Effects of cholesterol on (Na+,K+)-ATPase ATP hydrolyzing activity in bovine kidney

The (Na+,K+)-ATPase ATP hydrolyzing activity from rabbit kidney medulla basolateral membrane vesicles was studied as a function of the cholesterol content of the basolateral membranes. The cholesterol content of the membranes was modified by incubation with phospholipid vesicles. When the cholesterol content was increased above that found in the native membrane, the (Na+,K+)-ATPase ATP hydrolyzing activity was inhibited. When the cholesterol content was decreased from that found in the native membranes, the (Na+,K+)-ATPase ATP hydrolyzing activity was inhibited. Analogous effects were found with the K+-activated phosphatase activity of the same membrane vesicles. Therefore, at low cholesterol contents, cholesterol was stimulatory, and at high cholesterol contents, cholesterol was inhibitory. The structural specificity of this effect was tested by introducing lanosterol and ergosterol as 50% of the membrane sterol. Ergosterol was the least effective at supporting (Na+,K+)-ATPase ATP hydrolyzing activity, while lanosterol was more effective, but still not as effective as cholesterol.     

Structural-functional status of the cardiomyocyte cell membrane under experimental pathology

The results of research conducted on the cardiomyocytes plasma membranes structural and functional state under the experimental stress and atherosclerosis are displayed in this article. These experimental pathology is determined to be accompanied by some stereotypic quantitative and qualitative modifications occurred in the lipid matrix of the cardiomyocytes plasma membranes--increase of cholesterol content, decrease of phospholipids, accumulation of lisophospholipids and fatty acid. There are demonstrated results that the experimental stress has an atherogenic effect on the plasma membranes of cells by imputting the cholesterol into the membrane even in the intact animals with normal lipid metabolism. All these modifications are also accompanied by the activation of free-radical oxidation. All these changes are capable to lie in the basis of physical and chemical properties mechanism modification of membranes: modification of lipid matrix, change of viscosity, ion-transport properties of cardiomyocytes membranes, oppression of Na+, K(+)-ATPase activity.     

Na+,K+-ATPase activity of the subcellular fractions of the rat brain in aging

The Na+, K+-ATPase activity in the homogenate and in subcellular fractions of different parts of the brain of adult and old rats was studied in comparison. The content of cholesterol in the above fractions was also determined. In old age the Na+, K+-ATPase activity in the homogenate and microsomal fraction of the cerebral hemispheres' cortex decreases, while the Mg2+-ATPase activity in the cortex microsomal fraction increases. The age-related Na+, K+- and Mg2+-ATPase activity in the myelin of the stem in the synaptic plasma membranes of hemispheres and the brain stem remains unchanged whereas in the myelin fraction of hemispheres it grows. The content of cholesterol in the brain of old rats as compared with adult ones increases in the microsomal fraction and remains unchanged in synaptic membranes. The possible role of age-related modification of lipid component of plasma membranes in the above changes of Na+, K+-ATPase activity is discussed.     

A quantitative immunocytochemical study of Na+,K+-ATPase in rat hepatocytes after STZ-induced diabetes and dietary fish oil supplementation.

Because diabetes causes alterations in hepatic membrane fatty acid content, these changes may affect the Na+,K+-ATPase. In this study we documented the effects of streptozotocin (STZ)-induced diabetes on hepatic Na+,K+-ATPase catalytic alpha1-subunit and evaluated whether these changes could be normalized by fish oil supplementation. Two groups of diabetic rats received fish oil or olive oil supplementation. Both groups had a respective control group. We studied the localization of catalytic alpha1-subunit on bile canalicular and basolateral membranes using immunocytochemical methods and confocal laser scanning microscopy, and the Na+, K+-ATPase activity, membrane fluidity, and fatty acid composition on isolated hepatic membranes. A decrease in the alpha1-subunit was observed with diabetes in the bile canalicular membranes, without changes in basolateral membranes. This decrease was partially prevented by dietary fish oil. Diabetes induces significant changes as documented by enzymatic Na+,K+-ATPase activity, membrane fluidity, and fatty acid content, whereas little change in these parameters was observed after a fish oil diet. In conclusion, STZ-induced diabetes appears to modify bile canalicular membrane integrity and dietary fish oil partly prevents the diabetes-induced alterations.     

