A search for endogenous Na+,K+-ATPase inhibitor in acutely volume-expanded hog plasma led to lysophosphatidylcholine gamma-stearoyl

An Na+,K+-ATPase inhibitor possessing inhibitory activity against the specific binding of ouabain to Na+,K+-ATPase has been purified from the plasma of acutely saline-infused hogs. The purification was performed by a combination of Amberlite XAD-2 adsorption chromatography and five steps of high-pressure liquid chromatography (HPLC). Fast atom bombardment mass and proton nuclear magnetic resonance (NMR) spectrometric studies identified the purified substance as lysophosphatidylcholine gamma-stearoyl (LPCS). The ouabain-displacing activity in plasma, due to this compound, increased with time during saline infusion. The maximal level reached was approximately 12 times higher than that in the pre-infusion plasma sample. Lysophosphatidylcholines (LPCs) containing myristoyl, palmitoyl and oleoyl groups were also inhibitory to Na+,K+-ATPase and ouabain-binding to the enzyme. These LPCs were effective at 100 mumol/l concentrations in attaining 50% inhibition of the enzyme activity and ouabain-binding activity of Na+,K+-ATPase. These results suggest that LPCs containing long chain fatty acids could play an important role as a Na+,K+-ATPase inhibitors under volume-expanded conditions.     

Erythrocyte ghost Na+,K+-ATPase and blood pressure

To examine the relationship between body mass index, blood pressure, and the Na+,K+-adenosine triphosphatase (ATPase) system, we measured the erythrocyte ghost Na+,K+-ATPase and the erythrocyte Na+ concentration in 120 blacks and 127 whites (136 males and 111 females). Blacks showed a 13.9% higher erythrocyte Na+ (7.63 +/- 0.19 vs 6.70 +/- 0.11 [SEM] mEq/L; p = 0.0001) and a 16.1% lower erythrocyte ghost Na+,K+-ATPase activity (140.3 +/- 4.2 vs 167.3 +/- 4.7 nmol inorganic phosphate/mg protein/hr; p = 0.0002) than whites. Male subjects demonstrated a 6.4% higher erythrocyte Na+ (7.35 +/- 0.17 vs 6.91 +/- 0.14 mEq/L; p = 0.043) and an 11.5% lower Na+,K+-ATPase activity (145.7 +/- 3.7 vs 164.7 +/- 5.5 nmol inorganic phosphate/mg protein/hr; p = 0.0015) than female subjects. Significant (p less than 0.001) negative correlations were identified for the systolic, diastolic, and mean blood pressure levels and the erythrocyte ghost Na+,K+-ATPase. These findings were complemented by positive correlations for the blood pressure levels and erythrocyte Na+ concentrations. The body mass index was negatively correlated with erythrocyte ghost Na+,K+-ATPase and it accounted for 6.7%, 5.6%, and 6.1% of the variabilities in the systolic, diastolic, and mean blood pressure levels, respectively. Variabilities of 1.4% systolic, 12.3% diastolic, and 11.1% in mean arterial pressure were attributable to the erythrocyte ghost Na+,K+-ATPase activity. Provided that findings in erythrocytes also reflect the relative status of the vascular smooth muscle cell Na+,K+-ATPase, the predisposition of black, male, and obese persons to hypertension may relate, among other factors, to a lower activity of this enzyme system, which results in an increased vascular tone.     

