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.

Phosphorylated intermediate of the ouabain-insensitive, Na(+)-stimulated ATPase in rat kidney cortex and rainbow trout gills.

Several tissues from different animals, including the rat kidney and the freshwater rainbow trout gills, show an ouabain-insensitive, furosemide-sensitive, Na(+)-stimulated ATPase activity, which has been associated with the active control of the cell volume. This Na-ATPase is Mg(2+) dependent and it is inhibited by vanadate, which can be taken as an indication that this enzyme is a P-type ATPase. The P-type ATPases are known to form a phosphorylated intermediate during their catalytic cycle, where the phosphate binds an aspartyl residue at the enzyme's substrate site. In the current study, we partially characterized the phosphorylated intermediate of the ouabain-insensitive Na-ATPase of rat kidney cortex homogenates and that of gill microsomes from freshwater rainbow trout. While the kidney cortex homogenates, under our assay conditions, show both Na- and Na,K-ATPase activities, the gill microsomes, when assayed at pH 5.2, only show Na-ATPase activity. Both preparations showed a Mg(2+)-dependent, Na(+)-stimulated phosphorylated intermediate, which is enhanced by furosemide. Incubation of the phosphorylated enzyme with 0.6 N hydroxylamine (NH(2)OH) showed that it is acid-stable and sensitive to hydroxylamine, either when phosphorylated in the presence or absence of furosemide. Addition of ADP to the incubation medium drives the reaction cycle of the enzyme backward, diminishing its phosphorylation. Na(+) seems to stimulate both the phosphorylation and the dephosphorylation of the enzyme, at least for the Na-ATPase from gill microsomes. In a E1-E2 reaction cycle of the Na-ATPase, furosemide seems to be blocking the transition step from Na.E1 approximately P to Na.E2-P.

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.

Preeclampsia and Na,K-ATPase activity of red blood cell ghosts from neonatal and maternal blood.

We determined Na,K-ATPase activity and cholesterol/phospholipid ratio of maternal and cord red blood cell ghosts from either normotensive or preeclamptic pregnant women. The Na,K-ATPase activity of the red cell ghosts from neonatal blood is significantly lower (25-32%) as compared with the ATPase activity of the maternal red cell ghosts, regardless of the presence or not of preeclampsia. This diminution in Na,K-ATPase activity of the neonatal red blood cell ghosts could be due to an increase in the cholesterol/phospholipid molar ratio of the membrane. The Na,K-ATPase activity of the red blood cell ghosts from pregnant women was unaffected by preeclampsia; however, fetal red blood cell ghosts from infants of preeclamptic mothers showed a significantly lower ATPase activity (20%) than fetal red blood cell ghosts from infants of normotensive mothers. A low Na,K-ATPase activity in the neonatal red blood cells from mothers with preeclampsia could be an indication of an important modification of the physiological role of this enzyme.

Comparative study of the calcium adenosine triphosphatase of basal membranes of human placental trophoblasts from normotensive and preeclamptic pregnant women.

The Ca-ATPase activity of plasma membranes from human trophoblast is diminished in about 50% in preeclamptic women, as compared to normotensive pregnant women. This diminution is not due to changes in the behavior of the enzyme towards the free calcium concentration, the pH or the temperature of the incubation medium. Neither does it appear to be due to the presence of some condensing factor in the membranes, since the apparent energies of activation for the two tested ranges of temperature (10-20 and 20-37 degrees C) are similar for both normotensive and preeclamptic patients. The possibility of a diminution in the turnover rate of the Ca-ATPase in the plasma membranes from the preeclamptic patients is considered.

Ca-ATPase of human syncytiotrophoblast basal plasma membranes.

