External Osmolarity Research Articles

Osmotic modulation of slowly activating IKs in guinea-pig ventricular myocytes

The aims of the study were to determine the effects of anisosmotic bathing solution on selected properties of I(Ks), the slowly activating delayed-rectifier K(+) current important for repolarization of the action potential in cardiac cells. Guinea-pig ventricular myocytes were voltage-clamped using either the ruptured-patch or perforated-patch technique, and the amplitude, time course, and voltage dependence of I(Ks) were determined before [isosmotic (1T)] and during superfusion of hyposmotic (<1T) or hyperosmotic (>1T) bathing solution. Hyposmotic solution increased the amplitude of I(Ks), and hyperosmotic solution decreased it. Anisosmotic-induced changes in I(Ks) amplitude were complete in 2-5 min, well-maintained, reversible, and not accompanied by significant changes in I(Ks) time course and voltage dependence. There was little difference in the results obtained with the ruptured-patch technique and those obtained with the perforated-patch technique. The amplitude of I(Ks) was sensitive to small (±10%) changes in osmolarity, maximally increased by hyposmotic solution with T < 0.7, and strongly decreased by hyperosmotic solution with T > 1.5. Experimental data on a plot of relative (1T = 1.0) I(Ks) amplitude vs. the reciprocal of relative osmolarity are well-described by a Hill equation that has a lower asymptote of 0.0, an upper asymptote of 2.0, and a slope factor of 1.87 ± 0.07. Modulation of I(Ks) amplitude by anisosmotic solution is independent of patch configuration, unaccompanied by changes in current gating, and well-described by a Hill dose-response relation that predicts relatively strong responses of I(Ks) to small perturbations in external osmolarity.

Vacuolar two-pore K+ channels act as vacuolar osmosensors.

• Plant two-pore K(+) channels (TPKs) have been shown previously to play a role in vacuolar K(+) homeostasis. TPK activity is insensitive to membrane voltage, but regulated by cytoplasmic calcium and 14-3-3 proteins. This study reports that membrane stretch and osmotic gradients also alter the activity of TPKs from Arabidopsis, rice and barley, and that this may have a physiological relevance for osmotic homeostasis. • Mechanosensitivity was studied using patch clamp experiments and TPKs from Arabidopsis, rice and barley. In addition, the capability of TPKs to act as osmosensors was determined. By using protoplast disruption assays and intact plant survival assays, in genotypes that differed in TPK expression, the physiological relevance of TPK-based osmosensing was tested. • TPKs from all three species showed varying degrees of mechanosensitivity. TPK activity in channels from all three species was sensitive to trans-tonoplast osmotic gradients. TPK osmosensing is likely to proceed via the detection of small perturbations in membrane tension. Intact plant and protoplast assays showed that TPK-based osmosensing is important during exposure to rapid changes in external osmolarity. • Vacuolar TPK channels can act as intracellular osmosensors and rapidly increase channel activity during hypo-osmotic shock to release vacuolar K(+) .

Computational and Experimental Studies of Substrate Binding, Conformational Change and Importance of the Trimeric State in the Glycine Betaine Transporter BetP

The glycine betaine/sodium symporter BetP responds to changes in external osmolality by regulation of its transport activity. A recent X-ray structure of BetP confirms that it is a homotrimer and in this structure each protomer adopts an identical conformation, in which the pathway is occluded from both sides. Despite the availability of a wealth of experimental data for BetP, the structures of the alternate states (e.g., open to the outside of the cell), molecular mechanisms of substrate and Na+ binding and transport, as well as the functional implications of the trimeric state remain poorly understood. To address these questions, we carried out computational studies using a range of techniques to derive hypotheses that were then tested experimentally. First, to identify structural features of the alternate states, we developed a procedure for flexible fitting of the X-ray structure of BetP into a lower-resolution cryo-EM map of BetP in a more native lipid environment, in which the three protomers have different conformations. These results suggest that: (i) the protomers adopt distinct conformational states relevant to the transport cycle; and (ii) there is conformational coupling between the protomers. Second, we performed all-atom molecular dynamics simulations and in silico alanine scanning of BetP trimers in order to identify interface residues crucial for maintaining the trimeric state. Mutations of these residues to alanine were introduced experimentally revealing that the isolated monomers are functional, and that the trimeric state is important for the regulation and higher activity of the protein. Finally, using molecular modeling and biochemical experiments we identified two Na+ binding sites in BetP that could not be resolved in the 3.35 A resolution X-ray structure.

