Volume regulation by Amphiuma red blood cells. The membrane potential and its implications regarding the nature of the ion-flux pathways

After osmotic perturbation, the red blood cells of Amphiuma exhibited a volume-regulatory response that returned cell volume back to or toward control values. After osmotic swelling, cell-volume regulation (regulatory volume decrease; RVD) resulted from net cellular loss of K, Cl, and osmotically obliged H2O. In contrast, the volume-regulatory response to osmotic shrinkage (regulatory volume increase; RVI) was characterized by net cellular uptake of Na, Cl, and H2O. The net K and Na fluxes characteristic of RVD and RVI are increased by 1-2 orders of magnitude above those observed in studies of volume-static control cells. The cell membrane potential of volume-regulating and volume-static cells was measured by impalement with glass microelectrodes. The information gained from the electrical and ion-flux studies led to the conclusion that the ion fluxes responsible for cell-volume regulation proceed via electrically silent pathways. Furthermore, it was observed that Na fluxes during RVI were profoundly sensitive to medium [HCO3] and that during RVI the medium becomes more acid, whereas alkaline shifts in the suspension medium accompany RVD. The experimental observations are explained by a model featuring obligatorily coupled alkali metal-H and Cl-HCO3 exchangers. The anion- and cation-exchange pathways are separate and distinct yet functionally coupled via the net flux of H. As a result of the operation of such pathways, net alkali metal, Cl, and H2O fluxes proceed in the same direction, whereas H and HCO3 fluxes are cyclic. Data also are presented that suggest that the ion-flux pathways responsible for cell-volume regulation are not activated by changes in cell volume per se but by some event associated with osmotic perturbation, such as changes in intracellular pH.     

Cell volume regulation by Amphiuma red blood cells. The role of Ca+2 as a modulator of alkali metal/H+ exchange

In response to osmotic perturbation, the Amphiuma red blood cell regulates volume back to "normal" levels. After osmotic swelling, the cells lose K, Cl, and osmotically obliged H2O (regulatory volume decrease [RVD] ). After osmotic shrinkage, cell volume is regulated as a result of Na, Cl, and H2O uptake (regulatory volume increase [RVI] ). As previously shown (Cala, 1980 alpha), ion fluxes responsible for volume regulation are electroneutral, with alkali metal ions obligatorily counter-coupled to H, whereas net Cl flux is in exchange for HCO3. When they were exposed to the Ca ionophore A23187, Amphiuma red blood cells lost K, Cl, and H2O with kinetics (time course) similar to those observed during RVD. In contrast, when cells were osmotically swollen in Ca-free media, net K loss during RVD was inhibited by approximately 60%. A role for Ca in the activation of K/H exchange during RVD was suggested from these experiments, but interpretation was complicated by the fact that an increase in cellular Ca resulted in an increase in the membrane conductance to K (GK). To determine the relative contributions of conductive K flux and K/H exchange to total K flux, electrical studies were performed and the correspondence of net K flux to thermodynamic models for conductive vs. K/H exchange was evaluated. These studies led to the conclusion that although Ca activates both conductive and electroneutral K flux pathways, only the latter pathways contribute significantly to net K flux. On the basis of observations that A23187 did not activate K loss from cells during RVI (when the Na/H exchange was functioning) and that amiloride inhibited K/H exchange by swollen cells only when cells had previously been shrunk in the presence of amiloride, I concluded that Na/H and K/H exchange are mediated by the same membrane transport moiety.     

