Ouabain-insensitive, Na(+)-stimulated ATPase of several rat tissues: activity during a 24 h period.

Rhythmic daily changes in the Na,K-ATPase activity have been previously described for rat kidney cortex, showing two peaks: at 0900 h and 2100 h, and two valleys: at 1500 h and 0100 h -0300 h. The oscillations in Na,K-ATPase activity are produced by an inhibitor, which binds the enzyme and is present in the rat blood plasma at valley times and absent or at very low concentrations at peak times. Since it has been demonstrated that active Na(+) extrusion from the cells of several tissues depends not only on the Na,K-ATPase but also on the ouabain-insensitive Na-ATPase, we studied the activity of this latter enzyme of several rat tissues, i.e., kidney cortex, small intestine, liver, heart and red blood cells along the day. None of these tissues showed any variation of their Na-ATPase activity along the day. Preincubation of kidney cortex homogenates obtained at 0900 h, with blood plasma drawn at 0900 h and 1500 h, did not modify the Na-ATPase activity. Our results indicate that the Na-ATPase activity does not oscillate along the day. These results are in agreement with the idea that the Na-ATPase could partially compensate the Na(+) transport affected by oscillations of the Na,K-ATPase activity.

Functional and structural demonstration of the presence of Ca-ATPase (PMCA) in both microvillous and basal plasma membranes from syncytiotrophoblast of human term placenta.

It is known that human syncytiotrophoblast (hSCT) actively transports more than 80% of the Ca2+ that goes from maternal to fetal circulation. Transepithelial transport of Ca2+ is carried out through channels, transporters and exchangers located in both microvillous (MVM) and basal (BM) plasma membranes. The plasma membrane Ca-ATPase (PMCA) is the most important mechanism of Ca2+ homeostasis control in the human placenta. In this work, we reexamined the distribution of PMCA in isolated hSCT of term placenta. The PMCA activity was determined in isolated hSCT plasma membranes. A partial characterization of the PMCA activity was performed, including an evaluation of the sensitivity of this enzyme to an in vitro induced lipid peroxidation. Expression of the PMCA in hSCT plasma membranes and tissue sections was investigated using Western blots and immunohistochemistry, respectively. Our study demonstrates, for the first time, a correlation between the activity and structural distribution of PMCA in both MVM and BM of hSCT. It also demonstrates a higher PMCA activity and expression in MVM as compared to BM. Finally, PMCA4 seems to be preferentially distributed in both hSCT plasma membranes, while PMCA1 is shown to be present in the hSCT homogenate. However, the membrane fractions did not show any PMCA1 labeling. Our results must be taken into account in order to propose a new model for the transport of calcium across the hSCT.

Effect of placental hypoxia on the plasma membrane Ca-ATPase (PMCA) activity and the level of lipid peroxidation of syncytiotrophoblast and red blood cell ghosts.

Term placental villous fragments from normotensive pregnant women were incubated under hypoxia in order to induce lipid peroxidation of the placental plasma membranes and, consequently, to increase their release of lipid peroxide products into the incubation medium. The homogenates of the villous fragments were assayed for plasma membrane Ca-ATPase (PMCA) activity and TBARS. The incubation medium, after placental hypoxia, was used to incubate intact red blood cells (RBCs) from normotensive pregnant women. Similarly, intact RBCs from normotensive pregnant women were incubated with deproteinized blood plasma from normotensive pregnant women and women with preeclampsia. In all the cases, red cell ghosts were prepared from the incubated cells and assayed for PMCA and TBARS. The incubation of placental villous fragments under hypoxia led to an increase in the TBARS and a significant reduction in the PMCA activity of their homogenates, as compared to those of villous fragments incubated under normoxia. The exposure of intact RBCs from normotensive pregnant women either to the incubation medium of placental hypoxia or to deproteinized blood plasma from women with preeclampsia, caused a rise of the TBARS and a diminution of PMCA activity of the red cell ghosts. Inside-out vesicles were also prepared from intact RBCs incubated with the medium where the placental hypoxia was carried out. These vesicles were assayed for active calcium transport. Pretreatment of RBCs with the incubation medium of placental hypoxia led to a lower active calcium transport as compared to that of inside-out vesicles from RBCs without any preincubation. These results are in agreement with the idea that the RBCs can be peroxidized when passing through a highly oxidized medium, such as the placental intervillous space from women with preeclampsia. The peroxidized RBCs would contribute then to the propagation of lipid peroxidation from the placenta to nearby and far away tissues.

