Ionic regulation of the unicellular green algaDunaliella tertiolecta: Response to hypertonic shock

Summary The evolution of the volume, the Na⁺ and K⁺ contents and the glycerol and ATP contents were investigated after subjectingDunaliella tertiolecta cells to hypertonic shocks. It was found that the variations in the glycerol and the ion contents superimpose as the cell regulates its volume. Hypertonic shock induces a rapid increase (some minutes) in the Na⁺ influx and Na⁺ content followed by a decrease until a new steady value is reached after 30 min of cell transfer. The regulatory mechanism extruding Na⁺ out of the cells was dependent on the presence of K^– or Rb^– ions in the external medium. A transient pumping of K⁺ ions was found after subjecting the cells to a hypertonic shock. This increase in K⁺ content resulted from the transient increase in the K⁺ influxes. The K⁺ pumping mechanism was blocked by the absence of Ca⁺⁺ and Mg⁺⁺ ions in the external medium and was inhibited by DCCD, FCCP and DCMU, whereas ouabain, cyanide and PCMBS were ineffective. The increase in K⁺ content was observed if the hypertonic shock was induced by the addition of NaCl, glycerol or choline chloride. These results are interpreted on the basis of two distinct mechanisms: a Na^–/K^– exchange pump and a Na⁺ independent K⁺ pump. These ionic transfer mechanisms would participate in the osmoregulation ofDunaliella cells and would be of importance, particularly during the onset of the osmotic shock when glycerol synthesis is incomplete.     

Volume changes in activated human neutrophils: The role of Na+/H+ exchange

The apparent volume of neutrophils, as measured electronically with the Coulter counter, has been reported to increase upon treatment with chemotactic factors. The occurrence of a volume change was confirmed by forward angle light scattering and by isotopic measurements of intracellular water space in cells treated with 12-O-tetradecanoylphorbol 13, acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP). Cell swelling was associated with an increase in the osmotic content of the cells, determined from Boyle-van't Hoff plots, and with an increase in Na⁺ content, measured by flame photometry. The volume change was inhibited by replacement of extracellular Na⁺ with K⁺ or N-methyl-D-glucamine⁺, or by addition of amiloride. Swelling was also inhibited by the 5-N-substituted analogs of amiloride, which are potent specific inhibitors of the Na⁺/H⁺ antiport. This pathway is activated in neutrophils by both TPA and FMLP. Activation of Na⁺/H⁺ exchange, determined as a Na⁺-dependent and amiloride-sensitive cytoplasmic alkalinization, was also found when neutrophils were treated with hypertonic solutions. The hypertonic activation of the antiport was similarly followed by cell swelling, detectable by electronic sizing. The results indicate that activation of Na⁺/H⁺ exchange can lead to significant cell swelling in neutrophils.     

Influence of diet composition on feeding and water excretion by the tsetse fly, Glossina morsitans

Adult Glossina morsitans fed on aqueous salt solutions containing phagostimulant ATP in an in vitro feeding system gave an optimal feeding response only over a narrow pH range equivalent to that of vertebrate blood. There was much less discrimination on the basis of molar concentration.The rate and extent of water excretion by the fly was found to depend on the concentration of Na⁺ ions in the food medium: an active transport mechanism is indicated which enables water to pass from the meal through the anterior midgut wall and into the haemocoele. A favourable osmotic gradient assisted water transport in the presence of Na⁺ ions: the system could not operate efficiently in the presence of Na⁺ ions if the osmotic pressure of the food medium was higher than that of vertebrate blood, nor could it operate efficiently in any solution lacking Na⁺ ions.Normal transfer of a meal from the crop to the anterior midgut occurred only when the food medium was isotonic with vertebrate blood or in the presence of Na⁺ ions if hypotonic. Normal transfer of isotonic solutions was prevented in the presence of excess K⁺ ions, and hypertonic solutions were not transferred normally even in the presence of Na⁺ ions. Thus the rate of water excretion was reduced.Tsetse flies fed on blood in an in vitro feeding system excreted water at a significantly lower rate than flies fed on a living animal. Evidence suggests that this is due to a combined effect of changes in viscosity, effective ionic composition, and osmotic pressure, upon the normal rate and extent of food uptake and manipulation of the meal prior to digestion. The implications of this are discussed in terms of future developments of in vitro feeding techniques for haematophagous insects.     

