Initial Cell Volume Research Articles

Cryomicroscope investigation and thermodynamic modeling of the freezing of unfertilized hamster ova

Thermodynamic computer modeling was used to predict the freezing response of single-celled unfertilized hamster ova. The cell membrane transport characteristics were investigated, using a microscope diffusion chamber system. The mean osmotically inactive cell volume was determined to be 21.6% of the initial cell volume. An overall mean value of 0.8 ± 0.1 μm 3/gmm 2 · min · atm ( 18 ± 2.5 μm/sec) was determined for the membrane hydraulic coefficient, L p. The effect of the extracellular solute concentration on L p. was determined at room temperature (~23 °C). A thermodynamic computer model was used to predict the cell response to freezing. The predicted response was compared to the actual volumetric response observed during freezing on a temperature-controlled cryomicroscope conduction stage. The effect of the cooling rate on the nucleation temperature of unprotected ova and protected ova suspended in a 1.5 M DMSO solution was investigated. Overall mean nucleation temperatures of −13 and −57.1 °C were observed for unprotected and protected ova, respectively, where the mean nucleation temperature for protected ova was strongly cooling rate dependent.

L-929 cells under hyperosmotic conditions: volume changes.

Volume changes resulting from the addition of sorbitol to the environment of mouse L-929 cells were evaluated from cell diameter measurements. Over periods of 1 hour or less, this solute was effectively excluded from intracellular water. The reduction in cell volume was inversely related to sorbitol concentration up to levels of about 0.6 molal, above which no further significant reduction occurred. Reduced cell volumes were maintained for at least 1 hour without measurable volume regulation. The percentage of volume lost was independent of the initial cell volume and was quickly regained when physiological conditions were restored. However, cell volume was influenced strongly by cell density or by some variable related to it. L-cells store surface area when dehydrated, apparently by means of plasma membrane convolutions and microvilli, based on the rapid kinetics of reversible volume changes and on observations from scanning electron microscopy. These results are related to current views on the nature of intracellular organization.

Activation of Cl-dependent K transport in Ehrlich ascites tumor cells.

N-ethylmaleimide (NEM) treatment of steady-state Ehrlich cells induces a substantial net loss of cellular KCl and cell shrinkage. The majority of the initial K loss is Cl dependent. From estimates of membrane potential it is concluded that the NEM-induced KCl loss is electroneutral. The effect of NEM on H extrusion by cells in 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-containing medium showed that only an insignificant part of the K loss could be attributed to an activation of a K-H exchange system. Consequently, NEM appears to activate a K-Cl cotransport, which causes cell shrinkage. The anion preference of the K loss is Cl greater than Br much greater than SCN = NO3. NEM also seems to inhibit a Cl-dependent Na uptake previously described in shrunken cells. Addition of NEM to cells undergoing regulatory volume decrease after swelling in hyposmotic media results in a Cl-dependent acceleration of cell shrinkage, suggesting that a Cl-dependent component of K efflux is induced by NEM also in swollen cells. A Cl-dependent K efflux is also activated in Ca-depleted cells or at reduced extracellular pH after cell swelling. Under isotonic conditions activation of Cl-dependent K flux after Ca depletion or pH reduction could not be demonstrated. The combined results show that Ehrlich cells possess a latent K-Cl cotransport that becomes active after changes in the state of SH groups, regardless of the initial cell volume. A similar K-Cl cotransport is activated in hypotonically swollen cells after Ca depletion or after reduction of the extracellular pH.

Thermally defined cryomicroscopy and thermodynamic analysis in lymphocyte freezing

A cryomicroscope is described which provides the possibility of quantifying the volume loss of cells during freezing, detection of intracellular ice formation during cooling and warming, as well as the determination of viability as function of (constant) cooling rates. The basic mechanisms occurring in cryopreservation have been studied with this system using the human lymphocyte suspended in pure saline as a biological model system; experimentally observed exosmosis during freezing is compared to predictions from a thermodynamic model. Cell volume loss during freezing has been determined experimentally for cooling rates of 2.4, 12, 48, and 120 °K/min. Exosmosis also was calculated corresponding to various assumptions regarding the concentration dependence of the hydraulic permeability of the cells. Further calculations of exosmosis are performed for determining the effects of the initial cell volume. The temperatures and transition cooling rate ranges of intracellular ice formation have been determined. On the basis of exosmosis and a lethal level of intracellular salt concentration, a hypothetical relative optimum of the cooling rate is theoretically predicted and compared to the experiments.

