Na+-ATPase is a different entity from the (Na+ + K+)-ATPase in rat kidney basolateral plasma membranes

In this work, we present evidence in agreement with the hypothesis that there exist two Na+-stimulated ATPase activities in basolateral plasma membranes from rat kidney proximal tubular cells: (1) (Na+ + K+)-ATPase activity, which is inhibited by ouabain and by treating the membranes with trypsin, is insensitive to furosemide and reaches maximal activity upon treatment with SDS at an SDS/protein ratio of 1.6; (2) the Na+-ATPase activity, which is insensitive to ouabain and to trypsin treatment, is inhibited by furosemide and reaches maximal activity upon treatment with SDS at an SDS/protein ratio of 0.4.     

S-adenosyl-L-methionine modulates Na+ + K+-ATPase activity in rat colonic basolateral membranes.

Rat colonic basolateral membranes were incubated with S-adenosyl-L-[methyl-3H]methionine (0.3 mM) at 37 degrees C for 2 h at pH 9.0. This resulted in an increase in the specific activity of Na+ + K+-ATPase by 60%. Kinetic parameter analysis revealed a 2-fold increase in the Vmax. of this enzymatic activity, whereas the Km for ATP was unchanged. The methylation inhibitor S-adenosyl-L-homocysteine (2 mM) significantly reduced these S-adenosyl-L-methionine-stimulated increases in specific activity and the Vmax. of Na+ + K+-ATPase. S-Adenosyl-L-methionine treatment of basolateral membranes was also found to significantly increase the fluidity of these preparations, as assessed by steady-state fluorescence polarization techniques using the fluorophore 1,6-diphenyl-1,3,5-hexatriene; S-adenosyl-L-homocysteine (2 mM) again markedly reduced this S-adenosyl-L-methionine-induced increase in fluidity. While transmethylation reactions involving phospholipids, non-polar lipids and proteins were all found to exist in rat colonic basolateral membranes, based on a number of observations, the results of the present studies suggest that transmethylation of membrane phospholipids, but not membrane non-polar lipids or proteins, influenced the fluidity of basolateral membranes which, in turn, modified Na+ + K+-ATPase activity in these membranes.     

Immunocytochemical localization of Na+, K+-ATPase in the rat kidney

Summary To determine if rat kidney Na⁺, K⁺-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified lamb kidney medulla Na⁺, K⁺-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified lamb kidney Na⁺, K⁺-ATPase and rat kidney microsomes were treated with antiserum (1200), followed by [¹²⁵I]-Protein A and autoradiography, the rat kidney microsomes showed a prominent radioactive band coincident with the -subunit of the purified lamb kidney enzyme and a fainter radioactive band which corresponded to the -subunit. When the Na⁺, K⁺-ATPase antiserum was used for immunoperoxidase staining of paraffin and plastic sections of rat kidney fixed with Bouin's, glutaraldehyde, or paraformaldehyde, intense immunoreactive staining was present in the distal convoluted tubules, subcapsular collecting tubules, thick ascending limb of the loops of Henle, and papillary collecting ducts. Proximal convoluted tubules stained faintly, and the thin portions of the loops of Henle, straight descending portions of proximal tubules, and outer medullary collecting ducts did not stain. Staining was confined to basolateral surfaces of tubular epithelial cells. No staining was obtained with preimmune serum or primary antiserum absorbed with purified lamb kidney Na⁺, K⁺-ATPase, or with osmium tetroxide postfixation. We conclude that the basolateral membranes of the distal convoluted tubules and ascending thick limb of the loops of Henle are the major sites of immunoreactive Na⁺, K⁺-ATPase concentration in the rat kidney.Supported by Grant AM 17047 from NIH and by the Veterans Administration     

Immunocytochemical localization of Na+, K+-ATPase in the rat kidney

To determine if rat kidney Na+, K+-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified lamb kidney medulla Na+, K+-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified lamb kidney Na+, K+-ATPase and rat kidney microsomes were treated with antiserum (1:200), followed by [125I]-Protein A and autoradiography, the rat kidney microsomes showed a prominent radioactive band coincident with the alpha-subunit of the purified lamb kidney enzyme and a fainter radioactive band which corresponded to the beta-subunit. When the Na+, K+-ATPase antiserum was used for immunoperoxidase staining of paraffin and plastic sections of rat kidney fixed with Bouin's, glutaraldehyde, or paraformaldehyde, intense immunoreactive staining was present in the distal convoluted tubules, subcapsular collecting tubules, thick ascending limb of the loops of Henle, and papillary collecting ducts. Proximal convoluted tubules stained faintly, and the thin portions of the loops of Henle, straight descending portions of proximal tubules, and outer medullary collecting ducts did not stain. Staining was confined to basolateral surfaces of tubular epithelial cells. No staining was obtained with preimmune serum or primary antiserum absorbed with purified lamb kidney Na+, K+-ATPase, or with osmium tetroxide postfixation. We conclude that the basolateral membranes of the distal convoluted tubules and ascending thick limb of the loops of Henle are the major sites of immunoreactive Na+, K+-ATPase concentration in the rat kidney.     