Effect of anisotonic media on cell volume and electrolyte fluxes in slices of the dogfish (Squalus acanthias) rectal gland

Summary Osmotic responses of slices of dogfish rectal gland to hypotonic (urea-free) and hypertonic media were studied. Transfer of tissue from isotonic (890 mosM) to hypotonic (550 mosM) saline produced an osmotic swelling associated with a slow net uptake of cell K⁺ (and Cl^–) and a slow, two-component efflux of urea. Media made hypertonic (1180 mosM) by addition of urea or mannitol produced osmotic shrinkage with a net loss of KCl. The cell osmotic responses in hypotonic media were lower than predicted for an ideal osmometer. No volume regulatory responses were seen subsequent to the initial osmotic effects. The cation influx in hypotonic media lacked specificity: in the presence of 0.5 mM ouabain or in K⁺-free media a net influx of Na⁺ was found. At steady state, the cell membrane potential evaluated from the Nernst potentials of K⁺ and triphenylmethyl phosphonium⁺, was independent of medium tonicity, suggesting the membrane potential as a determinant in the cellular osmotic response. Zero-time⁸⁶Rb⁺ fluxes were measured:⁸⁶Rb⁺ influx was not affected by hypotonicity, implying an unchanged operation of the Na⁺–K⁺-ATPase. On the other hand,⁸⁶Rb⁺ efflux was significantly reduced at hypotonicity; this effect was transient, the efflux returning to the control value once the new steady state of cell volume had been reached. A controlled efflux system is therefore involved in the cell osmotic response. The absence of the volume regulatory phenomenon suggests that the cells are not equipped with a volume-sensing mechanism.Abbreviations and symbols DW dry weight - E extracellular (polyethylene glycol) space - E Nernst potential - H₂O_e H₂O_i tissue water, extra- and intracellular - TPMP ⁺ triphenyl methyl phosphonium salt - WW wet weight     

Hypertonicity primes malignant melanoma cells for apoptosis

The tumor environment critically influences responsiveness of cancer cells to chemotherapies, most of which activate the mitochondria-regulated (intrinsic) apoptotic cascade to kill malignant cells. Especially skin tumors encounter an environment with remarkable biophysical properties. Cutaneous accumulation of Na⁺ locally establishes osmotic pressure gradients in vivo (hypertonicity or hyperosmotic stress), but whether cutaneous hypertonicity is a factor that modulates the responsiveness of skin cancers to therapeutic apoptosis-induction has thus far not been investigated. Here, we show that hyperosmotic stress lowers the threshold for apoptosis induction in malignant melanoma, the deadliest form of skin cancer. Hypertonic conditions enforce addiction to BCL-2-like proteins to prevent initiation of the mitochondria-regulated (intrinsic) apoptotic pathway. Essentially, hyperosmotic stress primes mitochondria for death. Our work identifies osmotic pressure in the tumor microenvironment as a cell extrinsic factor that modulates responsiveness of malignant melanoma cells to therapy.     

Osmotic water transport with glucose in GLUT2 and SGLT

Carrier-mediated water cotransport is currently a favored explanation for water movement against an osmotic gradient. The vestibule within the central pore of Na⁺-dependent cotransporters or GLUT2 provides the necessary precondition for an osmotic mechanism, explaining this phenomenon without carriers. Simulating equilibrative glucose inflow via the narrow external orifice of GLUT2 raises vestibular tonicity relative to the external solution. Vestibular hypertonicity causes osmotic water inflow, which raises vestibular hydrostatic pressure and forces water, salt, and glucose into the outer cytosolic layer via its wide endofacial exit. Glucose uptake via GLUT2 also raises oocyte tonicity. Glucose exit from preloaded cells depletes the vestibule of glucose, making it hypotonic and thereby inducing water efflux. Inhibiting glucose exit with phloretin reestablishes vestibular hypertonicity, as it reequilibrates with the cytosolic glucose and net water inflow recommences. Simulated Na⁺-glucose cotransport demonstrates that active glucose accumulation within the vestibule generates water flows simultaneously with the onset of glucose flow and before any flow external to the transporter caused by hypertonicity in the outer cytosolic layers. The molar ratio of water/glucose flow is seen now to relate to the ratio of hydraulic and glucose permeability rather than to water storage capacity of putative water carriers.     

