Na+/H+ metabolic activity in the thrombocytes of spontaneously hypertensive rats

The sodium-proton exchange was determined in platelets of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The platelets were suspended in sodium propionate; the cytoplasmic acidification activated the exchanger and intracellular pH (the increasing) and volume of the platelets (the swelling) were registered. The activity of Na+/H+ exchange was inhibited by isopropyl amiloride. The platelets' volume and the exchange rate constant of SHR were increased on 30-40% as compared with those of WKY.     

A Na pump inhibitor from bovine posterior pituitary: purification, structure determination and its cardiovascular effect in rat.

We examined the hypothesis that hypothalamo-hypophysial tissue contains an endogenous Na pump inhibitor. From bovine posterior pituitary, we purified a substance which inhibits Rb uptake by human erythrocytes. This inhibitory activity was found in the eluate of 10% acetonitrile from a C18 flash column and purified by subsequent three steps of reversed-phase high-performance liquid chromatography (HPLC). Sequence analysis revealed that this substance was identical to joining peptide, one of the major products of proopiomelanocortin (POMC). This peptide had hypertensive and tachycardiac effects in spontaneously hypertensive rats (SHR) after central administration, with weak Na,K-ATPase inhibitory activity (IC50 = 0.5 mM).     

Increase in 12-lipoxygenase activity in platelets of spontaneously hypertensive rats

12-Lipoxygenase activity in platelets of spontaneously hypertensive rats was investigated. Enzyme activity was measured in the absence and the presence of reduced glutathione. In both assay conditions, 12-lipoxygenase activity in platelets of spontaneously hypertensive rats was significantly higher than that in platelets of normotensive rats. Since 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), a 12-lipoxygenase product of arachidonic acid in platelets, has been reported to be a potent chemoattractant for aortic smooth muscle cells, increase in biosynthesis of 12-HETE in platelets of spontaneously hypertensive rats might contribute to the explanation of pathogenesis of vascular disorder commonly found in hypertension patients.     

Inhibition of Na,K-ATPase-suppressive activity of translationally controlled tumor protein by sorting nexin 6

Translationally controlled tumor protein (TCTP) has both extra- and intracellular functions. Our group recently reported that TCTP interacts with Na,K-ATPase and suppresses its activity. Our studies led to the identification of sorting nexin 6 (SNX6) which binds with TCTP as a potential negative regulator of TCTP. SNX6 does not interact directly with any cytoplasmic domains of Na,K-ATPase. However, when overexpressed, it restores the Na,K-ATPase activity suppressed by TCTP. This was confirmed by measurements of purified plasma membrane Na,K-ATPase activity after incubation with recombinant TCTP and SNX6. SNX6 alone has no effect on Na,K-ATPase activity, but activates Na,K-ATPase via inhibition of TCTP. Inhibition of endogenous TCTP by the overexpression of SNX6 or knockdown of TCTP expression by siTCTP increased Na,K-ATPase activity above the basal level. The interaction between SNX6 and TCTP thus appears to regulate Na,K-ATPase activity.     

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.     

Regulatory role of nitric oxide on the cardiac Na, K-ATPase in hypertension

The present study was focused on regulatory role of nitric oxide on functional properties of the cardiac Na, K-ATPase in three various animal models of hypertension: spontaneously hypertensive male rats (SHR) with increased activity of nitric oxide synthase (NOS) by 60 % (Sh1), SHR with decreased activity of NOS by 40 % (Sh2) and rats with hypertension induced by L-NAME (40 mg/kg/day) with depressed activity of NOS by 72 % (LN). Studying the utilization of energy substrate we observed higher Na, K-ATPase activity in the whole concentration range of ATP in Sh1 and decreased activity in Sh2 and LN. Evaluation of kinetic parameters revealed an increase of Vmax value by 37 % in Sh1 and decrease by 30 % in Sh2 and 17 % in LN. The KM value remained unchanged in Sh2 and LN, but was lower by 38 % in Sh1 indicating increased affinity of the ATP binding site, as compared to controls. During the activation with Na+ we observed increased Vmax by 64 % and increased KNa by 106 % in Sh1. In Sh2 we found decreased Vmax by 40 % and increased KNa by 38 %. In LN, the enzyme showed unchanged Vmax with increased KNa by 50 %. The above data indicate a positive role of increased activity of NOS in improved utilization of ATP as well as enhanced binding of Na+ by the cardiac Na, K-ATPase.     

