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Preeclampsia (PE) is a pregnancy-specific syndrome with multisystem involvement which leads to fetal, neonatal, and maternal morbidity and mortality. A model of salt-loaded pregnant rats has been previously studied, sharing several pathological characteristics of preeclamptic women. In this study, it was compared the effects of the treatment with an oral magnesium salt, magnesium gluconate (Mg-gluconate), on the osmotic fragility of red blood cells, lipid peroxidation, and PMCA activity of placental homogenates and red blood cell ghosts in salt-loaded pregnant rats. Mg-gluconate has a higher antioxidant capacity than MgSO 4 due to the presence of several hydroxyl groups in the two anions of this salt. Salt-loaded pregnant rats received 1.8% NaCl solution ad libitum as a beverage during the last week of pregnancy. On day 22nd of pregnancy, the rats were euthanized and red blood cells and placenta were obtained. Salt-loaded pregnant rats showed an increased level of lipid peroxidation and a lowered PMCA activity in placental and red blood cell ghosts, as well as an increased osmotic fragility of their red blood cells. The treatment of the salt-loaded pregnant rats with Mg-gluconate avoids the rise in the level of lipid peroxidation and the concomitant lowering of the PMCA activity of their red blood cell membranes, reaching values similar to those from control pregnant rats. Also, this treatment prevents the increase of the osmotic fragility of their red blood cells, keeping values similar to those from control pregnant rats. Mg-gluconate seems to be an important candidate for the replacement of the MgSO 4 treatment of preeclamptic women., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rojas, Abad, Piñero, Medina, Chiarello, Proverbio and Marín.)

Magnesium sulfate (MgSO 4 ) has been used as a protector agent for many diseases related to oxidative stress. The effect of MgSO 4 on the oxidized lipid bilayer has not yet been studied using molecular dynamics calculations. In this work, the effects of oxidation were evaluated by using a POPC membrane model at different concentrations of its aldehyde (-CHO) and hydroperoxide (-OOH) derivatives with and without MgSO 4 . Several quantitative and qualitative properties were evaluated, such as membrane thickness, area per lipid, area compressibility modulus, snapshots after simulation finish, density distributions, time evolutions of oxidized group positions, and radial distributions of oxidized group concerning Mg. Results indicate that in the absence of MgSO 4 the mobility of oxidized groups, particularly -CHO, toward the surface interface is high. At a low oxidation level of the bilayer there is an increase in the compressibility modulus as compared to the unoxidized bilayer. MgSO 4 , at a low oxidation level, tends to lessen the oxidation effects by lowering the dispersion in the distribution of oxidized species toward the membrane surface and the water region. However, MgSO 4 does not change the trends of decreasing membrane thickness and area compressibility modulus and increasing area per lipid upon oxidation. In this regard, MgSO 4 diminishes the electrostatic long-distance attractive interactions between the oxidized groups and the charged headgroups of the interface, owing to the Mg +2 and SO 4 -2 screening effects and an electrostatic stabilization of the headgroups, preventing the pore formation, which is well-known to occur in oxidized membranes., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Preeclampsia is a heterogeneous pregnancy-specific syndrome associated with abnormal trophoblast invasion and endothelial dysfunction. Magnesium (Mg 2+ ) level may be normal or decreased in women with preeclampsia. However, the use of Mg 2+ salts, such as Mg 2+ sulphate, are useful in reducing the pathophysiological consequences of preeclampsia with severe features and eclampsia. Although the mechanism of action of this Mg 2+ salt is not well understood, the available evidence suggests a beneficial effect of Mg 2+ for the mother and foetus. The mechanisms include a lower level of soluble fms-like tyrosine kinase 1 and endoglin, blockage of brain N-methyl-D-aspartate receptors, decreased inflammation mediators, activation of nitric oxide synthases, blockage of arginases, and reduced free radicals level. The maintenance of Mg 2+ homeostasis in pregnancy is crucial for an appropriate pregnancy progression. Oral Mg 2+ salts can be used for this purpose which could result in mitigating the deleterious consequences of this syndrome to the mother, foetus, and newborn., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cd(2+) has been associated with decreased sperm motility in individuals exposed to this element, such as smokers. Among other factors, this lowered motility could be the result of inhibition exerted by Cd(2+) on the activity of the sperm ATPases associated with sperm motility. In this study, we evaluated the plasma membrane Ca(2+)-ATPase and the axonemal dynein-ATPase activities as well as sperm motility, in the presence of different free Cd(2+) concentrations in the assay media. It was found that spermatozoa incubated for 5 h in a medium containing 25 nm free Cd(2+) showed a significant inhibition of progressive motility, reaching values even lower at higher Cd(2+) concentrations. In addition, it was found that the activity of the plasma membrane Ca(2+)-ATPase reached maximal inhibition at 50 nm free Cd(2+), with a K50% inhibition of 18.3 nm free Cd(2+). The dynein-ATPase activity was maximally inhibited by 25 nm free Cd(2+) in the assay medium, with a K50% inhibition of 11.3 nm Cd(2+). Our results indicate that the decreased activity of the sperm ATPases might have a critical importance in the biochemical mechanisms underlying the decreased sperm motility of individuals exposed to Cd(2+)., (© 2015 Blackwell Verlag GmbH.)

