Thallium inhibition of ouabain-sensitive sodium transport and of the (Na+ plus K+)-ATPase in human erythrocytes.

The influence of Tl+ on Na+ transport and on the ATPase activity in human erythrocytes was studied. 0.1-1.0 mM Tl+ added to a K+-free medium inhibited the ouabain-sensitive self-exchange of Na+ and activated both the ouabain-sensitive 22Na outward transport and the transport related ATPase. 5-10mM external Tl+ caused inhibition of the ouabain-sensitive 22Na efflux as well as the (Na+ plus Tl+)-ATPase. Competition between the internal Na+ and rapidly penetrating thallous ions at the inner Na+-specific binding sites of the erythrocyte membrane could account for the inhibitory effect of Tl+. An increase of the internal Na+ concentration in erythrocytes or in ghosts protected the system against the inhibitory effect of high concentration of Tl+. A protective effect of Na+ was also demonstrated on the (Na+ plus Tl+)-ATPase of fragmented erythrocyte membranes studied at various Na+ and Tl+ concentrations.     

Active cation transport and Na+K+Mg ATPase of the monotreme erythrocytes

The erythrocytes of the echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus), which are practically devoid of intracellular ATP content (1), were examined for active Rb⁸⁶ influx and for the presence of Na+K+Mg ATPase. We found that intact erythrocytes of both species possess the ability to actively transport cations. Ouabain sensitive Rb⁸⁶ influx in the echidna was approximately 0.17 μmoles/ml cells × hr, whereas the platypus exhibited a higher value of 0.43 μmoles/ml cells × hr. Surprisingly, ouabain sensitive Na+K+Mg ATPase activity of isolated membranes was high amounting to some 15 to 25 fold higher than the human erythrocyte counterpart determined under identical conditions. These findings suggest that a trace amount of ATP is sufficient to maintain active cation transport across the monotreme cell membranes.     

The effect of the membrane skeleton of non-nucleated erythrocytes on the properties of transport ATPases]

Removal of spectrin and other proteins of membrane skeleton from rat erythrocyte membranes resulted in a significant loss of Na,K-ATPase and Ca-ATPase activities, and even more of respective phosphatase activities. At the same time the modulating influence of ATP and Ca2+ on the enzymes disappeared. These ATPase activities were reconstituted by addition of concentrated spectrin to spectrin-depleted membranes. The activating influence of Ca2+ on ouabain-resistant and ouabain-sensitive phosphatases in ghosts could be discovered only in the presence of ATP. The highest activities of both the phosphatases were revealed when both ATP (0.5 mM) and Ca2+ (10-30 mM) were present simultaneously in the incubation medium. These data show that the functioning of transport ATPases in non-nuclear erythrocyte membranes is related to the membrane skeleton: regulating influence of intracellular ATP and Ca2+ on enzymes seems to be realized through the proteins of the skeleton.     

Effects of sulfhydryl compounds on the inhibition of erythrocyte membrane Na+(-)K+ ATPase by ozone

Exposure of human erythrocyte membranes to ozone (5 mumol/10 min) resulted in the inhibition of erythrocyte membrane Na+(-)K+ ATPase (EC.3.6.1.39). It was determined that, the degree of enzyme inhibition in the directly ozone exposed membranes was greater than that of membranes obtained from ozone exposed intact erythrocytes. In the presence of varying concentrations (0-1.0 mM) of dithiotrethiol or mercaptoethanol Na+(-)K+ ATPase activities of both types of ozone exposed membranes were increased almost proportionally with the concentration of dithiotrethiol or mercaptoethanol however, the activities were still lower than the normal Na+(-)K+ ATPase value. The results indicate that, dithiotrethiol or mercaptoethanol prevent the enzyme inhibition by ozone in vitro. This suggests that the membrane thiol groups are primary targets for ozone and thereby preventing the oxidation of essential functional groups of enzyme protein.     

Method for the preparation of human erythrocyte membrane with low basal calcium ATPase, responsive to stimulation

A simple procedure for preparing erythrocyte membranes with low basal Ca2+ ATPase activity is described, which is stimulated several-fold by the addition of hemolysate in the incubation mixture. The cells are hemolyzed in hypotonic imidazole buffer and resulting membranes are washed with hypotonic phosphate buffer (pH 8.0) and the hemolyzing medium. The membrane preparations also have Mg2+-stimulated and Na+-K+-stimulated ATPase activities. The method allows the comparison of basal Ca2+ ATPase as well as hemolysate- or calmodulin-stimulated Ca2+ ATPase activities and thus may be useful in studying Ca2+ ATPase activity in various physiopathological conditions.     