Erythrocyte Na+,K+-ATPase and nasal potential in pseudohypoaldosteronism

OBJECTIVES: Pseudohypoaldosteronism type 1 (PHA1) is a rare inherited disorder characterized by salt-wasting due to target organ unresponsiveness to mineralocorticoids. PHA1 comprises two clinically and genetically distinct entities; isolated renal and systemic forms. DESIGN: The aim of this study was to investigate red blood cell (RBC) Na+,K+-ATPase activity and nasal potential difference (PD) in two pairs of unrelated dyzygous twins; one with the systemic form of the disease (PHA1-S) and the second with the isolated renal form (PHA1-R). Total and ouabain-sensitive ATPase activities were measured spectrophotometrically by a method that couples ATP hydrolysis with NADH oxidation. Maximal PD and response to amiloride perfusion were evaluated by a standard technique. RESULTS: In the twins with PHA1-S, persistently low activity of RBC Na+,K+-ATPase was found during a 6-year follow-up. Normalization of plasma renin activity (PRA) and plasma aldosterone was observed at the end of the first year of life. Maximal nasal PD was low and there was no significant response to amiloride. In the twins with PHA1-R, RBC Na+,K+-ATPase activity was very low at the time of diagnosis and normalized at the age of 6-8 months. PRA reverted gradually to normal values, whereas aldosterone levels remained high during the 6 years of follow-up. Maximal nasal PD and response to amiloride were normal. CONCLUSIONS: The observed differences in RBC Na+,K+-ATPase activity and nasal PD response to amiloride between the two pairs of twins support the contention of different basic pathogenic mechanisms in the two forms of PHA1.     

Monoclonal antibodies to rat Na+,K+-ATPase block enzymatic activity.

A panel of nine mouse monoclonal antibodies has been prepared against purified preparations of rat kidney Na+,K+-ATPase (EC 3.6.1.3). Selection for specific antibody was based upon the ability of crude hybridoma fluids to inhibit Na+-ATPase activity (using luciferase-linked ATPase assays) and upon antibody binding to both the purified kidney membrane enzyme and to glutaraldehyde-fixed hepatocytes by using standard enzyme-linked immunoadsorbent assays. After immunoaffinity purification, two of the antibodies (both of the IgG1 subclass) fully inhibit kidney and liver membrane Na+,K+-ATPase activity with Ki (apparent) values of 30 nM ("9-A5") and 600 nM ("9-B1"). Immunoblots demonstrate directly that three different 125I-labeled antibodies (6-4, 9-A5, and 9-B1) bind predominantly to a 94,000 Mr protein that comigrates in NaDodSO4/polyacrylamide gels with the fluorescein isothiocyanate-labeled alpha subunit of the Na+,K+-ATPase. Indirect immunofluorescence studies with these antibodies on paraformaldehyde-fixed liver slices reveal staining patterns congruent with bile canalicular membrane domains. These results together suggest that the antibodies exert inhibitory effects by recognizing alpha subunits of both liver and kidney Na+ pumps in their native conformations.     

Higher plasma Na+,K+-ATPase inhibitory activity in essential hypertensive patients

The ability of plasma to inhibit 86 rubidium uptake in rat aorta and to displace [3H]-ouabain from hog brain Na+,K+-ATPase was used as a measure of plasma Na+,K+-ATPase inhibitory activity in seven normotensive and eight hypertensive subjects. Rat aortae rings were incubated in oxygenated plasma containing 86 rubidium (2 microCi/mL) for 30 mins at 37 degrees C and uptake measured and expressed as mumol/kg wet weight/min. Plasma was extracted with a mixture of chloroform and methanol (2:1) and the extract separated by silicic acid column followed by thin layer chromatography and fractions assayed for ouabain displacement using digoxin as a standard. Total ouabain displacement was calculated as the sum of all fractions. There was a strong correlation between the two methods for total plasma Na+,K+-ATPase inhibitory activity (r = 0.761, P less than 0.01). There was a significant positive correlation between plasma Na+,K+-ATPase inhibitory activity and blood pressure in all subjects. Na+,K+-ATPase inhibitory activity was significantly higher in plasma of hypertensives by both methods (P less than 0.001). The increased Na+,K+-ATPase inhibitory activity in plasma from hypertensives was due to the nonesterified fatty acid, long chain acylcarnitine and diphosphatidylglycerol fractions.     

Multiple genes encode the human Na+,K+-ATPase catalytic subunit.

A human genomic library was constructed and screened with hybridization probes derived from sheep and rat cDNAs encoding the alpha and alpha(+) isoforms, respectively, of the Na+,K+-ATPase catalytic subunit. Genomic sequences spanning 150 kilobases were isolated. Four genes, designated alpha A, alpha B, alpha C, and alpha D, each 20-25 kilobases in length, were identified by restriction mapping, Southern blot hybridization analysis, and limited DNA sequencing. We present evidence that two of these genes, alpha A and alpha B, encode the alpha and alpha(+) isoforms, respectively. The other genes, alpha C and alpha D, one of which is physically linked to the alpha(+) gene, exhibit nucleotide and amino acid homology to Na+,K+-ATPase catalytic subunit cDNA sequences but do not correspond to any previously identified isoforms.     

