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.     

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 studies of rat outer medullary kidney tubules

Two reservations have previously made interpretation of biological 23Na NMR measurements difficult: the "size" of the extracellular space penetrated by the shift reagent and the possibility of a 60% reduction in the intensity of the NMR-visible 23Na signal due to quadrupolar interactions (Berendsen, H. J. C., and Edzes, H. T. (1973) Ann. N. Y. Acad. Sci. 204, 459-485; Civan, M. M., Degani, H., Margalit, Y., and Shporer, M. (1983) Am. J. Physiol. 245, C213-C219; Gupta, R. K., and Gupta, P. (1982) J. Magn. Reson. 47, 344-350). We have addressed both these issues using a suspension of rat outer medullary kidney tubules, nephron segments responsible for the fine control of total body volume and electrolyte balance. First, the extracellular space penetrated by the shift reagent dysprosium tripolyphosphate, as defined by the extracellular 23Na resonance, revealed a space similar to that which contained extracellular 35Cl- ions. Measurement of an extracellular 35Cl- space using 35Cl NMR was possible because the intracellular 35Cl- resonance was broadened beyond detection in the cells studied. Second, to characterize the reduction of the 23Na signal by quadrupolar interactions, the intracellular 23Na level was raised artificially by simultaneously inhibiting Na+ efflux and increasing the ion permeability of the plasma membrane. Under these conditions, NMR-observable intracellular Na+ reached a level which was approximately 81% of that in the medium, a level determined using chemical techniques. This observation would suggest that the resonance of the intracellular 23Na pool was not subject to a 60% reduction in signal intensity, as a result of nuclear quadrupolar interaction. The intracellular 23Na level measured, under basal conditions, was 23 +/- 2 mumol/ml of cell water (37 degrees C) (n = 3, S.D.) and was demonstrated to be responsive to a number of physiological stimuli. The level was temperature-sensitive. It was reduced by inhibitors of apical Na+ transport, furosemide and amiloride, and it was raised with (Na+ + K+)-ATPase inhibition. The furosemide and amiloride actions described would suggest that the Na+-transporting mechanisms sensitive to these agents (e.g. Na+/K+/Cl- cotransport system, Na+:H+ exchange system) contribute to the regulation of the intracellular Na+ level in the kidney tubular preparation studied.     

The effects of the extracellular manganese concentration and variation of the interpulse delay time in the CPMG sequence on water exchange time across erythrocyte membranes

There has been broad disagreement in the literature regarding the dependence of water exchange times (Te) across erythrocyte membranes studied by the 1H-NMR Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence on extracellular Mn2+ concentration. While some workers saw no change in Te with Mn2+, others reported a 35-50% decrease in Te with this extracellular paramagnetic relaxation agent. We present 1H-NMR evidence that a 30-50% change in Te can be produced by interdependence of the interpulse delay time of the CPMG pulse sequence and the external Mn2+ concentration. Such a large dependency is interpreted in terms of the diffusional effect as a major source. However, it is shown experimentally that if a large number of refocusing pi pulses are used, the observed transverse relaxation times are unaffected by Mn2+. Under these conditions excellent agreement of Te obtained in our study (13.0 +/- 0.64 ms (N = 36) at 21 degrees C) and that of 12.8 +/- 3.6 ms at 20-23 degrees C reported by the radiotracer method was found. Our findings suggest new and important implications for evaluating the previous reports of the 1H-NMR CPMG method concerning the [Mn2+] effect in the decrease of Te, and provide conditions where studies of water transport across erythrocyte membranes using this magnetic resonance method can be used with confidence.     

Proton nuclear magnetic resonance measurement of diffusional water permeability in suspended renal proximal tubules.