Influence of sterols and phospholipids on sarcolemmal and sarcoplasmic reticular cation transporters

We have examined the influence of different sterols and phospholipids on the activities of the cardiac sarcolemmal Na+-Ca2+ exchanger and Na+,K+-ATPase and the sarcoplasmic reticular Ca2+-ATPase in reconstituted proteoliposomes. When either the solubilized Na+-Ca2+ exchanger or the Na+,K+-ATPase is reconstituted into phosphatidylcholine (PC):phosphatidylserine (30:50 by weight) vesicles, high cholesterol levels (20% by weight) are required for activity to be expressed. This sterol requirement is highly specific for cholesterol. Several cholesterol analogues with minor structural changes are unable to support Na+-Ca2+ exchange or Na+,K+-ATPase activities. When solubilized sarcolemma is reconstituted into PC:cardiolipin vesicles, however, the requirement for cholesterol is lost. Substantial activity can be obtained in the complete absence of cholesterol or in the presence of several cholesterol analogues. Thus, sterol/protein interactions can be highly dependent on the phospholipid environment. In contrast, the skeletal muscle sarcoplasmic reticular Ca2+-ATPase functions equally well in the presence or absence of cholesterol after reconstitution into either PC:phosphatidylserine or PC:cardiolipin proteoliposomes. Phospholipid requirements of the transporters were also examined. The sarcolemmal Na+-Ca2+ exchanger, Na+,K+-ATPase, and the sarcoplasmic reticular Ca2+-ATPase all function optimally in the presence of phosphatidylserine or cardiolipin after reconstitution. Thus, the sarcolemmal cation transporters have similar sterol and phospholipid requirements and may have structural similarities in their hydrophobic regions. The sarcoplasmic reticular Ca2+ pump evolved in a low cholesterol membrane and has different lipid interactions. These findings may have general applicability to other plasma membrane and endoplasmic reticular enzymes.     

Modifications in the activities of membrane-bound enzymes during in vivo ageing of human and rabbit erythrocytes

Erythrocyte plasma membranes were isolated from a homogeneous population of human or rabbit erythrocytes fractionated into classes representing young, middle-age and old age in vivo. Lipid analyses of human erythrocyte plasma membranes reveal a decrease of the cholesterol to phospholipid molar ratio, followed by a marked decrease in the activities of the membrane-bound enzymes (Na+,K+)-stimulated ATPase, acetylcholinesterase and NAD+ase from young to old age. Such changes were not observed between young and middle-age rabbit erythrocytes. Incubation of rabbit young erythrocytes with phosphatidylcholine vesicles (liposomes) to obtain partial depletion of their membrane cholesterol, indicated that cholesterol depletion causes a statistically significant decrease of the (Na+,K+)-stimulated ATPase and acetylcholinesterase activities, but the NAD+ase activity remained almost unchanged. The biological significance of these data are discussed in terms of the differences and modifications in the interaction of membrane-bound enzymes with membrane lipids during in vivo ageing of erythrocytes.     

Effect of partial hepatectomy on the invertor mechanism of regulation of the Na+,K+-ATPase activity in hepatocytes of rats of various ages]

Age peculiarities of partial hepatectomy effect on the hepatocytes plasma membrane Na+, K(+)-ATPase activity and its insulin-induced stimulation has been studied. It has been shown that partial hepatectomy does not change basal Na+, K(+)-ATPase activity in adult rats. In old partial hepatectomised rats Na+, K(+)-ATPase activity is slightly higher than in control old rats, although this increase is not statistically significant. At the same time, partial hepatectomy acts differently on the insulin-induced Na+, K(+)-ATPase activation in adult and old rats. Insulin activates Na+, K(+)-ATPase at the same extent both in control and partial hepatectomized adult animals. In old hepatectomized rats, but not in old control animals, insulin stimulates Na+, K(+)-ATPase activity as well as. Thus hepatectomy "rejuvenates" old hepatocytes and results in recovery of invertor mechanism of Na+, K(+)-ATPase activation.     

