Mechanism of brain Na,K-ATPase activation by adrenaline]

Norepinephrine stimulates Na, K-ATPase from rat brain homogenates at concentrations of 10(-4)--10(-5) and 10(-7)--10(-8) M. A low concentration maximum is observed after 48 hrs of incubation at -20 degrees C and is not changed by the addition of alpha-tocopherol, glycerol and MAO inhibitor ipraside. The maximum observed at the mediator concentration equal to 10(-4)--10(-5) M is eliminated after treatment with EGTA. At all concentrations of norepinephrine the enzyme stimulation is removed by the alpha-adrenoblocker phentolamine. The activated enzyme reveals lower sensitivity to Ca2+ induced inhibition. The role of Ca2+ and conformational state of the membranes in the realization of the remote effect on the adrenoreceptor-Na, K-ATPase system is discussed.     

The effect of hypothermia on kinetic characteristics of the rat brain synaptic membrane Na,K-ATPase

The effect of profound hypothermia (acute or prolonged) on Km for ATP, Vm and strophanthine K affinity to Na,K-ATPase in the rat brain synaptosomal membranes was investigated. The temperature dependence of Na,K-ATPase activity in temperature range 5-40 degrees C was also studied. Hypothermia decreases Km and Vm, and increases affinity of strophanthine K to the enzyme. There are two linear sections in Arrhenius plots ofNa,K-ATPase activity. Hypothermia does not change position of the break point in Arrhenius plots. The mechanisms and biological significance of the changes revealed are discussed.     

Oligomerization of the Na,K-ATPase in cell membranes

The higher order oligomeric state of the Na,K-ATPase alphabeta heterodimer in cell membranes is the subject of controversy. We have utilized the baculovirus-infected insect cell system to express Na,K-ATPase with alpha-subunits bearing either His(6) or FLAG epitopes at the carboxyl terminus. Each of these constructs produced functional Na,K-ATPase alphabeta heterodimers that were delivered to the plasma membrane (PM). Cells were simultaneously co-infected with viruses encoding alpha-His/beta and alpha-FLAG/beta Na,K-ATPases. Co-immunoprecipitation of the His-tagged alpha-subunit in the endoplasmic reticulum (ER) and PM fractions of co-infected cells by the anti-FLAG antibody demonstrates that protein-protein associations exist between these heterodimers. This suggests the Na,K-ATPase is present in cell membranes in an oligomeric state of at least (alphabeta)(2) composition. Deletion of 256 amino acid residues from the central cytoplasmic loop of the alpha-subunit results in the deletion alpha-4,5-loop-less (alpha-4,5LL), which associates with beta but is confined to the ER. Co-immunoprecipitation demonstrates that when this inactive alpha-4,5LL/beta heterodimer is co-expressed with wild-type alphabeta, oligomers of wild-type alphabeta and alpha-4,5LL/beta form in the ER, but the alpha-4,5LL mutant remains retained in the ER, and the wild-type protein is still delivered to the PM. We conclude that the Na,K-ATPase is present as oligomers of the monomeric alphabeta heterodimer in native cell membranes.     

Effect of digitonin on the activity of Na,K-ATPase from bovine brain

The kinetic properties of intact and digitonin-treated Na,K-ATPase from bovine brain were studied. The temperature dependence curve for the rate of ATP hydrolysis under optimal conditions (upsilon 0) in the Arrhenius plots shows a break at 19-20 degrees. The temperature dependence curves for Km' and Km" have breaks at the same temperatures, while the Arrhenius plot for V is linear. The value of the Hill coefficient (nH) for ATP at 37 degrees is variable depending on ATP concentration, i. e. it is less than 1 at ATP concentrations below 50 mkM and is increased up to 3.2 at higher concentrations of the substrate. At high ATP concentrations the value of nH depends on temperature, falling down to 2.1 at 23 degrees and then down to 1 within the temperature range of 21-19 degrees. A further decrease in temperature does not significantly affect the nH value. Digitonin irreversibly inhibits Na, K-ATPase. ATP hydrolysis is more sensitive to the effect of the detergent than is nNPP hydrolysis, i. e. after complete inhibition of the ATPase about 40% of the phosphatase activity are retained. Treatment of Na,K-ATPase by digitonin results in elimination of the breaks in the Arrhenius plots for upsilon 0, Km' and Km", whereas the temperature dependence plot of V remains linear. Simultaneously digitonin eliminates the positive cooperativity of the enzyme for ATP. It is assumed that Na, K-ATPase from bovine brain is an oligomer of the (alpha beta) 4 type. Digitonin changes the type of interaction between the protomers within the oligomeric complex by changing the lipid environment of the enzyme or the type of protein -- lipid interactions.     

