Protein diffusion in the periplasm of E. coli under osmotic stress

The physical and mechanical properties of the cell envelope of Escherichia coli are poorly understood. We use fluorescence recovery after photobleaching to measure diffusion of periplasmic green fluorescent protein and probe the fluidity of the periplasm as a function of external osmotic conditions. For cells adapted to growth in complete medium at 0.14–1.02 Osm, the mean diffusion coefficient <D_peri> increases from 3.4 μm² s⁻¹ to 6.6 μm² s⁻¹ and the distribution of D_peri broadens as growth osmolality increases. This is consistent with a net gain of water by the periplasm, decreasing its biopolymer volume fraction. This supports a model in which the turgor pressure drops primarily across the thin peptidoglycan layer while the cell actively maintains osmotic balance between periplasm and cytoplasm, thus avoiding a substantial pressure differential across the cytoplasmic membrane. After sudden hyperosmotic shock (plasmolysis), the cytoplasm loses water as the periplasm gains water. Accordingly, <D_peri> increases threefold. The fluorescence recovery after photobleaching is complete and homogeneous in all cases, but in minimal medium, the periplasm is evidently thicker at the cell tips. For the relevant geometries, Brownian dynamics simulations in model cytoplasmic and periplasmic volumes provide analytical formulae for extraction of accurate diffusion coefficients from readily measurable quantities.     

Factors reducing and promoting the effectiveness of proline as an osmoprotectant in Escherichia coli K12

Proline accumulation in Escherichia coli is mediated by three proline porters. Proline catabolism is effected by proline porter I (PPI) and proline/delta 1-pyrroline carboxylate dehydrogenase. Proline did not accumulate cytoplasmically when E. coli was subjected to osmotic stress in minimal salts medium. Although PPI is induced when proline is provided as carbon or nitrogen source, its activity decreased following growth of the bacteria in minimal salts medium of high osmotic strength. Proline dehydrogenase was induced by proline in low or high osmotic strength media. Proline porter II (PPII) was both activated and induced in osmotically stressed bacteria, though the dependencies of the two responses on medium osmolarity differed. Osmotic downshift during the transport measurement decreased the uptake of proline, serine and glutamine by bacteria cultured in media of high osmotic strength. Thus, while osmotic upshift caused specific activation of PPII, osmotic downshift caused a non-specific reduction in amino acid uptake. Glycine betaine inhibited the uptake of [14C]proline via PPII and PPIII but not via PPI. The dependence of that inhibition on glycine betaine concentration was similar when PPII was uninduced, induced or activated by osmotic stress, or induced by amino acid limited growth. Thus PPII and PPIII, not PPI, contribute to the mechanism of osmoprotection by proline and glycine betaine. The tendency for exogenous proline to accumulate in the cytoplasm of bacteria exposed to osmotic stress would, however, be countered by increased proline catabolism.     

渗压震扰法释放Aeromonas sp.2016菌株外周间质中几丁质酶的研究

气单胞菌Aeromonassp．2016菌株能产生多种几丁质酶,其中的胞外酶C可能聚集于细胞外周胞质。为了避免破碎菌体而产生过多的杂蛋白,探索了用渗压震扰法(osmoticshock)来释放这部分酶。主要步骤是:先将菌体悬浮在20％蔗糖-0．03mol／LTris-HCI(pH8．0)高渗透压的溶液中,再快速转移到纯水低渗透压溶液中,产生瞬间渗压震荡,释放细胞外周胞质中的酶。结果表明,通过渗压震扰法释放出的酶纯度最高,比活力达到142．79U／g,比培养液上清液的54．46U／g和菌体破碎样品的14．66U／g分别高1．6倍和8．7倍,可用于纯化目的蛋白。     

Spin label studies on osmotically-induced changes in the aqueous cytoplasm of Phaeodactylum tricornutum