Altered Na+-K+-ATPase, cell Na+ and lipid profiles in canine arterial wall with chronic cigarette smoking

1. We evaluated the influence of cigarette smoking on arterial wall membranes, using Na+-K+-ATPase activity, free cholesterol (FC) and phospholipid (PL) contents as indices of membrane structural and functional integrity. 2. Segments of aorta, carotid and femoral arteries were obtained from normal dogs (controls) and dogs subjected to chronic cigarette smoking for 2 yr (12 cigarettes a day). 3. Na+-K+-ATPase activity was assessed in segments of carotid and femoral arteries using a ouabain-sensitive 86Rb uptake procedure for intact tissues. 4. Free cholesterol and phospholipids were separated, identified, and quantitated from extracts of aortic samples by means of two dimensional thin-layer chromatography. 5. Na+-K+-ATPase activity was reduced in the smoker group in both carotid and femoral arteries. This reduced enzyme activity was accompanied by a rise in cell Na+ levels at both arterial sites. 6. Aortic FC was elevated and the PL profile was altered in the smoker group; as a result, phosphatidylcholine was reduced, whereas lysophosphatidylcholine, phosphatidic acid, and cardiolipin were elevated. 7. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingolipid levels were unchanged. In addition, the FC/PL ratio was increased in the smokers. 8. Taken together, the changes in Na+-K+-ATPase activity, FC/PL ratio and phospholipid profiles observed are consistent with the hypothesis that chronic cigarette smoking causes a reorganization of the phospholipid bilayer in the smooth-muscle cell membrane of the arterial wall.     

The effect of antimembrane testicular antibodies on Na+, K(+)-ATPase activity in the testicles of rats of different ages]

The effect of large and small doses of rabbit antibodies specific to plasma membranes of the rat testicle cells has been studied in the experiments on Wistar rats of three age groups (preadolescent--aged 20 days, puberal--aged 5-7 months and old--aged 24-26 months). It is stated that incubation of plasma membranes by IgG fraction isolated from antimembrane testicular serum (IgG-ATCSm) in a large dose (43 g of protein G per 125 g of protein of membrane fraction) caused statistically reliable inhibition of Na+, K(+)-ATPase activity in the membranes of testicle cells of puberal and old rats. Preadolescent rats exhibit only a tendency to decrease the activity of this enzyme. Incubation of plasma membranes of testicle cells in rats of different age by small doses of IgG-ATCSm (0.43 g of protein G per 125 g of membrane protein) induced a statistically reliable increase of Na+, K(+)-ATPase activity in puberal and old animals and its slight increase in preadolescent rats. The IgG fraction isolated from normal rabbit serum (IgG-NRS) exerted a less pronounced effect on Na+, K(+)-ATPase activity parallel with retention of a tendency to a decrease of activity under the influence of large doses of the drug and to an increase with introduction of small doses.     

Formation of new high density glycogen-microtubule structures is induced by cardiac steroids

Cardiac steroids (CS), an important class of naturally occurring compounds, are synthesized in plants and animals. The only established receptor for CS is the ubiquitous Na(+),K(+)-ATPase, a major plasma membrane transporter. The binding of CS to Na(+),K(+)-ATPase causes the inhibition of Na(+) and K(+) transport and elicits cell-specific activation of several intracellular signaling mechanisms. It is well documented that the interaction of CS with Na(+),K(+)-ATPase is responsible for numerous changes in basic cellular physiological properties, such as electrical plasma membrane potential, cell volume, intracellular [Ca(2+)] and pH, endocytosed membrane traffic, and the transport of other solutes. In the present study we show that CS induces the formation of dark structures adjacent to the nucleus in human NT2 and ACHN cells. These structures, which are not surrounded by membranes, are clusters of glycogen and a distorted microtubule network. Formation of these clusters results from a relocation of glycogen and microtubules in the cells, two processes that are independent of one another. The molecular mechanisms underlying the formation of the clusters are mediated by the Na(+),K(+)-ATPase, ERK1/2 signaling pathway, and an additional unknown factor. Similar glycogen clusters are induced by hypoxia, suggesting that the CS-induced structural change, described in this study, may be part of a new type of cellular stress response.     

Activities of enzymes from canine kidney plasma membranes under effects of polyene antibiotics in vivo and in vitro]

The effects of amphotericin B drug containing sodium deoxycholate (DOC) and those of DOC and nistatin on the activities of Na+, K+-ATPase and 5'-nucleotidase of canine kidney plasma membranes were studied. It was found that the activities of Na+, K+-ATPase and 5'-nucleotidase were markedly inhibited only after intravenous injection of amphotericin B, whereas the other agents tested caused no changes in the enzyme activities. Similar results were obtained in vitro. In the presence of amphotericin B the activity of Na+, K+-ATPase was noticeably inhibited already at the antibiotic concentration of 0,1 mkg per mg of membrane protein. It was found that the injection of amphotericin B, DOC and nistatin did not qualitatively or quantitatively affect the phospholipid composition of the plasma membranes. This is indicative of the lack of correlation between the enzyme activities and changes in the phospholipid composition of the plasma membranes under effects of amphotericin B. The pyrimidine derivative--amygluracyl--markedly removes the inhibiting effect of amphotericin B on the enzyme activity of plasma membranes.     

