铯进入红细胞的NMR研究

在生理及生化研究中铯离子的行为受到极大的关注,因为可由它来阐述碱金属离子输运及酶活性等一些基本功能。当所研究的体系中没有K~+时,Cs离子有类似K~+的功能,它能激活Na/K ATPase。Cs进入细胞的速率及激活Na/K ATPase所要求的浓度与被研究系统的条件密切有关。本文用核磁共振谱方法测定红细胞内外的Cs~+及Cs~+进入细胞的速率。结果表明Cs-133的核磁共振谱是研究生物体系离子的分布及输运的极好工具。     

A 133Cs nuclear magnetic resonance study of endothelial Na(+)-K(+)-ATPase activity: can actin regulate its activity?

Using (133)Cs+ NMR, we developed a technique to repetitively measure, in vivo, Na(+)-K(+)-ATPase activity in endothelial cells. The measurements were made without the use of an exogenous shift reagent, because of the large chemical shift of 1.36 +/- 0.13 ppm between intra- and extracellular Cs+. Intracellularly we obtained a spin lattice relaxation time (T1) of 2.0 +/- 0.3 s, and extracellular T1 was 7.9 +/- 0.4 s. Na(+)-K+ pump activity in endothelial cells was determined at 12 +/- 3 nmol Cs+ x min(-1) x (mg Prot)[-1] under control conditions. When intracellular ATP was depleted by the addition of 5 mM 2-deoxy-D-glucose (DOG) and NaCN to about 5% of control, the pump rate decreased by 33%. After 80 min of perfusion with 5 mM DOG and NaCN, reperfusion with control medium rapidly reestablished the endothelial membrane Cs+ gradient. Using (133)Cs+ NMR as a convenient tool, we further addressed the proposed role of actin as a regulator of Na(+)-K+ pump activity in intact cells. Two models of actin rearrangement were tested. DOG caused a rearrangement of F-actin and an increase in G-actin, with a simultaneous decrease in ATP concentration. Cytochalasin D, however, caused an F-actin rearrangement different from that observed for DOG and an increase in G-actin, and cellular ATP levels remained unchanged. In both models, the Na(+)-K(+)-pump activity remained unchanged, as measured with (133)Cs NMR. Our results demonstrate that (133)Cs NMR can be used to repetitively measure Na(+)-K(+)-ATPase activity in endothelial cells. No evidence for a regulatory role of actin on Na(+)-K(+)-ATPase was found.     

Multinuclear NMR studies of the Langendorff perfused rat heart

The quantitation of intracellular sodium ion concentration [Na+]in perfused organs using NMR spectroscopy requires a knowledge of the extent of visibility of the 23Na resonance and of the intracellular volume of the organ. We have used a multinuclear NMR approach, in combination with the extracellular shift reagent dysprosium (III) tripolyphosphate, to determine the NMR visibility of intra- and extracellular 23Na and 35Cl ions, intracellular volume, and [Na+]in in the isolated Langendorff perfused rat heart. Based on a comparison of the extracellular volumes calculated using 2H and 23Na, 35Cl, or 59Co NMR of the perfused heart we conclude that resonances of extracellular sodium and chloride ions (including ions in interstitial spaces) are fully visible, contrary to assumptions in the literature. Furthermore, prolonged hypoxia or ischemia caused a dramatic increase in intracellular Na+ and [Na+] in rose to approach that in the external medium indicating full visibility of the intracellular 23Na resonance. Resonance intensities of intra- and extracellular 23Na ions, along with a knowledge of the extracellular space as a fraction of the total organ water space, yielded an average [Na+] in of about 10 mM (10 +/- 1.5 mM) for the rat heart at 37 degrees C. Double-quantum filtered 23Na NMR of the perfused rat heart in the absence and presence of paramagnetic reagents revealed, contrary to assumptions in the literature, that both intra- and extracellular sodium ions contribute to the detected signal.     