The effects of hyperosmotic stress and adaption on the aqueous cytoplasm of Phaeodactylum tricornutum have been studied with spin labels using 0.2M external Ni2+ to obtain spectra solely from labels within the cells. From partitioning of the TEMPO spin label between the internal aqueous phase and the membrane it is found that the internal volume of the cells decreased by approx. 50-60% in media of high osmotic strength (1.9 osmol/l). During the accumulation of proline in the cells (8.8 mg/ml packed cells) on incubation in the medium of high osmolarity for 3 days, the recovery of the volume was 80%. Further addition of proline to the medium resulted in an increase in the proline concentration in the cells (12.2 mg/ml packed cells) and a recovery in volume of 90%. Cells incubated in the absence of any nitrogen source showed very little recovery and were in a stressed state even in the absence of an osmotic gradient. From the rotational correlation times of the TEMPONE spin label it was found that the effective microviscosity in the cytoplasm of normal cells (approx. 3-8 cP) was considerably higher than that of the external medium (1 cP) and increased 1.5-2-fold under high osmotic stress (1.9 osmol/l). Adaption during the accumulation of proline only decreased the effective microviscosity by approx. 50% of the stressed-induced increase, a considerably smaller recovery than that of the cell volume.     

Intracellular localization of ATP:AMP phosphotransferase in Escherichia coli

ATP and AMP were immediately converted into ADP by intact cells of Escherichia coli in the presence of Mg2+, while ADP was also rapidly converted into ATP and AMP under the same conditions. Adenylate kinase was released when E. coli cells were converted to spheroplasts by treatment with lysozyme-EDTA or osmotic shock. Adenylate kinase activities detected in the cytoplasm, periplasm and membrane fractions were approximately 58%, 36% and 6% of the total cellular activity, respectively. These results indicate that adenylate kinase in E. coli occurs in the periplasm as well as the cytoplasm.     

Spin label studies on osmotically-induced changes in the aqueous cytoplasm of Phaeodactylum tricornutum

The effects of hyperosmotic stress and adaption on the aqueous cytoplasm of Phaeodactylum tricornutum have been studied with spin labels using 0.2 M external Ni²⁺ to obtain spectra solely from labels within the cells. From partitioning of the TEMPO spin label between the internal aqueous phase and the membrane it is found that the internal volume of the cells decreased by approx. 50–60% in media of high osmotic strength (1.9 osmol/l). During the accumulation of proline in the cells (8.8 mg/ml packed cells) on incubation in the medium of high osmolarity for 3 days, the recovery of the volume was 80%. Further addition of proline to the medium resulted in an increase in the proline concentration in the cells (12.2 mg/ml packed cells) and a recovery in volume of 90%. Cells incubated in the absence of any nitrogen source showed very little recovery and were in a stressed state even in the absence of an osmotic gradient. From the rotational correlation times of the TEMPONE spin label it was found that the effective microviscosity in the cytoplasm of normal cells (approx. 3–8 cP) was considerably higher than that of the external medium (1 cP) and increased 1.5–2-fold under high osmotic stress (1.9 osmol/l). Adaption during the accumulation of proline only decreased the effective microviscosity by approx. 50% of the stressed-induced increase, a considerably smaller recovery than that of the cell volume.     

Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity. Implications for protein-DNA interactions in vivo