Effect of calixarene-phosphonic acid on Na+, K+-ATPase activity in plasma membranes of the smooth-muscle cells

Effect of calix[4]arenes C-97, C-99, C-107, functionalized by fragments of alpha-hydroxy-phosphonic, alpha-aminophosphonic- and methylene-bisphosphonic acid on enzymatic activity of oubaine-sensitive Na+, K+-ATPase and oubaine-resistant basal Mg2+- ATPase (specific activity - 10.6 +/- 0.9 and 18.1 +/- 1.2 micromol Pi/h per 1 mg of protein, respectively; n = 7) was studied in experiments made on the suspension of myometrium cell plasma membranes treated by 0.1% solution of digitonin. It was found that calixarene-phosphonic acids in concentration of 100 microM inhibited enzymatic activity of Na+, K+-ATPase by 86-98% and did not practically affect activity of Mg2+-ATPase. These calixarenes were more efficient than oubaine in suppressing enzymatic activity of the sodium pump: in case of the effect of calixerenes the value of the appearence constant of inhibition I0.5 was < 0.1 microM. Calixarene-methylene-bisphosphonic acid (calixarene C-97; I0.5 =33 +/- 4 microM (n = 6) takes the most efficient inhibitory effect on Na+,K+-ATPase activity among the studied calixarenes. A phenomenon of negative cooperation: the Hill coefficient value etaH =0.1-0.5<1 is characteristic of both the inhibiting effect of calixarenes and oubaine. Reguliarities of calixarenes C-97 effect on enzymatic activity of Na+,K+-ATPase were studied. As it appeared its inhibiting effect cannot be caused by trivial factors - potentially possible binding of Mg ions by it and (or) this substance effect on Mg2+ interaction with ATP4- in the incubation medium. Calixerene C-97 does not also decrease the enzyme affinity for Mg ions or ATP. However this calixerenes decreases the affinity of Na+,K+-ATPase for Na ions (the value of activation constant K(Na+)) from 50 +/- 4 (control) to 76 +/- 6 microM in the control and under the effect of calixerene, respectively). A conclusion is made that calixerene C-97 is highly-efficient (with respect to oubaine) and selective (with respect to lack of its effect on basal Mg2+-ATPase) inhibitor of Na+,K+-ATPase of plasma membrane. In the practical aspect it may be used in concentration of 1-10 microM in biochemical membranology when testing and studying kinetic and catalytic properties of the sodium pump in case of such experimental model, as the plasma membrane fraction.     

Localization of Na+,K+-ATPase alpha-subunit to the sinusoidal and lateral but not canalicular membranes of rat hepatocytes

Controversy has recently developed over the surface distribution of Na+,K+-ATPase in hepatic parenchymal cells. We have reexamined this issue using several independent techniques. A monoclonal antibody specific for the endodomain of alpha-subunit was used to examine Na+,K+-ATPase distribution at the light and electron microscope levels. When cryostat sections of rat liver were incubated with the monoclonal antibody, followed by either rhodamine or horseradish peroxidase-conjugated goat anti-mouse secondary, fluorescent staining or horseradish peroxidase reaction product was observed at the basolateral surfaces of hepatocytes from the space of Disse to the tight junctions bordering bile canaliculi. No labeling of the canalicular plasma membrane was detected. In contrast, when hepatocytes were dissociated by collagenase digestion, Na+,K+-ATPase alpha-subunit was localized to the entire plasma membrane. Na+,K+-ATPase was quantitated in isolated rat liver plasma membrane fractions by Western blots using a polyclonal antibody against Na+,K+-ATPase alpha-subunit. Plasma membranes from the basolateral domain of hepatocytes possessed essentially all of the cell's estimated Na+,K+-ATPase catalytic activity and contained a 96-kD alpha-subunit band. Canalicular plasma membrane fractions, defined by their enrichment in alkaline phosphatase, 5' nucleotidase, gamma-glutamyl transferase, and leucine aminopeptidase had no detectable Na+,K+-ATPase activity and no alpha-subunit band could be detected in Western blots of these fractions. We conclude that Na+,K+-ATPase is limited to the sinusoidal and lateral domains of hepatocyte plasma membrane in intact liver. This basolateral distribution is consistent with its topology in other ion-transporting epithelia.     