Cell volume regulation in nonrenal epithelia

Cell volume regulation occurs in both tight, Na+-transporting epithelia (e.g., frog skin) and in leaky. NaCl-transporting epithelia (e.g. amphibian gallbladder). In tight epithelia volume regulation occurs only in response to cell swelling, i.e. only regulatory volume decrease (RVD) is observed, whereas in leaky epithelia cell volume regulation has been observed in response to osmotic challenges that either swell or shrink the cells. In other words, both RVD and regulatory volume increase (RVI) are present. Both volume regulatory responses involve stimulation of ion transport in a polarized fashion: in RVD the response is basolateral KCl efflux, whereas in RVI it is apical membrane NaCl uptake. The loss of KCl during RVD appears to result in most instances from increases in basolateral electrodiffusive K+ and Cl-permeabilities. In gallbladder, concomitant activation of coupled KCl efflux may also occur. The RVI response includes activation of apical membrane cation (Na+/H+) and anion (Cl-/HCO-3) exchangers. It is presently unclear whether the net ion fluxes resulting from activation of these transporters, during either RVD or RVI, account for the measured rates of restoration of cell volume. In gallbladder epithelium, RVD is inhibited by agents which disrupt microfilaments or interfere with the Ca2+-calmodulin system. These pharmacologic effects are absent in RVI. Some steps in the chain of events resulting in either RVI or RVD have been established, but the signals involved remain largely unknown. There is reason to suspect a role of intracellular pH in the case of RVI and of membrane insertion of transporters in the case of RVD, possibly with causal roles of both intracellular Ca2+ and the cytoskeleton in the latter.     

Volume regulation of Chinese hamster ovary cells in anisoosmotic media

Chinese hamster ovary (CHO) cells when suspended in anisoosmotic media regulate their volumes by the activation of specific ion transport pathways. In hypoosmotic media the cells first swell and then return to their isoosmotic volumes by the loss of cellular KCl and osmotically obliged water. This regulatory volume decrease (RVD) is insensitive to ouabain or bumetanide but is blocked by quinine, cetiedil and oligomycin C. Based on cell volume and membrane potential measurements under various experimental conditions, we conclude that hypoosmotic shock activates independent, conductive transport pathways for K+ and for Cl-, respectively. The anion pathway can also transport NO3- and SCN- but not gluconate- anions. Osmotic shrinkage of CHO cells does not produce a regulatory volume increase (RVI) unless the cells have previously undergone a cycle of RVD. RVI is a Na+-dependent, amiloride-sensitive, but ouabain- and oligomycin-insensitive process, probably involving a Na+-H+ exchange system. Internal acidification of isoosmotic cells by addition of a permeable weak acid also activates an amiloride-sensitive Na+-H+ exchange, producing a volume increase. Both RVD and RVI in CHO cells seem to involve molecular mechanisms similar to those described for the volume regulation of lymphocytes, indicating the prevalence of these phenomena in nucleated mammalian cells. Cultured CHO cell lines may provide a basis for a genetic characterization of the volume-regulatory transport pathways.     

Volume-regulating behavior of human platelets

Human platelets exposed to hypotonic media undergo an initial swelling followed by shrinking (regulatory volume decrease [RVD]). If the RVD is blocked, the degree of swelling is in accord with osmotic behavior. The cells could swell at least threefold without significant lysis. Two methods were used to follow the volume changes, electronic sizing and turbidimetry. Changes in shape produced only limited contribution to the measurements. The RVD was very rapid, essentially complete in 2 to 8 minutes, with a rate proportional to the degree of initial cell swelling. RVD involved a loss of KCl via volume-activated conductive permeability pathways for K+ and anions, presumably Cl-. In media containing greater than 50 mM KCl, the shrinking was inhibited and with higher concentrations was reversed (secondary swelling), suggesting that it is driven by the net gradient of K+ plus Cl-. The K+ pathway was specific for Rb+ and K+ compared to Li+ and Na+. The Cl- pathway accepted NO-3 and SCN- but not citrate or SO4(2-). In isotonic medium, the permeability of platelets to Cl- appeared to be low compared to that of K+. After hypotonic swelling both permeabilities were increased, but the Cl- permeability exceeded that of K+. The Cl- conductive pathway remained open as long as the cells were swollen. RVD was incomplete unless amiloride, an inhibitor of Na+/H+ exchange, was present or unless Na+ was replaced by an impermeant cation. In addition, acidification of the cytoplasm occurred upon cell swelling. This reduction in pHi appeared to activate Na+/H+ exchange, with a resultant uptake of Na+ and reduction in the rate and amount of shrinking. Like other cells, platelets responded to hypertonic shrinking with activation of Na+/H+ exchange, but regulatory volume increase was not detectable.     

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.     