Cytochemically assayable Na+,K+-ATPase inhibition by Milan hypertensive rat plasma

The ability of plasma from 3- and 9-week-old Milan hypertensive rats and their normotensive controls to inhibit Na+,K+-adenosine triphosphatase (ATPase) was studied using cytochemical bioassay techniques in fresh tissue. With a validated cytochemical bioassay that measures the capacity of biological samples to stimulate glucose-6-phosphate dehydrogenase activity in guinea pig proximal tubules as an indication of their capacity to inhibit Na+,K+-ATPase, the mean glucose-6-phosphate dehydrogenase-stimulating ability of the plasma of the 9-week-old Milan hypertensive rats and their normotensive controls was 586.0 +/- 88 and 23.4 +/- 8.3 U/ml (n = 7; p less than 0.001), while that of the 3-week-old Milan hypertensive rats (before the main rise in arterial pressure) and their normotensive controls was 99.9 +/- 27.4 and 7.8 +/- 1.8 U/ml (n = 7; p less than 0.001). With the use of a semiquantitative cytochemical assay that measures Na+,K+-ATPase activity directly, plasma from the adult hypertensive rats had a much greater capacity to inhibit Na+,K+-ATPase than the plasma of the control rats. The significantly raised levels found in the young hypertensive rats before the main rise in arterial pressure are consistent with the hypothesis that the rise in the ability of plasma to inhibit Na+,K+-ATPase is due to an inherited renal difficulty in excreting sodium.     

A possible role for Na+,K+-ATPase in regulating ATP-dependent endosome acidification.

Endosomes maintain a slightly acidic internal pH, which is directly responsible for their ability to ensure proper sorting of incoming receptors and ligands during endocytosis. At least two distinct subpopulations of endosomes can be distinguished, designated "early" and "late" on the basis of their kinetics of labeling with endocytic tracers. The subpopulations differ not only in their functions (rapid receptor recycling and transport to lysosomes, respectively) but also in their capacities for acidification in intact cells and in vitro. To investigate the possible basis for pH regulation in endosomes, we have studied the transport properties and ion permeabilities of early and late endosomes isolated from Chinese hamster ovary cells. Using endosomes selectively labeled with pH-sensitive endocytic tracers, we found that ATP-dependent acidification is electrogenic, being accompanied by the generation of an interior-positive membrane potential which opposes further acidification. While membrane potential and, consequently, acidification was controlled by the influx of permeant anions and efflux of protons and alkali cations, acidification was further modulated in Na+ and K+-containing buffers by the ouabain- and vanadate-sensitive Na+,K+-ATPase, which appears to be a functional component of the endosomal membrane. The data suggest that electrogenic Na+ transport due to Na+,K+-ATPase activity contributes to the interior-positive membrane potential, thereby reducing ATP-dependent H+ transport. Importantly, inhibition of acidification by Na+,K+-ATPase activity was found only in early endosomes, consistent with their limited acidification capacity relative to late endosomes and lysosomes.     

Relation of Na+, K(+)-ATPase to delayed motor nerve conduction velocity: effect of aldose reductase inhibitor, ADN-138, on Na+, K(+)-ATPase activity

The role of sorbitol, myo-inositol, and Na+, K(+)-adenosine triphosphatase (ATPase) activity on motor nerve conduction velocity (MNCV) in streptozotocin (STZ)-diabetic rats was studied. Reduction of MNCV and Na+, K(+)-ATPase in caudal nerves appeared after 3 weeks of diabetes, and at this time treatment with aldose reductase inhibitor (ARI), ADN-138 and 1% myo-inositol supplement was begun. One percent myo-inositol supplement for 3 weeks resulted in a significant increase in myo-inositol levels in diabetic nerves, but left MNCV and sorbitol levels unchanged. In contrast, treatment with ADN-138 for 3 weeks reduced sorbitol levels in diabetic nerves and resulted in significant increases in MNCV and Na+, K(+)-ATPase in the nerves. Since ADN-138 did not restore myo-inositol levels, the increase in Na+, K(+)-ATPase levels by ADN-138 treatment was independent of myo-inositol levels. Also, nerve Na+ levels in ADN-138-treated rats were reduced and the ratio of K+ to Na+ was raised, while 1% myo-inositol supplement did not affect them. These results suggest that treatment with ADN-138 elevates MNCV through a series of processes: ARI----reduction of sorbitol level----increase in Na+, K(+)-ATPase activity----correction of K+, Na+ imbalance----increase in MNCV.     