Isolation of the basal and lateral plasma membranes of rat kidney tubule cells.

A method was developed to isolate renal basolateral membranes from cortical kidney tubule cells of single rats. The isolated membrane fraction was characterized by the measurement of marker enzyme activities and by electron microscopy. 1. After centrifugation of crude plasma membranes on a discontinuous sucrose density gradient the basolateral membranes accumulated at a sucrose density of p= 1.14-1.15 g/ml. The yield was 147 mug membrane protein/g kidney wet weight. Protein recovery was 0.1%. 2. (Na+ + K+)-ATPase was enriched 22-fold from the homogenate. The recovery was 2.6%. The (Na+ + K+)/Mg2+-ATPase ratio was 4.1. 3. The contamination by brush borders was small. Alkaline phosphatase was 1.6-fold enriched and 0.2% was recovered. Aminopeptidase was 1-fold enriched with a recovery of 0.1%. The contamination by mitochondria, lysosomes and endoplasmic reticulum was negligible. 4. In electron micrographs the basolateral membranes showed a typical triple layered profile and were characterized by the presence of junctional complexes, gap junctions or tight junctions.     

Effect of hyperoxide radicals on bovine-erythrocyte membrane.

1. Bovine erythrocytes exposed to the action of an enzymic source of hyperoxide radicals (hypoxanthine + xanthine oxidase) exhibited hemolysis, which was prevented by the presence of hyperoxide dismutase. 2. Exposing bovine erythrocyte membranes to the source of hyperoxide radicals resulted in a decrease of (Mg2+ + Na+ + K+)ATPase activity which could be partially prevented by addition of hyperoxide dismutase. 3. The damage observed to erythrocyte membranes under the conditions applied is ascribed to toh formed in the Haber and Weiss reaction since a protection by OH scavengers was also observed.     

Mechanism of the change in erythrocyte osmotic resistance in rats exposed to valinomycin: the features seen in spontaneous hypertension

Introduction of valinomycin into erythrocyte incubation medium increased the cell stability to water-induced hemolysis. In these conditions the erythrocytes of spontaneously hypertensive and normotensive (control) rats release 63.2 +/- 1.5% and 80.9 +/- 1.6%, respectively, of the total hemoglobin content. Valinomycin effect is completely abolished with K+ substitution for Na+ and is independent of extracellular Ca2+ concentration. Valinomycin had no effect on human erythrocyte osmotic stability. It has been shown that valinomycin-induced kinetics of Na+ and K+ redistribution was different in human and rat erythrocytes. The distinctions are thought to be related to specific anion transport mediated by the third band protein--the main component of membrane cytoskeleton.     

Ca2+-Mg2+-ATPase activity in brain coated microvesicles purified on immunosorbents

Coated microvesicle fractions isolated from ox forebrain cortex by the ultracentrifugation procedure of Pearse (1) and by the modified, less time consuming method of Keen et al (2) had comparable Ca2+ +Mg2+ dependent ATPase activities (about 9 mumol/h per mg protein). The Na+ +K+ +Mg2+ dependent ATPase activity was 3.2 mumol/h per mg (+/- 1.0, S.D., n = 3) when microvesicles were prepared according to (1) and 1.5 mumol/h per mg (+/- 1.0, S.D., n = 3) when prepared according to (2). Oligomycin, ruthenium red, and trifluoperazine, inhibitors of Ca2+ transport in mitochondria and erythrocyte membranes had no effect on Ca2+ +Mg2+ dependent ATPase from any of the preparations. As demonstrated both by ATPase assays and electron microscopy, coated microvesicles could be bound to immunosorbents prepared with poly-specific antibodies against a coated microvesicle fraction obtained by the method of Pearse (1). The binding could be inhibited by dissolved coat protein using partially purified clathrin. The fraction of coated vesicles eluted from the immunosorbent was purified relative to the starting material as judged by electron microscopy. The Ca2+ +Mg2+ ATPase activity and calmodulin content was copurified with the coated microvesicles and the specific activity of Na+ +K+ +Mg2+ ATPase was decreased. Na+ +K+ +Mg2+ dependent ATPase activity in the coated microvesicle fraction could be ascribed to membranes with the appearance of microsomes. These membranes were also bound to the immunosorbents, but the binding was not influenced by clathrin. The capacity of the immunosorbents for these membranes was less than for the coated microvesicles, resulting in a decrease of Na+ +K+ +Mg2+ dependent ATPase activity in the eluted coated microvesicle fraction. It was concluded that Ca2+ +Mg2+ ATPase activity is not a contamination from plasma membrane vesicles or mitochondrial membranes but seems to be an integral part of the coated vesicle membrane.     

