Glutamate Induces a Calcineurin-Mediated Dephosphorylation of Na+,K+-ATPase that Results in Its Activation in Cerebellar Neurons in Culture

Abstract: In primary cultures of cerebellar neurons glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca²⁺ and Na⁺ into the neuron. To maintain Na⁺ homeostasis, the excess Na⁺ entering through the ion channel should be removed by Na⁺,K⁺-ATPase. It is shown that incubation of primary cultured cerebellar neurons with glutamate resulted in activation of the Na⁺,K⁺-ATPase. The effect was rapid, peaking between 5 and 15 min (85% activation), and was maintained for at least 2 h. Glutamate-induced activation of Na⁺,K⁺-ATPase was dose dependent: It was appreciable (37%) at 0.1 µ M and peaked (85%) at 100 µ M . The increase in Na⁺,K⁺-ATPase activity by glutamate was prevented by MK-801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase was reversed by phorbol 12-myristate 13-acetate, an activator of protein kinase C, indicating that activation of Na⁺,K⁺-ATPase is due to decreased phosphorylation by protein kinase C. W-7 or cyclosporin, both inhibitors of calcineurin, prevented the activation of Na⁺,K⁺-ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na⁺,K⁺-ATPase that was previously phosphorylated by protein kinase C. This dephosphorylation leads to activation of Na⁺,K⁺-ATPase.     

Inhibition of Rat Brain Microsomal Na+,K+-ATPase by S-Adenosylmethionine

Abstract: Rat brain microsomes were preincubated with S -adenosylmethionine (SAM), MgCl₂, and CaCl₂, then re-isolated, and the activity of Na⁺,K⁺-ATPase determined. SAM inhibited the Na⁺,K⁺-ATPase activity compared with microsomes subjected to similar treatment in the absence of SAM. A biphasic inhibitory effect was observed with a 50% decrease at a SAM concentration range of 0.4 μ M -3.2 μ M and a 70% reduction at a concentration range above 100 μ M . Inclusion of either S- adenosylhomocysteine or 3-deazaadenosine in the preincubations prevented the SAM inhibition of Na⁺,K⁺-ATPase activity. The inhibition by SAM appeared to be Mg²⁺- or Ca²⁺-dependent.     

Manipulation of Membrane Potential Modulates Malonate-Induced Striatal Excitotoxicity In Vivo

Abstract: Malonate is a reversible inhibitor of succinate dehydrogenase (SDH) that produces neurotoxicity by an N -methyl- d -aspartate (NMDA) receptor-dependent mechanism. We have examined the influence of pharmacological manipulation of membrane potential on striatal malonate toxicity in rats in vivo by analysis of lesion volume. Depolarization caused by coinjection of the Na⁺,K⁺-ATPase inhibitor ouabain or a high concentration of potassium greatly exacerbated malonate toxicity; this combined toxicity was blocked by the noncompetitive NMDA antagonist MK-801. The toxicity of NMDA was also exacerbated by ouabain. The overt toxicity of a high dose of ouabain (1 nmol) was largely prevented by MK-801. Coinjection of the K⁺ channel activator minoxidil (4 nmol) to reduce depolarization attenuated the toxicity of 1 µmol of malonate by ∼60% without affecting malonate-induced ATP depletion. These results indicate that membrane depolarization exacerbates malonate neurotoxicity and that membrane hyperpolarization protects against malonate-induced neuronal damage. We hypothesize that the effects of membrane potential on malonate toxicity are mediated through the NMDA receptor as a result of its combined agonist- and voltage-dependent properties.     