Ion and water shifts produced by ammonium chloride in high K and low K red cells

The effect of ammonium chloride on the cellular Na⁺, K⁺ and water has been examined in human and horse (high K), cow (medium K) and cat (low K) red cells. It was found that high K red cells, especially those of the horse, gained water an Na⁺, whereas the net movement of K⁺ was negligible. There was a correlation between the increase of cellular Na⁺ concentration and of the packed red cell volume. In contrast, the packed cell volume of low K red cells increased slightly or not at all, and Na⁺ ions leaked out from the cells. The high K cells had a lower Cl^? concentration and higher buffer capacity than the low K cells. The results obtained with the medium K (cow) cells usually lay between those of the other two cell types. In all the cases both the plasma and cell pH decreased resulting from the addition of ammonium chloride. The mechanism of movements of water and Na⁺ ions in high K cells remained unsolved, but the response of low K cells to ammonium chloride was near that of a cation exchange resin.     

A study of the effect of sorbitol on osmotic tolerance during partial desiccation of bovine sperm

The goal of the study was to improve the partial desiccation survival of bovine sperm by decreasing the dehydration induced osmotic injury. The protective role of sorbitol, a polyol, was investigated by (i) studying the osmotic behavior of sperm in hypertonic Tyrode’s buffer in the presence of sorbitol and trehalose, (ii) studying the effect of sorbitol and trehalose on sperm motility following partial dehydration. The osmotic behavior studies included the assessment of motility and volumetric responses in the presence of the additives. For the drying experiments, motility was assayed after drying the samples to different end water content followed by immediate rehydration. Compared to the effect of “intracellular + extracellular” trehalose alone, results showed a much improved motility in the presence of sorbitol and trehalose. While the drying results suggest an enhanced osmotolerance in the presence of sorbitol, the study of motility under hypertonic conditions combined with the sperm volume excursion experiments suggest that sorbitol imparts the enhancement by permeating into the cell cytoplasm.     

Effects of salt stress in relation to osmotic adjustment on sugarcane (<Emphasis Type="Italic">Saccharum officinarum</Emphasis> L.) callus cultures

In vitro responses of embryogenic sugarcane (Saccharum officinarum L.; cv. CoC-671) calli stressed with different levels of NaCl (0.0, 42.8, 85.6, 128.3, 171.1, 213.9 or 256.7 mM) were studied. The results showed that a significant decrease in callus growth and cell viability occurred with ≥85.6 mM NaCl. Higher amounts of free proline and glycine betaine were accumulated in NaCl-stressed calli. Although the leached and retained Na⁺ contents increased, the retained K⁺ content decreased with increasing levels of NaCl. Such a mechanism implies that sugarcane can be considered as a Na⁺-excluder. The accumulation of salt ions and osmolytes could play an important role in osmotic adjustment in sugarcane cells under salt stress.     

The Na+,K+,2Cl−-cotransport system in HeLa cells: Aspects of its physiological regulation

We have previously reported on the biochemical properties of a Na⁺,K⁺,2Cl^?-cotransport in HeLa cells and here we deal with aspects of its physiological regulation. Na⁺,K⁺,2Cl^?-cotransport in HeLa cells was studied by ⁸⁶Rb⁺ influx and ⁸⁶Rb⁺/²²Na⁺ efflux measurements. The effects of rat atrial natriuretic peptide (ANP), isoproterenol, and amino acids on ⁸⁶Rb⁺ flux, mediated by the bumet-anide-sensitive Na⁺, K⁺, 2Cl^?-cotransport system and the ouabain-sensitive Na⁺/K⁺-pump, were investigated. ANP reduced bumetanide-sensitive ⁸⁶Rb⁺ influx under isotonic as well as under hypertonic conditions. Similar decrease of bumetanide-sensitive ⁸⁶Rb⁺ influx was observed in the presence of 8-bromo-cGMP, while neither isoproterenol as a β-receptor agonist nor 8-bromo-cAMP-could alter bumetanide-sensitive ⁸⁶Rb⁺ influx. Furthermore, efflux of ⁸⁶Rb⁺ and ²²Na⁺ was greatly reduced in the presence of bumetanide and ANP. Together with our recent findings, showing functionally active, high affinity receptors for ANP on HeLa cells (Kort and Koch, Biochim. Biophys. Res. Commun. 168:148–154, 1990), this study indicates that ANP participates in the regulation of the Na⁺, K⁺, 2Cl^?-cotransport system in HeLa cells. Further measurements revealed that amino acids as present in the growth medium (Joklik's minimal essential medium) and the amino acid derivative α-methyl-aminoisobutyric acid (metAlB, 1 and 5 mM, respectively) also reduced Na⁺, K⁺, 2Cl^?-cotransport-mediated ⁸⁶Rb⁺ uptake and diminished the stimulatory effect of hypertonicity on the cotransporter. In addition, the Na⁺/K⁺-pump was markedly stimulated in the presence of amino acids, while neither ANP and 8-Br-cGMP nor isoproterenol and 8-Br-cAMP had a significant effect on the activity of the Na⁺/K⁺-pump.     

