Na,K-ATPase in kidneys of rats with hereditary hypertension induced by stress]

It has been found that Na, K-ATP-ase activity in microsomal fraction obtained from the medullar layer of kidneys of stress-sensitive hypertensive rats (SSHR) which were subjected to stress effects is lower by 20-40% than that in the Wistar rats. In hypertensive animals the stress (30-min immobilization) has led to a considerable increase in blood tension. Values I50 for ouabain and dependence of activity on the ratio of Na and K ions in the medium are similar in animals of both lines subjected to the stress. There are also no considerable differences in the protein composition of microsomal fraction from kidneys of rats of both lines. The effects which increase permeability of vesicules (channel-forming agent alamecytin, lubrol WX, freezing-thawing) activate Na,K-ATP-ase in the preparation from the kidneys of rats of the both lines. Under maximum activation there is a removal of differences in activity of the enzyme obtained from the tissues of the SSHR and Wistar animals after the stress action. Blood serum of SSHR rats after the stress inhibits purified Na,K-ATP-ase to the greater extent than the Wistar rat blood serum after the same effect. It is supposed that differences in Na,K-ATP-ase activity in microsomal fraction from the kidneys of rats of the above lines are stipulated by the differences in the "latent" ATP-ase activity.     

Activity of erythrocyte membrane Na,K-ATPase in rats with experimental botulism

The effect of type C botulinum toxin on Na, K, Mg-ATPase activities of erythrocyte membranes of white rats was studied in experiments in vivo and in vitro. The activity of Na, K, Mg-ATPase was found to be markedly inhibited in the preclinical period of poisoning, 2 hours after intraperitoneal injection of the toxin. In this case Mg-ATPase activity noticeably increased. In the presence of the development of a grave paralytic syndrome one day after intraperitoneal injection of the toxin, the activity of Na, K-ATPase of the erythrocyte membrane remained decreased as was the case in the preclinical period of poisoning, whereas the activity of Mg-ATPase returned to normal. The experiments in vitro with preincubation of erythrocyte membranes with botulinum toxin in the concentrations corresponding to the mean calculated ones in the experiments in vivo demonstrated inhibition of Na, K-ATPase. The magnitude of Mg-ATPase activity remained virtually unchanged in all the modifications of the experiments with boiled and native botulinum toxin. The in-vivo experiments with intraperitoneal injection of glutathione and unithiol to the pretreated animals attested to normalization of Na, K-ATPase in the preclinical period of poisoning, with this normalization being brought about by unithiol. In the in-vitro experiments with addition of unithiol or glutathione into the incubation medium, each of the donators of sulphhydryl groups prevented Na, K-ATPase inhibition with botulinum toxin.     

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.     

Short-term NO synthase inhibition and the Na+-binding properties of cardiac Na,K-ATPase

It is known that hypertension is accompanied by increased [Na+]i. The functional properties of Na,K-ATPase, which transports the Na+ out and K+ into myocardial cells during the relaxation phase, were investigated in the left ventricle (LV), septum (SV) and the right ventricle (RV) of anesthetized dogs with moderate acute blood pressure elevation elicited by short-term (4-hour) NO synthase inhibition. The NO-insufficiency was induced by administration of an L-arginine analogue, the N(G)-nitro-L-arginine methyl ester (L-NAME). Concerning the function of Na,K-ATPase under the conditions of lowered NO synthesis, we focused our attention to the binding of Na+ to the enzyme molecule. Activation of the enzyme by increasing Na+ concentrations revealed significant changes in both the maximal velocity (Vmax) and the affinity for Na+ (K(Na)) in all investigated heart sections. The Vmax increased by 27% in LV, by 87% in SV and by 58% in RV. The K(Na) value increased by 86% in LV, by 105% in SV and by 93% in RV, indicating an apparent decrease in the sensitivity of the Na+-binding site in the Na,K-ATPase molecule. This apparently decreased pump affinity for Na+ together with the increase of Vmax suggest that, during the short-term inhibition of NO synthesis, the Na,K-ATPase is capable of extruding the excessive Na+ from the myocardial cells more effectively at higher [Na+]i, as compared to the Na,K-ATPase of control animals.     

