Determinants of epithelial cell volume

Epithelial cell volume is determined by the concentration of intracellular, osmotically active solutes. The high water permeability of the cell membrane of most epithelia prevents the establishment of large osmotic gradients between the cell and the bathing solutions. Steady-state cell volume is determined by the relative rates of solute entry and exit across the cell membranes. Inhibition of solute exit leads to cell swelling because solute entry continues; inhibition of solute entry leads to cell shrinkage because solute exit continues. Cell volume is then a measure of the rate and direction of net solute movements. Epithelial cells are also capable of regulation of the rate of solute entry and exit to maintain intracellular composition. Feedback control of NaCl entry into Necturus gallbladder epithelial cells is demonstrable after inhibition of the Na,K-ATPase or reduction in the NaCl concentration of the serosal bath. Necturus gallbladder cells respond to a change in the osmolality of the perfusion solution by rapidly regulating their volume to control values. This regulatory behavior depends on the transient activation of quiescent transport systems. These transport systems are responsible for the rapid readjustments of cell volume that follow osmotic perturbation. These powerful transporters may also play a role in steady-state volume regulation as well as in the control of cell pH.     

Gallbladder epithelial cell hydraulic water permeability and volume regulation

The hydraulic water permeability (Lp) of the cell membranes of Necturus gallbladder epithelial cells was estimated from the rate of change of cell volume after a change in the osmolality of the bathing solution. Cell volume was calculated from computer reconstruction of light microscopic images of epithelial cells obtained by the "optical slice" technique. The tissue was mounted in a miniature Ussing chamber designed to achieve optimal optical properties, rapid bath exchange, and negligible unstirred layer thickness. The control solution contained only 80% of the normal NaCl concentration, the remainder of the osmolality was made up by mannitol, a condition that did not significantly decrease the fluid absorption rate in gallbladder sac preparations. The osmotic gradient ranged from 11.5 to 41 mosmol and was achieved by the addition or removal of mannitol from the perfusion solutions. The Lp of the apical membrane of the cell was 1.0 X 10(-3) cm/s . osmol (Posm = 0.055 cm/s) and that of the basolateral membrane was 2.2 X 10(-3) cm/s . osmol (Posm = 0.12 cm/s). These values were sufficiently high so that normal fluid absorption by Necturus gallbladder could be accomplished by a 2.4-mosmol solute gradient across the apical membrane and a 1.1-mosmol gradient across the basolateral membrane. After the initial cell shrinkage or swelling resulting from the anisotonic mucosal or serosal medium, cell volume returned rapidly toward the control value despite the fact that one bathing solution remained anisotonic. This volume regulatory response was not influenced by serosal ouabain or reduction of bath NaCl concentration to 10 mM. Complete removal of mucosal perfusate NaCl abolished volume regulation after cell shrinkage. Estimates were also made of the reflection coefficient for NaCl and urea at the apical cell membrane and of the velocity of water flow across the cytoplasm.     

Relationship between cell volume and ion transport in the early distal tubule of the Amphiuma kidney

The roles of apical and basolateral transport mechanisms in the regulation of cell volume and the hydraulic water permeabilities (Lp) of the individual cell membranes of the Amphiuma early distal tubule (diluting segment) were evaluated using video and optical techniques as well as conventional and Cl-sensitive microelectrodes. The Lp of the apical cell membrane calculated per square centimeter of tubule is less than 3% that of the basolateral cell membrane. Calculated per square centimeter of membrane, the Lp of the apical cell membrane is less than 40% that of the basolateral cell membrane. Thus, two factors are responsible for the asymmetry in the Lp of the early distal tubule: an intrinsic difference in the Lp per square centimeter of membrane area, and a difference in the surface areas of the apical and basolateral cell membranes. Early distal tubule cells do not regulate volume after a reduction in bath osmolality. This cell swelling occurs without a change in the intracellular Cl content or the basolateral cell membrane potential. In contrast, reducing the osmolality of the basolateral solution in the presence of luminal furosemide diminishes the magnitude of the increase in cell volume to a value below that predicted from the change in osmolality. This osmotic swelling is associated with a reduction in the intracellular Cl content. Hence, early distal tubule cells can lose solute in response to osmotic swelling, but only after the apical Na/K/Cl transporter is blocked. Inhibition of basolateral Na/K ATPase with ouabain results in severe cell swelling. This swelling in response to ouabain can be inhibited by the prior application of furosemide, which suggests that the swelling is due to the continued entry of solutes, primarily through the apical cotransport pathway.     