Glutamate Uptake Stimulates Na+,K+-ATPase Activity in Astrocytes via Activation of a Distinct Subunit Highly Sensitive to Ouabain

Abstract: The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na⁺-dependent transporter. Evidence had been provided that Na⁺,K⁺-ATPase might be involved in this process. We have now measured the activity of Na⁺,K⁺-ATPase in cultured astrocytes, using ouabain-sensitive ⁸⁶Rb uptake as an index. l -Glutamate increases glial Na⁺,K⁺-ATPase activity in a concentration-dependent manner with an EC₅₀ = 67 µ M . Both l - and d -aspartate, but not d -glutamate, produce a similar response, an observation that is consistent with an uptake-related effect rather than a receptor-mediated one. Under basal conditions, concentration-dependent inhibition of Na⁺,K⁺-ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain ( K _0.5 = 113 µ M ), compatible with the presence of an α₁ isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain ( K _0.5 = 20 n M ), thus revealing a high-affinity site akin to the α₂ isozyme. These results suggest that astrocytes possess a glutamate-sensitive isoform of Na⁺,K⁺-ATPase that can be mobilized in response to increased neuronal activity.     

Plasma ionic regulation and gill Na+/K+-ATPase changes during rapid transfer to sea water of yearling rainbow trout, Salmo gairdneri: time course and seasonal variation

Danish rainbow trout, Salmo gairdneri Richardson, (40–65 g) were transferred to 28%o sea water at intervals throughout the early spring and summer. Gill Na⁺/K⁺-ATPase of fish kept in fresh water surged distinctly during May. Simultaneously, a body silvering occurred and plasma concentrations of Cl⁻, Na⁺ and total thyroxine (T4) decreased. The seawater transfer-induced adaptive response in plasma electrolytes comprised a biphasic change, i.e., an adjustive peak phase and a regulatory phase lasting for 2 days and 1 week after transfer, respectively. Further, gill Na⁺/K⁺-ATPase activity increased to a new level after an initial lag phase of 2–3 days, but electrolyte regulation was mostly initiated prior to the adaptive change in ATPase activity. In spite of increasing pre-transfer freshwater Na⁺/K⁺-ATPase activity during the spring, the electrolyte peak level, the degree of muscle dehydration and the mortality of fish transferred to sea water increased from April to July. The apparent uncoupling of freshwater Na⁺/K⁺-ATPase activity and plasma electrolyte regulation in sea water is discussed in relation to smelting and prediction of hypo-osmoregulatory performance.     

Sucrose and ouabain effects on the kinetic properties of a membrane-bound (Na++ K++ Mg2+) ATPase in sugar beet roots

Kinetic studies of a microsomal (Na⁺+ K⁺+ Mg²⁺)ATPase from sugar beet roots ( Beta vulgaris L. cv. Monohill) show that sucrose influences the MgATPase in different ways depending on the presence of K⁺ and/or Na⁺ 1) In the presence of the substrate MgATP and Na⁺ the effect of sucrose follows simple Michaelis-Menten kinetics. 2) In the presence of substrate together with K⁺ or (K⁺+ Na⁺), sucrose has little effect on the ATPase activity. 3) In the presence of Na⁺, onabain acts as an uncompetitive inhibitor with respect to MgATP. 4) In the presence of K⁺ or (K⁺+ Na⁺), the inhibition by ouabain is somewhat depressed and shows non-linearity when 1/v is plotted versus 1/MgATP. 5) Sucrose and Na⁺ activate in a competitive way, so that a successive increase of the Na⁺ level decreases the activation by sucrose. Both K_m and V-values are thereby changed. 6) The sucrose activation in the presence of Na⁺ is also influenced by ouabain. It is, therefore, suggested that Na⁺ may regulate the interference between the Na⁺/K⁺ pump and a sucrose sensitive system.     