Osmotic tolerance of avian spermatozoa: influence of time, temperature, cryoprotectant and membrane ion pump function on sperm viability

Potential factors influencing sperm survival under hypertonic conditions were evaluated in the Sandhill crane (Grus canadensis) and turkey (Meleagridis gallopavo). Sperm osmotolerance (300-3000 mOsm/kg) was evaluated after: (1) equilibration times of 2, 10, 45 and 60 min at 4 °C versus 21 °C; (2) pre-equilibrating with dimethylacetamide (DMA) or dimethylsulfoxide (Me₂SO) at either 4 °C or 21 °C; and (3) inhibition of the Na⁺/K⁺ and the Na⁺/H⁺ antiporter membrane ionic pumps. Sperm viability was assessed using the eosin-nigrosin live/dead stain. Species-specific differences occurred in response to hypertonic conditions with crane sperm remaining viable under extreme hypertonicity (3000 mOsm/kg), whereas turkey sperm viability was compromised with only slightly hypertonic (500 mOsm/kg) conditions. The timing of spermolysis under hypertonic conditions was also species-specific, with a shorter interval for turkey (2 min) than crane (10 min) sperm. Turkey sperm osmotolerance was slightly improved by lowering the incubation temperature from 21 to 4 °C. Pre-equilibrating sperm with DMA reduced the incidence of hypertonic spermolysis only in the crane, at both room and refrigeration temperature. Inhibiting the Na⁺/K⁺ and the Na⁺/H⁺ antiporter membrane ion pumps did not impair resistance of crane and turkey spermatozoa to hypertonic stress; pump inhibition actually increased turkey sperm survival compared to control sperm. Results demonstrate marked species specificity in osmotolerance between crane and turkey sperm, as well as in the way temperature and time of exposure affect sperm survival under hypertonic conditions. Differences are independent of the role of osmotic pumps in these species.     

L-929 cells under hyperosmotic conditions

Changes in cell water content resulting from sorbitol addition to the environment of L-929 cells were evaluated gravimetrically using¹⁴C-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 infolding 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.     

Electrolyte and non-electrolyte distribution in the ehrlich ascites tumor cells during the cell cycle

In a previous study, evidence was presented for changes in the state of water and osmotically active solutes during the cell cycle. Total water was constant at 82% (w/w), while the fraction of water that was osmotically active decreased from a maximum during S to a minimum at mitosis. Total Na⁺, K⁺, and C1^? in milliequivalents per liter of cell water remained constant. Therefore, electrolytes are sequestered in the osmotically inactive water. Evidence is now presented that Na⁺ exists primarily as one compartment, with a second, slower compartment appearing during S and disappearing during G2. Na⁺ is completely exchangeable during the entire cell cycle. The distribution of other penetrating solutes was also investigated. When placed in hyperosmotic ethylene glycol solutions, cells first shrink, then swell to their original volumes. ¹⁴C-ethylene glycol distributes in 89% of cell water throughout the cell cycle. However, ¹⁴C-urea distributes in anywhere from 86–100% of the cell water, depending on the stage in the cell cycle. Both solutes are at chemical equilibrium in water in which they are distributed, but they differ in their effects on cell volume. The final volume at which cells equilibrate in urea varies with the concentration of urea in the environment and with time into the cell cycle. Results suggest a loss of osmotically active particles or decreased osmotic activity of urea.     