Principles of cell volume regulation

Cell volume is determined by the content of osmotically active solute (cell osmoles) and the osmolarity of the extracellular fluid. Cell osmoles consist of non-diffusible and diffusible solutes. A large fraction of the diffusible cation content balances negative charges on the non-diffusible solutes. The content of diffusible solutes is determined by the electrochemical gradients driving them across the plasma membrane and the availability and activity of transport pathways in the membrane. The classical view that the sodium pump offsets passive leaks must be modified to accommodate the contributions of a number of secondary active transport processes, as well as to allow for changes in cell nondiffusible osmoles and in their net negative charge. The behaviour of cells in anisosmotic media is often different from that predicted for a perfect osmometer. In many cases this is a consequence of changes in cell osmole content. However, caution is required in extrapolating from in vitro responses of isolated cells to large, acutely induced changes in medium osmolality to the responses of tissues in vivo to more subtle changes in extracellular osmolality.     

Epithelial cell volume modulation and regulation

Summary Epithelial cell volume is a sensitive indicator of the balance between solute entry into the cell and solute exit. Solute accumulation in the cell leads to cell swelling because the water permeability of the cell membranes is high. Similarly, solute depletion leads to cell shrinkage. The rate of volume change under a variety of experimental conditions may be utilized to study the rate and direction of solute transport by an epithelial cell. The pathways of water movement across an epithelium may also be deduced from the changes in cellular volume. A technique for the measurement of the volume of living epithelial cells is described, and a number of experiments are discussed in which cell volume determination provided significant new information about the dynamic behavior of epithelia. The mechanism of volume regulation of epithelial cells exposed to anisotonic bathing solution is discussed and shown to involve the transient stimulation of normally dormant ion exchangers in the cell membrane.     

Cellular volume homeostasis

All cells face constant challenges to their volume either through changes in intracellular solute content or extracellular osmolality. Cells respond to volume perturbations by activating membrane transport and/or metabolic processes that result in net solute loss or gain and return of cell volume to its normal resting state. This paper provides a brief overview of fundamental concepts of osmotic water flow across cell membranes, mechanisms of cell volume perturbation, the role of inorganic ions and organic osmolytes in cell volume regulation and the signaling mechanisms that regulate the activity of cell volume-sensitive transport and metabolic pathways.     

Roles of unfrozen fraction, salt concentration, and changes in cell volume in the survival of frozen human erythrocytes

The cause of slow freezing injury and the basis of the protection by solutes like glycerol are subjects of debate. During slow freezing, cells are sequestered in unfrozen channels between ice crystals that grow by removing pure water from the channels. As a consequence, the solute concentration in the channels rises and the volume of liquid in the channels progressively decreases. The rise in solute concentration, in turn, causes the cells to progressively shrink osmotically. Until recently cryobiologists have ascribed slow freezing injury to either the rise in solute (electrolyte) concentrations in the channels or to the consequent cell shrinkage, rather than to the decrease in the of the channels. Although ordinarily reciprocally coupled, it is possible to separate the composition of the channels from their size, or more precisely from the magnitude of the unfrozen fraction, by suspending cells in NaCl/cryoprotectant solutions in which the mole ratio of the two is held constant, but the molality of the NaCl is allowed to vary below and above isotonic. When human red cells are frozen in such solutions to temperatures that produce given NaCl concentrations (ms), but varying unfrozen fractions (U), survival at low U is found to be strongly dependent on U but independent of ms. At higher values of U, survival becomes inversely dependent on both ms and U. Although cell volume during freezing is independent of the NaCl tonicity in the solution, the cells in the several solutions differ in volume both prior to the onset of freezing and after the completion of thawing. We have now examined and compared the effect of returning the thawed cells to isotonic solutions and isotonic volume or nearly so, and find that there is little change in survival after exposure to low U, but that survival after exposure to high U values exhibits substantially increased sensitivity to ms, a sensitivity that is probably a manifestation of posthypertonic hemolysis. Low values of U were in general attained by the use of solutions with low tonicities of NaCl, and as a consequence cells frozen to low U values had larger volumes prior to freezing than cells frozen to higher U values. The significance of this confounding is discussed.     