A simple procedure for NMR measurements of intra- and extracellular sodium in intact tissues

The total Na+ and both the intra- and extracellular Na+ content of excised rat and frog tissues was quantitated by 23Na NMR at 95.51 MHz. An external capillary containing 33 mM Na7[Dy(P3O10)2], resonating about 30 ppm upfield relative to the 0.00 ppm of the intracellular Na+, was inserted into the tissues. The capillary was calibrated against a concentration range of pure NaCl solution, for measurement of intracellular Na+, and against the same concentrations of NaCl solutions containing 4-6 mM K7[Dy(P3O10)2] in 50 mM histidine. Cl and 100 mM choline. Cl, for measurement of extracellular Na+. Spectra were recorded on tissues first in the absence of the shift reagent for determination of the total Na+. After addition of a K7[Dy(P3O10)2] solution to the sample, the 23Na spectra were recorded immediately so that data accumulation was completed within 15 min. Under these conditions, the extracellular Na+ resonated from 10 to 20 ppm upfield relative to the intracellular Na+, and no loss in the intensity of the extracellular Na+ resonance occurred due to the lability of dysprosium(III)tripolyphosphate (cf. Matwiyoff et al., Magn. Reson. Med. 3: 164, 1986). The intra- and extracellular Na+ content of the tissue was calculated from the integrated areas of the respective Na+ resonances and that of the calibrated capillary, from the known weight of the tissue, and from the known volumes of the solutions added.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of a wide range of intracellular sodium concentrations in erythrocytes by 23Na nuclear magnetic resonance.

The accuracy of the 23Na nuclear magnetic resonance (NMR) method for measuring the sodium concentration in erythrocytes was tested by comparing the NMR results to those obtained by emission-flame photometry. Comparisons were made on aqueous solutions, hemolysates, gels, ghosts, and intact erythrocytes. The intra- and extracellular 23Na NMR signals were distinguished by addition of the dysprosium tripolyphosphate [Dy(PPP)7-2] shift reagent to the extracellular fluid. The intra- and extracellular volumes of ghosts and cells were determined by the isotope dilution method. Our results indicate that greater than 20% of the intracellular signal remains undetected by NMR in ghosts and cells. When the cells are hemolyzed, the amount of NMR-detectable sodium varies depending on the importance of gel formation. In hemolysates prepared by water addition, the NMR and flame photometry results are identical. The loss of signal in ghosts, cells, and undiluted hemolysates is attributed to partial binding of the Na+ ion to intracellular components, this binding being operative only when these components exist in a gel state. In a second part, 31P NMR was used to monitor the penetration of the shift reagent into the cells during incubation. Our data demonstrate that free Dy3+ can slowly accumulate inside the red cell.     

Determination of membrane potential and cell volume by 19F NMR using trifluoroacetate and trifluoroacetamide probes

The distribution of ionic species between intra- and extracellular compartments forms one basis for the determination of cell membrane potential. It is shown that fluorine-19 NMR studies of erythrocytes in the presence of trifluoroacetate, a stable, relatively nontoxic anion with pK = -0.3, provide a sensitive probe of membrane potential. Since such measurements are based on ion concentrations, the parallel use of the neutral analogue trifluoroacetamide to provide information on intra/extracellular volume ratios was also explored. In both cases, separate 19F resonances corresponding to intra- and extracellular ions were observed, with the intracellular resonance shifted downfield by approximately 0.2 ppm and the intracellular peak typically somewhat broader than the extracellular resonance. Studies with the band 3 anion-exchange inhibitor 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) indicate that both transmembrane diffusion and flux involving the band 3 anion exchanger contribute to the observed transport of the trifluoroacetate anion. Intra/extracellular volume ratios determined on the basis of trifluoroacetamide intensity ratios were in good agreement with determinations based on measured hematocrits. On the basis of the high sensitivity of 19F NMR and the capability of monitoring volume changes simultaneously, the time resolution for these measurements can approach the lifetime of intracellular trifluoroacetate ions and hence be limited by the trifluoroacetate flux rate.     