Evidence for chloride dependent potassium and water transport induced by hyposmotic stress in erythrocytes of the marine teleost,Opsanus tau

Red blood cells of the marine teleost,Opsanus tau (oyster toadfish), were characterized as to their normal hemoglobin, ion and water contents. Cells were exposed to ouabain containing, hyposmotic salt solutions (osmolarity reduced to 2/3 of normal) in which the cation or anion composition was varied. It was found that the initial cell volume expansion due to water influx was independent of the anion present. However, a secondary volume reduction was dependent on the presence of chloride or bromide anions. During volume reduction, cellular potassium and chloride ion contents fell by about equal amounts. Potassium loss was commensurate to the total amount of potassium ions detected extracellularly about 1.5h after the initial osmotic shock. No major changes were seen in the cellular sodium ion contents. When chloride ions within the cells and in the suspending medium were replaced by nitrate, iodide or thiocyanate, the cells failed to return to volumes close to those of isosmotically suspended controls, and the cellular potassium content also remained constant. In hypotonic potassium chloride the cells failed to extrude potassium chloride and water, and hence retained their expanded volume. Neither potassium loss nor volume decrease occurred in cells swollen in hypotonic sodium chloride media containing furosemide or 4,4′ diisothiocyano-2,2′-stilbene-disulfonic acid (DIDS). These two compounds are known inhibitors of monovalent cation cotransport and anion self exchange, respectively, in mammalian red cells. Hence toadfish red cells respond to osmotic swelling primarily by activation of an ouabain-insensitive, chloride dependent potassium transport system which is sensitive to inhibition by furosemide and DIDS.

Permeability of individual human erythrocytes to thiourea.

The osmotic swelling to haemolysis of individual red blood cells by isosmotic thiourea has been studied using microcine photography. 2. Crenation occurs immediately upon addition of isosmotic thiourea. The cell becomes a crenated sphere without volume decrease. 3. Subsequently, the cell volume increases linearly with time with maximum swelling occurring at about 102 sec which is 81% of the total haemolysis time. 4. At maximum swelling, the cell volume is 92% greater than the initial cell volume. This volume increase is about double that measured with other permeating substances. 5. The much larger maximum volume implies that thiourea increases the area of the cell membrane. This increase varies from 0 to 75% for individual cells, with a mean of 22%. 6. Membrane expansion varies inversely as the initial cell membrane area and cell volume (r=0-790). 7. Using the increased surface area, increased maximum volume and the swelling time, the mean permeability is calculated to be 5-52 X 10(-7) cm/sec (S.D. of mean=+/-1-19 X 10(-7) cm/sec). The distribution of permeabilities represents a normal distribution. 8. The pre-lytic potassium loss ranged from 0 to 36% with a mean value of 16-5%. This is consistent with values reported in the literature for slow haemolysis. As with other permeants the distribution is skewed towards lower values. 9. Membrane permeability of individual cells varies with the amount of membrane expansion observed. Coefficient of correlation between permeability and expansion index is 0-674. 10. There is no correlation between permeability and the reciprocal of the haemolysis time (r=-0-035). The correlation between permeability and the reciprocal of the swelling time is also poor (r=0-303), probably owing to the variability in membrane expansion by thiourea in individual cells. 11. As has been shown previously for faster permeants, the permeability coefficient cannot be calculated from the haemolysis time. Because thiourea alters the membrane area and the haemolytic volume, the coefficient cannot be calculated from the swelling time unless the changes in the membrane area are also taken into account.

Effects of tris and histidine on human erythrocytes and conditions influencing their mode of action

1. 1. The incubation of human erythrocytes in 0.172 M Tris · HCl, pH 7.6 buffer at 37° leads to (1) a pronounced cellular volume increase, (2) a preferential release of Na +, and (3) if continued sufficiently long, hemolysis. These effects are pH dependent and also are influenced to a considerable degree by such diverse reagents as NaCl, glucose, and histidine. In each instance, increasing levels of the latter compounds in a Tris · HCl incubation mixture led to diminished cellular volume increase and prolonged time of onset of hemolysis. 2. 2. Histidine solutions of 0.31 M, pH 7.5 caused a rapid and dramatic decrease in cellular volume of human erythrocytes and a concomitant rapid exit of cations. However, in a prolonged incubation, human erythrocytes slowly regained their cell volume as a result of histidine entry into the cell. Of considerable interest: Tris swollen cells undergo immediate shrinkage to far below the initial cell volume when incubated in histidine at 37 °C. Through repetition of this process two additional times, as much as 90–95% of the total cellular Na + and K + was removed without hemolysis. 3. 3. Human erythrocytes washed in 0.12 M MgCl 2 and then suspended in 0.31 M histidine, pH 7.5, lost upwards of 60% of their total Na + and 30% of their total K + after a 40 min incubation at 37 °C. However, when increasing amounts of 0.172 M Tris · HCl, pH 7.6 were added to the histidine suspension of cells, the release of K + was reduced to 5% but the release of Na + decreased only to 40% of the total cellular level. On the basis of these observations, it is evident that Tris exerts a preferential activity towards the efflux of Na + from the human erythrocyte, whereas histidine results in high efflux of K + and Na + from the cell.