替米沙坦对OK细胞Na+-K+ ATP酶活性的影响

目的探讨血管紧张素Ⅱ受体拮抗剂(ARB)替米沙坦和血管紧张素转换酶抑制剂(ACEI)苯那普利对负鼠近端小管上皮细胞(OK细胞)Na+-K+-ATP酶活性的影响.方法培养的OK细胞采用低渗方法制备细胞膜悬液,使用BCA-100蛋白质定量测定试剂盒测定膜蛋白;Na+-K+ ATP酶活性采用孔雀绿比色分析法测定释放的无机磷(Pi)含量,培养液中分别加入血管紧张素Ⅱ(Ang Ⅱ)、Ang Ⅱ+血管紧张素Ⅱ受体拮抗剂替米沙坦(Telmisartan)、Ang Ⅱ+血管紧张素转换酶抑制剂苯那普利(Benazepril),观察它们对OK细胞Na+-K+-ATP酶活性的影响.结果 (1)培养液中加入10-10 mol/L Ang Ⅱ组与对照组相比,OK细胞Na+-K+-ATP酶活性明显上升.(0.0972±0.0080 vs 0.0896±0.0065 μmol·L-1·mg pro-1·h-1, P<0.05)(2) 当培养液中同时加入10{10 mol/L Ang Ⅱ和10-9mol/L Telmisartan,与单加入10-10mol/L AngⅡ组相比,OK细胞Na+-K+-ATP酶活性明显降低.(0.0623±0.0053 vs 0.0972±0.0080 μmol·L-1·mg pro-1·h-1,P<0.05)(3)当培养液中同时加入10-10 mol/L AngⅡ和10-9 mol/L Benazepril,与单加入10-10 mol/L AngⅡ组相比,OK细胞Na+-K+-ATP酶活性无明显变化.(0.1027±0.0166 vs 0.0972±0.0080 μmol·L-1·mg pro-1·h-1, P>0.05).结论血管紧张素Ⅱ作为一种生长因子,不仅能刺激细胞增殖,又能调节近端小管的离子转运,增加Na+-K+-ATP酶活性;替米沙坦能抑制血管紧张素Ⅱ引起的OK细胞Na+-K+-ATP酶活性增加,而苯那普利则无此作用.     

Erythrocyte Na+, K+-ATPase activity in patients with congestive heart failure.

In order to determine if the Na+, K+-ATPase activity in erythrocyte membranes is altered in congestive heart failure, and to examine its clinical significance with respect to other clinical variables, erythrocyte Na+, K+-ATPase activity was measured in 51 patients with left ventricular ejection fractions <40% (coronary artery disease, n=26; dilated cardiomyopathy, n=25) and 24 control patients. Na+, K+-ATPase activity was lower in both coronary artery disease and dilated cardiomyopathy groups than control group even in the absence of digitalis use. There was a significant inverse correlation between Na+, K+-ATPase activity and plasma norepinephrine. The presence of non-sustained ventricular tachycardia was associated with a lower Na+, K+-ATPase activity in both groups with congestive heart failure without digitalis use than those without ventricular tachycardia. Plasma norepinephrine was higher in patients with non-sustained ventricular tachycardia than those without in the coronary artery disease group, but not in the dilated cardiomyopathy group. Na+, K+-ATPase activity may be helpful in predicting electrophysiologic instability in patients with heart failure.     

Ouabain-binding-site photoaffinity probes that label both subunits of Na+,K+-ATPase.

