锌对红细胞膜ATP酶活性影响的研究

从体内和体外 2方面研究锌对红细胞膜 Na+ ,K+ - ATP酶和 Ca2 + ,Mg2 + - ATP酶活性的影响。健康雄性 Wistar大鼠分别喂饲低锌、常量锌和高锌饲料 (饲料锌含量分别为 2 .2、2 8、12 8mg/kg) ,2 5天后各组随机取 8只动物腹主动脉取血后处死 ,测定血清锌含量和红细胞膜 Na+ ,K+ - ATP酶和 Ca2 + ,Mg2 + - ATP酶活性 ;各组剩余 6只动物改变其锌摄入量后继续喂饲 7天后 ,观察锌对红细胞膜 2种 ATP酶活性影响的敏感性。体外实验用新鲜制备的人红细胞膜 ,在不同浓度锌 (0、5、10、5 0、10 0和 5 0 0 μmol/L)溶液中温浴 ,观察 2种 ATP酶活性的变化趋势。结果表明 ,锌对红细胞膜的 2种 ATP酶活性均有影响 ,锌浓度过低或过高可抑制酶活性。与 Na+ ,K+ - ATP酶相比 ,Ca2 + ,Mg2 + - ATP酶对缺锌更敏感且补锌后不易恢复 ,但其对高锌有较强的耐受性。本次研究结果提示锌对于维持红细胞膜 ATP酶的正常活性有重要意义。     

Mercuric chloride-induced inhibition of different ATPases in the intestine of mudskipper, Boleophthalmus dentatus.

This paper deals with the toxicity of mercuric chloride to different ATPases in the intestine of mudskipper (Boleophthalmus dentatus). Mudskippers were exposed to four sublethal concentrations of mercuric chloride for three durations. The specific activities of Na+, K(+)-ATPase, Ca(2+)-ATPase, Mg(2+)-ATPase, Ca2+, HCO3(-)-ATPase, and Mg2+, HCO3(-)-ATPase were estimated. There was linear inhibition of all the enzymes with increasing mercuric chloride concentration as well as exposure duration. The Na+,K(+)-ATPase was found to be the enzyme most affected, followed by other ion-dependent ATPases. Inhibition of all the enzymes indicates severe damage to the intestinal cells, resulting in a blockage of the transport of substances across the membrane.     

Erythrocyte membrane (Na+, K+) and (Mg++, Ca++) activated adenosine triphosphatases in women using oral contraceptives--a preliminary report.

(Na⁺, K⁺) and (Mg⁺⁺, Ca⁺⁺) activated adenosine triphosphatases (Na⁺, K⁺ and Mg⁺⁺, Ca⁺⁺ ATPase, respectively) were studied in women who had used oral contraceptives (OC) for a period of 13 to 15 months. Erythrocyte and plasma Na⁺ and K⁺ were also determined in them. The values were compared with those obtained in age matched non-pregnant women and those in the third trimester of pregnancy. No significant differences could be seen in the activity of (Na+, K+) and (Mg++, Ca++) ATPases between the three groups. Erythrocyte as well as plasma Na+ and K+ contents (mEg/10¹² cells and mEq/litre plasma) in the women using OC were also in the normal range.     

Gossypol inhibition of Ca++ uptake and Ca++-ATPase in human ejaculated spermatozoal plasma membrane vesicles

Gossypol, a plant-derived polyphenolic compound known to exert contraceptive actions in men, inhibits Ca++-transport and Ca++-activated ATPase in isolated plasma membranes of ejaculated human sperm cells. It also inhibits the membrane bound Mg++- and Na+ + K+-dependent ATPases, 5'-nucleotidase and alkaline phosphatase systems. Ca++-ATPase inhibition by gossypol is non-competitive. It abolishes the discontinuity in Arrhenius expression of temperature dependence of Ca++-ATPase and increases the energy of activation. Phosphatidyl choline and Na+-deoxycholate inhibit Ca++-transport activity in the membrane vesicles. The apparent similarity of Ca++-transport inhibition by gossypol and phosphatidyl choline may indicate the possible capability of this compound to induce changes in the lipid microenvironment of the membranes, wherein the integral proteins operate. Inhibitory effect of gossypol on the plasma membrane Ca++-pump suggests that gossypol may affect sperm motility by a mechanism which is related to the structure and functions of the plasma membrane.     