Diffusional water permeability was measured in renal proximal tubule cell membranes by pulsed nuclear magnetic resonance using proton spin-lattice relaxation times (T1). A suspension of viable proximal tubules was prepared from rabbit renal cortex by Dounce homogenization and differential sieving. T1 measured in a tubule suspension (22% of exchangeable water in the intracellular compartment) containing 20 mM extracellular MnCl2 was biexponential with time constants 1.8 +/- 0.1 ms and 8.3 +/- 0.2 ms (mean +/- SD, n = 8, 37 degrees C, 10 MHz). The slower time constant, representing diffusional exchange of water between intracellular and extracellular compartments, increased to 11.6 +/- 0.6 ms (n = 6) after incubation of tubules with 5 mM parachloromercuribenzene sulfonate (pCMBS) for 60 min at 4 degrees C and was temperature dependent with activation energy Ea = 2.9 +/- 0.4 kcal/mol. To relate T1 data to cell membrane diffusional water permeabilities (Pd), a three-compartment exchange model was developed that included intrinsic decay of proton magnetization in each compartment and apical and basolateral membrane water transport. The model predicted that the slow T1 was relatively insensitive to apical membrane Pd because of low luminal/cell volume ratio. Based on this analysis, basolateral Pd (corrected for basolateral membrane surface convolutions) is 2.0 X 10(-3) cm/s, much lower than corresponding values for basolateral Pf (10-30 X 10(-3) cm/s) measured in the intact tubule and in isolated basolateral membrane vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H-NMR studies of the intracellular water of skeletal muscle fibers under various physiological conditions.

Intracellular water of frog skeletal muscle fibers has been studied under various physiological conditions by use of the 1H-nuclear magnetic resonance (NMR) technique. 1H-NMR spectra of muscle fibers had single peaks derived from the intracellular water. The spectra changed in a characteristic fashion when the fiber axis was aligned at various angles relative to the magnetic field of the NMR magnet. Further, the relaxation rates of the 1H-NMR spectra changed depending on the water content of muscle fibers, and in association with contraction and rigor formation of muscle fibers. The obtained results indicate that the intracellular water of muscle fibers is structured and aligned along the myofilaments, and further that the state of the intracellular water changes with physiological conditions.     

Osmotic mass transfer in the yeast Saccharomyces cerevisiae.

This paper reviews the passive mechanisms involved in the response of a yeast to changes in medium concentration and osmotic pressure. The results presented here were collected in our laboratory during the last decade and are experimentally based on the measurement of cell volume variations in response to changes in the medium composition. In the presence of isoosmotic concentration gradients of solutes between intracellular and extracellular media, mass transfers were found to be governed by the diffusion rate of the solutes through the cell membrane and were achieved within a few seconds. In the presence of osmotic gradients, mass transfers mainly consisting in a water flow were found to be rate limited by the mixing systems used to generate a change in the medium osmotic pressure. The use of ultra-rapid mixing systems allowed us to show that yeast cells respond to osmotic upshifts within a few milliseconds and to determine a very high hydraulic permeability for yeast membrane (Lp>6.10(-11) m x sec)-1) x Pa(-1)). This value suggested that yeast membrane may contain facilitators for water transfers between intra and extracellular media, i.e. aquaporins. Cell volume variation in response to osmotic gradients was only observed for osmotic gradients that exceeded the cell turgor pressure and the maximum cell volume decrease, observed during an hyperosmotic stress, corresponded to 60% of the initial yeast volume. These results showed that yeast membrane is highly permeable to water and that an important fraction of the intracellular content was rapidly transferred between intracellular and extracellular media in order to restore water balance after hyperosmotic stresses. Mechanisms implied in cell death resulting from these stresses are then discussed.     

The binding of nitrate to the human anion exchange protein (AE1) studied with 14N nuclear magnetic resonance