Interactions between growth, Cl− and Na+ uptake, and water relations of plants in saline environments. I. Slightly vacuolated cells

Abstract. Slightly vacuolated cells, i.e. microalgae and meristematic cells of vascular plants, maintain low Cl^? and Na⁺ concentrations even when exposed to a highly saline environment. The factors regulating the internal ion concentration are the relative rate of volume expansion, the membrane permeability to ions, the electrical potential, and the active ion fluxes. For ion species which are not actively transported, a formula is developed which relates the internal concentration to the rate of expansion of cell volume, the permeability of membranes to that ion, and the electrical potential. For example, when the external concentration of Cl^? is high, and Cl^? influx is probably mainly passive, the formula predicts that rapid growth keeps the internal Cl^? concentration lower than that in a non-growing cell with the same electrical potential; this effect is substantial if the plasmalemma has a low permeability to Cl^?. For ion species which are actively transported, the rate of pumping must be considered. For instance Na⁺ concentrations are kept low mainly by an efficient Na⁺ extrusion pump which works against the electric field across the membrane. The requirement for Na⁺ extrusion is related to the external Na⁺ concentration, the rate of expansion of cell volume, the membrane permeability, and the electrical potential. It is possible that microalgae have a more positive electrical potential than many other plant cells; if so, requirements for high rates of active Na⁺ extrusion will be lower. The required rates of Na⁺ extrusion are lower during rapid growth, provided that the permeability of the plasmalemma to Na⁺ is low. The energy required for the regulation of Cl^? and Na⁺ concentrations is low, especially in rapidly expanding cells where Na⁺ extrusion requires only 1–2% of the energy normally produced in respiration. The exclusion of these ions, however, must be accompanied by the synthesis of enough organic compounds to provide adequate osmotic solutes for the increases in volume accompanying growth. This process reduces the substrates available for respiration and synthesis of cell constituents, but the reduction is not prohibitively large—even for cells growing in 750 mol m^?3 NaCl, the carbohydrate accumulated as osmotic solute is only 10% of that consumed in respiration.     

Regulation of the paracellular Na+ and Cl- conductances by the NaCl-generated osmotic gradient in a manner dependent on the direction of osmotic gradients

In the present study, we investigated the effect of osmolality on the paracellular ion conductance (Gp) composed of the Na⁺ conductance (G_Na) and the Cl⁻ conductance (G_Cl). An osmotic gradient generated by NaCl with relatively apical hypertonicity (NaCl-absorption-direction) induced a large increase in the G_Na associated with a small increase in the G_Cl, whereas an osmotic gradient generated by NaCl with relatively basolateral hypertonicity (NaCl-secretion-direction) induced small increases in the G_Na and the G_Cl. These increases in the Gp caused by NaCl-generated osmotic gradients were diminished by the application of sucrose canceling the NaCl-generated osmotic gradient. The osmotic gradient generated by basolateral application of sucrose without any NaCl gradients had little effects on the Gp. However, this basolateral application of sucrose produced a precondition drastically quickening the time course of the action of the NaCl-generated osmotic gradient on the Gp. Further, we found that application of the basolateral hypotonicity generated by reduction of NaCl concentration shifted the localization of claudin-1 to the apical from the basolateral side. These results indicate that the osmotic gradient regulates the paracellular ion conductive pathway of tight junctions via a mechanism dependent on the direction of NaCl gradients associated with a shift of claudin-1 localization to the apical side in renal A6 epithelial cells.     

