Volume-activated taurine permeability in cells of the human erythroleukemic cell line K562

The effects of hypotonic shock on cell volume, taurine influx and efflux were examined in the human erythroleukemic cell line K562. Cells exposed to hypotonic solutions exhibited a regulatory volume decrease (RVD) following rapid increases in cell volume. Cell swelling was associated with a increased taurine influx and efflux. The volume-activated taurine pathway was Na⁺-independent, and increased in parallel with increasing cell volume. The chloride channel blocker, 2,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC), completely blocked the volume-activated taurine influx and efflux, while [dihydroin-denyl]oxy]alkanoic acids (DIOA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), an anion exchanger and anion channel blocker, respectively, also inhibited significantly. These results suggest that taurine transport is increased in response to hypotonic stress, which may be mediated via a volume-activated, DCDPC-sensitive anion channel. © 1996 Wiley-Liss, Inc.     

A mathematical model of the response of principal cells of collecting ducts to hypotonic shock

The regulatory decrease in the volume of principal cells of collecting ducts to hypoosmotic shock has been investigated experimentally and using the mathematical modeling. A mathematical model of the response of collecting duct principal cells to hypotonic shock has been constructed on the basis of the experimental time course of changes in cell volume measured by the fluorescent dye Calcein. It was shown that the regulatory decrease in volume under hypotonic conditions occurs via a marked release of osmolytes and is accompanied by a decrease in water permeability of the cell membrane. The mathematical modeling of transmembrane transport processes allowed us to quantitatively estimate the changes in membrane water permeability, which decreased tenfold, from 2 x 10(-1) cm/s to 2 x 10(-2) cm/s. It was also shown that the effective regulatory decrease in the volume of collecting duct principal cells in hypotonic medium results from a significant increase in membrane permeability for K+, Cl-, and organic anions.     

Mechanism of thiyl radical-catalyzed isomerization of unsaturated fatty acid residues in homogeneous solution and in liposomes

Taurine is an important osmolyte involved in cell volume regulation. During regulatory volume decrease it is released via a volume-sensitive organic osmolyte/anion channel. Several molecules have been suggested as candidates for osmolyte release. In this study, we chose three of these, namely ClC-2, ClC-3 and ICln, because of their expression in rat astrocytes, a cell type which is known to release taurine under hypotonic stress, and their activation by hypotonic shock. As all three candidates were also suggested to be chloride channels, we investigated their permeability for both chloride and taurine under isotonic and hypotonic conditions using the Xenopus laevis oocyte expression system. We found a volume-sensitive increase of chloride permeability in ClC-2-expressing oocytes only. Yet, the taurine permeability was significantly increased under hypotonic conditions in oocytes expressing any of the tested candidates. Further experiments confirmed that the detected taurine efflux does not represent unspecific leakage. These results suggest that ClC-2, ClC-3 and ICln either participate in taurine transport themselves or upregulate an endogenous oocyte osmolyte channel. In either case, the taurine efflux of oocytes not being accompanied by an increased chloride flux suggests that taurine and chloride can be released via two separate pathways.     

Effect of hypotonic shock on cultured pavement cells from freshwater or seawater rainbow trout gills

The effect of hypotonic shock on cultured pavement gill cells from freshwater (FW)- and seawater (SW)-adapted trout was investigated. Exposure to 2/3rd strength Ringer solution produced an increase in cell volume followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also induced a biphasic increase in cytosolic Ca(2+) with an initial peak followed by a sustained plateau. Absence of external Ca(2+) did not modify cell volume under isotonic conditions, but inhibited RVD after hypotonic shock. [Ca(2+)](i) response to hypotonicity was also partially inhibited in Ca-free bathing solutions. Similar results were obtained whether using cultured gill cells prepared from FW or SW fishes. When comparing freshly isolated cells with cultured gill cells, a similar Ca(2+) signalling response to hypotonic shock was observed regardless of the presence or absence of Ca(2+) in the solution. In conclusion, gill pavement cells in primary culture are able to regulate cell volume after a cell swelling and express a RVD response associated with an intracellular calcium increase. A similar response to a hypotonic shock was recorded for cultured gill cells collected from FW and SW trout. Finally, we showed that calcium responses were physiologically relevant as comparable results were observed with freshly isolated cells exposed to hypoosmotic shock.     