Ionic basis of volume regulation in mammalian cells following osmotic shock

Previous studies with mammalian cultured cells have shown that volume regulation in hypotonic medium requires active Na transport. In the present study, determinations of intracellular Na and K content were made in cultured mouse lymphoblasts during the process of swelling and subsequent shrinking (volume regulation) in hypotonic medium. Na and K content were measured in cells in which the shrinking phase was inhibited by the cardiac glycoside, ouabain. In osmotically-shocked cells, an initial permeability increase to K, and not Na, was observed, which allowed K to diffuse out rapidly, down its gradient. Na, meanwhile, rapidly flowed inward with water entry during the swelling process, and was later lost with the same kinetics as the cell shrinkage. This loss of Na was prevented in the presence of ouabain. The results imply that volume regulation is achieved by pumping Na gained during swelling out of the cells, while any K taken up by the pump is rapidly lost through a more permeable membrane. The loss of osmotically active Na, presumably with accompanying anions, allows water to passively diffuse down its osmotic gradient, reducing cell volume subsequent to the initial passive swelling, during which K was rapidly lost.     

Activation of ion transport pathways by changes in cell volume.

Swelling-activated K+ and Cl- channels, which mediate RVD, are found in most cell types. Prominent exceptions to this rule include red cells, which together with some types of epithelia, utilize electroneutral [K(+)-Cl-] cotransport for down-regulation of volume. Shrinkage-activated Na+/H+ exchange and [Na(+)-K(+)-2 Cl-] cotransport mediate RVI in many cell types, although the activation of these systems may require special conditions, such as previous RVD. Swelling-activated K+/H+ exchange and Ca2+/Na+ exchange seem to be restricted to certain species of red cells. Swelling-activated calcium channels, although not carrying sufficient ion flux to contribute to volume changes may play an important role in the activation of transport pathways. In this review of volume-activated ion transport pathways we have concentrated on regulatory phenomena. We have listed known secondary messenger pathways that modulate volume-activated transporters, although the evidence that volume signals are transduced via these systems is preliminary. We have focused on several mechanisms that might function as volume sensors. In our view, the most important candidates for this role are the structures which detect deformation or stretching of the membrane and the skeletal filaments attached to it, and the extraordinary effects that small changes in concentration of cytoplasmic macromolecules may exert on the activities of cytoplasmic and membrane enzymes (macromolecular crowding). It is noteworthy that volume-activated ion transporters are intercalated into the cellular signaling network as receptors, messengers and effectors. Stretch-activated ion channels may serve as receptors for cell volume itself. Cell swelling or shrinkage may serve a messenger function in the communication between opposing surfaces of epithelia, or in the regulation of metabolic pathways in the liver. Finally, these transporters may act as effector systems when they perform regulatory volume increase or decrease. This review discusses several examples in which relatively simple methods of examining volume regulation led to the discovery of transporters ultimately found to play key roles in the transmission of information within the cell. So, why volume? Because it's functionally important, it's relatively cheap (if you happened to have everything else, you only need some distilled water or concentrated salt solution), and since it involves many disciplines of experimental biology, it's fun to do.     

Cell volume regulation and signaling in 3T3-L1 pre-adipocytes and adipocytes: on the possible roles of caveolae, insulin receptors, FAK and ERK1/2

Caveolae have been implicated in sensing of cell volume perturbations, yet evidence is still limited and findings contradictory. Here, we investigated the possible role of caveolae in cell volume regulation and volume sensitive signaling in an adipocyte system with high (3T3-L1 adipocytes); intermediate (3T3-L1 pre-adipocytes); and low (cholesterol-depleted 3T3-L1 pre-adipocytes) caveolae levels. Using large-angle light scattering, we show that compared to pre-adipocytes, differentiated adipocytes exhibit several-fold increased rates of volume restoration following osmotic cell swelling (RVD) and osmotic cell shrinkage (RVI), accompanied by increased swelling-activated taurine efflux. However, caveolin-1 distribution was not detectably altered after osmotic swelling or shrinkage, and caveolae integrity, as studied by cholesterol depletion or expression of dominant negative Cav-1, was not required for either RVD or RVI in pre-adipocytes. The insulin receptor (InsR) localizes to caveolae and its expression dramatically increases upon adipocyte differentiation. In pre-adipocytes, InsR and its effectors focal adhesion kinase (FAK) and extracellular signal regulated kinase (ERK1/2) localized to focal adhesions and were activated by a 5 min exposure to insulin (100 nM). Osmotic shrinkage transiently inhibited InsR Y(146)-phosphorylation, followed by an increase at t=15 min; a similar pattern was seen for ERK1/2 and FAK, in a manner unaffected by cholesterol depletion. In contrast, cell swelling had no detectable effect on InsR, yet increased ERK1/2 phosphorylation. In conclusion, differentiated 3T3-L1 adipocytes exhibit greatly accelerated RVD and RVI responses and increased swelling-activated taurine efflux compared to pre-adipocytes. Furthermore, in pre-adipocytes, Cav-1/caveolae integrity is not required for volume regulation. Given the relationship between hyperosmotic stress and insulin signaling, the finding that cell volume regulation is dramatically altered upon adipocyte differentiation may be relevant for the understanding of insulin resistance and metabolic syndrome.     

