Cryoscopic studies in fluoride-Oxide-Silica systems: Part II. systems containing Mg2+, Ca2+, Ba2+ and Pb2+

The depressions of the freezing temperatures of MgF₂, CaF₂, and BaF₂ by adding 2MO · SiO₂, 3MO · 2SiO₂, MO · SiO₂, 2MO · 3SiO₂, and MO · 2SiO₂ where M = Mg, Ca and Ba, have been measured, as have the depressions of the freezing temperature of PbF₂ resulting from additions of 2Pb · SiO₂, 3PbO · 2SiO₂ and PbO · SiO₂. The variations of the activities of the fluorides with liquidus composition have been calculated. These are shown to be in good agreement with a proposed theoretical model of the constitutions of these melts. In the alkaline earth systems with MO/SiO₂ > 1.5 linear chain silicate ions and free F⁻ ions are postulated and in melts of MO/SiO₂ < 1.5 reaction between F⁻ and silicate ions to form polyfluorosilicate anions is postulated. These conclusions are in agreement with those drawn from infrared absorption studies of CaF₂-CaO-SiO₂ glasses. The activity behavior in the reciprocal systems MF₂-M′O · SiO₂ and M′F₂-MO · SiO₂ is explained in terms of polymerization and preferred ionic association effects within the melts. In the lead fluoride-silicate systems fluorination of the silicate ions occurs at PbO/SiO₂ = 2 and, in contrast with the alkali and alkaline earth systems, it appears that polyfluorosilicate anions and free O²⁻ anions can coexist in lead fluorosilicate melts.     

Kinetic studies of the effects of K+, Na+ and Li+ on the catalytic activity of human erythrocyte pyridoxal kinase

Kinetic studies were conducted to examine the effects of K+, Na+ and Li+ on human erythrocyte pyridoxal kinase (PK) activity. A dialyzed hemolysate served as the PK source. The substrates used were pyridoxal (PL) and ATP. Determination of the enzymatic activity was based on HPLC separation and fluorimetric detection of PL and pyridoxal 5'-phosphate as semicarbazone derivatives. In comparison to the poor activity of PK assayed without monovalent cation, all tested cations are activators. Among them, K+ is the most effective, improving both PK affinity for the substrates and maximal velocity. Na+ increases maximal velocity and PK affinity for ATP but decreases it for PL. Li+ is a poor activator which seems to modify the enzymatic mechanism from a random to an ordered sequential pattern with ATP bound before PL. Results suggest that K+ and Na+ bind to PK on the same site while Li+ binds on another site. This hypothesis and the mechanism of monovalent cation-PK interaction are compared to other well-known K(+)-activated enzymes.     

Overexpression of the Na+,K+-ATPase alpha1 subunit increases Na+,K+-ATPase function in A549 cells

Ron (the receptor for Macrophage Stimulating Protein) has never been implicated before in human malignancies or in cell transformation. In this report we show that Ron can acquire oncogenic potential by means of two amino acid substitutions-D1232V and M1254T-affecting highly conserved residues in the tyrosine kinase domain. The same mutations in Kit and Ret have been found associated with two human malignancies, mastocytosis and Multiple Endocrine Neoplasia type 2B (MEN2B), respectively. Both mutations caused Ron-mediated transformation of 3T3 fibroblasts and tumour formation in nude mice. Moreover, cells transformed by the oncogenic Ron mutants displayed high metastatic potential. The Ron mutant receptors were constitutively active and the catalytic efficiency of the mutated kinase was higher than that of wild-type Ron. Oncogenic Ron mutants enhanced activation of the Ras/MAPK cascade with respect to wild type Ron, without affecting the JNK/SAPK pathway. Expression of Ron mutants in 3T3 fibroblasts led to different patterns of tyrosine-phos-phorylated proteins. These data show that point mutations altering catalytic properties and possibly substrate specificity of the Ron kinase may force cells toward tumorigenesis and metastasis.     