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.     

Study of the reaction of kidney collecting duct principal cells to hypotonic shock. Experiment and mathematical model

The reaction of rat kidney collecting duct principal cells to hypotonic shock was studied. The changes in cell relative volume were measured using fluorescent dye calcein, and a mathematical model based on our experimental results was developed. It was shown that regulatory volume decrease is mainly provided by significant release of osmolytes from the cell and decrease of the plasma membrane water permeability. Using our model, we calculated the membrane water permeability and found it to decrease from 2 · 10⁻¹ to 2 · 10⁻² cm/s. We conclude that for effective RVD to occur, a dramatic increase in the membrane permeability to K⁺, Cl⁻ and organic anions is necessary.     

Cell volume regulation in renal cortical cells

Both proximal renal tubule cells and cultured Madin-Darby canine kidney (MDCK) cells are capable of regulating their volume in hypotonic media. Regulatory cell volume decrease in proximal straight tubules is impaired by barium, amiloride and acetazolamide and depends on the presence of bicarbonate and of sodium, whereas it is unaffected by complete removal of extracellular chloride. The observations may point to parallel loss of potassium through potassium channels as well as of bicarbonate and sodium via a bicarbonate-sodium cotransport. Alternatively, potassium/hydrogen ion exchange or potassium bicarbonate cotransport could be involved. In MDCK cells, exposure to hypotonic media apparently leads to the activation of an anion channel, while potassium conductance is rather decreased. In both proximal tubules and MDCK cells, volume regulatory decrease is possibly triggered by leucotrienes, which may be released during cell swelling. Cell volume is altered in a variety of conditions even at isotonic extracellular fluid and cell volume-regulatory mechanisms are likely to participate in regulation of renal transepithelial transport.     

Cholesterol depletion facilitates recovery from hypotonic cell swelling in CHO cells

The maintenance of cell volume homeostasis is critical for preventing pathological cell swelling that may lead to severe cellular dysfunction or cell death. Our earlier studies have shown that volume-regulated anion channels that play a major role in the regulation of cell volume are facilitated by a decrease in cellular cholesterol suggesting that cholesterol depletion should also facilitate regulatory volume decrease (RVD), the ability of cells to recover from hypotonic swelling. In this study, we test this hypothesis using a novel methodology developed to measure changes in cell volume using a microfluidics chamber. Our data show that cholesterol depletion of Chinese Hamster Ovary (CHO) significantly facilitates the recovery process, as is apparent from a faster onset of the RVD (162±10 s. vs. 114±5 s. in control and cholesterol depleted cells respectively) and a higher degree of volume recovery after 10 min of the hypotonic challenge (41%±6% vs. 65%±6% in control and cholesterol depleted cells respectively). In contrast, enriching cells with cholesterol had no effect on the RVD process. We also show here that similarly to our previous observations in endothelial cells, cholesterol depletion significantly increases the stiffness of CHO cells suggesting that facilitation of RVD may be associated with cell stiffening. Furthermore, we also show that increasing cell stiffness by stabilizing F-actin with jasplakinolide also facilitates RVD development. We propose that cell stiffening enhances cell mechano-sensitivity, which in turn facilitates the RVD process.     