The contribution of magnetic susceptibility effects to transmembrane chemical shift differences in the 31P NMR spectra of oxygenated erythrocyte suspensions

Triethyl phosphate, dimethyl methylphosphonate, and the hypophosphite ion all contain the phosphoryl functional group. When added to an oxygenated erythrocyte suspension, the former compound gives rise to a single 31P NMR resonance, whereas the latter compounds give rise to separate intra- and extracellular 31P NMR resonances. On the basis of experiments with intact oxygenated cell suspensions (in which the hematocrit was varied) and with oxygenated cell lysates (in which the lysate concentration was varied), it was concluded that the chemical shifts of the intra- and extracellular populations of triethyl phosphate differ as a consequence of the diamagnetic susceptibility of intracellular oxyhemoglobin but that this difference is averaged by the rapid exchange of the compound across the cell membrane. The difference in the magnetic susceptibility of the intra- and extracellular compartments contributes to the observed separation of the intra- and extracellular resonances of dimethyl methylphosphonate and hypophosphite. The magnitude of this contribution is, however, substantially less than that calculated using a simple two-compartment model and varies with the hematocrit of the suspension. Furthermore, it is insufficient to fully account for the transmembrane chemical shift differences observed for dimethyl methylphosphonate and hypophosphite. An additional effect is operating to move the intracellular resonances of these compounds to a lower chemical shift. The effect is mediated by an intracellular component, and the magnitude of the resultant chemical shift variations depends upon the chemical structure of the phosphoryl compound involved.     

Nuclear magnetic resonance monitoring of sodium in biological tissues

In recent year, the 23Na nuclear magnetic resonance (NMR) technique has been applied to the study of biological tissues. The advantages of this method are noninvasiveness and good sensitivity. The resonances of the intra- and extra-cellular sodium can be separated by the addition of shift reagents to the extracellular compartment. The method has been mostly applied to cell suspensions, kidney tubules, glands, and small organs. Owing to line-broadening effects, the NMR visibility of the intracellular sodium is reduced to 40% in most cases but can be lower or higher. Time-dependent measurements are possible with adequate life-supporting equipment, allowing the determination of transport parameters. 23Na relaxation times are short in tissues (below 50 ms) and highly dependent on the medium composition. The application of the 23Na NMR technique to intact organs can be hampered by the difficulty of getting a good distribution of the shift reagent in the extracellular milieu. A summary of the studies performed is presented with specific examples to illustrate typical applications.     

23Na NMR study of intracellular sodium ions in Dictyostelium discoideum amoeba

The intracellular sodium concentration in the amoebae from the slime mold Dictyostelium discoideum has been studied using 23Na NMR. The 23Na resonances from intracellular and extracellular compartments could be observed separately in the presence of the anionic shift reagent Dy(PPPi)7-2 which does not enter into the amoebae and thus selectively affects Na+ in the extracellular space. 31P NMR was used to control the absence of cellular toxicity of the shift reagent. The intracellular Na+ content was calculated by comparison of the intensities of the two distinct peaks arising from the intra- and extracellular spaces. It remained low (0.6 to 3 mM) in the presence of external Na+ (20 to 70 mM), and a large Na+ gradient (20- to 40-fold) was maintained. A rapid reloading of cells previously depleted of Na+ was readily measured by 23Na NMR. Nystatin, an antibiotic known to perturb the ion permeability of membranes, increased the intracellular Na+ concentration. The time dependence of the 23Na and 31P NMR spectra showed a rapid degradation of Dy(PPPi)7-2 which may be catalyzed by an acid phosphatase.     

Characterization of transmembrane chemical shift differences in the 31P NMR spectra of various phosphoryl compounds added to erythrocyte suspensions

Trimethyl phosphate, dimethyl methylphosphonate, diethyl methylphosphonate, trimethylphosphine oxide, and the hypophosphite, phenylphosphinate, and diphenylphosphinate ions all contain the phosphoryl functional group. When added to an intact erythrocyte suspension at 20 degrees C, each of the compounds gave rise to separate intra- and extracellular 31P NMR resonances, and the separation between the two resonances of each compound varied with the mean cell volume. The differences between the intra- and extracellular chemical shifts were shown to be primarily attributable to the effects of hemoglobin. The presence of hemoglobin inside the cell gave rise to a significant difference in the magnetic susceptibilities of the two compartments. In addition, it exerted a large susceptibility-independent chemical shift effect, the magnitude of which was dependent upon the chemical structure of the phosphoryl compound involved. A number of other intra- and extracellular components were also shown to cause chemical shift variations, smaller than those arising from hemoglobin but nonetheless significant. The cell volume dependence of the transmembrane chemical shift differences therefore reflected not only the cell volume dependence of the intracellular hemoglobin concentration but also the changing concentration of the other solutes in the two compartments. In addition to their cell volume dependence, the transmembrane chemical shift differences varied with temperature. In the case of the nonelectrolytes this reflected not only the temperature dependence of the mechanism(s) responsible for the susceptibility-independent shift effects but also the temperature dependence of the rates at which the compounds traversed the cell membrane.     