Increased brain Na, K-ATPase activity of rats under stress

The activities of Na, K- and Mg-dependent ATPases were measured in crude synaptosomal fractions isolated from the rat brain gray matter. Prolonged (6 h) exposure to emotional painful stress stimulated Na, K-ATPase activity by 40% without affecting that of Mg-ATPase. Preliminary injection of the free radical scavenger ionol presented Na, K-ATPase activation, thus suggesting the involvement of lipid peroxidation initiated in brain tissues under stress in acceleration of NA-pump function. However, model studies with lipid peroxidation induced in vitro by an ascorbate-dependent system in a membranous suspension demonstrated an opposite effect, i. e. fast inhibition of Na, K-ATPase. Possible reasons for the different effects of lipid peroxidation in vivo under stress and on Na, K-ATPase activity in vitro are discussed. It is concluded that activation of Na K-ATPase is a mechanism which is responsible for acceleration of reflex conditioning and for the maintenance of the conditioned reflexes in stress-exposed animals.     

Acetylcholine induced reciprocal changes in the Na, K-ATPase and acetylcholinesterase activity of nerve and membrane preparations of Na, K-ATF-azy]

ATPase and cholinesterase activities in the homogenate of the frog nerve and membrane Na,K-ATPase preparation of the bovine brain were investigated. Preliminary treatment of the nerve and the preparation by acetylcholine solution (10(-6)--10(-7) M) enhanced their Na,K-ATPase activity and reduced their cholinesterase activity. Possible mechanisms of this phenomenon are discussed.     

Comparative characteristics of the properties of erythrocyte Na,K-ATPase in man and the carp Cyprinus carpio

Studies have been made on some of the properties of Na,K-ATPase of a nuclear erythrocytes of man and nuclear erythrocytes of the carp Cyprinus carpio. Human erythrocytes yielded the enzymic activity only after their treatment by a detergent Twin-20; under optimal conditions, it amounted to 2.6 mcmole /ml of erythrocytes per 1 h. In carp erythrocytes, Na,K-ATPase activity could be detected without detergent treatment, being 10-fold higher under optimal conditions than that in human erythrocytes. Repetitive washing of carp erythrocytes from the plasma (for more than 3 times), significantly increased their viscosity and resulted in spontaneous hemolysis. Simultaneously , the activity of Na,K-ATPase increased 2-10 times depending on the composition of incubation media. Under these conditions, the pattern of changes in the enzymatic activity, resulting from shifts in Mg2+ and EDTA concentrations, was altered. The presence of latent Na,K-ATPase activity in the erythrocytes in explained by a low permeability of membranes to ATP and ions. Exogeneous ATP cannot be utilized by the enzyme in the intact human erythrocytes, whereas intact carp erythrocytes exhibit significant permeability to the exogeneous substrate. It is suggested that in vivo this fact may be of physiological importance.     

Effect of albumin on the Na, K-ATPase activity in the rat erythrocytes during immobilization stress

Immobilisation stress (IMS) led to a 42% decrease in erythrocyte Na, K-ATPase activity in rats. Pre-treatment of the "stressed" erythrocytes with human serum albumin (HSA) and 1-day exposition of the HSA prior to the IMS led to stabilising of enzyme activity at the control level. Absence of inhibiting effect of non-protein supernatants of the blood plasma of stressed rats on enzyme activity of normal erythrocytes was shown in presence of the HSA both in vitro and in vivo. The mechanism of the HSA protective effect on the Na,K-ATPase activity of erythrocytes in the IMS, is discussed.     