Separation and characterization of Na+,K(+)-ATPase containing vesicles

Na+,K(+)-ATPase was reconstituted in vesicles prepared by a dialysis method. Ion-exchange chromatography was used to obtain well characterized fractions from the inhomogeneous vesicle preparation. Lipid and protein content was determined by optical methods during the elution process. It was possible to separate fractions with distinct enzymatic and transport activities. A protocol was set up, which allowed to calculate the average number of 5-IAF labeled ion pumps per vesicle in the different fractions. The dependence of the number of protein molecules per vesicle was studied as function of the initial protein concentration added to the lipid solution before dialysis. The transport activity disappears completely at very low protein concentrations (3.3 micrograms protein per mg lipid). This observation is in favor of the proposal discussed in the literature, that the heterodimer (alpha beta)2 is the transport-active form of the Na+,K(+)-ATPase. The presented method can be applied to all reconstituted vesicle preparations in which the proteins can be labeled quantitatively with a fluorescence dye.     

Na+,K+-ATPase inhibitors in rat urine: origins and physiological significance

To identify the origins and structures of mammalian tissue-derived Na+,K+-ATPase inhibitors, we investigated the tissue distribution of inhibitors in rats. Among many tissues tested, urine was found to contain high levels of many inhibitors. The structures of the two major inhibitors were identified as neoconvalloside and periplogenin monorhamnoside, which are derivatives of strophanthidin. Urinary levels of these inhibitors, however, decreased considerably after changing the diet from the regular diet to purified synthetic diet, suggesting that the majority of the urinary inhibitors are of dietary origin. Investigation of the ingredients of the diet further revealed that alfalfa meal and ground oats are the major sources of these cardiac glycosides. As to the physiological relevance of the cardiac glycosides, a low concentration (1-50 nM) of ouabain dose-dependently enhanced aldosterone secretion from adrenal glomerulosa cells by an increase in local renin release. Ouabain was also found to be involved in AT2 receptor-specific expression in rat PC12W cells through an increment in intracellular Na+. These results suggest that Na+,K+-ATPase inhibitors, regardless of the source, are involved in the regulation of blood pressure.     

Dephosphorylation kinetics of pig kidney Na+,K+-ATPase

The kinetics of K+-stimulated dephosphorylation of the Na+,K+-ATPase were investigated at pH 7.4, 24 degrees C, and an ATP concentration of 1.0 mM via the stopped-flow technique using the fluorescent label RH421. Two different mixing procedures were used: (a) premixing with ATP to allow phosphorylation to go to completion, followed by mixing with KCl; and (b) simultaneous mixing with ATP and KCl. Using mixing procedure (a), the dephosphorylation rate constant of enzyme complexed with K+ ions could be determined directly to be 190 s-1).     

Coordinate interaction between ATP-sensitive K+ channel and Na+, K+-ATPase modulates ischemic preconditioning

BACKGROUND: We reported that digoxin abolishes the infarct size (IS)-limiting effect of ischemic preconditioning (IPC). Because ATP-sensitive K+ (KATP) channels are involved in IPC, we studied whether Na+,K+-ATPase and KATP channels functionally interact, thereby modulating IPC. METHODS AND RESULTS: Rabbits received 30 minutes of coronary artery occlusion followed by 3 hours of reperfusion. IPC was elicited by 5 minutes of occlusion followed by 10 minutes of reperfusion. The IS, expressed as a percentage of the area at risk, was 40.2+/-2.8% in control and 39.8+/-5.0% in digoxin pretreatment rabbits. Both IPC and pretreatment with cromakalim, a KATP channel opener, reduced IS to 11.8+/-1.8% and 13.4+/-2.6% (P<0. 05 versus control). Digoxin abolished the reduction in IS induced by IPC (33.5+/-3.3%), whereas it did not change that induced by cromakalim (18.8+/-3.0%). In patch-clamp experiments, digoxin was found to inhibit the opening of KATP channels in single ventricular myocytes in which ATP depletion had been induced by metabolic stress. In contrast, digoxin had little effect on the channel opening induced by cromakalim. Moreover, the inhibitory action of digoxin on channel activities was dependent on subsarcolemmal ATP concentration. CONCLUSIONS: The IS-limiting effect of IPC is modulated by an interaction between KATP channels and Na+,K+-ATPase through subsarcolemmal ATP.     

