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1.
Previous evidence from this laboratory indicated that catecholamines and brain endogenous factors modulate Na+, K+-ATPase activity of the synaptosomal membranes. The filtration of a brain total soluble fraction through Sephadex G-50 permitted the separation of two fractions-peaks I and II-which stimulated and inhibited Na+, K+-ATPase, respectively (Rodríguez de Lores Arnaiz and Antonelli de Gomez de Lima, Neurochem. Res.11, 1986, 933). In order to study tissue specificity a rat kidney total soluble was fractionated in Sephadex G-50 and kidney peak I and II fractions were separated; as control, a total soluble fraction prepared from rat cerebral cortex was also processed. The UV absorbance profile of the kidney total soluble showed two zones and was similar to the profile of the brain total soluble. Synaptosomal membranes Na+, K+- and Mg2+-ATPases were stimulated 60–100% in the presence of kidney and cerebral cortex peak I; Na+, K+-ATPase was inhibited 35–65% by kidney peak II and 60–80% by brain peak II. Mg2+-ATPase activity was not modified by peak II fractions. ATPases activity of a kidney crude microsomal fraction was not modified by kidney peak I or brain peak II, and was slightly increased by kidney peak II or brain peak I. Kidney purified Na+, K+-ATPase was increased 16–20% by brain peak I and II fractions. These findings indicate that modulatory factors of ATPase activity are not exclusive to the brain. On the contrary, there might be tissue specificity with respect to the enzyme source.  相似文献   

2.
Double quantum and triple quantum filtered 23Na nuclear magnetic resonance techniques were used to characterise in detail the isotropic and anisotropic binding and dynamics of intra- and extracellular Na+ in different cellular systems, in the absence and presence of Li+. The kinetics of Li+ influx by different cell types was evaluated. At steady state, astrocytes accumulated more Li+ than red blood cells (RBCs), while a higher intracellular Li+ concentration was found in chromaffin than in SH-SY5Y cells. Anisotropic and isotropic motions were detected for extracellular Na+ in all cellular systems studied. Isotropic intracellular Na+ motions were observed in all types of cells, while anisotropic Na+ motions in the intracellular compartment were only detected in RBCs. 23Na triple quantum signal efficiency for intracellular Na+ was SH-SY5Y > chromaffin > RBCs, while the reverse order was observed for the extracellular ions. 23Na double quantum signal efficiency for intracellular Na+ was non-zero only in RBCs, and for extracellular Na+ the order RBCs > chromaffin > SH-SY5Y cells was observed. Li+ loading generally decreased intracellular Na+ isotropic movements in the cells, except for astrocytes incubated with a low Li+ concentration and increased anisotropic intracellular Na+ movements in RBCs. Li+ effects on the extracellular signals were more complex, reflecting Li+/Na+ competition for isotropic and anisotropic binding sites at the extracellular surface of cell membranes and also at the surface of the gel used for cell immobilisation. These results are relevant and contribute to the interpretation of the in vivo pharmacokinetics and sites of Li+ action.  相似文献   

3.
7Li and 23Na NMR spectra of the respective cations in gelatin and ι-carrageenan gels containing cryptand-[2.1.1] (for Li+) or cryptand-[2.2.2] (for Na+) displayed two transitions: the one at higher frequency corresponded to the cation surrounded by gel, the other to cation inside its appropriately sized cryptand. While binding to cryptands yielded much broader lines and shorter T 1 relaxation times, anisotropic splitting in first order 7Li or 23Na NMR spectra was not detected. Stretching the gels resulted in increasing the anisotropic electric field gradient tensor; thus, the NMR transitions of the cation in the gel were split (removal of degeneracy) to display its characteristic 3:4:3 triplet for spin = 3/2 nuclei. The transitions of the cryptand-bound cations (Li+-cryptand-[2.1.1] and Na+-cryptand-[2.2.2]) showed different extents of interaction with the electric field gradient tensor depending on the composition of the gel matrix. The NMR signal for 7Li+-cryptand-[2.1.1] in stretched gelatin gel showed a five-fold increased splitting as compared to the 7Li+ signal in the reference gel. In stretched ι-carrageenan gels, no anisotropic splitting from the cryptand-bound Li+ was recorded. Steady-state irradiation envelopes or z-spectra showed evidence of Li+ exchange between isotropic (cryptand) and anisotropic (gel) sites only at higher temperatures (55 °C). For Na+ bound to the cryptand-[2.2.2], anisotropic splitting (three-fold smaller compared with the 23Na signal in the reference gel) was only recorded in stretched ι-carrageenan gels, whereas gelatin gels showed only anisotropic splitting for the 23Na signal in the reference gel.  相似文献   

