Osmotic injury of PC-3 cells by hypertonic NaCl solutions at temperatures above 0 degrees C

Cell injury due to osmotic dehydration, which is regarded as a major cause of injury during freeze-thaw processes, was examined closely using a perfusion microscope. Human prostatic adenocarcinoma cells (PC-3), which were put in a chamber, were subjected to hyperosmotic stresses by perfusing NaCl solutions of varying concentrations into the chamber. Cells were exposed to 2.5 and 4.5M NaCl solutions for 1-60 min by changing the concentrations at 0.2, 1, and 10 M/min. Decrease in cell viability was biphasic: the viability decreased first after the increase in NaCl concentration due to dehydration and then after return to isotonic conditions due to rehydration. Rehydration was substantially more responsible for cell injury than dehydration, which was marked at lower NaCl concentrations and lower temperatures. Injury resulting from contraction was negligible at the 2.5 M NaCl solution. While the hypertonic cell survival, which was determined without a return to isotonic conditions, was almost independent of time of exposure to hyperosmotic concentrations, the post-hypertonic survival after returning to isotonic conditions decreased with increasing exposure time, suggesting that the rehydration-induced injury was a consequence of time-dependent alteration of the plasma membrane. The post-hypertonic survival was lower for higher NaCl concentrations and higher temperatures, which was qualitatively consistent with previous studies. Effects of the rate of concentration change on the post-hypertonic cell survival were observed at 4.5 M; the highest rate of survival was obtained by slower increase and faster decrease in the NaCl concentration. However, the effect was negligible at 2.5 M.     

Cold and hyperosmolar fluids in canine trachea: vascular and smooth muscle tone and albumin flux

We have studied the effects of liquids of various osmolalities and temperatures on the tracheal vasculature, smooth muscle tone, and transepithelial albumin flux. In 10 anesthetized dogs a 10- to 13-cm length of cervical trachea was cannulated to allow instillation of fluids into its lumen. The cranial tracheal arteries were perfused at constant flow, with monitoring of the perfusion pressures (Ptr) and the external tracheal diameter (Dtr). Control fluid was Krebs-Henseleit solution (KH) with NaCl added to result in a 325-mosM solution (isotonic). Hypertonic solutions were KH with NaCl (warm hypertonic) or glucose (hypertonic glucose) added to result in a 800-mosM solution. All solutions were at 38 degrees C, with isotonic and the hypertonic NaCl solutions also given at 18 degrees C (cold isotonic and cold hypertonic). Fluorescent labeled albumin was given intravenously, and the change in fluorescence in the fluid was measured during each 15-min period. Changing from warm isotonic to cold isotonic decreased Dtr and Ptr. Changing from warm isotonic to warm hypertonic or hypertonic glucose decreased Ptr with no change in Dtr. The cold hypertonic responses were not different from cold isotonic responses. Warm hypertonic solution increased albumin flux into the tracheal lumen over a 15-min period to three times that of the control period, persisting for 15 min after replacement with warm isotonic solution. Cooling induces a vasodilation and smooth muscle contraction of the trachea, whereas hypertonic solutions result in vasodilation and, if osmolality is increased with NaCl, an increase in albumin flux into the tracheal lumen.     

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (P_f) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the ‘PfFit’), to yield P_f.     

About the characters of thermoprotection in influence of osmolites on bacteria

The influence of the length of staying of Escherichia coli B/r cells in hypertonic NaCl solution before heating at 52 and 60 degrees C on the magnitude of salt thermoprotection was investigated. In addition, the dependence of the isotonic and thermoprotective NaCl concentrations on the exposure temperature was investigated. It was shown that the volume of cell osmotic thermoprotection was independent on the length of preliminary staying of microorganisms in hypertonic NaCl solution. It was also shown that the magnitude of isotonic and thermoprotective osmolite concentrations increased with the increase in the exposure temperature. The analysis of the data obtained and published in literature indicates that the compensating mechanism is involved in salt bacteria thermoprotection rather than the dehydratation one.     