The mechanism of the hemolytic activity of polyene antibiotics

The kinetics of the filipin-, amphotericin B- and nystatin-induced hemolysis of human erythrocytes were investigated. Filipin-induced hemolysis is of the damage type. It is an all-or-none process, partly inhibited by Ca2+ or Ba2+ but not by Mg2+, Na+ or SO42-. The hemolytic activity of filipin is explained by the formation of large aggregates within the erythrocyte membrane in the form of large perforations, permeable to substances of low molecular weight as well as to macromolecules, including hemoglobin. In isotonic KCl solution, both amphotericin B and nystatin, at low concentrations, form smaller aggregates within the membranes. As a result, the permeability of the membranes to KCl increases and hemolysis occurs. However, the kinetics of the hemolysis induced by the two polyenes is complex. The process shows some features of the permeability type and some of the damage type. It is suggested that amphotericin B and nystatin may simultaneously form a number of transport systems, differing in their molecular organisation and hemolytic activity. Their participation in erythrocyte membrane permeability can be modified by small changes in membrane organisation and the chemical composition of the incubation medium. In isotonic solutions of divalent cation chlorides, and at higher antibiotic concentration, additional aggregates, allowing divalent cations to permeate, appear. These structures do not permit SO4(2-) to permeate.     

Effect of arachidonic acid on duodenal enterocyte ATPases

Duodenal ion transport processes are supported by ATPase enzymes in basolateral membranes of the enterocyte. In vivo studies have shown that long term n-6 poly-unsaturated fatty acid (PUFA) supplementation in rats causes increases in intestinal Ca absorption, coupled with a higher total calcium balance and bone calcium content. The present in vitro study was undertaken to test the effect of arachidonic acid (AA), a highly unsaturated (and thus physiologically potent) member of the n-6 PUFA family, on ATPases in enterocyte basolateral membranes isolated with a sorbitol density gradient procedure. This paper presents results which show that AA inhibits Na+,K+-ATPase in a dose-dependent manner (-67% of basal activity at a concentration of 30 microg/ml, P < 0.005) but that this effect is not mediated by protein kinase C, as shown by the use of the protein kinase C blocker calphostin (0.5 microM). Indomethacin (IDM) at 0.1 mM, a cyclo-oxygenase blocker, could also not reverse the inhibitory effect of AA on Na+,K+-ATPase. Ca2+-ATPase, on the other hand, is not affected significantly (-10%, P > 0.05) by arachidonic acid at 30 microg/ml.     

Mode of interaction of the single a subunit with the multimeric c subunits during the translocation of the coupling ions by F1F0 ATPases.

We have recently isolated a mutant (aK220R, aV264E, aI278N) of the Na+-translocating Escherichia coli/Propionigenium modestum ATPase hybrid with a Na+-inhibited growth phenotype on succinate. ATP hydrolysis by the reconstituted mutant ATPase was inhibited by external (N side) NaCl but not by internal (P side) NaCl. In contrast, LiCl activated the ATPase from the N side and inhibited it from the P side. A similar pattern of activation and inhibition was observed with NaCl and the ATPase from the parent strain PEF42. We conclude from these results that the binding sites for the coupling ions on the c subunits are freely accessible from the N side. Upon occupation of these sites, the ATPase becomes more active, provided that the ions can be further translocated to the P side through a channel of the a subunit. If by mutation of the a subunit this channel becomes impermeable for Na+, N side Na+ ions specifically inhibit the ATPase activity. These conclusions were corroborated by the observation that proton transport into proteoliposomes containing the mutant ATPase was abolished by N side but not by P side Na+ ions. In contrast, LiCl affected proton translocation from either side, similar to the sidedness effect of Na+ ions on H+ transport by the parent hybrid ATPase. If the ATPase carrying the mutated a subunit was incubated with 22NaCl and ATP, 1 mol 22Na+/mol enzyme was occluded. With the parent hybrid ATPase, 22Na+ occlusion was not observed. The occluded 22Na+ could be removed from its tight binding site by 20 mM LiCl, while incubation with 20 mM NaCl was without effect. Li+ but not Na+ is therefore apparently able to pass through the mutated a subunit and make the entrapped Na+ ions accessible again to the aqueous environment. These results suggest an ion translocation mechanism through F0 that in the ATP hydrolysis mode involves binding of the coupling ions from the cytoplasm to the multiple c subunits, ATP-driven rotation to bring a Na+, Li+, or H+-loaded c subunit into a contact site with the a subunit and release of the coupling ions through the a subunit channel to the periplasmic surface of the membrane.     