Manipulation of cell volume and membrane pore comparison following single cell permeabilization with 60- and 600-ns electric pulses

Intense nanosecond-duration electric pulses (nsEP) open stable nanopores in the cell membrane, followed by cell volume changes due to water uptake or expulsion, as regulated by the osmolality balance of pore-impermeable solutes inside and outside the cell. The size of pores opened by either fifty 60-ns EP (~13 kV/cm) or five, 600-ns EP (~6 kV/cm) in GH3 cells was estimated by isoosmotic replacement of bath NaCl with polyethylene glycols and sugars. Such replacement reduced cell swelling or resulted in transient or sustained cell shrinking in response to EP. depending on the availability of pores permeable to the test solute. Unexpectedly, solute substitutions showed that for the same integral area of pores opened by 60- and 600-ns treatments (as estimated by cell volume changes), the pore sizes were similar. However, the 600-ns exposure triggered significantly higher cell uptake of propidium. We concluded that 600-ns EP opened a greater number of larger (propidium-permeable pores), but the fraction of the larger pores in the entire pore population was insufficient to contribute to cell volume changes. For both the 60- and 600-ns exposures, cell volume changes were determined by pores smaller than 0.9 nm in diameter; however, the diameter increased with increasing the nsEP intensity.     

The volume of mitochondria-rich cells of frog skin epithelium

Summary The pathway for movement of chloride ions across frog skin is not well understood. Mitochondria-rich (MR) cells have been proposed as the route for chloride across the skin. To test this hypothesis we studied the MR cells of the skin of the frog,Rana pipiens, by quantitative light microscopic determination of cell volume. MR cell volume was influenced by changes in the chloride concentration or osmolality of the outside bathing solution. MR cells shrank about 23% when all chloride was removed from the outside (mucosal) bathing solution. MR cells were also shown to be responsive to changes in the osmolality of either the mucosal or serosal bath. Osmotically-induced swelling caused by dilution of the serosal bath resulted in volume regulatory decrease. These results are consistent with the hypothesis that MR cells constitute the pathway for chloride movement across frog skin.     

The physico-chemical mechanism of mediated transport. II. Osmotic and isosmotic volume flow

The process of volume change of cells subject to osmotic shocks or isosmotic entrance of permeant solute is formulated on the basis of the accepted structure for the plasma membrane and a physico-chemical approach similar to that recently developed. The effect of relevant parameters is discussed and theoretical equilibrium values for the variables are calculated in connection with water and permeant solute permeability determinations. Although a sorption-diffusional mechanism for solute and/or water volume flow within the membrane is assumed in both cases, the kinetics of volume change is shown to be totally different between them. In the isosmotic process a fixed relationship, given by the total solute concentration, is shown to exist between the permeant solute and volume fluxes to the cell, thereby implying a definite value for the volume fraction of water in the migration pathway, higher than 90%. The bi-phase osmotic regulatory response caused by permeant solute is simulated on the basis of an osmotic and isosmotic processes in series, showing good agreement with general behavior. Finally, an explanation to the problem of volume flow and forces in connection with a diffusional mechanism in biological and artificial membranes, is presented.     

Cell volume increase and extracellular Ca2+ are needed for hyposmotically induced prolactin release in tilapia

In thetilapia (Oreochromis mossambicus), as in many euryhalineteleost fish, prolactin (PRL) plays a central role in freshwater adaptation, acting on osmoregulatory surfaces to reduce ion and waterpermeability and increase solute retention. Consistent with theseactions, PRL release is stimulated as extracellular osmolality isreduced both in vivo and in vitro. In the current experiments, aperfusion system utilizing dispersed PRL cells was developed forpermitting the simultaneous measurement of cell volume and PRL release.Intracellular Ca²⁺ was monitored using fura 2-loaded cellsunder the same conditions. When PRL cells were exposed to hyposmoticmedium, an increase in PRL cell volume preceded the increase in PRLrelease. Cell volume increased in proportion to decreases of 15 and30% in osmolality. However, regulatory volume decrease was clearlyseen only after a 30% reduction. The hyposmotically induced PRLrelease was sharply reduced in Ca²⁺-deleted hyposmoticmedium, although cell volume changes were identical to those observedin normal hyposmotic medium. In most cells, a rise in intracellularCa²⁺ concentration ([Ca²⁺]_i)during hyposmotic stimulation was dependent on the availability ofextracellular Ca²⁺, although small transient increases in[Ca²⁺]_i were sometimes observed uponintroduction of Ca²⁺-deleted media of the same or reducedosmolality. These results indicate that an increase in cell size is acritical step in the transduction of an osmotic signal into PRL releaseand that the hyposmotically induced increase in PRL release is greatlydependent on extracellular Ca²⁺.