Digestive cells from Mytilus galloprovincialis show a partial regulatory volume decrease following acute hypotonic stress through mechanisms involving inorganic ions

The response of isolated digestive cells of the digestive gland of Mytilus galloprovincialis to hypotonic shock was studied using videometric methods. The isolated cells exposed to a rapid change (from 1100 to 800 mosmol kg^?1) of the bathing solution osmolality swelled but thereafter underwent a regulatory volume decrease (RVD), tending to recover the original size. When the hypotonic stress was applied in the presence of quinine and glibenclamide, known inhibitors of swelling activated ion channels, the cells did not exhibit an RVD response; in addition, they showed a larger increase in size in respect to control cells. These observations suggest that the digestive cells of the digestive gland have the machinery to cope with the hyposmotic shock allowing them to exhibit a small but significant RVD preventing an excessive increase in cell size. The pharmacological treatment of digestive cells during the RVD experiments suggests that cell volume is regulated by K⁺ and Cl^? efflux followed by an obliged water efflux from the cell. The involvement of organic osmolytes such as taurine and betaine seems to be excluded by NMR measurement on digestive cells. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterization of regulatory volume decrease in the THP-1 and HL-60 human myelocytic cell linesn

Exposure to hypotonic stress produces a transient increase in cell volume followed by a regulatory volume decrease (RVD) in both THP-1 and HL-60 cells. In contrast, cells exposed to hypotonic stress in a high K/low Na Hanks' solution not only failed to volume regulate, but displayed a secondary swelling. Thus, while an outward K gradient was required ful KVD, the secondary swelling indicated that hypotonic stress increased permeability in the absence of a negative membrane potential. The K channel blocker quinine (1–4 mM) blocked RVD in both cell types. Gramicidin's ability to overcome the quinine block of RVD indicated that RVD is mediated by a quinine-sensitive cation transport mechanism that is independent of the swelling-induced anion transport mechanism. Barium (1–4 mM), another K channel blocker, slowed the rate of RVD, while 4-aminopyridine, charybdotoxin, tetraethylammonium chloride, tetrabutylammonium chloride, and gadolinium had no effect on RVD. Furthermore, RVD was not mediated by calcium-activated conductances, since it occurred normally in Ca-free medium, in medium containing cadmium, and in BAPTA-loaded cells. Gramicidin produced little or no volume change in isotonic medium, suggesting that basal C1 permeability of both THP-1 and HL-60 cells is low. However, swelling induced an anion efflux pathway that is permeable to both chloride and bromide, but is impermeable to methanesulfonate and glutamate. The anion channel blocker 3,5-diiodosalicylic acid (DISA) antagonized RVD in both cell types. In conclusion, RVD in THP-1 and HL-60 cells is mediated by independent anion and cation transport mechanisms that involve both a DISA-sensitive anion pathway and a quinine-inhibitable K efflux pathway, neither of which requires increases in intra-cellular calcium to be activated. © 1994 wiley-Liss, Inc.     

Cell volume regulation following hypotonic shock in hepatocytes isolated from Sparus aurata

The response of isolated hepatocytes of Sparus aurata to hypotonic shock was studied by the aid of videometric and light scattering methods. The isolated cells exposed to a rapid change (from 370 to 260 mOsm/kg) of the osmolarity of the bathing solution swelled but thereafter underwent a decrease of cell volume tending to recovery the original size. This homeostatic response RVD (regulatory volume decrease) was inhibited in the absence of extracellular Ca²⁺ and in the presence of TMB8, an inhibitor of Ca²⁺ release from intracellular stores. It is likely that Ca²⁺ entry through verapamil sensitive Ca²⁺-channels, probably leading to a release of Ca²⁺ from intracellular stores, is responsible for RVD since the blocker impaired the ability of the cell to recover its volume after the hypotonic shock. RVD tests performed in the presence of various inhibitors of different transport mechanisms, such as BaCl₂, quinine, glybenclamide and bumetanide as well as in the presence of a KCl activator, NEM, led us to suggest that the recovery of cell volume in hypotonic solution is accomplished by an efflux of K⁺ and Cl^? through conductive pathways paralleled by the operation of the KCl cotransport, followed by an obliged water efflux from the cells.     

