Non-osmotic Volume Research Articles

How sterols affect protoplasts plasma membrane water permeability and their volume under osmotic shock.

Protoplasts isolated from Arabidopsis leaves were used to study the initial stages of the plant cell response to osmotic stress. The role of sterols in these processes was investigated by their extraction from the protoplast plasma membrane in the presence of the oligosaccharide - methyl-β-cyclodextrin (MβCD). Depletion of membrane sterols caused by MβCD treatment did not alter protoplast volume under isosmotic conditions; however, volumes changed significantly when protoplasts were exposed to osmotic stress. Estimation of the plasma membrane water permeability coefficient (Pos), calculated from the initial rate of protoplast osmotic shrinkage, showed that control suspension is characterized by a high dispersion of the Pos values. However, Pos became more homogeneous after plasma membrane sterol depletion. Protoplasts were stained with FM 1-43 to assess how sterol extraction affects vesicular transport under osmotic shock. In order to determine the protoplast non-osmotic volume (Vb) steady-state volumes at different external osmolarities were fitted with linear dependences of the Boyle-van't Hoff (BVH) plot. It was found that sterol extraction is accompanied by a change in the slope of the BVH plot and a decrease in the apparent Vb. Several possible mechanisms behind the change in the protoplast volume and plasma membrane Pos regulation by sterols under osmotic stress are discussed.

Volume Regulation and Nonosmotic Volume of Individual Human Platelets Quantified by High-Speed Scanning Ion Conductance Microscopy.

Platelets are anucleate cells that play an important role in wound closure following vessel injury. Maintaining a constant platelet volume is critical for platelet function. For example, water-induced swelling can promote procoagulant activity and initiate thrombosis. However, techniques for measuring changes in platelet volume such as light transmittance or impedance techniques have inherent limitations as they only allow qualitative measurements or do not work on the single-cell level. Here, we introduce high-speed scanning ion conductance microscopy (HS-SICM) as a new platform for studying volume regulation mechanisms of individual platelets. We optimized HS-SICM to quantitatively image the morphology of adherent platelets as a function of time at scanning speeds up to 7 seconds per frame and with 0.1 fL precision. We demonstrate that HS-SICM can quantitatively measure the rapid swelling of individual platelets after a hypotonic shock and the following regulatory volume decrease (RVD). We found that the RVD of thrombin-, ADP-, and collagen-activated platelets was significantly reduced compared with nonactivated platelets. Applying the Boyle-van't Hoff relationship allowed us to extract the nonosmotic volume and volume fraction on a single-platelet level. Activation by thrombin or ADP, but not by collagen, resulted in a decrease of the nonosmotic volume, likely due to a release reaction, leaving the total volume unaffected. This work shows that HS-SICM is a versatile tool for resolving rapid morphological changes and volume dynamics of adherent living platelets.

Upgrading the Measurement of Membrane Hydraulic Conductivity and the Osmotically Inactive Volume of Protoplasts for Evaluating the Freshness of Postharvest Leafy Vegetables

HighlightsA method to simultaneously determine membrane water permeability (Lp) and nonosmotic volume (Vb) is proposed.The proposed method revealed that Vb was neither proportional nor constant to the protoplast size.Individually determined Vb improved accurate estimation of Lp.During storage, Lp increased and Vb decreased in spinach leaves, which may well indicate produce freshness.Abstract. Membrane water permeability is of great importance to better understand the mechanism of water loss; however, its correct understanding and measurement is still an issue. We proposed and tested the efficacy of a numerical analytic approach for the simultaneous determination of the hydraulic conductivity (Lp) and relative osmotically inactive volume (b) of protoplasts isolated from spinach mesophyll tissue. The new approach revealed that the osmotically inactive volume was neither proportional nor constant to the protoplast size and differed for each protoplast. Usage of individually determined b for the computation provided the best fitting performance for determining the Lp, whereas collective b decreased the accuracy of regression and resulted in the underestimation of Lp. Our approach also has the advantage of a shorter period of hyperosmotic challenge, and as such, is suitable for observing fragile protoplasts. We found a gradual reduction in b and a linear increase in Lp during four days of storage at 20°C, likely due to degradation of internal components and dysfunction of the cell membrane, respectively. We also discuss the potential need to consider tissue specificity and possible damage by protoplast isolation to conduct shelf-life research of leafy vegetables. Keywords: Mesophyll protoplast, Nonosmotic volume, Numerical determination, Spinacia oleracea, Water loss, Water permeability.