Effect of acetylcholine on the cerebral microsomal Na, K-ATPase of rats of different ages

Na, K- and Mg-ATPase activity of the cerebral cortex microsomal fraction has been studied and compared in adult and old rats. The activity of Na, K-ATPase decreases while that of Mg-ATPase increases with age. The total ATPase activity remains unchanged. The effect of acetylcholine on ATPase activity has been found to be age-dependent.     

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.     

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.     

Gender difference in functional properties of Na,K-ATPase in the heart of spontaneously hypertensive rats

The aim of present study was the investigation of functional properties of the cardiac Na,K-ATPase in 16 weeks old male and female spontaneously hypertensive rats (SHR). The Na,K-ATPase activity in the presence of increasing concentrations of ATP, as well as Na(+) was lower in SHR of both genders, as compared to respective normotensive controls. Evaluation of kinetic parameters revealed a significant decrease of the maximum velocity (V(max)) in males (30% for ATP-activation, 40% for Na(+)-activation), as well as in females (24% for ATP, 29% for Na(+)), indicating a hypertension-induced diminution of the number of active enzyme molecules in cardiac sarcolemma. Insignificant changes were observed in the value of Michaelis-Menten constant (K(m)) in both cases. The concentration of sodium that gives half-maximal reaction velocity (K(Na)), increased by 38% in male and by 70% in female SHR. This impairment in the affinity of the Na(+)-binding site together with decreased number of active Na,K-ATPase molecules are probably responsible for the deteriorated enzyme-function in hearts of SHR. Direct comparison of SHR of both genders showed, that the enzyme from female hearts seems to be adapted better to hypertension as documented by its increased activity as a consequence of improved ability to bind and utilize ATP, as suggested by 32% decrease of K(m) value in females. In addition, the enzyme from female hearts is able to increase its activity (by 41%) in the presence of increasing sodium concentration even in the range where the enzyme from male hearts is already saturated.     

Changes in the activity of acetylcholinesterase and Na,K-ATPase in human erythrocytes irradiated with X-rays

The response of human erythrocytes to X-rays in the dose range from 40 Gy to 600 Gy was determined on the basis of changes in the activities of AChE and ATPase. The Na,K-ATPase activity increased above the control value at doses below 200 Gy, while at the doses higher than 200 Gy, it decreased, reaching 96% of the control value at a dose of 600 Gy. In the range of doses up to 200 Gy, the AChE activity, expressed as Vmax, did not change. At higher doses, it fell drastically, reaching 33% of the control value at a dose of 600 Gy. Simultaneously, the enzyme substrate affinity decreased at 200 Gy, and then started to increase at lower values of Vmax. The obtained results suggest that under appropriate conditions, low doses of radiation may have the opposite effects to high doses.     

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.     

Phosphorylation of the Na,K-ATPase and the H,K-ATPase

Phosphorylation is a widely used, reversible means of regulating enzymatic activity. Among the important phosphorylation targets are the Na⁺,K⁺- and H⁺,K⁺-ATPases that pump ions against their chemical gradients to uphold ionic concentration differences over the plasma membrane. The two pumps are very homologous, and at least one of the phosphorylation sites is conserved, namely a cAMP activated protein kinase (PKA) site, which is important for regulating pumping activity, either by changing the cellular distribution of the ATPases or by directly altering the kinetic properties as supported by electrophysiological results presented here. We further review the other proposed pump phosphorylations.     

Organophosphate inhibition of avian salt gland Na, K-ATPase activity

1. Adult black ducks (Anas rubripes) were given freshwater or saltwater (1.5% NaCl) for 11 days and half of each group was also given an organophosphate (17 p.p.m. fenthion) in the diet on days 6-11. 2. After 11 days, ducks drinking saltwater had lost more weight and had higher plasma Na and uric acid concentrations and osmolalities than birds drinking freshwater. 3. Saltwater treatment stimulated the salt gland to increased weight and Na, K-ATPase activity. 4. Fenthion generally reduced plasma and brain cholinesterase activity and depressed cholinesterase and Na, K-ATPase activities in salt glands of birds drinking saltwater.     

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.