Inward-directed current generated by the Na+,K+ pump in Na(+)- and K(+)-free medium

In Na(+)- and K(+)-free solution, an inward-directed current can be detected in Xenopus oocytes, which is inhibited by cardiac glycosides and activated by ATP. Therefore, it is assumed to be generated by the Na+,K+ pump. At negative membrane potentials, the pump current increases with more negative potentials and with increasing [H+] in the external medium. This current is not observed when Mg2+ instead of Ba2+ is the only divalent cation present in the bath medium, and it does not depend on whether Na+ or K+ is present internally. At 5 to 10 mM Na+ externally, maximum pump-generated current is obtained while no current can be detected in presence of physiological [Na+]. It is suggested that in low-Na+ and K(+)-free medium the Na+,K+ pump molecule can either form a conductive pathway that is permeable to Ba2+ or protons or operate in its conventional transport mode accepting Ba2+ as a K+ congener. A reversed pump mode or an electrogenic uncoupled Na(+)-efflux mode is excluded.     

Na+,K+-ATPase activity in cultured bovine retinal pigment epithelium

Ibogaine (Endabuse) is a psychoactive indole alkaloid found in the West African shrub, Tabernanthe iboga. This drug interrupts cocaine and amphetamine abuse and has been proposed for treatment of addiction to these stimulants. However, the mechanism of action that explains its pharmacological properties is unclear. Since previous studies demonstrated differential effects of psychotomimetic drugs (cocaine and methamphetamine) on neuropeptides such as neurotensin (NT), the present study was designed to determine: (1) the effects of ibogaine on striatal, nigral, cortical, and accumbens neurotensin-like immunoreactivity (NTLI); (2) the effects of selective dopamine antagonists on ibogaine-induced changes in NT concentrations in these brain areas; and (3) the effects of ibogaine pretreatment on cocaine-induced changes in striatal, nigral, cortical and accumbens NTLI content. Ibogaine treatments profoundly affected NT systems by increasing striatal, nigral, and accumbens NTLI content 12 h after the last drug administration. In contrast, NTLI concentrations were not significantly increased in the frontal cortex after ibogaine treatment. The ibogaine-induced increases in NTLI in striatum, nucleus accumbens and substantia nigra were blocked by coadministration of the selective D1 receptor antagonist, SCH 23390. The D2 receptor antagonist, eticlopride, blocked the ibogaine-induced increase in nigral NTLI, but not in striatum and nucleus accumbens. Ibogaine pretreatment significantly blocked the striatal and nigral increases of NTLI resulting from a single cocaine administration. Whereas many of the responses by NT systems to ibogaine resembled those which occur after cocaine, there were also some important differences. These data suggest that NT may contribute to an interaction between ibogaine and the DA system and may participate in the pharmacological actions of this drug.     

Bufodienolides as endogenous Na+, K+-ATPase inhibitors: biosynthesis in bovine and rat adrenals

The biosynthesis of digitalis-like compounds (DLC) was determined in bovine and rat adrenal homogenates by following changes in the concentration of DLC using three independent sensitive bioassays: inhibition of [3H]-ouabain binding to red blood cells and competitive ouabain and bufalin ELISA. The amounts of DLC in bovine and rat adrenal homogenates, as measured by the two first bioassays, increased with time when the mixtures were incubated under tissue culture conditions. These results suggest that Na+, K+-ATPase inhibitors which interact with ouabain antibodies, but not those which interact with bufalin antibodies, are synthesized in bovine and rat adrenals.     