Physiochemical Modeling of Vesicle Dynamics upon Osmotic Upshift

We modeled the relaxation dynamics of (lipid) vesicles upon osmotic upshift, taking into account volume variation, chemical reaction kinetics, and passive transport across the membrane. We focused on the relaxation kinetics upon addition of impermeable osmolytes such as KCl and membrane-permeable solutes such as weak acids. We studied the effect of the most relevant physical parameters on the dynamic behavior of the system, as well as on the relaxation rates. We observe that 1) the dynamic complexity of the relaxation kinetics depends on the number of permeable species; 2) the permeability coefficients (P) and the weak acid strength (pKa-values) determine the dynamic behavior of the system; 3) the vesicle size does not affect the dynamics, but only the relaxation rates of the system; and 4) heterogeneities in the vesicle size provoke stretching of the relaxation curves. The model was successfully benchmarked for determining permeability coefficients by fitting of our experimental relaxation curves and by comparison of the data with literature values (in this issue of Biophysical Journal). To describe the dynamics of yeast cells upon osmotic upshift, we extended the model to account for turgor pressure and nonosmotic volume.

The Water to Solute Permeability Ratio Governs the Osmotic Volume Dynamics in Beetroot Vacuoles.

Plant cell vacuoles occupy up to 90% of the cell volume and, beyond their physiological function, are constantly subjected to water and solute exchange. The osmotic flow and vacuole volume dynamics relies on the vacuole membrane -the tonoplast- and its capacity to regulate its permeability to both water and solutes. The osmotic permeability coefficient (Pf) is the parameter that better characterizes the water transport when submitted to an osmotic gradient. Usually, Pf determinations are made in vitro from the initial rate of volume change, when a fast (almost instantaneous) osmolality change occurs. When aquaporins are present, it is accepted that initial volume changes are only due to water movements. However, in living cells osmotic changes are not necessarily abrupt but gradually imposed. Under these conditions, water flux might not be the only relevant driving force shaping the vacuole volume response. In this study, we quantitatively investigated volume dynamics of isolated Beta vulgaris root vacuoles under progressively applied osmotic gradients at different pH, a condition that modifies the tonoplast Pf. We followed the vacuole volume changes while simultaneously determining the external osmolality time-courses and analyzing these data with mathematical modeling. Our findings indicate that vacuole volume changes, under progressively applied osmotic gradients, would not depend on the membrane elastic properties, nor on the non-osmotic volume of the vacuole, but on water and solute fluxes across the tonoplast. We found that the volume of the vacuole at the steady state is determined by the ratio of water to solute permeabilites (Pf/Ps), which in turn is ruled by pH. The dependence of the permeability ratio on pH can be interpreted in terms of the degree of aquaporin inhibition and the consequently solute transport modulation. This is relevant in many plant organs such as root, leaves, cotyledons, or stems that perform extensive rhythmic growth movements, which very likely involve considerable cell volume changes within seconds to hours.

Biophysical Assessment of Human Aquaporin-7 as a Water and Glycerol Channel in 3T3-L1 Adipocytes

The plasma membrane aquaporin-7 (AQP7) has been shown to be expressed in adipose tissue and its role in glycerol release/uptake in adipocytes has been postulated and correlated with obesity onset. However, some studies have contradicted this view. Based on this situation, we have re-assessed the precise localization of AQP7 in adipose tissue and analyzed its function as a water and/or glycerol channel in adipose cells. Fractionation of mice adipose tissue revealed that AQP7 is located in both adipose and stromal vascular fractions. Moreover, AQP7 was the only aquaglyceroporin expressed in adipose tissue and in 3T3-L1 adipocytes. By overexpressing the human AQP7 in 3T3-L1 adipocytes it was possible to ascertain its role as a water and glycerol channel in a gain-of-function scenario. AQP7 expression had no effect in equilibrium cell volume but AQP7 loss of function correlated with higher triglyceride content. Furthermore it is also reported for the first time a negative correlation between water permeability and the cell non-osmotic volume supporting the observation that AQP7 depleted cells are more prone to lipid accumulation. Additionally, the strong positive correlation between the rates of water and glycerol transport highlights the role of AQP7 as both a water and a glycerol channel and reflects its expression levels in cells. In all, our results clearly document a direct involvement of AQP7 in water and glycerol transport, as well as in triglyceride content in adipocytes.