Shrinkage-induced Activation of the Na+/H+ Exchanger in Ehrlich Ascites Tumor Cells: Mechanisms Involved in the Activation and a Role for the Exchanger in Cell Volume Regulation

Amiloride-sensitive, Na⁺-dependent, DIDS-insensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells. This was assessed using the fluorescent pH-sensitive probe 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel increase in the amiloride-sensitive unidirectional Na⁺ influx is also observed. This indicates that hypertonic challenge activates a Na⁺/H⁺ exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pH _i , but the overall picture confirms the results from cell suspensions. Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is strongly reduced in ATP-depleted cells. Furthermore, it is inhibited by chelerythrine and H-7, inhibitors of protein kinase C (PKC). In contrast, Calyculin A, an inhibitor of protein phosphatases PP1 and PP2A, stimulates shrinkage-induced alkalinization. Osmotic activation of the exchanger is unaffected by removal of calcium from the experimental medium, and by buffering of intracellular free calcium with BAPTA. At 25 mm HCO⁻ ₃, but not in nominally HCO⁻ ₃-free medium, Na⁺/H⁺ exchange contributes significantly to regulatory volume increase in Ehrlich cells. Under isotonic conditions, the Na⁺/H⁺ exchanger is activated by ionomycin, an effect which may be secondary to ionomycin-induced cell shrinkage. Received: 2 March 1995/Revised: 29 September 1995     

Water and ion regulation in cicadas in relation to xylem feeding

Cicadas feed on xylem fluid. This is hypotonic to the haemolymph and contains high concentrations of potassium, sodium, calcium, magnesium, chloride, and phosphate ions. The urine contains the same ions in the same proportions but in slightly lower concentrations. Amino acids and sucrose are present in xylem fluid and traces of amino acids are also found in urine.Water is rapidly shunted from foregut to hindgut via the filter chamber. Injection of xylem fluid into the oesophagus results in an immediate tenfold increase in flow rate in the ileum. The osmotic pressure of xylem fluid in the filter chamber rapidly rises whilst the osmotic pressure in the anterior part of the ileum rapidly falls.Absorption of nutrients and ions into the haemolymph probably occurs in the conical segment and anterior tubular midgut. Storage excretion of divalent ions occurs in the mid-midgut and ions may be transported from the haemolymph into the posterior tubular midgut.The Malpighian tubules secrete a fluid slightly hypertonic to blood containing K⁺ (42 mM/l.] and Na⁺ (14 mM/l.).The osmotic pressures within the internal Malpighian tubules and internal midgut in the filter chamber are considerably higher than the osmotic pressure of the xylem fluid when it first enters the filter chamber proper. Passive osmosis will occur and water will be shunted into the ileum.Reabsorption of K⁺ and Na⁺ occurs in the ileum.     

Ion movements induced by transcellular osmosis inNitella flexilis

Summary Movements of K⁺, Na⁺, and Cl^}- ions during transcellular osmosis were studied in internodal cells ofNitella flexilis. Much K⁺ was released from the endosmotic cell part, but only a little from the exosmotic cell part. The amount of K⁺ released depended on the osmotic gradient driving transcellular osmosis. Movement of Na⁺ was hardly detected. Cl^}- was released in nearly the same amounts as K⁺. Release of K⁺ from the endosmotic cell half was stimulated remarkably by lowering the temperature from 20 to 1 °C, and also by lowering the internal osmotic pressure but inhibited by raising it.The dependence of K⁺ release on osmotic gradient, internal osmotic pressure and temperature can be explained by their effects on membrane depolarization and membrane resistance (Hayama et al. 1978). We concluded thatP _K remained unchanged, whileP _Cl increased a great deal in the endosmotic cell part.     

Intracellular compartmentation of ions in salt adapted tobacco cells

Na⁺ and Cl⁻ are the principal solutes utilized for osmotic adjustment in cells of Nicotiana tabacum L. var Wisconsin 38 (tobacco) adapted to NaCl, accumulating to levels of 472 and 386 millimolar, respectively, in cells adapted to 428 millimolar NaCl. X-ray microanalysis of unetched frozen-hydrated cells adapted to salt indicated that Na⁺ and Cl⁻ were compartmentalized in the vacuole, at concentrations of 780 and 624 millimolar, respectively, while cytoplasmic concentrations of the ions were maintained at 96 millimolar. The morphometric differences which existed between unadapted and salt adapted cells, (cytoplasmic volume of 22 and 45% of the cell, respectively), facilitated containment of the excited volume of the x-ray signal in the cytoplasm of the adapted cells. Confirmation of ion compartmentation in salt adapted cells was obtained based on kinetic analyses of ²²Na⁺ and ³⁶Cl⁻ efflux from cells in steady state. These data provide evidence that ion compartmentation is a component of salt adaptation of glycophyte cells.