Eel ATPase activity as biomarker of thiobencarb exposure

European eels (Anguilla anguilla) were exposed to a sublethal thiobencarb concentration of 0.22 mg/L in a flow-through system for 96 h. Mg(2+) and Na(+)-K(+) adenosine triphosphatase (ATPase) activities were evaluated in gill and muscle tissues at 2, 12, 24, 48, 72, and 96 h of thiobencarb exposure. Gill ATPase activities were rapidly inhibited from 2h of contact onward. Highest inhibition was registered for Na(+), K(+)-ATPase (85%) from 2 to 12h. Both Mg(2+) and total ATPase were inhibited (>73%) during the first hours of toxicant exposure. At the end of the exposure period (96 h) ATPase activities were still different from those of the controls (>50%). Significant inhibition was detected in Na(+), K(+)-ATPase activity (80%) in muscle tissue after 2h and it was maintained over the entire exposure time. However, Mg(2+)-ATPase and total ATPase showed only perturbations after 2 h of exposure. Eels were exposed to 0.22 mg/L of thiobencarb for 96 h and then a recovery period in herbicide-free water was allowed for 192 h. Gill and muscle samples were removed at 8, 24, 72, 96, 144, and 192 h and ATPase activity was evaluated. Following 144 h of recovery, Mg(2+)- and Na(+), K(+)-ATPase activities, as well as total ATPase activity, in gills of those animals previously exposed to 0.22 mg/L of thiobencarb were still significantly different compared to controls. Thiobencarb seems to act to alter the ionic profiles. Since ion-dependent ATPases are known to regulate the influx and efflux of ions across the membrane to maintain the physiological requirements of the cells, the inhibition of Na(+), K(+)-ATPase probably induced osmoregulatory perturbations. On the other hand, thiobencarb exposure causes increases in the muscle water content of A. anguilla. The results indicated that water content increased significantly (>100% higher than the controls) during the first 24 h of exposure.     

Reduction and inactivation of the sarcoplasmic Ca(2+)-ATPase by 2-mercaptoethanol--contrast to the (Na+,K+) ATPase.

The sarcoplasmic Ca(2+)-ATPase was reduced with 300 mM 2-mercaptoethanol at elevated temperatures (40-45 degrees C) with a concomitant loss of ATPase activity. The reduction and inactivation of the Ca(2+)-ATPase proceeded rapidly in the absence of Ca2+. The Ca(2+)-ATPase was also inactivated with 2-mercaptoethanol in the presence of diluted SDS (0.4 mg/ml) even at 20 degrees C. In contrast to the (Na+, K+) ATPase, the inactivated Ca(2+)-ATPase in the presence of diluted SDS was sedimented by the centrifugation at 100,000 x g for 30 min.     

Effects of manganese on rat brain microsomal Mg2+-Na+-K+-ATPase: in vivo and in vitro studies

The effect of manganese on brain microsomal Mg2+-Na+K+-ATPase was examined both in vitro and in vivo. Daily intraperitoneal administration of MnCl2 . 4H2O (Mn2+, 6 mg/kg) to the rats for a period of 90 days produced 10% (P less than 0.05) inhibition in the activity of Mg2+-ATPase, and 72 and 63% increases in the contents of manganese and copper, respectively, in the microsomal fraction of brain. In in vitro studies, lower concentrations of Mn2+ activated while higher concentrations inhibited the activity of brain microsomal ATPase. Addition of equal concentrations of Mn2+ + Cu2+ (8 mM) in vitro produced 8% inhibition in the activity of Mg2+-ATPase and 83% inhibition in Na+-K+-ATPase. Free Cu2+ ions were able to antagonize the effect of Mn2+ on ATPase in vitro and inhibited the activity of Mg2+-Na+-K+-ATPase with more pronounced effect of Na+-K+-ATPase. The lack of change in the activity of Na+-K+-ATPase in the brain microsomes of rats administered manganese, in spite of a significant increase in copper, could not be explained. It is, however, evident that a manganese-induced elevation in brain copper was not responsible for initiating biochemical changes in manganese neurotoxicity.     