Rectification of the Water Permeability in COS-7 Cells at 22, 10 and 0°C

The osmotic and permeability parameters of a cell membrane are essential physico-chemical properties of a cell and particularly important with respect to cell volume changes and the regulation thereof. Here, we report the hydraulic conductivity, Lp, the non-osmotic volume, Vb, and the Arrhenius activation energy, Ea, of mammalian COS-7 cells. The ratio of Vb to the isotonic cell volume, Vc iso, was 0.29. Ea, the activation energy required for the permeation of water through the cell membrane, was 10,700, and 12,000 cal/mol under hyper- and hypotonic conditions, respectively. Average values for Lp were calculated from swell/shrink curves by using an integrated equation for Lp. The curves represented the volume changes of 358 individually measured cells, placed into solutions of nonpermeating solutes of 157 or 602 mOsm/kg (at 0, 10 or 22°C) and imaged over time. Lp estimates for all six combinations of osmolality and temperature were calculated, resulting in values of 0.11, 0.21, and 0.10 µm/min/atm for exosmotic flow and 0.79, 1.73 and 1.87 µm/min/atm for endosmotic flow (at 0, 10 and 22°C, respectively). The unexpected finding of several fold higher Lp values for endosmotic flow indicates highly asymmetric membrane permeability for water in COS-7. This phenomenon is known as rectification and has mainly been reported for plant cell, but only rarely for animal cells. Although the mechanism underlying the strong rectification found in COS-7 cells is yet unknown, it is a phenomenon of biological interest and has important practical consequences, for instance, in the development of optimal cryopreservation.

Membrane permeability coefficients of murine primary neural brain cells in the presence of cryoprotectant

Neural cells isolated from the brain have a number of research and clinical applications, including transplantation to patients with neurodegenerative conditions. Tissue supply is one of the major limiting factors to clinical transplantation. Cryopreservation of primary neural cells would improve supply, aid in organisation of transplantation surgery and facilitate research. To date, cryopreservation using standard methods has resulted in reduced yield and/or viability of primary neural tissue. In order to optimise freezing protocols specifically for such cells, the non-osmotic volume ( V b), water permeability ( L p) and permeability to cryoprotectant ( P cpa) were determined. Murine foetal brain tissue from the ganglionic eminence (GE), ventral mesencephalon (VM), or neocortical mantle (Ctx) was trypsinised to a single cell suspension. To determine V b, cell volume was measured after exposure to anisotonic solutions of sucrose (150–1500 mOsmol/kg). L p (μm/min.atm) and P cpa (μm/s) were determined for GE cells by measuring cell volume during exposure to 1.5 mol/l cryoprotectant. Cell volume was determined using an electronic particle counting method. V b was 27% for Ctx and GE, and 30% for VM. The osmotic response of GE cells was similar in the presence of propane-1,2-diol and dimethyl sulphoxide. In the presence of ethylene glycol, cell volume decrease was greater on initial exposure to cryoprotectant and recovery slower. Differences in L p, but not P cpa, were found between cryoprotectants. The present results provide key parameters for optimisation of freezing protocols for cryopreservation of primary foetal brain tissues for application in neural cell transplantation.

Water Transport during Freezing of Human Dermal Fibroblast as Affected by Various Freezing Rates

In order to predict optimal freezing processes for the cryopreservation of human dermal fibroblasts, knowledge of fundamental cryobiological characteristics including cell osmotic characteristics is required. In this study, the osmotic characteristics of human dermal fibroblasts were experimentally measured. The cell volume was 5100 μm3, and the relative diameter was 21 μm, at isotonic osmolality. The cell volume change was also measured after the cells were exposed and equilibrated to different anisosmotic conditions. It was found that the cell volume was a linear function of the reciprocal of the extracellular osmolality (Boyle–van't Hoff plot). The nonosmotic volume of the cell was 36% of the isotonic volume of the cells. Then these parameters including the nonosmotic volume of the cell were used in the water transport model to simulate water transport of fibroblasts at various freezing rates between 0.01–50°C/min.