The binding of [14N]nitrate to the human erythrocyte anion transport protein, AE1, was studied using 14N nuclear magnetic resonance spectroscopy (14N-NMR). The line-width at half-height of the 14NO3- resonance increased in direct proportion to the concentration of erythrocyte ghost protein. Addition of the AE1 specific inhibitor 4,4'-dinitrostilbene-2,2'-disulfonate markedly reduced this line-broadening, indicating that the broadening was predominantly due to a specific interaction between nitrate and AE1. The dependence of the AE1 specific line-broadening on nitrate concentration had a first-order dissociation constant KD of 6.9 +/- 0.9 mM. In contrast, Cl- interaction with AE1 studied by 35Cl-NMR showed a chloride concentration-dependent line-broadening with a KD of 74 +/- 10 mM, indicating that AE1 has a higher affinity for nitrate than for chloride. Bicarbonate and chloride were found to be competitive inhibitors of the AE1 specific 14NO3- line-broadening (94 +/- 6% and 101 +/- 3% inhibition, respectively). Based on the concentration dependence of inhibition and using a model of competitive inhibition, the KD of bicarbonate binding to AE1 was estimated to be 5.4 +/- 1.3 mM. Nitrate is a structural analog of bicarbonate, making the interaction of nitrate with AE1 a good model for the bicarbonate-AE1 interaction. The 14N-NMR nitrate binding assay, along with the 35Cl-NMR binding assay now in use, will provide a powerful tool for studying the structure of the AE1 binding site for both physiologic substrates, bicarbonate and chloride.     

Integrity of intracellular domain of Notch ligand is indispensable for cleavage required for release of the Notch2 intracellular domain

The biological activity of the soluble form of the Notch ligand (sNL) and requirement of the intracellular domain (ICD) of the Notch ligand have been debated. Here we show that soluble Delta1 (sD1) activates Notch2 (N2), but much more weakly than full-length Delta1 (fD1). Furthermore, tracing the N2 molecule after sD1 stimulation revealed that sD1 has a defect in the cleavage releasing ICD of N2 (intracellular cleavage), although it triggers cleavage in the extracellular domain of N2. This represents the molecular basis of the lower activity of sD1 and suggests the presence of an unknown mechanism regulating activation of the intracellular cleavage. The fact that Delta1 lacking its ICD (D1Delta(ICD)) exhibits the phenotype similar to that exhibited by sD1 indicates that the ICD of D1 (D1(ICD)) is involved in such an as yet unknown mechanism. Furthermore, the findings that D1Delta(ICD) acts in a dominant-negative fashion against fD1 and that the signal-transducing activity of sD1 is enhanced by antibody-mediated cross-linking suggest that the multi merization of Delta1 mediated by D1(ICD) may be required for activation of the N2 intracellular cleavage.     

Secretion of chondroitin SO4 by monolayer cultures of chick embryo chondrocytes

Monolayer cultures of chick embryo tibial chondrocytes incorporate 35SO42- into chondroitin SO4 which is rapidly secreted from the cells into two extracellular pools. Part of the extracellular chondroitin SO4 is recovered in a soluble form in the culture medium, and the remainder is associated with the cell matrix from which it is released by isotonic trypsinization. At 38 degrees C labeled chondroitin SO4 appears in the cell matrix fraction within 5 min after addition of 35SO42- and in the culture medium fraction 15 min after 35SO42- is added. The intracellular pool of labeled chondroitin SO4 reaches a steady state level of 150 to 200 pmol of bound SO4 per 10(6) cells in 60 min, while the cell matrix and medium fractions increase at rates of 3 and 1 nmol of bound SO4 per h per 10(6) cells, respectively. After 4 h of labeling, less than 20% of the newly synthesized cell-associated chondroitin SO4 is in the intracellular fraction. By labeling cells for 15 min at 25 degrees C 80% of the cell-associated chondroitin 35SO4 is obtained in the intracellular fraction. This material is chased without lag into both the cell matrix fraction and the medium fraction. A mixture of NaF and NaCN, both at 30 mM, lowers the cellular ATP level to 15% of normal and blocks secretion of the intracellular chondroitin SO4 into both extracellular fractions. Colchicine at 10(-6) M gives a partial inhibition of both synthesis and secretion of chondroitinSO4. Sucrose density gradient sedimentation analysis of the intracellular chondroitin SO4 and the two extracellular fractions shows that all three fractions contain both a heavy and light proteoglycan fraction. The intracellular light proteoglycan fraction is secreted preferentially into the culture medium where it represents 30% of the total culture medium pool. The ratio of 6-sulfated GalNAc to 4-sulfated GalNAc in the heavy proteochondroitin SO4 fraction is approximately twice that found for the light fraction.     

