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.     

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.     

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.     

NMR studies of intracellular sodium ions in mammalian cardiac myocytes

The unambiguous measurement of intracellular sodium ion [Na+]i by the noninvasive NMR technique offers a new opportunity to monitor precisely the maintenance and fluctuations of [Na+]i levels in intact cells and tissues. The anionic frequency shift reagent, dysprosium (III) tripolyphosphate, which does not permeate intact cells, when added to suspensions of intact adult rat cardiac myocytes, alters the NMR frequency of extracellular sodium ions, [Na+]o, leaving that of intracellular ions, [Na+]i, unaffected. Using 23Na NMR in conjunction with this shift reagent, we have determined NMR-visible intracellular Na+ ion concentration in a suspension of isolated cardiac myocytes under standard conditions with insulin and Ca2+ in the extracellular medium to be 8.8 +/- 1.2 mmol/liter of cells (n = 4). This value is comparable to that measured by intracellular ion-selective microelectrodes in heart tissue. Cardiac myocytes incubated for several hours in insulin-deficient, Ca2+-containing medium prior to NMR measurement exhibited a somewhat lower [Na+]i value of 6.9 +/- 0.5 mmol/liter of cells (n = 3). Reversible Na+ loading of the cells by manipulation of extracellular calcium levels is readily measured by the NMR technique. Incubation of myocytes in a Ca2+-free, insulin-containing medium causes a 3-fold increase in [Na+]i to a level of 22.8 +/- 2.6 mmol/liter of cells (n = 10). In contrast to cells with insulin, insulin-deficient myocytes exhibit a markedly lower level of [Na+]i of only 14.6 +/- 2.0 mmol/liter of cells (n = 4) in Ca2+-free medium. These observations suggest that insulin may stimulate a pathway for Na+ influx in heart cells.     

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.     

23Na and flame photometric studies of the NMR visibility of sodium in rat muscle.

23Na nuclear magnetic resonance spectroscopy (NMR) is increasingly being used to study Na+ gradients and fluxes in biological tissues. However, the quantitative aspects of 23Na NMR applied to living systems remain controversial. This paper compares sodium concentrations determined by 23Na NMR in intact rat hindlimb (n = 8) and excised rat gastrocnemius muscle (n = 4) with those obtained by flame photometric methods. In both types of samples, 90% of the sodium measured by flame photometry was found to be NMR-visible. This is much higher than previously reported values. The NMR measurements for intact hindlimb correlated linearly with the flame photometric measurements, implying that one pool of sodium, predominantly extracellular, is 100% visible. From measurements on excised muscle, in which extracellular space is more clearly defined, the NMR visibility of intracellular Na+ was calculated to be 70%, assuming an extracellular space of 12% of the total tissue water volume and an extracellular NMR visibility of 100%. 23Na transverse relaxation measurements were carried out using a Hahn spin echo on both intact hindlimb (n = 1) and excised muscle (n = 2) samples. These showed relaxation curves that could each be described adequately using two relaxation times. The rapidly relaxing component showed a T2 value of 3-4 ms and the slowly relaxing component a T2 of 21-37 ms. A spin lattice relaxation (T1) measurement on intact hindlimb yielded a value of 51 ms. These relatively long relaxation times show that the quadrupolar relaxation effect of Na+ complexing to large macromolecules or being otherwise motionally restricted is relatively weak. This is consistent with the high NMR visibilities reported here.     

Double-quantum-filtered 23Na NMR study of intracellular sodium in the perfused liver.