Measuring the Osmotic Water Permeability of the Plant Protoplast Plasma Membrane: Implication of the Nonosmotic Volume

Starting from the original theoretical descriptions of osmotically induced water volume flow in membrane systems, a convenient procedure to determine the osmotic water permeability coefficient (P (os)) and the relative nonosmotic volume (beta) of individual protoplasts is presented. Measurements performed on protoplasts prepared from pollen grains and pollen tubes of Lilium longiflorum cv. Thunb. and from mesophyll cells of Nicotiana tabacum L. and Arabidopsis thaliana revealed low values for the osmotic water permeability coefficient in the range 5-20 microm.s(-1) with significant differences in P (os), depending on whether beta is considered or not. The value of beta was determined using two different methods: by interpolation from Boyle-van't Hoff plots or by fitting a solution of the theoretical equation for water volume flow to the whole volume transients measured during osmotic swelling. The values determined with the second method were less affected by the heterogeneity of the protoplast samples and were around 30% of the respective isoosmotic protoplast volume. It is therefore important to consider nonosmotic volume in the calculation of P (os) as plant protoplasts behave as nonideal osmometers.

Differences for bound water content as estimated by pressure–volume and adsorption isotherm curves

Pressure–volume ( PV) and adsorption isotherm (AI) curves were constructed on fresh and dry tissues of durum wheat respectively, to assess the relation between the water status of living leaves and the properties of water that is bound (BW) with different strength to ionic, polar or hydrophobic sites of macromolecules. The leaves were collected from six genotypes grown in field, in 2 years. The amounts of the non-osmotic BW fraction, free water and osmotic potential at full turgor were determined by pressure–volume ( PV) curves. Three parameters that relate to the amounts of the weakly and strongly bound water (quantitative BW properties) and five parameters related to tissues-binding strength for the same water fractions (qualitative BW properties) were calculated by AI-curves. The non-osmotic volume of PV-curves doesn’t correspond to the water fraction bound to the charged or polar sites of macromolecules as determined by AI-curves. The qualitative parameters, in contrast to quantitative parameters, may be affected by common physical-chemical factors: the changes in the amount of strongly bound water in tissues were independent from those in the weakly bound fraction; in contrast an increase in tissue affinity for strongly bound water implied a simultaneous increase in the affinity for weakly bound water. The qualitative properties of bound water may be particularly important for drought adaptation in durum wheat, being associated with the solute potential and with the succulence degree of the leaves.

Cryopreservation of umbilical cord blood: 1. Osmotically inactive volume, hydraulic conductivity and permeability of CD34 + cells to dimethyl sulphoxide

Umbilical cord blood (UCB) is an accepted treatment for the reconstitution of bone marrow function following myeloablative treatment predominantly in children and juveniles. Current cryopreservation protocols use methods established for bone marrow and peripheral blood progenitors cells that have largely been developed empirically. Such protocols can result in losses of up to 50% of the nucleated cell population: losses unacceptable for cord blood. The design of optimal cryopreservation regimes requires the development of addition and elution protocols for the chosen cryoprotectant; protocols that minimise damaging osmotic transients. The biophysical parameters necessary to model the addition and elution of dimethyl sulphoxide to and from cord blood CD34 + cells have been established. An electronic particle counting method was used to establish the volumetric response of CD34 + cells to changes in osmolality of the suspending medium. The non-osmotic volume of the cell was 0.27 of the cells isotonic volume. The permeation kinetics of CD34 + cells to water and dimethyl sulphoxide were investigated at two temperatures, +1.5 and +20 °C. Values for the hydraulic conductivity were 3.2×10 −8 and 2.8×10 −7 cm/atm/s, respectively. Values for the permeability of dimethyl sulphoxide at these temperatures were 4.2×10 −7 and 7.4×10 −6 cm/s, respectively. Clonogenic assays indicated that the ability of CD34 + cells to grow and differentiate was significantly impaired outside the limits 0.6–4× isotonic. Based on the Boyle van’t Hoff plot, the tolerable limits for cell volume excursion were therefore 45–140% of isotonic volume. The addition and elution of cryoprotectant was modelled using a two-parameter model. Current protocols for the addition of cryoprotectant based on exposure at +4 °C would require additional time for complete equilibration of the cryoprotectant. During the elution phase current protocols are likely to cause CD34 + cells to exceed tolerable limits. The addition of a short holding period during elution reduces the likelihood of this occurring.