The uptake of manganese ions and the apparent thermal transition of the erythrocyte membranes

The nuclear magnetic resonance manganese doping technique is currently used for the determination of the water diffusional exchange time through human erythrocyte membranes. An apparent thermal transition at 26 degrees C was noticed at 18-30 mM manganese doping in the suspending solution. An analysis in terms of a two-phase nuclear spin exchanging system revealed that apparent thermal transitions are expected to occur in the upper and lower temperature regions. They represent a shift from intermediate exchange rates where water diffusion through the membrane is dominant to either fast or slow exchange rates where proton relaxation is the controlling process. The lower temperature apparent transition may be altered by the intracellular manganese concentration; the lower the Mn2+ concentration the lower the transition. Also according to this interpretation only a fraction of the erythrocytes are significantly permeated by Mn2+. The upper transition depends on the Mn2+ concentration in the extracellular volume; it decreases with decreasing Mn2+ concentration.     

Fused cells of frog proximal tubule: II. Voltage-dependent intracellular pH

Experiments were performed in intact proximal tubules of the doubly perfused kidney and in fused proximal tubule cells of Rana esculenta to evaluate the dependence of intracellular pH (pHi) on cell membrane potential applying pH-sensitive and conventional microelectrodes. In proximal tubules an increase of the K+ concentration in the peritubular perfusate from 3 to 15 mmol/liter decreased the peritubular cell membrane potential from -55 +/- 2 to -38 +/- 1 mV paralleled by an increase of pHi from 7.54 +/- 0.02 to 7.66 +/- 0.02. The stilbene derivative DIDS hyperpolarized the cell membrane potential from -57 +/- 2 to -71 +/- 4 mV and led to a significant increase of the K+-induced cell membrane depolarization, but prevented the K+-induced intracellular alkalinization. Fused proximal tubule cells were impaled by three microelectrodes simultaneously and cell voltage was clamped stepwise while pHi changes were monitored. Cell membrane hyperpolarization acidified the cell cytoplasm in a linear relationship. This voltage-induced intracellular acidification was reduced to about one-third when HCO-3 ions were omitted from the extracellular medium. We conclude that in proximal tubule cells pHi depends on cell voltage due to the rheogenicity of the HCO-3 transport system.     

Role of Ca2+ and aquaporin-2 in regulation of basolateral water permeability of collecting ducts in the rat kidney

The role of AQP2,3 and intracellular calcium in vasopressin-induced increase in the water permeability of the basolateral cell membrane in microdissected rat kidney OMCD was studied. It was shown that increase in the water permeability of the basolateral membranes correlated with increase in the content of AQP2 and AQP3 in the membrane fraction isolated from outer kidney medulla. Preliminary loading of cells with BAPTA-AM which binds intracellular Ca2+ abolished the increase in the water permeability and prevented the rise of the AQP2 content in response to dDAVP. BAPTA was ineffective to block the enhancement of AQP2 content in membrane fraction in presence of dDAVP. These results suggest that the increase in intracellular calcium activity and the enhanced content of AQP2 in plasma membrane are important for the antidiuretic effect of dDAVP.     

Intra- and extracellular carbohydrates in plant cell cultures investigated by 1H-NMR

With the aim of quantifying intra- and extracellular carbohydrates media and cell-extracts from a Tabernaemontana divaricata plant cell-suspension culture were investigated with ¹H-NMR.For suppression of the solvent peak the Meiboom-Gill modification of the Carr-Purcell (CPMG) spin-echo sequence was used after addition of a paramagnetic relaxation agent (Mn²⁺) to the sample. Several aspects of this method were optimized (the manganese concentration, the interpulse delay and the number of spin-echo cycles) so as to obtain a rapid and easy method in which no pretreatment of media or cell-extracts was needed. Besides the speed and ease of the method, also the direct identification of carbohydrates and other main components is an advantage.The exhaustion of extracellular carbohydrates was found to coincide with the maximum amount of intracellular carbohydrates. The intracellular carbohydrates, i.e. glucose and fructose, were consumed at a low rate, during several weeks.Abbreviations ¹H-NMR proton nuclear magnetic resonance - 2,4-D 2,4-dichlorophenoxyacetic acid     