Ethanol may stimulate or inhibit (Na+ + K+)-ATPase, depending upon Na+ and K+ concentrations

The influence of varying the ratios of [Na+]/[K+] on the effects of alcohol (500 mg/dl) on brain (Na+ + K+)-ATPase, using a commercial porcine enzyme preparation, showed that, generally, activity was stimulated by ethanol when [Na+] less than [K+], but inhibited when [Na+] greater than [K+] (with sum kept constant at 150 mM). In addition, when [Na+]/[K+] was 15/90 mM, representative of normal intracellular levels, ethanol (500 mg/dl) stimulated the porcine enzyme, but inhibited it when [Na+]/[K+] was 144/6 mM, representative of normal extracellular levels. Similarly, in freshly prepared enzyme from highly purified rat brain synaptic membranes, ethanol (100, 300, and 450 mg/dl) stimulated when [Na+]/[K+] was 15/88 mM (representing intracellular levels), but inhibited when [Na+]/[K+] was 142/4 mM (extracellular levels).     

The effect of L-cysteine and glutathione on inhibition of Na+, K+-ATPase activity by aspartame metabolites in human erythrocyte membrane

BACKGROUND: Reports have implicated Aspartame (N-L-a-aspartyl-L-phenylalanine methyl ester, ASP) in neurological problems. AIM: To evaluate Na(+), K(+)-ATPase activities in human erythrocyte membranes after incubation with the ASP metabolites, phenylalanine (Phe), methanol (MeOH) and aspartic acid (Asp). METHODS: Erythrocyte membranes were obtained from 12 healthy individuals and were incubated at 37 degrees C for 1 h with the sum or each of the ASP metabolites separately, which are commonly measured in blood after ASP ingestion. Na(+), K(+)-ATPase and Mg(2+)-ATPase activities were measured spectrophotometrically. RESULTS: Membrane Mg(2+)-ATPase activity was not altered. The sum of ASP metabolite concentrations corresponding to 34, 150 or 200 mg/kg of the sweetener ingestion resulted in an inhibition of the membrane Na(+), K(+)-ATPase by -30, -40, -48%, respectively. MeOH concentrations of 0.14, 0.60 or 0.80 mM decreased the enzyme activity by -25, -38, -43%, respectively. Asp concentrations of 2.80, 7.60 or 10.0 mM inhibited membrane Na(+), K(+)-ATPase by -26, -40, -46%, respectively. Phe concentrations of 0.14, 0.35 or 0.50 mM reduced the enzyme activity by -24, -44, -48%, respectively. Preincubation with L-cysteine or reduced glutathione (GSH) completely or partially restored the inhibited membrane Na(+), K(+)-ATPase activity by high or toxic ASP metabolite concentrations. CONCLUSIONS: Low concentrations of ASP metabolites had no effect on Na(+), K(+)-ATPase activity. High or abuse concentrations of ASP hydrolysis products significantly decreased the membrane enzyme activity, which was completely or partially prevented by L-cysteine or reduced GSH.     

The effect of diet on total antioxidant status, erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in patients with classical galactosaemia