Regulation of fetal Na+/K+-ATPase in rat kidney by corticosteroids

The enzymatic differentiation of various tissues is under hormonal control in the perinatal period. Since the regulation of Na+/K+-ATPase has not been explored prenatally, the aim of this study was to determine the corticosteroid sensitivity of sodium pump maturation in the fetal period. Na+/K+-ATPase activity was both measured in kidney homogenates of fetal rats and localized by in-situ histochemistry. Sodium pump activity was first quantifiable on day 18 of fetal development as 1.4 +/- 0.17 mumol Pi/h per mg protein, and was increased 3.4-times by day 22 of gestation. While the Na+/K+-ATPase activity was the most intense in cortical tubules at an earlier fetal age (18th and 19th day), the reaction product in the medullary tubules increased with fetal age, becoming highly intense on the 21st and 22nd day of gestation. From the 18th to 21st day of fetal development homogenate Na+/K+-ATPase activity increased as a function of chronologic age. While mineralocorticoids were without any effect on Na+/K+-ATPase activity, on the last day of the fetal development, the glucocorticoid dexamethasone proved to be successful in stimulating enzyme activity in corticosteroid-suppressed animals. According to our results, glucocorticoid hormones seem to be operating as an endogenous driving force for sodium pump maturation at the end of fetal development.     

Changes in Na+-K+-ATPase activity influence cell attachment to fibronectin

Most vital cellular functions aredependent on a fine-tuned regulation of intracellular ion homeostasis.Here we have demonstrated, using COS cells that were untransfected ortransfected with wild-type rat ouabain-resistantNa⁺-K⁺-ATPase, that partial inhibition ofNa⁺-K⁺-ATPase has a dramatic influence oncell attachment to fibronectin. Ouabain dose-dependently decreasedattachment in untransfected cells and in cells expressing wild-typeNa⁺-K⁺-ATPase, but not in cells expressingouabain-insensitive Na⁺-K⁺-ATPase, whereasinhibition of Na⁺-K⁺-ATPase by loweringextracellular K⁺ concentration decreased attachment in allthree cell types. Thirty percent inhibition ofNa⁺-K⁺-ATPase significantly attenuatedattachment. Na⁺-K⁺-ATPase inhibition caused asustained increase in the intracellular Ca²⁺ concentrationthat obscured Ca²⁺ transients observed in untreated cellsduring attachment. Inhibitors of Ca²⁺ transporterssignificantly decreased attachment, but inhibition ofNa⁺/H⁺ exchanger did not. Ouabain reduced focaladhesion kinase autophosphorylation but had no effect on cell surfaceintegrin expression. These results suggest that the level ofNa⁺-K⁺-ATPase activity strongly influences cellattachment, possibly by an effect on intracellular Ca²⁺.

     

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.     

Na+/K+-ATPase activity in rat erythrocytes after prolonged starvation

Na⁺/K⁺-ATPase (sodium, potassium adenosine triphosphatase, EC 3.6.3.9) activity has been studied in whole erythrocytes from rats over time of total food deprivation for 1, 3, 5, 7–8, and 10–12 days with free access to water. Changes in Na⁺/K⁺-ATPase activity have been found to be phase-specific, i.e., associated with periods of certain metabolism level. After the hunger state and accommodation to endogenous nutrition (phases 0-I), from the 3rd to the 7th–8th day a period of compensated accommodation begins (phase II characterized by a stable euglycemic state, while the level of plateau of protein losses and hormonal stimulation are achieved). The Na⁺/K⁺-ATPase activity changes during the phase II were insignificant (p > 0.05), but potassium loss was observed in erythrocytes and blood plasma from the 5th day of starvation onwards. The phase III (the 10th–12th days) is an onset of the terminal period characterized by the lower activities of Na⁺/K⁺-ATPase (ouabain-sensitive activity) and Mg²⁺-ATPase (ouabain-independent activity) and by reduced sodium plasma levels that previously had remained virtually unchanged. There are considered possible causes of the observed decrease in the Na⁺/K⁺-ATPase activity during prolonged starvation, such as aging of the circulating erythrocyte population (the absence of reticulocytes and young erythrocytes), depletion of cell energy resources (hypoglycemia and glycopenia), effect of endogenous ouabain, and endotoxemia.     