Functional analysis and tissue-specific expression of Drosophila Na+,K+-ATPase subunits

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 beta subunits of Na+,K+-ATPase from various other species. In this study we have isolated a new Drosophila Na+,K+-ATPase alpha subunit cDNA clone (PSalpha; GenBank accession no. AF044974) and demonstrate expression of functional Na+,K+-ATPase activity when PSalpha 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.     

A Glu329-->Gln variant of the alpha-subunit of the rat kidney Na+,K(+)-ATPase can sustain active transport of Na+ and K+ and Na+,K(+)-activated ATP hydrolysis with normal turnover number

An allelic variant of the ouabain-insensitive rat kidney Na+,K(+)-ATPase alpha 1-isoform was identified by chance in a cDNA library. The variant differed from the wild-type rat kidney Na+,K(+)-ATPase by a single G-to-C base substitution in the cDNA, which on amino acid level gave rise to a glutamine in place of the glutamate residue Glu329 previously suggested as a likely donator of oxygen ligands for Na+ and K+ binding. The variant cDNA was transfected into COS-1 cells and the transfectants expanded with success into stable cell lines that were able to grow in the presence of a concentration of ouabain highly cytotoxic to the parental cells containing only the endogenous COS-1 cell Na+,K(+)-ATPase. Under these conditions, the viability of the cells depended on the cation transport mediated by the ouabain-insensitive Glu329-->Gln variant, whose cDNA was shown by polymerase chain reaction amplification to be stably integrated into the COS-1 cell genome. The maximum specific ATP hydrolysis activity of isolated plasma membranes of the Glu329-->Gln variant did not differ significantly from that of plasma membranes containing the wild type. A method was established for measurement of the phosphorylation capacity of the expressed Glu329-->Gln variant and wild-type enzyme, and it was thereby demonstrated that the variant had a turnover number similar if not identical to that of the wild-type.     

Complexes of carboxymethylcellulose in water. Part 2. Co2+ and Al3+ remediation studies of wastewaters with Co2+, Al3+, Cu2+, VO2+ and Mo6+

The properties of carboxymethylcellulose (CMC) complexes and Co²⁺ and Al³⁺ are reported in this work. The complexing power of CMC was greater to Al³⁺ than to Co²⁺, although it was not possible to determine some of the equilibrium constants. The infrared (IR) spectroscopy and thermal analysis helped in showing the existence of these complexes in the solid state. The films observed by scanning electron microscopy (SEM) provided a certainty that the chains of the biopolymer were not extensively broken by the use of strong mineral acid employed in some of the experimental steps of this study.Two water solutions — bidistilled and deionized water and an Iraí River, Curitiba, PR (Brazil) sample — were obtained by adding metal salts of Al³⁺, Co²⁺, Cu²⁺, Mo⁶⁺(initially in the form of molybdate) and VO²⁺ to which CMC was later added as a remediation agent. At different times, aliquots of those water samples were analyzed for their metal contents and showed ability to sequester different percentages of each of the metal ions, therefore, rendering the water samples within the Brazilian and Spanish standards for potable water (varying from < 0.3 to 5 mg/L depending on toxicity). The CMC complexes could be recovered by mechanical removal at the pH where these complexes are not very soluble. This process can be applied to municipal wasterwater treatment plants as CMC is a more cost-effective and non toxic alternative material than commercial employed alum. The metals can be recycled after the decomplexing process from the recovered solid complexes and with the additional benefit of using CMC that it will leave no trace of Al³⁺ ions in the water rising from the use of alum.     

Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.     