Impairment of Na+,K+-ATPase activity following enterotoxigenic Campylobacter jejuni infection: changes in Na+, Cl− and 3-O-methyl-D-glucose transport in vitro, in rat ileum

Abstract Unidirectional fluxes of Na⁺, Cl⁻ and 3-O-methyl-D-glucose (3-MG) were measured in vitro across Campylobacter jejuni live culture-infected and control rat ileal short-circuited tissues by the Using Chamber technique. Net secretion of Na⁺ and enhanced secretion of Cl⁻ ions was observed in the infected animals ( P < 0.001, n =6) as compared to the net absorption of Na⁺ and marginal secretion of Cl⁻ ions in the control animals. There was a significant decrease in the mucosal-to-serosal fluxes of 3-MG in C. jejuni -infected rat ileum. The specific Na⁺,K⁺-ATPase activity when measured biochemically in the membrane-rich fraction of enterocytes was found to be significantly lower (58%) in the infected group as compared to the control group ( P < 0.001). Our results therefore suggest that infection with an enterotoxigenic C. jejuni inhibits the Na⁺,K⁺-ATPase activity in rat enterocytes. The impairment of Na⁺,K⁺-ATPase activity thus appears to induce a secondary change in Na⁺,Cl⁻ and 3-MG transport in vitro in rat ileum.     

Functional Analysis and Tissue-Specific Expression of Drosophila Na+,K+-ATPase Subunits

Abstract: We have previously purified and characterized a nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana [nerve antigen (NRV)] and identified two separate genes coding for three different proteins. All three proteins share homology with the β subunits of Na⁺,K⁺-ATPase from various other species. In this study we have isolated a new Drosophila Na⁺,K⁺-ATPase α subunit cDNA clone (PSα; GenBank accession no. AF044974) and demonstrate expression of functional Na⁺,K⁺-ATPase activity when PSα mRNA is coinjected into Xenopus oocytes along with any of the three different Nrv mRNAs. Western blotting, RNase protection assays, and immunocytochemical staining of adult fly sections indicate that NRV2 is expressed primarily in the nervous system. Staining is most intense in the brain and thoracic ganglia and is most likely associated with neuronal elements. NRV1 is more broadly expressed in muscle and excretory tissue and also shows diffuse distribution in the nervous system. Similar to other species, Drosophila expresses multiple isoforms of Na⁺,K⁺-ATPase subunits in a tissue- and cell type-specific pattern. It will now be possible to use the advantages of Drosophila molecular and classical genetics to investigate the phenotypic consequences of altering Na⁺,K⁺-ATPase expression in various cell and tissue types.     

Involvement of Na+,K+-ATPase in the Mitogenic Effect of Insulin-Like Growth Factor-I on Cultured Rat Astrocytes

Abstract: We have previously reported that insulin/insulin-like growth factor (IGF)-I induced the α1 isoform of Na⁺,K⁺-ATPase in cultured astrocytes. In this study the effects of insulin/IGF-I on Na⁺,K⁺-ATPase activity and cell proliferation were examined in astrocytes cultured under the various conditions, to test the possible involvement of the enzyme activity in the mitogenic action of IGF-I on astrocytes. Insulin increased Na⁺,K⁺-ATPase activity and stimulated cell proliferation in subconfluent astrocytes (cultured for 7–14 days in vitro). In contrast, these effects were not observed in confluent cells (cultured for 28 days). Furthermore, insulin stimulated neither the enzyme activity nor [³H]thymidine incorporation in astrocytes preincubated in fetal calf serum-free medium for 2 days (quiescent cells) and treated with dibutyryl cyclic AMP (differentiated cells). The increases in Na⁺,K⁺-ATPase activity and expression of the α1 mRNA preceded the mitogenic effect. ¹²⁵I-IGF-I binding experiment showed that all the cells used here had similar binding characteristics. The insulin-induced increase in enzyme activity was not affected by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and it was observed even in Ca²⁺-free medium. The stimulation by IGF-I of [³H]thymidine incorporation was attenuated by ouabain and a low external K⁺ level. These findings suggest that stimulation of Na⁺,K⁺-ATPase activity is involved in the mitogenic action of IGF-I on cultured astrocytes.     

Expression of Na+, K+-ATPase α-Subunit in Animalized and Vegetalized Embryos of the Sea Urchin, Hemicentrotus pulcherrimus.