     

Osmotic response of mammalian cells: Effects of permeating cryoprotectants on nonsolvent volume

The nonsolvent volume, b, of a cell permits calculation of cell water volume from measurements of total cell volume, and, consequently, it is used extensively in the determination of membrane permeability coefficients for water and solutes and also in simulations of water and solute fluxes during freezing of cells. The nonsolvent volume is most commonly determined from the ordinate intercept of plots of cell volume as a function of the reciprocal of extracellular nonpermeating solute concentration (so-called Boyle-van't Hoff plots). Once derived, b is often assumed to be constant even under conditions that may differ markedly from those under which it was determined. Our aim was to investigate whether this assumption was valid when cells were exposed to the cryoprotectants glycerol, dimethyl sulphoxide (Me₂SO), or propane-1,2-diol. Rabbit corneal keratocytes, a fibroblastic cell type, were exposed to 10% (v/v) cryoprotectant for 30 min at 22°C in solutions containing a range of nonpermeating solute concentrations. Cell volumes were determined by an electronic particle sizer and mode volume plotted as an inverse function of the concentration of nonpermeating solute. The cells behaved as osmometers under all conditions studied, but we found no evidence to suggest that the nonsolvent volume of cells was altered by Me₂SO or propane-1,2-diol. Glycerol, however, reduced the slope of the Boyle-van't Hoff plot, but this could be ascribed to the failure of the cells to equilibrate fully with the glycerol over the 30 min exposure time; thus, b was unaffected by glycerol. It may be assumed, therefore, that the nonsolvent volume was not influenced by the presence inside cells of any of these nonelectrolyte cryoprotectants. © 1996 Wiley-Liss, Inc.     

Kinetics of glutaraldehyde fixation of erythrocytes: size, deformability, form, osmotic and hemolytic properties.

Red blood cells interact with glutaraldehyde (GA) in a complex kinetic pattern of events. At a given GA concentration in phosphate buffered saline (PBS), the sequence of cell 'volume' response, as measured by resistive pulse spectroscopy (RPS), includes: an immediate response to the overall solution osmolality; a constant volume, latent phase; a rapid swelling phase; an intermediate constant volume phase; and a shrinkage phase to a final steady state volume. The final volume depends on fixative solution osmolality; for GA concentrations between 0.05% and 0.25% w/v, fixative osmolalities of less than 355 mosM, including 'isotonic', or greater than 355 mosM, lead to final cell volumes greater or less than native, respectively. Cell-membrane deformability decreases continuously and monotonically with time, as assessed by RPS. The rate of fixation is a direct function of GA concentration, in accordance with a derived empirical expression. The measured kinetic responses are related to considerations of cell size, deformability, and form, and to mechanisms involved in abrupt osmotic hemolysis.     

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.     

Preservation of resuspended red cell concentrate. Red cell volume and hemolysis

Sucrose in a concentration of 30 to 50 mmol/l preservation solution (10-20 mmol/l red cell concentrate (RCC) and 3-5 mmol per unit RCC) and an ionic strength of about 0.16 avoid changes of red cell volume during 6 weeks of storage. Increasing sucrose concentrations up to 80 mmol/l RCC decrease the hemolysis. But a sucrose concentration of only 10 mmol/l RCC causes an acceptable low hemolysis rate of 0.25% after 35 days of storage in PCV FENWAL plastic bags. Sucrose can be replaced by mannitol or sorbitol at the same final concentration. Changes in red cell metabolism and viability will not be expected.     

May active solute flux control the cell volume in the steady state?

The dynamics of a bioreactor with a variable volume and an active solute flux based on the thermodynamics of irreversible processes and stability analysis was studied. The active solute flux may control both the bioreactor volume and the hydrostatic pressure as well as the concentration of the solute inside the cell in the steady state. The range of the active solute flux is limited by amplitudes (j1(0), J2(0] of the active transport depending on the membrane transport parameters. The dynamic system is stable for j0 greater than jth0.     

The mean red cell volume in long distance runners

Red cell indices were determined in 6 well trained runners before and after a 100 km race, and Coulter Counter (CC) determinations compared with calculated values derived from centrifuged hematocrit (ctrf), red cell count (CC) and hemoglobin measurements. The following changes were observed immediately after the race, as compared to values 3 days before: MCV(ctrf) decreased by 4.9% (p less than 0.001), MCV(CC) increased by 1.9% (p less than 0.05), MCHC(ctrf) increased by 4% and MCHC(CC) decreased by 3%. The increase in MCV(CC) suggests that intraerythrocyte osmolality was increased, this probably leading to swelling of the cells induced by a shift of water from the diluting Coulter Counter solution into the red cells prior to the MCV measurement. The decrease in MCV(ctrf) immediately after the race was not correlated with the increase in plasma osmolality. This suggests that plasma osmolality alone was not the key factor for regulation of red cell volume. The changes in MCV(ctrf), which contributed to a surprising stability of the hematocrit value and plasma volume, might represent a physiological principle for the maintenance of a favourable blood viscosity.     