Stopped-flow kinetic investigations of conformational changes of pig kidney Na+,K+-ATPase

The kinetics of Na+-dependent partial reactions of the Na+,K+-ATPase were investigated via the stopped-flow technique using the fluorescent labels RH421 and BIPM. After the enzyme is mixed with MgATP, both labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau1 approximately 180 s-1 (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3 --> E2P(Na+)3 + ADP). The rate of the phosphorylation reaction measured by the acid quenched-flow technique was 190 s-1 at 100 microM ATP, suggesting that phosphorylation controls the kinetics of the RH421 signal and that the conformational change is very fast (>/=600 s-1). The rate of the RH421 signal was optimal at pH 7.5. The Na+ concentration dependence of 1/tau1 showed half-saturation at a Na+ concentration of 8-10 mM with positive cooperativity involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high affinity ATP binding site determined from the ATP concentration dependence of 1/tau1 was 7.0 (+/-0.6) microM, while the apparent Kd for the low affinity site and the rate constant for the E2 to E1 conformational change evaluated in the absence of Mg2+ were 143 (+/-17) microM and 相似文献   

Modifications induced by acylphosphatase in the functional properties of heart sarcolemma Na+,K+ pump

When hepatic stellate cells (Ito cells, fat-storing cells) were incubated with adrenomedullin, they underwent relaxation as monitored by the silicone-rubber membrane method; 43%, 65% and 87% of stellate cells relaxed 5, 10 and 20 min, respectively, after addition of 10(-6) M adrenomedullin. Adrenomedullin also triggered the dissociation of F-actin and induced transformation of stellate cells to dendritic cell-like structure. When incubated with 10(-6) M of adrenomedullin for 30 min, cellular levels of cAMP increased from the basal value of 10.2 +/- 1.4 to 107 +/- 2.8 pmol/2 x 10(5) cells without affecting cGMP levels. The reaction occurred dose-dependently and was inhibited by an antagonist of calcitonin gene-related peptide. Adrenomedullin had negligible effects on DNA and protein synthesis in proliferating stellate cells. Thus, adrenomedullin is a potent relaxing peptide to hepatic stellate cells and may contribute to the regulation of sinusoidal microcirculation.     

Transformations in α 2+γ titanium aluminide alloys containing molybdenum: Part I. Solidification behavior

The solidification structures of 12 alloys in the Ti-Al-Mo system with Al contents ranging from 44 to 50 at. pct and Mo contents ranging from 2 to 6 at. pct have been characterized metallographically and, for composition gradients, by electron probe microanalysis. All alloys solidify dendritically through sequences that fall into four distinct categories: alloys that solidify completely into the β phase field, alloys that solidify first into an L+β phase field and finally into an α+β phase field through an L+β+γ region, alloys that solidify first into a L+β phase field and finally form α+β+γ structures, and alloys that solidify into a L+α phase field. Postsolidification transformations occur as consequence of transitions from the high-temperature β, α+β, and α+γ phase fields to low-temperature α+β+γ or β+γ region. A variety of phase distributions result, such as Widmanstätten α ₂+B2 structures from the α → α+β reaction followed by ordering, lamellar α ₂+γ structures from the α → α+γ reaction followed by ordering of the α phase, eutectoid B2+γ structures from the α → B2+γ reaction, and platelike γ structures from the β → β+γ reaction. The former are observed in Al lean alloys, while the latter are present in Mo rich alloys.     

Purification and characterization of a Na+, K+ ATPase inhibitor found in an endotoxin of Leptospira interrogans

We showed previously that the glycolipoprotein fraction prepared from Leptospira interrogans inhibited the Na+,K+ ATPase enzyme purified from brain or kidney and in isolated nephron segments (M. Younes-Ibrahim, P. Burth, M. V. Castro Faria, B. Buffin-Meyer, S. Marsy, C. Barlet-Bas, L. Cheval, and A. Doucet, C. R. Acad. Sci. Paris Ser. III 318:619-625, 1995). In the present communication, we have demonstrated that unsaturated fatty acids such as oleic and palmitoleic acids, which are adsorbed to this fraction, are effective inhibitors of the enzyme.