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1.
The determination of ATP-hydrolytic activity of Na pump does not always reveal the enzyme damage in vivo. The method assessing Na, K-ATPase molecular conformational stability in the rat heart sarcolemma based on thermal denaturation is suggested. After a prolonged emotional-painful stress (EPS) the activity of Na, K-ATPase dropped by 20%, as the rate of its thermal denaturation in the range of 50-60 degrees C increased 2-3-fold. Thermodynamic calculations have demonstrated a decrease in Ea, delta H and delta S* of Na, K-ATPase thermal denaturation process after EPS. An analogous enzyme damage was found after the activation of lipid peroxidation in sarcolemma membrane suspension. These results imply that essential changes in intra- and supra-molecular properties of Na, K-ATPase under EPS may be detected by thermal denaturation. Lipid peroxidation is a most likely reason for EPS-induced Na pump damage.  相似文献   

2.
Chronic emotional pain stress in rats causes disturbances of the cardiovascular system function (increase in arterial pressure and in heart rate), typical of neuroses-like state, and changes of the vegetative nervous system reactivity tested with functional load by two-hour hypokinesis. Increase in spleen weight is observed as well as a tendency to adrenals weight increase, a decrease of Na, K-ATPase activity and activation of lipid peroxidation in cortical and hippocampal homogenates. Administration of F-801 antioxidant according to therapeutic scheme after the end of stress action, restores normal function of the cardiovascular system, normal reactivity of the vegetative nervous system, decreases adrenals weight and increases the weight of thymus and also normalizes ATPase activity and the level of lipid peroxidation. A backward correlation dependence of the Na, K-ATPase activity on the level of malondialdehyde in the brain tissue has been established.  相似文献   

3.
The effects of carnosine on erythrocyte membrane Na,K-ATPase and isolated enzyme in vitro as well as on membrane Na,K-ATPase activity and lipid peroxidation (LPO) in chronic heart failure (CHF) and acute myocardial infarction (AMI) have been studied. CHF and AMI have been shown to be associated with significant inhibition of the erythrocyte membrane Na,K-ATPase activity and LPO activation. Marked activation of erythrocyte membrane Na,K-ATPase by carnosine in comparison with the isolated enzyme has been established. The ability of carnosine to induce Na,K-ATPase activation and prevent membrane depolarization indicates that the dipeptide may be a useful tool in the pathogenetic therapy of CFH and AMI.  相似文献   

4.
Na,K-ATPase (ATP phosphohydrolase EC 3.6.1.3) activity was determined in homogenates of cortical brain slices after incubation in normal Krebs-Ringer phosphate medium at 1 atm oxygen pressure. After 10 min of incubation Na,K-ATPase activity was reduced by approximately 50%. Longer incubation did not cause further change in activity. The presence of 0.1 mM-MnCl2 in the medium offered significant protection, while an excursion to 10 atm oxygen pressure caused further inactivation. Measurements of malonaldehyde levels suggest that the inhibition of Na,K-ATPase is a result of lipid peroxidation. The evidence indicates that brain slices incubated under standard conditions suffer considerable oxidative damage.  相似文献   

5.
The mechanism of light-induced changes in the activity of Na,K-ATPase from plasma membranes (PM) of photoreceptor cells was studied in vitro. Illumination resulted in inhibition of the ATPase activity and an increase of 18O exchange between water and Pi. The maximum light effect was revealed when the PM contained both the inner segments of the rods (RIS) and rod outer segments (ROS) of the photoreceptor cells. Lipid peroxidation stimulated by the FeSO4+ascorbate system induced a decrease of the ATPase activity. Antioxidants (ionol, Na2SeO3, vitamin E) prevented the effect of the lipid peroxidation products on NA,K-ATPase and the photoinduced changes of the enzyme activity. It is supposed that the photoinduced changes of the Na,K-ATPase activity in vitro are due to lipid peroxidation of photoreceptor PM.  相似文献   