4.
The recent work of Cope on 23Na magnetic resonance studies of frog muscle has been repeated with the view of investigating certain objections which can be raised concerning the original studies. The present work leads to the conclusion that Cope's results concerning bound sodium are essentially correct in that a large fraction of the 23Na present does not contribute normally to a detectable nuclear magnetic resonance (NMR) signal. This “missing” signal can be detected at high radio-frequency intensity however, and a signal-saturation study distinctly reveals its presence.  相似文献   

5.
Shporer and Civan (Biochim. Biophys. Acta (1974) 354, 291–304) reported the effect of magnetic-field strength on the NMR relaxation times of 23Na in frog skeletal muscle. From these data, they estimated the correlation time τc for bound 23Na whose tumbling is severely restricted, and they suggested that the fraction of bound 23Na does not exceed some few percent of the total 23Na population. However, a step in their theoretical approach seems oversimplified. With an improved approach, we obtained an effective τc of 4–9 ns for bound 23Na. This value is some 10 times shorter than the corresponding value estimated by them from the same data. On the other hand, their conclusion concerning the amount of bound 23Na seems to remain valid. The origin of the observed difference between the two transverse relaxation times of tissue 23Na is also discussed.  相似文献   

6.
Plasma membranes were islotaed from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5′-nucleotidase and (Na++K+)-ATPase were used. The yield of plasma membrane was 0.6–0.9 mg protein per g wet weight of liver. The recovery of 5′-nucleotidase and (Na++K+)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the acitvity of glucose-6 phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5′-nucleotidase, alkaline phosphatase, (Na++K+)-ATPase and Mg2+-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na++K+)-ATPase and Mg2+-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphatase was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.  相似文献   

7.
The effects of the cardiac glycoside dihydroouabain (DHO), and the ericaceous toxin grayanotoxin-I (GTX-I) on myocardial cellular sodium (Nai) concentrations were investigated using sodium-23 nuclear magnetic resonance (23Na NMR) spectroscopy at 30°C in isolated perfused guinea-pig hearts. The Nai NMR signals from perfused Langendorff heart preparations were obtained by the modified inversion recovery (IR) method based on the previous observation that the spin-lattice relaxation time (T1) of the Nai (25 or 34 msec at 8.46 Tesla (T)) is much faster than that of extracellular sodium (64 msec at 9.4 T). Nai was estimated from the calibration curve of the frequency area of the23Na NMR FT spectra plotted against the standard Na concentration. The Nai concentration of the heart increased concomitantly with the positive inotropic effects (PIE) of DHO, GTX-I and monensin (MON). The cumulative sequential addition of DHO (5×10–6 M), GTX-I (7×10–8 M) and MON (5×10–6 M), each of which alone induced no appreciable PIE, produced a 22% elevation in Nai concentration relative to that of the control (100%) accompanying a PIE of 44%. The mechanism of this Nai elevation induced by combinational addition of DHO, GTX-I and MON may be mediated as follows: GTX-I increases the net Na-influxvia Na+ channels; DHO inhibits the pumping out of Na+ from the cell; and MON transports external Na+ into the cell, acting as a sodium ionophore. Consequently, these drugs act synergistically to increase the Nai, thereby increasing the intracellular Ca2+ concentrationvia Na+–Ca2+ exchange.  相似文献   

8.
Shporer and Civan (Biochim. Biophys. Acta (1974) 354, 291–304) reported the effect of magnetic-field strength on the NMR relaxation times of 23Na in frog skeletal muscle. From these data, they estimated the correlation time τc for bound 23Na whose tumbling is severely restricted, and they suggested that the fraction of bound 23Na does not exceed some few percent of the total 23Na population. However, a step in their theoretical approach seems oversimplified. With an improved approach, we obtained an effective τc of 4–9 ns for bound 23Na. This value is some 10 times shorter than the corresponding value estimated by them from the same data. On the other hand, their conclusion concerning the amount of bound 23Na seems to remain valid. The origin of the observed difference between the two transverse relaxation times of tissue 23Na is also discussed.  相似文献   

9.

Objective

Na+ can be stored in muscle and skin without commensurate water accumulation. The aim of this study was to assess Na+ and H2O in muscle and skin with MRI in acute heart failure patients before and after diuretic treatment and in a healthy cohort.

Methods

Nine patients (mean age 78 years; range 58–87) and nine age and gender-matched controls were studied. They underwent 23Na/1H-MRI at the calf with a custom-made knee coil. Patients were studied before and after diuretic therapy. 23Na-MRI gray-scale measurements of Na+-phantoms served to quantify Na+-concentrations. A fat-suppressed inversion recovery sequence was used to quantify H2O content.