Objective: To evaluate the osmotic fragility of red blood cells and the level of lipid peroxidation, the Ca(2+)-ATPase activity of red cell ghosts and placental homogenates from salt-loaded pregnant rats., Methods: Salt-loaded pregnant rats received 1.8% NaCl solution ad libitum as a beverage for seven days, starting on 15th day of pregnancy. Then, it was evaluated the level of lipid peroxidation and the Ca(2+)-ATPase activity of placental homogenates and red blood cell ghosts from control and experimental rats. Furthermore, the osmotic fragility of the red blood cells was evaluated by measuring the lysis of these cells when incubated with a NaCl solution with different osmolarities., Results: It was found that placental homogenates and red blood cell ghosts from experimental pregnant rats showed an increased level of lipid peroxidation and a lowered Ca(2+)-ATPase activity, as compared to control pregnant rats. They also presented an increased osmotic fragility of their red blood cells., Conclusions: Salt-loaded pregnant rats showed, similar to preeclamptic women, an increased level of lipid peroxidation and a lowered Ca(2+)-ATPase activity in placental and red blood cells membranes, as well as an increased osmotic fragility of the red blood cells.

Human sperm has several mechanisms to control its ionic milieu, such as the Na,K-ATPase (NKA), the Ca-ATPase of the plasma membrane (PMCA), the Na(+)/Ca(2) (+)-exchanger (NCX) and the Na(+)/H(+)-exchanger (NHE). On the other hand, the dynein-ATPase is the intracellular motor for sperm motility. In this work, we evaluated NKA, PMCA, NHE, NCX and dynein-ATPase activities in human sperm and investigated their correlation with sperm motility. Sperm motility was measured by Computer Assisted Semen Analysis. It was found that the NKA activity is inhibited by ouabain with two Ki (7.9 × 10(-9) and 9.8 × 10(-5) M), which is consistent with the presence of two isoforms of α subunit of the NKA in the sperm plasma membranes (α1 and α4), being α4 more sensitive to ouabain. The decrease in NKA activity is associated with a reduction in sperm motility. In addition, sperm motility was evaluated in the presence of known inhibitors of NHE, PMCA and NCX, such as amiloride, eosin, and KB-R7943, respectively, as well as in the presence of nigericin after incubation with ouabain. Amiloride, eosin and KB-R7943 significantly reduced sperm motility. Nigericin reversed the effect of ouabain and amiloride on sperm motility. Dynein-ATPase activity was inhibited by acidic pH and micromolar concentrations of Ca(2) (+). We explain our results in terms of inhibition of the dynein-ATPase in the presence of higher cytosolic H(+) and Ca(2) (+), and therefore inhibition of sperm motility., (© 2015 Society for Reproduction and Fertility.)