6.
Iron is a key element in cell function; however, its excess in iron overload conditions can be harmful through the generation of reactive oxygen species (ROS) and cell oxidative stress. Activity of Na,K-ATPase has been shown to be implicated in cellular iron uptake and iron modulates the Na,K-ATPase function from different tissues. In this study, we determined the effect of iron overload on Na,K-ATPase activity and established the role that isoforms and conformational states of this enzyme has on this effect. Total blood and membrane preparations from erythrocytes (ghost cells), as well as pig kidney and rat brain cortex, and enterocytes cells (Caco-2) were used. In E1-related subconformations, an enzyme activation effect by iron was observed, and in the E2-related subconformations enzyme inhibition was observed. The enzyme's kinetic parameters were significantly changed only in the Na+ curve in ghost cells. In contrast to Na,K-ATPase α2 and α3 isoforms, activation was not observed for the α1 isoform. In Caco-2 cells, which only contain Na,K-ATPase α1 isoform, the FeCl3 increased the intracellular storage of iron, catalase activity, the production of H2O2 and the expression levels of the α1 isoform. In contrast, iron did not affect lipid peroxidation, GSH content, superoxide dismutase and Na,K-ATPase activities. These results suggest that iron itself modulates Na,K-ATPase and that one or more E1-related subconformations seems to be determinant for the sensitivity of iron modulation through a mechanism in which the involvement of the Na, K-ATPase α3 isoform needs to be further investigated.  相似文献   

7.
Using the fluorescent probe technique, it was shown that activation of lipid peroxidation decreases the value of transmembrane potential of rat brain synaptosomes. Depolarization of synaptosomes may be due to the impairment of the "barrier" properties of synaptosomal membranes and the decrease in Na,K-ATPase activity. alpha-Tocopherol and its model derivative devoid of the phytol chain--2,2,5,7,8-pentamethyl-6-oxychromanol--stabilize the transmembrane potential value during inhibition of lipid peroxidation. alpha-Tocopherol acetate causes no stabilizing or inhibiting effects. Unlike 2,2,5,7,8-pentamethyl-6-oxychromanol, alpha-tocopherol exerts a structuralizing action which manifests itself in the stabilization of the synaptosomal membrane potential during incomplete inhibition of lipid peroxidation. The previously established ability of alpha-tocopherol to protect synaptosomes from the damaging action of phospholipases and the experimental results of this work permit to regard vitamin E as a universal stabilizer of brain synaptosomal membranes.  相似文献   

8.
Hypercapnia has been shown to impair alveolar fluid reabsorption (AFR) by decreasing Na,K-ATPase activity. Extracellular signal-regulated kinase pathway (ERK) is activated under conditions of cellular stress and has been known to regulate the Na,K-ATPase. Here, we show that hypercapnia leads to ERK activation in a time-dependent manner in alveolar epithelial cells (AEC). Inhibition of ERK by U0126 or siRNA prevented both the hypercapnia-induced Na,K-ATPase endocytosis and impairment of AFR. Moreover, ERK inhibition prevented AMPK activation, a known modulator of hypercapnia-induced Na,K-ATPase endocytosis. Accordingly, these data suggest that hypercapnia-induced Na,K-ATPase endocytosis is dependent on ERK activation in AEC and that ERK plays an important role in hypercapnia-induced impairment of AFR in rat lungs.  相似文献   

9.
Recently, cardiotonic steroids (CTS) have been shown to lead to the activation of Na,K-ATPase at low concentrations in brain, promoting neuroprotection against ischemia. We report here the results of the use of digoxin and its semisynthetic derivatives BD-14, BD-15, and BD-16 against partial chemical ischemic induction followed by reperfusion in murine neuroblastoma cells neuro-2a (N2a). For chemical ischemic induction, sodium azide (5 mM) was used for 5 hours, and then reperfusion was induced for 24 hours. Na,K-ATPase activity and protein levels were analyzed in membrane preparation of N2a cells pretreated with the compounds (150 nM), in the controls and in induced chemical ischemia. In the Na,K-ATPase activity and protein levels assays, the steroids digoxin and BD-15 demonstrated a capacity to modulate the activity of the enzyme directly, increasing its levels of expression and activity. Oxidative parameters, such as superoxide dismutase (SOD) activity, lipid peroxidation (thiobarbituric acid reactive substance), glutathione peroxidase (GPx), glutathione (GSH) levels, hydrogen peroxide content, and the amount of free radicals (reactive oxygen species) during induced chemical ischemia were also evaluated. Regarding the redox state, lipid peroxidation, hydrogen peroxide content, and GPx activity, we have observed an increase in the chemical ischemic group, and a reduction in the groups treated with CTS. SOD activity increased in all treated groups when compared to control and GSH levels decreased when treated with sodium azide and did not change with CTS treatments. Regarding the lipid profile, we saw a decrease in the content of phospholipids and cholesterol in the chemical ischemic group, and an increase in the groups treated with CTS. In conclusion, the compounds used in this study demonstrate promising results, since they appear to promote neuroprotection in cells exposed to chemical ischemia.  相似文献   