Results

Plasma Na+-levels did not change during therapy. Mean Na+-concentrations in muscle and skin decreased after furosemide therapy (before therapy: 30.7±6.4 and 43.5±14.5 mmol/L; after therapy: 24.2±6.1 and 32.2±12.0 mmol/L; p˂0.05 and p˂0.01). Water content measurements did not differ significantly before and after furosemide therapy in muscle (p = 0.17) and only tended to be reduced in skin (p = 0.06). Na+-concentrations in calf muscle and skin of patients before and after diuretic therapy were significantly higher than in healthy subjects (18.3±2.5 and 21.1±2.3 mmol/L).

Conclusions

23Na-MRI shows accumulation of Na+ in muscle and skin in patients with acute heart failure. Diuretic treatment can mobilize this Na+-deposition; however, contrary to expectations, water and Na+-mobilization are poorly correlated.  相似文献   

10.
H Monoi 《Biophysical journal》1976,16(12):1349-1355
Effects of alkali cations on the nuclear magnetic resonance intensity of 23Na were studied in rat liver homogenate. The loss in the resonance intensity of 23Na in the homogenate was able to be divided into two components, one of which is abolished by the addition of Cs+ ("Cs-sensitive component"), the other being insensitive to Cs+ ("Cs-insensitive component"). Both components were sensitive to guanidinium ion. In a pH range of 7.4-4.9, the Cs-sensitive component varied remarkably, but the Cs-insensitive component remained virtually unchanged. The sequence of effectiveness of alkali cations (300 mmol/kg sample) in restoring the fractional intensity of 23Na was: Cs approximately Na greater than Li approximately Rb greater than K. It was suggested that the sequences of effectiveness of alkali cations in abolishing the two components are quite different from each other. The present results were examined within the framework of a simple model. Within this framework, the results suggest that there occur, in particulate fractions, sites whose affinity for Cs+ is sufficiently lower than that for Na+.  相似文献   

11.
Glycogen in sea urchin eggs is found in both the precipitate and the supernatant fractions obtained by adding perchloric acid to the egg homogenate. Glycogen in the acid-insoluble fraction is apparently protein-bound (bound glycogen) while the acid-extractable form (free glycogen) seems to bind with less protein. The greatest amount of bound glycogen is found in the particulate fraction obtained by centrifugation of the egg homogenate at 10,000g for 30 minutes. The supernatant fraction obtained by centrifugation at 105,000g for two hours contained the largest amount of free glycogen of all the fractions obtained. The bound glycogen decreases and the free glycogen increases markedly following fertilization, while the total level of glycogen does not change. The glycogen release from the bound state occurs in vitro and the rate of release is higher in fertilized eggs than in unfertilized eggs. Polyamines (putrecine, spermidine, and spermine) cause an increase in the rate of glycogen release in the egg homogenate. cAMP, AMP, and ADP exert no effect on glycogen release in vitro, whereas ATP slightly enhances the rate of glycogen release. Na+ and K+ hardly accelerate the rate of glycogen release, and divalent cations, such as Ca2+ and Mg2+, cause an increase in the rate of glycogen release.  相似文献   

12.
Activities of carbonic anhydrase and Na+,K+-ATPase in tissue homogenates and in subcellular fractions from different brain regions were studied in inherited primary hypothyroid (hyt/hyt) mice. The body weight, the weight of different brain regions, and the plasma thyroxine and triiodothyronine levels of hyt/hyt mice were significantly lower than those of the age-matched hyt/+ controls. In tissue homogenates of cerebral cortex, brain stem and cerebellum of hypothyroid mice, the activity of carbonic anhydrase (units/mg protein) was 59.2, 57.6, and 43.2%, and the activity of Na+,K+-ATPase (nmol Pi/mg protein/min) was 73.7, 74.4 and 68.7%, respectively, of that in corresponding regions of euthyroid littermates. The decrease in enzyme activity in tissue homogenates was also reflected in different subcellular fractions. In cerebral cortex and brain stem, carbonic anhydrase activity in cytosol, myelin and mitochondrial fractions of hypothyroid mice was about 45–50% of that in euthyroid mice, while in cerebellum the carbonic anhydrase activity in these subcellular fractions of hyt/hyt mice was only 33–38% of that in hyt/+ controls. Na+,K+-ATPase activity in myelin fraction of different brain regions of hyt/hyt mice was about 34–42% of that in hyt/+ mice, while in mitochondria, synaptosome and microsome fractions were about 44–52, 46–53, and 66–68%, respectively of controls. These data indicate that the activity of both carbonic anhydrase and Na+,K+-ATPase was affected more in the myelin than other subcellular fractions and more in the cerebellum than cerebral cortex and brain stem by deficiency of thyroid hormones. A reduction in the activity of transport enzymes in brain tissues as a result of thyroid hormone deficiency during the critical period of development may underlie permanent nervous disorders in primary hypothyroidism.  相似文献   