The coupling of Na,K-ATPase with the sodium channels in the plasma membranes of the brain synaptosomes of rats]

Effect of neurotoxins veratrine (100 micrograms/ml) and tetrodotoxin (1 microM) on the binding of 3H-ouabain (10(-8) M) with Na,K-ATPase of intact synaptosomes and isolated synaptic membranes was studied. The persistent opening of sodium channels in synaptosomes by veratrine results in an increase of specific binding of the labeled ligand by 20%. A similar effect was caused by Na/H exchanger monensin. Destruction of microtubules with vinblastine and colchicine has no influence on veratrine action, while depolymerization of microfilaments with cytochalasin B reverses the neurotoxin effect. In isolated synaptic membranes veratrine and tetrodotoxin stimulate ouabain binding, the absolute veratrine-induced increment being several times higher in the presence of ATP than in its absence. Since the closed vesicles of any type are not permeable to ATP and ouabain, it means that in the isolated membranes an interaction between sodium channels and Na,K-ATPase molecules takes place. In intact nerve endings such a mechanism may be operative along with the known ways of control of sodium pump and its ouabain-binding site.     

18 O-exchange during ATP and n-nitrophenylphosphate hydrolysis by Na, K-ATPase from bovine brain]

The reaction of the oxygen isotope exchange (18O-exchange) was studied in the course of the Na, K-ATPase reaction. It was shown that the intermediary and direct 18O-exchanges occurred in the system in the presence of both ATP and p-NPP. These findings are indicative of the same intermediate during the hydrolytic process in both cases. The intermediary 18O-exchange was activated by N-ethylmaleimide, hydroxylamine and 2.0--1.5 18O atoms, respectively. The detection of 18O-exchange Ouabain had no effect on the exchange. The levels of intermediary 18O-exchange during ATP and p-NPP hydrolyses were equal to 1.3--1.4 and 2.0--1.5 18O atoms, respectively. The detection of 18O-exchange reactions at the intermediary steps of both ATP and p-NPP hydrolyses implies the identity of certain stages in the destruction of these substrates by Na, K-ATPase.     

The role of conformational changes in the functioning of brain Na,K-ATPase]

In the experiments with enzyme preparations of Na,K-ATPase from normal brain tissue (NBT) and tumorous brain tissue (TBT) the following data were established: 1) the cooperativity of Na,K-ATPase with Na+ from NBT is temperature-dependent, the Hill coefficient (nH) at 37, 27.0-30.5 and 20-22 degrees C being 1.80 +/- 0.07, 1.30 +/- 0.09 and 1.10 +/- 0.08, respectively; the cooperativity of Na+ with Na,K-ATPase from TBT was absent; 2) the cooperativity for ouabain (nH-1.30 +/- 0.05) was revealed only in the case of Na-pump from TBT; 3) the protective effect of ATP against the inhibitory action of pCMB is temperature-dependent and differs significantly in enzyme preparations from NBT and TBT; 4) the parameters of the temperature inactivation of enzyme preparations at 45-52 degrees C, especially the change of entropy (delta S*) were different in the case of NBT and TBT; 5) a peptide fraction isolated from sheep brain differently inhibited the Na,K-ATPase from NBT and TBT. In conclusion, these data demonstrate that there are significant differences in functioning of Na,K-ATPase from NBT and TBT, and that besides lipid-protein interactions the local domenic conformational changes in the enzyme molecule may play a definite role in these differences.     

Inhibition of Na,K-ATPase from chick brain by polyamines.

