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.     

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.     

Coupling between the sodium and proton gradients in respiring Escherichia coli cells measured by 23Na and 31P nuclear magnetic resonance

The relationship between the steady-state sodium gradient (delta pNa) and the protonmotive force developed by endogenously respiring Escherichia coli cells has been studied quantitatively, using 23Na NMR for measurement of intracellular and extracellular sodium concentrations, 31P NMR for measurement of intracellular and extracellular pH, and tetraphenylphosphonium distribution for measurement of membrane potential. At constant protonmotive force, the sodium concentration gradient was independent of extracellular concentrations over the measured range of 4-285 mM, indicating that intracellular sodium concentration is not regulated. The magnitude of delta pNa was measured as a function of the composition and magnitude of the protonmotive force. At external pH values below 7.2, delta pNa was parallel to delta pH but showed no simple relationship to the membrane potential; above pH 7.2 the parallel relationship began to diverge, with delta pH continuing to decrease but delta pNa starting to level off or increase. Although plots of delta pNa versus delta pH had slopes of close to 1, the value of delta pNa consistently exceeded that of delta pH by approximately 0.4 units, indicating a partially electrogenic character to the putative H+/Na+ antiport. The apparent stoichiometry was 1.13 +/- 0.01 at external pH below 7.2. The possible significance of this nonintegral stoichiometry is discussed according to a model in which two distinct integral stoichiometries (possibly 1H+/1Na+ and 2H+/1Na+) are available with some relative probability; the model predicts futile cycling of sodium ions and a dissipative proton current. In the course of this study, we discovered that the magnitude of the pH gradient developed by the cells was osmolarity-dependent, yielding steady-state intracellular pH values that varied from 7.1 at 100 mosm to 7.7 at 800 mosm.     

Sarcolemma phospholipid structure investigated by immunogold electron microscopy and (31)P NMR spectroscopy with lanthanide ions.

The biological functions of plasma membranes depend greatly on the biophysical properties resulting from protein and phospholipid structure. We investigated the phospholipid structure of the normal sarcolemma membrane, which is known to be highly dysfunctional in myopathies. Combining electron microscopy and (31)P nuclear magnetic resonance (NMR) spectroscopy on isolated sarcolemma vesicles, we find that (i) the sarcolemma vesicles maintain the in-vivo cellular sidedness, (ii) the phospholipid mobility is close to that observed in model membranes (similar lateral diffusion coefficients and spin-lattice T(1) relaxation times). Using broad-band and magic angle spinning (31)P NMR spectroscopy with lanthanide ions (Pr(3+)), it is possible to quantify the distribution of phospholipids between internal and external membrane layers, showing that the trans-bilayer distribution is highly asymmetrical.     

A study of the conformation of 5S RNA by 31P NMR.

Only a small number of resolved, single phosphorous, phosphodiester resonances are observed in the 31P spectrum of the 5S rRNA from E. coli. Its spectrum is much simpler than that of a tRNA (Gueron, M. and Shulman, R.G. (1975) Proc. Natl. Acad. Sci. 72, 3482-3484), which suggests that 5S RNA does not have a tightly folded, tRNA-like, tertiary structure. The resolved resonances in the 5S spectrum originate in loops D and E, near bases 88 and 76, respectively.     

23Na and 31P NMR studies of the effects of salt stress on the freshwater cyanobacterium Synechococcus 6311

We have used 23Na and 31P nuclear magnetic resonance (NMR) spectroscopy to elucidate some of the bioenergetic changes that occur in the freshwater cyanobacterium Synechococcus 6311 after a transition from growth medium (Na concentration 0.01 M) to medium containing 0.5 M NaCl. 23Na NMR analysis showed Na rapidly penetrates the cells under dark aerobic conditions; cells grown for several days in high salt medium, however, reestablish a low internal sodium content, comparable to control cells. For 31P NMR analysis, a system was devised to aerate and illuminate cell suspensions during spectral acquisition. The NMR spectra showed that when cells are presented with 0.5 M NaCl (final concentration), nucleotide triphosphate peaks decrease, the inorganic phosphate peak increases, and the cytoplasmic pH transiently increases from 7.4 to 7.9. Pyrophosphate added to cell suspensions is hydrolyzed to inorganic phosphate apparently by an extracellular phosphatase, allowing external and internal pools of inorganic phosphate to be distinguished. Nucleotide triphosphate levels fall almost as much when cells are incubated in darkness as under anoxia, indicating that both respiration and photosynthesis contribute to the maintenance of intracellular ATP levels. Cells grown in high salt medium for several generations exhibited a pattern of 31P metabolites similar to control cells, except that they produced more (and more intense) peaks in the monoester phosphate region, presumably signals from sugar phosphates.     

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.     

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.     