Structural-functional changes in the synaptic membranes of the cerebral cortex of rats during electric stimulation in vitro]

Electric stimulation (EC) of a suspension of native synaptic membranes of rat brain cortex in the Krebs-Ringer-glucose medium revealed Ca-dependent inhibition of Na+, K+-ATPase and inhibition of transport Ca-activated, Mg-dependent ATPase. The effects observed are not induced by a change in the SH-groups of the membrane proteins and are removed by an addition of total lipids of the brain (membrane protein: lipid = 5:1) or 0.35 mM novocaine. Cyclic 3',5'-AMP in concentrations of 0.1--1.0 mM causes an inhibition (up to 50%) of Na+, K+-ATPase of native synaptic membranes. The Na+, K+-ATPase activity of purified membrane preparations is not changed either by the cyclic nucleotide, or by EC. It is assumed that depolarization of excitable membranes results in structural changes, mediated by the activation of protein kinase, and manifesting themselves as labilization of protein-lipid ratios.     

Comparative study of calixarene effect on Na+, K+ -ATPase activity in plasma membrane of contractile and mobile cells

The paper is devoted to comparative analysis of the influence of a new class of macrocyclic compounds - calixarens on enzymatic activity of two ATP-hydrolase systems localized in the plasmatic membrane of contractile (myocytes of the uterus) and mobile (spermatozoids) cells--Na+, K+ -ATPase and basal Mg2+ -ATPase. The experiments performed on plasmatic membrane suspensions of myometrium and spermatozoids treated with detergent the authors studied the influence of calixarens C-97, C-99, C-107 (identified by the codes), functionalized with fragments of alpha-hydroxyphosphonic, alpha-aminophosphonic and methylenbisphosphonic acids accordingly, on enzymatic activity. The results have shown that C-97 and C-107 calixarenphosphonic acids in 100 microM concentration (97-99%) inhibit Na+, K+ -ATPase activity in both cases almost completely. C-99 (100 microM) calixaren appeared to be less effective with regard of its influence on the enzymatic systems under study: in the case of plasmatic membranes of myometrium suspension the activity of Na+, K+ -ATPase was decreased by 84-88%, and in the case of spermatozoids suspension--just by 15-20% of the control. All the studied calixarens (for both objects) in the maximal concentration (100 microM) practically did not influence the activity of basal Mg2+ -ATP-ase. The calixarens inhibited the enzymatic activity of Na+, K+ -ATPase more effectively than ouabain: in the first case the value of apparent inhibition constant I(0,5) was 25-100 nM, and in the second case--20-100 microM. The inhibition influence of calixarens on Na+, K+ -ATPase activity is characterized by the phenomenon of negative cooperativity (Hill's coefficient nH <1); the influence of ouabain in the case of plasmatic membranes of myometrium suspension is also characterized by negative cooperativity (nH < 1), and in case of spermatozoids suspension--by positive cooperativity (nH >1). The above results show that the studied calixarens are effective inhibitors of Na+, K+ -ATPase plasmatic membrane of contractive and mobile cells (C-97, C-99, C-107 calixarens in case of myocytes of uterus, and C-97, C-107 calixarens in case of spermatozoids).     

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.     

Increased oxidative stress and decreased activities of Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase in the red blood cells of the hibernating black bear

During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P < 0.0001). Similarly, MDA content of erythrocyte membranes was significantly increased during hibernation (P < 0.025). The activity of Ca(2+)/Mg(2+)-ATPase in the erythrocyte membrane was significantly decreased in the hibernating state as compared to the active state. Na(+)/K(+)-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression.     

