Marinobufagenin,resibufogenin and preeclampsia

The bufodienolides are cardiac glycosides which have the ability to inhibit the enzyme, Na⁺/K⁺ ATPase (sodium potassium adenosine triphosphatase). They are cardiac inotropes, cause vasoconstriction (and, potentially, hypertension) and are natriuretic. Evidence has accrued over time which supports the view that they are mechanistically involved in volume expansion-mediated hypertension. In this communication, the authors summarize data which support the view that the bufodienolides and, in particular, marinobufagenin (MBG) are involved in the pathogenesis of preeclampsia. In a rat model of the syndrome, MBG causes hypertension, proteinuria, intrauterine growth restriction and increased weight gain. All of these phenotypic characteristics are prevented by an antagonist to MBG, resibufogenin (RBG). The “preeclamptic” animals also develop a vascular leak syndrome, resulting in hemoconcentration. Abnormalities in the MAPK (mitogen-activated protein kinase) system play a role in the mechanism by which MBG produces the abnormalities in the pregnant rat. Studies to discover the relevance of these findings to human preeclampsia are currently underway in several laboratories and clinics.     

Leptin-induced increase in blood pressure and markers of endothelial activation during pregnancy in Sprague Dawley rats is prevented by resibufogenin,a marinobufagenin antagonist

Levels of leptin and marinobufagenin (MBG), a cardiotonic steroid, are elevated in the serum of women with pre-eclampsia. Besides this, leptin administration to pregnant rats increases systolic blood pressure (SBP), urinary protein excretion and serum markers of endothelial activation. The link between leptin and MBG is unknown and it is also unclear if leptin-induced increases in blood pressure and proteinuria in the pregnant rat could be prevented by an MBG antagonist. To ascertain this link, this study investigated the effect of resibufogenin (RBG), a marinobufagenin antagonist, on leptin-induced increases in blood pressure and proteinuria during pregnancy in rats.Four groups of Sprague-Dawley rats, aged 12 weeks, were given either normal saline (CONTROL) or 120 μg/kg/day of leptin (LEP), or 120 μg/kg/day of leptin+30 μg/kg/day of resibufogenin (L + RBG) or 30 μg/kg/day of resibufogenin (RBG) from Day 1–20 of pregnancy. Systolic blood pressure and urinary protein excretion (UPE) were measured during the study period. Animals were euthanized on day 21 of pregnancy and vascular cell adhesion molecule 1, (VCAM-1), soluble intracellular cell adhesion molecule 1 (sICAM-1), E-selectin and endothelin-1 (ET-1) were estimated in the serum.SBP, UPE, VCAM-1, sICAM-1 and ET-1 were significantly higher only in the LEP group when compared with those in CONT and in L + RBG and RBG groups.The prevention by RBG of leptin-induced increases in SBP, proteinuria, and endothelial activation during pregnancy seem to suggest a potential role for MBG in leptin-induced adverse effects on blood pressure, urinary protein excretion and endothelial activity during pregnancy in the rat.     

Examination of the cellular mechanisms by which marinobufagenin inhibits cytotrophoblast function

Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid involved in the inhibition of Na(+)/K(+)-ATPase. Increased plasma levels have been reported in patients with volume expansion-related hypertension. We have recently demonstrated that MBG impairs first trimester cytotrophoblast (CTB) cell proliferation, migration, and invasion, which may play a role in the development of preeclampsia. However, whether apoptosis contributes to altered CTB cell function by MBG remains unknown. Using the human extravillous CTB cell line SGHPL-4, we examined the effect of MBG and a similar Na(+)/K(+)-ATPase inhibitor, ouabain, on the phosphorylation status of Jnk, p38, and Src. Additionally, we measured apoptosis by caspase 9 and 3/7 activity and by annexin-V staining. We also investigated interleukin-6 (IL-6) secretion with or without p38 and Jnk inhibition. MBG significantly increased the phosphorylation of Jnk, p38, and Src and increased the expression of caspase 9 and 3/7 indicating the activation of apoptosis. MBG treatment also stimulated the expression of the early apoptosis marker, annexin-V, which was prevented by Jnk and p38 inhibition. MBG also stimulated the secretion of IL-6, which was attenuated by p38 inhibition. Ouabain had similar effects to those of MBG, suggesting that the apoptotic effects on CTB cells may be mediated by inhibition of Na(+)/K(+)-ATPase. In conclusion, the MBG-induced impairment of CTB function occurs via activation of Jnk, p38, and Src leading to increased apoptosis and IL-6 secretion. These observations may have clinical applicability with respect to the therapy of preeclampsia.     