The water-accessible volumes, the amounts of all significant osmolytes, and the protein concentration in the cytoplasm of aerobically grown Escherichia coli K-12 have been determined as a function of the osmolarity of the minimal growth medium. The volume of cytoplasmic water (Vcyto) decreases linearly with increasing osmolarity from 2.23(+/- 0.12) microliters/mg dry weight in cells grown at 0.10 OSM to 1.18(+/- 0.06) microliters/mg dry weight at 1.02 OSM. Above 0.28 OSM, growth rate decreases linearly with increasing osmolarity. The growth rate extrapolates to zero at an osmolarity of approximately 1.8, corresponding to an estimated Vcyto of 0.5(+/- 0.2) microliters/mg dry weight. Measurements of Vcyto in titrations of non-growing cells with the plasmolyzing agent NaCl were used to obtain volumes of "bound" water (presumably water of macromolecular hydration) and cytoplasmic osmotic coefficients for cells grown in medium of low (0.10 OSM) and moderate (0.28 OSM) osmolarity. The volume of bound water Vb is similar in the two osmotic conditions (Vb = 0.40(+/- 0.04) microliters/mg dry wt), and corresponds to approximately 0.5 g H2O/g cytoplasmic macromolecule. Since Vcyto decreases with increasing osmolarity, whereas Vb appears to be independent of osmolarity, water of hydration becomes a larger fraction of Vcyto as the osmolarity of the growth medium increases. Growth appears to cease at the osmolarity where Vcyto is approximately equal to Vb. K+ and glutamate (Glu-) are the only significant cytoplasmic osmolytes in cells grown in medium of low osmolarity. The amount of K+ greatly exceeds that of Glu-. Analysis of cytoplasmic electroneutrality indicates that the cytoplasm behaves like a concentrated solution of the K+ salt of cytoplasmic polyanions, in which the amount of additional electrolyte (K+ Glu-) increases with increasing osmolarity. As the osmolarity of the growth medium becomes very low, the cytoplasm approaches an electrolyte-free K+-polyanion solution. In vivo osmotic coefficients were determined from the variation of Vcyto with external osmolarity in plasmolysis titrations of non-growing cells. The values obtained (phi = 0.54(+/- 0.06) for cells grown at 0.10 OSM and phi = 0.71(+/- 0.11) at 0.28 OSM) indicate a high degree of non-ideality of intracellular ions arising from coulombic interactions between K+ and cytoplasmic polyanions. Analysis of these osmotic coefficients using polyelectrolyte theory indicates that the thermodynamic activity of cytoplasmic K+ increases from approximately 0.14 M in cells grown at an external osmolarity of 0.10 OSM to approximately 0.76 M at 1.02 OSM.(ABSTRACT TRUNCATED AT 400 WORDS)     

Osmoregulatory function of large vacuoles found in notochordal cells of the intervertebral disc running title: an osmoregulatory vacuole

The nucleus pulposi of many species contain residual cells from the embryonic notochord, which exhibit a very unusual appearance (large vacuoles occupying approximately 80% of the cell volume, surrounded by an actin cytoskeleton). While the vacuoles have been qualitatively described, their composition and function has remained elusive. Given that these cells are believed to generate and experience significant osmotic pressures in both the notochord and intervertebral disc, we hypothesized that the vacuoles may serve as osmoregulatory organelles. Using both experimental and theoretical means, we demonstrated that the vacuoles contain a low-osmolality solution, generated via ion pumps on the vacuolar membrane. During hypotonic stress the vacuoles release their contents into the cytoplasm, diluting the cytoplasm and restoring the osmotic balance across the cell membrane. Thus the vacuoles function to regulate the cell volume and tonicity during rapid osmotic stress, protecting the cells from potentially damaging swelling pressures.     

Changes in surface area of intact guard cells are correlated with membrane internalization

Guard cells must maintain the integrity of the plasma membrane as they undergo large, rapid changes in volume. It has been assumed that changes in volume are accompanied by changes in surface area, but mechanisms for regulating plasma membrane surface area have not been identified in intact guard cells, and the extent to which surface area of the guard cells changes with volume has never been determined. The alternative hypothesis-that surface area remains approximately constant because of changes in shape-has not been investigated. To address these questions, we determined surface area for intact guard cells of Vicia faba as they underwent changes in volume in response to changes in the external osmotic potential. We also estimated membrane internalization for these cells. Epidermal peels were subjected to external solutions of varying osmotic potential to shrink and swell the guard cells. A membrane-specific fluorescent dye was used to identify the plasma membrane, and confocal microscopy was used to acquire a series of optical paradermal sections of the guard cell pair at each osmotic potential. Solid digital objects representing the guard cells were created from the membrane outlines identified in these paradermal sections, and surface area, volume, and various linear dimensions were determined for these solid objects. Surface area decreased by as much as 40% when external osmotic potential was increased from 0 to 1.5 MPa, and surface area varied linearly with volume. Membrane internalization was approximated by determining the amount of the fluorescence in the cell's interior. This value was shown to increase approximately linearly with decreases in the cell's surface area. The changes in surface area, volume, and membrane internalization were reversible when the guard cells were returned to a buffer solution with an osmotic potential of approximately zero. The data show that intact guard cells undergo changes in surface area that are too large to be accommodated by plasma membrane stretching and shrinkage and suggest that membrane is reversibly internalized to maintain cell integrity.     