4"'-Diazomalonyldigitoxin and its isomer, 3"'-diazomalonyldigitoxin, have been synthesized at high specific radioactivity and used as photolabels for the Na,K-ATPase (ATP phosphohydrolase, EC 3.6.1.3) purified from Electrophorus electricus. Photoaffinity labeling experiments using both type I and type II complexes of enzyme with both photolabels showed ouabain-protectable labeling of the alpha as well as the beta subunit. These data suggest that, in the purified eel enzyme, the alpha and beta subunits are in intimate contact, at least in the region of the third digitoxose of the "sugar-specific" binding site.     

Age-related oxidative inactivation of Na+, K+-ATPase in rat brain crude synaptosomes

The study was undertaken to examine the status of Na(+), K(+)-ATPase in aged rat brain and to verify if any alteration of this enzyme in aged brain could be related to an oxidative damage. The crude synaptosomes from rat brain were exposed in vitro to an oxidative stress in the form of a combination of Fe(2+) (100 microM) and ascorbate (2 mM) for up to 2 h when increased lipid peroxidation (nearly four-fold), extensive protein carbonyl formation and a marked decrease of Na(+), K(+)-ATPase activity (approximately 88%) were observed. All these changes were prevented by the presence of a chain-breaking anti-oxidant, butylated hydroxytoluene (0.2 mM), in the incubation mixture. When the same crude synaptosomal membranes from the young (4-6 months) and aged (18-22 months) rat brains were analysed, a significant reduction of Na(+), K(+)-ATPase activity (nearly 48%) along with significantly elevated levels of lipid peroxidation products and protein carbonyls could be detected in the aged animals in comparison to young ones. The latter data in combination with the results of in vitro experiments imply that the age-related decline of rat brain Na(+), K(+)-ATPase activity is presumably the consequence of an enhanced oxidative damage in aging brain     

Electroporation-mediated gene transfer of the Na+,K+ -ATPase rescues endotoxin-induced lung injury

RATIONALE: Acute lung injury and acute respiratory distress syndrome are common clinical syndromes resulting largely from the accumulation of and inability to clear pulmonary edema, due to injury to the alveolar epithelium. Gene therapy may represent an important alternative for the treatment and prevention of these diseases by restoring alveolar epithelial function. We have recently developed an electroporation strategy to transfer genes to the lungs of mice, with high efficiency and low inflammation. OBJECTIVES: We asked whether electroporation-mediated transfer of genes encoding subunits of the Na+,K+ -ATPase could protect from LPS-induced lung injury or be used to treat already injured lungs by up-regulating mechanisms of pulmonary edema clearance. METHODS: Plasmids were delivered to the lungs of mice using transthoracic electroporation. Lung injury was induced by intratracheal administration of LPS (4 mg/kg body weight). Biochemical, cellular, and physiologic measurements were taken to assess gene transfer and lung injury. MEASUREMENTS AND MAIN RESULTS: Improvements in wet-to-dry ratios, pulmonary effusions, bronchoalveolar lavage protein levels and cellularity, alveolar fluid clearance, and respiratory mechanics were seen after delivery of plasmids expressing Na+,K+ -ATPase subunits, but not control plasmids, in LPS-injured lungs. Delivery of plasmids expressing Na+,K+ -ATPase subunits both protected from subsequent lung injury and partially reversed existing lung injury by these measures. CONCLUSIONS: These results demonstrate that electroporation can be used effectively in healthy and injured lungs to facilitate gene delivery and expression. To our knowledge, this is the first successful use of gene delivery to treat existing lung injury, and may have future clinical potential.     

Alanine Prevents the Decrease of Na+,K+-ATPase Activity in Experimental Phenylketonuria

Our objective was to investigate the effect of alanine administration on Na⁺,K⁺-ATPase activity in cerebral cortex of rats subjected to chemically-induced phenylketonuria. Wistar rats were treated from the 6th to the 28th day of life with subcutaneous injections of either 2.6 mol alanine or 5.2 mol phenylalanine plus 2.6 mol -methylphenylalanine per g body weight or phenylalanine plus -methylphenylalanine plus alanine in the same doses or equivalent volumes of 0.15 M saline. The animals were killed on the 29th or 60th day of life. Synaptic plasma membrane from cerebral cortex was prepared for Na⁺,K⁺-ATPase activity determination. The results showed that alanine injection prevents the decrease of Na⁺,K⁺-ATPase activity in animals subjected to experimental phenylketonuria. Therefore, in case the same effects are achieved with ingested alanine, it is possible that alanine supplementation may be an important dietary adjuvant for phenylketonuric patients.     

