The cytoskeleton and cell volume regulation

Although the precise mechanisms have yet to be elucidated, early events in osmotic signal transduction may involve the clustering of cell surface receptors, initiating downstream signaling events such as assembly of focal adhesion complexes, and activation of, e.g. Rho family GTPases, phospholipases, lipid kinases, and tyrosine- and serine/threonine protein kinases. In the present paper, we briefly review recent evidence regarding the possible relation between such signaling events, the F-actin cytoskeleton, and volume-regulatory membrane transporters, focusing primarily on our own work in Ehrlich ascites tumer cells (EATC). In EATC, cell shrinkage is associated with an increase, and cell swelling with a decrease in F-actin content, respectively. The role of the F-actin cytoskeleton in cell volume regulation in various cell types has largely been investigated using cytochalasins to disrupt F-actin and highly varying effects have been reported. Findings in EATC show that the effect of cytochalasin treatment cannot always be assumed to be F-actin depolymerization, and that, moreover, there is no well-defined correlation between effects of cytochalasins on F-actin content and their effects on F-actin organization and cell morphology. At a concentration verified to depolymerize F-actin, cytochalasin B (CB), but not cytochalasin D (CD), inhibited the regulatory volume decrease (RVD) and regulatory volume increase (RVI) processes in EATC. This suggests that the effect of CB is related to an effect other than F-actin depolymerization, possibly its F-actin severing activity.     

Importance of cytoskeletal elements in volume regulatory responses of trout hepatocytes

The role of cytoskeletal elements in volume regulation was studied in trout hepatocytes by investigating changes in F-actin distribution during anisotonic exposure and assessing the impact of cytoskeleton disruption on volume regulatory responses. Hypotonic challenge caused a significant decrease in the ratio of cortical to cytoplasmic F-actin, whereas this ratio was unaffected in hypertonic saline. Disruption of microfilaments with cytochalasin B (CB) or cytochalasin D significantly slowed volume recovery following hypo- and hypertonic exposure in both attached and suspended cells. The decrease of net proton release and the intracellular acidification elicited by hypotonicity were unaltered by CB, whereas the increase of proton release in hypertonic saline was dramatically reduced. Because amiloride almost completely blocked the hypertonic increase of proton release and cytoskeleton disruption diminished the associated increase of intracellular pH (pH(i)), we suggest that F-actin disruption affected Na(+)/H(+) exchanger activity. In line with this, pH(i) recovery after an ammonium prepulse was significantly inhibited in CB-treated cells. The increase of cytosolic Na(+) under hypertonic conditions was not diminished but, rather, enhanced by F-actin disruption, presumably due to inhibited Na(+)-K(+)-ATPase activity and stimulated Na(+) channel activity. The elevation of cytosolic Ca(2+) in hypertonic medium was significantly reduced by CB. Altogether, our results indicate that the F-actin network is of crucial importance in the cellular responses to anisotonic conditions, possibly via interaction with the activity of ion transporters and with signalling cascades responsible for their activation. Disruption of microtubules with colchicine had no effect on any of the parameters investigated.     

