Characterization of Regulatory Volume Behavior by Fluorescence Quenching in Human Corneal Epithelial Cells

An in-depth understanding of the mechanisms underlying regulatory volume behavior in corneal epithelial cells has been in part hampered by the lack of adequate methodology for characterizing this phenomenon. Accordingly, we developed a novel approach to characterize time-dependent changes in relative cell volume induced by anisosmotic challenges in calcein-loaded SV40-immortalized human corneal epithelial (HCE) cells with a fluorescence microplate analyzer. During a hypertonic challenge, cells shrank rapidly, followed by a temperature-dependent regulatory volume increase (RVI), τ_c = 19 min. In contrast, a hypotonic challenge induced a rapid (τ_c = 2.5 min) regulatory volume decrease (RVD). Temperature decline from 37 to 24°C reduced RVI by 59%, but did not affect RVD. Bumetanide (50 μM), ouabain (1 mM), DIDS (1 mM), EIPA (100 μM), or Na⁺-free solution reduced the RVI by 60, 61, 39, 32, and 69%, respectively. K⁺, Cl⁻ channel and K⁺-Cl⁻ cotransporter (KCC) inhibition obtained with either 4-AP (1 mM), DIDS (1 mM), DIOA (100 μM), high K⁺ (20 mM) or Cl⁻-free solution, suppressed RVD by 42, 47, 34, 52 and 58%, respectively. KCC activity also affects steady-state cell volume, since its inhibition or stimulation induced relative volume alterations under isotonic conditions. Taken together, K⁺ and Cl⁻ channels in parallel with KCC activity are important mediators of RVD, whereas RVI is temperature-dependent and is essentially mediated by the Na⁺-K⁺-2Cl⁻ cotransporter (Na⁺-K⁺-2Cl⁻) and the Na⁺-K⁺ pump. Inhibition of K⁺ and Cl⁻ channels and KCC but not Na⁺-K⁺-2Cl⁻ affect steady-state cell volume under isotonic conditions. This is the first report that KCC activity is required for HCE cell volume regulation and maintenance of steady-state cell volume.     

Adaptation by Corneal Epithelial Cells to Chronic Hypertonic Stress Depends on Upregulation of Na:K:2Cl Cotransporter Gene and Protein Expression and Ion Transport Activity

We examined the ability of SV40-immortalized human and rabbit corneal epithelial cells (HCEC and RCEC, respectively) to adapt to chronic hypertonic stress. Under isotonic conditions, in the presence of 50 μm bumetanide, proliferation measured as ³H-thymidine incorporation declined in RCEC and HCEC by 8 and 35%, respectively. After 48 hr exposure to 375 mOsm medium, RCEC proliferation fell by 19% whereas in HCEC it declined by 45%. Light scattering behavior demonstrated that both cell lines mediate nearly complete regulatory volume increase (RVI) responses to an acute hypertonic (375 mOsm) challenge, which in part depend on bumetanide-sensitive Na-K-2Cl cotransporter (NKCC) activity. Following exposing RCEC for 48 hr to 375 mOsm medium, their RVI response to an acute hypertonic challenge was inhibited by 17%. However, in HCEC this response declined by 68%. During exposure to 375 mOsm medium for up to 24 hr, only RCEC upregulated NKCC gene and protein expression as well as bumetanide-sensitive ⁸⁶Rb influx. These increases are consistent with the smaller declines in RVI and proliferation capacity occurring during this period in RCEC than in HCEC. Therefore, adaptation by RCEC to chronic hypertonic stress is dependent on stimulation of NKCC gene and protein expression and functional activity. On the other hand, under isotonic conditions, HCEC RVI and proliferation are more dependent on NKCC activity than they are in RCEC. Received: 7 March 2000/Revised: 18 May 2000     

Fluoxetine Inhibits K+ Transport Pathways (K+ Efflux, Na+-K+-2Cl− Cotransport, and Na+ Pump) Underlying Volume Regulation in Corneal Endothelial Cells