Effect of alloxan diabetes on (Na+ + K+)-activated ATPase in brush border membrane of the mucosal cell of rat small intestine]

In order to elucidate a possible relationship between (Na+ + K+)-activated ATPase and intestinal absorption of actively transported monosaccharides enzyme activity was measured in mucosal cells from alloxan diabetic rats. The general effect of increasing capacity of active, Na+-dependent transport processes in diabetes mellitus is associated with a significantly enhanced (Na+ +K+)-activated ATPase activity in mucosal homogenate from diabetic animals. To study the localization of these effects within the cell we isolated purified brush borders and their substructures. To enable a comparison to be made between preparation procedures of diabetic and control animals the fractions were controlled by electronmicroscopy and by measuring the sucrase activity. In the purified brush border fraction of alloxan treated rats there was no significant increase in (Na+ + K+)-activated ATPase activity. Based on these results we conclude that the (Na+ + K+)-activated ATPase in the basolateral membranes was increased in alloxan diabetes, and it seems very likely that this enzyme is involved in the regulation of Na+-dependent transport processes.     

Marinobufagenin is an upstream modulator of Gadd45a stress signaling in preeclampsia

Preeclampsia (PE) is a hypertensive disorder of pregnancy, in which marinobufagenin (MBG), a circulating cardiotonic steroid, is increased. The Gadd45a stress sensor protein is an upstream modulator of the pathophysiological changes observed in PE. However, the effects of MBG on Gadd45a stress signaling remain unknown. We examined the expression of Gadd45a, the sFlt-1 receptor, and p38, as well as caspase 3 and 8 activities in placental samples from four groups of rats. These were: normal pregnant (NP, n = 8); pregnant rats which received weekly injections of desoxycorticosterone acetate and 0.9% saline as their drinking water (PDS, n = 9); normal pregnant rats injected with MBG (NPM, n = 8); and PDS rats injected with resibufogenin (RBG), an in vivo antagonist of MBG (PDSR, n = 8). Utilizing human cytotrophoblast (CTB) cells, we examined the effect of MBG on these stress signaling proteins in vitro. Placental Gadd45a expression, caspase 3 and 8 activities, sFlt-1 concentrations, and sFlt-1 receptor expression were significantly higher in PDS and NPM compared to NP and PDSR rats. Gadd45a protein was significantly upregulated in the CTB cells when MBG was present in concentrations ≥1 nM. Treatment with MBG (≥ 1 nM) also significantly arrested cell cycle progression and activated the expression of the Gadd45a-mediated stress signaling proteins. Inhibition of Gadd45a through RNAi-mediation attenuated MBG-induced CTB cell stress signaling. In conclusion, MBG is involved in the alteration in Gadd45a stress signaling both in vivo and in vitro and RBG prevents these changes when administered in vivo.     

Alterations in the activities of hepatic plasma-membrane and microsomal enzymes during liver regeneration.

