Cell volume regulation and swelling-activated chloride channels

Maintenance of a constant volume is essential for normal cell function. Following cell swelling, as a consequence of reduction of extracellular osmolarity or increase of intracellular content of osmolytes, animal cells are able to restore their original volume by activation of potassium and chloride conductances. The loss of these ions, followed passively by water, is responsible for the homeostatic response called regulatory volume decrease (RVD). Activation of a chloride conductance upon cell swelling is a key step in RVD. Several proteins have been proposed as candidates for this chloride conductance. The status of the field is reviewed, with particular emphasis on ClC-3, a member of the ClC family which has been recently proposed as the chloride channel involved in cell volume regulation.     

Role of water channels in the regulation of the volume of principal cells of rat kidney collecting ducts in hypoosmotic medium

The effect of plasma membrane water permeability on the rate of changes in the volume of principal cells of collecting ducts of the outer substantia medullaris under conditions of hypoosmotic shock has been studied. Changes in cell volume were studied by the fluorescent method. It was shown that the hypotonic shock induced a rapid increase in the cell volume with the characteristic time that depended on plasma membrane water permeability. The decrease in volume occurred much more slowly, and the rate of volume decrease directly correlated with the rate of swelling. The inhibition of potassium transport by barium chloride decreased the rate of volume restoration, without affecting substantially the duration of the swelling phase. The inhibition of mercury-sensitive water channels by mercury caused a significant increase in the time of both cell swelling and volume restoration. It was concluded that the state of water channels largely determines the rate of the regulatory response of epithelial cells of collecting ducts to hypoosmotic shock and affects the exchange of cell osmolites.     

THE SWELLING OF GELATIN AND THE VOLUME OF SURROUNDING SOLUTION

The swelling of isoelectric gelatin added to various volumes of acid of different concentration at 5°C. has been determined. The swelling is determined only by the concentration of the supernatant solution at equilibrium and is independent of the volume of acid. Similar experiments with unpurified gelatin show that in this case, owing to the presence of neutral salts the swelling is a function of the volume as well as the concentration of acid. Both results are predicted by the Procter-Wilson-Loeb theory of the swelling of gelatin.     

The mechanism of mitochondrial swelling. V. Permeability of mitochondria to alkali metal salts of strong acid anions

Mitochondria do not swell appreciably when suspended in media containing the chlorides or bromides of alkali metal or ammonium ions. On the other hand, extensive swelling takes place when mitochondria are suspended in ammonium or sodium acetate. These findings have been widely interpreted to mean that the mitochondrial membrane is impermeable to chloride and bromide ions. However, the resistance of the mitochondria to volume changes is not necessarily a valid criteria of impermeability to a given ion pair. Such a conclusion presumes the as yet untested assumptions that (1) permeability to the ion pair is pair is always the rate-limiting step in swelling, and (2) permeability to the ion pair is equivalent to the driving force for water influx. We have conducted experiments addressed to the question of mitochondrial permeability by methods (tracer exchange diffusion) which are independent of volume changes. Our findings indicate that the mitochondrial membrane is very readily penetrated by alkali metal chloride and bromide salts. Further, we have concluded that the resistance to swelling in such media is associated with a lack of driving force.     

SWELLING OF ERYTHROCYTES IN SOLUTIONS OF AMMONIUM SALTS

Two rather simple equations have been derived, which make it possible to express in a single number the result of a series of determinations of the volume of erythrocytes swelling in solutions of ammonium salts. In all experiments made with several combinations of different concentrations of permeating and non-permeating salts, the curves calculated from the equations have covered the points found by experiment.     