The effect of calcium ions on cell volume and motility of bovine spermatozoa.

SummaryCa2+ was added to a buffered medium containing various combinations of K+, fructose, CN-, ouabain, and BSA. Constant osmolarity, pH, and temperature of the incubation media were maintained. Without K+ in the medium, Ca2+ limited the duration of rapid swelling of spermatozoa by causing early onset of shrinkage to about 90% of initial cell volume. Shrinkage occurred sooner as the Ca2+ level increased, and was associated with a fuzzy microscopic appearance of the sperm cells. Addition of 2.25 to 3.0 mM K+, and to a lesser extent 1.0 mM of fructose, delayed the onset of swelling and subsequent shrinkage associated with Ca2+ alone. This delay also was observed in a variety of media which promoted good sperm motility. BSA was able to counteract the effects of Ca2+, but this was abolished by addition of CN-. Fructose overcame the CN- effect, indicating that the process is energy related. It appears likely that Ca2+ enters the cell only after energy sources are depleted, and its presence there causes struc...

Factors stabilizing bull sperm cell volume and prolonging motility at high dilution

Sperm cell volume changes were monitored electronically, and motility was observed microscopically during incubation at 35 °C in chemically defined media isosmotic with 0.15 M NaCl and buffered at pH 7.0. Neither 2.0 mM Pi nor 10 mM Mg 2+ had any marked effects on cell volume stability or motility when used with different levels of fructose and K +. Both 3.0 mM K + and 10 mM fructose significantly ( P < 0.05) delayed the onset of cellular swelling, but this delay and the effects on motility were minimal in overcoming the ‘dilution effect’. EDTA (0.05–1.0 mM) had a marked stabilizing effect on cell volume and essentially doubled the duration of motility. When EDTA was combined with fructose the effects were more beneficial than with either one alone. BSA (1 mg/ml) also stabilized cell volume in the presence and absence of fructose, and markedly prolonged vigorous sperm motility. BSA and EDTA tended to be additive in effect. CN −markedly improved the otherwise limited beneficial effects of fructose on sperm volume stability and motility even in the presence of oligomycin. Without fructose, CN − caused early swelling and an apparent eventual cell lysis. CN − could not abolish the beneficial effects of BSA in the presence of fructose or those of BSA or EDTA during preincubation without fructose. Oligomycin blocked the beneficial response to EDTA or BSA in the absence of fructose, but it did not prevent the improvement and extension of motility caused by EDTA, BSA or CN − when fructose was included. Oligomycin caused immediate swelling followed by cell shrinkage to less than the initial cell volume. The onset of shrinkage could be delayed by fructose and further delayed if CN −, EDTA or BSA was also included. The delay in onset of shrinkage was accompanied by prolonged motility. Spermatozoa, immobilized by preincubation in a fructose-free media containing CN −, could be activated by the addition of fructose up to the time swelling began. Both BSA and EDTA extended this period during which motility could be restored.

Factors affecting the hemolytic action of "lysolecithin" upon rabbit erythrocytes.

1. Lysolipid was prepared by the action of snake venom on egg yolk, and a study was made of the factors affecting its hemolytic action upon rabbit erythrocytes. 2. Lysis proceeded very rapidly at first, then ceased within a few minutes at room temperature. A given amount of lysin appeared to hemolyze a fixed number of cells, under specified conditions. 3. The more dilute erythrocyte suspensions required relatively more lysin per cell, for 50 per cent hemolysis of the suspension. There may be an equilibrium between the lysin dissolved in the medium and that adsorbed on the cells. 4. The degree of hemolysis for varying lysin concentrations was measured, and the cells showed a typical distribution of resistance to hemolysis. 5. As the temperature was lowered lysis was more extensive. Adsorption of the lysin on the cell surface was apparently increased. 6. The resistance of the erythrocytes to lysis increased slightly as the pH was raised from 5.5 to 7.8. 7. Resistance to lysis was independent of the tonicity of the medium and of initial cell volume. The magnitude of the cell surface was probably the determining factor. 8. A marked shrinkage of the erythrocytes was observed in the presence of calcium ions and lysin, but not in the absence of the lysin. 9. Hemolytic resistance curves obtained by the Wilbrandt technique were of the "colloid-osmotic" type. However, there was no evidence of prolytic loss of potassium ions. 10. Hypotonic fragility of the cells was slightly increased in the presence of the lysin. The rate of penetration of thiourea was greatly increased.