Bacteria Regulate Turgor Pressure in Order to Grow

The extreme concentrations of chemicals in a bacterium's cytoplasm generate an osmotic pressure that inflates the cell. It is thought that E. coli use a number of interconnected systems to adapt to changes in external pressure, allowing them to maintain turgor and live in environments that range more than two-hundred-fold in external osmolality. To date, how this adaptation is achieved and why pressure is required for survival have been poorly addressed. Previous measurements of osmoregulation in bacteria have been unable to directly probe a single cell's adaptation, focusing instead on the activity of various transporters, or changes in population growth rates. I will show that different mechanisms used by bacteria can be explored using fluorescence imaging to monitor changes in cell shape during adaptation on a single cell level with a time resolution on the order of seconds. This type of measurement allows the different adaptation pathways to be studied individually as well as in defined groups. Furthermore, I will demonstrate that using a number of interconnected systems; bacteria actively regulate their turgor pressure to a preset value, despite changes in their local environment. This precise value of the turgor pressure is required for cell growth and I will make direct connections between pressure adaptation and growth rate in challenging environments.

Quantification of cell volume changes upon hyperosmotic stress in Saccharomyces cerevisiae

Cell volume is a biophysical property, which is of great importance for quantitative characterisations of biological processes, such as osmotic adaptation. It also is a crucial parameter in the most common type of mathematical description of cellular behaviour-ordinary differential equation (ODE) models, e.g. the integrative model of the osmotic stress response in baker's yeast (E. Klipp, B. Nordlander, R. Kruger, P. Gennemark and S. Hohmann, Nat. Biotechnol., 2005, 23, 975-982). Until recently only rough estimates of this value were available. In this study we measured the mean volume of more than 300 individual yeast cells (Saccharomyces cerevisiae). We quantitatively characterised the dependence between the relative cell volume and the concentration of osmoticum in the cell surrounding. We also followed the recovery of the cellular volume over time, as well as the influence of increased external osmolarity on the nuclear volume. We found that cell shrinkage caused by shifts in the external osmolarity is proportional to the stress intensity only up to 1000 mM NaCl. At this concentration the yeast cells shrink to approximately 55% of their unstressed volume and this volume is maintained even in the case of further osmolarity increase. We observed that returning to the initial, unstressed volume takes more than 45 minutes for stress concentrations exceeding 100 mM NaCl and that only cells treated with the latter concentration are able to fully regain their initial size within the course of the experiment. We postulate that the cytoplasm plays a protective role for the nucleus by buffering the changes in volume caused by external osmolarity shifts. In conclusion, we quantitatively characterised the dynamics of cell volume changes caused by hyperosmotic stress, providing an accurate description of a biophysical cell property, which is crucial for precise mathematical simulations of cellular processes.

The yfeR gene of Salmonella enterica serovar Typhimurium encodes an osmoregulated LysR-type transcriptional regulator

A genetic screening for osmoregulated genes allowed us to identify the yfeR gene of Salmonella enterica serovar Typhimurium. The yfeR gene product encodes a novel LysR-type transcriptional regulator (LTTR), the expression of which decreases when external osmolarity increases. Out of the adjacent gene yfeH, YfeR modulates expression of several genes that may be required for optimal growth under low osmolarity conditions.

Intra- and extracellular osmotic regulation in the hololimnetic Caridea and Anomura: a phylogenetic perspective on the conquest of fresh water by the decapod Crustacea