Assessment of the effects of cetylpyridium chloride on water content of the collagen-keratocyte matrix of the mammalian corneal stroma ex vivo

The effects of cationic surfactants on the time-dependent increases in hydration of the corneal stroma were investigated to assess if the contribution of the proteoglycans could be titrated and how it might relate to the maximum and minimum swelling properties of the corneal stroma. From recent post-mortem eyes from adult sheep, square (8 x 8 mm) samples of corneal stroma were prepared and incubated in isotonic neutral pH mixed salts solution with added glucose, or pure water, at 37 degrees C. The time-dependent changes in wet mass were assessed over 24 h in the absence or presence of 0. 001-2% w/v cetylpyridium chloride (CPC) or benzalkonium chloride (BAC). The rate and magnitude of stromal swelling was reduced in a concentration-dependent fashion by the surfactants. In mixed salts solution, 100% inhibition of swelling could be achieved at 2% CPC and BAC. In pure water, the relative swelling was much more substantial and could only be attenuated by CPC.     

Exposure of the Isolated Frog Skin to High Potassium Concentrations at the Internal Surface : II. Changes in epithelial cell volume,resistance and response to antidiuretic hormone

Isolated frog skin epithelia undergo marked, but reversible swelling when the external skin surface is bathed with conventional NaCl Ringer''s and the internal surface with KCl Ringer''s solutions. In 2 hours, epithelial thickness increased by over twofold. When NaCl Ringer''s was replaced on both sides of the skin, volume returned to control levels in less than 1 hour. When sulfate, rather than chloride, was the predominant anion, exposure of the internal surface to high potassium concentrations did not evoke changes in epithelial cell volume. With both KCl and K₂SO₄ Ringer''s, an immediate drop in DC resistance across the skin occurred. This was followed by partial recovery. Both the immediate drop and partial recovery were unrelated to changes in volume. A slow, sustained secondary drop in resistance was observed with KCl but not K₂SO₄ Ringer''s. This slower drop was associated temporally with swelling. When epithelial cell swelling occurred (i.e. with KCl Ringer''s), the characteristic response of the skin to vasopressin was abolished. However, with sulfate as anion, vasopressin elicited an increase in short-circuit current and/or in cell volume despite high internal potassium concentrations. It is concluded that epithelial swelling increased the permeability of the sodium-selective barrier at the external surface of the cells; and the possibility exists that stretching of cell membranes altered dimensions of pathways through which Na and water move, thereby mimicking the effects of vasopressin.     

Salinity tolerance of Gambusia affinis (Baird & Girard) and Heteropneustes fossilis (Bloch)

The present paper describes the results of a study on the salinity tolerance of two freshwater fishes, Gambusia affinis (Baird & Girard) and Heteropneustes fossilis (Bloch). The two species were treated with various concentrations of sea water and three most common salts found in the saline waters of Iraq, namely sodium chloride, calcium chloride and potassium chloride. The mortality rates following these treatments were recorded. Both species showed the highest degree of resistance for sea water and sodium chloride, and the lowest degree of tolerance to potassium chloride. Gambusia affinis was more resistant to these salts and sea water than H. fossilis .     

Volume and pH regulation in agnathan erythrocytes: comparisons between the hagfish,Myxine glutinosa,and the lampreys,Petromyzon marinus and Lampetra fluviatilis

The responses of hagfish (Myxine glutinosa) and lamprey (Lampetra fluviatilis and Petromyzon marinus) erythrocytes to osmotic swelling in hypoosmotic medium and to acid-base disturbances induced by ammonium chloride prepulse were studied. The erythrocytes of hagfish regulated neither cell volume after osmotic swelling nor intracellular pH after acidification. In contrast, the erythrocytes of lamprey lost potassium and chloride after osmotic swelling, whereby their volume recovered. Furthermore, the red cell pH of lamprey recovered from experimental acidification in a nominally bicarbonate-free medium in the presence of sodium, confirming that the pathway involved is sodium/proton exchange.Abbreviation DMO 5,5-dimethyloxazolidine-2,4-dione     

Ouabain-insensitive salt and water movements in duck red cells. II. The role of chloride in the volume response