Volume regulation in leukocytes: Requirement for an intact cytoskeleton

Animal cells regulate their volume by controlling the flux of ions across their plasma membrane. Recent evidence suggests that ion channels and pumps are physically associated with, and may be regulated by components of the cytoskeleton. To elucidate the role of elements of the cytoskeleton in volume regulation, we studied the effects of cytoskeletal disrupting agents on regulatory volume decrease (RVD) in three different leukocyte types: Jurkat lymphoma cells, HL-60 cells, and human peripheral blood neutrophils. Cell volume was measured in two ways: (i) electronically with a Coulter counter and (ii) by forward light scattering in a flow cytometer. Exposure of all leukocyte types to hypotonic medium (200 mOsm) resulted in an immediate increase in cell volume followed by a regulatory decrease to baseline by 20 min. In the presence of the microtubule disrupting agents, colchicine and nocodazole, RVD was totally inhibited which corresponded to loss of microtubules as determined by immunofluorescence. Similarly, RVD was inhibited in Jurkat cells incubated with the actin binding agents, cytochalasin B (CB) or D (CD). In contrast, in HL-60 cells and human neutrophils, RVD was unaffected by treatment with either CB or CD. While cytochalasins are generally thought of as microfilament disrupting agents, their primary action is to prevent F-actin polymerization. The extent of ensuing microfilament disruption depends in part on the rate of filament turnover. In an attempt to understand the differential effects of the cytochalasins on RVD, the F-actin content of the different cells was determined by NBD-phallacidin staining and flow cytometry. Pretreatment with CB or CD resulted in profound actin disassembly in Jurkat cells (relative fluorescence index RFI: 1.0 control vs. 0.21 ± 0.01 for CB and 0.48 ± 0.02 for CD). However, the cytochalasins did not induce net disassembly in either HL-60 cells or human neutrophils. To study the effects of an increase in F-actin on volume regulation, neutrophils were treated with the chemoattractant f-Met-Leu-Phe or with an antibody (Ab) to β2 integrins followed by a cross-linking secondary Ab. Despite an increase in F-actin in both circumstances, RVD remained intact. Taken together, these results suggest that both microtubules and microfilaments are important in volume regulation. © 1995 Wiley-Liss, Inc.     

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.     

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.     

KCNQ channels are involved in the regulatory volume decrease response in primary neonatal rat cardiomyocytes

Cardiomyocytes may experience significant cell swelling during ischemia and reperfusion. Such changes in cardiomyocyte volume have been shown to affect the electrical properties of the heart, possibly leading to cardiac arrhythmia. In the present study the regulatory volume decrease (RVD) response of neonatal rat cardiomyocytes was studied in intact single cells attached to coverslips, i.e. with an intact cytoskeleton. The potential contribution of KCNQ (Kv7) channels to the RVD response and the possible involvement of the F-actin cytoskeleton were investigated. The rate of RVD was significantly inhibited in the presence of the KCNQ channel blocker XE-991 (10 and 100 microM). Electrophysiological experiments confirmed the presence of an XE-991 sensitive current and Western blotting analysis revealed that KCNQ1 channel protein was present in the neonatal rat cardiomyocytes. Hypoosmotic cell swelling changes the structure of the F-actin cytoskeleton, leading to a more rounded cell shape, less pronounced F-actin stress fibers and patches of actin. In the presence of cytochalasin D (1 microM), a potent inhibitor of actin polymerization, the RVD response was strongly reduced, confirming a possible role for an intact F-actin cytoskeleton in linking cell swelling to activation of ion transport in neonatal rat cardiomyocytes.     

Regulatory volume decrease (RVD) by isolated and in situ bovine articular chondrocytes

Articular chondrocytes in vivo are exposed to a changing osmotic environment under both physiological (static load) and pathological (osteoarthritis) conditions. Such changes to matrix hydration could alter cell volume in situ and influence matrix metabolism. However the ability of chondrocytes to regulate their volume in the face of osmotic perturbations have not been studied in detail. We have investigated the regulatory volume decrease (RVD) capacity of bovine articular chondrocytes within, and isolated from the matrix, before and following acute hypotonic challenge. Cell volumes were determined by visualising fluorescently-labelled chondrocytes using confocal laser scanning microscopy (CLSM) at 21 degrees C. Chondrocytes in situ were grouped into superficial (SZ), mid (MZ), and deep zones (DZ). When exposed to 180mOsm or 250mOsm hypotonic challenge, cells in situ swelled rapidly (within approximately 90 sec). Chondrocytes then exhibited rapid RVD (t(1/2) approximately 8 min), with cells from all zones returning to approximately 3% of their initial volume after 20 min. There was no significant difference in the rates of RVD between chondrocytes in the three zones. Similarly, no difference in the rate of RVD was observed for an osmotic shock from 280 to 250 or 180mOsm. Chondrocytes isolated from the matrix into medium of 380mOsm and then exposed to 280mOsm showed an identical RVD response to that of in situ cells. The RVD response of in situ cells was inhibited by REV 5901. The results suggested that the signalling pathways involved in RVD remained intact after chondrocyte isolation from cartilage and thus it was likely that there was no role for cell-matrix interactions in mediating RVD.     

