ATPases, ion exchangers and human sperm motility.

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.

Na⁺, K⁺-ATPase and Ca²âº-ATPase activities in basal and microvillous syncytiotrophoblast membranes from preeclamptic human term placenta.

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.

Preeclampsia, placenta, oxidative stress, and PMCA.

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.

Effect of ultraviolet C irradiation on human sperm motility and lipid peroxidation.

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.

Effect of magnesium sulfate on the osmotic fragility and lipid peroxidation of intact red blood cells from pregnant women with severe preeclampsia.

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.

Biochemical identification of dynein-ATPase activity in human sperm.

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.

Ca-ATPase activity of human red cell ghosts: preeclampsia, lipid peroxidation and MgSO4.

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.

The plasma membrane Ca2+-ATPase protein from red blood cells is not modified in preeclampsia.

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.

Lipid peroxidation and Ca-ATPase activity of basal plasma membranes of syncytiotrophoblast from normotensive pregnant women.

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.

Effect of magnesium sulfate on the calcium-stimulated adenosine triphosphatase activity and lipid peroxidation of red blood cell membranes from preeclamptic women.

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.

Sulfato de magnesio ¿una panacea? / Magnesium sulfate: a panace?

El magnesio es un catión importante que juega un rol esencial en muchas funciones fisiológicas como: procesos metabólicos que requieren energía; síntesis de proteínas; mantenimiento de la integridad de membranas celulares y subcelulares; excitabilidad neuromuscular y, contracción muscular. La principal fuente de Mg es la dieta y los órganos responsables de mantener las concentraciones plasmáticas de Mg dentro de los límites normales son el intestino delgado y el riñón. El Mg actualmente es empleado para fines terapéuticos y se administra en forma de sales, entre las cuales cabe destacar al sulfato de Mg. Esta sal posee una gran variedad de efectos benéficos sobre el organismo, entre los cuales podemos mencionar un efecto antioxidante, propiedades neuro y cardioprotectoras, efecto anticonvulsivante, propiedades broncodilatadoras y efectos vasodilatadores, entre otras. En los últimos años, el uso del sulfato de Mg para fines terapéuticos ha tomado considerable auge y pareciera representar una panacea en el área clínica. Así, el sulfato de Mg es administrado para una gran cantidad de desórdenes clínicos, entre los cuales podemos destacar: preeclampsia y partos pre-términos, desórdenes cardiovasculares, procesos de isquemia cerebral, asma, migrañas, y síndrome Irukandji entre otros. Al ser administrado en las concentraciones consideradas como terapéuticas, basadas en la experiencia clínica, el sulfato de Mg posee mínimos efectos tóxicos

Efecto del etanol sobre las actividades de ATPasas de Na+ y de Na+, K+ de membranas plasmáticas laterobasales de células de túbulo proximal de riñón de ratas viejas / Effect of ethanol on the Na+ and Na+, K+ ATPase activities of basolateral plasma membranes of kidney proximal tubular cells of old rats

Existen dos ATPasas estimuladas por sodio en las membranas de células de túbulo proximal renal: La ATPasa de Na+,K+ y la Na+. La actividad de ambas enzimas disminuye con el envejecimiento. OBJETIVO: Comparar el efecto del alcohol (etanol) sobre las enzimas mencionadas en ratas jóvenes y viejas. MATERIALES Y METODOS: Se prepararon homogenizados de corteza renal y fracciones enriquesidas en membranas plasmáticas laterobasales de células de túbulo proximal renal procedentes de ratas macho Sprague-Dawley jóvenes y viejas. Se determinó las actividades ATPásicas respectivas en las preparaciones mediante un método espectrofotométrico tanto en ausencia como en presencia de etanol. RESULTADOS: Se encontró que, mientras la ATPasa de sodio, potasio era menos sensible al etanol, la ATPasa de sodio era más sensible al etanol en ratas viejas comparadas con jóvenes. CONCLUSIONES:Los hallazgos indican una clara diferenciación entre las dos ATPasas y permiten especular que la ingestión continua de etanol podría influenciar con más fuerza el funcionamiento de la ATPasa de sodio en personas viejas que en jóvenes...

A blood plasma inhibitor is responsible for circadian changes in rat renal Na,K-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.

Lipid peroxidation and active calcium transport in inside-out vesicles of red blood cells from preeclamptic women.

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.

Calcium-ATPase activity of red blood cell ghosts from preeclamptic women, antepartum and postpartum.

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.

Preeclampsia and calcium-ATPase activity of plasma membranes from human myometrium and placental trophoblast.

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.

Preeclampsia and calcium-ATPase activity of red cell ghosts from neonatal and maternal blood.

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.

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 percent ethanol on the Na+ and on the Na+, K(+)-ATPase activities. The differential effect of the alcohol on the two ATPases (approximately 40 percent inhibition of the Na(+)-ATPase and approximately 10 percent 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 percent ethanol inhibits the two ATPases in a totally different way

Modulación de la bomba de Na en celulas de tubulo proximal de riñon de rata / Modulation of the Na-pump in rat kidney proximal tubular cells

en el presente trabajo se presenta evidencia indicando qeu uno de los dos mecanismos activos de expulsión de Na+ demostrables en células de túbulo proximal de riñon de mamífero, la Bomba de Na (que expulsa de las células sodio acompañado por cloruro y agua), es modulada en su actividad por el volumen celular, en el sentido de que un aumento de volumen celular, se traduce en un aumento de su actividad. Por otro lado, el otro mecanismo de expulsión activa de Na, la bomba de Na, K (que expulsa de las células sodio intercambiado por potasio), es totalmente indiferente a los cambios de volumen que puedan experimentar las células. Este efecto modulador del volumen que puedan experimentar las células. En efecto modulador del volumen celular sobre la actividad de la Bomba de Na, puede ser demostrado tanto "in vitro" como "in vivo"