Analytical isolation of plasma membranes of intestinal epithelial cells: identification of Na, K-ATPase rich membranes and the distribution of enzyme activities.

A procedure was developed for the analytical isolation of brush border and basal lateral plasma membranes of intestinal epithelial cells. Brush border fragments were collected by low speed centrifugation, disrupted in hypertonic sorbitol, and subjected to density gradient centrifugation for separation of plasma membranes from nuclei and core material. Sucrase specific activity in the purified brush border plasma membranes was increased fortyfold with respect to the initial homogenate. Basal lateral membrane were harvested from the low speed supernatant and resolved from other subcellular components by equilibrium density gradient centrifugation. Recovery of Na, K-ATPase activity was 94%, and 61% of the recovered activity was present in a single symmetrical peak. The specific activity of Na, K-ATPase was increased twelvefold, and it was purified with respect to sucrase, succinic dehydrogenase, NADPH-cytochrome c reductase, nonspecific esterase, beta-glucuronidase, DNA, and RNA. The observed purification factors are comparable to results reported for other purification procedures, and the yield of Na, K-ATPase is greater by a factor of two than those reported for other procedures which produce no net increase in the Na, K-ATPase activity. Na, K-ATPase rich membranes are shown to originate from the basal lateral plasma membranes by the patterns of labeling that were produced when either isolated cells or everted gut sacs were incubated with the slowly permeating reagent 35S-p-(diazonium)-benzenesulfonic acid. In the former case subsequently purified Na, K-ATPase rich and sucrase rich membranes are labeled to the same extent, while in the latter there is a tenfold excess of label in the sucrase rich membranes. The plasma membrane fractions were in both cases more heavily labeled than intracellular protein. Alkaline phosphatase and calcium-stimulated ATPase were present at comparable levels on the two aspects of the epithelial cell plasma membrane, and 25% of the acid phosphatase activity was present on the basal lateral membrane, while it was absent from the brush border membrane. Less than 6% of the total Na, K-ATPase was present in brush border membranes.     

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.     

Regulation of caveolin-1 membrane trafficking by the Na/K-ATPase

Here, we show that the Na/K-ATPase interacts with caveolin-1 (Cav1) and regulates Cav1 trafficking. Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface. These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase α1 subunit. Moreover, knockdown of the Na/K-ATPase increases basal levels of active Src and stimulates endocytosis of Cav1 from the plasma membrane. Microtubule-dependent long-range directional trafficking in Na/K-ATPase–depleted cells results in perinuclear accumulation of Cav1-positive vesicles. Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi. Thus, the Na/K-ATPase regulates Cav1 endocytic trafficking and stabilizes the Cav1 plasma membrane pool.     

Analytical isolation of plasma membranes of intestinal epithelial cells: Identification of Na,K-ATPase rich membranes and the distribution of enzyme activities

Summary A procedure was developed for the analytical isolation of brush border and basal lateral plasma membranes of intestinal epithelial cells. Brush border fragments were collected by low speed centrifugation, disrupted in hypertonic sorbitol, and subjected to density gradient centrifugation for separation of plasma membranes from nuclei and cole material. Sucrase specific activity in the purified brush border plasma membrane was increased fortyfold with respect to the initial homogenate. Basal lateral membrane were harvested from the low speed supernatant and resolved from other subcellular components by equilibrium density gradient centrifugation. Recovery of Na, K-ATPase activity was 94%, and 61% of the recovered activity was present in a single symmetrical peak. The specific activity of Na, K-ATPase was increased twelvefold, and it was purified with respect to sucrase, succinic dehydrogenase, NADPH-cytochromec reductase, nonspecific esterase, -glucoronidase, DNA, and RNA. The observed purification factors are comparable to results reported for other purification procedures, and the yield of Na, K-ATPase is greater by a factor of two than those reported for other procedures which produce no net increase in the Na, K-ATPase activity.Na, K-ATPase rich membranes are shown to originate from the basal lateral plasma membranes by the patterns of labeling that were produced when either isolated cells or everted gut sacs were incubated with the slowly permeating reagent³⁵S-p-(diazonium)-benzenesulfonic acid. In the former case subsequently purified Na, K-ATPase rich and sucrase rich membranes are labeled to the same extent, while in the latter there is a tenfold excess of label in the sucrase rich membranes. The plasma membrane fractions were in both cases more heavily labeled than intracellular protein.Alkaline phosphatase and calcium-stimulated ATPase were present at comparable levels on the two aspects of the epithelial cell plasma membrane, and 25% of the acid phosphatase activity was present on the basal lateral membrane, while it was absent from the brush border membrane. Less than 6% of the total Na, K-ATPase was present in brush border membranes.     

