Rottlerin Inhibits (Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>)-ATPase Activity in Brain Tissue and Alters <Emphasis Type="SmallCaps">d</Emphasis>-Aspartate Dependent Redistribution of Glutamate Transporter GLAST in Cultured Astrocytes

The naturally occurring toxin rottlerin has been used by other laboratories as a specific inhibitor of protein kinase C-delta (PKC-δ) to obtain evidence that the activity-dependent distribution of glutamate transporter GLAST is regulated by PKC-δ mediated phosphorylation. Using immunofluorescence labelling for GLAST and deconvolution microscopy we have observed that d-aspartate-induced redistribution of GLAST towards the plasma membranes of cultured astrocytes was abolished by rottlerin. In brain tissue in vitro, rottlerin reduced apparent activity of (Na⁺, K⁺)-dependent ATPase (Na⁺, K⁺-ATPase) and increased oxygen consumption in accordance with its known activity as an uncoupler of oxidative phosphorylation (“metabolic poison”). Rottlerin also inhibited Na⁺, K⁺-ATPase in cultured astrocytes. As the glutamate transport critically depends on energy metabolism and on the activity of Na⁺, K⁺-ATPase in particular, we suggest that the metabolic toxicity of rottlerin and/or the decreased activity of the Na⁺, K⁺-ATPase could explain both the glutamate transport inhibition and altered GLAST distribution caused by rottlerin even without any involvement of PKC-δ-catalysed phosphorylation in the process.     

Plasma methylglyoxal and glyoxal are elevated and related to early membrane alteration in young, complication-free patients with Type 1 diabetes

The reactive aldehydes methylglyoxal and glyoxal, arise from enzymatic and non-enzymatic degradation of glucose, lipid and protein catabolism, and lipid peroxidation. In Type 1 diabetes mellitus (T1DM) where hyperglycemia, oxidative stress, and lipid peroxidation are common, these aldehydes may be elevated. These aldehydes form advanced glycation end products (AGEs) with proteins that are implicated in diabetic complications. We measured plasma methylglyoxal and glyoxal in young, complication-free T1DM patients and assessed activity of the ubiquitous membrane enzyme, Na⁺/K⁺ ATPase. A total of 56 patients with TIDM (DM group), 6–22 years, and 18 non-diabetics (ND group), 6–21 years, were enrolled. Mean plasma A1C (%) was higher in the DM group (8.5 ± 1.3) as compared to the ND group (5.0 ± 0.3). Using a novel liquid chromatography-mass spectrophotometry method, we found that mean plasma methylglyoxal (nmol/l) and glyoxal levels (nmol/l), respectively, were higher in the DM group (841.7 ± 237.7, 1051.8 ± 515.2) versus the ND group (439.2 ± 90.1, 328.2 ± 207.5). Erythrocyte membrane Na⁺/K⁺ ATPase activity (nmol NADH oxidized/min/mg protein) was elevated in the DM group (4.47 ± 0.98) compared to the ND group (2.16 ± 0.59). A1C correlated with plasma methylglyoxal and glyoxal, and both aldehydes correlated with each other. A high correlation of A1C with Na⁺/K⁺ ATPase activity, and a regression analysis showing A1C as a good predictor of activity of this enzyme, point to a role for glucose in membrane alteration. In complication-free patients, increased plasma methylglyoxal, plasma glyoxal, and erythrocyte Na⁺/K⁺ ATPase activity may foretell future diabetic complications, and emphasize a need for aggressive management.     

The Identification of the Sodium Pump

The identification of the sodium potassium pump as a Na⁺, K⁺-ATPase is described.     

The Identification of the Sodium Pump

The identification of the sodium potassium pump as a Na⁺, K⁺-ATPase is described.     