FURTHER STUDIES ON THE KINETICS OF OSMOSIS IN LIVING CELLS

Using unfertilized eggs of Arbacia punctulata as natural osmometers an attempt has been made to account for the course of swelling and shrinking of these cells in anisotonic solutions by means of the laws governing osmosis and diffusion. The method employed has been to compute permeability of the cell to water, as measured by the rate of volume change per unit of cell surface per unit of osmotic pressure outstanding between the cell and its medium. Permeability to water as here defined and as somewhat differently defined by Northrop is approximately constant during swelling and shrinking, at least for the first several minutes of these processes. Permeability is found to be independent of the osmotic pressure of the solution in which cells are swelling. Water is found to leave cells more readily than it enters, that is, permeability is greater during exosmosis than during endosmosis.     

Solute Accumulation in Plant Cells: III. Treatments which Arrest and Restore the Course of Absorption and Growth in Cultured Carrot Explants1

Prior papers have dealt with the range of the growth responsesof carrot explants to the composition of the ambient media andhow these affected the solute concentration and compositionof the tissue. They have also dealt with the sequential eventsalong the time course of growth of the tissue explant. Thispaper presents results and conclusions derived from experimentswhich exploit changes in the growing explants when their normalcourse of growth and solute uptake is interrupted by exposingthem sequentially to different ambient media. After explants were induced to grow during an initial 6 days,they were placed in a minimal nutrient medium which lacked growthstimuli and salts, notably potassium ions. Thus the internalsalt concentrations of the cells declined as they continuedto divide and enlarge at a reduced rate and their osmotic valuewas maintained by storage of organic solutes (sugar). The subsequentresponses of these ‘low salt’ cells to differentnutrient regimes were studied. When salts were resupplied, growth was stimulated somewhat andthe osmotic value of the cells increased as salts were accumulated;with a renewed full nutrient medium and a full complement ofgrowth substances, the cultures re-embark upon their growthand attain the average cell size and composition as if theyhad not been reversibly arrested. Thus reversible trends insolute composition of cells may be superimposed upon their normaldevelopmental course by alternately withholding and restoringthe stimuli to their growth and by changing the balance betweenorganic and inorganic solutes supplied in the medium. The emphasis is on the control of osmotic value in the cellsas they enlarge and mature and this over-rides the changes inthe solutes they receive (salts or sugars) via the ambient medium.The effects here observed were induced by sequential changesin the culture medium, but these obviously relate to similarresponses of cells in the intact plant body as their growthand solute supply is modified through interactions between organsas the plant grows.     

New osmoregulated beta(1-3),beta(1-6) glucosyltransferase(s) in Azospirillum brasilense.

A linear beta(1-3),beta(1-6) glucan was detected in the periplasm of Azospirillum brasilense cells growing in a medium of low osmotic strength. This glucan was produced in vitro by purified bacterial inner membranes with UDP-glucose as the sugar donor in the presence of Mg2+. Growth in a high-osmotic-strength medium strongly reduced the amount of this glucan accumulated in the periplasmic space, and the inhibition was associated with a reduction in the enzymatic activity of the beta(1-3),beta(1-6) glucosyltransferase(s).     