Lack of Threshold for Anisotonic Cell Volume Regulation

Most cells possess mechanisms that are able to detect cellular volume shifts and to signal the initiation of appropriate volume regulatory responses. However, the identity and characteristics of the detecting mechanism remain obscure. In this study, we explored the influence of hypertonic and hypotonic challenges of varying magnitude on the characteristics of the ensuing regulatory volume increase (RVI) and regulatory volume decrease (RVD) of cultured bovine corneal endothelial cells (CBCECs). The main question we asked was whether a threshold of stimulation existed that would unleash a regulatory response. CBCECs (passage 1–3) were seeded on rectangular glass coverslips and grown for 1–2 days. We used a procedure based on detection of light scattering to monitor the transient volume changes of such plated cells when subjected to osmotic challenge. The osmometric responses were asymmetric: cells shrank faster than they swelled (by a factor of 3). Complete volume regulatory responses took 10–12 min. Bumetanide (50 μM) resulted in incomplete (50%) RVI. We found no threshold as the cells examined responded to hypertonic and hypotonic stimuli as low as 1%. There was some gradation as stimuli of <4% resulted in incomplete volume regulation. The degree of activation of the volume responses grew as an exponential buildup with the strength of the anisotonic challenge. We discuss how our observations are consistent with volume sensing mechanisms based on both ionic strength and the cytoskeleton.     

Stimulation of regulatory volume decrease (RVD) by isolated bovine articular chondrocytes following F-actin disruption using latrunculin B

Articular chondrocytes are exposed to significant changes in extracellular osmolarity during normal joint activity, which can lead to changes in cell volume and metabolism of the extracellular matrix (ECM). Chondrocytes can respond to cell swelling/shrinking by volume regulatory pathways, but the signalling pathways are poorly understood although a role for the cytoskeleton is frequently implicated. Here, we have investigated the effects of disruption of the chondrocyte F-actin cytoskeleton on the recovery of cell volume by RVD. The cytoskeleton was perturbed using the relatively specific agent latrunculin B (5 microM; 30 min) and loss of F-actin integrity quantified using fluorescent phalloidin-labelling and confocal laser scanning microscopy (CLSM). Imaging of isolated chondrocytes labelled with Fura-2 to measure the fluorescence associated with cell volume changes, showed that the extent of hypo-osmotic swelling was unaffected by latrunculin B treatment. Two categories of the chondrocyte RVD response were observed: 'fast' RVD where at 3 min post-osmotic challenge there was a recovery in cell fluorescence of >or=80%, whereas other cells exhibited 'slow' RVD. Latrunculin B increased the proportion of chondrocytes demonstrating 'fast' RVD by approximately 10 fold and reduced those cells showing 'slow' RVD. An inhibitor of chondrocyte RVD (REV 5901) had no significant effect on the integrity of the cytoskeleton showing that the RVD response could be inhibited independent of the state of the F-actin cytoskeleton. These results suggest that the intact cortical F-actin cytoskeleton has a restraining effect on the RVD response of isolated bovine articular chondrocytes.     

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.     

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.     

Relation between cytoskeleton,hypo-osmotic treatment and volume regulation in Ehrlich ascites tumor cells

Summary Pretreatment with cytochalasin B, which is known to disrupt microfilaments, significantly inhibits regulatory volume decrease (RVD) in Ehrlich ascites tumor cells, suggesting that an intact microfilament network is a prerequisite for a normal RVD response. Colchicine, which is known to disrupt microtubules, has no significant effect on RVD. Ehrlich cells have a cortical three-dimensional, orthogonal F-actin filament network which makes the cells look completely black in light microscopy following immunogold/silver staining using anti-actin antibodies. After addition of cytochalasin B, the stained cells get lighter with black dots localized to the plasma membrane and appearance of multiple knobby protrusions at cell periphery. Also, a significant decrease in the staining of the cells is seen after 15 min of RVD in hypotonic medium. This microfilament reorganization appears during RVD in the presence of external Ca²⁺ or Ca²⁺-ionophore A23187. It is, however, abolished in the absence of extracellular calcium, with or without prior depletion of intracellular Ca²⁺ stores. An effect of increased calcium influx might therefore be considered. The microfilament reorganization during RVD is abolished by the calmodulin antagonists pimozide and trifluoperazine, suggesting the involvement of calmodulin in the process. The microfilament reorganization is also prevented by addition of quinine. This quinine inhibition is overcome by addition of the K⁺ ionophore valinomycin.     