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.     

Effect of volume changes on ouabain-insensitive net outward cation movements in human red cells

Summary The effect of cell volume changes in human red cells on ouabain-insensitive net outward cation movements through 1) the Na–K and Li–K cotransport, 2) the Li–Na counter-transport system and 3) the furosemide-insensitive Na, K and Li pathway was studied. Cell volume was altered by changing a) the internal cation content (isosmotic method) or b) the external osmolarity of the medium (osmotic method). Na–K and Li–K cotransport were measured as the furosemide-sensitive Na or Li and K efflux into (Na, Li and K)-free (Mg-sucrose replacement) medium from cells loaded to contain approximately equal concentrations of Na and K, or a constant K/Li concentration ratio of 91, respectively. Li–Na countertransport was assayed as the Na-stimulated Li efflux from Li-loaded cells and net furosemide-insensitive outfluxes in (Na, Li and K)-free media containing 1mm furosemide. Swelling of cells by the isosmotic, but not by the osmotic method reduced furosemide-sensitive Na and Li but not K efflux by 80 and 86%, respectively. Changes in cell volume by both methods had no effect on Li–Na countertransport. The effects of cell volume changes were measured on the rate constants of ouabain- and furosemide-insensitive cation fluxes and were found to be complex. Isosmotic shrinkage more than doubled the rate constants of Na and Li efflux but did not affect that of K efflux. Osmotic shrinkage increased the K efflux rate constant by 50% only in cells loaded for countertransport. Isosmotic cell swelling specifically increased the K⁺ efflux rate constants both in cells loaded for cotransport and countertransport assays while no effect was observed in cells swollen by the osmotic method. Thus, the three transport pathways responded differently to changes in cell volume, and, furthermore, responses were different depending on the method of changing cell water content.     

Regulatory volume decrease in alveolar macrophages: cation loss is not correlated with changes in membrane recycling

Alveolar macrophages regain their normal volume after swelling in hypo-osmotic solutions. This process, termed regulatory volume decrease (RVD), is initiated 3-5 minutes after exposure of cells to hypo-osmotic solutions, and by 30 min, near-normal volumes are attained. Volume decrease does not occur at 0 degrees C or in solutions in which Na+ has been replaced by K+, or Cl- by the impermeant anion gluconate. These results, as well as direct measurement of intracellular cations, indicate that decreases in cell volume result primarily from the loss of K+ and Cl- and are similar to RVD in lymphocytes. Kinetic analysis of cation loss, both by directly measuring changes in intracellular cation content and by assaying rubidium efflux, showed that cation loss occurred immediately upon media dilution. The rate of cation loss fit first-order kinetics and preceded both the initiation of volume decrease and the maximum increase in surface receptor number. These results suggest that the cation transporters responsible for RVD are located at the cell surface and that regulation of activity is not dependent on alterations in membrane movement.     

Activation of Na+/H+ exchange in lymphocytes by osmotically induced volume changes and by cytoplasmic acidification