Characterization of volume-sensitive organic osmolyte efflux and anion current in Xenopus oocytes

Cell swelling activates an outwardly rectifying anion current in numerous mammalian cell types. An extensive body of evidence indicates that the channel responsible for this current is the major pathway for volume regulatory organic osmolyte loss. Cell swelling also activates an outwardly rectifying anion current in Xenopus oocytes. Unlike mammalian cells, oocytes allow the direct study of both swelling-activated anion current and organic osmolyte efflux under nearly identical experimental conditions. We therefore exploited the unique properties of oocytes in order to examine further the relationship between anion channel activity and swelling-activated organic osmolyte transport. Swelling-activated anion current and organic osmolyte efflux were studied in parallel in batches of oocytes obtained from single frogs. The magnitude of swelling-activated anion current and organic osmolyte efflux exhibited a positive linear correlation. In addition, the two processes had similar pharmacological characteristics and activation, rundown and reactivation kinetics. The present study provides further strong support for the concept that the channel responsible for swelling-activated Cl⁻ efflux and the outwardly rectifying anion conductance is also the major pathway by which organic osmolytes are lost from vertebrate cells during regulatory volume decrease. Received: 22 April 1996/Revised: 18 December 1996     

The Role of Calcium in the Volume Regulation of Rat Lacrimal Acinar Cells

Earlier studies have suggested a role for Ca²⁺ in regulatory volume decrease (RVD) in response to hypotonic stress through the activation of Ca²⁺-dependent ion channels (Kotera & Brown, 1993; Park et al., 1994). The involvement of Ca²⁺ in regulating cell volume in rat lacrimal acinar cells was therefore examined using a video-imaging technique to measure cell volume. The trivalent cation Gd³⁺ inhibited RVD, suggesting that Ca²⁺ entry is important and may be via stretch-activated cation channels. However, Fura-2 loaded cells did not show an increase in [Ca²⁺] _i during exposure to hypotonic solutions. The absence of any changes in [Ca²⁺] _i resulted from the buffering of cytosolic Ca²⁺ by Fura-2 during hypotonic shock and therefore inhibition of RVD. The intracellular Ca²⁺ chelator, BAPTA, also inhibited the RVD response to hypotonic shock. An increase in [Ca²⁺] _i induced by either acetylcholine or ionomycin, was found to decrease cell volume under isotonic conditions in lacrimal acinar cells. Cell shrinkage was inhibited by tetraethylammonium ion, an inhibitor of Ca²⁺-activated K⁺ channels. On the basis of the presented data, we suggest an involvement of intracellular Ca²⁺ in controlling cell volume in lacrimal acinar cells. Received: 20 February 1998/Revised: 1 May 1998     

Volume-induced increase of anion permeability in human lymphocytes

Peripheral blood mononuclear cells (PBM) readjust their volumes after swelling in hypotonic media. This regulatory volume decrease (RVD) is associated with a loss of cellular K+ and is thought to be promoted by an increased permeability to this ion. In contrast, no change in volume was observed when K+ permeability of PBM in isotonic media was increased to comparable or higher levels using valinomycin. Moreover, valinomycin-induced 86Rb+ loss in K+-free medium was considerably slower than in K+-rich medium. These results suggest that anion conductance limits net salt loss in isotonic media. Direct measurements of relative conductance confirmed that in volume-static cells, anion conductance is lower than that of K+. In volume-regulating cells depolarization occurred presumably as a result of increased anion conductance. Accordingly, the efflux of 36Cl from PBM was markedly increased by hypotonic stress. Since both membrane potential and intracellular 36Cl concentration are reduced in hypotonically swollen cells, the increased efflux is probably due to a change in Cl- permeability. Anions and cations seem to move independently through the volume-induced pathways: the initial rate of 86Rb uptake in swollen cells was not affected by replacement of external Cl- by SO=4; conversely, 36Cl fluxes were unaffected by substitution of K+ by Na+. The data indicate that anion conductance is rate-determining in salt and water loss from PBM. An increase in anion conductance is suggested to be the critical step of RVD of human PBM.     