Differences in the Requirements for Cryopreservation of Porcine Aortic Smooth Muscle and Endothelial Cells

One of the basic requirements for the production of tissue-engineered constructs is an effective means of storing both the constructs and the cells that will be used to make them. This paper reports on the cryopreservation of porcine aortic smooth muscle and endothelial cells intended for the production of model vascular constructs. We first determined the cell volume, nonosmotic volume, and the permeability parameters for water and the cryoprotectant dimethyl sulfoxide (Me(2)SO) in these cells at 2-4 degrees and 22 degrees C. The following results were obtained: Table unavailable in HTML format. Using a cell culture assay, both cell types were shown to tolerate threefold changes in cell volume, in either direction, without significant injury. Although these data suggested that single-step methods for the introduction and removal of 10% w/w Me(2)SO should be effective, an additional mannitol dilution step was adopted in order to reduce the time required for removal of the Me(2)SO. Following cooling at 0.3, 1, or 10 degrees C/min and storage at less than -160 degrees C, the survival of porcine aortic smooth muscle cell suspensions, measured by a cell culture assay, was inversely related to cooling rate; at 0.3 degrees C/min, recovery was >80%. The survival rate for aortic endothelial cells was directly related to cooling rate over the range tested and was >80% at 10 degrees C/min.

Mechanisms Involved in Diurnal Changes of Osmotic Potential in Grapevines under Drought Conditions

Summary The aim of this study was the quantitative determination of the contribution of various possible mechanisms involved in diurnal changes of osmotic potential in grapevine leaves ( Vitis vinifera L., cv. Victoria ) under water stress conditions. Results of this study revealed that dehydration accounted for 36 % of the diurnal changes in osmotic potential in stressed plants. The relative contribution of changes in non-osmotic volume was negative. Net solute accumulation accounted for 73 % of the diurnal changes, indicating the occurrence of an active osmotic adjustment during the day in stressed plants. There was evidence that compounds other than sugars contributed to diurnal osmotic adjustment in stressed grapevines.

Osmotic shock of fertilized mouse ova.

The effect of osmotic changes on fertilized mouse ova was studied by measuring their survival, defined as development into hatching blastocysts, after exposure to various concentrations of ethanediol (ethylene glycol). In addition, a Boyle-van't Hoff plot was derived from exposing ova to hypotonic and hypertonic solutions ranging from 0.1 to 2.8 osmol. Volume of ova was inversely proportional to osmolality over this range. Extrapolation of this relationship yielded a nonosmotic volume of the ova of 22.5%. Eighty-five per cent or more of the ova survived exposure to this wide range of concentrations and developed into blastocysts. The rate of development of ova exposed to anisotonic solutions was the same as that of controls. Ova underwent osmotic shock when abruptly diluted out of concentrated solutions of ethanediol with an isotonic solution. Their survival was highly dependent on the ethanediol concentration with which they had equilibrated before dilution, and the manner, rate and temperature of dilution. The longer the exposure to ethanediol the greater was the sensitivity of the ova to osmotic shock, reflecting permeation of ethanediol into the ova. Osmotic shock could be alleviated by dilution at a high temperature, and prevented by the use of sucrose as an osmotic buffer at 37 degrees C. Identification of the variables that influence osmotic shock of ova will be helpful in the systematic study of their cryopreservation.

Osmotic Adjustment in Sorghum

Osmotic adjustment, defined as a lowering of osmotic potential (psi(pi)) due to net solute accumulation in response to water stress, has been considered to be a beneficial drought tolerance mechanism in some crop species. The objective of this experiment was to determine the relative contribution of passive versus active mechanisms involved in diurnal psi(pi) changes in sorghum (Sorghum bicolor L. Moench) leaf tissue in response to water stress. A single sorghum hybrid (cv ATx623 x RTx430) was grown in the field under variable water supplies. Water potential, psi(pi), and relative water content were measured diurnally on expanding and the uppermost fully expanded leaves before flowering and on fully expanded leaves during the grain-filling period. Diurnal changes in total osmotic potential (Deltapsi(pi)) in response to water stress was 1.1 megapascals before flowering and 1.4 megapascals during grain filling in comparison with 0.53 megapascal under well-watered conditions. Under water-stressed conditions, passive concentration of solutes associated with dehydration accounted for 50% (0.55 megapascal) of the diurnal Deltapsi(pi) before flowering and 47% (0.66 megapascal) of the change during grain filling. Net solute accumulation accounted for 42% (0.46 megapascal) of the diurnal Deltapsi(pi) before flowering and 45% (0.63 megapascal) of the change during grain filling in water-stressed leaves. The relative contribution of changes in nonosmotic volume (decreased turgid weight/dry weight) to diurnal Deltapsi(pi) was less than 8% at either growth stages. Water stress did not affect leaf tissue elasticity or partitioning of water between the symplasm and apoplasm.