The effect of unilateral ureteral obstruction on renal function in pigs measured by diffusion-weighted MRI

The objective of this study was to examine the effect of unilateral ureteral obstruction on the apparent diffusion coefficient (ADC) in pig kidney. Changes in ADC is suggested to reflect changes in the ratio of extracellular to intracellular volume. Thirteen pigs were allocated into three groups: 1) pigs subjected to acute unilateral ureteral obstruction (AUO) (n = 3), 2) pigs subjected to chronic partial unilateral obstruction (CPUO) (n = 3), and 3) control pigs (n = 7). The extra- to intracellular volume ratio was indirectly measured in both the ipsilateral obstructed kidney and contralateral non-obstructed kidney by the ADC of the renal tissue using diffusion-weighted echo-planar magnetic resonance imaging. ADC was 2.07 +/- 0.27 x 10(-3) mm2/s in the cortex and 2.10 +/- 0.24 x 10(-3) mm2/s in the medulla of normal control kidneys. In the obstructed kidney from the AUO group the ADC of the medulla was significantly reduced 24 hours after occlusion of the ureter (1.65 +/- 0.05 x 10(-3) mm2/s vs 2.10 +/- 0.24 x 10(-3) mm2/s; p < 0.05). Similarly ADC decreased slightly in the cortex of the ipsilateral kidney. In contrast, ADC of the ipsilateral kidney of CPUO pigs was increased both in the renal medulla (3.13 +/- 0.21 x 10(-3) mm2/s vs. 2.10 +/- 0.24 x 10(-3) mm2/s; p < 0.05) and cortex (3.09 +/- 0.14 x 10(-3) mm2/s vs. 2.07 x 10(-3) mm2/s, p < 0.05). In conclusion, the results of the present study suggest that diffusion weighted imaging (ADC) may be a useful parameter to incorporate when identifying whether a ureteric obstruction is acute or chronic.     

New Insights on Human Skeletal Muscle Tissue Compartments Revealed by In Vivo T2 NMR Relaxometry

The spin-spin (T2) relaxation of ¹H-NMR signals in human skeletal muscle has been previously hypothesized to reveal information about myowater compartmentation. Although experimental support has been provided, no consensus has yet emerged concerning the attribution of specific anatomical compartments to the observed T2 components. Potential application of a noninvasive tool that might offer such information urges the quest for a definitive answer to this question. The purpose of this work was to obtain new information that might help elucidate the mechanism of T2 distribution in muscle. To do so, in vivo T2 relaxation data was acquired from the soleus of eight healthy volunteers using a localized Carr-Purcell-Meiboom-Gill technique. Each acquisition contained 1000 echoes with an interecho spacing of 1 ms. Data were acquired from each subject under different vascular filling preparations expected to change exclusively the extracellular water fraction. Two exponential components were systematically observed: an intermediate component (T2 ∼ 32 ms) and a long component (100 < T2 < 210 ms). The relative fraction and T2 value characterizing the long component systematically increased after progressive augmentation of extracellular water volume. Characteristic relaxation behavior for each vascular filling condition was analyzed with a two-site exchange model and a three-site two-exchange model. We show that a two-site exchange model can only predict the observations for small exchange rates, much more representative of transendothelial than transcytolemmal exchange regimes. The three-site two-exchange model representing the intracellular, interstitial, and vascular spaces was capable of precisely predicting the observations for realistic transcytolemmal and transendothelial exchange rates. The estimated intrinsic relative fractions of each of these compartments corroborate with estimations from previous works and strongly suggest that the T2 relaxation from water within the intracellular and interstitial spaces is described by the intermediate component, whereas the long component represents water within the vascular space.     