A marked increase in the Na⁺, K⁺-ATPase activity of sea urchin embryos occurred following an elevation of its mRNA level, revealed by Northern blotting analysis, in developmental period between the swimming blastula and the late gastrula stage. cDNA clone of Na⁺, K⁺-ATPase α-subunit, obtained from γgt10 cDNA library of sea urchin gastrulae, was digested with EcoRl ad Hindlll. The obtained 268 bp cDNA fragment, hybridized to a 4.6 Kb RNA, was used as probe for Northern blotting analysis. The level of Na⁺, K⁺-ATPase mRNA was higher in embryo-wall cell fraction isolated from late gastrulae (ectoderm cells) than the level in the bag fraction, containing mesenchyme cells (mesoderm cells) and archenteron (endoderm cells). The activity of Na⁺, K⁺-ATPase and the level of its mRNA were higher in animalized embryos obtained by pulse treatment with A23187 for 3 hr, starting at the 8–16 cell stage and were considerably lower in vegetalized embryos induced by 3 hr treatment with Li⁺ than that in normal embryos at the post gastrula corresponidng stage. Augmentation of Na⁺, K⁺-ATPase gene expression can be regarded as a marker for ectoderm cell differentiation at the post gastrula stage, which results from determination of cell fate in prehatching period.     

Probable Role of Allylisothiocyanate-Sensitive H+, K+-ATPase in Spicule Calcification in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

In embryos of the sea urchin, Hemicentrotus pulcherrimus , as well as in cultured cells derived from isolated micromeres, spicule formation was inhibited by allylisothiocyanate, an inhibitor of H⁺, K⁺-ATPase, at above 0.5 μM and was almost completely blocked at above 10 μM. Amiloride, an inhibitor of Na⁺, H⁺ antiporter, at above 100 μM exerted only slight inhibitory effect, if any, on spicule formation. Intravesicular acidification, determined using [ dimethylamine -¹⁴C]-aminopyrine as a pH probe, was observed in the presence of ATP and 200 mM KCl in microsome fraction obtained from embryos at the post gastrula stage, at which embryos underwent spicule calcification. Intravesicular acidification and K⁺-dependent ATPase activity were almost completely inhibited by allylisothiocyanate at 10 μM. Allylisothiocyanate-sensitive ATPase activity was found mainly in the mesenchyme cells with spicules isolated from prisms. H⁺, K⁺-ATPase, an H⁺ pump, probably mediates H⁺ release to accelerate CaCO₃ deposition from Ca²⁺, CO₂ and H₂O in the primary mesenchyme cells. Intravesicular acidification was stimulated by valinomycin at the late gastrula and the prism stages but not at the pluteus stage. K⁺ permeability probably increases after the prism stage to activate H⁺ release.     

Effects of phloridzin, metavanadate and oligomycin on membrane bound (Na++ K++ Mg2+)ATPase activity in sugar beet roots

To clarify the reaction mechanism of a (Na⁺+ K⁺+ Mg²⁺)ATPase activity in sugar beet roots ( Beta vulgaris L. cv. Monohill) phloridzin, oligomycin (inhibitors of animal ATPases) and metavanadate (NH₄VO₃) have been used. Kinetic studies showed that: 1) Phloridzin inhibition is uncompetitive with respect to MgATP and not influenced by Na⁺ or K⁺. 2) This inhibition is only found in preparations made in the absence of sucrose. 3) Oligomycin and vanadate inhibit the ATPase in different ways. Omission of sucrose from the preparation medium favours vanadate inhibition but suppresses oligomycin inhibition. 4) The kinetic pattern of the Na⁺ activation of the ATPase differs in preparations made in the absence and presence of sucrose, but that of K⁺ activation is the same. – These results indicate that inclusion as against omission of sucrose from the preparation medium causes a conformational change of the membrane fragments/vesicles, which then expose different surfaces to the surrounding medium.     