Coexisting independent sodium-sensitive and sodium-insensitive mechanisms of genetic hypertension in spontaneously hypertensive rats (SHR)

Some essential hypertensive patients and genetic hypertensive rat strains have less than the normal levels of Mg2+ tightly bound to the plasma membranes of their erythrocytes and other cells, i.e., the magnesium binding defect (MgBD). This binding defect appears to cause increased passive permeability of the membrane to Na+ and thereby its increased intracellular concentration, particularly if the Na+-extrusion enzyme systems of the cell are also defective. The Na+-Ca2+ exchange system in the cell membrane exports Na+ and imports Ca2+, increasing the tone of the smooth muscle cell and thus producing hypertension (HTn). This HTn is Na+-sensitive. Evidence supporting this postulate was obtained by determining the intraerythrocyte total concentrations of Na+, Ca2+, K+, and Mg2+ in two strains of spontaneously hypertensive rats (SHR and SS/Jr rats, having the MgBD together with the other requisites of the Na+-sensitive pathway) and their respective controls (WKY and SR/Jr rats, in which this complete pathway is absent). The Na+ and Ca2+ concentrations in the hypertensive rats were increased, and that of K+ was decreased. The concentrations of these cations were very similar in the two hypertensive strains. The level of membrane tightly bound Ca2+ in SHR erythrocyte membranes was significantly higher than those in the other three rat strains, which were not statistically different from each other. These results support previously reported evidence of the existence of a novel HTn-generating mechanism in the SHR rat, in which the intracellular Ca2+ concentration is increased as the result of the enhanced diffusion of this ion into the cell and the accompanying deficiency of the Ca2+ extrusion enzyme systems. This pathway is therefore Na+-insensitive, i.e., Ca2+-sensitive.     

Chronic nicotine modifies skeletal muscle Na,K-ATPase activity through its interaction with the nicotinic acetylcholine receptor and phospholemman

Our previous finding that the muscle nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase interact as a regulatory complex to modulate Na,K-ATPase activity suggested that chronic, circulating nicotine may alter this interaction, with long-term changes in the membrane potential. To test this hypothesis, we chronically exposed rats to nicotine delivered orally for 21-31 days. Chronic nicotine produced a steady membrane depolarization of ～3 mV in the diaphragm muscle, which resulted from a net change in electrogenic transport by the Na,K-ATPase α2 and α1 isoforms. Electrogenic transport by the α2 isoform increased (+1.8 mV) while the activity of the α1 isoform decreased (-4.4 mV). Protein expression of Na,K-ATPase α1 or α2 isoforms and the nAChR did not change; however, the content of α2 subunit in the plasma membrane decreased by 25%, indicating that its stimulated electrogenic transport is due to an increase in specific activity. The physical association between the nAChR, the Na,K-ATPase α1 or α2 subunits, and the regulatory subunit of the Na,K-ATPase, phospholemman (PLM), measured by co-immuno precipitation, was stable and unchanged. Chronic nicotine treatment activated PKCα/β2 and PKCδ and was accompanied by parallel increases in PLM phosphorylation at Ser(63) and Ser(68). Collectively, these results demonstrate that nicotine at chronic doses, acting through the nAChR-Na,K-ATPase complex, is able to modulate Na,K-ATPase activity in an isoform-specific manner and that the regulatory range includes both stimulation and inhibition of enzyme activity. Cholinergic modulation of Na,K-ATPase activity is achieved, in part, through activation of PKC and phosphorylation of PLM.     