Regulation of Na+ transport in brown adipose tissue.

In order to test the hypothesis that Na+, K+-ATPase (Na+,K+-dependent ATPase) is involved in the noradrenaline-mediated stimulation of respiration in brown adipose tissue, the effects of noradrenaline on Na+,K+-ATPase in isolated brown-fat-cell membrane vesicles, and on 22Na+ and K+ (86Rb+) fluxes across the membranes of intact isolated cells, were measured. The ouabain-sensitive fraction of the K+-dependent ATPase activity in the isolated membrane-vesicle preparation was small and was not affected by the presence of noradrenaline in the incubation media. The uptake of 86Rb+ into intact hormone-sensitive cells was inhibited by 80% by ouabain, but it was insensitive to the presence of noradrenaline. 22Na+ uptake and efflux measured in the intact cells were 8 times more rapid than the 86Rb+ fluxes and were unaffected by ouabain. This indicated the presence of a separate, more active, transport system for Na+ than the Na+,K+-ATPase. This is likely to be a Na+/Na+ exchange activity under normal aerobic conditions. However, under anaerobic conditions, or conditions simulating anaerobiosis (2 mM-NaCN), the unidirectional uptake of Na+ increased dramatically, while efflux was unaltered.     

Na+ATPase of the mammalian erythrocyte membrane. Reversibility of phosphorylation at 0 degrees.

When human erythrocyte membranes are phosphorylated with a very low concentration of [gamma-32P]ATP (0.02 muM) at 0 degrees, and then EDTA is added, rapid disappearance of the phosphoenzyme intermediate of Na+ATPase is observed. The initial rapid phase of phosphoenzyme disappearance is, for the most part, not associated with P1 release and its rate constant, kD, is severalfold greater than the ratio of Na+ATPase activity to phosphoenzyme intermediate, v:EP, at steady state. It is concluded that this rapid disappearance of phosphoenzyme is due to resynthesis of ATP via reversal of phosphorylation. In contrast, rapid reversal is not observed when excess nonradioactive ATP is added to reduce E32P formation, provided Mg2+ is present; however, K+ added with the ATP stimulates reversal. Rapid reversal following EDTA addition is unlikely also when higher ATP concentrations (greater than or equal to 10(-6) M) are used to phosphorylate the enzyme since, at higher ATP, kD congruent to v:EP. The results are compatible with the concept that the Na+ATPase enzyme is composed of two or more catalytic subunits, in which ATP at one catalytic site can regulate the reactivity at another site.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Ca2+-mediated activation of human erythrocyte membrane Ca2+-ATPase

Ca2+-ATPase of human erythrocyte membranes, after being washed to remove Ca2+ after incubation with the ion, was found to be activated. Stimulation of the ATPase was related neither to fluidity change nor to cytoskeletal degradation of the membranes mediated by Ca2+. Activation of the transport enzyme was also unaffected by detergent treatment of the membrane, but was suppressed when leupeptin was included during incubation of the membranes with Ca2+. Stimulation of the ATPase by a membrane-associated Ca2+-dependent proteinase was thus suggested. Much less 138 kDa Ca2+-ATPase protein could be harvested from a Triton extract of membranes incubated with Ca2+ than without Ca2+. Activity of the activated enzyme could not be further elevated by exogenous calpain, even after treatment of the membranes with glycodeoxycholate. There was also an overlap in the effect of calmodulin and the Ca2+-mediated stimulation of membrane Ca2+-ATPase. While Km(ATP) of the stimulated ATPase remained unchanged, a significant drop in the free-Ca2+ concentration for half-maximal activation of the enzyme was observed.     