Changes in organic solutes,volume, energy state,and metabolism associated with osmotic stress in a glial cell line: A multinuclear NMR study

Diffusion-weighted in vivo¹H-NMR spectroscopy of F98 glioma cells embedded in basement membrane gel threads showed that the initial cell swelling to about 180% of the original volume induced under hypotonic stress was followed by a regulatory volume decrease to nearly 100% of the control volume in Dulbecco's modified Eagle's medium (DMEM) but only to 130% in Krebs-Henseleit buffer (KHB, containing only glucose as a substrate) after 7 h. The initial cell shrinkage to approx. 70% induced by the hypertonic stress was compensated by a regulatory volume increase which after 7 h reached almost 100% of the control value in KHB and 75% in DMEM.¹H-,¹³C-and³¹P-NMR spectroscopy of perchloric acid extracts showed that these volume regulatory processes were accompanied by pronounced changes in the content of organic osmolytes. Adaptation of intra- to extracellular osmolarity was preferentially mediated by a decrease in the cytosolic taurine level under hypotonic stress and by an intracellular accumulation of amino acids under hypertonic stress. If these solutes were not available in sufficient quantities (as in KHB), the osmolarity of the cytosol was increasingly modified by biosynthesis of products and intermediates of essential metabolic pathways, such as alanine, glutamate and glycerophosphocholine in addition to ethanolamine. The cellular nucleoside triphosphate level measured by in vivo³¹P-NMR spectroscopy indicated that the energy state of the cells was more easily sustained under hypotonic than hypertonic conditions.To whom to address reprint requests.     

Swelling-activated pathways in human T-lymphocytes studied by cell volumetry and electrorotation

Small organic solutes, including sugar derivatives, amino acids, etc., contribute significantly to the osmoregulation of mammalian cells. The present study explores the mechanisms of swelling-activated membrane permeability for electrolytes and neutral carbohydrates in Jurkat cells. Electrorotation was used to analyze the relationship between the hypotonically induced changes in the electrically accessible surface area of the plasma membrane (probed by the capacitance) and its permeability to the monomeric sugar alcohol sorbitol, the disaccharide trehalose, and electrolyte. Time-resolved capacitance and volumetric measurements were performed in parallel using media of different osmolalities containing either sorbitol or trehalose as the major solute. Under mild hypotonic stress in 200 mOsm sorbitol or trehalose solutions, the cells accomplished regulatory volume decrease by releasing cytosolic electrolytes presumably through pathways activated by the swelling-mediated retraction of microvilli. This is suggested by a rapid decrease of the area-specific membrane capacitance C(m) (microF/cm2). The cell membrane was impermeable to both carbohydrates in 200 mOsm media. Whereas trehalose permeability remained also very poor in 100 mOsm medium, extreme swelling of cells in a strongly hypotonic solution (100 mOsm) led to a dramatic increase in sorbitol permeability as evidenced by regulatory volume decrease inhibition. The different osmotic thresholds for activation of electrolyte release and sorbitol influx suggest the involvement of separate swelling-activated pathways. Whereas the electrolyte efflux seemed to utilize pathways preexisting in the plasma membrane, putative sorbitol channels might be inserted into the membrane from cytosolic vesicles via swelling-mediated exocytosis, as indicated by a substantial increase in the whole-cell capacitance C(C) (pF) in strongly hypotonic solutions.     