We acquired double-quantum-filtered 23Na NMR spectra from perfused liver, using a range of tau values from 0.2 to 24 ms, where tau is the separation between the first and second pi/2 pulses in the radio-frequency pulse sequence. For each tau value we compared the amplitude of the double-quantum-filtered 23Na NMR signal acquired from intracellular sodium ions when the liver was perfused with buffer containing the "shift reagent" Dy(PPP)2 to the amplitude of the total double-quantum-filtered 23Na NMR signal acquired when the liver was perfused with buffer containing no Dy(PPP)2. For tau < or = 4 ms, the average ratio of the two amplitudes was 0.98 +/- 0.03 (mean +/- SEM). For tau > or = 8 ms, the average ratio was significantly less than 1. These results demonstrate that double-quantum-filtered 23Na NMR signals acquired from perfused liver using short tau values arise almost exclusively from intracellular sodium ions, but double-quantum-filtered 23Na NMR signals acquired from perfused liver using long tau values contain contributions from both intracellular and extracellular sodium ions. This conclusion suggests that multiple-quantum-filtered 23Na NMR spectroscopy will be useful in studying intracellular sodium levels in the perfused liver, and possibly in the intact liver in vivo.     

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.     

NMR studies of intracellular sodium ions in amphibian oocytes, ovulated eggs, and early embryos

23Na NMR, in combination with an anionic paramagnetic shift reagent dysprosium bis(tripolyphosphate), has been used to study intracellular Na+ in Rana oocytes, ovulated eggs, and early cleavage embryos. The technique allows accurate and simultaneous determination of both extracellular space and intracellular Na+ concentration. In prophase-arrested, follicle-enclosed oocytes, only about 17% of the total oocyte Na+ (approximately 40 mmol/kg of cells) was NMR-visible. Homogenizing oocytes in 0.24 M sucrose did not significantly affect the 23Na resonance. About 30% of the total oocyte Na+ was associated with the yolk platelets isolated at room temperature by differential centrifugation. NMR analysis, however, did not yield a detectable 23Na signal from these intact platelets. Thus, while yolk platelets are rich in Na+, this Na+ does not contribute to the oocyte 23Na NMR signal. Denuded oocytes, obtained by removing the follicular epithelium, gained about 10 mmol of total Na+/kg of cells and exhibited a comparable increase in NMR-visible Na+, suggesting the existence of compartments with varying degree of NMR visibility within the oocyte. Partially relaxed 23Na Fourier transform NMR spectra revealed the existence of at least two major intracellular compartments of NMR-visible Na+ with different magnetic environments and relaxation behavior in denuded oocytes. Since platelet Na+ appears to be NMR-invisible, one of the two observed compartments may be the nucleus. Progesterone action on the amphibian oocyte caused measurable changes in NMR-visible Na+. By ovulation (second metaphase), there is a gain in total egg Na+, and the NMR-visible Na+ is also increased. Following fertilization, however, there is some loss of total cell Na+ but, by the 2-4 cell stage, about 70% of the total Na+ becomes NMR-visible. These results indicate that a sizable fraction of the Na+ in follicle-enclosed, prophase oocyte is sequestered and located in NMR-invisible compartments and that changes in NMR-visible intracellular Na+ occur following hormonal and developmental stimuli.     

Measurement of intracellular sodium concentration and sodium transport in Escherichia coli by 23Na nuclear magnetic resonance

Escherichia coli is known to actively extrude sodium ions, but little is known concerning the concentration gradient it can develop. We report here simultaneous measurements, by 23Na NMR, of intracellular and extracellular Na+ concentrations of E. coli cells before and after energization. 23Na spectra in the presence of a paramagnetic shift reagent (dysprosium tripolyphosphate) consisted of two resonances, an unshifted one corresponding to intracellular Na+ and a shifted one corresponding to Na+ in the extracellular medium, including the periplasm. Extracellular Na+ was found to be completely visible despite the presence of a broad component in its resonance; intracellular Na+ was only 45% visible. Measurements of Na+ were made under aerobic and glycolytic conditions. Na+ extrusion and maintenance of a stable low intracellular Na+ concentration were found to correlate with the development and maintenance of proton motive force, a result that is consistent with proton-driven Na+/H+ exchange as a means of Na+ transport. In both respiring and glycolyzing cells, at an extracellular Na+ concentration of 100 mM, the intracellular Na+ concentration observed (4 mM) corresponded to an inwardly directed Na+ gradient with a concentration ratio of about 25. The kinetics of Na+ transport suggest that rapid extrusion of Na+ against its electrochemical gradient may be regulated by proton motive force or intracellular pH.     