We have studied regulatory volume responses of cultured bovine corneal endothelial cells (CBCEC) using light scattering. We assessed the contributions of fluoxetine (Prozac) and bumetanide-sensitive membrane ion transport pathways to such responses by determining K⁺ efflux and influx. Cells swollen by a 20% hypo-osmotic solution underwent a regulatory volume decrease (RVD) response, which after 6 min restored relative cell volume by 98%. Fluoxetine inhibited RVD recovery; 20 μm by 26%, and 50 μm totally. Fluoxetine had a triphasic effect on K⁺ efflux; from 20 to 100 μm it inhibited efflux 2-fold, whereas at higher concentrations the efflux first increased to 1.5-fold above the control value, and then decreased again. Cells shrunk by a 20% hyperosmotic solution underwent a regulatory volume increase (RVI) which also after 6 min restored the cell volume by 99%. Fluoxetine inhibited RVI; 20 μm by 25%, and 50 μm completely. Bumetanide (1 μm) inhibited RVI by 43%. In a Cl⁻-free medium, fluoxetine (50–500 μm) progressively inhibited bumetanide-insensitive K⁺ influx. The inhibitions of RVI and K⁺ influx induced by fluoxetine 20 to 50 μm were similar to those induced by 1 μm bumetanide and by Cl⁻-free medium. A computer simulation suggests that fluoxetine can interact with the selectivity filter of K⁺ channels. The data suggest that CBCEC can mediate RVD and RVI in part through increases in K⁺ efflux and Na-K-2Cl cotransport (NKCC) activity. Interestingly, the data also suggest that fluoxetine at 20 to 50 μm inhibits NKCC, and at 100–1000 μm inhibits the Na⁺ pump. One possible explanation for these findings is that fluoxetine could interact with K⁺-selective sites in K⁺ channels, the NKC cotransporter and the Na⁺ pump.     

Effects of alterations in endothelial cell volume on transendothelial albumin permeability

We examined the effects of alterations in endothelial cell volume on transendothelial albumin permeability. Studies were done using a confluent monolayer of bovine pulmonary artery endothelial cells grown on gelatinized microporous filters. When endothelial cells were exposed to media made hypertonic with 200 mM mannitol, the intracellular volume (measured with 14C-urea) decreased twofold and remained decreased over a 30-minute time-span, thus showing no significant regulatory volume increase (RVI) within this time period. When endothelial cells were exposed to hypotonic media, intracellular volume rapidly doubled within 2 minutes, and then decreased to baseline values within 10 minutes in spite of the sustained hypotonic environment, a process known as regulatory volume decrease (RVD). We also measured the transendothelial flux of 125I-albumin with the cells exposed to the same osmotic changes. We observed that only under hypertonic conditions was there a significant change in the 125I-albumin permeability. These results indicate that the pulmonary artery endothelial cells in culture alter their cell volume when exposed to variations in the osmotic environment, and also show RVD in response to hypotonic conditions but no RVI within 40 minutes after exposure to hypertonic conditions. The transendothelial albumin permeability did not change under hypotonic conditions but increased under hypertonic conditions. Thus, endothelial cells shrinkage may be an important mechanism of increased endothelial macromolecule permeability. These volume changes may occur in endothelial cells in situ and have a role in inducing alterations in the transendothelial permeability to proteins.     

Volume Regulation of Nerve Terminals

Pinched-off presynaptic nerve terminals (synaptosomes) possess significant regulatory volume increase (RVI) and regulatory volume decrease (RVD) capabilities. Following a swelling induced by a hypotonic challenge, the synaptosomes regulate their volume and adjust it, in 2 min, to within 5% of its initial value (RVD) at an initial rate of -0.77 +/- 0.10%/s (mean +/- SEM). Following a shrinking induced by a hypertonic challenge, the synaptosomes also regulate their volume at an initial rate of 0.18 +/- 0.02%/s (RVI), resulting in a new steady state, reached within 5-10 min, with a synaptosomal volume below the original volume. The omission of Na+ or K+ ions from the extrasynaptosomal medium reduces the initial rate of RVI by 72.5 and 66.5%, respectively. The "loop diuretics" bumetanide and furosemide significantly inhibited the RVI of the synaptosomes. In contrast, ouabain, amiloride, or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid did not have any significant effect on RVI parameters. Furthermore, bumetanide-sensitive 86Rb uptake by rat brain synaptosomes was stimulated threefold by a hypertonic perturbation of 30%. Thus we conclude that the RVI of synaptosomes is mainly due to a stimulation of the Na+, K+, Cl- co-transport system induced by the synaptosomal shrinking following the hypertonic challenge.     