After swelling in hypotonic solutions, peripheral blood mononuclear cells (PBM) shrink toward their original volumes. Upon restoration of isotonicity, the cells initially shrink but then regain near-normal size again. This regulatory volume increase (RVI) is abolished by removal of Na+o or Cl-o or by addition of amiloride. RVI is unaffected by removal of K+o or by ouabain and is only partially inhibited by 1 mM furosemide. As a result of increased influx, the cells gain both Na+ and K+ during reswelling. In contrast, only Na+ content increases in the presence of ouabain. Amiloride largely eliminates the changes in the content of both cations. Using diS-C3-(5), no significant membrane potential changes were detected during RVI, which suggests that the fluxes are electroneutral. The cytoplasmic pH of volume-static cells was measured with 5,6-dicarboxyfluorescein. After acid loading, the addition of extracellular Na+ induced an amiloride-inhibitable alkalinization, which is consistent with Na+/H+ exchange. Cytoplasmic pH was not affected by cell shrinkage itself, but an internal alkalinization, which was also amiloride sensitive and Na+ dependent, developed during reswelling. In isotonic lightly buffered solutions without HCO-3, an amiloride-sensitive acidification of the medium was measurable when Na+ was added to shrunken PBM. K+ was unable to mimic this effect. The observations are compatible with the model proposed by Cala (J. Gen. Physiol. 1980. 76:683-708), whereby an electroneutral Na+o/H+i exchange is activated by osmotic shrinking. Cellular volume gain occurs as Cl-o simultaneously exchanges for either HCO-3i or OH-i. Na+i is secondarily replaced by K+ through the pump, but this step is not essential for RVI.     

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.     

Ouabain-insensitive salt and water movements in duck red cells. I. Kinetics of cation transport under hypertonic conditions

Duck red cells in hypertonic media experience rapid osmotic shrinkage followed by gradual reswelling back toward their original volume. This uptake of salt and water is self limiting and demands a specific ionic composition of the external solution. Although ouabain (10(-4)M) alters the pattern of cation accumulation from predominantly potassium to sodium, it does not affect the rate of the reaction, or the total amount of salt or water taken up. To study the response without the complications of active Na-K transport, ouabain was added to most incubations. All water accumulated by the cells can be accounted for by net salt uptake. Specific external cation requirements for reswelling include: sufficient sodium (more than 23 mM), and elevated potassium (more than 7 mM). In the absence of external potassium cells lose potassium without gaining sodium and continue to shrink instead of reswelling. Adding rubidium to the potassium- free solution promotes an even greater loss of cell potassium, yet causes swelling due to a net uptake of sodium and rubidium followed by chloride. The diuretic furosemide (10(-3)M) inhibits net sodium uptake which depends on potassium (or rubidium), as well as inhibits net sodium uptake which depends on sodium. As a result, cell volume is stabilized in the presence of this drug by inhibition of shrinkage, at low, and of swelling at high external potassium. The response has a high apparent energy of activation (15-20 kcal/mol). We propose that net salt and water movements in hypertonic solutions containing ouabain are mediated by direct coupling or cis-interaction, between sodium and potassium so that the uphill movement of one is driven by the downhill movement of the other in the same direction.     

Volume regulation in intertidalProcephalothrix spiralis (Nemertina) andClitellio arenarius (Oligochaeta)

Summary Intertidal worms such as the nemertine,P. spiralis, and the oligochaete,C. arenarius, are exposed to fluctuating salinity conditions in their natural habitat. Both species are volume regulators. To determine the influence of neurosecretory and neuroglandular mechanisms on regulatory volume increase (RVI) in hyperosmotic media, control and decerebrated worms of both species were (1) exposed to 70% SW followed by return to 100% SW [70% SW (2, 12 or 24 h) 100% SW (12 h)] or (2) exposed to a fluctuating salinity regimen [70% SW (2 h) 100% SW (12 h) repeat]. The effects of fluctuating salinity on regulatory volume decrease (RVD), in hypoosmotic media, were examined simultaneously.RVI was manifested either as a shrinkage limitation or a net volume gain. In both cases it was accompanied by increases in Na and Cl but not K content (moles/gram solute free dry weight=g s.f.d.w.). Repeated return to full-strength seawater potentiated the RVI response in controlC. arenarius only. Whole animal volume control is a result of extracellular as well as intracellular volume regulation. InC. arenarius, decerebration prevented RVI by apparently inhibiting extracellular volume control through reduction in ion uptake. It also resulted in reduced capacity to maintain a hyperosmotic gradient across the body wall which further supports an effect of decerebration on ion uptake at the integument or gut. InP. spiralis, in which whole animal volume regulation is primarily an intracellular phenomenon, decerebration did not affect RVI.RVD (swelling limitation and net volume loss) was, in all worms, accompanied by a decrease in Na and Cl but not K content (moles/g s.f.d.w.). Repeated exposure to 70% SW reduced the RVD response inP. spiralis and in controlC. arenarius. Decerebration eliminated extracellular swelling limitation and delayed net volume loss inC. arenarius but had no measurable effect onP. spiralis (corroborating previous results; Ferraris and Schmidt-Nielsen 1982a, b).It appears that neuroendocrine factors primarily stimulate extracellular volume regulation in RVI as well as in RVD, but have no effect on intracellular volume regulation in these worms. When the worms are exposed to repeated changes in medium osmolality RVD is reduced in both species, while RVI is enhanced in controlC. arenarius only. Thus, while both extracellular and intracellular RVD are reduced, only extracellular RVI is potentiated.Abbreviations g s.f.d.w. gram solute free dry weight - NPS ninhydrin positive substances - RVD regulatory volume decrease - RVI regulatory volume increase - SW seawater     