We investigate extra- and intracellular osmoregulatory capability in two species of hololimnetic Caridea and Anomura: Macrobrachium brasiliense, a palaemonid shrimp, and Aegla franca, an aeglid anomuran, both restricted to continental waters. We also appraise the sharing of physiological characteristics by the hololimnetic Decapoda, and their origins and role in the conquest of fresh water. Both species survive salinity exposure well. While overall hyperosmoregulatory capability is weak in A. franca and moderate in M. brasiliense, both species strongly hyporegulate hemolymph [Cl(-)] but not osmolality. Muscle total free amino acids (FAA) increase slowly but markedly in response to the rapid rise in hemolymph osmolality consequent to hyperosmotic challenge: 3.5-fold in A. franca and 1.9-fold in M. brasiliense. Glycine, taurine, arginine, alanine and proline constitute ≈85% of muscle FAA pools in fresh water; taurine, arginine, alanine each contribute ≈22% in A. franca, while glycine predominates (70%) in M. brasiliense. These FAA also show the greatest increases on salinity challenge. Muscle FAA titers correlate strongly (R=0.82) with hemolymph osmolalities across the main decapod sub/infraorders, revealing that marine species with high hemolymph osmolalities achieve isosmoticity of the intra- and extracellular fluids partly through elevated intracellular FAA concentrations; freshwater species show low hemolymph osmolalities and exhibit reduced intracellular FAA titers, consistent with isosmoticity at a far lower external osmolality. Given the decapod phylogeny adopted here and their multiple, independent invasions of fresh water, particularly by the Caridea and Anomura, our findings suggest that homoplastic strategies underlie osmotic and ionic homeostasis in the extant freshwater Decapoda.

Bacterial stimulus perception and signal transduction: Response to osmotic stress

When exposed to osmotic stress from the environment, bacteria act to maintain cell turgor and hydration by responding both on the level of gene transcription and protein activity. Upon a sudden decrease in external osmolality, internal solutes are released by the action of membrane embedded mechanosensitive channels. In response to an osmotic upshift, the concentration of osmolytes in the cytoplasm is increased both by de novo synthesis and by active uptake. In order to coordinate these processes of osmoregulation, cells are equipped with systems and mechanisms of sensing physical stimuli correlated to changes in the external osmolality (osmosensing), with pathways to transduce these stimuli into useful signals which can be processed in the cell (signal transduction), and mechanisms of regulating proper responses in the cell to recover from the environmental stress and to maintain all necessary physiological functions (osmoregulation). These processes will be described by a number of representative examples, mainly of osmoreactive transport systems with a focus on available data of their molecular mechanism.

Biopolymer mediated trehalose uptake for enhanced erythrocyte cryosurvival

A biopolymer has been shown to facilitate efficient delivery of trehalose, a bioprotectant normally impermeable to the phospholipid bilayer, into ovine erythrocytes. Cellular uptake of trehalose was found to be dependent on polymer pendant amino acid type and degree of grafting, polymer concentration, pH, external trehalose concentration, incubation temperature and time. Optimization of these parameters yielded an intracellular trehalose concentration of 123 ± 16 m m and concomitant improvement of erythrocyte cryosurvival of up to 20.4 ± 5.6%. Intracellular trehalose was shown to impart cellular osmoprotection up to an external osmolarity of 230 mOsm and increased osmotic sensitivity above this threshold. Biopolymer mediated membrane permeability was shown to be rapidly and completely reversible via washing with phosphate buffered saline.

Effects of osmotic challenges on membrane potential in human articular chondrocytes from healthy and osteoarthritic cartilage

Changes in external osmolarity arise from variations in mechanical loads on joints and may affect the homeostasis of chondrocytes, which are the only cell type responsible for matrix turnover. Accordingly, variations in membrane potential may affect cartilage production. The present study assessed the effects of variations in external osmolarity on membrane potential and the possible mechanisms responsible for this response. Membrane potential was measured by the patch clamp whole-cell technique using human articular chondrocytes freshly isolated from healthy and osteoarthritic cartilage. The membrane potential was -39±4 mV in articular human chondrocytes from healthy cartilage and -26±4 mV in those from osteoarthritic cartilage. Increasing the osmolarity produced a reversible hyperpolarization mediated by K+ efflux through BKCa channels in both groups of chondrocytes, but the response in osteoarthritic cells was significantly reduced; no other K+ pathways were involved in this effect. Alternatively, decreasing the osmolarity elicited depolarization in healthy chondrocytes but did not produce any response in chondrocytes from osteoarthritic cartilage. The depolarization was dependent on Na+ influx through Gd3+-sensitive stretch-activated cation channels and was independent of external Ca2+. The differential responses observed in chondrocytes from osteoarthritic cartilage suggest that disregulation on the responses to external osmolarity may be involved in the process that leads to the alterations in the cartilage structure observed in osteoarthritis.