Different properties of two brain extracts separated in sephadex G-50 that modify synaptosomal ATPase activities

We have previously reported that Na⁺,K⁺-ATPase of nerve ending membranes is stimulated by catecholamines only in the presence of a brain soluble fraction. The filtration of this soluble fraction through Sephadex G-50 permitted the separation of two extracts of maximal UV absorbance (peaks I and II) which showed different effects on ATPases. Peak I stimulated both Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities and peak II inhibited Na⁺,K⁺-ATPase activity. We have now studied the activity of ATPases in the presence of the whole eluate obtained from the Sephadex G-50 column. It was observed that maximal effects on ATPases were obtained with peaks I and II. Peak I and peak II fractions were unable to modify the activity of acetylcholinesterase or 5-nucleotidase present in the synaptosomal membranes. The stimulatory effect of peak I on ATPases was concentration dependent (up to 1100), it was stable at different pHs and it was reverted by catecholamines. The inhibitory effect of peak II on Na⁺,K⁺-ATPase was concentration dependent (up to 150,000), it was stable only at acid pH, and it was partially reverted by catecholamines. These findings indicate that the factors responsible for the effects of peaks I and II have different properties and that their actions on ATPases show enzyme specifity.     

The effects of captopril and losartan on erythrocyte membrane Na+/K+- ATPase activity in experimental diabetes mellitus

Diabetes mellitus induces a decrease in sodium potassium-adenosine triphosphatase (Na⁺/K⁺- ATPase) activity in several tissues in the rat and red blood cells (RBC) and nervous tissue in human patients. This decrease in Na⁺/K⁺- ATPase activity is thought to play a role in the development of long term complications of the disease. Angiotensin enzyme inhibitors (ACEi) and angiotensin-II receptor antagonists (ARBs) reduce proteinuria and retard the progression of renal failure in patients with IDDM and diabetic rats. We investigated the effects of captopril and losartan, which are used in the treatment of diabetic nephropathy, on Na⁺/K⁺- ATPase activity. Captopril had an inhibitory effect on red cell plasma membrane Na⁺/K⁺ ATPase activity, but losartan did not. Our study draws attention to the inhibitory effect of captopril on Na⁺/K⁺ ATPase activity. Micro and macro vascular complications are preceeding mortality and morbidity causes in diabetes mellitus. There is a strong relationship between the decrease in Na⁺/K⁺ ATPase activity and hypertension. The non-sulphydryl containing ACEi and ARBs must be the choice of treatment in hypertensive diabetic patients and diabetic nephropathy.     

Positive Darwinian selection results in resistance to cardioactive toxins in true toads (Anura: Bufonidae)

Members of the Family Bufonidae, true toads, are famous for their endogenously synthesized cardioactive steroids that serve as defensive toxins. Evolution of resistance to these toxins is not understood. We sequenced a key region of the toxin''s binding site in the Na⁺/K⁺ ATPase for relevant taxa representing Hyloidea (including bufonids), Ranoidea and Archaeobatrachia and tested for positive selection in a phylogenetic context. Bufonidae were distinct from other Hyloidea at 4–6 of 12 sites and, with one exception, had a homologous amino acid sequence. Melanophryniscus stelzneri had a distinct sequence, consistent with other independent evidence for a differentiated toxin. Tests within Bufonidae detected positive selection within the binding region, providing, to our knowledge, the first evidence of this type for positive selection within Amphibia. There was no evidence for positive selection on Bufonidae or M. stelzneri lineages. Sequence change in Leptodactylus ocellatus, a leptodactylid predator of Bufonidae, provides a molecular basis for predator resistance possibly associated with gene duplication.     

Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytesin vitro andin vivo

Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K⁺ or Rb⁺. In pure Na⁺ media, furosemide accelerated cell Na⁺ gain and retarded cellular K⁺ loss. External K⁺ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb⁺ accelerated the Na⁺ gain like K⁺, but did not affect the K⁺ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes mediates an equimolar extrusion of Na⁺ and K⁺ in Na⁺ media (Na⁺/K⁺ cotransport), a 1:1 K⁺/K⁺ (K⁺/Rb⁺) and Na⁺/Na⁺ exchange progressively appearing upon increasing external K⁺ (Rb⁺) concentrations to 5mm. The effect of furosemide (or external K⁺/Rb⁺) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na⁺ media, or with a cell swelling, indicating that the furosemide-sensitive Na⁺/K⁺ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K⁺ or Rb⁺. Thein vivo red cell K⁺ content was negatively correlated to the rate of furosemide-sensitive K⁺ (Rb⁺) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K⁺ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na⁺ content showed no correlation to the activity of the furosemide-sensitive transport system.     