13.
The neurological disorders familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are caused by mutations of Na+,K+-ATPase α2 and α3 isoforms, expressed in glial and neuronal cells, respectively. Although these disorders are distinct, they overlap in phenotypical presentation. Two Na+,K+-ATPase mutations, extending the C terminus by either 28 residues (“+28” mutation) or an extra tyrosine (“+Y”), are associated with FHM2 and RDP, respectively. We describe here functional consequences of these and other neurological disease mutations as well as an extension of the C terminus only by a single alanine. The dependence of the mutational effects on the specific α isoform in which the mutation is introduced was furthermore studied. At the cellular level we have characterized the C-terminal extension mutants and other mutants, addressing the question to what extent they cause a change of the intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) in COS cells. C-terminal extension mutants generally showed dramatically reduced Na+ affinity without disturbance of K+ binding, as did other RDP mutants. No phosphorylation from ATP was observed for the +28 mutation of α2 despite a high expression level. A significant rise of [Na+]i and reduction of [K+]i was detected in cells expressing mutants with reduced Na+ affinity and did not require a concomitant reduction of the maximal catalytic turnover rate or expression level. Moreover, two mutations that increase Na+ affinity were found to reduce [Na+]i. It is concluded that the Na+ affinity of the Na+,K+-ATPase is an important determinant of [Na+]i.  相似文献   

14.
The research on complex I has gained recently a new enthusiasm, especially after the resolution of the crystallographic structures of bacterial and mitochondrial complexes. Most attention is now dedicated to the investigation of the energy coupling mechanism(s). The proton has been identified as the coupling ion, although in the case of some bacterial complexes I Na+ has been proposed to have that role. We have addressed the relation of some complexes I with Na+ and developed an innovative methodology using 23Na NMR spectroscopy. This allowed the investigation of Na+ transport taking the advantage of directly monitoring changes in Na+ concentration. Methodological aspects concerning the use of 23Na NMR spectroscopy to measure accurately sodium transport in bacterial membrane vesicles are discussed here. External-vesicle Na+ concentrations were determined by two different methods: 1) by integration of the resonance frequency peak and 2) using calibration curves of resonance frequency shift dependence on Na+ concentration. Although the calibration curves are a suitable way to determine Na+ concentration changes under conditions of fast exchange, it was shown not to be applicable to the bacterial membrane vesicle systems. In this case, the integration of the resonance frequency peak is the most appropriate analysis for the quantification of external-vesicle Na+ concentration. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).  相似文献   

15.
Short-term (2–30 min) cyclic stretch activates the Na pump in cultured aortic smooth muscle cells (ASMCs). This effect of stretch involves the phosphotidylinositol 3-kinase (PI 3-kinase) participation. Presently, we investigated whether this stimulation is the result of translocation of Na+,K+-ATPase from endosomes to the plasma membrane. ASMCs were stretched 20% for 5 min using the Flexercell Strain Unit. The plasma membrane and endosome fractions were isolated and Western blotted to localize the Na+,K+-ATPase α-1-subunit protein. Membrane marker enzyme, 5′ nucleotidase activity, and the early and recycling endosome markers Rab4 and Rab11 were used to verify the enrichment of these fractions. Stretch increased Na+,K+-ATPase α-1 expression in plasma membrane fractions and decreased it in endosomes. PI 3-kinase inhibitors LY294002 and wortmannin blocked the stretch-induced translocation of the Na+,K+-ATPase α-1-subunit. Rab4 and Rab11 were enriched in the endosomal fraction, whereas 5′ nucleotidase activity was enriched in the plasma membrane fraction. We conclude that stimulation of the Na pump activity by shortterm cyclic stretch is the result, at least in part, of transport of the α-subunit of the enzyme from endosomes to the plasma membrane.  相似文献   