The Response of Single E. Coli Cells to Changes in External Osmolarity

The extreme concentrations of chemicals in a bacterium's cytoplasm generate a large osmotic pressure that inflates the cell. Using a number of interconnected systems, bacteria actively regulate their turgor pressure to resist changes in their local environment. In response to osmotic shock and changes in internal ion concentration, the osmosensory transporters ProP/U, BetT/U, TrkAH and KdpFABC transport external chemicals such as proline, choline and potassium into the cell, whereas the mechano-sensitive channels MscS and MscL export solutes from the cell in response to increased membrane strain. Although each has been shown to play a role in the regulation of turgor, details of how the different systems are coordinated by a cell is poorly understood. Previous measurements of osmoregulation in bacteria have been unable to directly probe the adaptation of turgor pressure, focusing instead on the activity of various transporters, or the change in cellular survival rates. Here we move beyond these limited measurements using AFM and fluorescence imaging to monitor turgor pressure and cell volume adaptation on a single cell level with a time resolution on the order of seconds. To explore different mechanisms used by bacteria, we moved exponentially growing cells from LB medium to an iso-osmotic buffered medium and allowed the cells to adapt. We subsequently challenged the cells with varying levels of sucrose (as an external osmolyte) and potassium or proline. We measured the dependence of the adaptation time and adaptation level on different amounts of extracellular potassium or proline and the magnitude of the osmotic shock. This type of measurement allows us to uncouple the different adaptation pathways and to study them individually and in small groups to quantify their function and interactions.

Rescue of Postcompaction-Stage Mouse Embryo Development from Hypertonicity by Amino Acid Transporter Substrates That May Function as Organic Osmolytes1

Early preimplantation embryos are sensitive to external osmolarity and use novel mechanisms to accumulate organic osmolytes and thus control their cell volumes and maintain viability. However, these mechanisms are restricted to the cleavage stages of development, and it was unknown whether postcompaction embryos use organic osmolytes. Mouse embryos developing from the 8-cell stage formed blastocoel cavities in vitro at osmolarities up to 360 mOsM. Above this range, several putative organic osmolytes (alanine, glutamine, glycine, and beta-alanine) rescued blastocyst development, but several effective osmoprotectants in cleavage-stage embryos (such as betaine and proline) did not. At physiological osmolarities, each of these compounds resulted in significantly larger blastocysts. This was not due to increased cell numbers, which were unaffected in blastocysts by osmolarity in the range where blastocyst size was rescued by potential organic osmolytes, although cell number was decreased at higher osmolarities and was rescued by each osmolyte. The effective osmolytes were accumulated intracellularly by embryos developing in vitro from the 8-cell stage to blastocysts. However, unlike conventional organic osmolytes in somatic cells or those in cleavage-stage embryos, their intracellular concentrations were not increased with increasing external osmolarity. With the exception of beta-alanine, which is taken up via the beta-amino acid transport system, the effective osmolytes were transported by the B(0,+) system, which becomes highly active in blastocysts. The intracellular accumulation of these osmolytes in postcompaction embryos thus appears to support optimal development and blastocyst expansion at physiological osmolarities and may contribute to the embryo's ability to withstand stress.

Involvement of volume‐sensitive Cl− channels in the proliferation of human subcutaneous pre‐adipocytes

1. Obesity is a significant challenge in terms of public health and preventive medicine. Inhibition of pre-adipocyte proliferation is believed to be important in the proposed anti-obesity mechanism. The aim of the present study was to examine the interplay between Cl(-) channels and their possible involvement in the proliferation of undifferentiated human pre-adipocytes. 2. Pre-adipocytes were isolated from human abdominal subcutaneous adipose tissue. Membrane ion currents were recorded using the whole-cell patch-clamp technique. Expression of the Cl(-) channel ClC-3 gene and protein was determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot, respectively. Cell proliferation was evaluated using the [(3)H]-thymidine incorporation assay. 3. Electrophysiological recordings revealed a volume-sensitive Cl(-) current (I(Cl.vol)) expressed in pre-adipocytes that was activated under hyposmotic conditions (external osmolarity decreased by 80%) and inhibited by the Cl(-) channel blocker tamoxifen. 4. Expression of the ClC-3 channel gene and protein was confirmed by RT-PCR and western blot analysis. Blocking I(Cl.vol) with tamoxifen supressed the proliferation of pre-adipocytes in a concentration-dependent manner. 5. Collectively, the results of the present study indicate that the volume-sensitive Cl(-) channel participates in regulation of the proliferation of human subcutaneous pre-adipocytes.