OBJECTIVE: Classical galactosaemia is characterized by high levels of galactose-1-phosphate (Gal-1-P), galactose and galactitol. In vitro studies have shown modulation of the rat brain Na+,K+-ATPase and Mg2+-ATPase activities by Gal-1-P. The aim of this study was to evaluate the erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in galactosaemic patients and to correlate them to Gal-1-P, total antioxidant status (TAS) and membrane protein content (PC). PATIENTS AND METHODS: Nine patients (N=9) originally on "loose diet" (group B) were requested to follow their diet strictly (group A). Twelve healthy children were the controls (group C). The activities of the enzymes, TAS and Gal-1-P in blood were determined spectrophotometrically. In the in vitro study, erythrocyte membranes from controls were preincubated with Gal-1-P (300 microM), and then with l-cysteine (0.83 mM) or reduced glutathione (0.83 mM) whereas these from the patients with the antioxidants only. RESULTS: Na+,K+-ATPase, Mg2+-ATPase, TAS and PC were significantly (P<0.001) reduced (0.31+/-0.03, 1.7+/-0.2 micromol Pi/hxmg protein, 0.89+/-0.02 mmol/l, 36.8+/-2.0 g/l, respectively) in group B as compared with those of group A (0.58+/-0.06, 2.5+/-0.2 micromol Pi/hxmg protein, 1.41+/-0.11 mmol/l, 51.5+/-3.1g/l, respectively) and controls (0.67+/-0.05, 3.2+/-0.2 micromol Pi/hxmg protein, 1.65+/-0.12 mmol/l, 64.0+/-3.5 g/l, respectively). Gal-1-P levels in group B was significantly higher than those in group A and controls. Positive correlation coefficients were found between the enzyme activities, PC and TAS whereas Gal-1-P inversely correlated to the enzyme activities. Incubation of the erythrocyte membranes from the patients with the antioxidants failed to restore the activities of inhibited enzymes, whereas the inhibition by Gal-1-P in controls was reversed. CONCLUSIONS: High blood Gal-1-P concentrations resulted in low TAS and PC. The inhibition of Na+,K+-ATPase and Mg2+-ATPase may be due to the presence of free radicals and/or the elevated Gal-1-P.     

Inhibition of (Na+, K+)-ATPase by fluoride: evidence for a membrane adaptation to ethanol

Fluoride ions inhibit several membrane enzymes in a manner that is dependent on membrane fluidity. Inhibition of (Na+, K+)-ATPase by fluoride ions may be a model for membrane effects on (Na+, K+)-ATPase. Therefore, we have examined properties of fluoride inhibition relative to interactions with ethanol and to ligands that alter sensitivity of (Na+, K+)-ATPase to ethanol. Fluoride ion reduced the K0.5 and Hill coefficient for K+ activation of p-nitrophenylphosphatase. Ethanol decreased the Hill coefficient and apparent affinity for inhibition of phosphatase activity by fluoride ion while dimethylsulfoxide had the opposite effects. Chronic ethanol treatment in vivo, which produced behavioral tolerance, had effects on fluoride inhibition opposite to those of ethanol in vitro. Inhibition by fluoride therefore may provide a useful marker for physiologic or pharmacologic conditions that alter regulation of (Na+, K+)-ATPase by membrane properties.     

拟除虫菊酯类杀虫剂对大鼠脑突触体ATPase活性的影响

In vitro Effect of several pyrethroids on rat brain synaptosomal ATPase activities was investigated. No significant changes in Na+, K+-ATPase and oligomycin-insensitive Mg2+-ATPase activities were observed under present experimental conditions, but all pyrethroids tested caused significant inhibition of oligomycin-sensitive Mg2+-ATPase activity with certain concentration dependence. The results suggest a possibility that pyrethroids may alter the cellular energy metabolism of the nervous system.     

Effect of cadmium on transmembrane Na+ and K+ transport systems in human erythrocytes.

The effects of cadmium (Cd2+) on Na+,K(+)-ATPase in disrupted human erythrocyte membranes and on various transmembrane Na+ and K+ transport systems in intact erythrocyte suspensions were studied. Cadmium2+ inhibited the erythrocyte Na+,K(+)-ATPase enzyme with a 50% inhibition at a Cd2+ concentration of 6.25 microM. The Cd2+ inhibition in the human erythrocyte was non-competitive with respect to Na+,K+, and ATP. Cadmium2+ exerted no acute effect, however, on the Na+,K(+)-ATPase pump activity as measured by the ouabain sensitive 86Rb uptake or Na+ efflux in intact red blood cells. Cadmium2+ also inhibited the Ca2+ dependent K+ channels in human red blood cells, whereas it had no effect on Na+,K+ cotransport, Na+,Li+ countertransport, anion carrier, and the number of active Na+ pump units. The data indicate that in human erythrocytes under acute conditions Cd2+ exerts an inhibitory effect on Na+,K(+)-ATPase enzyme in disrupted erythrocytes and the Ca2+ stimulated K+ efflux in intact red blood cells without affecting the Na+ pump, Na+,K+ cotransport, and Na+,Li+ countertransport activity.     