Active sodium transport in basolateral plasma membrane vesicles from rat kidney proximal tubular cells.

Inside-out vesicles prepared with basolateral plasma membranes from rat kidney proximal tubular cells can accumulate Na+ actively in two ways. Mode 1, which is K+-independent, is ouabain-insensitive and is inhibited by furosemide and mode 2, which is K+-dependent, is inhibited by ouabain and is insensitive to furosemide. The presence of Mg2+ and ATP in the incubation medium is essential for both modes of Na+ uptake to proceed and in both cases, the nucleotide is hydrolyzed during the process. These results are consistent with the idea of the existence, in these membranes, of two Na+ pumps: one, which can work in the absence of K+ (Na+ pump) and another, which needs K+ to work (Na+ + K+ pump).

Effect of Ca2+ on the ouabain-insensitive, active Na+ uptake in inside-out basolateral plasma membrane vesicles from rat kidney proximal tubular cells.

The ouabain-insensitive, active Na+ uptake of inside-out vesicles prepared with basolateral plasma membranes from rat kidney proximal tubular cells can be increased by the presence of micromolar concentrations of Ca2+ in the assay medium. The concomitant ATP hydrolysis associated with the Na+ uptake is also increased by the presence of Ca2+. The Na+ uptake and the concomitant ATP hydrolysis are inhibited by 2 mM furosemide. The effect of Ca2+ is not due to the activity of an Na+-Ca2+ exchanger. The present results are in accordance with our previous model (Proverbio, F., Proverbio, T. and Marín, R. (1982) Biochim. Biophys. Acta 688, 757-763) in which we proposed that Ca2+ seems to modulate the activity of the ouabain-insensitive Na+ pump, in two different ways: (1) in a strong association with the membranes in which Ca2+ (stable component) is essential for the pump activity and (2) in a weak association with the membranes in which Ca2+ (labile component) can be quickly and easily removed by reducing the free Ca2+ concentration of the assay medium to values lower than 1 microM. The Ka for Ca2+ (for the labile component) is around 5 microM. The Ca2+ modulation of the ouabain-insensitive Na+ pump is an indication that Ca2+ could regulate the magnitude of the Na+ extrusion accompanied by Cl- and water present in rat kidney proximal tubular cells.

Inside-out basolateral plasma membrane vesicles from rat kidney proximal tubular cells.

A method for preparation of highly purified basolateral plasma membranes from rat kidney proximal tubular cells is reported. These membranes were assayed for the presence of vesicles as well as for their orientation. (Na+ + K+)-ATPase activity and [3H]ouabain binding studies with membranes treated with or without SDS revealed that the preparation consisted of almost 100% vesicles. The percentage of inside-out vesicles was found to be approx. 70%. This percentage was determined measuring the (Na+ + K+)-ATPase activity in K+-loaded vesicles and in membranes treated with or without trypsin and SDS. These membranes represent a very efficient tool to assay the correlation between active transport and ATPase activities in basolateral plasma membranes from rat kidney proximal tubular cells.

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.

Ouabain-insensitive Na+-stimulated ATPase activity of basolateral plasma membranes from guinea-pig kidney cortex cells. II. Effect of Ca2+.