This paper describes the effect of external chloride on the typical swelling response induced in duck red cells by hypertonicity or norepinephrine. Lowering chloride inhibits swelling and produces concomitant changes in net movements of sodium and potassium in ouabain-treated cells, which resemble the effect of lowering external sodium or potassium. Inhibition is the same whether chloride is replaced with gluconate or with an osmotic equivalent of sucrose. Since changes in external chloride also cause predictable changes in cell chloride, pH, and water, these variables were systematically investigated by varying external pH along with chloride. Lowering pH to 6.60 does not abolish the response if external chloride levels are normal, although the cells are initially swollen due to the increased acidity. Cells deliberately preswollen in hypotonic solutions with appropriate ionic composition can also respond to norepinephrine by further swelling. These results rule out initial values of cell water, chloride, and pH as significant variables affecting the response. Initial values of the chloride equilibrium potential do have marked effect on the direction and rate of net water movement. If chloride is lowered by replacement with the permeant anion, acetate, E(Cl) is unchanged and a normal response to norepinephrine, which is inhibited by furosemide, is observed. Increasing internal sodium by the nystatin technique also inhibits the response. A theory is developed which depicts that the cotransport carrier proposed in the previous paper (W.F. Schmidt and T.J. McManus. 1977b. J. Gen. Physiol. 70:81-97) moves in response to the net electrochemical potential difference driving sodium and potassium across the membrane. Predictions of this theory fit the data for both cations and anions.     

Electromechanics and Volume Dynamics in Nonexcitable Tissue Cells

Cell volume regulation is fundamentally important in phenomena such as cell growth, proliferation, tissue homeostasis, and embryogenesis. How the cell size is set, maintained, and changed over a cell’s lifetime is not well understood. In this work we focus on how the volume of nonexcitable tissue cells is coupled to the cell membrane electrical potential and the concentrations of membrane-permeable ions in the cell environment. Specifically, we demonstrate that a sudden cell depolarization using the whole-cell patch clamp results in a 50% increase in cell volume, whereas hyperpolarization results in a slight volume decrease. We find that cell volume can be partially controlled by changing the chloride or the sodium/potassium concentrations in the extracellular environment while maintaining a constant external osmotic pressure. Depletion of external chloride leads to a volume decrease in suspended HN31 cells. Introducing cells to a high-potassium solution causes volume increase up to 50%. Cell volume is also influenced by cortical tension: actin depolymerization leads to cell volume increase. We present an electrophysiology model of water dynamics driven by changes in membrane potential and the concentrations of permeable ions in the cells surrounding. The model quantitatively predicts that the cell volume is directly proportional to the intracellular protein content.     

Comparative efficacy of chlorophyllin in reducing cytotoxicity of some heavy metals

Summary The potential of chlorophyllin in reducing clastogenicity was studied against two concentrations of each of three potent metallic clastogens (cesium chloride, mercuric chloride and cobalt chloride) in bone marrow cells of mice in vivo. The respective salts and chlorophyllin were administered orally to mice by gavaging in different combinations. Simultaneous administration of chlorophyllin with both concentrations of each salt reduced the clastogenic effects in the order Cs > Hg > Co. Chlorophyllin could not decrease the clastogenic effects when administered 2 h before the salts.     

Water channels in platelet volume regulation

The regulation of platelet volume significantly affects its function. Because water is the major molecule in cells and its active transport via water channels called aquaporins (AQPs) have been implicated in cellular and organelle volume regulation, the presence of water channels in platelets and their potential role in platelet volume regulation was investigated. G-protein-mediated AQP regulation in secretory vesicle swelling has previously been reported in neurons and in pancreatic acinar cells. Mercuric chloride has been demonstrated to inhibit most AQPs except AQP6, which is stimulated by the compound. Exposure of platelets to HgCl(2)-induced swelling in a dose-dependent manner, suggesting the presence of AQP6 in platelets. Immunoblot analysis of platelet protein confirmed the presence of AQP6, and also of G(αo), G(αi-1) and G(αi-3) proteins. Results from this study demonstrate for the first time that in platelets AQP6 is involved in cell volume regulation via a G-protein-mediated pathway.     