Role of the F-actin cytoskeleton in the RVD and RVI processes in Ehrlich ascites tumor cells.

The role of the F-actin cytoskeleton in cell volume regulation was studied in Ehrlich ascites tumor cells, using a quantitative rhodamine-phalloidin assay, confocal laser scanning microscopy, and electronic cell sizing. A hypotonic challenge (160 mOsm) was associated with a decrease in cellular F-actin content at 1 and 3 min and a hypertonic challenge (600 mOsm) with an increase in cellular F-actin content at 1, 3, and 5 min, respectively, compared to isotonic (310 mOsm) control cells. Confocal visualization of F-actin in fixed, intact Ehrlich cells demonstrated that osmotic challenges mainly affect the F-actin in the cortical region of the cells, with no visible changes in F-actin in other cell regions. The possible role of the F-actin cytoskeleton in RVD was studied using 0. 5 microM cytochalasin B (CB), cytochalasin D (CD), or chaetoglobosin C (ChtC), a cytochalasin analog with little or no affinity for F-actin. Recovery of cell volume after hypotonic swelling was slower in cells pretreated for 3 min with 0.5 microM CB, but not in CD- and ChtC-treated cells, compared to osmotically swollen control cells. Moreover, the maximal cell volume after swelling was decreased in CB-treated, but not in CD- or Chtc-treated cells. Following a hypertonic challenge imposed using the RVD/RVI protocol, recovery from cell shrinkage was slower in CB-treated, but not in CD- or Chtc-treated cells, whereas the minimal cell volume after shrinkage was unaltered by either of these treatments. It is concluded that osmotic cell swelling and shrinkage elicit a decrease and an increase in the F-actin content in Ehrlich cells, respectively. The RVD and RVI processes are inhibited by 0.5 microM CB, but not by 0.5 microM CD, which is more specific for actin.     

Osmotic stress resistance imparts acquired anti-apoptotic mechanisms in lymphocytes

Apoptosis is a stochastic, physiological form of cell death that is characterized by unique morphological and biochemical properties. A defining feature of apoptosis in all cells is the apoptotic volume decrease or AVD, which has been considered a passive component of the cell death process. Most cells have inherent volume regulatory increase (RVI) mechanisms to contest an imposed loss in cell size, however T-cells are unique in that they do not have a RVI response. We utilized this property to explore potential regulatory roles of a RVI response in apoptosis. Exposure of immature T-cells to hyperosmotic stress resulted in a rapid, synchronous, and caspase-dependent apoptosis. Multiple rounds of osmotic stress followed by recovery of cells in normal media resulted in the development of a population of cells that were resistant to osmotic stress induced apoptosis. These cells were also resistant to other apoptotic stimuli that activate via the intrinsic cell death pathway, while remaining sensitive to extrinsic apoptotic stimuli. Interestingly, these osmotic stress resistant cells showed no increase in anti-apoptotic proteins, and released cytochrome c from their mitochondria following exposure to intrinsic apoptotic stimuli. The osmotic stress resistant cells developed a RVI response, and inhibition of the RVI restored sensitivity to apoptotic agents. Analysis of apoptotic signaling pathways showed a sustained increase in phospho-AKT, whose inhibition also prevented an RVI response resulting in apoptosis. These results define a critical role of volume regulation mechanisms in apoptotic resistance.     

Effect of anisotonic media on volume,ion and amino-acid content and membrane potential of kidney cells (MDCK) in culture