Differences in Ca2+-mediation of hypotonic and Na+-nutrient regulatory volume decrease in suspensions of jejunal enterocytes

We determined differences in the Ca2+ signalling of K+ and Cl- conductances required for Regulatory Volume Decrease (RVD) in jejunal villus enterocytes passively swollen (0.5 or 0.95.isotonic) compared with swelling because of the absorption of D-glucose (D-Glc) or L-Alanine (L-Ala). Cell volume was measured using electronic cell sizing. In nominally Ca(2+)-free medium containing EGTA (100 microM) RVD after 0.5 or 0.95.isotonic challenge was prevented. L-Ala swelling and subsequent RVD was influenced in Ca(2+)-free medium. Villus cells were incubated with 10 microM of the acetomethoxy derivative of 1,2.bis (2-aminophenoxy) ethane N,N,N1,N1 tetracetic acid (BAPTA-AM) and RVD after 0.5.isotonic swelling or L-Ala swelling was prevented. Niguldipine (0.1 microM), nifedipine (5 microM), diltiazem (100 microM), Ni2+, and Co2+ (1 mM) all prevented hypotonic RVD but had no effect on RVD after L-Ala addition. Charybdotoxin (25 nM) a potent inhibitor of Ca(2+)-activated K+ channels, had no effect on hypotonic RVD but prevented RVD of villus cells swollen by D-Glc. We used the calmodulin antagonists, naphthalene sulfonamide derivatives W-7 and W-13, to assess calmodulin activation of K+ and Cl- conductance in these two models. L-Ala swelling and subsequent RVD was not influenced by 25 microM W-7; hypotonic RVD was prevented by 25 microM W-7 or 100 microM W-13. The W-13 inhibition of RVD was by-passed with 0.5 microM gramicidin. Our data show that hypotonic RVD requires extracellular Ca2+ and that the K+ conductance activated is not charybdotoxin sensitive but requires calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory volume decrease in cultured kidney cells (A6): role of amino acids

Volume regulation was studied in A6 epithelia grown on permeable supports by measuring cell thickness (Tc) while simultaneously recording short circuit current (ISC) and transepithelial conductance (Gt). Lowering the tonicity of the basolateral solution (pi b) from 250 or 215 to 140 mOsm/kg elicited a rapid rise in Tc followed by a regulation of the cell volume towards control. This decrease in Tc displays the characteristics of the regulatory volume decrease (RVD). Upon restoring the isoosmotic conditions, Tc decreased rapidly below its control value. A post RVD regulatory volume increase (RVI) as described for other cell types was not observed. The subsequent reduction of the basolateral osmolality increased Tc to the level recorded at the end of the first hypoosmotic pulse. Because cell content was not altered during the isoosmotic period the second hypoosmotic challenge was isotonic with the cell and did therefore not evoke an RVD. However, the cell did not lose its ability to volume regulate since an RVD could be elicited by further reduction of pi b from 140 to 100 mOsm/kg. The possibility of an involvement of amino acids in the RVD was tested. The amount of amino acids in the cell as well as excreted in the bath was determined by amino acid analysis. Millimolar concentrations of threonine, serine, alanine, glutamate, glycine and aspartate were found in the cell extract. The cellular amino acid concentration was 28.8 +/- 0.4 mM. The amounts of glycine, aspartate and glutamate excreted from the cell during the hypotonic treatment were significantly larger than in control conditions. The excretion of these amino acids during hypotonicity decreased the cellular amino acid concentration by 8.4 +/- 0.2 mM. This quantity cannot completely account for the RVD during the first hypotonic challenge. The addition of glycine, aspartate and glutamate to the bathing solutions, although used at concentrations higher than intracellularly, did not reduce RVD. On the contrary, this maneuver increased the amplitude of the RVD following both hypoosmotic pulses. This result suggests a stimulatory role of the amino acids on the processes responsible for the RVD.     