Effect of HMG-CoA reductase inhibitors on the erythrocyte membrane]

We studied 10 patients affected by primary hypercholesterolemia treated with placebo for 1 month and with simvastatin (20 mg die) for 6 months during a double-blind clinical trial. At 1-month intervals we determined the following parameters in the serum: total and HDL-cholesterol, triglycerides, apolipoprotein A1 and B. At the same time intervals, we also determined the cholesterol and phospholipid concentration, the Na+/K+ ATPase activity and the fluidity of the erythrocyte membranes. Our results demonstrated the following modifications in the erythrocyte membranes during simvastatin treatment: 1) an initial increase in the cholesterol concentration and in the cholesterol/phospholipid ratio, with a significant decrease only after 4 months; 2) a similar behaviour of membrane fluidity, with an initial decrease and an elevation after 4 months; 3) an increase in the Na+/K+ ATPase activity only after 4 months. We hypothesize that simvastatin not only inhibits the hepatic synthesis of cholesterol, but also modifies the cholesterol exchange between plasma and the erythrocyte membrane.     

Release of the component of Streptococcus faecalis Na(+)-ATPase from the membranes

The Na(+)-stimulated ATPase activity of Streptococcus faecalis was lost by washing the membranes with ethylenediaminetetraacetic acid (EDTA). ATPase activities of both the EDTA extract and the stripped membranes did not show any stimulation by Na+ ions. However, the Na(+)-stimulated ATPase was readily reconstituted by an incubation of these fractions combined. It was only reconstituted from the fractions prepared under the condition that the Na(+)-ATPase is amplified, and not from those boiled or digested by trypsin. Thus, the component of Na(+)-ATPase of this organism is capable of being released from the membranes.     

Stimulation of Na+,K+-ATPase activity in certain membranes of the rat central nervous system (CNS) by acute administration of desipramine (DMI)

1. The activities of ATPase in rat CNS were studied 3 hr after administration of the noradrenaline uptake inhibitor, desipramine (DMI: 10 mg.kg-1, i.p.). Na+K+-ATPase activity significantly increased after DMI in the whole particulate from hypothalamus and mesencephalus but no changes in frontal cortex or in pons-medulla oblongata areas were found. This increase was prevented when the animals were pretreated with the noradrenergic neurotoxic N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). 2. Purified membrane fractions from hypothalamus were obtained by differential and sucrose gradient centrifugation (0.8-1.2 M sucrose). It was observed that after DMI, Na+,K+-ATPase activity increased only in the membranous fraction lying at 0.9 M sucrose. 3. Mg2+- or Ca2+-ATPase activities were not modified by DMI treatment. 4. Citalopram, a specific serotonergic uptake inhibitor, did not affect ATPase activities. 5. The results obtained could indicate that DMI acute administration selectively stimulates Na+,K+-ATPase activity of certain membranes of the CNS after an increase in the concentration of the noradrenergic neurotransmitter in the synaptic gap.     

Modulation of rat erythrocyte antioxidant defense system by buthionine sulfoximine and its reversal by glutathione monoester therapy

The protective effects of glutathione monoester (GME) on buthionine sulfoximine (BSO)-induced glutathione (GSH) depletion and its sequel were evaluated in rat erythrocyte/erythrocyte membrane. Animals were divided into three groups (n=6 in each): control, BSO and BSO+GME group. Administration of BSO, at a concentration of 4 mmol/kg bw, to the albino rats resulted in depletion of blood GSH level to about 59%. GSH was elevated several folds in the GME group as compared to the control (P<0.05) and BSO (P<0.001) groups. Decreased concentration of vitamin E was found in the erythrocyte membrane isolated from BSO-administered animals. Antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) were also found to be altered due to BSO-induced GSH depletion in blood erythrocytes. The SOD and CAT activities in BSO group were significantly lower (P<0.001) than the other groups. Lipid peroxidation index and malondialdehyde (MDA) levels in erythrocytes and their membranes were increased to about 45% and 40%, respectively. The activities of Ca2+ ATPase, Mg2+ ATPase and Na+K+ ATPase were lower than those of control group (P<0.05), whereas the activities of these enzymes were found to be restored to normal followed by GME therapy (P<0.05). Cholesterol, phospholipid and C/P ratio and some of the phospholipid classes like phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin were significantly (P<0.05) altered in the erythrocyte membranes of BSO-administered rats compared with those of control group. These parameters were restored to control group levels in GME-treated group. Oxidative stress may play a major role in the BSO-mediated gamma glutamyl cysteine synthetase (gamma-GCS) inhibition and hence the depletion of GSH. In conclusion, our findings have shown that antioxidant status decreased and lipid peroxidation increased in BSO-treated rats. GME potentiates the RBC and blood antioxidant defense mechanisms and decreases lipid peroxidation.