10.
Young, adult, and old rats were used to study the effect of age on the integrity and functioning of brain synaptosomes. An evaluation was made of the differences in lipid composition, membrane fluidity, Na+, K(+)-ATPase activity, and susceptibility to in vitro lipid peroxidation. There was an age-related increase in synaptosomal free fatty acids, with no modification in acyl chain composition, and a decrease in membrane phospholipids which increased the cholesterol/phospholipid mole ratio. With altered lipid composition, there was a corresponding age-dependent decrease in membrane fluidity, a reduction of Na+, K(+)-ATPase activity, and an overall greater susceptibility to in vitro lipid peroxidation. Furthermore, lipid peroxidation promoted strong modifications of the membrane fluidity, lipid composition, and Na+,K(+)-ATPase activity just as aging did, thus indicating a possible contribution of oxidative damage to ageing processes. The cases studied revealed that the greater responsiveness of old membranes to in vitro lipid peroxidation resulted in the highest degree of membrane alteration, indicating that all pathological states known to promote a peroxidative injury can have even more dramatic consequences when they take place in old brain.  相似文献   

11.
A study was made of the influence of ionizing radiation of 0.31 C/kg on the kinetic parameters showing the activity of brain Na, K-ATPase preparation to be a function of ion-regulator concentration. The use of the new method for the analysis of the enzyme cation centers permitted to estimate that whole-body irradiation of rats with the above dose did not cause in vitro a substantial change in the pattern of Na, K-ATPase activation by Na and K ions.  相似文献   

12.
Digitalis-like compounds (DLCs), specific inhibitors of Na,K-ATPase, are implicated in cellular signaling. Exposure of cell cultures to ouabain, a well-known DLC, leads to up- or down regulation of various processes and involves activation of Src kinase. Since Na,K-ATPase is the only known target for DLC binding an in vitro experimental setup using highly purified Na,K-ATPase from pig kidney and commercially available recombinant Src was used to investigate the mechanism of coupling between the Na,K-ATPase and Src. Digoxin was used as a representative DLC for inhibition of Na,K-ATPase. The activation of Src kinase was measured as the degree of its autophosphorylation. It was observed that in addition to digoxin, Src activation was dependent on concentrations of other specific ligands of Na,K-ATPase: Na(+), K(+), vanadate, ATP and ADP. The magnitude of the steady-state ATPase activity therefore seemed to affect Src activation. Further experiments with an ATP regenerating system showed that the ATP/ADP ratio determined the extent of Src activation. Thus, our model system which represents the proposed very proximal part of the Na,K-ATPase-Src signaling cascade, shows that Src kinase activity is regulated by both ATP and ADP concentrations and provides no evidence for a direct interaction between Na,K-ATPase and Src.  相似文献   

13.
Cornelius F 《Biochemistry》2001,40(30):8842-8851
The effects of phospholipid acyl chain length (n(c)), degree of acyl chain saturation, and cholesterol on Na,K-ATPase reconstituted into liposomes of defined lipid composition are described. The optimal acyl chain length of monounsaturated phosphatidylcholine in the absence of cholesterol was found to be 22 but decreased to 18 in the presence of 40 mol % cholesterol. This indicates that the hydrophobic matching of the lipid bilayer and the transmembrane hydrophobic core of the membrane protein is a crucial parameter in supporting optimal Na,K-ATPase activity. In addition, the increased bilayer order induced by both cholesterol and saturated phospholipids could be important for the conformational mobility of the Na,K-ATPase changing the distribution of conformations. Lipid fluidity was important for several parameters of reconstitution, e.g., the amount of protein inserted and the orientation in the liposomes. The temperature dependence of the Na,K-ATPase as well of the Na-ATPase reactions depends both on phospholipid acyl chain length and on cholesterol. Cholesterol increased significantly both the enthalpy of activation and entropy of activation for Na,K-ATPase activity and Na-ATPase activity of Na,K-ATPase reconstituted with monounsaturated phospholipids. In the presence of cholesterol the free energy of activation was minimum at a lipid acyl chain length of 18, the same that supported maximum turnover. In the case of ATPase reconstituted without cholesterol, the minimum free energy of activation and the maximum turnover both shifted to longer acyl chain lengths of about 22.  相似文献   