A marked increase in the activities of rat liver plasma-membrane (Na+ + K+)-stimulated ATPase and microsomal Ca2+-stimulated ATPase was observed 18h after partial hepatectomy. Lipid analyses for both membrane preparations reveal that in partially hepatectomized rats the cholesterol and sphingomyelin content are decreased with a subsequent decrease in the cholesterol/phospholipid molar ratio compared with those of sham-operated animals. Changes in the allosteric properties of plasma-membrane (Na+ + K+)-stimulated ATPase by F- (as reflected by changes in the Hill coefficient) indicated a fluidization of the lipid bilayer of both membrane preparations in 18 h-regenerating liver. The amphipathic dodecyl glucoside incorporated into the hepatic plasma membranes evoked a marked increase in the (Na+ + K+)-stimulated ATPase and 5'-nucleotidase activities. The lack of effect of the glucoside on the Lubrol-PX-solubilized 5'-nucleotidase indicates that changes in the activities of the membrane-bound enzymes caused by the glucoside are due to modulation of the membrane fluidity. Dodecyl glucoside appears to increase the membrane fluidity, evaluated through changes in the Hill coefficient for plasma-membrane (Na+ + K+)-stimulated ATPase. The biological significance of these data is discussed in terms of the differences and changes in the interaction of membrane-bound enzymes with membrane lipids during liver regeneration.     

Cardiotonic steroids differentially affect intracellular Na+ and [Na+]i/[K+]i-independent signaling in C7-MDCK cells

Recently, we reported that ouabain kills renal epithelial and vascular endothelial cells independently of elevation of the [Na(+)](i)/[K(+)](i) ratio. These observations raised the possibility of finding cardiotonic steroids (CTS) that inhibit the Na(+),K(+) pump without attenuating cell survival and vice versa. To test this hypothesis, we compared CTS action on Na(+),K(+) pump, [Na(+)](i) content, and survival of Madin-Darby canine kidney cells. At a concentration of 1 microM, ouabain and other tested cardenolides, as well as bufadienolides such as bufalin, cinobufagin, cinobufotalin, and telobufotoxin, led to approximately 10-fold inhibition of the Na(+),K(+) pump, a 2-3-fold decrease in staining with dimethylthiazol-diphenyltetrazolium (MTT), and massive death indicated by detachment of approximately 80% of cells and caspase-3 activation. In contrast, Na(+),K(+) pump inhibition and elevation of [Na(+)](i) seen in the presence of 3 microM marinobufagenin (MBG) and marinobufotoxin did not affect MTT staining and cell survival. Inhibition of the Na(+),Rb(+) pump in K(+)-free medium was not accompanied by a decline of MTT staining and cell detachment but increased sensitivity to CTS. In K(+)-free medium, half-maximal inhibition of (86)Rb influx was observed in the presence of 0.04 microM ouabain and 0.1 microM MBG, whereas half-maximal detachment and decline of MTT staining were detected at 0.03 and 0.004 microM of ouabain versus 10 and 3 microM of MBG, respectively. Both ouabain binding and ouabain-induced [Na(+)](i),[K(+)](i)-independent signaling were suppressed in the presence of MBG. Thus, our results show that CTS exhibit distinctly different potency in Na(+),K(+) pump inhibition and triggering of [Na(+)](i)/[K(+)](i)-independent signaling, including cell death.     

Reduction of Na/K-ATPase potentiates marinobufagenin-induced cardiac dysfunction and myocyte apoptosis