Osmotic regulation of prolactin secretion. Possible role of chloride

The role of osmotic pressure in the exocytosis of prolactin from rat pituitary tumor (GH) cells in culture was investigated. Reducing the osmotic strength of the medium from 300 mosm to 150 mosm by removal of NaCl did not alter basal secretion of prolactin but inhibited secretion stimulated by thyrotropin-releasing hormone (TRH) and forskolin. Both basal and stimulated secretion of prolactin were inhibited by increasing the osmotic strength of the medium with NaCl (IC50 at approximately 500 mosm). The stimulated release of hormone from GH-cells was independent of sodium and unaffected by replacement of sodium ion with tetramethylammonium or choline, or by addition of 500 nM tetrodotoxin. Secretagogue-stimulated release was, however, dependent upon chloride. Exchange of medium chloride with benzoate or isethionate significantly inhibited the stimulated release of prolactin (IC50 at approximately 60 mM exchange) regardless of the secretagogue utilized (phorbol ester, forskolin, depolarization plus BAY K8644, or TRH). Exchange of medium chloride with either isethionate or benzoate reduced cell volume by 10% compared to 60% for sucrose and mannitol, suggesting that inhibition of secretion by isethionate exchange was not a result of increased intracellular osmotic pressure. Complete exchange of medium chloride with isethionate did not alter equilibrium [3H]methyl-TRH binding, resting internal [Ca2+], or the [Ca2+]i response to depolarization and TRH as measured with intracellularly trapped Fura 2. Chloride removal did not change resting internal pH and recovery from an acid load as measured by the intracellular pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The stimulated secretion of prolactin was also inhibited by exchange of chloride with isethionate in normal pituitary cells in primary culture and the ability of normal cells to respond to the dopamine agonist bromocryptine was not affected by the exchange. These results suggest that exocytosis of prolactin from GH-cells and normal pituitary cells in culture is an osmotically driven process that is chloride-dependent. Stimulated release is more chloride-dependent than constitutive release. The inhibitory effect of isethionate substitution occurs after signal transduction and is distinct from the site of dopamine inhibition of prolactin release.     

Cell volume dependence of 1H spin-echo NMR signals in human erythrocyte suspensions. The influence of in situ field gradients

The 1H spin-echo NMR signal amplitudes and intensities of low molecular weight solutes in the cytoplasm and extracellular fluid of suspensions of human erythrocytes were shown to depend on the osmotic pressure of the media. At low osmotic pressure (220 mosM/kg) freeze-thaw lysis of the cells resulted in signal enhancement which was greatest for extracellular molecules, but both intra- and extracellular species were almost equally enhanced at 580 mosM/kg. This effect is due to field gradients formed at cell boundaries as a result of differences in magnetic susceptibility between the medium and the cytoplasm. T2 values measured using the Carr-Purcell-Meiboom-Gill pulse sequence, with tau = 0.0003 s, depended little on cell volume and absolute changes in volume magnetic susceptibility were also small. The mean field gradients, calculated from data obtained on cell suspensions at different osmotic pressures, were in the range 0.25-1.98 G/cm and 0.89-2.09 G/cm for intra- and extracellular compartments, respectively. The maintenance of isotonicity of the extracellular fluid during metabolic studies of cell suspensions is important in order to avoid artefacts in the determination of metabolite concentrations when using the spin-echo technique. Conversely it may be possible to perform transport measurements using spin-echo NMR to monitor the cell volume changes which occur during the transmembrane migration of molecules.     