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)     

23Na NMR study of Fibrobacter succinogenes S85: comparison of three chemical shift reagents and calculation of sodium concentration using ionophores

In order to measure intracellular sodium concentrations in resting cells of Fibrobacter succinogenes S85 by (23)Na NMR spectrometry, two methodological aspects were studied. First, three different shift reagents (Dy(PPP(i))(7-)(2), Tm(DOTP)(5-), and Dy(TTHA)(3-)) were tested for their ability to separate internal and external (23)Na NMR resonances. Their toxicity toward F. succinogenes cells was evaluated by in vivo(13)C NMR experiments. Tm(DOTP)(5-) was found to be the most efficient shift reagent while being nontoxic. Second, a new methodology was developed to calculate intracellular sodium concentration in F. succinogenes by using ionophores. This approach avoided the problem of intracellular volume measurement and that of sodium visibility determination.     

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%.     

Study of ion translocation by respiratory complex I. A new insight using (23)Na NMR spectroscopy

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 (23)Na 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 (23)Na 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).     

Changes of intracellular Na~ concentration in erythrocytes caused by pulsed electrical field

Changes of sodium ionic concentration of human erythrocytes applied to pulsed electrical field (PEF) were studied by using shift reagent and NMR spectroscopy. The results show that the concentration of intracellular Na increases with the increasing intensity of PEF when the erythrocytes are applied to PEF with higher intensities. The relationship between intracellular Na concentrations and the intensities of PEF does not follow linear or exponen-tial behavior. As the intensities increase, the intracellular Na concentrations increase even faster by an exponential curve. However under effects of PEF at lower intensities, intracellular Na concentration decreases. Ouabain can in-hibit the decrease of intracellular Na concentration, and the inhibition increases with the increasing concentration of ouabain, suggesting that Na , K -ATPase on cell membrane can be activated by PEF at lower intensities. Direct measurement of activities of the enzyme by using Malachite green method has confirmed this observatio     

23Na, 19F, 35Cl and 31P multinuclear nuclear magnetic resonance studies of perfused rat kidney

The concentration of intracellular sodium [Na+]i has been measured in the perfused rat kidney using 23Na nuclear magnetic resonance (NMR) in combination with the extracellular shift reagent Dy(PPPi)7-(2). The data show 100% visibility of Na+ in interstitial spaces. A measurement of the resonance intensities of intra- and extracellular 23Na ions along with a knowledge of the extracellular space as a fraction of the total kidney water space yielded an average [Na+]i of 27 +/- 2 mM for the kidney at 37 degrees C. After prolonged ischemia [Na+]i rose to approach that in the external medium. In the absence of 5% albumin in the perfusion medium, the linewidth of the 35Cl resonance of an adult kidney (45 Hz) was about twofold larger than that of the medium alone (25 Hz). In contrast, the linewidth of 35Cl resonance of an adult kidney perfused with an albumin-containing medium (82 Hz) was only about 27% of that from the medium alone (300 Hz). We interpret this effect to be due to compartmentation of albumin in the extracellular space such that the interstitial space is not freely accessible to albumin. However, for a developing, immature kidney from a growing animal, perfused with an albumin-containing medium, the linewidth of the 35Cl resonance (233 Hz) was only slightly less than that of the medium alone (300 Hz), indicating a much greater albumin permeability of the capillary walls. 19F NMR of a perfused adult kidney, loaded with the membrane-impermeant intracellular calcium indicator 5FBAPTA, yielded a value of 256 nM for [Ca2+]i. Induction of ischemia for 10 min caused the [Ca2+]i to rapidly rise to 660 nM, which could not be fully reversed by reperfusion, suggesting irreversible injury.     