Objective: The aim of this study is to evaluate the effect of preeclampsia on the level of lipid peroxidation, activity and expression of both plasma membrane Ca(2+)- and Na(+), K(+)-ATPases in syncytiotrophoblast., Methods: The level of lipid peroxidation was estimated by measuring TBARS. ATPase activities were quantified by a colorimetric method measuring the amount of inorganic phosphate during the assay. Expression of Ca(2+)- and Na(+), K(+)-ATPases in syncytiotrophoblast plasma membranes and term placenta tissue sections was investigated using Western blot and immunohistochemistry, respectively., Results: Our results show a higher level of lipid peroxidation of syncytiotrophoblast plasma membranes from preeclamptic, as compared to uncomplicated pregnant women. Preeclampsia also significantly reduced the activity of Ca(2+)- and Na(+), K(+)-ATPases; however, expression of both ATPases was unaffected., Conclusion: Our findings suggest that the reduction of Ca(2+)- and Na(+), K(+)-ATPase activities during preeclampsia could be at least partially due to an increased level of lipid peroxidation of the syncytiotrophoblast plasma membranes.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In the current study the possible relationship between the Ca(2+)/Mg(2+) ratio of human syncytiotrophoblast plasma membranes and their lipid peroxidation and Ca(2+)-ATPase activity was determined. Syncytiotrophoblast plasma membranes of placental explants cultured under hypoxia increased their lipid peroxidation and Ca(2+) content, diminished their Ca(2+)-ATPase activity, and kept their Mg(2+) content unchanged. Membranes preincubated with different concentrations of Ca(2+) increased their Ca(2+) content without changes in their Mg(2+) content. There is a direct relationship between Ca(2+) content and lipid peroxidation of the membranes, as well as an inverse relationship between their Ca(2+) content and Ca(2+)-ATPase activity. On the contrary, preincubation of membranes with different concentrations of Mg(2+) showed a higher Mg(2+) content without changing their lipid peroxidation and Ca(2+)-ATPase activity. Explants cultured under hypoxia in the presence of 4 mM MgSO4 showed similar values of lipid peroxidation and Ca(2+)-ATPase activity of their membranes compared to those of explants cultured under normoxia. Increased Ca(2+) content of the membranes by interacting with negatively charged phospholipids could result in destabilizing effects of the membrane structure, exposing hydrocarbon chains of fatty acids to the action of free radicals. Mg(2+) might exert a stabilizing effect of the membranes, avoiding their exposure to free radicals.

Objective: The aim of this study is to summarize the reported evidence on the possible relationship between preeclampsia, placenta, oxidative stress and plasma membrane Ca-ATPase (PMCA) activity, responsible for fine control of intracellular calcium concentration., Methods: Literature search was conducted in MEDLINE/PubMed and several unpublished results from our laboratory were included., Results: Lipid peroxidation in placental and red blood cell plasma membranes during preeclampsia and a concomitant diminution of their PMCA activity are described., Conclusions: Uteroplacental hypoperfusion raises lipid peroxidation by-products in the blood plasma that could alter structure and functionality of the cell membranes of the endothelium and several tissues.

Purpose: Ultraviolet C (UVC) irradiation of aqueous solutions is known to be a good source of reactive oxygen species (ROS). The aim of this study is to examine the effect of increasing doses of UVC irradiation, in the presence and absence of the antioxidant butylated hydroxytoluene (BHT), on human sperm motility and lipid peroxidation of its membranes., Materials and Methods: Human sperm samples were irradiated with UVC light (254 nm) for different periods of time. A computer-assisted semen analysis of sperm motility was carried out after UV irradiation. The percentage of motile sperm (%MOT), progressive motility, straight line velocity (VSL), curvilinear velocity (VCL) and the percentage of linearity (%LIN) were evaluated. The level of lipid peroxidation of sperm membranes was estimated by measurement of the thiobarbituric acid reactive substances (TBARS)., Results: UVC irradiation of human spermatozoa produced a diminution of the sperm motility (%MOT, progressive motility, VSL, VCL, %LIN), viability and, concomitantly, an increase of the level of lipid peroxidation of the sperm membranes. The observed effects of the UVC irradiation were prevented by addition of the antioxidant BHT, indicating that the effects of UVC on the tested sperm parameters are mediated by an important rise in lipid peroxidation of the sperm membrane., Conclusion: Lipid peroxidation of the human sperm plasma membrane leads to a decrease in the sperm motility (%MOT, progressive motility, VSL, VCL, %LIN) and viability. The protective effect of BHT on the UVC-irradiated sperm cells indicates the effects of ROS on sperm function.

Trypanosoma evansi is the causative agent of equine trypanosomoses. The disease is characterized by fever, anemia, and cachexia. Peroxidative damage of the red blood cells caused by the parasite, may contribute to the pathogenesis of the anemia seen in trypanosomoses. Consequently, we evaluated the hematocrit, the osmotic fragility of the red blood cells, the level of lipid peroxidation and the activity of the Ca-ATPase of red blood cell ghosts from rats experimentally infected with T. evansi. After 72 h inoculation, the hematocrit decreased from 49.5% to 33%; the osmotic fragility of the red blood cells was approximately 40% higher as compared to the healthy animals; and the red blood cell ghosts showed a higher level of lipid peroxidation and a lower Ca-ATPase activity than the red cell ghosts from the healthy animals. In vitro incubations of red blood cells from healthy animals with T. evansi, produced also a significant increase of the osmotic fragility of the red blood cells., (Copyright 2009 Elsevier Inc. All rights reserved.)

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., (2009 Elsevier Masson SAS. All rights reserved.)