In the present work, a Mg(2+)-dependent, Ca(2+)-stimulated ATPase activity was determined and characterized in purified preparations of syncytiotrophoblast basal (fetal facing) plasma membranes, and its characteristics were compared to those of the active Ca(2+)-transport already demonstrated in this tissue. Similar to the active Ca(2+)transport, the Ca-ATPase is Mg(2+)-dependent, is stimulated by calmodulin, and is inhibited by vanadate. The K(m) for Ca(2+)activation is 0.25+/- 0.02microM, a value near to that described for calcium active transport in this tissue. Consequently, the Ca-ATPase activity of human syncytiotrophoblast basal plasma membrane described in this paper could be responsible for the active extrusion of calcium from the syncytiotrophoblast toward the fetal circulation.

Ca-ATPase of human myometrium plasma membranes.

We determined and characterized the Mg2+-dependent, Ca2+-stimulated ATPase (Ca-ATPase) activity in cell plasma membranes from the myometrium of pregnant women, and compared these characteristics to those of the active Ca2+-transport already demonstrated in this tissue. Similarly to the Ca2+-transport system, the Ca2+-ATPase is Mg2+-dependent, stimulated by calmodulin, and inhibited by vanadate. The Km for Ca2+ activation is 0.40 microM, very similar to that found for active calcium transport, i.e. 0.25 microM. Consequently, this Ca2+-ATPase can be responsible for the active calcium transport across the plasma membranes of smooth muscle cells.

Preeclampsia, lipid peroxidation, and calcium adenosine triphosphatase activity of red blood cell ghosts.

OBJECTIVE: We evaluated the effect of lipid peroxidation on the calcium adenosine triphosphatase activity of red blood cell ghosts from normotensive pregnant women and compared it with the adenosine triphosphatase activity and lipid peroxidation in preeclampsia. STUDY DESIGN: Ten nulliparous normotensive and 10 nulliparous preeclamptic pregnant women (38 to 39 weeks of gestation) were used as blood donors. Preeclampsia was diagnosed on the basis of blood pressure (>140/90 mm Hg) and proteinuria (>0.5 gm of urinary protein per day). Red blood cell ghosts were prepared for both groups and used for calcium adenosine triphosphatase activity and lipid peroxidation determinations. Control ghosts (normotensive) were irradiated with ultraviolet light for different lengths of time. RESULTS: Calcium adenosine triphosphatase activity of red blood cell ghosts from normotensive women is sensitive to lipid peroxidation. The lipid peroxidation of red blood cell ghosts from preeclamptic women is higher than that from normotensive women. CONCLUSION: The diminution of the calcium adenosine triphosphatase activity with preeclampsia could be explained by the sensitivity of this adenosine triphosphatase to lipid peroxidation.

Inhibitory effect of ethanol on the Na(+)-ATPase activity of rat kidney proximal tubular cell plasma membranes.

The inhibitory effect of 2% ethanol (400 mM) in the incubation medium on several characteristics of the Na(+)-ATPase of basolateral plasma membranes from rat kidney proximal tubular cells was investigated. Ethanol did not change the Km of the enzyme for Mg2+, ATP or Na+; it did not change either the optimal pH or temperature values of the incubation medium for the enzyme to act and finally, it did not affect the apparent energy of activation of the enzyme. It was also found that 2% ethanol produced stronger inhibition of the ATPase when it is in an activated or stimulated state, than when it is working at its lower basal level. The presented results can be explained by assuming that 2% ethanol in the incubation medium inhibits Na(+)-ATPase activity by affecting the enzyme structure as well as its activating mechanism.

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.

Na,K-ATPase activity in red blood cells from patients with Chediak-Higashi syndrome.

Na,K-ATPase activity of red blood cells from Chediak-Higashi syndrome (CHS) patients and relatives (gene heterozygous) was determined and compared to that of control, healthy, individuals. The enzyme activity was found to be strongly diminished in the CHS patients and slightly lower in their relatives. This reduced activity was due to a lower turnover number of the Na, K-ATPase as well as a decreased number of pumps. The reduced enzyme activity observed in these patients could be the result of an abnormal cell membrane fluidity, and the lowered number of Na, K-pumps could be explained as a consequence of an altered or deficient cell machinery caused by the CHS gene.

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.

Preeclampsia and calcium adenosine triphosphatase activity of red blood cell ghosts.