Membrane Anomalies in Huntington''s Disease Fibroblasts

Plasma membranes, microsomes, and mitochondria were isolated from paired, passage number matched, cultured human fibroblasts. The cells were obtained from skin biopsies of Huntington's disease (HD) subjects and from sex and age matched controls. All fibroblasts were cultured in identical media for three to seven passages. Enrichment of surface marker enzymes such as Na+,K+-ATPase indicated a 10-fold purification of the isolated plasma membrane. The specific activity of Na+,K+-ATPase was 62 and 82% greater in the crude homogenate and isolated plasma membrane, respectively, of HD fibroblasts than in control fibroblasts. The specific activity of plasma membrane Na+,K+-ATPase was correlated with lipid composition and with membrane structure as determined by measurement of the rotational relaxation time and limiting anisotropy of fluorescence probe molecules. Major alterations in the structure of the plasma membranes in HD fibroblasts were not noted. The rotational relaxation time and limiting anisotropy of 1,6-diphenyl-1,3,5-hexatriene and of trans-parinaric acid were not significantly different between the plasma membrane, microsomes, or mitochondria of HD versus those of control fibroblasts. trans-Parinaric acid demonstrated the coexistence of fluid and solid domains in all three subcellular membrane fractions of the normal and HD skin fibroblasts. Lastly, both trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene displayed characteristic breakpoints in Arrhenius plots of absorbance corrected fluorescence in plasma membranes, microsomes, and mitochondria. In all cases, similar breakpoint temperatures, indicative of phase alterations, were noted near 20 degrees and 30 degrees C. These breakpoints were unaltered in HD. In summary, the data do not support the concept of major membrane structural defects in HD.     

Relationship between (Na + K)-ATPase activity, lipid peroxidation and fatty acid profile in erythrocytes of hypertensive and normotensive subjects

Oxidative stress may play a role in the pathogenic mechanism of essential hypertension. Lipid peroxidation can alter the cellular structure of membrane-bound enzymes by changing the membrane phospholipids fatty acids composition. We investigated the relationship between (Na + K)-ATPase activity, lipid peroxidation, and erythrocyte fatty acid composition in essential hypertension. The study included 40 essential hypertensive and 49 healthy normotensive men (ages 35–60 years). Exclusion criteria were obesity, dyslipidemia, diabetes mellitus, smoking, and any current medication. Patients underwent 24-h ambulatory blood pressure monitoring and blood sampling. Lipid peroxidation was measured in the plasma and erythrocytes as 8-isoprostane or malondialdehyde (MDA), respectively. Antioxidant capacity was measured as ferric reducing ability of plasma (FRAP) in the plasma and as reduced/oxidized glutathione (GSH/GSSG ratio) in erythrocytes. (Na + K)-ATPase activity and fatty acids were determined in erythrocyte membranes. Hypertensives had higher levels of plasma 8-isoprostane, erythrocyte MDA, and relative percentage of saturated membrane fatty acids, but lower plasma FRAP levels, erythrocyte GSH/GSSG ratio, (Na + K)-ATPase activity and relative percentage of unsaturated membrane fatty acids, compared with normotensives. Day-time systolic and diastolic blood pressures correlated positively with lipid peroxidation parameters, but negatively with (Na + K)-ATPase activity. These findings suggest that the modulation of (Na + K)-ATPase activity may be associated with changes in the fatty acid composition induced by oxidative stress and provide evidence of a role for this enzyme in the pathophysiology of essential hypertension.     

Association of kidney and parotid Na+, K(+)-ATPase microsomes with actin and analogs of spectrin and ankyrin

Kidney Na+,K(+)-ATPase has been recently shown to bind erythroid ankyrin and to colocalize with ankyrin at the basolateral cell surface of kidney epithelial cells. These observations suggest that Na+,K(+)-ATPase is linked via ankyrin to the spectrin/actin-based membrane cytoskeleton. In the present study we show that Na+,K(+)-ATPase and analogs of spectrin, ankyrin and actin copurify from detergent extracts of pig kidney and parotid gland membranes. Actin, spectrin and ankyrin were extracted from purified Na+,K(+)-ATPase microsomes at virtually identical conditions as their counterparts from the erythrocyte membrane, i.e., 1 mM EDTA (spectrin, actin) and 1 M KCl (ankyrin). Visualization of the stripped proteins by rotary shadowing revealed numerous elongated spectrin-like dimers (100 nm) and tetramers (215 nm), a fraction of which (17%) was associated with globular (10 nm) ankyrin-like particles. Like erythrocyte ankyrin, kidney ankyrin was cleaved into a soluble 72 kDa fragment and a membrane-bound 90 kDa fragment. Consistent with our previous immunocytochemical findings on the pig kidney, Na+,K(+)-ATPase and ankyrin were found to be colocalized at the basolateral plasma membrane of striated ducts and acini of the pig parotid gland. The present findings confirm and extend the recently proposed concept that in polarized epithelial cells Na+,K(+)-ATPase may serve as major attachment site for the spectrin-based membrane cytoskeleton to the basolateral cell domain. Connections of integral membrane proteins to the cytoskeleton may help to place these proteins at specialized domains of the cell surface and to prevent them from endocytosis.