Summary Effects of anisotonic media on a monolayer of confluent kidney cells in culture (MDCK) were studied by measuring: cell thickness and cross-section changes, ion and amino-acid content and membrane potential. The volume was also determined with cells in suspension. When cells in a monolayer were incubated in hypotonic media, the lateral and the apical membranes were rapidly stretched. Afterwards the lateral membranes returned to their initial state while the apical membranes remained stretched. This partial regulatory volume decrease (RVD) was verified with cells in suspension. RVD was accompanied by a loss of K⁺, Cl^– and amino acids, but there was no loss of inorganic phosphate. Also a transient hyperpolarization of the membrane potential was observed, suggesting an increase of the K⁺ conductance during RVD. Upon restoring the isotonic medium, a regulatory volume increase (RVI) was observed accompanied by a rapid Na⁺ and Cl^– increase and followed by a slow recovery of the initial K⁺ and Na⁺ content while amino acids remained at their reduced content. A transient depolarization of the membrane potential was measured during this RVI, suggesting that Na⁺ and Cl^– conductance could have increased. In hypertonic media, only a small and slow RVI was observed accompanied by an increase in K⁺ and Cl^– content but without any change of membrane potential. Quinine partly inhibited RVD in hypotonic media with cells in a monolayer while inhibiting RVD completely with cells in suspension. Incubation during four hours in a Ca²⁺ free medium had no effect on RVD. Furosemide and amiloride had no effect on RVD and RVI. Volume regulation, RVD or RVI, was not affected by replacing Cl^– by nitrate. When cells in a monolayer were incubated in a hypotonic K₂SO₄ medium, no RVD was observed. From these results, it seems that MDCK cells in a confluent monolayer regulate their volume by activating specific ion and amino-acid transport pathways. Selective K⁺ and Na⁺ conductances are activated during RVD and RVI, while the activated anion conductance has a low selectivity. The controlling mechanism might not be the free intracellular Ca²⁺ concentration.     

Regulatory and necrotic volume increase in boar spermatozoa

Spermatozoa of many species initially respond to hypotonicity as perfect osmometers. Thereafter they undergo a regulatory process resulting in a decrease in cell volume, similar to that reported for somatic cells. Regulatory volume increase (RVI), a complementary process which is assumed to occur following initial shrinkage of sperm volume after exposure to a hypertonic medium, has not yet been described in detail for spermatozoa. In this study, we investigated whether spermatozoa are able to regulate their volume after hypertonic stress and whether this ability is maintained in preserved sperm. Cell volume changes were recorded using electronic cell sizing. Sperm response to the ion channels blockers quinidine, tamoxifen, and dydeoxyforskolin, and to protein kinase/phosphatase inhibitors lavendustin, staurosporine, and vanadate was studied to investigate possible mechanisms of RVI. Annexin V staining was used in combination with propidium iodide to determine whether hypertonic stress may induce apoptosis. Overall protein tyrosine phosphorylation under hypertonic conditions was measured via flow cytometry using antiphosphotyrosine antibody. Spermatozoa exposed to hypertonic stress initially responded with an abundant subpopulation according to the perfect osmometer model and recovered their volume from this shrinkage after 20 min. RVI was inhibited by quinidine and tamoxifen, which indicates the involvement of the important cellular ions sodium and chloride in this process. Volume regulatory ability was essentially maintained during storage of liquid semen. However, the response of the sperm population was heterogeneous. A second population raised, containing spermatozoa with larger volumes, which demonstrated irregularities in the volume response with respect to osmotic challenge, ion channel blockers, and storage. Under hypertonic conditions, both protein kinase inhibitors (PKI) led to increased isotonic volumes and to elevated initial relative volumes and subsequent volume decrease. RVI was inhibited by the vanadate. Hypertonic stress did not result in an increase in early apoptotic cells, but produced a shift toward late necrotic cells. Substitution of sodium and chloride by choline and sulfate resulted in decreased isotonic volume of sperm treated with lavendustin. Tyrosine phosphorylation levels were reduced after 20 min under hypertonic conditions. It was concluded that RVI is regulated via a protein tyrosine kinase-dependent pathway, and that dephosphorylation occurs when volume regulation is required. The necrotic volume increase (NVI) is associated with the accumulation of sodium and chloride following uncontrolled opening of the channels. The ability to regulate volume after exposure to hypertonic conditions is important for sperm functionality and can have practical applications in spermatological diagnostics and cryopreservation.     