Transcription levels of CHS5 and CHS4 genes in Paracoccidioides brasiliensis mycelial phase, respond to alterations in external osmolarity, oxidative stress and glucose concentration

The complete sequence of Paracoccidioides brasiliensis CHS5 gene, encoding a putative chitin synthase revealed a 5583 nt open reading frame, interrupted by three introns of 82, 87 and 97 bp (GenBank Accession No EF654132). The deduced protein contains 1861 amino acids with a predicted molecular weight of 206.9 kDa. Both its large size and the presence of a N-terminal region of approx. 800 residues with a characteristic putative myosin motor-like domain, allow us to include PbrChs5 into class V fungal chitin synthases. Sequence analysis of over 4 kb from the 5′ UTR region in CHS5, revealed the presence of a previously reported CHS4 gene in P. brasiliensis, arranged in a head-to-head configuration with CHS5. A motif search in this shared region showed the presence of stress response elements (STREs), three binding sites for the transcription activators Rlm1p (known to be stimulated by hypo-osmotic stress) and clusters of Adr1 (related to glucose repression). A quantitative RT-PCR analysis pointed to changes in transcription levels for both genes following oxidative stress, alteration of external osmolarity and under glucose-repressible conditions, suggesting a common regulatory mechanism of transcription.

A Systems-Level Analysis of Perfect Adaptation in Yeast Osmoregulation

Negative feedback can serve many different cellular functions, including noise reduction in transcriptional networks and the creation of circadian oscillations. However, only one special type of negative feedback (“integral feedback”) ensures perfect adaptation, where steady-state output is independent of steady-state input. Here we quantitatively measure single-cell dynamics in the Saccharomyces cerevisiae hyperosmotic shock network, which regulates membrane turgor pressure. Importantly, we find that the nuclear enrichment of the MAP kinase Hog1 perfectly adapts to changes in external osmolarity, a feature robust to signaling fidelity and operating with very low noise. By monitoring multiple system quantities (e.g., cell volume, Hog1, glycerol) and using varied input waveforms (e.g., steps and ramps), we assess the network location of the mechanism responsible for perfect adaptation in a minimally invasive manner. We conclude that the system contains only one effective integrating mechanism, which requires Hog1 kinase activity and regulates glycerol synthesis but not leakage.

A Systems-Level Analysis of Perfect Adaptation in Yeast Osmoregulation

Negative feedback can serve many different cellular functions, including noise reduction in transcriptional networks and the creation of circadian oscillations. However, only one special type of negative feedback ("integral feedback") ensures perfect adaptation, where steady-state output is independent of steady-state input. Here we quantitatively measure single-cell dynamics in the Saccharomyces cerevisiae hyperosmotic shock network, which regulates membrane turgor pressure. Importantly, we find that the nuclear enrichment of the MAP kinase Hog1 perfectly adapts to changes in external osmolarity, a feature robust to signaling fidelity and operating with very low noise. By monitoring multiple system quantities (e.g., cell volume, Hog1, glycerol) and using varied input waveforms (e.g., steps and ramps), we assess in a minimally invasive manner the network location of the mechanism responsible for perfect adaptation. We conclude that the system contains only one effective integrating mechanism, which requires Hog1 kinase activity and regulates glycerol synthesis but not leakage.