The regulation of total creatine content in a myoblast cell line

Total cellular creatine content is an important bioenergetic parameter in skeletal muscle. To understand its regulation we investigated creatine transport and accumulation in the G8 cultured skeletal myoblast line. Like other cell types, these contain a creatine transporter, whose activity, measured using a radiolabelling technique, was saturable (Km = 110 ± 25 M) and largely dependent on extracellular [Na⁺]. To study sustained influences on steady state creatine concentration we measured total cellular creatine content using a fluorimetric method in 48 h incubations. We found that the total cellular creatine content was relatively independent of extracellular creatine concentration, consistent with high affinity sodium-dependent uptake balanced by slow passive efflux. Accordingly, in creatine-free incubations net creatine efflux was slow ( 5 ± 1 % of basal creatine content per day over 6 days), while creatine content in 48 h incubations was reduced by 28 ± 13% of control by the Na⁺,K⁺-ATPase inhibitor ouabain. Creatine accumulation after 48 h was stimulated by treatment with the mixed - and -adrenergic agonist noradrenaline, the -adrenergic agonist isoproterenol, the ₂-agonist clenbuterol and the cAMP analogue N⁶,2-O-dibutyryladenosine 3,5-cyclic monophosphate, but was unaffected by the ₁ adrenergic agonist methoxamine. The noradrenaline enhancement of creatine accumulation at 48 h was inhibited by the mixed - and -antagonist labetalol and by the -antagonist propranolol, but was unaffected by the ₂ antagonist phentolamine; greater inhibition was caused by the ₂ antagonist butoxamine than the ₁ antagonist atenolol. Creatine accumulation at 48 h was increased to 230 ± 6% of control by insulin and by 140 ± 13% by IGF-I (both at 3 nM). Creatine accumulation at 48 h was also increased to 280 ± 40% of control by 3,3,5-triiodothyronine (at 70 M) and to 220 ± 35% of control by amylin (60 nM). As 3,3,5-triiodothyronine, amylin and isoproterenol all stimulate the Na⁺,K⁺-ATPase, we suggest that they stimulate Na⁺-creatine cotransport indirectly by increasing the transmembrane [Na⁺] concentration gradient and membrane potential.Abbreviations IGF-I insulin-like growth factor I - IGF-II insulin-like growth factor II - T₃ 3,3,5-triiodothyronine - CGRP calcitonin gene-related peptide     

Quantitation of Na+/K+-ATpase and glucose transporter isoforms in rat adipocyte plasma membrane by immunogold labeling

We have quantitated and studied the topology of isoforms of the Na⁺/K⁺-ATPase and of the glucose transporter in rat adipocyte plasma membranes.Adipocytes were incubated with or without insulin for 15 min. Sheets of native plasma membrane, with the cytoplasmic face exposed, were prepared by adsorption to EM grids. Grids were incubated in parallel with monoclonal antibodies against the Na⁺/ K⁺-ATPase isoforms 1 and 2, and the glucose transporter isoforms GLUT1 and GLUT4, followed by immunogold labeling, negative staining and quantitation by counting of the gold particles in electron micrographs. In addition, the distribution of glucose transporters and Na⁺/K⁺-ATPase isoforms in subcellular membrane fractions prepared by an established fractionation procedure was monitored by Western blotting.We found that the Na⁺/K⁺-ATPases and the glucose transporters were confined to the planar part of the plasma membrane, without association to caveolar invaginations.The vast majority of the Na⁺/K⁺-ATPase molecules in the adipocyte plasma membrane were of the 2 isoform; GLUT4 was the dominating glucose transporter isoform.The total number of Na⁺/K⁺-ATPase molecules labeled in the plasma membrane was 3.5×10⁵ per cell, independent of insulin stimulation. Concomitantly, insulin increased GLUT4 labeling sevenfold to a value of 3.5×10⁵ per cell.The authors wish to thank Ulla Blankensteiner, Jonna Harpøth and Lisette Hansen for their skillful technical assistance and Birgit Risto for excellent work with the photographic prints. The 1 and 2 antibodies were kindly donated by K.W. Sweadner, Boston, and the F18 and F27 antibodies were granted by Peer N. Jørgensen, Novo-Nordisk, Bagsværd, Denmark. This work was supported by The NOVO Foundation, The Nordisk Insulin Foundation, The Danish Diabetes Foundation, The P. Carl Petersen Foundation and the Foundation of 17-12-81.     