Regulatory volume increase (RVI) by in situ and isolated bovine articular chondrocytes

Metabolism of the matrix by chondrocytes is sensitive to alterations in cell volume that occur, for example, during static loading and osteoarthritis. The ability of chondrocytes to respond to changes in volume could be important, and this study was aimed at testing the hypothesis that chondrocytes can regulate their volume following cell shrinking by regulatory volume increase (RVI). We used single cell fluorescence imaging of in situ bovine articular chondrocytes, cells freshly isolated into 280 or 380 mOsm, or 2-D cultured chondrocytes loaded with calcein or fura-2, to investigate RVI and changes to [Ca2+]i during shrinkage. Following a 42% hyperosmotic challenge, chondrocytes rapidly shrunk, however, only approximately 6% of the in situ or freshly isolated chondrocytes demonstrated RVI. This contrasted with 2D-cultured chondrocytes where approximately 54% of the cells exhibited RVI. The rate of RVI was the same for all preparations. During the 'post-RVD/RVI protocol', approximately 60% of the in situ and freshly isolated chondrocytes demonstrated RVD, but only approximately 5% showed RVI. There was no relationship between [Ca2+]i and RVI either during hyperosmotic challenge, or during RVD suggesting that changes to [Ca2+]i were not required for RVI. Depolymerisation of the actin cytoskeleton by latrunculin, increased RVI by freshly isolated chondrocytes, in a bumetanide-sensitive manner. The results showed that in situ and freshly isolated articular chondrocytes have only limited RVI capacity. However, RVI was stimulated by treating freshly isolated chondrocytes with latrunculin B and following 2D culture of chondrocytes, suggesting that cytoskeletal integrity plays a role in regulating RVI activity which appears to be mediated principally by the Na+ - K+ -2Cl- cotransporter.     

Cell swelling activates cloned Ca(2+)-activated K(+) channels: a role for the F-actin cytoskeleton

Cloned Ca(2+)-activated K(+) channels of intermediate (hIK) or small (rSK3) conductance were expressed in HEK 293 cells, and channel activity was monitored using whole-cell patch clamp. hIK and rSK3 currents already activated by intracellular calcium were further increased by 95% and 125%, respectively, upon exposure of the cells to a 33% decrease in extracellular osmolarity. hIK and rSK3 currents were inhibited by 46% and 32%, respectively, by a 50% increase in extracellular osmolarity. Cell swelling and channel activation were not associated with detectable increases in [Ca(2+)](i), evidenced by population and single-cell measurements. In addition, inhibitors of IK and SK channels significantly reduced the rate of regulatory volume decrease (RVD) in cells expressing these channels. Cell swelling induced a decrease, and cell shrinkage an increase, in net cellular F-actin content. The swelling-induced activation of hIK channels was strongly inhibited by cytochalasin D (CD), in concentrations that caused depolymerization of F-actin filaments, indicating a role for the F-actin cytoskeleton in modulation of hIK by changes in cell volume. In conclusion, hIK and rSK3 channels are activated by cell swelling and inhibited by shrinkage. A role for the F-actin cytoskeleton in the swelling-induced activation of hIK channels is suggested.     