An osmosensitive voltage‐gated K+ current in rat supraoptic neurons

The magnocellular neurosecretory cells of the hypothalamus (MNCs) regulate their electrical behaviour as a function of external osmolality through changes in the activity of osmosensitive ion channels. We now present evidence that the MNCs express an osmosensitive voltage-gated K(+) current (the OKC). Whole-cell patch-clamp experiments on acutely isolated MNCs were used to show that increases in the external osmolality from 295 to 325 mosmol/kg cause an increase in a slow, tetraethylammonium-insensitive outward current. The equilibrium potential for this current is close to the predicted E(K) in two different concentrations of external K(+). The OKC is sensitive to block by Ba(2+) (0.3 mm), and by the M-type K(+) current blockers linopirdine (150 microm) and XE991 (5 microm), and to enhancement by retigabine (10 microm), which increases opening of M-type K(+) channels. The OKC is suppressed by muscarine (30 microm) and is decreased by the L-type Ca(2+) channel blocker nifedipine (10 microm), but not by apamin (100 nm), which blocks SK-type Ca(2+)-dependent K(+) currents. Reverse transcriptase-polymerase chain reaction and immunocytochemical data suggest that MNCs express several members of the K(V)7 (KCNQ) family of K(+) channels, including K(V)7.2, 7.3, 7.4 and 7.5. Extracellular recordings of individual MNCs in a hypothalamic explant preparation demonstrated that an XE991- and retigabine-sensitive current contribute to the regulation of MNC firing. Our data suggest that the MNCs express an osmosensitive K(+) current that could contribute to the regulation of MNC firing by external osmolality and that could be mediated by K(V)7/M-type K(+) channels.

Characterization of Spermidine Transport System in a Cyanobacterium,Synechocystis sp. PCC 6803

The transport of spermidine into a cyanobacterium, Synechocystis sp. PCC 6803, was characterized by measuring the uptake of 14C-spermidine. Spermidine transport was shown to be saturable with an apparent affinity constant (Km) value of 67 microM and a maximal velocity (Vmax) value of 0.45 nmol/min/mg protein. Spermidine uptake was pHdependent with the pH optimum being 8.0. The competition experiment showed strong inhibition of spermidine uptake by putrescine and spermine, whereas amino acids were hardly inhibitory. The inhibition kinetics of spermidine transport by putrescine and spermine was found to be noncompetitive with Ki values of 292 and 432 microM, respectively. The inhibition of spermidine transport by various metabolic inhibitors and ionophores suggests that spermidine uptake is energy-dependent. The diminution of cell growth was observed in cells grown at a high concentration of NaCl. Addition of a low concentration of spermidine at 0.5 mM relieved growth inhibition by salt stress. Upshift of the external osmolality generated by either NaCl or sorbitol caused an increased spermidine transport with about 30- 40% increase at 10 mosmol/kg upshift.

The Hydrogel Nature of Mammalian Cytoplasm Contributes to Osmosensing and Extracellular pH Sensing

Cytoplasm is thought to have many hydrogel-like characteristics, including the ability to absorb large amounts of water and change volume in response to alterations in external environment, as well as having limited leakage of ions and proteins. Some gel-like behaviors have not been rigorously confirmed in mammalian cells, and others should be examined under conditions where gel volume can be accurately monitored. Thus, possible contributions of cytoplasm hydrogel properties to cellular processes such as volume sensing and regulation remain unclear. We used three-dimensional imaging to measure volume of single substrate-attached cells after permeabilization of their plasma membrane. Permeabilized cells swelled or shrinked reversibly in response to variations of external osmolality. Volume changes were 3.7-fold greater than observed with intact cells, consistent with cytoplasm's high water-absorbing capacity. Volume was maximal at neutral pH and shrunk at acidic or alkaline pH, consistent with pH-dependent changes of protein charge density and repulsive forces within cellular matrix. Volume shrunk with increased Mg2+ concentration, as expected for increased charge screening and ionic crosslinking effects. Findings demonstrate that mammalian cytoplasm resembles hydrogel and functions as a highly sensitive osmosensor and extracellular pH sensor. Its high water-absorbing capacity may allow rapid modulation of local fluidity, macromolecular crowding, and activity of intracellular environment.

Organic vs inorganic

In order to survive hyperosmotic stress bacteria should adjust their cell turgor to altered conditions by increasing the intracellular osmolality. The classical view is that bacterial osmotic adjustment is achieved via accumulation of so-called “compatible solutes” – some organic osmolytes which can be accumulated in the cytosol at high concentrations without interfering with cell metabolism. In our recently published paper,11 we have shown that in the absence of osmolytes in the environment uptake of inorganic ions (and, specifically, K+) is central to osmotic adjustment in E. coli under hyperosmotic stress conditions. Here we show that optimal E. coli growth, observed at 2% NaCl, corresponds to an osmotic balance between external and internal osmolality within bacterial cells. This is achieved by the regulation of net K+ fluxes across the bacterial membrane. We suggest that the role of compatible solutes in osmotic adjustment in bacteria is indirect and confined to the fine tuning of a number of ion channels and transporters in order to achieve osmotic balance.