Effects of osmotic stress on gamete size, rhizoid initiation and germling growth in fucoid algae

Effects of osmotic stress on gamete size were studied for Fucus vesiculosus; spermatozoids behaved as osmometers when the osmotic pressure of the external artificial seawater medium (ASP12S) was changed. The non-osmotic volume was approximately 44% of the spermatozoid volume in full-strength ASP12S. Unfertilized eggs behaved as osmometers but with a relatively small change in volume. The apparent non-osmotic volume of the egg was approximately 93% of the volume in full-strength ASP12S. The significance of these observations and their possible implications for fertilization and cross-fertilization in situ is discussed. Rhizoid initiation in zygotes of Fucus spiralis and F. vesiculosus decreased with increasing salinity. A decrease also occurred when F. vesiculosus was incubated in low salinity media, though this was not the case in F. spiralis. Subsequent growth patterns of the germlings of both fucoid algae were similar, although F. vesiculosus had a higher growth rate than F. spiralis. Both species showe...

Osmotic adjustment and the accumulation of organic solutes in whole cells and protoplasts of Saccharomyces cerevisiae.

In the presence of a suitable carbon source, whole cells and protoplasts of Saccharomyces cerevisiae synthesized glycerol as a compatible organic solute in response to increased external osmotic pressure. Boyle-van't Hoff plots showed that protoplasts, and non-turgid cells, exhibited a linear relationship between volume and the external osmotic pressure (i.e. they behaved as near-ideal osmometers), and that both protoplasts and cells have a component which is not osmotically responsive--the non-osmotic volume (NOV). Glycerol levels in whole cells and protoplasts were elevated by increased external osmotic pressure over a similar time-scale to the period of exponential cell growth, reaching a maximum value at 6-12 h and declining thereafter. This suggests that the restoration of turgor pressure in whole cells was not the sole regulator of glycerol accumulation. Stationary phase whole cells had negligible levels of intracellular glycerol after growth in a medium of raised osmotic pressure. However, intracellular trehalose synthesis in these cells began earlier and reached a higher maximum level than in basal medium. Once exponential growth had stopped, cell turgor and internal osmotic pressure decreased somewhat. These new, lower values may be determined by the extent of trehalose accumulation in stationary phase cells.

The Use of Hordeum Leaf Protoplasts to Study Non-osmotic Volume during Cell Development

Summary Volume changes of barley ( Hordeum vulgare L. cv. Patty) protoplasts, isolated from different developmental regions of primary leaves of barley seedlings, were measured after incubation in a range of sorbitol concentrations of different osmotic potentials. The presence or absence of KCl in the external protoplast medium had no significant effect on protoplast volume changes in short or long-term incubations. In short-term incubations of 5 minutes, protoplasts prepared from the whole of the leaf blade behaved as osmometers and showed volume changes in response to changing external osmotic potential. In long-term incubations of 8 hours similar protoplasts showed further changes in volume suggesting that a regulation of volume had occurred. The non-osmotic volumes (NOV) of protoplasts from three different developmental regions of the primary leaf were estimated by observing the short-term volume changes. The protoplasts were incubated in sorbitol solutions of different osmotic potentials and the Boyle-van't Hoff relationship applied to the results. NOV s of 5000, 2400, and 800 μm 3 were obtained for protoplasts from 60–75, 15–30 and 0–5 mm respectively above the basal intercalary meristem of the primary leaf. Relative contributions of plastids and nuclei to NOV were compared to the developmental status of the cell. It is suggested that NOV may be a useful concept to reflect the developmental status of leaf cells.

Osmotic significance of glycerol accumulation in exponentially growing yeasts

Natural-abundance 13C-nuclear magnetic resonance spectroscopy has shown glycerol to be the major osmotically significant low-molecular-weight solute in exponentially growing, salt-stressed cells of the yeasts Saccharomyces cerevisiae, Zygosaccharomyces rouxii, and Debaromyces hansenii. Measurement of the intracellular nonosmotic volume (i.e., the fraction of the cell that is osmotically unresponsive) by using the Boyle-van't Hoff relationship (for nonturgid cells, the osmotic volume is directly proportional to the reciprocal of the external osmotic pressure) showed that the nonosmotic volume represented up to 53% of the total cell volume; the highest values were recorded in media with maximum added NaCl. Determinations of intracellular glycerol levels with respect to cell osmotic volumes showed that increases in intracellular glycerol may counterbalance up to 95% of the external osmotic pressure due to added NaCl. The lack of other organic osmotica in 13C-nuclear magnetic resonance spectra indicates that inorganic ions may constitute the remaining component of intracellular osmotic pressure.