Differential regulation of Na+,K+-ATPase and the Na+-coupled glucose transporter in hypertensive rat kidney

Several Na⁺ transporters are functionally abnormal in the hypertensive rat. Here, we examined the effects of a high-salt load on renal Na⁺,K⁺-ATPase and the sodium-coupled glucose transporter (SGLT1) in Dahl salt-resistant (DR) and salt-sensitive (DS) rats. The protein levels of Na⁺,K⁺-ATPase and SGLT1 in the DS rat were the same as those in the DR rat, and were not affected by the high-salt load. In the DS rat, a high-salt load decreased Na⁺,K⁺-ATPase activity, and this decrease coincided with a decrease in the apparent Mechaelis constant (K_m) for ATP, but not with a change of maximum velocity (V_max). On the contrary, a high-salt load increased SGLT1 activity in the DS rat, which coincided with an increase in the V_max for α-methyl glucopyranoside. The protein level of phosphorylated tyrosine residues in Na⁺,K⁺-ATPase was decreased by the high-salt load in the DS rat. The amount of phosphorylated serine was not affected by the high-salt load in DR rats, and could not be detected in DS rats. On the other hand, the amount of phosphorylated serine residues in SGLT1 was increased by the high-salt load. However, the phosphorylated tyrosine was the same for all samples. Therefore, we concluded that the high-salt load changes the protein kinase levels in DS rats, and that the regulation of Na⁺,K⁺-ATPase and SGLT1 activity occurs via protein phosphorylation.     

Effect of prostaglandin F2 alpha on Na+-K+-ATPase activity in luteal membranes

Slices of rat corpora lutea (CL) incubated with prostaglandin F2 alpha (PGF2 alpha) in Krebs-Hensenleit (K-H) Ringer solution showed a decrease in Na+-K+-ATPase activity after 60 min of incubation. However, PGF2 alpha in vitro did not alter Na+-K+-ATPase activity of isolated luteal membrane fractions. Following PGF2 alpha-induced in vivo luteal regression, reduction of Vmax and elevation of the activation energy above transition temperature of the lipid phase of the membrane occurred without changes in Km, optimum pH and transition temperature. These results suggest that reduction of Na+-K+-ATPase activity after PGF2 alpha treatment may be due to reduction in the number of enzyme molecules or to masking of the active site of the enzyme without any change in enzyme characteristics. In addition, a change in membrane-bound enzyme activity may be an early step in PGF2 alpha-induced luteolysis.     

Regucalcin increases Ca2+-ATPase activity and ATP-dependent calcium uptake in the microsomes of rat kidney cortex

The effect of regucalcin, a Ca²⁺-binding protein, on Ca²⁺ transport system in rat renal cortex microsomes was investigated. The presence of regucalcin (10^-8 to 10^-6 M) in the reaction mixture caused a significant increase in Ca²⁺-ATPase activity and ATP-dependent⁴⁵ Ca²⁺ uptake in the microsomes. Regucalcin (10^-7 M) increased Ca²⁺-ATPase activity independently of increasing concentrations of CaCl_{_2}. The microsomal Ca²⁺-ATPase activity and⁴⁵ Ca²⁺ uptake were markedly decreased by the presence of vanadate (0.1 mM) or N-ethylmaleimide (NEM; 5 mM) in the absence or presence of regucalcin. Dithiothreitol (DTT; 5 mM) markedly elevated Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake in the microsomes. The DTT effects were not further enhanced by regucalcin (10^-7 M). Meanwhile, the microsomal Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake were significantly decreased by the presence of dibutyryl cyclic AMP (DcAMP; 10^-5 and 10^-3 M) or inositol 1,4, 5-trisphosphate (IP3; 10^-7 and 10^-5 M). The effect of regucalcin (10^-7 M) on Ca²⁺ ATPase activity and ⁴⁵Ca²⁺ uptake was weakened in the presence of DcAMP or IP₃. The present results demonstrate that regucalcin has a stimulatory effect on ATP-dependent Ca²⁺ uptake in the microsomes of rat renal cortex due to acting on the thiol groups of Ca²⁺-ATPase.     