Objective: To evaluate the osmotic fragility and level of lipid peroxidation of red blood cells from pregnant women with severe preeclampsia, treated or not with MgSO(4)., Methods: Osmotic fragility and lipid peroxidation of red blood cells was evaluated in 11 normotensive pregnant women and eleven pregnant women with severe preeclampsia., Results: MgSO(4) therapy, either in vivo or in vitro, leads to a reduction of the osmotic fragility and the level of lipid peroxidation of red blood cells from pregnant women with severe preeclampsia., Conclusions: Interaction of MgSO(4) with free radicals, by avoiding an excessive lipid peroxidation of the red blood cell membrane, would protect the membrane structure, avoiding in this way the increase in osmotic fragility.

Previous evidence established that a sequestered form of adenosine triphosphate (ATP pools) resides in the membrane/cytoskeletal complex of red cell porous ghosts. Here, we further characterize the roles these ATP pools can perform in the operation of the membrane's Na(+) and Ca(2+) pumps. The formation of the Na(+)- and Ca(2+)-dependent phosphointermediates of both types of pumps (E(Na)-P and E(Ca)-P) that conventionally can be labeled with trace amounts of [gamma-(3)P]ATP cannot occur when the pools contain unlabeled ATP, presumably because of dilution of the [gamma-(3)P]ATP in the pool. Running the pumps forward with either Na(+) or Ca(2+) removes pool ATP and allows the normal formation of labeled E(Na)-P or E(Ca)-P, indicating that both types of pumps can share the same pools of ATP. We also show that the halftime for loading the pools with bulk ATP is 10-15 minutes. We observed that when unlabeled "caged ATP" is entrapped in the membrane pools, it is inactive until nascent ATP is photoreleased, thereby blocking the labeled formation of E(Na)-P. We also demonstrate that ATP generated by the membrane-bound pyruvate kinase fills the membrane pools. Other results show that pool ATP alone, like bulk ATP, can promote the binding of ouabain to the membrane. In addition, we found that pool ATP alone functions together with bulk Na(+) (without Mg(2+)) to release prebound ouabain. Curiously, ouabain was found to block bulk ATP from entering the pools. Finally, we show, with red cell inside-outside vesicles, that pool ATP alone supports the uptake of (45)Ca by the Ca(2+) pump, analogous to the Na(+) pump uptake of (22)Na in this circumstance. Although the membrane locus of the ATP pools within the membrane/cytoskeletal complex is unknown, it appears that pool ATP functions as the proximate energy source for the Na(+) and Ca(2+) pumps.

Dynein-ATPase is the intracellular motor for sperm motility. In the present work we assayed the dynein-ATPase activity in an axoneme-containing fraction of human sperm, free of plasma membranes, in normozoospermic and asthenozoospermic donors. Axoneme-containing fractions were isolated from semen samples obtained from healthy donors with either normozoospermia or asthenozoospermia, as indicated by a sperm motility lower than 50% (WHO grade a + b). The dynein-ATPase activity was assayed and partially characterized. The dynein-ATPase activity in the axoneme-containing fractions was identified as Mg2+-dependent ATPase activity inhibited by 10 microM vanadate. This inhibition was not seen when the assay was done in the presence of 1 mM norepinephrine. The dynein-ATPase activity is Mg2+-dependent, Li+-sensitive, and insensitive to 2 mM ouabain, 1 microM oligomycin, and 1 microM thapsigargin. The dynein-ATPase activity was significantly lower (p < 0.001) for asthenozoospermic donors as compared to normozoospermic donors. This is a straightforward dynein-ATPase assay that can be used to obtain data of functional interest in clinical or experimental settings.

The increased level of lipid peroxidation of red blood cells during preeclampsia is considered to be responsible for the diminished Ca-ATPase activity in these cells. The level of lipid peroxidation and the Ca-ATPase activity of red blood cells from preeclamptic women, return to their normal values after in vivo and in vitro treatment with MgSO4 for 24 h. In order to evaluate whether or not cell intactness is essential for these changes, we used either intact red blood cells or red cell ghosts from normotensive pregnant women. The intact red blood cells were treated with Fenton's reagent and then incubated with 4 mM MgSO4. The red cells ghosts were irradiated with UV light and afterwards incubated with MgSO4 at 4 degrees C. Lipid peroxidation and Ca-ATPase activity were determined for all the preparations. Both, Fenton's reagent and UV irradiation increased the level of lipid peroxidation and diminished the Ca-ATPase activity of the red cell membranes. Incubation of the cells treated with Fenton's reagent, or the ghosts irradiated with UV, with 4 mM MgSO4, returned Ca-ATPase activity and lipid peroxidation levels to normal values. The presence of MgSO4 blocked the effects in the ghosts of UV irradiation. MgSO4 seems to better protect the red cell membrane against lipid peroxidation than other SO4= and Cl- salts. These results indicate that the changes in the lipid peroxidation of the red cell ghosts and their Ca-ATPase activity are a result of changes to the cell membranes.