14.
The lipid peroxidation (LPO) level between in the adult and old rats brain and liver was determined as to be essentially undiffering. Stress activated the LPO independence the age of animals and tissues investigated. The concentration changes of LPO products testify to it. In the adult rats under the stress capability of tissues to induction in vitro ferment and ascorbat-depending LPO, in comparison with the control, decreases, at old--does not change in the brain and considerably grows in the liver. Stress is accompanied by an oppression of Na, K-ATP-ase PM activity of hepatocytes, more expressed in the old animals.  相似文献   

15.
Disturbances of Na,K-ATPase activity are implicated in the pathophysiology of cerebral ischemia. Previous experiments have shown that EGb 761 protects NaK-ATPase activity against one hour of cerebral ischemia. In the brain however, the 3 isoenzymes responsible for Na,K-ATPase activity may be differentially affected by various times of ischemia. In the present study, we investigated the effect of a longer period of ischemia, and the protection provided by a pre-treatment with EGb 761 on each of the 3 cerebral NaK-ATPase isoenzymes. In control and EGb 761 pre-treated mice exposed to a 6 hr unilateral occlusion of the middle cerebral artery, Na,K-ATPase activity was decreased by 60% and lipid peroxidation was increased by 40% in the ipsilateral (ischemic) cortex compared to the contralateral one. In parallel, membrane integrity was altered. The alteration of NaK-ATPase activity, as a whole, resulted from a decrease in the activity of the 3 isoenzymes. The two isoenzymes of high ouabain affinity however, had their affinities decreased while the sensitivity of the lowest affinity isoenzyme was increased. Pre-treatment with EGb 761 abolished the differences observed between ipsi- and contralateral cortex, with the exception of the change in ouabain affinity of the low affinity isoenzyme. Ischemia also induced changes in Na,K-ATPase isoenzyme ouabain affinities in the contralateral cortex that where not prevented by EGb 761.  相似文献   

16.
Depressant drugs are considered to exert their pharmacological effects as a result of membrane interactions determined by their physico-chemical properties. In this study, a correlation was found between lipid solubility and potency of various local anaesthetics, antihistamines, tricyclic antidepressants and phenothiazine tranquilizers as inhibitors of the Na, K-ATPase activity of a microsomal membrane fraction from bovine brain cortex. Depressant drugs such as chlorpromazine, which have the greatest lipid solubilities, were competitive inhibitors of Na activation but noncompetitive toward K activation, whereas drugs such as tetracaine with lower lipid solubilities were competitive inhibitors of K activation but noncompetitive toward Na activation. Drugs with intermediate lipid solubilities were mixed competitive-noncompetitive inhibitors of both Na and K activation. Both chlorpromazine and tetracaine competitively inhibited cation activation by a heterotropic allosteric mechanism, probably mediated through membrane conformational changes. Whereas quaternization or a decrease in the incubation pH interfered with the ability of chlorpromazine to inhibit Na activation in a competitive fashion, these changes did not affect the ability of tetracaine to compete with K activation. In addition Mn, Ca and phosphatidyl serine were very effective non-competitive antagonists of chlorpromazine inhibition of Na, K-ATPase, whereas these agents competitively antagonized tetracaine inhibition to a lesser extent. These data suggest that the more lipid soluble phenothiazines penetrate into and react in hydrophobic areas of the membrane microenvironment, resulting in a membrane perturbation which interferes with Na activation. On the other hand the less lipid soluble local anaesthetics probably act at superficial sites near the membrane surface, resulting in a different membrane perturbation which interferes with the K activation mechanism. It is suggested that lipid solubility may be a significant factor in determining selectivity in the membrane interactions of various pharmacological agents and hence differences in pharmacological activity among different classes of depressant drugs.  相似文献   