Na-NMR Studies of the Intracellular Sodium Ion Concentration in the Halotolerant Alga Dunaliella salina

The Intracellular Na⁺ concentration in the halotolerant alga Dunaliella salina was measured in intact cells by ²³Na-NMR spectroscopy, utilizing the dysprosium tripolyphosphate complex as a sodium shift reagent, and was found to be 88 ± 28 millimolar. Intracellular sodium ion content and intracellular volume were the same, within the experimental error, in cells adapted to grow in media containing between 0.1 and 4.0 molar NaCl. These values assume extracellular and intracellular NMR visibilities of the ²³Na nuclei of 100 and 40%, respectively. The relaxation rate of intracellular sodium was enhanced with increasing salinity of the growth medium, in parallel to the intracellular osmosity due to the presence of glycerol, indicating that Na⁺ ions and glycerol are codistribbuted within the cell volume.     

23Na-nuclear magnetic resonance investigation of gramicidin-induced ion transport through membranes under equilibrium conditions.

A technique for investigating the gramicidin-facilitated transport of Na+ ions across lipid bilayers of large unilamellar vesicles under the condition of ionic equilibrium has been developed using a combination of heat incubation of the gramicidin with the vesicles and 23Na-nuclear magnetic resonance (NMR) spectroscopy. Isolation of the two 23Na-NMR signals from the intra- and extravesicular Na+ with the shift reagent, dysprosium (III) tripolyphosphate, allows the equilibrium flux of Na+ through the gramicidin channels to be detected and treated as a two-site exchange process. This study indicates that the transport of Na+ through gramicidin channels is second order with respect to the gramicidin concentration.     

Evidences on sodium ions compartmentalization in biological systems due to pathological states. A noninvasive NMR study

Noninvasive nuclear magnetic resonance was used to measure the relaxation decay curves of naturally occurring 23Na ions in several biological systems. Experimental results showed an increase of membrane bound population for pathologic samples as compared with control. The bound sodium population was put in evidence using singular value decomposition method. Thus, the singular values that are obtained without any a priori from the fitting the relaxation decay curves are a new parameter in characterizing the cellular state. In the presence of artificial biological membranes, 23Na bound strongly to membranes containing phosphatidylcholine (PC) and phosphatidylserine (PS), but not to membranes consisting of only PC. A large bound population also appeared in the presence of apoptotic epithelial cells, which are known to translocate PS to the cell surface. A role for PS was confirmed by showing that sodium binds to the surface of epithelial cells infected with Chlamydia psittaci, and the amplitude of the bound population increases with a time-course similar to the appearance of PS on the surface of dying cells. Finally, this approach could distinguish between normal perfused liver and liver undergoing ischemia, due most likely to the exposure of surface PS on apoptotic and necrotic cells in the damaged tissue. Taken together, these studies demonstrate that the analysis of 23Na relaxation decay curves could reveal the presence of cells undergoing apoptosis and/or necrosis in living tissues. Noninvasive 23Na NMR measurements could thus be envisioned for controlling the quality of organs before transplantation, for the detection of asymptomatic infections that result in death of the host cell or inflammation of the tissue, and for characterizing the efficiency of novel apoptosis-inducing drugs to treat cancer.     

Direct High-resolution Nuclear Magnetic Resonance Studies of Cation Transport in Vivo: Na+ Transport in Yeast Cells

A new nuclear magnetic resonance (NMR) method for monitoring transmembrane metal cation transport is reported. It is illustrated with a study of Na⁺ efflux from Na⁺-rich yeast cells. The technique involves the use of an anionic paramagnetic shift reagent, present only outside the cells, to induce a splitting of the sodium-23 NMR peak, in this case, into components representing intra- and extracellular Na⁺. The time course of the efflux is in good agreement with the literature and can be well fitted with a double exponential decay expression. Splitting of the lithium-7 NMR signal from a suspension of Li⁺-rich respiratory-deficient, petite yeasts is also reported.