Effect of cadmium on transmembrane Na+ and K+ transport systems in human erythrocytes.

The effects of cadmium (Cd2+) on Na+,K(+)-ATPase in disrupted human erythrocyte membranes and on various transmembrane Na+ and K+ transport systems in intact erythrocyte suspensions were studied. Cadmium2+ inhibited the erythrocyte Na+,K(+)-ATPase enzyme with a 50% inhibition at a Cd2+ concentration of 6.25 microM. The Cd2+ inhibition in the human erythrocyte was non-competitive with respect to Na+,K+, and ATP. Cadmium2+ exerted no acute effect, however, on the Na+,K(+)-ATPase pump activity as measured by the ouabain sensitive 86Rb uptake or Na+ efflux in intact red blood cells. Cadmium2+ also inhibited the Ca2+ dependent K+ channels in human red blood cells, whereas it had no effect on Na+,K+ cotransport, Na+,Li+ countertransport, anion carrier, and the number of active Na+ pump units. The data indicate that in human erythrocytes under acute conditions Cd2+ exerts an inhibitory effect on Na+,K(+)-ATPase enzyme in disrupted erythrocytes and the Ca2+ stimulated K+ efflux in intact red blood cells without affecting the Na+ pump, Na+,K+ cotransport, and Na+,Li+ countertransport activity.     

Influence of thymic fraction 5 on erythrocyte (Na+ + K+)-ATPase activity in hypercholesterolemic rabbits

The effects of thymus fraction 5 injection on cholesterol, triglyceride and phospholipid levels in plasma and erythrocytes as well as on membrane-bound (Na+ + K+)-ATPase activity were investigated in rabbits fed a high-cholesterol diet for 3 months. While cholesterol feeding caused an increase in plasma and erythrocytes phospholipid levels, (Na+ + K+)-ATPase activity was found to be reduced. After high-cholesterol diet, the rabbits were given normal diet with or without thymosin F5 injections every other day for 21 days. Thymosin F5 treatment caused a significant decrease in plasma and erythrocyte phospholipid levels whereas membrane-bound (Na+ + K+)-ATPase activity was increased significantly. It is concluded that hypercholesterolemic lesions and the decreased erythrocyte ATPase activity may be eliminated by thymic extracts in rabbits.     

A relationship between gill silver accumulation and acute silver toxicity in the freshwater rainbow trout: support for the acute silver biotic ligand model

Rainbow trout were exposed to a range of silver concentrations (as AgNO3) in flowing synthetic soft water (0.05 mM Na+, 0.05 mM Cl-, 0.05 mM Ca2+, 0.02 mM Mg2+, 0.02 mM K+, pH 7.0, approximately 0.7 mg C/L dissolved organic carbon, 10 mg CaCO3/L, 10 +/- 2 degrees C) to investigate a possible relationship between short-term gill silver accumulation (3 h or 24 h) and acute silver toxicity (96-h mortality). We also investigated potential relationships between gill silver accumulation and inhibition of Na+ uptake plus inhibition of gill Na+K(+)-adenosine triphosphatase (ATPase) activity. The 96-h median lethal concentration (LC50) values were 13.3 microg total Ag L(-1) and 3.3 microg dissolved Ag L(-1). A relationship was demonstrated between 3-h and 24-h gill silver accumulation and 96-h mortality. A relationship also was demonstrated between gill silver accumulation and inhibition of Na+ uptake at 24 h of exposure. No relationship between gill silver accumulation and inhibition of gill Na+K(+)-ATPase activity was found. The 96-h median lethal gill accumulation (LA50) values of 129 (at 3 h) and 191 ng g(-1) (at 24 h) and a conditional equilibrium binding constant of 8.0 for Ag+ binding to the gills were calculated. These observations support use of the silver biotic ligand model (BLM) as a regulatory tool to predict acute silver toxicity.     