Decreases in cardiac Na/K-ATPase have been documented in patients with heart failure. Reduction of Na/K-ATPase α1 also contributes to the deficiency in cardiac contractility in animal models. Our previous studies demonstrate that reduction of cellular Na/K-ATPase causes cell growth inhibition and cell death in renal proximal tubule cells. To test whether reduction of Na/K-ATPase in combination with increased cardiotonic steroids causes cardiac myocyte death and cardiac dysfunction, we examined heart function in Na/K-ATPase α1 heterozygote knock-out mice (α1(+/-)) in comparison to wild type (WT) littermates after infusion of marinobufagenin (MBG). Adult cardiac myocytes were also isolated from both WT and α1(+/-) mice for in vitro experiments. The results demonstrated that MBG infusion increased myocyte apoptosis and induced significant left ventricle dilation in α1(+/-) mice but not in their WT littermates. Mechanistically, it was found that in WT myocytes MBG activated the Src/Akt/mTOR signaling pathway, which further increased phosphorylation of ribosome S6 kinase (S6K) and BAD (Bcl-2-associated death promoter) and protected cells from apoptosis. In α1(+/-) myocytes, the basal level of phospho-BAD is higher compared with WT myocytes, but MBG failed to induce further activation of the mTOR pathway. Reduction of Na/K-ATPase also caused the activation of caspase 9 but not caspase 8 in these cells. Using cultures of neonatal cardiac myocytes, we demonstrated that inhibition of the mTOR pathway by rapamycin also enabled MBG to activate caspase 9 and induce myocyte apoptosis.     

Ion-sparing diuresis by 2,3-dibenzylbutane-1,4-diol, a synthetic mammalian-lignan derivative.

Diuretic properties of a synthetic lignan, 2,3-dibenzylbutane-1,4-diol (hattalin), and a naturally occurring arctigenin were examined in BALB/c male mice and Wistar male rats. Intra peritoneal administration of hattalin (50 mg/kg) in mice increased urine volume by 1.7-3.1 fold that of placebo-treated animals 40-260 min after administration (p less than 0.05 vs control). In contrast, 100 mg/kg of arctigenin had no effect on urine volume in mice. Hattalin (100 mg/kg), arctigenin (100 mg/kg), or furosemide (50 mg/kg) as a positive control was administered orally to rats, and accumulated urine volume was measured for up to 6-12 h. The urine volume of animals administered with hattalin showed 1.4-1.5 fold that of placebo-treated animals after 2-6 h of administration (P less than 0.05, n = 10). On the other hand, arctigenin showed no significant effect on urine volume for up to 12 h after administration (n = 8). The urine volume in animals administered with furosemide (n = 10) was 2.0-3.0 fold that of placebo-treated animals (P less than 0.01). Furosemide increased total Na+, K+, or Cl- excretion by 1.9, 1.8 or 2.2 fold, respectively, when compared with placebo-treated controls (P less than 0.01), whereas hattalin decreased Na+ excretion by 3.6 times (P less than 0.01), K+ excretion by 1.4 times (not significant), and Cl- excretion by 3.1 times (P less than 0.01). Serum Na+ and K+ levels did not change in both furosemide- and hattalin-administered rats, however, serum Cl- levels in these animals significantly decreased (P less than 0.01) when compared with controls. The results suggest that the diuretic property of hattalin is due to a novel mechanism which is different from that of furosemide or other diuretics modifying the ion-exchange at the uriniferous tubules.     

Studies on the microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase system in rat ventral prostate

A Mg(2+)+Na(+)+K(+)-stimulated adenosine triphosphatase (ATPase) preparation was isolated from rat ventral prostate by flotation of microsomal membranes in high-density sucrose solutions. The reaction medium for optimum Na(+)+K(+)-stimulated ATPase activity was found to be: Na(+), 115mm; K(+), 7-10mm; Mg(2+), 3mm; ATP, 3mm; tris buffer, pH7.4 at 38 degrees , 20mm. The average DeltaP(i) (Mg(2+)+Na(+)+K(+) minus Mg(2+)+Na(+)) was 9mumoles/mg. of protein/hr., representing a 30% increase over the Mg(2+)+Na(+)-stimulated ATPase activity. At high concentrations, K(+) was inhibitory to the enzyme activity. Half-maximal inhibition of Na(+)+K(+)-stimulated ATPase activity was elicited by ouabain at 0.1mm. The preparation exhibited phosphatase activity towards ribonucleoside triphosphates other than ATP. However, stimulation of P(i) release by Na(+)+K(+) was observed only with ATP as substrate. The apparent K(m) for ATP for Na(+)+K(+)-stimulated activity was about 0.3x10(-3)m. Ca(2+) inhibited only the Na(+)+K(+)-stimulated ATPase activity. Mg(2+) could be replaced by Ca(2+) but then no Na(+)+K(+) stimulation of ATPase activity was noticed. The addition of testosterone or dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) in vitro at 0.1-10mum under a variety of experimental conditions did not significantly increase the Na(+)+K(+)-stimulated ATPase activity. The enzyme preparations from prostates of orchidectomized rats, however, exhibited a drastic decrease in the specific activity of Na(+)+K(+)-stimulated ATPase; these changes were prevented in the orchidectomized rats by injection of testosterone propionate.     