Effect of colchicine on estrogen action. I. Inhibition of 17 beta-estradiol-induced water and potassium uptake in the immature rat uterus

The effects of colchicine on 17 beta-estradiol-induced water and electrolyte uptake in the uterus of the immature rat have been examined 6 h after treatment with this estrogen. Estradiol stimulates an increase in total uterine Na+, K+ and water while intracellular Na+ and K+ concentrations remain relatively unchanged. Assuming the sodium space is equivalent to the extracellular space, the extracellular fluid compartment increases about 84% in response to estradiol. Similarly, the intracellular compartment increases by about 62%. The uptake of water into the cellular compartment may be a direct response to a stimulation of K+ accumulation by uterine cells. Colchicine inhibits both estradiol-induced rise in intracellular potassium and both intra- and extracellular water.     

一种直接观测完整细胞内外钠离子浓度的NMR技术

借助位移试剂(Shift Reagent)测细胞内外阳离子浓度的NMR方法是近十年发展的新技术,可直接观测完整活细胞和组织内外的阳离子浓度,具有非破坏性连续性观察的优点.本实验室用自己制备的位移试剂测试NaCl溶液,人红细胞和豚鼠红细胞内Na~+浓度.获得良好的~(23)Na NMR谱的共振分离.     

23Na and 39K nuclear magnetic resonance studies of perfused rat hearts. Discrimination of intra- and extracellular ions using a shift reagent.

High-resolution 23Na and 39K nuclear magnetic resonance (NMR) spectra of perfused, beating rat hearts have been obtained in the absence and presence of the downfield shift reagent Dy(TTHA)3- in the perfusing medium. Evidence indicates that Dy(TTHA)3- enters essentially all extracellular spaces but does not enter intracellular spaces. It can thus be used to discriminate the resonances of the ions in these spaces. Experiments supporting this conclusion include interventions that inhibit the Na+/K+ pump such as the inclusion of ouabain in and the exclusion of K+ from the perfusing medium. In each of these experiments, a peak corresponding to intracellular sodium increased in intensity. In the latter experiment, the increase was reversed when the concentration of K+ in the perfusing medium was returned to normal. When the concentration of Ca2+ in the perfusing medium was also returned to normal, the previously quiescent heart resumed beating. In the beating heart where the Na+/K+ pump was not inhibited, the intensity of the intracellular Na+ resonance was less than 20% of that expected. Although the data are more sparse, the NMR visibility of the intracellular K+ signal appears to be no more than 20%.     

Metabolomic, proteomic and biophysical analyses of Arabidopsis thaliana cells exposed to a caesium stress. Influence of potassium supply

The incorporation and localisation of 133Cs in a plant cellular model and the metabolic response induced were analysed as a function of external K concentration using a multidisciplinary approach. Sucrose-fed photosynthetic Arabidopsis thaliana suspension cells, grown in a K-containing or K-depleted medium, were submitted to a 1 mM Cs stress. Cell growth, strongly diminished in absence of K, was not influenced by Cs. In contrast, the chlorophyll content, affected by a Cs stress superposed to K depletion, did not vary under the sole K depletion. The uptake of Cs was monitored in vivo using 133Cs NMR spectroscopy while the final K and Cs concentrations were determined using atomic absorption spectrometry. Cs absorption rate and final concentration increased in a K-depleted external medium; in vivo NMR revealed that intracellular Cs was distributed in two kinds of compartment. Synchrotron X-ray fluorescence microscopy indicated that one could be the chloroplasts. In parallel, the cellular response to the Cs stress was analysed using proteomic and metabolic profiling. Proteins up- and down-regulated in response to Cs, in presence of K+ or not, were analysed by 2D gel electrophoresis and identified by mass spectrometry. No salient feature was detected excepting the overexpression of antioxidant enzymes, a common response of Arabidopsis cells stressed whether by Cs or by K-depletion. 13C and 31P NMR analysis of acid extracts showed that the metabolome impact of the Cs stress was also a function of the K nutrition. These analyses suggested that sugar metabolism and glycolytic fluxes were affected in a way depending upon the medium content in K+. Metabolic flux measurements using 13C labelling would be an elegant way to pursue on this line. Using our experimental system, a progressively stronger Cs stress might point out other specific responses elicited by Cs.     