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.

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.

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.

Aim: To investigate if interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha) are able to stimulate the level of lipid peroxidation of sperm membranes, either alone or in the presence of leukocytes., Methods: Semen samples from normozoospermic donors were prepared by density gradient. The sperms were exposed to the indicated cytokines, at physiological and infection-inflammation concentrations, in the absence or presence of leukocytes. Lipid peroxidation of the sperm membranes was determined by measuring malondialdehyde (MDA) and 4-hydroxialkenals (HAE) formation., Results: TNF-alpha, IL-8 and IFN-gamma increased the level of sperm membrane lipid peroxidation when tested at physiological concentrations. At infection-inflammation concentrations, only IL-8 was able to produce a higher effect. When assayed in the presence of leucocytes, IL-8 and TNF-alpha showed a higher effect at infection-inflammation concentrations than at physiological concentrations. Finally, IL-8 showed a higher effect in the presence of leukocytes than in their absence at both physiological and infection-inflammation concentrations. TNF-alpha also showed a higher effect when assayed in the presence of leukocytes than in their absence, but only at infection-inflammation concentrations. There was no effect of IL-6 or IL-10 in any of the tested conditions., Conclusion: Several pro-inflammatory cytokines at physiological concentrations increase the level of lipid peroxidation of sperm membranes, which could be important for the sperm fecundation process. However, infection-inflammation concentrations of some cytokines, such as IL-8 and TNF-alpha, either alone or in the presence of leukocytes, could drive the lipid peroxidation of the spermatozoa plasma membrane to levels that can affect the sperm fertility capacity.

Plasma membrane Ca2+-ATPase activity diminishes by about 50% in red blood cells during preeclampsia. We investigated whether the number of Ca2+-ATPase molecules is modified in red cell membranes from preeclamptic pregnant women by measuring the specific phosphorylated intermediate of this enzyme. Also, we isolated the Ca2+-ATPase protein from both normotensive and preeclamptic pregnant women and estimated its molecular weight, and its cross-reactions with specific polyclonal and monoclonal (5F10) antibodies against it. We measured the Ca2+-ATPase activity in a purified state and the effect of known modulators of this ATPase. It was found that the phosphorylated intermediate associated with PMCA is similar for red cell ghosts from normotensive and preeclamptic women, suggesting a similar number of ATPase molecules in these membranes. The molecular weight of the Ca2+-ATPase is around 140 kDa for both normotensive and preeclamptic membranes, and its cross-reactions with specific antibodies is similar, suggesting that the protein structure remains intact in preeclampsia. Calmodulin, ethanol, or both calmodulin plus ethanol, stimulated the Ca2+-ATPase activity to the same extent for both normotensive and preeclamptic preparations. Our results showed that the reduced Ca2+-ATPase activity of the red cell membranes from preeclamptic women is not associated with a defective enzyme, but rather with a high level of lipid peroxidation.

Background: The Ca-ATPase activity of the plasma membranes of several tissues of preeclamptic pregnant women is significantly reduced when compared with the values of normotensive pregnant women. This has been explained considering the raise in the level of lipid peroxidation of the plasma membranes with preeclampsia. In this work we studied the effect of lipid peroxidation of syncytiotrophoblast basal (fetal facing) plasma membranes from normotensive pregnant women, on their level of Ca-ATPase activity., Methods: The syncytiotrophoblast basal (fetal facing) plasma membranes from normotensive pregnant women were isolated and irradiated with ultraviolet (UV) light (254 nm). The membranes were then assayed for Ca-ATPase activity and lipid peroxidation by TBARS., Results: The UV irradiation raises the level of lipid peroxidation of the membranes, producing a concomitant inhibition of their Ca-ATPase activity. Presence of the antioxidant butylated hydroxytoluene during the UV irradiation of the membranes prevents increase in their level of lipid peroxidation and hence the inhibition of their Ca-ATPase activity., Conclusion: These results give a strong support to the hypothesis that the lowered Ca-ATPase activity already described for plasma membranes of several tissues of preeclamptic women is the consequence of the increased level of lipid peroxidation shown by these membranes., (Copyright (c) 2006 S. Karger AG, Basel.)