Sensitive assay and kinetic property of urinary inhibitor of Na+, K+-ATPase in sodium-loaded patients with essential hypertension

A sensitive assay method to evaluate the inhibitor of Na+, K+-ATPase in human urine was developed by measuring the inorganic phosphate liberated from ATP in vitro using Na+, K+-ATPase from porcine cerebral cortex. Ouabain inhibited the Na+, K+-ATPase by competing with the potassium ion (an apparent Ki = 2.6 +/- 0.89 X 10(-8) M, n = 8) under the condition of 100 mM NaCl, 4.5 mM MgSO4 and 0.56 mM ATP. The apparent Km value of KCl was 0.4 mM. Factors inhibiting Na+, K+-ATPase were detected in the post-salt fraction on Sephadex G-15 chromatography following the ethanol extraction of lyophilized fresh urine of sodium loaded human subjects (300 meq Na+/day, for 4 days) with essential hypertension. Two active fractions around the 400 daltons following salt were eluted on Sephadex G-15 chromatography. The slower eluted factor competed kinetically with potassium ion, but the inhibitory activity was lost within two days during storage at 4 degrees C. The faster-eluted inhibitor lost its activity within a day. These results indicate that the unstable inhibiting factors of Na+, K+-ATPase exist in human urine and one of these factors inhibits ouabain sensitive Na+, K+-ATPase by binding to the potassium binding site (or very close to it), which exists at the outer surface of the cell membrane of this enzyme.     

Peroxynitrite induced decrease in Na^＋, K^＋-ATPase activity is restored by taurine

AIM: Peroxynitrite (ONOO-) is a powerful oxidant shown to damage membranes. In the present study, the effect of taurine on changes of liver plasma membrane Na+, K+-ATPase induced by ONOO- was investigated. METHODS: Liver plasma membrane was exposed to ONOO-with or without taurine. Na+, K+-ATPase activity and lipid peroxidation as thiobarbituric acid reactive substances (TBARS) levels were measured. RESULTS: Different concentrations of ONOO- (100, 200, 500, and 1 000 μmol/L) were found to decrease liver plasma membrane Na+, K+-ATPase activity significantly. The depletion of enzyme activity was not concentration dependent. Effects of different concentrations of taurine on liver plasma membrane Na+, K+-ATPase activity were also measured. Taurine did not cause any increase in enzyme activity. When plasma membranes were treated with 200 μmol/L ONOO- with different concentrations of taurine, a restoring effect of taurine on enzyme activity was observed. TBARS levels were also measured and taurine was found to decrease the elevated values. CONCLUSION: Taurine is observed to act as an antioxidant of ONOO- to decrease lipid peroxidation and thus affect liver plasma membrane Na+, K+-ATPase by restoring its activity.     

Ovariectomy increases Na+, K+-ATPase, acetylcholinesterase and catalase in rat hippocampus

In the present work we investigated the effect of ovariectomy on Na+, K+-ATPase and acetylcholinesterase (AChE) activities in rat hippocampus. We also studied some parameters of oxidative stress, namely total radical-trapping antioxidant potential (TRAP), thiobarbituric acid-reactive substances (TBA-RS), as well as the antioxidant enzyme activities superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities. Our hypothesis is that ovariectomy might cause alterations in essential enzyme activities necessary to brain normal functioning and that these chances could be caused by oxidative stress. Female adult Wistar rats were divided into three groups: (1) naive (control); (2) sham-operated; and (3) ovariectomized. Thirty days after ovariectomy rats were sacrificed. Results showed that rats subjected to ovariectomy presented a significant increase in Na+, K+-ATPase, AChE and CAT activities, but did not change the oxidative stress parameters studied when compared to sham or naive rats. Since ovariectomy mimics postmenopausal changes, our findings showing alteration in the activities of brain Na+, K+-ATPase, AChE and CAT may be related to problems in postmenopausal women.