Permeability of the Cell Envelope and Osmotic Behavior in Saccharomyces cerevisiae

Bakers' yeast (Saccharomyces cerevisiae) was equilibrated with distilled water and then packed into standardized pellets by centrifugation. The fractional space (S value) that was accessible to passive permeation was probed with a variety of mono- and divalent salts, mono- and disaccharides, polyols, substrates and products of beta-fructofuranosidase (EC 3.2.1.26) and acid phosphatase (EC 3.1.3.2), and a cross-linked polymer of sucrose (Ficoll 400). A simple but very reproducible method was developed to measure pellet volume. At the limit of zero osmolality for bathing medium, the interstitial space was 0.223 ml/ml of pellet, and the aqueous volume of cell envelopes was 0.117 ml/ml of pellet. Thus the cell envelope for this yeast, under these conditions, was approximately 15% of the total cell volume. At a finite osmolality, the space in a yeast pellet that was accessible to salt was accounted for by the sum of initial interstitial space, the volume of the cell envelopes, and the volume of water abstracted from the cells by osmosis. Plots of S value versus osmolality were linear for uncharged probes and curvilinear for all salts. When Ficoll and potassium thiocyanate were presented to the yeast in admixture, the S values for the salt increased continuously over the range of osmolality studied. However, the S values for Ficoll 400 (which did not penetrate the cell wall) were lower by an amount equilivalent to the cell envelopes; they increased in parallel with the S curve for salt up to 1.15 osmol/kg and then plateaued. The results support the concept of incipient plasmolysis at 1.15 osmol/kg, and the separation of protoplasm from the cell wall is indicated with more concentrated solutions. Such cells were still viable if slowly diluted in distilled water, but they were injured by the shock of rapid dilution. However, shocking the cells did not release beta-fructofuranosidase into the medium. The complete accessibility of salts toward killed cells was demonstrated with yeast that had been pretreated with heat, organic solvents, or glutaraldehyde.     

Effects of hyperosmotic solutions on the filament lattice of intact frog skeletal muscle.

The effect of increasing the osmotic strength of the extracellular solution on the fifament lattice of living frog sartorius and semitendinosus muscle has been studied using low-angle x-ray diffraction to measure the lattice spacing. As the extracellular osmotic strength is increased, the filament lattice shrinks like an osmometer until a minimal spacing between the thick filaments is reached. This minimal spacing varies from 20 to 31 nm, depending on the sarcomere length. Further increase in the osmotic strength produces little further shrinkage. The osmotic shrinkage curve indicates, for both muscles, an osmotically-inactive volume of approximately 30% of the volume in normal Ringer's solution. Shrinkage appears to be independent of temperature and the type of particle used to increase the osmotic strength (glucose, sucrose, small ions). The rate at which osmotic equilibruim is reached depends on muscle size, being slower for greater muscle diameters. Equilibrium spacings are approached exponentially with time constants ranging from 20 to 60 min. Independent of osmotic equilibrium, the lattice tends to shrink slowly by approximately 3% over the first few hours after dissection, probably because of a leakage of K+ ions from inside the muscle cells. This can be partly prevented by using an extracellular solution which contains a higher concentration of K+ ions or which is hypoosmotic. The volume of the muscle filament lattice (1.155d10(2) . S) is constant over a very wide range of sarcomere lengths, and is equal to approximately 3.6 x 10(6) nm3 for a range of amphibian muscle types.     

Electrorotation of single yeast cells at frequencies between 100 Hz and 1.6 GHz.

The determination of complete electrorotation spectra of living cells has been made possible by the development of a quadrature generator and an electrode assembly that span the frequency range between 100 Hz and 1.6 GHz. Multiple spectra of single cells of the yeast Saccharomyces cerevisiae have been measured at different medium conductivities ranging from 0.7 to 550 microS cm-1. A spherical four-shell model was applied that simulated the experimental data well and disclosed the four-layer structure of the cell envelope attributed to the plasma membrane, the periplasmic space, and a thick inner and a thin outer wall region. Below 10 kHz an additional rotation effect was found, which changed its direction depending on the ionic strength of the medium. This is supposed to be connected with properties of the cell surface and its close vicinity. From the four-shell simulation the following physical properties of cell compartments could be derived: specific capacitance of plasma membrane (0.76 microF cm-2), periplasmic space (0.5 microF cm-2), and outer wall region (0.1 microF cm-2). The conductivity of cytoplasm, plasma membrane, and inner wall region were found to vary with medium ionic strength from 9 to 12 mS cm-1, 5.8 nS cm-1 to approximately 50 nS cm-1, and 6 microS cm-1 to 240 microS cm-1, respectively.     