7Li and 23Na NMR studies of transmembrane cation transport mediated by ionophore lasalocid A

Ion transport across phospholipid vesicles was studied by ⁷Li and ²³Na-NMR using an aqueous anionic paramagnetic shift reagent, dysprosium nitrilotriacetate [Dy(NTA)₂]³⁻, mediated by ionophores, lasalocid A and A23187. The intra- and extracellular ⁷Li and ²³Na-NMR signals were well separated (20 Hz) at mM concentration of the shift reagent. The observed data on the rate constant for lithium transport across DPPC vesicles at various concentrations of the ionophores indicated that lasalocid A is a more efficient carrier for lithium ion compared with the sodium ion transport by this ionophore, while A23187 was not specific to either of the ions (Li or Na). ©1998 European Peptide Society and John Wiley & Sons, Ltd.     

An Na-23 and P-31 NMR investigation of sonicated cardiolipin sodium salt in aqueous medium

Na-23 and P-31 nuclear magnetic resonance was used to investigate the structure and dynamics of Na+ trapped in the enclosed aqueous spaces of unilamellar vesicles of Cardiolipin sodium salt. After sonicated Cardiolipin sodium salt forms a clear solution in aqueous medium and gives rise to a narrow, isotropic P-31 NMR line. This line was attributed to the presence of either vesicles or more complex liquid crystalline structures showing superposed-signals for inner and outer phosphate groups. The use of paramagnetic shift reagent Dy(PPPi)2 made it possible to observe only the line of the phosphate groups arranged within the structures. From an analysis of the spin-lattice relaxation parameters of Na-23 at 21.04 MHz and 52.6 MHz, the correlation time tau = 1.3 x 10(-9) sec was estimated. It was then possible to calculate the value of 1.9 MHz for the quadrupolar coupling parameter. These findings were interpreted in terms of the occurrence of specific bindings between Na+ and the phosphate moieties within the unilamellar structures of Cardiolipin.     

31P and 23Na nuclear magnetic resonance studies of resting and stimulated mast cells

Exocytosis induced by crosslinking the type I receptor for Fc epsilon domains present on rat mucosal mast cells (RBL-2H3-line) requires the influx of Ca2+ ions and is markedly influenced by the concentration of monovalent cations (K+, Na+ and protons) in their medium. We investigated the role of these ions in coupling the immunological stimulus to secretion using NMR spectroscopy to monitor simultaneously intracellular pH, ATP and Na+ concentrations and the secretory response of living adherent mast cells. Using this methodology we observed that: (i) ATP concentration and intracellular pH are highly regulated and no changes could be resolved in them upon stimulation and during exocytosis. (ii) In the absence of potassium ions in the cells' medium, a decrease is observed in the intracellular pH and ATP concentration and an increase in the Na+ concentration. (iii) From the influx of extracellular Na+ following inhibition of the Na+, K(+)-ATPase by ouabain, we estimated the inward Na+ current of resting cells to 5 x 10(7) ions/(cell.s). This value does not vary by more than 10% during exocytosis.     

Metabolic changes of the Antarctic green alga Prasiola crispa subjected to water stress investigated by in vivo 31P NMR

The energy status and the phosphate metabolism of Prasiola crispduring and after desiccation stress was investigated by in vivo³¹P NMR. The effect of desiccation was simulated by additionof the nonionic osmoticum PEG 200 (polyethylene glycol). Photosynthesisand respiration were effectively inhibited under these conditions.The most notable changes in the in vivo ³¹P NMR spectra werean increase in the cytoplasmic inorganic phosphate signal afterPEG stress, a decrease in the polyphosphates and a lowfieldshift of the core polyphosphate signal followed by an appearanceof extracellular inorganic phosphate. Cytoplasmic pH remainedalmost constant during stress. After a return to control conditions,photosynthesis and respiration recovered within 4 h as wellas the concentrations of the phosphorus metabolites. An as yetunassigned phosphate signal increased in the phosphodiesterregion of the NMR spectra. Simultaneousty, the polyphosphatesignal recovered in intensity and chemical shift. It is suggestedthat phosphate metabolism and complexation of cations to polyphosphatesmay play an important role in the distinct desiccation toleranceof P. crispa. Key words: In vivo ³¹P NMR, Prasiola crispa, desiccation tolerance, polyphosphates     

Effect of head-group structure and counterion condensation on phase equilibria in anionic phospholipid-water systems studied by 2H, 23Na, and 31P NMR and X-ray diffraction