Volume changes in hemolytic systems containing resorcinol,taurocholate, and saponin

Simultaneous measurement of hemolysis, the volume of the intact cells, and the K lost from the intact cells of systems containing resorcinol, sodium taurocholate, and saponin shows that the volume increases may be conspicuously small while the K losses are large, and that the volume increases are un-equal for equal K losses produced by different lysins. In higher concentrations of the same lysins, the critical volume for hemolysis is a function of the nature of the lysin and of its concentration. It is impossible to say whether these observations are compatible with the current "dual mechanism" and "colloid osmotic" hypotheses of hemolysis, in which the swelling of the cell is supposed to result from the lysin having made it cation-permeable. The difficulty to be overcome is that the theory cannot be developed to describe volume changes in finite time unless we know what assumptions to make about the mobilities of K and Na, the forces driving them into and out of the cell, etc. The experimental results do not suggest, however, that any simple set of assumptions would be satisfactory. The conditions which regulate the upper limit of the swelling, i.e. the point at which a swelling phenomenon becomes a hemolytic phenomenon, are functions of the nature of the lysin and sometimes of its concentration. They require to be specified by an independent statement, over and above any statement which may be made about the rate at which swelling occurs in the system. The simplest view of the situation is that the conditions which regulate the critical volume and those which regulate the rate of swelling are both functions, as yet undefined, of the reaction which takes place between the lysin and the structural components of the red cell.     

Ultrastructural Alterations of Erwinia carotovora subsp. atroseptica Caused by Treatment with Aluminum Chloride and Sodium Metabisulfite

Aluminum and bisulfite salts inhibit the growth of several fungi and bacteria, and their application effectively controls potato soft rot caused by Erwinia carotovora. In an effort to understand their inhibitory action, ultrastructural changes in Erwinia carotovora subsp. atroseptica after exposure (0 to 20 min) to different concentrations (0.05, 0.1, and 0.2 M) of these salts were examined by using transmission electron microscopy. Plasma membrane integrity was evaluated by using the SYTOX Green fluorochrome that penetrates only cells with altered membranes. Bacteria exposed to all aluminum chloride concentrations, especially 0.2 M, exhibited loosening of the cell walls, cell wall rupture, cytoplasmic aggregation, and an absence of extracellular vesicles. Sodium metabisulfite caused mainly a retraction of plasma membrane and cellular voids which were more pronounced with increasing concentration. Bacterial mortality was closely associated with SYTOX stain absorption when bacteria were exposed to either a high concentration (0.2 M) of aluminum chloride or prolonged exposure (20 min) to 0.05 M aluminum chloride or to a pH of 2.5. Bacteria exposed to lower concentrations of aluminum chloride (0.05 and 0.1 M) for 10 min or less, or to metabisulfite at all concentrations, did not exhibit significant stain absorption, suggesting that no membrane damage occurred or it was too weak to allow the penetration of the stain into the cell. While mortality caused by aluminum chloride involves membrane damage and subsequent cytoplasmic aggregation, sulfite exerts its effect intracellularly; it is transported across the membrane by free diffusion of molecular SO₂ with little damage to the cellular membrane.     

Effect of salt and water stress on RNA synthesis in the excised embryo axis of germinating mung bean (Phaseolus aureus ROXB).

The rate of synthesis of RNA was studied under salt and water stresses created by the addition of four different salts and polyethylene glycol-6000 (PEG-6000) in excised embryo axis of mung bean using^l4C-uracil. Iso-osmotio concentrations of salts and PEG resulted in differential effect on RNA synthesis. PEG inhibited, whereas salt stimulated incorporation of labelled uracil into RNA. Ionic differences on RNA synthesis were very clear as synthesis of RNA was more affected with sulphate than chloride and with sodium than potassium ions.     