The effect of the treatment with magnesium sulfate (MgSO(4)) on Ca-ATPase activity and level of lipid peroxidation of red blood cells from preeclamptic pregnant women was examined because it is known that these parameters are affected with preeclampsia. Red cell ghosts from 11 normotensive and 11 preeclamptic pregnant women, before and after treatment with MgSO(4), were assayed for Ca-ATPase activity and level of lipid peroxidation, determined as TBARS or conjugated dienes. It was found that the Ca-ATPase activity is significantly lower and the level of lipid peroxidation is significantly higher in the preeclamptic women with no treatment, as compared to normotensive pregnant women. Both parameters return to normal values after the MgSO(4) therapy. These results can be mimicked by in vitro preincubation with MgSO(4) of intact red blood cells from preeclamptic pregnant women, without any treatment. Our data indicate that MgSO(4) treatment of preeclamptic pregnant women modifies both the Ca-ATPase activity and the level of lipid peroxidation of their red blood cell membranes, reaching values similar to those of normotensive pregnant women. The diminution of the level of lipid peroxidation by MgSO(4), can account for the increase in Ca-ATPase activity.

Rhythmic changes in activity following a circadian schedule have been described for several enzymes. The possibility of circadian changes in Na,K-ATPase activity was studied in homogenates of rat kidney cortex cells. Male Sprague-Dawley rats were kept on a schedule of 12h light (06:00-18:00 h) and 12 h darkness (18:00-06:00 h) for 2 weeks. At the end of the conditioning period, one rat was killed every 2 h, until completion of a 24 h cycle. Outermost kidney cortex slices were prepared, homogenized and assayed for Na,K-ATPase activity. The whole procedure was repeated six times. Na,K-ATPase activity shows an important oscillation (2 cycles/24 h). Peak activities were detected at 09:00 and 21:00 h, whereas the lowest activities were detected at 15:00 and 01:00-03:00 h. The highest activity was 40+/-3 nmoles Pi mg protein(-1)min(-1) (09:00 h), and the lowest was 79+/-3 nmoles Pi mg protein(-1)min(-1) (15:00 h). The amount of the Na+-stimulated phosphorylated intermediate is the same for the 09:00 h and 15:00 h homogenates. Preincubation of 09:00 h kidney cortex homogenates with blood plasma drawn from rats at either 03:00 h or 15:00 h, significantly inhibited their Na,K-ATPase activity. This inhibition was not seen when the preincubation was carried out with either 09:00 h or 21:00 h blood plasma. The striking oscillation (2 cycles/24 h) of the Na,K-ATPase activity of rat kidney cortex cells is ascribed to the presence of an endogenous inhibitor in blood plasma.

We previously reported that in preeclampsia Ca-ATPase activity diminishes about 50% in red blood cells, myometrium and syncitiotrophoblast plasma membranes. In this work, we measured the active Ca++ uptake by inside-out vesicles of human red blood cells from preeclamptic and normotensive pregnant women. Active calcium uptake by the vesicles was diminished by 49+/-3% in the preeclamptic women as compared to the gestational controls ( 8.06 +/- 0.11 nmol Ca++/mg protein min, gestational controls; 4.08 +/- 0.1 nmol Ca++/mg protein min, preeclamptics). This lowered calcium uptake correlates well with the lowered Ca-ATPase activity found in the red blood cells ghosts of the preeclamptic women (17.05 +/- 0.96 nmol Pi/mg protein min, gestational controls; 8.85 +/- 0.45 nmol Pi/mg protein min, preeclamptics). The reduced calcium uptake and Ca-ATPase activity of the red cell membranes both appear to be associated with a high level of lipid peroxidation. Thus there is a diminution in the active transport of calcium in the red blood cells of preeclamptic women. If this also occurs in other cell types of the preclamptic women, it could result in an increase in their cytosolic calcium concentration which might be responsible, in part, for some of the symptoms of this disease.

Objective: In an effort to evaluate the prognosis of threatened abortion, we established the ratio of serum human chorionic gonadotropin (hCG), as measured by bioassay or radioimmunoassay techniques, of samples from patients with threatened abortion or normal pregnancy., Study Design: Peripheral blood samples from patients in their first trimester of pregnancy with threatened abortion (n=24), or normal pregnancy (n=12), were assayed for progesterone (RIA), and for immunoactive (DELFIA) and bioactive (mouse Leydig cell testosterone production assay) hCG., Results: Serum progesterone was not statistically different between the threatened-continuing and the threatened-miscarried groups. The ratio of hCG bioactive/hCG immunoactive (B/I) was significantly lower for the patients of the threatened group that experienced abortion. The B/I ratio for the control and threatened-continuing patients was similar., Conclusion: The hCG bioactive/hCG immunoactive ratio could be a good indicator of the prognosis of threatened abortion.