The phase equilibria, hydration, and sodium counterion association for the systems DOPA-2H2O, DOPS-2H2O, DOPG-2H2O, and DPG-2H2O were investigated with 2H, 23Na, and 31P NMR and X-ray diffraction. The following one-phase regions were found in the DOPA-water system: a reversed hexagonal liquid-crystalline (HII) phase up to about 35 wt % water and a lamellar liquid-crystalline (L alpha) phase between about 55 and 98 wt % water. The area per DOPA molecule was 36-65 A2 in the HII phase (10-40 wt % water) and 69 A2 in the L alpha phase (60 wt % water). DOPS and DOPG with 10-98 wt % water, and DPG with 20-95 wt % water formed an L alpha phase at temperatures between 25 and 55 degrees C. At temperatures above 55 degrees C, DPG with 20 and 30 wt % water formed a mixture of L alpha, HII, and cubic liquid-crystalline phases, the mole percent of lipid forming nonlamellar phases being smaller at 30 wt % water than at 20 wt % water. DPG with 10 wt % water probably formed a mixture of an L alpha phase and at least one nonlamellar liquid-crystalline phase at 25 and 35 degrees C, and a pure HII phase at 45 degrees C and higher temperatures. At water concentrations above about 50 wt % the 23Na quadrupole splitting was constant for all four lipid-water systems studied, implying that the counterion association to the charged lipid aggregates did not change upon dilution. These experimental observations can be described with an ion condensation model but not with a simple equilibrium model. The fraction of counterions located close to the lipid-water interface was calculated to be greater than 95%. The 2H and 23Na NMR quadrupole splittings of 2H2O and sodium counterions, respectively, indicate that the molecular order in the polar head-group region decreases for the L alpha phase in the order DOPA approximately DPG greater than DOPS greater than DOPG.     

Cation binding in Na,K-ATPase, investigated by 205Tl solid-state NMR spectroscopy

Cation binding to Na,K-ATPase is characterized in native membranes at room temperature by solid-state NMR spectroscopy using the K(+) congener (205)Tl. It has been demonstrated that the signals from occluded Tl(+) and nonspecifically bound Tl(+) can be detected and distinguished by NMR. Effects of dipole-dipole coupling between (1)H and (205)Tl in the occlusion sites show that the ions are rigidly bound, rather than just occluded. Furthermore, a low chemical shift suggests occlusion site geometries with a relatively small contribution from carboxylate and hydroxyl groups. Nonspecific binding of Tl(+) is characterized by rapid chemical exchange, in agreement with the observed low binding affinity.     

Metabolic control principles and 31P NMR

31P NMR is a unique research tool for studying metabolism during exercise. This communication gives the underlying theory and experimental data for obtaining the transfer characteristic relating work and NMR-determined metabolic paramenters, particularly the ratio of free inorganic phosphate to phosphocreatine (Pi/PCr). Furthermore, illustrations of the types of transfer characteristics observed in different individuals and different training regimens can be obtained, including both hyperbolic (Michaelis-Menten) and sigmoid transfer characteristics. With a hyperbolic transfer characteristic, oxygen delivery appears to be adequate to support the exercise regimen over the range studied (up to 50% of Vmax), whereas with a sigmoid transfer characteristic, there appears an imprint of limited oxygen delivery on the kinetics from rest up to 50% of Vmax. These two types of transfer functions are appropriate to explain the transition to anaerobic metabolism (anaerobic threshold), with a hyperbolic transfer characteristic representing a graded transition; and a sigmoid transfer characteristic representing an abrupt transition. A sigmoid transfer characteristic may involve greater lactate accumulation at submaximal exercise levels, and in both types of transfer characteristic sufficient aerobic lactate is formed to meet the metabolic demand for pyruvate. The results suggest an important role for 31 P NMR in studies of energy-related metabolites as a means of determining exercise performance in terms of regulation of tissue oxidative metabolism and oxygen delivery to tissue.     

High-pressure 31P NMR study of dipalmitoylphosphatidylcholine bilayers.

High-pressure 31P NMR was used for the first time to investigate the effects of pressure on the structure and dynamics of the phosphocholine headgroup in pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) multilamellar aqueous dispersions and in DPPC bilayers containing the positively charged form of the local anesthetic tetracaine (TTC). The 31P chemical shift anisotropies, delta sigma, and the 31P spin-lattice relaxation times, T1, were measured as a function of pressure from 1 bar to 5 kbar at 50 degrees C for both pure DPPC and DPPC/TTC bilayers. This pressure range permitted us to explore the rich phase behavior of DPPC from the liquid-crystalline (LC) phase through various gel phases such as gel I (P beta'), gel II (L beta'), gel III, gel IV, gel X, and the interdigitated, Gi, gel phase. For pure DPPC bilayers, pressure had an ordering effect on the phospholipid headgroup within the same phase and induced an interdigitated Gi gel phase which was formed between the gel I (P beta') and gel II (L beta') phases. The 31P spin-lattice relaxation time measurements showed that the main phase transition (LC to gel I) was accompanied by the transition between the fast and slow correlation time regimes. Axially symmetric 31P NMR lineshapes were observed at pressures up to approximately 3 kbar but changed to characteristic axially asymmetric rigid lattice lineshapes at higher pressures (3.1-5.1 kbar).(ABSTRACT TRUNCATED AT 250 WORDS)