The ouabain-insensitive, Mg2+-dependent, Na+-stimulated ATPase activity present in fresh basolateral plasma membranes from guinea-pig kidney cortex cells (prepared at pH 7.2) can be increased by the addition of micromolar concentrations of Ca2+ to the assay medium. The Ca2+ involved in this effect seems to be associated with the membranes in two different ways: as a labile component, which can be quickly and easily 'deactivated' by reducing the free Ca2+ concentration of the assay medium to values lower than 1 microM; and as a stable component, which can be 'deactivated' by preincubating the membranes for periods of 3-4 h with 2 mM EDTA or EGTA. Both components are easily activated by micromolar concentrations of Ca2+. The Ka of the system for Na+ is the same, 8 mM, whether only the stable component or both components, stable and labile, are working. In other words, the activating effect of Ca2+ on the Na+-stimulated ATPase is on the Vmax, and not on the Ka of the system for Na+. The activating effect of Ca2+ may be related to some conformational change produced by the interaction of this ion with the membranes, since it can also be obtained by resuspending the membranes at pH 7.8 or by ageing the preparations. Changes in the Ca2+ concentration may modulate the ouabain-insensitive, Na+-stimulated ATPase activity. This modulation could regulate the magnitude of the extrusion of Na+ accompanied by Cl- and water that these cells show, and to which the Na+-ATPase has been associated as being responsible for the energy supply of this mode of Na+ extrusion.

Cell volume-sensitive Na+-ATPase activity in rat kidney cortex cell membranes.

A ouabain-insensitive, K+-independent, sodium pump, has been demonstrated in guinea-pig and rat kidney proximal tubular cells. This pump is thought to be distinct from the ouabain-sensitive Na+/K+ pump. We present evidence here indicating the modulation of the biochemical expression of the Na+ pump, i.e. the ouabain-insensitive Na+-ATPase, by the cell volume in rat kidney proximal tubular cells. Thus, basolateral plasma membranes from swollen cells show a ouabain-insensitive Na+-ATPase activity 10-times higher than that in membranes from control cells. If the swollen cells recover their volume, the activity decreases ten times to control values. The ouabain-sensitive Na+/K+-ATPase is not affected by changes in the cell volume.

Partial characterization of the ouabain-insensitive, Na+-stimulated ATPase activity of kidney basal-lateral plasma membranes.

The present paper characterizes the Na+-stimulated ATPase activity present in basal-lateral plasma membranes from guinea-pig kidney proximal tubular cells. These characteristics are compared with those of the (Na+ + K+)-stimulated ATPase activity, and they are: (A) Na+-ATPase activity: (1) requires Mg2+; (2) may be activated by mu molar quantities of Ca2+; (3) optimal ratio Mg:ATP = 5:1-2 and Ka for Mg:ATP = 3:0.60 mM; (4) Ka for Na+:8 mM; (5) does not require K+; (6) is only stimulated by Na+ and Li+ (in a lower extent); (7) is similarly stimulated by the Na+ salt of different anions; (8) hydrolyzes only ATP; (9) optimal temperature: 47 degrees C; (10) optimal pH: 6.9; (11) is ouabain insensitive; (12) is totally inhibited by 1.5 mM ethacrynic acid, 2 mM furosemide and 0.75 mM triflocin. (B) (Na+ + K+)-ATPase activity: (1) also requires Mg2+; (2) is inhibited by Ca2+; (3) optimal ratio Mg:ATP = 1.25:1 and Ka for Mg:ATP = 0.50: 0.40 mM; (4) Ka for Na+: 14 mM (data not shown); (5) needs K+ together with Na+; (6) K+ may be substituted by: Rb+ greater than NH+4 greater than Cs+; (7) is anion insensitive; (8) hydrolyzes mostly ATP and to a lesser extent GTP, ITP, UTP, ADP, CTP; (9) optimal temperature: 52 degrees C; (10) optimal pH: 7.2; (11) 100% inhibited by 1 mM ouabain; (12) 63% inhibited by 1.5 mM ethacrynic acid, 10% inhibited by 2 mM furosemide and insensitive to 0.75 mM triflocin.

Magnesium sulfate affords protection against oxidative damage during severe preeclampsia.