Dexamethasone modulates the activity of the eel branchial Na+/K+ATPase in both chloride and pavement cells

In fish, gills actively accumulate ions in freshwater (FW) with Na+ absorption taking place at the level of pavement cells, and excrete monovalent ions, mainly Na+ and Cl-, through the chloride cells in sea water (SW). The Na+/K+ATPase plays a crucial role in the functionality of osmoregulatory cells and we showed previously that angiotensin II modulates its activity in the eel gill (1). We here show the effects of synthetic steroid dexamethasone (DEX) on the activity of Na+/K+ATPase in both gill pavement and chloride cells from FW- and SW-adapted animals. Results showed that in the chloride cells 100 nM DEX provoked a significant increment in the activity of Na+/K+ATPase in both SW- and FW-adapted animals. This effect was greatest at 2 hours in SW, and at 6 hours in FW. The increment in the activity of the Na+/K+ATPase was dose-dependent in both environmental adaptations. Conversely, in pavement cells from FW-adapted eels 100 nM DEX decremented the activity of Na+/K+ATPase (4-fold reduction after 6 hour incubations), while in SW, DEX increased the enzyme activity of about 25% at 2 hours, and of about 55% at 6 hours. These results are consistent with the different physiological roles that pavement and chloride cells have in the two different adaptive conditions.     

Stimulation of Na+,K+-ATPase activity in certain membranes of the rat central nervous system (CNS) by acute administration of desipramine (DMI)

1. The activities of ATPase in rat CNS were studied 3 hr after administration of the noradrenaline uptake inhibitor, desipramine (DMI: 10 mg.kg-1, i.p.). Na+K+-ATPase activity significantly increased after DMI in the whole particulate from hypothalamus and mesencephalus but no changes in frontal cortex or in pons-medulla oblongata areas were found. This increase was prevented when the animals were pretreated with the noradrenergic neurotoxic N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). 2. Purified membrane fractions from hypothalamus were obtained by differential and sucrose gradient centrifugation (0.8-1.2 M sucrose). It was observed that after DMI, Na+,K+-ATPase activity increased only in the membranous fraction lying at 0.9 M sucrose. 3. Mg2+- or Ca2+-ATPase activities were not modified by DMI treatment. 4. Citalopram, a specific serotonergic uptake inhibitor, did not affect ATPase activities. 5. The results obtained could indicate that DMI acute administration selectively stimulates Na+,K+-ATPase activity of certain membranes of the CNS after an increase in the concentration of the noradrenergic neurotransmitter in the synaptic gap.     

Ganglioside protection of the erythrocyte membranes in myocardial ischemia]

Myocardial ischemia was shown to lead to modification of structural and functional organization of rat erythrocyte membranes. Thus, it was found that the activity of Na+, K+-ATP-ase markedly decreased, while accumulation of LPO products and of lysophosphatidylcholine (lyso--PC) took place in erythrocyte membranes of rats subjected to myocardial ischemia. Using nonpenetrating modifier trinitrobenzosulfonic acid, an increase in the content of modified phosphatidylethanolamine in erythrocyte membranes of ischemic rats was revealed as compared to the membranes of control animals. The intravenous administration of gangliosides (30 mg/kg) resulted in partial normalization of Na+, K+(-)ATPase activity, of LPO product and lysoPC content and of transbilayer distribution of lipids.     