Calcium and cytoskeleton signaling during cell volume regulation in isolated nematocytes of Aiptasia mutabilis (Cnidaria: Anthozoa)

Cell volume regulation has not been completely clarified in Coelenterates. The present investigation focuses on cell volume regulation under anisosmotic conditions, both hyposmotic and hypertonic, and on the underlying signals in nematocytes isolated from the Coelenterate Aiptasia mutabilis living in sea water. Nematocytes, once isolated from acontia, that were submitted to either hyposmotic (35%) and hypertonic shock (45%) show RVD and RVI capabilities, respectively. In order to ascertain the role of Ca2+ in triggering such regulatory mechanisms and the possible involvement of cytoskeleton components, tests were performed by employing either Ca2+ free conditions, Gd3+ as Ca2+ channel blockers, TFP as calmodulin inhibitor, colchicine as microtubule inhibitor and cytochalasin B as microfilament polymerization inhibitor. Results show that isolated nematocytes of A. mutabilis can regulate their volume upon both hyposmotic and hypertonic challenge. Ca2+ both from external medium and from internal stores is needed to perform RVD mechanisms, whereas, intracellular Ca2+ seems to be mainly involved in RVI. Moreover cytoskeletal components may play an important role since a significant RVD and RVI inhibition was observed in treated cells. On the basis of our observations further studies are warranted to further verify the role of signals, including phosphatases and phosphorylases, in cell volume regulation of primitive eukaryotic cells.     

Dysfunction of regulatory volume increase is a key component of apoptosis

Sustained cell shrinkage is a major hallmark of apoptotic cell death. In apoptotic cells, whole cell volume reduction, called apoptotic volume decrease (AVD), proceeds until fragmentation of cells. Under non-apoptotic conditions, human epithelial HeLa cells exhibited a slow regulatory volume increase (RVI) after osmotic shrinkage induced by exposure to hypertonic solution. When AVD was induced by treatment with a Fas ligand, TNF-alpha or staurosporine, however, it was found that HeLa cells failed to undergo RVI. When RVI was inhibited by combined application of Na+/H+ exchanger (NHE) and anion exchanger blockers, hypertonic stress induced prolonged shrinkage followed by caspase-3 activation in HeLa cells. Hypertonicity also induced apoptosis in NHE1-deficient PS120 fibroblasts, which lack the RVI response. When RVI was restored by transfection of these cells with NHE1, hypertonicity-induced apoptosis was completely prevented. Thus, it is concluded that RVI dysfunction is indispensable for the persistence of AVD and induction of apoptosis.     

Apoptosis, cell volume regulation and volume-regulatory chloride channels

Apoptosis occurs in response to various stimuli under physiological and pathological circumstances. A major hallmark of the programmed cell death is normotonic shrinkage of cells. Induction of the apoptotic volume decrease (AVD) was found to precede cytochrome c release, caspase-3 activation and DNA laddering. A broad-spectrum caspase inhibitor blocked these biochemical apoptotic events but failed to block the AVD. The normotonic AVD induction was coupled to facilitation of the regulatory volume decrease (RVD), which is attained by parallel operation of Cl- and K+ channels, under hypotonic conditions. Both the AVD induction and RVD facilitation were prevented by application of a blocker of volume-regulatory Cl- or K+ channels. Furthermore, apoptotic cell death was rescued by channel blocker-induced prevention of AVD. Thus, it is concluded that the AVD is produced under normotonic conditions by a mechanism similar, though without preceding swelling, to RVD and represents an early prerequisite to apoptotic events leading to cell death. It was previously reported that hypertonic stress triggers apoptosis in cell types that lack the regulatory volume increase (RVI) mechanism. Taken together, it is suggested that 'disordered' or altered cell volume regulation is associated with apoptosis.     

Volume changes of an endothelial cell monolayer on exposure to anisotonic media

The osmotic process plays an important role in controlling the distribution of water across cell membranes and thus the cell volume. A system was designed to detect the volume changes of an endothelial cell monolayer when cells were exposed to media with altered osmolalities. Electrodes housed in a flow chamber measured the resistance of ionic media flowing over a cultured cell layer. Assuming the cell membrane acts as an electrical insulator, volume changes of the cell layer can be calculated from the corresponding changes in chamber resistance. The media used in the experiments had osmolalities in the range 120-630 mmol/kg. When cells were exposed to hypertonic media, there was rapid shrinkage with an approximate 30% reduction in total cell volume for a twofold increase in osmolality. On exposure to hypotonic media, the cells initially swelled with an approximate 20% volume increase for a decrease in osmolality by half. With sustained exposure to low osmolality media, there was a gradual and partial return of cell volume towards isotonic values that started 10 minutes after and was complete within 30 minutes of the osmolality alteration. This finding suggests regulatory volume decrease (RVD); however, no regulatory volume increase (RVI) was observed with the continued exposure to hypertonic media over 45 minutes.     