INTRODUCTION: MgSO4 is the drug of choice to prevent seizures in preeclamptic pregnant women, but its mechanism of action at the molecular level remains an enigma. In previous works, we found that treating preeclamptic women with MgSO4 reduces the lipid peroxidation of their red blood cell membranes to normal levels and leads to a significant reduction in the osmotic fragility of the red blood cells that is increased during preeclampsia. In addition, the increase in lipid peroxidation of red cell membranes induced by the Fenton reaction does not occur when MgSO4 is present. METHODS: The antioxidant protection of MgSO4 was evaluated in UV-C-treated red blood cell ghosts and syncytiotrophoblast plasma membranes by measuring their level of lipid peroxidation. The interaction of MgSO4 with free radicals was assessed for its association with the galvinoxyl radical, the quenching of H2O2-induced chemiluminescence and its effect on sensitized peroxidation of linoleic acid. RESULTS: a) MgSO4 protected red blood cell ghosts and the syncytiotrophoblast plasma membranes of normotensive pregnant women against lipid peroxidation induced by UV-C irradiation. b) MgSO4 does not seem to scavenge the galvinoxyl free radical. c) The quenching of the H2O2-enhanced luminol chemiluminescence is increased by the presence of MgSO4. d) The peroxidation of linoleic acid is significantly blocked by MgSO4. DISCUSSION: MgSO4 may provide protection against oxidative damage of plasma membranes through interactions with alkyl radicals.

Na-pump activity in rat kidney cortex cells and its relationship with the cell volume.

The present work was undertaken to evaluate whether changes in cell water content of rat kidney cortex cells can modulate the transport activity of the ouabain-insensitive Na pump as they modulate the ouabain-insensitive Na+-ATPase. It was found that there is a close relationship between the cell volume and activity of the Na pump, whereas Na,K-pump activity is not affected by variations in cell volume. When the cell water content is low, Na-pump activity (Na+ transport and Na+-ATPase activity) is minimal. Increases in cell water content produce a concomitant increase in Na-pump activity.

Ouabain-insensitive, Na-ATPase activity in pure suspensions of rat kidney proximal tubules.

The present work was undertaken to evaluate the distribution of the Na-ATPase activity in the different components of the rat kidney cortex. Suspensions of glomeruli, proximal and distal tubules were prepared following a collagenase digestion of outermost kidney cortex slices and a separation on a Percoll gradient. It was found that the Na-ATPase activity is higher in the fraction enriched in proximal tubules. The fraction enriched in glomeruli and in distal tubules show also a Na-ATPase activity, but it is lower.

Effect of a high Na+ diet on cell volume and Na(+)-stimulated ATPase activities of rat kidney membranes.

Proximal tubular cells from kidneys of male rats chronically fed with an isotonic NaCl solution, show a volume increase which is dependent on the length of the treatment with NaCl, when compared with control rats. Parallel to the cell volume increase, there is an increase of the ouabain-insensitive Na-ATPase activity, whereas the ouabain-sensitive Na,K-ATPase activity remains unchanged. These results establish a clear relationship between a chronic Na-diet, kidney cell volume and Na-ATPase activity.

Ouabain-sensitive Na+,K(+)-ATPase in the plasma membrane of Leishmania mexicana.

The mechanism responsible for the regulation of intracellular Na+ and K+ concentrations in trypanosomatids is unknown. In higher eukaryotes a ouabain-sensitive Na+,K(+)-ATPase located in the plasma membrane is the main mechanism for the regulation of the intracellular concentrations of Na+ and K+, while in trypanosomatids there are conflicting evidences about the existence of this type of ATPase. By the use of a highly enriched plasma membrane fraction, we showed that an ouabain-sensitive Na+,K(+)-ATPase is present in L. mexicana. The affinity of the enzyme for Na+ and K+ is similar to that reported for the mammalian Na+,K(+)-ATPase, showing also the same kinetic parameters regarding the relative concentration of those cations that give the optimal activity. Vanadate (10 microM) fully inhibits the ATPase activity, suggesting that the enzyme belongs to the P-type family of ionic pumps. The enzyme is sensitive to ouabain and other cardiac glycosides. These cardiac glycosides do not show any appreciable effect on the higher Mg(2+)-ATPase activity present in the same preparation. By the use of [3H]ouabain, we also show in this report that the binding of the inhibitor to the enzyme was specific. Taken together, these results demonstrate that an ouabain-sensitive Na+,K(+)-ATPase is present in the plasma membrane of Leishmania mexicana. Therefore, this Na+,K(+)-ATPase should participate in the intracellular regulation of these cations in Leishmania.