Hydrodynamics and cell volume oscillations in the pollen tube apical region are integral components of the biomechanics of Nicotiana tabacum pollen tube growth

Pollen tube growth is localized at the apex and displays oscillatory dynamics. It is thought that a balance between intracellular turgor pressure (hydrostatic pressure, reflected by the cell volume) and cell wall loosening is a critical factor driving pollen tube growth. We previously demonstrated that water flows freely into and out of the pollen tube apical region dependent on the extracellular osmotic potential, that cell volume changes reflect changes in the intracellular pressure, and that cell volume changes differentially induce, increases or decreases in specific phospholipid signals. This article shows that manipulation of the extracellular osmotic potential rapidly induces modulations in pollen tube growth rate frequencies, demonstrating that changes in the intracellular pressure are sufficient to reset the pollen tube growth oscillator. This indicates a direct link between intracellular hydrostatic pressure and pollen tube growth. Altering hydrodynamic flow through the pollen tube by replacing extracellular H₂O with ²H₂O adversely affects both cell volume and growth rate oscillations and induces aberrant morphologies. Normal growth and cell morphology are rescued by replacing ²H₂O with H₂O. Further studies revealed that the cell volume oscillates in the pollen tube apical region. These cell volume oscillations were not from changes in cell shape at the tip and were detectable up to 30 μm distal to the tip (the longest length measured). Cell volume in the apical region oscillates with the same frequency as growth rate oscillations but surprisingly the cycles are phase-shifted by 180°. Raman microscopy yields evidence that hydrodynamic flow out of the apex may be part of the biomechanics that drive cellular expansion. The combined results suggest that hydrodynamic loading/unloading in the apical region induces cell volume oscillations and has a role in driving cell elongation and pollen tube growth.     

Water permeability of chlorella cell membranes by nuclear magnetic resonance: measured diffusion coefficients and relaxation times

Measurement by two nuclear magnetic resonance (NMR) techniques of the mean residence time τ_a of water molecules inside Chlorella vulgaris (Beijerinck) var. “viridis” (Chodot) is reported. The first is the Conlon and Outhred (1972 Biochim Biophys Acta 288: 354-361) technique in which extracellular water is doped with paramagnetic Mn²⁺ ions. Some complications in application of this technique are identified as being caused by the affinity of Chlorella cell walls for Mn²⁺ ions which shortens the NMR relaxation times of intra- and extracellular water. The second is based upon observations of effects of diffusion on the spin echo of intra- and extracellular water. Echo attenuation of intracellular water is distinguished from that of extracellular water by the extent to which diffusive motion is restricted. Intracellular water, being restricted to the cell volume, suffers less echo attenuation. From the dependence of echo amplitude upon gradient strength at several values of echo time, the mean residence time of intracellular water can be determined. From the mean residence time of intracellular water, the diffusional water permeability coefficient of the Chlorella membrane is calculated to be 2.1 ± 0.4 × 10⁻³ cm sec⁻¹.     

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.     

Estimation of segmental muscle volume by bioelectrical impedance spectroscopy.

This study validated bioelectrical impedance spectroscopy (BIS) with Cole-Cole modeled measurements of calf and arm segmental water volume and volume changes during 72 h of simulated microgravity and caloric restriction by using magnetic resonance imaging (MRI) muscle volume as a criterion method. MRI and BIS measurements of calf and upper arm segments were made in 18 healthy men and women [age, 29 +/- 8 (SD) yr; height, 171 +/- 11 cm; mass, 71 +/- 16 kg] before and after the intervention. Muscle volume of arm and leg segments by MRI was on average 15 +/- 10 and 14 +/- 8% lower, respectively, than the estimated total water volume by BIS (P < 0.01), but their correlations were excellent (r = 0.96 and r = 0.93, respectively). MRI- vs. BIS-predicted volume changes were a decrease of 49 +/- 68 vs. 41 +/- 62 ml in the calf and a decrease of 18 +/- 23 vs. 11 +/- 24 ml in the arm, respectively (P > 0.05 for both). BIS detected the extracellular water shifts in the calf resulting from the head-down tilt treatment, but the underfeeding protocol was not of sufficient duration or intensity to produce limb intracellular water changes detectable by BIS. BIS was highly correlated with segmental muscle volume and tracked changes associated with head-down tilt. Further research, however, is needed to determine whether BIS can accurately access separate changes in intracellular and extracellular volume.