17.
The effect of desoxycorticosterone (DOC) on Na, K-ATPase activity was studied in vivo and in vitro on microsomal rat brain fractions. An hour after intramuscular administration of DOC a noticeable increase in the enzyme activity was observed. Preincubation of microsomal brain fractions with 5 and 15 mkg/ml of DOC caused a decrease in Na, K-ATPase activity, with the results evident 3-5 minutes after the addition of the hormone into the incubation medium. The idea of a two-phase hormonal effect is suggested. It is likely that desoxycorticosterone effect is realized both by the direct influence, on Na, K-ATPase of the brain plasma membrane and by the influence on the biosynthesis.  相似文献   

18.
Activity of the Na/K-ATPase from rat brain synaptic membranes is inhibited by NA (noradrenaline). However, during fractionation of cytozole from nerve endings, two non-homogeneous peaks are found (SF(a), 60-100 kD and SF( i ),;10 kD), which influence the Na/K-ATPase activity, both directly and SF(a) NA-dependently. Joint action of NA and synaptic factors (SF(a) and SF(i)) on the Na/K-ATPase, represents a sum of four different processes: 1) NA, without synaptic factors, inhibits the Na/K-ATPase; 2) At low SF(a) concentrations NA-dependent Na/K-ATPase activatory mechanism is evident; 3) At high SF(a) concentrations NA-independent Na/K-ATPase is activated; 4) The low-molecular SF(i) protein inhibits the Na/K-ATPase. Regulation of the Na/K-ATPase activity by NA, SF(a) and SF( i), obtained in similar conditions from two weeks old and one year old rats, is different. In older rats SF(i) is characterized with strong Na/K-ATPase inhibition; in younger rats SF(i) does not change the Na/K-ATPase activity. The NA- and SF(i) -dependent inhibition and activation ratio is different in young and elder rats. In two week olds NA/SF(i) activatory mechanism is stronger, while in one year olds NA-dependent inhibition of the Na/K-ATPase is prevailing. These experimental data indicate that regulation of the Na/K-ATPase activity has an important role in synaptic transmission and that this process has noteworthy, albeit presently unknown, functional importance in integrative activity of the brain.  相似文献   

19.
The effect of insulin on the activity of Na, K-ATPase was studied in rat brain microsomes. Addition of insulin to the incubation medium in a dose of 0.18 U/ml coupled with strophanthine did not change the enzyme activity. The raising of the hormone dose to 0.36 U/ml was accompanied by inhibition of the enzyme activity. The incubation duration (10 and 30 min) did not influence the Na-pump. Preincubation of brain microsomes with insulin for 5 min significantly activated Na, K-ATPase. It has been thus demonstrated that insulin is capable of influencing the activity of Na, K-ATPase of rat brain microsomes in vitro. The effect obtained depends both on the dose of the hormone introduced into the incubation medium and the experimental conditions.  相似文献   

20.
In this study, renal tissue, subdivided into the cortex and medulla of Wistar rats subjected to a cafeteria diet (CAF) for 24 days or to normal diet, was used to analyze whether the renal enzyme Na,K-ATPase activity was modified by CAF diet, as well as to analyze the α1 subunit of renal Na,K-ATPase expression levels. The lipid profile of the renal plasma membrane and oxidative stress were verified. In the Na,K-ATPase activity evaluation, no alteration was found, but a significant decrease of 30% in the cortex was detected in the α1 subunit expression of the enzyme. There was a 24% decrease in phospholipids in the cortex of rats submitted to CAF, a 17% increase in cholesterol levels in the cortex, and a 23% decrease in the medulla. Lipid peroxidation was significantly increased in the groups submitted to CAF, both in the cortical region, 29%, and in the medulla, 35%. Also, a reduction of 45% in the glutathione levels was observed in the cortex and medulla with CAF. CAF showed a nearly two-fold increase in glutathione peroxidase (GPX) activity in relation to the control group in the cortex and a 59% increase in the GPx activity in the medulla. In conclusion, although the diet was administered for a short period of time, important results were found, especially those related to the lipid profile and oxidative stress, which may directly affect renal function.  相似文献   

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