Loss of intracellular glycerol from Dunaliella by electroporation at constant osmotic pressure: subsequent restoration of glycerol content and associated volume changes

The effect of electroporation on Dunaliella tertiolecta at constant osmotic pressure (or water activity) was investigated. The following metabolic and physiological parameters were determined: extracellular and intracellular glycerol content, soluble protein content, photosynthetic oxygen evolution, mitochondrial oxygen uptake, cell volume and cell density. Electroporation conditions are described that released about 10% of intracellular glycerol to the external medium with minimal apparent effects on metabolism. Glycerol release originated from most cells rather than by total rupture of a small proportion of cells. Cell volume, measured on motile cells by video microscopy, reduced by 23% immediately after electroporation. Cell density did not increase. The uptake of mannitol, the major solute in the electroporation medium, was less than 20% of glycerol release. Following electroporation, the intracellular glycerol content and the cell volume both returned to pre-electroporation values after about 30min. Because the cells were maintained at constant external osmotic pressure throughout the procedure, it is concluded that the regulatory mechanism responsible for setting the intracellular glycerol content does not sense external osmotic pressure per se. These findings are consistent with a mechanism that senses a parameter linked directly to cell volume to set the intracellular glycerol content.     

Cell volume dependence of 1H spin-echo NMR signals in human erythrocyte suspensions: The influence of in situ field gradients

The ¹H spin-echo NMR signal amplitudes and intensities of low molecular weight solutes in the cytoplasm and extracellular fluid of suspensions of human erythrocytes were shown to depend on the osmotic pressure of the media. At low osmotic pressure (220 mosM/kg) freeze-thaw lysis of the cells resulted in signal enhancement which was greatest for extracellular molecules, but both intra- and extracellular species were almost equally enhanced at 580 mosM/kg. This effect is due to field gradients formed at cell boundaries as a result of differences in magnetic susceptibility between the medium and the cytoplasm. T₂ values measured using the Carr-Purcell-Meiboom-Gill pulse sequence, with τ = 0.0003 s, depended little on cell volume and absolute changes in volume magnetic susceptibility were also small. The mean field gradients, calculated from data obtained on cell suspensions at different osmotic pressures, were in the range 0.25–1.98 G/cm and 0.89–2.09 G/cm for intra- and extracellular compartments, respectively. The maintenance of isotonicity of the extracellular fluid during metabolic studies of cell suspensions is important in order to avoid artefacts in the determination of metabolite concentrations when using the spin-echo technique. Conversely it may be possible to perform transport measurements using spin-echo NMR to monitor the cell volume changes which occur during the transmembrane migration of molecules.     

L-929 cells under hyperosmotic conditions. Water, Na+, and K+

Changes in cell water content resulting from sorbitol addition to the environment of L-929 cells were evaluated gravimetrically using 14C-labeled polyethylene glycol as a probe of extracellular space. Reductions in cell water were proportional to sorbitol supplements up to 0.6 molal, above which no further measurable decrease occurred. No volume regulation occurred for at least 1 h but the percentage of cell water lost was quickly regained when physiological conditions were restored. The amount of cell water lost because of a given hyperosmotic exposure was found to exceed the loss of cell volume. That discrepancy could be the result of an overestimation of extracellular space and/or an underestimation of cell volume reduction as a result of in-folding of the cell surface. Na+ and K+ were also measured in cells of variable water content and volume: no significant change occurred in the amounts of these ions per cell, but large increases in total cell concentration resulted from hyperosmotic exposure. The sum of Na+ and K+ concentrations exceeds the total osmotic pressure of the medium indicating that an appreciable fraction of Na+ and K+ must be bound to fixed charges within the cells. The results are evaluated in the context of intracellular organization.