Molecular and functional characterization of choline transporter in rat renal tubule epithelial NRK-52E cells

Homeostatic regulation of the plasma choline concentration depends on the effective functioning of a choline transporter in the kidney. However, the nature of the choline transport system in the kidney is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake in the rat renal tubule epithelial cell line NRK-52E. Choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (K_m) of 16.5 μM and a maximal velocity (V_max) of 133.9 pmol/mg protein/min. The V_max value of choline uptake was strongly enhanced in the absence of Na⁺ without any change in K_m values. The increase in choline uptake under Na⁺-free conditions was inhibited by Na⁺/H⁺ exchanger (NHE) inhibitors. Choline uptake was inhibited by the choline uptake inhibitor hemicholinium-3 (HC-3) and organic cations, and was decreased by acidification of the extracellular medium and by intracellular alkalinization. Collapse of the plasma membrane H⁺ electrochemical gradient by a protonophore inhibited choline uptake. NRK-52E cells mainly express mRNA for choline transporter-like proteins (CTL1 and CTL2), and NHE1 and NHE8. CTL1 protein was recognized in both plasma membrane and mitochondria. CTL2 protein was mainly expressed in mitochondria. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in NRK-52E cells and is responsible for choline uptake. This choline transport system uses a directed H⁺ gradient as a driving force, and its transport functions in co-operation with NHE8. Furthermore, the presence of CTL2 in mitochondria provides a potential site for the control of choline oxidation.     

离子转运蛋白调控钝齿棒杆菌离子和pH稳态促进L-精氨酸合成

L-精氨酸是一种半必需氨基酸,广泛应用于食品、制药、饲料等行业。【目的】当前对L-精氨酸生产菌株的研究,极少涉及离子转运领域。在本研究中,发现在发酵时适量添加外源K~+有利于促进钝齿棒杆菌(Corynebacterium crenatum) SYPA5-5合成L-精氨酸。【方法】在C. crenatum SYPA5-5发酵培养基外源添加0.5 g/L和2.5 g/L的K_3PO_4,取对数期发酵样品进行转录组数据分析,挖掘出K~+转运相关的阳离子转运ATP酶CTAP1以及单价阳离子/H~+逆转运蛋白Mrp1A,研究其在C. crenatum SYPA5-5快速合成L-精氨酸阶段,对菌株生长及L-精氨酸合成的影响。【结果】对基因ctap1和mrp1分别进行敲除和过表达,深入研究突变株对L-精氨酸合成的影响。研究发现同时过表达离子转运蛋白CTAP1和Mrp1A更有利于胞内离子、pH稳态和渗透压调节,最终提高L-精氨酸的产量。在补料分批发酵中分别过表达Mrp1A、CTAP1以及同时过表达Mrp1A和CTAP1的菌株L-精氨酸产量分别达到61.4 g/L、63.9 g/L和65.3 g/L,产率分别为0.383 g/g、0.392 g/g和0.395 g/g,比C. crenatum SYPA5-5分别提高了34.9%、38.0%和39.1%。【结论】CTAP1是特异性的K~+转运ATP酶,可以将培养基中的K~+运输到胞内。同时Mrp1A可将胞内K~+和Na~+等单价阳离子运输到胞外,将胞外H~+运输至胞内,中和胞内L-精氨酸所导致的碱性环境,从而维持胞内pH稳定。CTAP1和Mrp1A的研究为解析离子转运机制和L-精氨酸合成之间的联系奠定了基础。     