The amounts of the polyamines putrescine, spermine and spermidine as well as the Na,K-ATPase activity have been determined in the developing chick brain. The amounts of spermine and spermidine per gram fresh weight do not change significantly, the amount of putrescine declines until the 17th day of incubation after which an increase takes place. Spermine is able to inhibit the Na,K-ATPase from chick brain competitively. Half maximal inhibition is achieved at 4 X 10(-5) mol/1 spermine. This polyamine functions as an allosteric inhibitor; the Hill coefficient is 2.2 +/- 0.3. A regulatory effect of spermine on the Na,K-ATPase from chick brain is discussed. In contrast to spermine 1 mmol/1 spermidine inhibits the Na,K-ATPase only slightly, while 1 mmol/1 putrescine does not inhibit the Na,K-ATPase at all.     

Enzymatic micromethod for determining Na, K-ATPase activity in rat cerebral cortex membranes]

A method for the assay of Na,K-ATPase activity of unpurified synaptosomal fraction obtained from the microquantities (2--3 mg of fresh tissue) of the rat cerebral cortex is described. This method is based on the fluorimetric determination of ADP formed in the course of ATPase reaction. The method is highly sensitive and may be used to determine the membrane preparations Na,K-ATPase activity with the protein content of 0.05--10.0 microgram per sample.     

Effects of paradoxical sleep deprivation on the activity of opiate receptors isolated from synaptic membranes of the rat brain]

The specific binding of 3H-naloxone with opiate receptors isolated from brain synaptic membranes of control and paradoxical sleep deprived rats were studied. This extreme state was shown to reduce the naloxone-binding activity of synaptic membranes by 35% and isolated receptors by 25-28%. The values of Kd and Bmax were calculated for isolated opiate receptors at different stages of purification. Considerable decrease of 3H-naloxone binding sites density (2 times) in the isolated opiate receptors with simultaneous increase of their affinity (3-4,5 times) was found following 24 hours paradoxical sleep deprivation. These findings suggest development of fixed alterations in the structure and functions of integral receptor proteins under extreme influences.     

Selective inhibition of brain Na,K-ATPase by drugs

The effect of drugs from the class of cardiac (methyldigoxin, verapamil, propranolol), antiepileptic (carbamazepine), sedative (diazepam) and antihistaminic (promethazine) drugs on Na,K-ATPase activity of plasma membranes was studied in rat brain synaptosomes. Methyldigoxin in a concentration of 0.1 mmol/l inhibits enzyme activity by 80 %. Verapamil, propranolol and promethazine in concentrations of 20, 20 and 2 mmol/l respectively, entirely inhibit the ATPase activity. Carbamazepine and diazepam in concentrations of 0.02-60 mmol/l have no effect on the activity of this enzyme. According to the drug concentrations that inhibit 50 % of enzyme activity (IC(50)), the potency can be listed in the following order: methyldigoxin promethazine verapamil ? propranolol. From the inhibition of commercially available purified Na,K-ATPase isolated from porcine cerebral cortex in the presence of chosen drugs, as well as from kinetic studies on synaptosomal plasma membranes, it may be concluded that the drugs inhibit enzyme activity, partly by acting directly on the enzyme proteins. Propranolol, verapamil and promethazine inhibitions acted in an uncompetitive manner. The results suggest that these three drugs may contribute to neurological dysfunctions and indicate the necessity to take into consideration the side effects of the investigated drugs during the treatment of various pathological conditions.     

Age-related characteristics of the Na+,K+-ATPase activity of synaptic membranes from the rat brain

The study of albino rats aged 6-7 months and 25-27 months revealed the age-related increase of maximal activity (V) of Na+, K+-ATPase of synaptosomal plasma membranes, separated from the cerebral cortex, while the level of Km remained stable. It is shown that in old rats as compared to the adult ones the affinity of Na+, K+-ATPase to sodium ions increases and the character of the ATP hydrolysis schedule changes in the presence of different ration of ions-activators. There are no significant changes in the inhibiting effect of strophantidin K on Na+, K+-ATPase activity of synaptosomal plasma membranes.     

Effect of mineralocorticoids on the Na, K-ATPase activity of the rat brain

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.     

