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In the present work, a Mg [sup 2++] -dependent, Ca [sup 2+] -stimulatedATPase activity was determined and characterized in purified preparationsof syncytiotrophoblast basal (fetal facing) plasma membranes, and its characteristicswere compared to those of the active Ca [sup 2+] -transport already demonstratedin this tissue. Similar to the active Ca [sup 2+] transport, the Ca-ATPaseis Mg [sup 2+] -dependent, is stimulated by calmodulin, and is inhibitedby vanadate. The K[sub m] for Ca [sup 2+] activation is 0.25±0.02µM, a value near to that described for calcium active transportin this tissue. Consequently, the Ca-ATPase activity of human syncytiotrophoblastbasal plasma membrane described in this paper could be responsible for theactive extrusion of calcium from the syncytiotrophoblast toward the fetalcirculation. [ABSTRACT FROM AUTHOR]

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 ouabaininsensitive 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. [ABSTRACT FROM AUTHOR]

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.

The central aim of this study was to investigate whether male Wistar rats chronically fed a high-fat diet (HFD) over 106 days present high levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), and Na+ and Ca2+ transport alterations in the left ventricle, together with dyslipidemia and decreased glucose tolerance, and to investigate the influence of Ang-(3–4). The rats became moderately overweight with an expansion of visceral adiposity. Na+-transporting ATPases, sarco-endoplasmic reticulum Ca2+-ATPase (SERCA2a), and the abundance of Angiotensin II receptors were studied together with lipid and glycemic profiles from plasma and left-ventricle echocardiographic parameters fractional shortening (FS) and ejection fraction (EF). IL-6 and TNF-α increased (62% and 53%, respectively), but returned to normal levels with Angiotensin-(3–4) administration after 106 days. Significant lipidogram alterations accompanied a decrease in glucose tolerance. Angiotensin II receptors abundance did not change. (Na+ + K+)ATPase and ouabain-resistant Na+-ATPase were downregulated and upregulated, respectively, but returned to normal values upon Angiotensin-(3–4) administration. SERCA2a lost its ability to respond to excess ATP. Echocardiography showed no changes in FS or EF. We conclude that being overweight causes an increase in Ang-(3–4)-sensitive IL-6 and TNF-α levels, and ion transport alterations in the left ventricle that could evolve into future heart dysfunction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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.

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.

Aim: Preeclampsia (PE) is a dangerous condition that affects 3-5% of pregnancies and has a substantial risk of death and morbidity for both mothers and newborns. The processes behind the etiology of PE are not entirely known, despite the fact that it is the primary cause of illness and death among mothers globally. In order to further understand the correlations between these parameters, this study will look at the levels and presence of anti-carbonicanhydrase (CA) I and II antibodies, total oxidant capacity (TOC), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) in early PE. Material and Method: The research analyzed 30 pregnant women with early PE and 30 normal pregnant women as the control group. Serum levels of anti-CAI (pg/mL), anti-CAII (ng/mL), MDA (nmol/mL), TOS (U/mL), T-AOC (U/mL) were measured and compared between the two groups. Results: Significant variations were noted in the amount of anti-CA I, anti-CA II, MDA, TOS, and T-AOC (both p<0.05) between the control group and the early PE group. More specifically, oxidative stress indicators were changed and increased levels of anti-CA I and anti-CA II were seen in the early PE group in comparison with the control group. Conclusion: The findings show that elevated amounts of anti-CAI and anti-CAII antibodies may serve as predictive markers for early PE. The significant differences in oxidative stress parameters further support the oxidative stress involvement in the pathogenesis of early PE. However, more extensive Research is required to validate these results and clarify the mechanisms underlying PE. [ABSTRACT FROM AUTHOR]

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: 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.

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.

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.

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.

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 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.