Loss of cell volume regulation during metabolic inhibition in renal epithelial cells (A6): role of intracellular pH

In renalischemia, tubular obstruction induced by swelling of epithelialcells might be an important mechanism for reduction of the glomerularfiltration rate. We investigated ischemic cell swelling byexamining volume regulation of A6 cells during metabolic inhibition(MI) induced by cyanide and 2-deoxyglucose. Changes in cell volume weremonitored by recording cell thickness (T_c). Intracellular pH (pH_c) measurements were performed with thepH-sensitive probe 5-chloromethyl-fluoresceine diacetate.T_c measurements showed that MI increases cellvolume. Cell swelling during MI is proportional to the rate ofNa⁺ transport and is not followed by a volume regulatoryresponse. Furthermore, MI prevents the regulatory volume decrease (RVD) elicited by a hyposmotic shock. MI induces a pronounced intracellular acidification that is conserved during a subsequent hypotonic shock. Atransient acidification induced by a NH₄Cl prepulse causes a marked delay of the RVD in response to a hypotonic shock. On theother hand, acute lowering of external pH to 5, simultaneously with thehypotonic shock, allowed the onset of RVD. However, this RVD wascompletely arrested ~10 min after the initiation of the hyposmoticchallenge. The inhibition of RVD appears to be related to thepronounced acidification that occurred within this time period. Incontrast, when external pH was lowered 20 min before the hyposmoticshock, RVD was absent. These data suggest that internal acidificationinhibits cellular volume regulation in A6 cells. Therefore, theintracellular acidification associated with MI might at least partlyaccount for the failure of volume regulation in swollen epithelial cells.

     

Tyrosine kinase inhibition affects skate anion exchanger isoform I alterations after volume expansion

Upon exposure to hypotonic medium, skate red blood cells swell and then reduce their volume by releasing organic osmolytes and associated water. The regulatory volume decrease is inhibited by stilbenes and anion exchange inhibitors, suggesting involvement of the red blood cell anion exchanger skAE1. To determine the role of tyrosine phosphorylation, red blood cells were volume expanded with and without prior treatment with the tyrosine kinase inhibitor piceatannol. At the concentration used, 130 microM, piceatannol nearly completely inhibits p72(syk), a tyrosine kinase previously shown to phosphorylate skAE1 (M. W. Musch, E. H. Hubert, and L. Goldstein. J Biol Chem 274: 7923-7928, 1999). Hyposmotic-induced volume expansion stimulated association of p72(syk) with a light membrane fraction of skate red blood cells. Piceatannol did not inhibit this association but decreased hyposmotically stimulated increased skAE1 tyrosine phophorylation. Movement of skAE1 from an intracellular to a surface detergent-resistant membrane domain and tetramer formation were not inhibited by piceatannol treatment. Two effects of hyposmotic-induced volume expansion, decreased band 4.1 binding and increased ankyrin, were both inhibited by piceatannol. These results suggest that at least one event requiring p72(syk) activation is pivotal for hyposmotic-induced increased transport; however, steps that do not require tyrosine phosphorylation may also play a role.     

Comparison of the Osmolyte Transport Properties Induced by trAE1 versus IClswell in Xenopus Oocytes

During cell swelling, cells release organic osmolytes via a volume-activated channel as part of the regulatory volume decrease. The erythrocyte membrane protein AE1 (band 3), has been shown to be involved in regulatory volume responses of fish erythrocytes. Previous studies showed that the expression of trout AE1 in Xenopus laevis oocytes induces band 3 anion exchange activity and organic osmolyte channel activity. However, an endogenous swelling-activated anion channel, IClswell, is present in Xenopus oocyte membranes. Therefore, it is not yet known whether a new organic osmolyte channel is formed or whether the endogenous channel, IClswell, is activated when trout AE1 is expressed in the oocytes. The purpose of this study was to determine whether the expression of trout AE1 in Xenopus oocytes leads to the formation and membrane insertion of a new organic osmolyte channel or activates IClswell. To differentiate between the two possibilities, we compared the time courses, pH profiles and inhibitor sensitivities of both trout AE1 and IClswell. The results of taurine-uptake experiments show that the time courses and pH levels for optimum expression of trout AE1 and IClswell differ significantly. The inhibitor sensitivities of the organic osmolyte channel mediated by trout AE1 and IClswell are also significantly different, strongly suggesting that the expression of trout AE1 in Xenopus oocytes does not activate IClswell, but rather forms a new organic osmolyte channel.