Ultrastructural localization of Na+,K+-ATPase in rat and rabbit kidney medulla

Na+,K+-ATPase was localized at the ultrastructural level in rat and rabbit kidney medulla. The cytochemical method for the K+-dependent phosphatase component of the enzyme, using p-nitrophenylphosphate (NPP) as substrate, was employed to demonstrate the distribution of Na+, K+- ATPase in tissue-chopped sections from kidneys perfusion-fixed with 1% paraformaldehyde-0.25% glutaraldehyde. In other outer medulla of rat kidney, ascending thick limbs (MATL) were sites of intense K+-dependent NPPase (K+-NPPase) activity, whereas descending thick limbs and collecting tubules were barely reactive. Although descending thin limbs (DTL) of short loop nephrons were unstained, DTL from long loop nephrons in outer medulla were sites of moderate K+-NPPase activity. In rat inner medulla, DTL and ascending thin limbs (ATL) were unreactive for K+-NPPase. In rabbit medulla, only MATL were sites of significant K+-NPPase activity. The specificity of the cytochemical localization of Na+,K+-ATPase at reactive sites in rat and rabbit kidney medulla was demonstrated by K+-dependence of reaction product deposition, localization of reaction product (precipitated phosphate hydrolyzed from NPP) to the cytoplasmic side of basolateral plasma membranes, insensitivity of the reaction to inhibitors of nonspecific alkaline phosphatase, and, in the glycoside-sensitive rabbit kidney, substantial inhibition of staining by ouabain. The observed pattern of distribution of the sodium transport enzyme in kidney medulla is particularly relevant to current models for urine concentration. The presence of substantial Na+,K+-ATPase in MATL is consistent with the putative role of this segment as the driving force for the countercurrent multiplication system in the outer medulla. The absence of significant activity in inner medullary ATL and DTL, however, implies that interstitial solute accumulation in this region probably occurs by passive processes. The localization of significant Na+,K+-ATPase in outer medullary DTL of long loop nephrons in the rat suggests that solute addition in this segment may occur in part by an active salt secretory mechanism that could ultimately contribute to the generation of inner medullary interstitial hypertonicity and urine concentration.     

Characterization of a beta-actinin-like protein in purified non-muscle cell membranes. Its activity on (Na+ + K+)-ATPase

Treatment by EDTA of purified plasma membranes from MF2S cells (a variant of the murine plasmacytoma MOPC 173) solubilized proteins and increased by a 1000-fold the sensitivity of (Na+ + K+)-ATPase to ouabain. When added back with Ca2+ to treated plasma membranes, these EDTA-solubilized proteins restored the initial sensitivity of the enzyme to its inhibitor. We report the purification of a protein of Mr 32000, isolated from the EDTA-treated membrane supernatant. This protein was purified by a one-step procedure involving a preparative polyacrylamide gel electrophoresis without detergent. In the presence of Ca2+ it was able to restore the original sensitivity to ouabain of (Na+ + K+)-ATPase from EDTA-treated membrane. This protein was shown to be similar to the beta-actinin described by Maruyama by the following criteria: (1) molecular weight and amino acid composition; (2) cross-reactivity with their respective antisera; (3) in the presence of Ca2+ the same quantitative biological activity on ouabain sensitivity of the (Na+ + K+)-ATPase. A possible interaction between beta-actinin, calmodulin and membrane-bound (Na+ + K+)-ATPase is discussed.     

Low-affinity Na+ sites on (Na+ +K+)-ATPase modulate inhibition of Na+-ATPase activity by vanadate

Na+-ATPase activity is extremely sensitive to inhibition by vanadate at low Na+ concentrations where Na+ occupies only high-affinity activation sites. Na+ occupies low-affinity activation sites to reverse inhibition of Na+-ATPase and (Na+, K+)-ATPase activities by vanadate. This effect of Na+ is competitive with respect to both vanadate and Mg2+. The apparent affinity of the enzyme for vanadate is markedly increased by K+. The principal effect of K+ may be to displace Na+ from the low-affinity sites at which it activates Na+-ATPase activity.     

Vanadate inhibition of rat cerebral cortex Na+, K+-ATPase during postnatal development

The Na⁺, K⁺-ATPase activity and its response to vanadate inhibition was investigated in cerebral cortex homogenates of 7-, 12- and 18-day-old rats. The enzyme was inhibited by vanadate in a dose-dependent manner in all these age groups. Furthermore, there was a different sensitivity towards vanadate during postnatal development; the concentration of V⁺⁵ needed for 50% inhibiton of Na⁺, K⁺-ATPase was 1.1×10^–6M, 2×10^–7M and 4.4×10^–7M for 7-, 12- and 18-day-old rats, respectively. It is suggested that the different sensitivity of Na⁺, K⁺-ATPase towards vanadate inhibition during postnatal development might be due to age-dependent changes in the ratio of various cell types.Special Issue dedicated to Dr. O. H. Lowry.