Na+, K+, Cl− cotransport and its regulation in Ehrlich ascites Tumor cells. Ca2+/Calmodulin and protein kinase C dependent pathways

Summary Net Cl^– uptake as well as unidirectional³⁶Cl influx during regulatory volume increase (RVI) require external K⁺. Half-maximal rate of bumetanide-sensitive³⁶Cl uptake is attained at about 3.3mm external K⁺. The bumetanide-sensitive K⁺ influx found during RVI is strongly dependent on both Na⁺ and Cl^–. The bumetanide-sensitive unidirectional Na⁺ influx during RVI is dependent on K⁺ as well as on Cl^–. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na⁺, K⁺ and Cl^–. In the presence of ouabain and Ba⁺ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na⁺, 0.8 K⁺, 2.0 Cl^– or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K⁺ and Cl^– flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na⁺, K⁺, 2Cl^– cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K⁺ influx is activated. During hypotonic conditions a small bumetanide-sensitive K⁺ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca²⁺ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K⁺ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na⁺, K⁺, Cl^– cotransport involves both Ca²⁺/calmodulin and protein kinase C.     

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     

Regulatory volume decrease in the presence of HCO3- by single osteosarcoma cells UMR-106-01.

The technique for the simultaneous recording of cell volume changes and pHi in single cells was used to study the role of HCO3- in regulatory volume decrease (RVD) by the osteosarcoma cells UMR-106-01. In the presence of HCO3-, steady state pHi is regulated by Na+/H+ exchange, Na+ (HCO3-)3 cotransport and Na(+)-independent Cl-/HCO3- exchange. Following swelling in hypotonic medium, pHi was reduced from 7.16 +/- 0.02 to 6.48 +/- 0.02 within 3.4 +/- 0.28 min. During this period of time, the cells performed RVD until cell volume was decreased by 31 +/- 5% beyond that of control cells (RVD overshoot). Subsequently, while the cells were still in hypotonic medium, pHi slowly increased from 6.48 +/- 0.02 to 6.75 +/- 0.02. This increase in pHi coincided with an increase in cell volume back to normal (recovery from RVD overshoot or hypotonic regulatory volume increase (RVI)). The same profound changes in cell volume and pHi after cell swelling were observed in the complete absence of Cl- or Na+, providing HCO3- was present. On the other hand, depolarizing the cells by increasing external K+ or by inhibition of K+ channels with quinidine, Ba2+ or tetraethylammonium prevented the changes in pHi and RVD. These findings suggest that in the presence of HCO3-, RVD in UMR-106-01 cells is largely mediated by the conductive efflux of K+ and HCO3-. Removal of external Na+ but not Cl- prevented the hypotonic RVI that occurred after the overshoot in RVD. Amiloride had no effect, whereas pretreatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) strongly inhibited hypotonic RVI. Thus, hypotonic RVI is mediated by a Na+(out)-dependent, Cl(-)-independent and DIDS-inhibitable mechanism, which is indicative of a Na+(HCO3-)3 cotransporter. This is the first evidence for the involvement of this transporter in cell volume regulation. The present results also stress the power of the new technique used in delineating complicated cell volume regulatory mechanisms in attached single cells.     

An In Vitro Model of Skeletal Muscle Volume Regulation

Introduction

Hypertonic media causes cells to shrink due to water loss through aquaporin channels. After acute shrinkage, cells either regulate their volume or, alternatively, undergo a number of metabolic changes which ultimately lead to cell death. In many cell types, hypertonic shrinkage is followed by apoptosis. Due to the complex 3D morphology of skeletal muscle and the difficulty in obtaining isolated human tissue, we have begun skeletal muscle volume regulation studies using the human skeletal muscle cell line TE671RD. In this study we investigated whether hypertonic challenge of the human skeletal muscle cell line TE671RD triggered cell death or evoked a cell volume recovery response.