Ouabain-sensitive H,K-ATPase functions as Na,K-ATPase in apical membranes of rat distal colon

Na,K-ATPase activity has been identified in the apical membrane of rat distal colon, whereas ouabain-sensitive and ouabain-insensitive H,K-ATPase activities are localized solely to apical membranes. This study was designed to determine whether apical membrane Na,K-ATPase represented contamination of basolateral membranes or an alternate mode of H,K-ATPase expression. An antibody directed against the H, K-ATPase alpha subunit (HKcalpha) inhibited apical Na,K-ATPase activity by 92% but did not alter basolateral membrane Na,K-ATPase activity. Two distinct H,K-ATPase isoforms exist; one of which, the ouabain-insensitive HKcalpha, has been cloned. Because dietary sodium depletion markedly increases ouabain-insensitive active potassium absorption and HKcalpha mRNA and protein expression, Na, K-ATPase and H,K-ATPase activities and protein expression were determined in apical membranes from control and sodium-depleted rats. Sodium depletion substantially increased ouabain-insensitive H, K-ATPase activity and HKcalpha protein expression by 109-250% but increased ouabain-sensitive Na,K-ATPase and H,K-ATPase activities by only 30% and 42%, respectively. These studies suggest that apical membrane Na,K-ATPase activity is an alternate mode of ouabain-sensitive H,K-ATPase and does not solely represent basolateral membrane contamination.     

Factors determining the functional activity of Na,K-ATPase]

A comparative estimation was made of modifications of Na,K-ATPase and Mg-ATPase parameters in the process of phylogenesis and as a result of sudden thermal selection. On the basis of our own and literary data a suggestion was put forward about the availability of quite different ways of thermal adaptation in ATP-hydrolyzing enzymes associated with different physiological functions.     

Investigation of the effect of phospholipase on Na,K-ATPase activity]

Temperature dependence of bovine brain NA,K-ATPase before and after the short-term treatment of enzyme preparations with phospholipases A, C and D is investigated. Arrhenius plots of the temperature dependence of the reaction rate catalysed by Na,K-ATPase are non-linear, they have an inflection at the region of about 20 degrees C. The treatment of the enzyme with phospholipase A makes the inflection more smooth, phospholipase D shifts the inflection by 4 degrees C to lower temperature and simultaneously activates Na,K-ATPase. Phospholipase C sharply changes the Arrhenius curve and makes it linear. The data obtained are discussed with respect to the role of phospholipids in the formation of membrane bilayer and in the regulation of Na,K-ATPase activity.     

Some properties of glial membrane Na, K-ATPase]

Na,K-ATPase activity in glial membranes is rather low that in the nerve ending membranes, but is characterized by the same kind of Na+/K+-dependence. Glial Na,K-ATPase is insensitive to acetylcholine (ACh), 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA) while norepinephrine activates Na,K-ATPase at low concentrations and inhibits it at high concentrations. Participation of Na,K-ATPase in the regulatory mechanisms of the neuron-neuroglia relations is discussed.     

Localization of the phlorizin site on Na, K-ATPase in red cell membranes.

Phlorizin at 2 X 10(-4) M inhibited Na+ and Rb+-activated ATPase activities in human red cell membranes by 43%. It inhibited the 86Rb uptake activity of erythrocytes by only 15%. 86Rb uptake into resealed ghosts was inhibited strongly when phlorizin and ATP were preloaded in the ghosts before resealing. Na,K-ATPase activity in the resealed ghosts was also inhibited in the presence of phlorizin inside but not outside the ghosts. These findings suggested that the phlorizin site is located inside the cell.     

Incorporation of Na,K-ATPase into human erythrocyte membranes using liposomes

A procedure for incorporation of isolated cattle brain Na,K-ATPase into erythrocyte membranes by proteoliposomes has been elaborated. The Na,K-ATPase activity of proteoliposome-treated human erythrocytes containing incorporated Na,K-ATPase does not exceed that of control erythrocytes. In the erythrocyte membrane the incorporated enzyme exists in a functionally active state and retains the vector properties of the Na+-pump. Exogenous ATP stimulates 22Na influx and 86Rb efflux in and from the erythrocytes.