In vivo measurements of intra- and extracellular Na+ and water in the brain and muscle by nuclear magnetic resonance spectroscopy with shift reagent.

The introduction of new paramagnetic shift reagents in the nuclear magnetic resonance (NMR) method has made it possible to distinguish intra- and extracellular ions in tissues or organs in vitro. We measured the intra- and extracellular 23Na and 1H in vivo in the gerbil brain and skeletal muscle by NMR spectroscopy employing the shift reagent, dysprosium triethylenetetraminehexaacetate (Dy[TTHA]3-). Without Dy(TTHA)3-, the 23Na and 1H signals were seen only as single peaks, but gradual intravenous infusion of Dy(TTHA)3- separated these signals into two peaks, respectively. The unshifted peaks reflected the intracellular 23Na and 1H signals, while the shifted peaks reflected the extracellular signals. In the brain spectra, an additional small peak, which represented intravascular signals, was detected and its intensity increased after injection of papaverine hydrochloride. The present method is advantageous over the microelectrode technique because of its nondestructiveness and its capability for obtaining intra- and extracellular volume information from measurements of the 1H spectra, the peaks of which reflect the intra- and extracellular water amounts. The intracellular Na+ increase associating with increased cellular volume after ouabain in the muscle was clearly visualized by this method. The technique is clearly of use for physiological and pathophysiological studies of organs.     

Competition between Li+ and Mg2+ for ATP in human erythrocytes. A 31P NMR and optical spectroscopy study

We have investigated the influence of Li+ on free intracellular Mg2+ concentration in human erythrocytes by 31P NMR and optical absorbance spectroscopies. In red cells loaded with 3 mM intracellular Li+, the chemical shift separation between the alpha- and beta-phosphate resonances of MgATP2- was approx. 0.9 ppm larger than that observed in Li+-free red cells. By analyzing the interaction of each red cell component with Mg2+ and Li+, we found that Mg2+ is displaced in part from MgATP2- upon addition of Li+ and that the released Mg2+ is bound to the red cell membrane causing an overall decrease in free intracellular Mg2+ concentration.     

Symmetry and asymmetry of permeation through toxin-modified Na+ channels.

Single Na+ channels from rat skeletal muscle were inserted into planar lipid bilayers in the presence of either 200 nM batrachotoxin (BTX) or 50 microM veratridine (VT). These toxins, in addition to their ability to shift inactivation of voltage-gated Na+ channels, may be used as probes of ion conduction in these channels. Channels modified by either of the toxins have qualitatively similar selectivity for the alkali cations (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). Biionic reversal potentials, for example, were concentration independent for all ions studied. Na+/K+ and Na+/Rb+ reversal potentials, however, were dependent on the orientation of the ionic species with respect to the intra- or extracellular face of the channel, whereas Na+/Li+ biionic reversal potentials were not orientation dependent. A simple, four-barrier, three-well, single-ion occupancy model was used to generate current-voltage relationships similar to those observed in symmetrical solutions of Na, K, or Li ions. The barrier profiles for Na and Li ions were symmetric, whereas that for K ions was asymmetric. This suggests the barrier to ion permeation for K ions may be different than that for Na and Li ions. With this model, these hypothetical energy barrier profiles could predict the orientation-dependent reversal potentials observed for Na+/K+ and Na+/Rb+. The energy barrier profiles, however, were not capable of describing biionic Na/Li ion permeation. Together these results support the hypothesis that Na ions have a different rate determining step for ion permeation than that of K and Rb ions.     