Methods

The cellular volume of TE671RD cells was calculated from the 2D surface area. Cell death was assessed by both the trypan blue live/dead assay and the TUNEL assay.

Results

Medium osmolality was increased by addition of up to 200mM sucrose. Addition of 200mM sucrose resulted in mean cell shrinkage of 44±1% after 30mins. At later time points (2 and 4 hrs) two separate cell subpopulations with differing mean cell volume became apparent. The first subpopulation (15±2% of the total cell number) continued to shrink whereas the second subpopulation had an increased cell volume. Cell death was observed in a small proportion of cells (approximately 6-8%).

Conclusion

We have established that a substantial proportion of TE671RD cells respond to hypertonic challenge with RVI, but that these cells are resistant to hypertonicity triggered cell death.     

Mechanosensitive Calcium Entry and Mobilization in Renal A6 Cells

Using spectrofluorescence imaging of fura-2 loaded renal A6 cells, we have investigated the generation of the cytosolic Ca²⁺ signal in response to osmotic shock and localized membrane stretch. Upon hypotonic exposure, the cells began to swell prior to a transient increase in [Ca²⁺] _i and the cells remained swollen after [Ca²⁺] _i had returned towards basal levels. Exposure to 2/3rd strength Ringer produced a cell volume increase within 3 min, followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also produced a transient increase in [Ca²⁺] after a delay of 22 sec. Both the RVD and [Ca²⁺] _i response to hypotonicity were inhibited in a Ca²⁺-free bathing solution and by gadolinium (10 μm), an inhibitor of stretch-activated channels. Stretching the membrane by application of subatmospheric pressure (-2 kPa) inside a cell-attached patch-pipette induced a similar global increase in [Ca²⁺] _i as occurred after hypotonic shock. A stretch-sensitive [Ca²⁺] _i increase was also observed in a Ca²⁺-free bathing solution, provided the patch-pipette contained Ca²⁺. The mechanosensitive [Ca²⁺] _i response was by gadolinium (10 μm) or Ca²⁺-free pipette solutions, even when Ca²⁺ (2 mm) was present in the bath. Long-term (>10 min) pretreatment of the cells with thapsigargin inhibited the [Ca²⁺] _i response to hypotonicity. These results provide evidence that cell swelling or mechanical stimulation can activate a powerful amplification system linked to intracellular Ca²⁺ release mechanisms. Received: 3 August 1998/Revised: 19 November 1998     

Volume-sensitive tyrosine kinases regulate liver cell volume through effects on vesicular trafficking and membrane Na+ permeability

In liver cells, the influx of Na+ mediated by nonselective cation (NSC) channels in the plasma membrane contributes importantly to regulation of cell volume. Under basal conditions, channels are closed; but both physiologic (e.g. insulin) and pathologic (e.g. oxidative stress) stimuli that are known to stimulate tyrosine kinases are associated with large increases in membrane Na+ permeability to approximately 80 pA/pF or more. Consequently, the purpose of these studies was to evaluate whether volume-sensitive tyrosine kinases mediate cell volume increases through effects on the activity or distribution of NSC channel proteins. In HTC hepatoma cells, decreases in cell volume evoked by hypertonic exposure increased total cellular tyrosine kinase activity approximately 20-fold. Moreover, hypertonic exposure (320-400 mosM) was followed after a delay by NSC channel activation and partial recovery of cell volume toward basal values (regulatory volume increase (RVI)). The tyrosine kinase inhibitors genistein and erbstatin prevented both NSC channel activation and RVI. Similarly, hypertonic exposure resulted in an increase in p60(c-src) activity, and intracellular dialysis with recombinant p60(c-src) led to activation of NSC currents in the absence of an osmolar gradient. Utilizing FM1-43 fluorescence, exposure to hypertonic media caused a rapid increase in the rate of exocytosis of approximately 40% (p < 0.01), and genistein inhibited both exocytosis and channel activation. These findings indicate that volume-sensitive increases in p60(c-src) and/or related tyrosine kinases play a key role in the regulation of membrane Na+ permeability, suggesting that increases in the NSC conductance may be mediated in part through rapid recruitment of a distinct pool of channel-containing vesicles.