Axonal Contact Regulates Expression of α2 and β2 Isoforms of Na+,K+-ATPase in Schwann Cells: Adhesion Molecules and Nerve Regeneration

Abstract: Three isoforms of catalytic α subunits and two isoforms of β subunits of Na⁺,K⁺-ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na⁺,K⁺-ATPase was highly resistant to ouabain. The ouabain-resistant α1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na⁺,K⁺-ATPase activity. After sciatic nerve injury, the α3 and β1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, α2 and β2 isoform expression and Na⁺,K⁺-ATPase activity sensitive to pyrithiamine (a specific inhibitor of the α2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that α3 and β1 isoforms are exclusive for the axon and α2 and β2 isoforms are exclusive for the Schwann cell, although axonal contact regulates α2 and β2 isoform expressions. Because the β2 isoform of Na⁺,K⁺-ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/β2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/β2 may act as an adhesion molecule in peripheral nerve regeneration.     

Heterogeneous Na+ Sensitivity of Na+,K+-ATPase Isoenzymes in Whole Brain Membranes

Abstract: The Na⁺ sensitivity of whole brain membrane Na⁺,K⁺-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 m M Na⁺, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10⁻⁵ and 10⁻⁷ M ), we were able to discriminate Na⁺ sensitivity of Na⁺, K⁺-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, α1, exhibited high Na⁺ sensitivity [ K _a of 3.88 ± 0.25 m M Na⁺ and a Hill coefficient ( n ) of 1.98 ± 0.13]; the ouabain high-affinity isoform, α2, had two Na⁺ sensitivities, a high ( K _a of 4.98 ± 0.2 m M Na⁺ and n of 1.34 ± 0.10) and a low ( K _a of 28 ± 0.5 m M Na⁺ and an n of 1.92 ± 0.18) Na⁺ sensitivity activated above a thresh old (22 ± 0.3 m M Na⁺); and the ouabain very-high-affinity isoform, α3, was resolved by two processes and appears to have two Na⁺ sensitivities (apparent K _a values of 3.5 and 20 m M Na⁺). We show that Na⁺ dependence in the absence of ouabain is the result of at least of five Na⁺ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.     

Dexamethasone Increases Adrenalectomy-Depressed Na+,K+-ATPase mRNA and Ouabain Binding in Spinal Cord Ventral Horn

Abstract: The Na⁺,K⁺-ATPase plays a key role in the regulation of ion fluxes and membrane repolarization in the CNS. We have studied glucocorticoid effects on biosynthesis of the Na⁺,K⁺-ATPase and on ouabain binding in the ventral horn of the spinal cord using intact rats, adrenalectomized (ADX) rats, and ADX rats receiving dexamethasone (ADX + DEX) during 4 days. Cryostat sections from spinal cords were incubated with a ³⁵S-oligonucleotide coding for the α₃-subunit or a ³H-cDNA coding for the β₁-subunit of the Na⁺,K⁺-ATPase using in situ hybridization techniques. In ventral horn motoneurons, grain density per cell and grain density per area of some for both probes were slightly reduced in ADX rats but significantly increased in the ADX + DEX group, using ANOVA and the Bonferroni's test. Statistical analysis of frequency histograms of neuronal densities further indicated a significant shift to the right for intact rats compared with ADX rats for both probes. Concomitantly, [³H]ouabain binding to membrane preparations from ventral horns was reduced in ADX rats and restored to normal by DEX administration. No effect of adrenalectomy or DEX treatment was obtained in the dorsal horn. In conclusion, glucocorticoids positively modulate the mRNA for the α₃-subunit and the β₁-subunit of the Na⁺,K⁺-ATPase and recover ouabain binding to normal values. The increments of the synthesis and activity of an enzyme affecting membrane repolarization and synaptic neurotransmission are consistent with the alleged stimulatory effect of glucocorticoids on spinal cord function.     