16.
23Na NMR relaxation rate measurements show that Na+ binds specificially to phosphatidylserine vesicles and is displaced partially from the binding site by K+ and Ca2+ but to a considerably less extent by tetraethylammonium ion. The data indicate that tetraethylammonium ion affects the binding of Na+ only slightly, by affecting the surface potential through its presence in the double layer, without competing for a phosphatidylserine binding site. Values for the intrinsic binding constant for the Na+-phosphatidylserine complex that would be consistent with the competition experiments (and the dependence of the relaxation rate on concentration of free Na+) fall in the range 0.4–1.2 M?1 with a better fit towards the higher values. We conclude that in the absence of competing cations in solution an appreciable fraction of the phosphatidylserine sites could be associated with bound Na+ at 0.1 M Na+ concentration.  相似文献   

17.
The previously reported class of potent inorganic inhibitors of Na,K-ATPase, named MCS factors, was shown to inhibit not only Na,K-ATPase but several P-type ATPases with high potency in the sub-micromolar range. These MCS factors were found to bind to the intracellular side of the Na, K-ATPase. The inhibition is not competitive with ouabain binding, thus excluding its role as cardiac-steroid-like inhibitor of the Na,K-ATPase. The mechanism of inhibition of Na,K-ATPase was investigated with the fluorescent styryl dye RH421, a dye known to report changes of local electric fields in the membrane dielectric. MCS factors interact with the Na,K-ATPase in the E1 conformation of the ion pump and induce a conformational rearrangement that causes a change of the equilibrium dissociation constant for one of the first two intracellular cation binding sites. The MCS-inhibited state was found to have bound one cation (H+, Na+ or K+) in one of the two unspecific binding sites, and at high Na+ concentrations another Na+ ion was bound to the highly Na+-selective ion-binding site.  相似文献   

18.
Abstract— The activities of (Na+ K+)-ATPase and its proposed partial reactions, K +-pNPPase and Na +-dependent phosphorylation, all increase tenfold relative to microsomal protein between 5 days prior to birth and 60 days postnatally in NaI-treated rat cerebral microsomes, and all reach half of their adult values between the fifth and tenth postnatal day. These increases are concurrent with the most rapid changes in cerebral wet weight. Increases in the amount of the related phosphorylatable polypeptide during development. as estimated by densitometry of Coomassie-stained polyacrylamide gels after electrophoresis of constant amounts of microsomal protein dissolved in sodium dodecylsulfate, parallel the increments in levels of Na +-dependent phosphorylation. The fraction of total phosphorylation that is Na +-dependent increases steadily during development. suggesting a precursor role for some of the Na +-independent fraction. The results are consistent with a single biosynthetic control for the enzymatic sites critical to the partial reactions of (Na +-K +)-ATPase. No changes in turnover number or affinity for substrate or ligands were found during development. Little similarity was noted among the age-related changes of Mg 2+ -ATPase activity. Mg 2+ -paranitrophenylphosphatase activity, and Na+-independent phosphorylation levels. The most rapid changes in (Na+-K+)-ATPase take place during the period corresponding to glial proliferation and neuronal arborization.  相似文献   

19.
The transport of Na+ in mature Eurycea oocytes was studied by quantitative radioautography of 22Na+ using techniques suitable for localization of diffusible solutes, together with conventional extractive techniques. Intracellular Na+ consisted of three kinetic fractions: a cytoplasmic fast fraction of about 8.5 µeq/ml H2O; a cytoplasmic slow fraction of about 58.7 µeq/ml H2O; and a nuclear fast fraction of about 11.1 µeq/ml H2O. A nuclear slow fraction, if it exists, does not exceed 5% of the cytoplasmic. The fast fractions represent freely diffusible Na+ in the two compartments; the nuclear solvent space is 1.3 times the cytoplasmic. The flux of both fast fractions is determined by the permeability of the cortical membrane, with neither the nuclear membrane nor diffusion in the cytoplasm detectably slowing the flux. The cytoplasmic slow fraction is interpreted to represent Na+ bound to nondiffusible constituents which are excluded from the nucleus; these may be yolk platelets, although the widespread observation of Na+ binding in other cells, and the high Na+/K+ selectivity, argues against simple ion-binding to the yolk phosphoprotein.  相似文献   

20.
Dopamine inhibits Mg2+,Na+,K+- and Na+,K+-ATPase activities but does not modify Mg2+-ATPase activity of nerve ending membranes isolated from rat cerebral cortex. In the presence of the soluble fraction of brain, dopamine activates total, Na+,K+-, and Mg2+-ATPases. Dopamine stimulation of nerve ending membrane ATPases is achieved when soluble fractions of brain, kidney, or liver are used. On the other hand, dopamine effects are not observed on kidney or heart ATPase preparations. These results indicate tissue specificity of dopamine effects with respect to the enzyme source; there is no tissue specificity for the requirement of the soluble fraction to achieve stimulation of ATPases by dopamine.  相似文献   

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