Differential effects of ethanol on two synaptic membrane Ca2+ transport systems

The Na+-Ca2+ exchange activity in synaptic plasma membranes is inhibited by very low concentrations of ethanol (less than 25 mM). The high affinity Mg2+- and ATP-dependent Ca2+ transport in highly purified synaptic membranes is much less sensitive to inhibition by ethanol, with no statistically significant inhibition observed until an ethanol concentration of nearly 800 mM was used. Manipulations of the lipid environment designed to increase membrane fluidity enhanced the activity of the Na+-Ca2+ exchange system but inhibited the ATP-dependent Ca2+ pump. The differential responses of the two synaptic plasma membrane Ca2+ transporting systems to such modifications of membrane structure suggest that these two ion transport processes differ in the extent to which their activity is dependent on the lipid microenvironment in which they reside. Thus, the effects of ethanol on the Na+-Ca2+ antiporter may represent a fairly selective inhibitory process.     

Maternal ethanol consumption: effect on (Na+-K+)-ATPase in rat offspring

The activity of Mg2+-activated, ouabain-sensitive adenosine triphosphatase, (Na+-K+)-ATPase, was determined in homogenates of hypothalamus, cortex, cerebellum, and brain stem from the 19-day-old offspring of rats that were pair-fed control or (6.6%, v/v) ethanol liquid diets on a chronic basis prior to parturition. In the offspring of both control and ethanol-fed rats the specific activity of (Na+-K+)-ATPase was significantly (p less than 0.01) greater in the cortex than it was in the hypothalamus, brain stem or cerebellum (hypothalamus approximately brain stem approximately cerebellum). When the offspring of ethanol-fed and control rats were compared we observed no significant (p greater than 0.05) differences in the activity of (Na+-K+)-ATPase in any of the four brain regions examined. In addition, the results of kinetic analyses of cortical (Na+-K+)-ATPase were similar in the 19-day-old offspring of ethanol-fed rats and those whose mothers consumed either the control liquid diet or standard laboratory chow. The results of these studies suggest that the activity of the plasma membrane enzyme, (Na+-K+)-ATPase, was not affected in the 19-day-old offspring of ethanol-fed rats.     

Predicting acute zinc toxicity for Daphnia magna as a function of key water chemistry characteristics: development and validation of a biotic ligand model

The individual effect of different major cations (Ca2+, Mg2+, Na+, K+, and H+) on the acute toxicity of zinc to the waterflea Daphnia magna was investigated. The 48-h median effective concentration (EC50) in the baseline test medium (i.e., a standard medium with very low ion concentrations) was about 6 microM (Zn2+). An increase of Ca2+ (from 0.25 mM to 3 mM), Mg2+ (from 0.25 mM to 2 mM), and Na+ activity (from 0.077 mM to 13 mM) reduced zinc toxicity by a factor of 6.3, 2.1, and 3.1, respectively. No further toxicity reduction was observed when Ca2+ and Mg2+ activities exceeded 3.0 and 2.0 mM, respectively. Both K+ and H+ did not significantly alter zinc toxicity (expressed as Zn2+ activity). From these data, conditional stability constants for Ca2+ (log K = 3.24), Mg2+ (log K = 2.97), Na+ (log K = 2.16), and Zn2+ (log K = 5.31) were derived and incorporated into a biotic ligand model (BLM) predicting acute zinc toxicity to D. magna in surface waters with different water quality characteristics. Validation of the developed BLM using 17 media with different pH, hardness, and dissolved organic carbon (DOC) content resulted in a significant correlation coefficient (R2 = 0.76) between predicted and observed 48-h EC50. Eighty-eight percent of the predictions were within a factor of 1.3 of the observed 48-h EC50.     

Erythrocyte lipid peroxidation and (Na+ + K+)-ATPase activity in cholesterol fed rabbits

Cholesterol, phospholipid and lipid peroxide levels in plasma and erythrocytes as well as membrane-bound (Na+ + K+)-ATPase activity were determined in rabbits fed a high-cholesterol diet for 3 months. While cholesterol feeding caused an increase in lipid peroxide levels, (Na+ + K+)-ATPase activity was found to be reduced. According to this, we can assume that, in high-cholesterol fed rabbits, elevated lipid peroxides may be one of the responsible factors for the decreased erythrocyte (Na+ + K+)-ATPase activity.