Red Cell Size and Resistance to Osmotic Hemolysis

In explanation of the fact that the red cells of different mammals show different “fragilities”, i. e., different resistances to osmotic hemolysis, it has been pointed out that the extent dA to which the least resistant cell membrane can be stretched without there being a loss of pigment depends on the mean initial cell volume V, so that the ratio dA/V is substantially constant. This purely experimental result can be interpreted as meaning that molecules adjacent to the thin (perhaps bi-molecular) layer upon which semi-permeability depends can enter it when it is stretched, thereby allowing of a certain amount of stretching before lysis occurs (Ponder1).∗ Another well known fact is that different red cells of the same animal show different “fragilities”, so that a resistance distribution of roughly symmetrical form arises with respect to tonicity. One would expect that here too the resistance might be related to the initial cell volume, and it is with this point that this note is concerned.The volume whic...

STUDIES ON OSMOTIC EQUILIBRIUM AND ON THE KINETICS OF OSMOSIS IN LIVING CELLS BY A DIFFRACTION METHOD

1. Osmotic equilibrium and kinetics of osmosis of living cells (unfertilized eggs of Arbacia punctulata) have been studied by a diffraction method. This method consists of illuminating a suspension of cells by parallel monochromatic light and measuring, by means of telescope and scale, the angular dimensions of the resulting diffraction pattern from which the average volume of the cells may be computed. The method is far less laborious and possesses several advantages over direct measurement of individual cells. The average size of a large number of cells is obtained from a single measurement of the diffraction pattern and thus individual variability is averaged out. The observations can be made at intervals of a few seconds, permitting changes in volume to be followed satisfactorily. During the measurements the cells are in suspension and are constantly stirred. 2. Volumes of cells in equilibrium with solutions of different osmotic pressure have been determined. In agreement with our previous experiments, based upon direct microscope measurements, we have confirmed the applicability of the law of Boyle-van't Hoff to these cells; that is to say, the product of volume and pressure has been found to be approximately constant if allowance be made for the volume of osmotically inactive material of the cell contents. The volume of osmotically inactive material was found to be, on the average, 12 per cent of the initial cell volume; in eggs from different animals this value ranged from 6 to 20 per cent. 3. Permeability to water of the Arbacia egg has been found to average, at 22 degrees C., 0.106 cubic micra of water per square micron of cell surface, per minute, per atmosphere of difference in osmotic pressure. 4. Permeability to ethylene glycol has been found to average, at 24 degrees C., 4.0 x 10(-15) mols, per square micron of cell surface, per minute, for a concentration difference of 1 mol per liter. This is in agreement with the values reported by Stewart and Jacobs.

Studies on Osmotic Equilibrium and on the Kinetics of Osmosis in living Cells by a Diffraction Method

1. Osmotic equilibrium and kinetics of osmosis of living cells (unfertilized eggs of Arbacia punctulata) have been studied by a diffraction method. This method consists of illuminating a suspension of cells by parallel monochromatic light and measuring, by means of telescope and scale, the angular dimensions of the resulting diffraction pattern from which the average volume of the cells may be computed. The method is far less laborious and possesses several advantages over direct measurement of individual cells. The average size of a large number of cells is obtained from a single measurement of the diffraction pattern and thus individual variability is averaged out. The observations can be made at intervals of a few seconds, permitting changes in volume to be followed satisfactorily. During the measurements the cells are in suspension and are constantly stirred. 2. Volumes of cells in equilibrium with solutions of different osmotic pressure have been determined. In agreement with our previous experiments, based upon direct microscope measurements, we have confirmed the applicability of the law of Boyle-van't Hoff to these cells; that is to say, the product of volume and pressure has been found to be approximately constant if allowance be made for the volume of osmotically inactive material of the cell contents. The volume of osmotically inactive material was found to be, on the average, 12 per cent of the initial cell volume; in eggs from different animals this value ranged from 6 to 20 per cent. 3. Permeability to water of the Arbacia egg has been found to average, at 22 degrees C., 0.106 cubic micra of water per square micron of cell surface, per minute, per atmosphere of difference in osmotic pressure. 4. Permeability to ethylene glycol has been found to average, at 24 degrees C., 4.0 x 10(-15) mols, per square micron of cell surface, per minute, for a concentration difference of 1 mol per liter. This is in agreement with the values reported by Stewart and Jacobs.