Aim: To relate the presence of anti-Chlamydial trachomatis IgA in semen with sperm lipid membrane peroxidation and changes in seminal parameters., Methods: Semen samples of the male partners of 52 couples assessed for undiagnosed infertility were examined for the presence of IgA antibody against C. trachomatis. The level of sperm membrane lipid peroxidation was estimated by determining the malondialdehyde (MDA) formation., Results: Sperm membrane of infertile males with positive IgA antibodies against C. trachomatis showed a higher level of lipid peroxidation than that of infertile males with negative IgA antibody (P<0.05). There was a positive correlation (P<0.01) between the level of C. trachomatis antibody and the magnitude of sperm membrane lipid peroxidation. All the other tested semen parameters were found to be similar in the two groups., Conclusion: The activation of immune system by C. trachomatis may promote lipid peroxidation of the sperm membrane. This could be the way by which C. trachomatis affects fertility.

Objective: We determined calcium-activated adenosine triphosphatase (Ca-ATPase) activity and thiobarbituric acid-reactive substances (TBARS) of plasma membranes from myometrium and placental trophoblast of normotensive and preeclamptic pregnant women., Methods: Samples of myometrium were obtained by uterine biopsies taken upon delivery by cesarean section from nulliparous normotensive and preeclamptic pregnant women. Placentas were obtained after full term vaginal delivery from either normotensive or preeclamptic women. Plasma membrane fractions were prepared from both myometrium and placenta and assayed for Ca-ATPase activity and TBARS., Main Outcome Measure(s): We expected to find a higher level of TBARS and, consequently, a lower activity of Ca-ATPase of the plasma membrane fractions obtained from both myometrium and placenta of preeclamptic women., Results: The Ca-ATPase activity of myometrium and placental trophoblast from preeclamptic women was about 50% lower than that from normotensive women, while the TBARS were higher., Conclusions: A reduced Ca-ATPase activity, caused by an increased level of TBARS, may result in an increase in the cytosolic calcium concentration in the vascular smooth muscle cells of preeclamptic women and thus partially explain the high blood pressure developed by these patients.

Objective: We determined the calcium activated adenosine triphosphatase (Ca-ATPase) activity and level of lipid peroxidation thiobarbituric acid-reactive substances (TBARS) of red blood cell ghosts in the antepartum and postpartum of normotensive and preeclamptic pregnant women., Methods: Samples of venous blood were obtained by venipuncture of nulliparous normotensive and preeclamptic pregnant women antepartum and two, four, six, and 20 weeks postpartum. The red blood cell ghosts were prepared and assayed for Ca-ATPase activity and TBARS., Main Outcome Measure(s): We expected to find a return to normal values of both Ca-ATPase activity and TBARS level of the red blood cell ghosts, modified in the preeclamptic pregnant women, during their puerperium., Results: The Ca-ATPase activity of red cell ghosts from preeclamptic women, antepartum, is lower than that of normotensive pregnant women. The ATPase activity returns to normal values during the first six weeks of postpartum. The level of TBARS of red cell ghosts from preeclamptic women follows a pattern that is inversely proportional to the one of the Ca-ATPase activity., Conclusions: Preeclampsia produces a significant diminution of the Ca-ATPase activity and an increase in the levels of TBARS in the erythrocytes. As soon as all the symptoms of preeclampsia disappear in the postpartum, both Ca-ATPase activity and TBARS return to normal values.

Objective: Determination of Ca-ATPase activity and thiobarbituric acid-reactive substances (TBARS) of red cell ghosts from neonatal and maternal blood of normotensive and preeclamptic women., Methods: Venous blood was obtained by venipuncture of six nulliparous normotensive and six preeclamptic pregnant women. After cesarean delivery, blood samples were obtained from the umbilical cord of the same patients. Red blood cell ghosts were prepared and assayed for Ca-ATPase activity and TBARS., Main Outcome Measure(s): We expected to find a higher level of TBARS, and consequently, a lower activity of the Ca-ATPase activity of the neonatal samples from preeclamptic mothers., Results: The preeclamptic condition produces a significant diminution of the Ca-ATPase activity in both maternal and neonatal red blood cell ghosts (p<0.001 in both cases). The TBARS of the red cell ghosts are significantly increased with preeclampsia in mothers and their neonates (p<0.001 and p<0.005, respectively). The Ca-ATPase activity of red cell ghosts from neonatal blood is significantly lower than the corresponding values of red cell ghosts from both normotensive and preeclamptic women (p<0.001 in both cases)., Conclusions: Preeclamptic mothers and their neonates show a diminished Ca-ATPase activity and an increased level of TBARS in their red blood cell ghosts.