OBJECTIVE: The current work was undertaken to study the calcium adenosine triphosphatase activity of red blood cell membranes from pregnant women with preeclampsia. STUDY DESIGN: Six normotensive and six preeclamptic pregnant women at 38 to 39 weeks of gestation were studied. The diagnosis of preeclampsia was made on the basis of blood pressure (> 140/90 mm Hg), proteinuria (> 0.5 gm of urinary protein per day), or edema. Hemoglobin-free red blood cell ghosts were prepared from the heparinized blood samples and were used to determine the calcium adenosine triphosphatase activity. RESULTS: It was found that the calcium adenosine triphosphatase activity of preeclamptic women is diminished by about 50% compared with that of normotensive pregnant women. CONCLUSION: A diminution of the calcium adenosine triphosphatase activity of erythrocytes in preeclampsia might be an indication that the in vivo activity of the calcium pump of these cells is diminished, which could, in turn, drive the cells to increase their cytoplasmic free calcium concentration.

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

In the present work it was investigated the effect of 2% ethanol on the Na+ and on the Na+, K(+)-ATPase activities. The differential effect of the alcohol on the two ATPases (approximately 40% inhibition of the Na(+)-ATPase and approximately 10% inhibition of the Na+, K(+)-ATPase), is not due to a higher degree of denaturalization of the enzyme, nor to a faster effect of ethanol on the Na(+)-than on the Na+, K(+)-ATPase. Our results show that ethanol affects the selectivity of the Na+, K(+)-ATPase for Na+ and/or for K+, enhancing the Na+ affinity for the K+ sites, and/or reducing the K+ affinity for its own sites. This effect was not seen for the Na(+)-ATPase, indicating that 2% ethanol inhibits the two ATPases in a totally different way.

[Modulation of Na pump in proximal kidney tubules cells of the rat]. / Modulación de la bomba de Na en células de túbulo proximal de riñón de rata.

The present paper shows evidence indicating that one of the active mechanisms of sodium extrusion shown to be present in basolateral plasma membranes of the proximal tubular cells of mammalian kidney, the Na-pump (which extrudes sodium from the cells accompanied by chloride and water), is modulated in its activity by the cell volume. An increase of the cell volume, produces a concomitant increase of the activity of the Na-pump. On the other hand, the activity of the Na, K-pump, which extrudes sodium from the cells in exchange for potassium, is totally independent of the cell volume. The modulating effect of the cell volume on the activity of the Na-pump, can be either demonstrated under "in vitro" or "in vivo" conditions.

[Lipid peroxidation and ATPase activities]. / Peroxidacion lipidica y actividades ATPásicas.

In the present paper we summarize the theoretical aspects of the process of lipid peroxidation of the plasma membranes from cells of animal tissues. Also, we review the activity of plasma membrane ATPases, i.e., The Na,K-, Na- and Ca-ATPases, and how this modulation could play a role in some diseases.

Partial characterization of the inhibitory effect of lipid peroxidation on the ouabain-insensitive Na-ATPase of rat kidney cortex plasma membranes.

The present work evaluates the effect of lipid peroxidation on the ouabain-insensitive Na-ATPase of basolateral plasma membranes from rat kidney proximal tubular cells as an indirect way to study the lipid dependence of this enzyme. An inverse relationship between lipid peroxidation and Na-ATPase activity was found. This effect was due neither to a change in the optimal Km of the system for Na+ nor for the substrate Mg:ATP, nor the optimal pH value of the medium. The optimal temperature value, however, was shifted toward a higher value. There was also an increase of the apparent energy of activation in the region of temperatures above the transition point (20 degrees C) with increase in lipid peroxidation. Peroxidized membranes incubated with phosphatidylcholine from soybean restored their Na-ATPase activity. On the other hand, the Na-ATPase activity was sensitive to oleoly lysophosphatidylcholine. These results suggest that lipid peroxidation might be affecting the Na-ATPase activity through either an increase of peroxidized phospholipids, which might change the membrane fluidity of the lipid microenvironment of the ATPase molecules, or through a direct effect of lysophospholipids released during the lipid peroxidation.