Location and ion-binding of membrane-associated valinomycin, a proton nuclear magnetic resonance study

Valinomycin, incorporated in small unilamellar vesicles of perdeuterated dimyristoylphosphatidylcholine, reveals several well-resolved 1H-NMR resonances. These resonances were used to examine the location, orientation and ion-binding of membrane-bound valinomycin. The order of affinity of membrane-bound valinomycin for cations is Rb+ greater than K+ greater than Cs+ greater than Ba2+, and binding is sensitive to surface change. The exchange between bound and free forms is fast on the NMR time scale. The intrinsic binding constants, extrapolated to zero anion concentration, are similar to those determined in aqueous solution. Rb+ and K+ show 1:1 binding to valinomycin, whereas the stoichiometry of Cs+ and Ba2+ is not certain. Paramagnetic chemical shift reagents and nitroxide spin label relaxation probes were used to study the location and orientation of valinomycin in the membrane. Despite relatively fast exchange of bound cations, the time average location of the cation-free form of valinomycin is deep within the bilayer under the conditions of these experiments. Upon complexation to K+, valinomycin moves closer to the interfacial region.     

NMR measurement of intracellular water volume in rat kidney proximal tubules

Knowledge of cell water volume is essential for the measurement of concentrations of intracellular ions and metabolites in kidney proximal tubules. We have developed a method which utilizes 35Cl-NMR as a measure of extracellular volume and 2H-NMR, in combination with a membrane-impermeable shift-reagent [Dy-DTPA]2-, as a measure of the ratio of intra- and extracellular water volumes. Measurement of extracellular volume by 35Cl-NMR is possible, since the resonance of intracellular 35Cl is too broad to be detectable in kidney cells. The 2H-NMR measurement exploits the fact that only extracellular water is in direct contact with [Dy-DTPA]2-. However, rapid exchange of water across the cell membrane results in only a single 2H2O resonance at a chemical shift which is a weighted average of the shifted extra- and unshifted intracellular water resonances. Expression of the extracellular volume as a fraction of the total volume by fCl and as a fraction of the total water-volume by fD, permits the calculation of the fractional cell-water content fw = [(1/fD)-1]/[(1/fCl)-1]. This approach was applied to proximal tubular suspensions prepared from the rat kidney. The water content was found to be 76.9 +/- 1.8% (n = 6) at 37 degrees C. Increasing extracellular osmolality from 295 to 390 mOsm/kg H2O, by addition of mannitol, decreased the water content by 21%. Our results are in good agreement with those obtained by the gravimetric method.     

Cesium toxicity in Arabidopsis

Cesium (Cs) is chemically similar to potassium (K). However, although K is an essential element, Cs is toxic to plants. Two contrasting hypotheses to explain Cs toxicity have been proposed: (1) extracellular Cs+ prevents K+ uptake and, thereby, induces K starvation; and (2) intracellular Cs+ interacts with vital K(+)-binding sites in proteins, either competitively or noncompetitively, impairing their activities. We tested these hypotheses with Arabidopsis (Arabidopsis thaliana). Increasing the Cs concentration in the agar ([Cs](agar)) on which Arabidopsis were grown reduced shoot growth. Increasing the K concentration in the agar ([K](agar)) increased the [Cs](agar) at which Cs toxicity was observed. However, although increasing [Cs](agar) reduced shoot K concentration ([K](shoot)), the decrease in shoot growth appeared unrelated to [K](shoot) per se. Furthermore, the changes in gene expression in Cs-intoxicated plants differed from those of K-starved plants, suggesting that Cs intoxication was not perceived genetically solely as K starvation. In addition to reducing [K](shoot), increasing [Cs](agar) also increased shoot Cs concentration ([Cs](shoot)), but shoot growth appeared unrelated to [Cs](shoot) per se. The relationship between shoot growth and [Cs](shoot)/[K](shoot) suggested that, at a nontoxic [Cs](shoot), growth was determined by [K](shoot) but that the growth of Cs-intoxicated plants was related to the [Cs](shoot)/[K](shoot) quotient. This is consistent with Cs intoxication resulting from competition between K+ and Cs+ for K(+)-binding sites on essential proteins.