Cell volume regulation in liver

The maintenance of liver cell volume in isotonic extracellular fluid requires the continuous supply of energy: sodium is extruded in exchange for potassium by the sodium/potassium ATPase, conductive potassium efflux creates a cell-negative membrane potential, which expelles chloride through conductive pathways. Thus, the various organic substances accumulated within the cell are osmotically counterbalanced in large part by the large difference of chloride concentration across the cell membrane. Impairment of energy supply leads to dissipation of ion gradients, depolarization and cell swelling. However, even in the presence of ouabain the liver cell can extrude ions by furosemide-sensitive transport in intracellular vesicles and subsequent exocytosis. In isotonic extracellular fluid cell swelling may follow an increase in extracellular potassium concentration, which impairs potassium efflux and depolarizes the cell membrane leading to chloride accumulation. Replacement of extracellular chloride with impermeable anions leads to cell shrinkage. During excessive sodium-coupled entry of amino acids and subsequent stimulation of sodium/potassium-ATPase by increase in intracellular sodium activity, an increase in cell volume is blunted by activation of potassium channels, which maintain cell membrane potential and allow for loss of cellular potassium. Cell swelling induced by exposure of liver cells to hypotonic extracellular fluid is followed by regulatory volume decrease (RVD), cell shrinkage induced by reexposure to isotonic perfusate is followed by regulatory volume increase (RVI). Available evidence suggests that RVD is accomplished by activation of potassium channels, hyperpolarization and subsequent extrusion of chloride along with potassium, and that RVI depends on the activation of sodium hydrogen ion exchange with subsequent activation of sodium/potassium-ATPase leading to the respective accumulation of potassium and bicarbonate. In addition, exposure of liver to anisotonic perfusates alters glycogen degradation, glycolysis and probably urea formation, which are enhanced by exposure to hypertonic perfusates and depressed by hypotonic perfusates.     

Cytochalasin B and the sialic acids of Ehrlich ascites cells

The effect of cytochalasin B (CB) on the electrophoretic mobility and density of ionized sialic acid groups at the surface of Ehrlich ascites cells was examined together with a biochemical assay of the total sialic acid content of treated and control cells. Sialic acid assays indicated that CB-treated cells had a greater amount of total sialic acid and sialic acid sensitive to neuraminidase than control cells/cell. Equal amounts of sialic acid were removable by neuraminidase treatment from control cells and cells pretreated with neuraminidase and subsequently cultured with CB. The electrophoresis results showed a decrease in electrophoretic mobility in the presence of CB which could be reversed by growth in CB-free medium. Neuraminidase treatment did not make a significant additional reduction in the mobility of CB-treated cells. CB also prevented the recovery of electrophoretic mobility of neuraminidase treated cells. The results suggest that while CB does not inhibit sialic acid synthesis, it does alter the expression of ionized sialic acid groups at the electrokinetic surface. CB-containing culture media could be re-utilized several times suggesting that CB is not significantly bound or metabolized by Ehrlich ascites cells.     

Intracellular Delivery of Carbohydrates into Mammalian Cells through Swelling-activated Pathways

Volume changes of human T-lymphocytes (Jurkat line) exposed to hypotonic carbohydrate-substituted solutions of different composition and osmolality were studied by videomicroscopy. In 200 mOsm media the cells first swelled within 1–2 min and then underwent regulatory volume decrease (RVD) to their original isotonic volume within 10–15 min. RVD also occurred in strongly hypotonic 100 mOsm solutions of di- and trisaccharides (trehalose, sucrose, raffinose). In contrast to oligosaccharide media, 100 mOsm solutions of monomeric carbohydrates (glucose, galactose, inositol and sorbitol) inhibited RVD. The complex volumetric data were analyzed with a membrane transport model that allowed the estimation of the hydraulic conductivity and volume-dependent solute permeabilities. We found that under slightly hypotonic stress (200 mOsm) the cell membrane was impermeable to all carbohydrates studied here. Upon osmolality decrease to 100 mOsm, the membrane permeability to monomeric carbohydrates increased dramatically (apparently due to channel activation caused by extensive cell swelling), whereas oligosaccharide permeability remained very poor. The size-selectivity of the swelling-activated sugar permeation was confirmed by direct chromatographic measurements of intracellular sugars. The results of this study are of interest for biotechnology, where sugars and related compounds are increasingly being used as potential cryo- and lyoprotective agents for preservation of rare and valuable mammalian cells and tissues.This revised version was published online in June 2005 with a corrected cover date.