Effect of zinc deficiency on enzyme activities in rat and pig erythrocyte membranes

There is need for a reliable index of zinc status in humans. Considering the importance of zinc in membrane function, activities of erythrocyte membrane enzymes have been measured in animals of low and normal zinc status as possible indices. Immature rats and neonatal pigs were fed low and adequate zinc diets; the latter was fed both ad libitum and restricted so as to control for food intake effects. Low rates of gain and plasma zinc concentrations demonstrated that animals fed the low zinc diets were of low zinc status. Erythrocyte membranes were prepared and assayed for Na,K-ATPase, 5'-nucleotidase, and calcium-ATPase activities. Na,K-ATPase activity was not affected by zinc status, but 5'-nucleotidase was significantly lower in deficient animals of both species than in controls, whose food intake was restricted to maintain comparable weight (2.76 vs 3.94 nmol/hr/mg of protein in rats and 60.5 vs 119 in pigs). The basal calcium-ATPase activities were also decreased by low zinc status in both species. Addition of calmodulin in vitro stimulated activity two-fold to four-fold and resulted in the same maximal activities for all treatments. The results show that erythrocyte membrane 5'-nucleotidase activity is an index of zinc status in these species. It is suggested that the decreased membrane calcium-ATPase activity in zinc deficiency is caused by a defect in calmodulin metabolism.     

Effect of complement proteins C5b-9 on blood platelets. Evidence for reversible depolarization of membrane potential

The carbocyanine dye 3,3'-dipropylthiodicarbocyanine iodide has been used to investigate changes in membrane potential (Em) which occur upon binding of complement proteins C5b-9 to the plasma membrane of blood platelets. Gel-filtered platelets exposed to C5b6 and C7 in serum-free medium show no change in Em from that of controls, as indicated by either 3,3,'-dipropylthiodicarbocyanine iodide fluorescence or by the distribution of [14C]tetraphenylphosphonium bromide. Addition of complement proteins C8 and C9 to the C5b67 platelets results in partial depolarization of Em, which spontaneously repolarizes to basal levels within 15-20 min at 37 degrees C. Under these conditions, C5b-9-treated platelets show no increase in lysis over complement-free controls. Isotonic replacement of external sodium by either potassium or choline alters both the rate and extent of membrane depolarization and inhibits the platelets' capacity to repolarize after C5b-9 assembly. Repolarization of Em to basal levels is also completely blocked by addition of ouabain, confirming that this recovery is mediated by the plasma membrane Na+/K+ pump. These results demonstrate that membrane binding of the C5b-9 proteins can induce a transient change in Em when bound to the plasma membrane at a sublytic concentration, providing a mechanism for target cell activation by these potentially cytolytic proteins.     

Effect of thyroid hormone on the distribution and activity of Na, K-ATPase in ventricular myocardium

Employing detergent-free sucrose-density gradient fractionation method we isolated cholesterol-rich lighter membrane fractions containing ∼10% of protein, ∼30% of cholesterol in membranes of ventricular myocardium. Cholesterol-rich lighter membrane fractions contain >70% of Na, K-ATPase and caveolins 1 and 3 and <10% of β-actin. Treatment of hypothyroid rats with T₃ increased the relative abundance of both α1 and β1 Na, K-ATPase subunits in total membranes by 4- to 5-fold (with no change in caveolin-3), and resulted in 1.9-fold increase in enzyme activity. T₃-induced Na, K-ATPase subunits were preferentially distributed to the lighter fractions (#s 4, 5 and 6); and increased abundance of α1 and β1 were 34-70% and 43-68%, respectively. We conclude that the activity of Na, K-ATPase is not uniform in cardiac membranes, and while a significant amount of Na, K-ATPase is present in cardiac cholesterol-rich membrane fractions, the intrinsic activity is significantly less than the enzyme present in relatively cholesterol-poor membranes.     

Action of 3-beta adrenolytics on plasma renin activity measured by radioimmunology in genetically hypertensive rats]

Spontaneously hypertensive rats (SHR) have basal levels of plasma renin activity (PRA) lower than the ones observed in normal Sprague Dawley rats. Three beta blocking agents are orally administered to unanesthetized spontaneously hypertensive and normotensive rats. Propranolol and S 464 reduce PRA in spontaneously hypertensive and normotensive control rats. Pindolol do not lower PRA in normotensive rats but increases levels of PRA in spontaneously hypertensive rats. These results are discussed.     

Aldosterone directly induces Na, K-ATPase alpha 1-subunit mRNA in the renal cortex of rat

The change of blood pressure and the induction of Na, K-ATPase alpha 1-subunit mRNA have been investigated in the renal cortex of aldosterone-treated hypertensive rat. The increase of blood pressure by aldosterone-treatment for 25 days was decreased by the treatment of amiloride or spironolactone. The level of Na, K-ATPase alpha 1-subunit mRNA of the renal cortex in aldosterone-treated rat was increased than that in the control, and its increase was repressed by treatment of spironolactone, but not altered by the treatment of amiloride. This result suggests that the increase of Na, K-ATPase alpha 1-subunit mRNA in the renal cortex of aldosterone-treated hypertensive rat may be related with the direct induction of Na, K-ATPase mRNA without the increase of Na-traffic through Na-channel.     