Na+/K+ ATPase and its functional coupling with Na+/Ca2+ exchanger in mouse embryonic stem cells during differentiation into cardiomyocytes

Cardiomyocytes derived from mouse embryonic stem (mES) cells have been demonstrated to exhibit a time-dependent expression of ion channels and signal transduction pathways in electrophysiological studies. However, ion transporters, such as Na+/K+ ATPase (Na+ pump) or Na+/Ca2+ exchanger, which play crucial roles for cardiac function, have not been well studied in this system. In this study, we investigated the functional expression of Na+/K+ ATPase and Na+/Ca2+ exchanger in mES cells during in vitro differentiation into cardiomyocytes, as well as the functional coupling between the two transporters. By measuring [Na+]i and Na+ pump current (Ip), it was shown that an ouabain-high sensitive Na+/K+ ATPase was expressed functionally in undifferentiated mES cells and these activities increased during a time course of differentiation. Using RT-PCR, the expression of mRNA for alpha1-subunit and alpha3-subunit of the Na+/K+ ATPase could be detected in both undifferentiated mES cells and derived cardiomyocytes. In contrast alpha2-subunit mRNA could be detected only in derived cardiomyocytes but not in undifferentiated mES cells. mRNA for the Na+/Ca2+ exchanger 1 isoform (NCX1) could be detected in undifferentiated mES cells and its expression levels seemed to gradually increase throughout the differentiation accompanied by increasing its Ca2+ extrusion function. At the middle stages of differentiation (after 10-day induction), more than 75% derived cardiomyocytes exhibited [Ca2+]i oscillations by blocking of Na+/K+ ATPase, suggesting the functional coupling with Na+/Ca2+ exchanger. From these results and RT-PCR analysis, we conclude that alpha2-subunit Na+/K+ ATPase mainly contributes to establish the functional coupling with NCX1 at the middle stages of differentiation of cardiomyocytes.     

Blockade of early and late retinal biochemical alterations associated with diabetes development by the selective bradykinin B1 receptor antagonist R-954

The chronic hyperglycemia measured alongside diabetes development is associated with significant long-term damage and failure of various organs. In the present study it was shown that hyperglycemia induced early and long term increases in nitric oxide (NO) levels, kallikrein activity and vascular capillary permeability measured as plasma extravasation, and decreases of Na/K ATPase activity in diabetic rat retina 4 and 12 weeks after streptozotocin (STZ) injection. Treatment of the animals for 5 consecutive days with a novel selective bradykinin B(1) receptor (BKB(1)-R) antagonist R-954 (2mg/kg s.c) at the end of the 4 and 12 week periods highly reduced NO, kallikrein and capillary permeability and increased Na/K ATPase activity in the retina. These results suggest that the BKB(1)-R receptor subtype is over-expressed during the streptozotocin-induced development of diabetes in rat retina as evidenced by the inhibitory effects of the BKB(1)-R antagonist R-954 on NO, kallikrein and vascular permeability increases as well as Na/K ATPase decreases. The beneficial role of the BKB(1)-R antagonist R-954 for the treatment of the diabetic retinopathy is also suggested.     

Sodium/potassium adenosine triphosphatase alpha-subunit and alpha-subunit mRNA levels in early rabbit embryos

Sodium/potassium adenosine triphosphatase (Na+/K+ ATPase) and Na+/K+ ATPase mRNA content of rabbit embryos during preimplantation development were evaluated. Changes in Na+/K+ ATPase alpha-subunit content were detected with Western blotting using polyclonal antiserum against guinea pig Na+/K+ ATPase. Total RNA samples from rabbit embryos were analyzed by using Northern blots hybridized with random primer-labeled cDNA for Na+/K+ ATPase alpha-subunit from sheep kidney. Northern blots exhibited a single mRNA band (3.65 kilobases) in sheep kidneys and rabbit embryos. Between Day 4 and Day 6 of development, Na+/K+ ATPase alpha-subunit mRNA content increased 35-fold whereas Na+/K+ ATPase alpha-subunit content increased 22-fold. The similar increase in Na+/K+ ATPase alpha-subunit mRNA and alpha-subunit content in rabbit embryos suggests that Na+/K+ ATPase is partly regulated at the mRNA level during blastocyst expansion.     