Regulation of cell volume via microvillar ion channels

A novel mechanism of cellular volume regulation is presented, which ensues from the recently introduced concept of transport and ion channel regulation via microvillar structures (Lange K, 1999, J Cell Physiol 180:19-35). According to this notion, the activity of ion channels and transporter proteins located on microvilli of differentiated cells is regulated by changes in the structural organization of the bundle of actin filaments in the microvillar shaft region. Cells with microvillar surfaces represent two-compartment systems consisting of the cytoplasm on the one side and the sum of the microvillar tip (or, entrance) compartments on the other side. The two compartments are separated by the microvillar actin filament bundle acting as diffusion barrier ions and other solutes. The specific organization of ion and water channels on the surface of microvillar cell types enables this two-compartment system to respond to hypo- and hyperosmotic conditions by activation of ionic fluxes along electrochemical gradients. Hypotonic exposure results in swelling of the cytoplasmic compartment accompanied by a corresponding reduction in the length of the microvillar diffusion barrier, allowing osmolyte efflux and regulatory volume decrease (RVD). Hypertonic conditions, which cause shortening of the diffusion barrier via swelling of the entrance compartment, allow osmolyte influx for regulatory volume increase (RVI). Swelling of either the cytoplasmic or the entrance compartment, by using membrane portions of the microvillar shafts for surface enlargement, activates ion fluxes between the cytoplasm and the entrance compartment by shortening of microvilli. The pool of available membrane lipids used for cell swelling, which is proportional to length and number of microvilli per cell, represents the sensor system that directly translates surface enlargements into activation of ion channels. Thus, the use of additional membrane components for osmotic swelling or other types of surface-expanding shape changes (such as the volume-invariant cell spreading or stretching) directly regulates influx and efflux activities of microvillar ion channels. The proposed mechanism of ion flux regulation also applies to the physiological main functions of epithelial cells and the auxiliary action of swelling-induced ATP release. Furthermore, the microvillar entrance compartment, as a finely dispersed ion-accessible peripheral space, represents a cellular sensor for environmental ionic/osmotic conditions able to detect concentration gradients with high lateral resolution. Volume regulation via microvillar surfaces is only one special aspect of the general property of mechanosensitivity of microvillar ionic pathways.     

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.     

The Response of Duck Erythrocytes to Nonhemolytic Hypotonic Media : Evidence for a volume-controlling mechanism

Duck erythrocytes were incubated in hypotonic media at tonicities which do not produce hemolysis. The cells'' response can be divided into two phases: an initial rapid phase of osmotic swelling and a second more prolonged phase (volume regulatory phase) in which the cells shrink until they approach their initial isotonic volume. Shrinkage associated with the volume regulatory phase is the consequence of a nearly isosmotic loss of KCl and water from the cell. The potassium loss results from a transient increase in K efflux. There is also a small reduction in Na permeability. Changes in cell size during the volume regulatory phase are not altered by 10^-4 M ouabain although this concentration of ouabain does change the cellular cation content. The over-all response of duck erythrocytes is considered as an example of "isosmotic intracellular regulation," a term used to describe a form of volume regulation common to euryhaline invertebrates which is achieved by adjusting the number of effective intracellular osmotic particles. The volume regulatory phase is discussed as the product of a membrane mechanism which is sensitive to some parameter associated with cell volume and is capable of regulating the loss of potassium from the cell. This mechanism is able to regulate cell size when the Na-K exchange, ouabain-inhibitable pump mechanism is blocked.