Identification and biochemical localization of a Na-K-Cl cotransporter in the human placental cell line BeWo

Several transport systems mediating the placental transport of Na, K and Cl have been described, but whether the trophoblast membrane also expresses a Na-K-Cl cotransporter that mediates the coupled movement of all three ions remains unclear. Here we show that BeWo cells, a human trophoblastic cell line, exhibit bumetanide-sensitive (86)Rb (a K surrogate) uptake. Entry via this route accounts for approximately 17% of the (86)Rb influx with the remainder being mediated largely via the Na,K-ATPase. The activity of the bumetanide-sensitive transporter was rapidly elevated (>40%) upon subjecting cells to an acute hyperosmotic challenge signifying a potential role in cell volume regulation. Antibodies to the Na-K-Cl cotransporter identified a single band of approximately 200 kDa on Western blots of fractionated BeWo membranes. This immunoreactivity colocalized with that of the Na,K-ATPase (a basal membrane marker), but was absent from membranes enriched with placental alkaline phosphatase (an apical membrane marker). These findings show for the first time, that a Na-K-Cl cotransporter is expressed in a human placental cell line which may be involved in regulating trophoblast cell volume.     

Kidney mitochondrial respiration in protein- and energy-malnourished rats

Several lines of evidence show a close association between plasma membrane Na,K-ATPase and mitochondrial respiration. Extending the observation in human erythrocyte membrane (6), Na,K-ATPase activity has been shown to be elevated in kidney microsomal preparations from protein- and energy-malnourished rats (10). Kidney mitochondrial respiration was studied in these rats under various conditions of assay. Sucrose was used as a modifier of mitochondrial morphology and volume to study its effect on these mitochondria. Mitochondrial state 3 respiration was increased by 35% in protein-deficient rats (P less than 0.02). Vmax(ADP) of state 3 respiration was increased by about 47% in protein- as well as energy-restricted rats. Mitochondria from protein- and energy-deficient rats were more tightly coupled as compared to those from control group. Km apparent for (ADP) and (Pi) were elevated in protein- and energy-malnourished rats. The magnitude of increase was much more in energy-deficient rats. Morphological differences between the mitochondria from two dietary manipulations were reflected in differences in the responses of state 3 respiration, Km(ADP), state 4 respiration, and respiratory control ratios to changing sucrose concentrations. This increase in mitochondrial respiration parallels the increased Na,K-ATPase activity in these rats. Increased Km (ADP and Pi) for mitochondrial respiration are perhaps in response to increased availability of these metabolites in the cytosol. The sucrose effect, in addition, distinguishes the morphological differences in mitochondrial membrane due to protein or energy deficiencies. In conclusion, these results, to a great extent, support an association between the activity of Na,K-ATPase and mitochondrial respiration. The study of mechanism(s) which could contribute to the enhancement of mitochondrial respiration will be of general importance to the understanding of regulation of mitochondrial oxidative phosphorylation, and is of particular interest to us.     

The age-related characteristics of the hormonal regulation of Na+, K(+)-ATPase activity in the kidney cortex of rats]

The aldosterone binding in isolated distal convoluted and cortical collecting tubules of renal nephrons and the influence of hormonal induction on the Na, K-ATPase activity in membrane fraction of kidney cortex were studied in 10-day- and 2-month-old rats. No reliable difference in aldosterone-specific binding was revealed (0.26 +/- 0.04 and 0.22 +/- 0.03 fmol/mm of tubule length, respectively, at the age of 10 days and 2 months). It was found that Na, K-ATPase activity increased with age from 0.39 +/- 0.06 to 0.72 +/- 0.10 mumol Pi/mg of protein.1 hour.100 microliters. Aldosterone induction caused approximately a 3-fold increase of the enzyme activity in both age groups comparing to the control level. Co-induction of aldosterone and spironolactone resulted in a 50% decrease of Na, K-ATPase activity in adult rats, but did not influence that in young rats. The revealed age-related differences in the mechanism of hormonal Na, K-ATPase regulation are supposed to underlie the absence of physiological reaction of the kidney to aldosterone in early postnatal ontogenesis.