Abnormal Ca2(+)-ATPase activity in erythrocytes of non-insulin-dependent diabetic rats

Non-insulin-dependent diabetic (NIDD) rats have an increased Ca2(+)-ATPase activity in their kidney basolateral membranes. We find that a similar increased activity occurs in erythrocytes of the NIDD animals. This alteration in membrane ATPase activity appears to be specific for the Ca2(+)-ATPase as (Na(+) + K+) and Mg2(+)-ATPase and Na, K and Mg concentrations in the erythrocyte were not affected by the diabetic condition in these animals. Thus, abnormalities in membrane Ca2(+)-ATPase activity in the NIDD rats are not restricted to one tissue and appear to be a generalized pathology in the NIDD animals.     

Altered Na+-K+ ATPase immunoreactivity within GABAergic neurons in the gerbil hippocampal complex induced by spontaneous seizure and vigabatrin treatment

In the present study, the expression of Na(+)-K(+) ATPase in the gerbil hippocampus associated with various sequelae of spontaneous seizures were investigated in order to identify the roles of Na(+)-K(+) ATPase in the epileptogenesis and the recovery mechanisms in these animals. The population of Na(+)-K(+) ATPase immunoreactive neurons and Na(+)-K(+) ATPase immunodensity were significantly lower in the pre-seizure group of SS gerbils than those in SR gerbils. At 30-min postictal, the Na(+)-K(+) ATPase immunoreactivity was significantly elevated in the hippocampal complex. At 3-h postictal, the Na(+)-K(+) ATPase immunoreactivity in the hippocampus was declined, as compared to the 30-min postictal. At 12h after seizure on-set, Na(+)-K(+) ATPase expression was re-enhanced in the all regions of the hippocampal complex including the dentate hilus. Following administration of vigabatrin Na(+)-K(+) ATPase expression was also increased. The present data suggest that altered Na(+)-K(+) ATPase expression may contribute the regulation of the seizure activity in this animal.     

Sodium/potassium adenosine triphosphatase alpha- and beta-subunit and alpha-subunit mRNA levels during mouse embryo development in vitro

Changes in sodium/potassium adenosine triphosphatase (Na+/K+ ATPase) and Na+/K+ ATPase mRNA content during preimplantation mouse embryo development were determined. Western blotting, using polyclonal antiserum against guinea pig Na+/K+ ATPase, was used to detect changes in Na+/K+ ATPase alpha- and beta-subunit content during mouse embryo development. Total RNA from mouse embryos was analyzed using Northern and slot blots hybridized with random-primer-labeled cDNA for Na+/K+ ATPase alpha-subunit from sheep kidney. Northern blots exhibited a single mRNA band (3.65 kb) in sheep and mouse kidneys and mouse embryos. Although Na+/K+ ATPase alpha-subunit mRNA content of mouse embryos increased 45-fold between Day 1 and Day 4 of development, Na+/K+ ATPase alpha-subunit content remained constant, and beta-subunit content increased 9-fold. The Na+/K+ ATPase alpha-subunit and alpha-subunit mRNA content did not increase in a similar manner. The results suggest that, in mouse embryos, blastocoel formation is not triggered by an increase in Na+/K+ ATPase alpha-subunit content. Changes in beta-subunit content may be important in regulating Na+/K+ ATPase activity and blastocoel formation.