Inter-domain motions of the N-domain of the KdpFABC complex, a P-type ATPase, are not driven by ATP-induced conformational changes

P-type ATPases are involved in the active transport of ions across biological membranes. The KdpFABC complex (P-type ATPase) of Escherichia coli is a high-affinity K+ uptake system that operates only when the cell experiences osmotic stress or K+ limitation. Here, we present the solution structure of the nucleotide binding domain of KdpB (backbone RMSD 0.17 A) and a model of the AMP-PNP binding mode based on intermolecular distance restraints. The calculated AMP-PNP binding mode shows the purine ring of the nucleotide to be "clipped" into the binding pocket via a pi-pi-interaction to F377 on one side and a cation-pi-interaction to K395 on the other. This binding mechanism seems to be conserved in all P-type ATPases, except the heavy metal transporting ATPases (type IB). Thus, we conclude that the Kdp-ATPase (currently type IA) is misgrouped and has more similarities to type III ATPases. The KdpB N-domain is the smallest and simplest known for a P-type ATPase, and represents a minimal example of this functional unit. No evidence of significant conformational changes was observed within the N-domain upon nucleotide binding, thus ruling out a role for ATP-induced conformational changes in the reaction cycle.     

Distribution of Glutamate Transporter GLAST in Membranes of Cultured Astrocytes in the Presence of Glutamate Transport Substrates and ATP

Neurotransmitter l-glutamate released at central synapses is taken up and “recycled” by astrocytes using glutamate transporter molecules such as GLAST and GLT. Glutamate transport is essential for prevention of glutamate neurotoxicity, it is a key regulator of neurotransmitter metabolism and may contribute to mechanisms through which neurons and glia communicate with each other. Using immunocytochemistry and image analysis we have found that extracellular d-aspartate (a typical substrate for glutamate transport) can cause redistribution of GLAST from cytoplasm to the cell membrane. The process appears to involve phosphorylation/dephosphorylation and requires intact cytoskeleton. Glutamate transport ligands l -trans-pyrrolidine-2,4-dicarboxylate and dl-threo-3-benzyloxyaspartate but not anti,endo-3,4-methanopyrrolidine dicarboxylate have produced similar redistribution of GLAST. Several representative ligands for glutamate receptors whether of ionotropic or metabotropic type, were found to have no effect. In addition, extracellular ATP induced formation of GLAST clusters in the cell membranes by a process apparently mediated by P2 receptors. The present data suggest that GLAST can rapidly and specifically respond to changes in the cellular environment thus potentially helping to fine-tune the functions of astrocytes. The authors J.-W. Shin and K. T. D. Nguyen have contributed equally.     

Sargachromanols as inhibitors of Na+/K+ ATPase and isocitrate lyase

Sargachromanols A-P (1-16), 16 meroterpenoids of the chromene class isolated from the brown alga Sargassum siliquastrum, were evaluated for their inhibitory activities toward Na⁺/K⁺ ATPase from porcine cerebral cortex and isocitrate lyase (ICL) from Candida albicans. These studies led to the identification of compounds 4, 6, 8, and 12 as potent Na⁺/K⁺ ATPase inhibitors. Compounds 12, 13, and 16 exhibited moderate ICL inhibitory activity. Compound 12 also showed weak antibacterial activity. The preliminary structure-activity relationship of these compounds is described to elucidate the essential structural requirements.     

Hormonal control of the follicular epithelium during vitellogenin uptake

Summary

Among the many functions of follicle cells in the insect ovary is the regulation of the entrance into the follicle of the vitellogenin circulating in the hemolymph. The vitellogenin enters the follicle via large spaces which appear between the follicle cells. The appearance of these spaces (patency) is a result of a reduction in volume of the follicle cells caused by the action of juvenile hormone which activates a juvenile-hormone-sensitive Na⁺K⁺ ATPase via a pathway involving protein kinase C. A putative juvenile hormone receptor protein has been identified in membranes from follicle cells. An antigonadotropin, a small neuropeptide, antagonizes the action of juvenile hormone on the follicle cells.