Objective: To compare hCG levels, obtained by biologic and immunologic means, in women with normal pregnancies and women with preeclampsia., Methods: Peripheral blood samples from women in the third trimester with preeclampsia (n = 30) or normal pregnancies (n = 30) were assayed for immunoactive and bioactive hCG (mouse Leydig cell testosterone production assay)., Results: Serum bioactive hCG levels tended to be lower than normal, and immunoactive hCG levels tended to be higher in women with preeclampsia, but the differences were not statistically significant. However, the ratio of bioactive to immunoactive hCG was significantly lower than normal for preeclamptic women (0.70 +/- 0.28 vs. 1.15 +/- 0.35 for normotensive pregnant women [mean +/- standard deviation], P <.001)., Conclusion: The ratio of bioactive to immunoreactive serum hCG is lower among preeclamptic than among normotensive pregnant women.

Concentrations of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in seminal fluid, as well as levels of sperm lipid membrane peroxidation, were investigated in fertile and infertile men. Semen samples, obtained by masturbation from 37 infertile and 14 fertile men, were examined for the presence of TNF-alpha and IL-6. The level of lipid peroxidation of the sperm membrane was measured by determining malondialdehyde (MDA) formation. The correlation between the IL-6 and the TNF-alpha concentrations in seminal plasma with the levels of lipid peroxidation of the sperm membranes was statistically evaluated. The IL-6 concentration in seminal plasma of infertile men was significantly higher than that of fertile men (p < .05). Similarly, the level of membrane lipid peroxidation was higher for the semen of infertile men than that of fertile men (p < .001). A significant positive correlation was found between IL-6 levels in seminal plasma and membrane sperm lipid peroxidation (p < .002), but not between this parameter and TNF-alpha levels in seminal plasma. These findings suggest a possible association between IL-6 seminal plasma levels and lipid peroxidation of sperm membrane. Stimulation of reactive species production by human sperm and leucocytes, induced by the high levels of IL-6, could explain these results.

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.

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., (Copyright 2001 S. Karger AG, Basel.)

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.

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.

In the present paper, we show the existence of a furosemide-sensitive Na(+)-stimulated, Mg(2+)-dependent ATPase activity in cell lysates of Malpighian tubular cells from Rhodnius prolixus, which could be the biochemical expression of the Na(+)-pump. The main characteristics of this activity are: (1) K0.5 for Na+ = 1.49 +/- 0.18 mM, (2) Vmax = 2.8 +/- 0.1 nmol inorganic orthophosphate (Pi).mg prot-1.min-1, (3) it is fully abolished by 2 mM furosemide, (4)it is insensitive to ouabain concentrations up to 10(-2) M, (5) it is sensitive to the presence of vanadate in the incubation medium indicating it to be a P-type ATPase, and (6) it is stimulated by nanomolar concentrations of Ca2+ in the incubation medium.

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.

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.

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.

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.

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.

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.

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.

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.

1. Two Na(+)-stimulated ATPase activities were determined in gill homogenates from squid, shrimp and teleost fish; in kidney slice homogenates from teleost fish, bullfrog, toad, iguana, chicken, duck, rat, pig and cow, as well as in homogenates from rat small intestinal cells, brain cortex and liver slices. The two Na(+)-stimulated ATPase activities, the Na- and the Na,K-ATPase, showed a different behavior toward K+ and ouabain. 2. The ouabain-insensitive, K(+)-independent, Na-ATPase activity for all the studied homogenates was completely inhibited by 2 mM furosemide. 3. An increase in cell volume of the kidney, brain cortex and liver slice preparations, as well as of the rat small intestinal cells, produced a concomitant increase of the ouabain-insensitive Na-ATPase.

Rats chronically fed for 15 months with an isotonic NaCl solution show a volume increase of their kidney proximal tubular cells as well as of the ouabain-insensitive, Na-stimulated ATPase activity of basolateral plasma membranes from these cells. If the experimental rats are changed to tap water after 15 months of the high sodium diet, the results are partially reversed. Thus, the cell volume and the Na-ATPase activity of their basolateral plasma membranes decrease, reaching values closer to those of control rats. There is more Na+ and Cl- in the cells from rats under the high Na(+)-diet than in the cells from control rats. On the other hand, when the rats on the high sodium diet are changed to control conditions (drinking tap water), their Na+ and Cl- contents decrease, approaching control values. It is proposed that the Na-ATPase activity is modulated "in vivo" by the cell volume.