Characterization of the Na+, K+-ATPase activity of basolateral plasma membranes of kidney proximal tubular cells from young and old rats.

Several characteristics of the Na+, K+-ATPase activity of basolateral plasma membranes of kidney proximal tubular cells from young (3 months) and old (24 months) rats were studied. In both cases, the ATPase activity reached optimum values under the following conditions: Mg2+:ATP concentrations (mM) 5:5 (apparent Km 0.5 mM); Na+ concentration 50 mM (apparent Km 18 mM); K+ concentration 20 mM (apparent Km 2.5 mM); pH 7.2; temperature 52 degrees. The values of the apparent energy of activation of the system were similar for young and old rats in the temperature range 20-52 degrees but were 55% higher for the old rats in the temperature range 10-20 degrees.

ATPase activities in kidney basolateral plasma membranes of young and old rats.

The present work studied the turnover rate of (Na+ + K+)-ATPase as well as Mg2+- and Na+- ATPase activities in basolateral plasma membranes from kidney cortex cells of young and old rats. It was found that, as for the homogenates, the turnover rate of the (Na+ + K+)-ATPase was diminished by aging in about 40%. The Mg2+-ATPase activities on the other hand, were similar for the rat kidneys of young and old, in both the homogenates as well as the basolateral plasma membrane fractions.

Effect of ethanol on the Na(+)- and the Na+,K(+)-ATPase activities of basolateral plasma membranes of kidney proximal tubular cells.

The Na(+)- and the Na+,K(+)-ATPase activities of basolateral plasma membranes from rat kidney proximal tubular cells were affected differentially by ethanol. Moreover, at concentrations of ethanol that can be reached in vivo in the blood plasma (50 mM) there was a significant effect on the Na(+)-ATPase activity and practically no effect on the Na+,K(+)-ATPase activity.

High sodium diet and Na+-stimulated ATPase activities in basolateral plasma membranes from rat kidney proximal tubular cells.

The ouabain-insensitive, Na+-stimulated ATPase activity of kidney proximal tubular cells from rats fed a high Na+ diet for 4 months was increased approximately 70% when compared with control (normal diet) rats. The higher ATPase activity was not due to a change in the affinity of the system toward ATP, Mg2+ or Na+. This increase in Na+-ATPase activity may be due to either a higher number of pumps or to a higher turnover rate of the enzyme or both. The ouabain-sensitive, Na+, K+-stimulated ATPase activity, on the other hand, did not change with the high sodium diet. These results can be taken as evidence that the Na+,K+-ATPase and the Na+-ATPase of basolateral plasma membranes of proximal tubular cells from rat kidney are two different entities.

Ouabain-insensitive, Na(+)-stimulated ATPase activity in rabbit cardiac sarcolemma.

The rabbit cardiac sarcolemma shows an ouabain, Na,K-stimulated ATPase activity and an ouabain-insensitive, Na-stimulated ATPase activity. The Na-ATPase has the following characteristics: (i) It is also stimulated by other monovalent cations. (ii) It is inhibited by 2 mM Furosemide and by 2 mM ethacrynic acid. (iii) It reaches maximal values (Vmax) at around 20 mM Na+. (iv) The apparent Km is around 5 mM. Except for the monovalent cation stimulation, the main characteristics of this ATPase are very similar to those of the ouabain-insensitive, Na-stimulated ATPase of mammalian kidneys.