Osmotic responses of unicellular blue-green algae (cyanobacteria): changes in cell volume and intracellular solute levels in response to hyperosmotic treatment

Abstract Changes in cell volume and solute content upon hyperosmotic shock have been studied for six unicellular blue-green algae (cyanobacteria): Synechococcus PCC 6301, PCC 6311; Synechocystis PCC 6702, PCC 6714, PCC 6803 and PCC 7008. The extent of change in volume was shown to be dependent upon the solute used to establish the osmotic gradient, with cells in NaCl showing a reduced shrinkage when compared to cells in media containing added sorbitol and sucrose. Uptake of extracellular solutes during hyperosmotic shock was observed in Synechocystis PCC 6714, with maximum accumulation of external solutes in NaCl and minimum solute uptake in sucrose solutions. Conversely, solute loss from the cells (K⁺ and amino acids) was greatest in sucrose-containing media and least in NaCl. The results show that these blue-green algae do not behave as ‘ideal osmometers’ in media of high osmotic strength. It is proposed that short-term changes in plasmalemma permeability in these organisms may be due to transient membrane instability resulting from osmotic imbalance between the cell and its surrounding fluid at the onset of hyperosmotic shock.     

Effects of preparative procedures on the volume and content of resealed red cell ghosts

The effects of variations in preparative procedures on the volume and content of resealed red cell ghosts have been investigated. Following hypotonic lysis at 0 degrees C, and after a variable delay time (td), concentrated buffer was added to restore isotonicity; resealing was then induced by incubation at 37 degrees C for one hour. Using this procedure, both the resealed ghost volume and the residual hemoglobin (Hb) content decreased for increasing td. If ghosts were maintained at 0 degree C (i.e., no 37 degrees C incubation), they remained nearly spherical until isotonicity was restored. Their volume then fell abruptly, but subsequently increased toward an intermediate level. The fall in volume was greater and the final level achieved was smaller for longer delay times. At 0 degree C, return to isotonicity also halted the otherwise gradual loss of residual Hb from unsealed ghosts. In addition, ghosts with internal osmolality of 40 to 300 mosmol/kg were prepared by adding different amounts of concentrated buffer before resealing for one hour at 37 degrees C. Under these conditions, the final ghost volume was inversely related to the resealing osmolality (i.e., lower osmolality yielded a larger volume). Ghost volume also increased, along with Hb content, if the quantity or concentration of the red cell suspension added to the lysing medium was increased. We conclude that resealed ghost volume is influenced by the ratio of lysate to resealing medium osmolality and by the colloid osmotic pressure of the residual ghost Hb. These data indicate methods by which ghosts with desired characteristics can be prepared, and have potential application for studies of ghost mechanical and biophysical behavior.     

Hybridoma cell behaviour in continuous culture under hyperosmotic stress

In this paper, we propose an alternative strategy to the ones proposed before (Oh et al., 1993; Øyaas et al., 1994a) to get real increases of global final antibody titer and production at hyperosmotic stress, by reducing the detrimental effect of such a stress on cell growth, and conserving the stimulating effect on antibody production. It consists of cultivating the cells in continuous culture and increasing the osmolality stepwise. In this way, the cells could progressively adapt to the higher osmolality at each step and antibody titers could be nearly doubled at 370 and 400 mOsm kg-1, compared to the standard osmolality of 335 mOsm kg-1. Surprisingly, the stimulation of antibody production was not confirmed for higher osmolalities, 425 and 450 mOsm kg- 1, despite the minor negative effect on cell growth. Intracellular IgG analysis by flow cytometry revealed at these osmolalities a significant population of non-producing cells. However, even when taking into account this non-producing population, a stimulating effect on antibody production could not be shown at these highest osmolalities. It seems to us that osmolality has a significant effect on the appearance of these non-producing cells, since they were not observed in continuous cultures at standard osmolality, of comparable duration and at an even higher dilution rate. The appearance of the non-producing cells coincides furthermore with modifications of the synthesised antibody, as shown by electrophoretic techniques. It is however not really clear if these two observations reflect actually the same phenomenon. Hyperosmolality affects the cell behaviour in continuous culture in multiple ways, independently of the growth rate, counting all at least partially for the observed stimulation of antibody production: acceleration of the amino acid, and in particular the glutamine metabolism, increase of the cell volume, increase of the intracellular pH and accumulation of cells in the G1 cell cycle phase.