The AQP-3 water channel is a pivotal modulator of glycerol-induced chloride channel activation in nasopharyngeal carcinoma cells

Aquaporin (AQP) and chloride channels are ubiquitous in virtually all living cells, playing pivotal roles in cell proliferation, migration and apoptosis. We previously reported that AQP-3 aquaglyceroporin and ClC-3 chloride channels could form complexes to regulate cell volume in nasopharyngeal carcinoma cells. In this study, the roles of AQP-3 in their hetero-complexes were further investigated. Glycerol entered the cells via AQP-3 and induced two different Cl⁻ currents through cell swelling-dependent or -independent pathways. The swelling-dependent Cl⁻ current was significantly inhibited by pretreatment with CuCl₂ and AQP-3-siRNA. After siRNA-induced AQP-3 knock-down, the 140 mM glycerol isoosmotic solution swelled cells by 22% (45% in AQP-3-intact cells) and induced a smaller Cl⁻ current; this current was smaller than that activated by 8% cell volume swelling, which induced by the 140 mM glycerol hyperosmotic solution in AQP-3-intact cells. This suggests that the interaction between AQP-3 and ClC-3 plays an important role in cell volume regulation and that AQP-3 may be a modulator that opens volume-regulated chloride channels. The swelling-independent Cl⁻ current, which was activated by extracellular glycerol, was reduced by CuCl₂ and AQP-3-siRNA pretreatment. Dialyzing glycerol into cells via the pipette directly induced the swelling-independent Cl⁻ current; however this current was blocked by AQP-3 down-regulation, suggesting AQP-3 is essential for the opening of chloride channels. In conclusion, AQP-3 is the pathway for water, glycerol and other small solutes to enter cells, and it may be an essential modulator for the gating of chloride channels.     

THE INFLUENCE OF ELECTROLYTES ON THE SOLUTION AND PRECIPITATION OF CASEIN AND GELATIN

1. Colloids have been divided into two groups according to the ease with which their solutions or suspensions are precipitated by electrolytes. One group (hydrophilic colloids), e.g., solutions of gelatin or crystalline egg albumin in water, requires high concentrations of electrolytes for this purpose, while the other group (hydrophobic colloids) requires low concentrations. In the latter group the precipitating ion of the salt has the opposite sign of charge as the colloidal particle (Hardy''s rule), while no such relation exists in the precipitation of colloids of the first group. 2. The influence of electrolytes on the solubility of solid Na caseinate, which belongs to the first group (hydrophilic colloids), and of solid casein chloride which belongs to the second group (hydrophobic colloids), was investigated and it was found that the forces determining the solution are entirely different in the two cases. The forces which cause the hydrophobic casein chloride to go into solution are forces regulated by the Donnan equilibrium; namely, the swelling of particles. As soon as the swelling of a solid particle of casein chloride exceeds a certain limit it is dissolved. The forces which cause the hydrophilic Na caseinate to go into solution are of a different character and may be those of residual valency. Swelling plays no rôle in this case, and the solubility of Na caseinate is not regulated by the Donnan equilibrium. 3. The stability of solutions of casein chloride (requiring low concentrations of electrolytes for precipitation) is due, first, to the osmotic pressure generated through the Donnan equilibrium between the casein ions tending to form an aggregate, whereby the protein ions of the nascent micellum are forced apart again; and second, to the potential difference between the surface of a micellum and the surrounding solution (also regulated by the Donnan equilibrium) which prevents the further coalescence of micella already formed. This latter consequence of the Donnan effect had already been suggested by J. A. Wilson. 4. The precipitation of this group of hydrophobic colloids by salts is due to the diminution or annihilation of the osmotic pressure and the P.D. just discussed. Since low concentrations of electrolytes suffice for the depression of the swelling and P.D. of the micella, it is clear why low concentrations of electrolytes suffice for the precipitation of hydrophobic colloids, such as casein chloride. 5. This also explains why only that ion of the precipitating salt is active in the precipitation of hydrophobic colloids which has the opposite sign of charge as the colloidal ion, since this is always the case in the Donnan effect. Hardy''s rule is, therefore, at least in the precipitation of casein chloride, only a consequence of the Donnan effect. 6. For the salting out of hydrophilic colloids, like gelatin, from watery solution, sulfates are more efficient than chlorides regardless of the pH of the gelatin solution. Solution experiments lead to the result that while CaCl₂ or NaCl increase the solubility of isoelectric gelatin in water, and the more, the higher the concentration of the salt, Na₂SO₄ increases the solubility of isoelectric gelatin in low concentrations, but when the concentration of Na₂SO₄ exceeds M/32 it diminishes the solubility of isoelectric gelatin the more, the higher the concentration. The reason for this difference in the action of the two salts is not yet clear. 7. There is neither any necessity nor any room for the assumption that the precipitation of proteins is due to the adsorption of the ions of the precipitating salt by the colloid.     

