by insulin.  相似文献   

17.
In this study we prepared sarcolemmal fractions from bovine and rat hearts; their Na+K+ ATPase activities, measured in the presence of saponin to unmask latent Na+K+ ATPase, were 59.4 and 48.8 µ mol Pi/mg protein · h, respectively. The rate of Na+dependent Ca2+ uptake was linear for the first 10 s and a plateau was reached in 3 min. Oxidation by free radical generation either with H2O2, FeSO4 plus DTT or xanthine oxidase plus hypoxanthine stimulated Na+/Ca2+ exchange in a time-dependent manner. The stimulation was abolished by deferoxamine or o-phenanthroline. By contrast, oxidation by HOCI inhibited Na+/Ca2+ exchange in proportion to its concentration, and this inhibition was antagonized by DTT. DTT alone had no effect on the exchange. Insulin stimulated Na+/Ca2+ exchange, its maximal effect was attained after 30min incubation with 100 µ units/ml. N-ethylmaleimide inhibited the exchange both in the presence and in the absence of insulin. Sarcolemmal fractions prepared from hearts of alloxan-treated, acutely diabetic rats showed a significant decrease in Na+/Ca2+ exchange. Addition of insulin in vitro significantly stimulated Na+/Ca2+ exchange of both diabetic and control groups. The results indicate that sarcolemmal Na+/Ca2+ exchange function is modulated by oxidation-reduction states and by the presence of insulin.  相似文献   

18.
The sodium/proton antiport system of Escherichia coli has been characterized by the effect of Na+ on the pH gradient established by respiration in everted membrane vesicles. The system has equal affinity for Na+ and Li+. Between pH 7 and 9 dissipation of Δψ, membrane potential, has no effect on the affinity for Na+ but decreases the V of the antiport reaction. Uptake of 22Na+ by everted membrane vesicles was observed using flow dialysis.  相似文献   

19.
Right-side-out plasma membrane vesicles were isolated from wheat roots using an aqueous polymer two-phase system. The purity and orientation of the vesicles were confirmed by marker enzyme analysis. Membrane potential (Ψ)-dependent 22Na+ influx and sodium/proton (Na+/ H+) antiport-mediated efflux across the plasma membrane were studied using these vesicles. Membrane potentials were imposed on the vesicles using either K+ gradients in the presence of valinomycin or H+ gradients. The ΔΨ was quantified by the uptake of the lipophilic cation tetraphenylphosphonium. Uptake of Na+ into the vesicles was stimulated by a negative ΔΨ and had a Km for extrav-esicular Na+ of 34.8 ± 5.9 mol m3. The ΔΨ-dependent uptake of Na+ was similar in vesicles from roots of hexaploid (cv. Troy) and tetraploid (cv. Langdon) wheat differing in a K+/Na+ discrimination trait, and was also unaffected by growth in 50 mol m?3 NaCl. Inhibition of ΔΨ-dependent Na+ uptake by Ca2+ was greater in the hexaploid than in the tetraploid. Sodium/proton antiport was measured as Na+-dependent, amiloride-inhibited pH gradient formation in the vesicles. Acidification of the vesicle interior was measured by the uptake of 14C-methylamine. The Na+/H+ antiport had a Km, for intravesicular Na+ of between 13 and 19 mol m?3. In the hexaploid, Na+/H+ antiport activity was greater when roots were grown in the presence of 50 mol m?3NaCl, and was also greater than the activity in salt-grown tetraploid wheat roots. Antiport activity was not increased in a Langdon 4D chromosome substitution line which carries a trait for K+/Na+ discrimination. It is concluded that neither of the transport processes measured is responsible for the Na+/K+ discrimination trait located on the 4D chromosome of wheat.  相似文献   

20.
Salinity stress is one of the most serious factors limiting the productivity of agricultural crops. A possible survival strategy of plants under saline conditions is to sequester excess Na+ in the vacuole by vacuolar Na+/H+ antiport using a pH gradient generated by H+-ATPasc (EC 3.6.1.35) and H+-Pyrophosphatase (H+-PPase; EC 3.6.1.1) to maintain a higher K+/Na+ ratio in cytoplasm. The effect of exogenously applied polyamines (PAs) in stabilizing root tonoplast integrity and function against salt stress in the barley (Hordeum vulgare L.) seedlings was investigated. The NaCl-induced reductions in the contents of phospholipids and PAs in tonoplast vesicles isolated from barely seedling roots, as well as the activities of H+-ATPase, H+-PPase and vacuolar Na+/H+ antiport were all partially restored by the application of 0.5 mM putrescine and 0.5 mM spermidine, especially the former. The above results indicated that one of the mechanisms involved in attenuating salt injury in barley seedlings by exogenous PAs application was to maintain tonoplast integrity and function under saline conditions. Moreover, the possible mechanism involved in counteracting detrimental effects of salt on the barley seedlings by the application of exogenous PAs was discussed.  相似文献   

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