Salinity effects on oxygen consumption, gill Na+, K+-ATPase and ion regulation in juvenile coho salmon

The metabolic response of juvenile coho salmon Oncorhynchus kisutch to different salinities was examined, using whole-animal oxygen consumption rates and gill Na⁺, K⁺-ATPase activities as indicators of osmoregulatory energetics. Coho salmon smolts were acclimated to fresh water (FW), isosmotic salinity (ISO, 10‰) and sea water (SW, 28‰) and were sampled for up to 6 weeks for plasma levels of cortisol, glucose and ions (Na⁺, K⁺, Cl⁻), gill Na⁺, K⁺-ATPase activity and oxygen consumption rates. Following an initial adjustment period, plasma constituents in SW fish returned to near-FW values, indicating that the fish were acclimated to SW by day 21. Gill Na⁺, K⁺-ATPase activities on days 21 and 42 were lowest in ISO, higher in FW and highest in SW. This result is consistent with the idea that less energy would be required to maintain ion balance in an isosmotic environment, where the ionic gradients between extracellular fluid and water would be minimal. Oxygen consumption rates of swimming fish (1 body length s⁻¹), however, did not differ significantly between the three test salinities after 6 weeks. The results of this study suggest that the metabolic response of juvenile salmonids to changes in salinity is dependent on life-history stage (e.g. fry v . smolt), and that oxygen consumption rates do not necessarily reflect osmoregulatory costs.     

Changes in the Activities of H+, K+-ATPase and Na+, K+-ATPase in Cultured Cells Derived from Micromeres of Sea Urchin Embryos with Special Reference to Their Roles in Spicule Rod Formation

In cultured cells derived from micromeres isolated at the 16-cell stage of sea urchin embryos, the activity of H⁺, K⁺-ATPase became detectable after 15 hr of culture, when the cells started to form spicules, and then increased reaching a plateau from 25 hr of culture. The Na⁺, K⁺-ATPase activity of isolated micromeres increased to a maximum at 20 hr of culture and thereafter decreased gradually. Allylisothiocyanate, an inhibitor of H⁺, K⁺-ATPase, caused a decrease in intracellular pH (pHi) accompanied by blockage of ⁴⁵Ca deposition in spicule rods in spicule-forming cells at 30 hr of culture. Ouabain and amiloride had scarcely any effect on the pHi or ⁴⁵, deposition. In cultured cells exposed to nifedipine, which blocked ⁴⁵Ca deposition in spicule rods, allylisothiocyanate did not cause any decrease in pHi. These results show that H⁺, which is generated in the overall reaction to produce CaCO₃ from Ca²⁺ and HCO₃⁻, is probably released from the cells mainly in the reaction catalyzed by H⁺, K⁺-ATPase to maintain successive production of CaCO₃.     

Ionic regulation and Na+–K+-ATPase activity in gills and kidney of the freshwater stingray Paratrygon aiereba living in white and blackwaters in the Amazon Basin

During low-water period, freshwater stingray Paratrygon aiereba collected in the whitewater (WW) of the River Amazon showed higher urea content, osmolality, Na⁺ and Cl⁻ concentrations in plasma and perivisceral fluid than those caught in blackwater (BW) of the River Negro. Gills and kidney Na⁺–K⁺-ATPase activities were significantly lower in WW than in BW fish. The high level of kidney Na⁺–K⁺-ATPase activity in P. aiereba may minimize ion loss and generate diluted solute-free urine in ion-poor BW environment.     

Ionic regulation and Na+,K+-ATPase activity in gills and kidney of the Antarctic aglomerular cod icefish exposed to dilute sea water

When Notothenia neglecta was exposed to diluted, half strength, sea water for 6 h or 10 days, serum concentrations of Cl^-, Na⁺, K⁺ and Mg²⁺ did not differ from those of sea water controls. This indicates that the fish were capable of both short- and long-term regulation. Renal Na⁺,K⁺-ATPase activity decreased after a 6 h exposure to diluted sea water, but there were no differences between diluted sea water and controls after 10 days of exposure.