Ultrastructural alterations of Erwinia carotovora subsp. atroseptica caused by treatment with aluminum chloride and sodium metabisulfite

Aluminum and bisulfite salts inhibit the growth of several fungi and bacteria, and their application effectively controls potato soft rot caused by Erwinia carotovora. In an effort to understand their inhibitory action, ultrastructural changes in Erwinia carotovora subsp. atroseptica after exposure (0 to 20 min) to different concentrations (0.05, 0.1, and 0.2 M) of these salts were examined by using transmission electron microscopy. Plasma membrane integrity was evaluated by using the SYTOX Green fluorochrome that penetrates only cells with altered membranes. Bacteria exposed to all aluminum chloride concentrations, especially 0.2 M, exhibited loosening of the cell walls, cell wall rupture, cytoplasmic aggregation, and an absence of extracellular vesicles. Sodium metabisulfite caused mainly a retraction of plasma membrane and cellular voids which were more pronounced with increasing concentration. Bacterial mortality was closely associated with SYTOX stain absorption when bacteria were exposed to either a high concentration (0.2 M) of aluminum chloride or prolonged exposure (20 min) to 0.05 M aluminum chloride or to a pH of 2.5. Bacteria exposed to lower concentrations of aluminum chloride (0.05 and 0.1 M) for 10 min or less, or to metabisulfite at all concentrations, did not exhibit significant stain absorption, suggesting that no membrane damage occurred or it was too weak to allow the penetration of the stain into the cell. While mortality caused by aluminum chloride involves membrane damage and subsequent cytoplasmic aggregation, sulfite exerts its effect intracellularly; it is transported across the membrane by free diffusion of molecular SO2 with little damage to the cellular membrane.     

Swelling of Root Cell Walls as an Indicator of Their Functional State

The swelling capacity of cell walls isolated from different parts of lupine root was investigated. The water content in fragments of intact roots (Q) and swelling coefficient of standardized samples of cell walls (K^cw) were determined, and the dependences of Q and Kcw on the distance from the root tip (L) were plotted. It was shown that the change in Q value along the stretch of the lupine root reaches its maximum at distances of 1.5-6 cm or 7-12 cm from the root tip in 7-day-old and 14-day-old seedlings, respectively, whereas the K^cw value distribution over the root length is virtually invariable. In the radial direction, both the Q and K^cw values in cortex tissues are about twice higher than in the central cylinder. In our opinion, the changes of both Q and K^cw in the radial direction are associated with different degrees of cross-linking between polymer chains in cell wall structures of root cortex and central cylinder. The results of measurement of the K^cw value are consistent with the widely accepted mechanisms of water transport in roots in the radial direction. These data show that water transport through apoplast to the border between the cortex and central cylinder is accompanied by an increase in the resistance to water flow. Among other factors, this increase is due to a greater degree of cross-linking between cell wall polymers in the central cylinder. The results of measurement of the swelling coefficient of standardized cell wall samples in water and in 10 mM KCl at different pH values show that the swelling capacity of root cell walls varies according to the physicochemical properties of synthetic ion exchangers. Cell walls shrink (cell wall volume decreases) as ion concentration in solution increases and pH decreases. This causes an increase in the hydraulic resistance (or a decrease in the hydraulic conductivity) of apoplast. It was concluded that swelling is determined by the physicochemical properties of the cell wall, whereas the change in the swelling capacity induced by variation of external or internal conditions is an element of the mechanism of regulation of volume water flow in roots.