Surface changes induced by osmotic stress and its influence on the glycerol permeability in lipid bilayers

The penetration rate of glycerol across lipid bilayers can be assayed dispersing liposomes filled with a 0.1 M glucose solution in an isotonic or a hypertonic solution of glycerol. The kinetic of glycerol permeation is found to be different in each of those cases. Liposomes dispersed above the phase transition temperature in hypertonic solutions show an increase in the surface polarization as measured by means of merocyanine 540. Under this condition, the permeation of glycerol shows a two-step kinetic which is indicative of a non-fickean diffusion process. In contrast, liposomes dispersed in isotonic solutions of the permeant show a fickean behavior. The changes in polarization of the membrane interface are ascribed to variations in the surface potential due to the osmotic collapse and the glycerol concentration in contact with the outer surface. The permeability of polar molecules can, in consequence, be considered as a function of the surface potential of the liposome which is congruent with previous data in literature reporting that water permeability increases as a function of the zeta potential of liposomes shrunken in hypertonic solutions.     

Caffeine- and Potassium-Induced Contractures of Frog Striated Muscle Fibers in Hypertonic Solutions

The effect of hypertonic solutions on the caffeine- and KCl-induced contractures of isolated fibers of frog skeletal muscle was tested. Hypertonic solutions, twice the normal osmotic strength, prepared by adding NaCl or sucrose, potentiate the caffeine-induced contractures. The fibers may develop tensions of 3.6 kg/cm² of fiber transverse section. The same hypertonic medium reduced the peak tension of KCl-induced contractures. Thus the hypertonic condition does not affect the contractile mechanism itself. These findings give further support to the view that the differential effect of hypertonic solution is on the excitation-contraction coupling mechanism. Extracellular calcium is not essentially required for the first few of a series of caffeine-induced contractures either in hypertonic or in isotonic solutions.     

Hemolysis of human red blood cells by freezing and thawing in solutions containing polyvinylpyrrolidone: relationship with postthypertonic hemolysis and solute movements

An investigation was made into the effects of the presence of polyvinylpyrrolidone (PVP) on changes in human red blood cells suspended in hypertonic solutions, on posthypertonic hemolysis, and on freezing at temperatures down to ?12 °C.PVP is very effective at reducing hemolysis when the red blood cells are frozen at temperatures down to ?12 °C. However, the membranes of the cells recovered on thawing have become very permeable to sodium and potassium ions and there is a much increased hemolysis if the cells are resuspended in an isotonic solution of sodium chloride.The presence of PVP does not affect the dehydration of the cells or the development of a change in membrane permeability when the cells are shrunken in hypertonic solutions at 0 °C. Neither does its presence in the hypertonic solution reduce the extent of posthypertonic hemolysis at 4 °C (as measured by the hemolysis on resuspension in an isotonic solution of sodium chloride), but it is more effective than sucrose at reducing hemolysis when present in the resuspension solution. It is concluded that the PVP is able to prevent swelling and hemolysis of cells which are very permeable to cations by opposing the colloid osmotic pressure due to the hemoglobin. However, this does not explain how PVP is able to protect cells against freezing damage at high cooling rates, and a mechanism by which it might do this is discussed.     

Osmoregulation in red blood cells of Bufo viridis

The effect of hyperosmotic solution of NaCl, urea and mannitol on Bufo viridis red blood cells were studied. The percentage of water content in B. viridis red blood cells decreased significantly in NaCl and mannitol hypertonic solutions compared to urea hypertonic solution. The urea concentration found in red blood cells in a urea hypertonic solution was significantly higher than in red blood cells acclimated to NaCl and mannitol hypertonic solutions. The Na+ concentration was significantly lower in red blood cells immersed in urea hypertonic solution than in red blood cells immersed in hypertonic NaCl and mannitol solutions. However, the K+ concentration increased at a similar rate in three different hypertonic solutions.     

The effect of media tonicity on Escherichia coli resistance to heating with different gradient

The influence of NaCl water solutions and glycerine hypertonic concentration on the survival of bacteria Escherichia coli B/r heated with different values of heat drop was investigated. It was shown that the transfer of cell suspensions from isotonic conditions to media with raised osmotic pressure, preliminarily heated up to 60 degrees C, and the following heating at this temperature inhibited differences in cell sensitivity to heating at different heat drop. Unlike, it was found that the transfer of cell suspensions from isotonic conditions to hypertonic media before and after heating at 60 degrees C increased differences in resistance of these microorganisms to heating at different heat drop. It is proposed that different resistance of bacteria to damaging action of hyperthermia at different heat drop, and a modified influence of hypertonic solutions on these differences may be due to heat induced destabilization of cell osmotic homeostasis. The extent of expression of this destabilization may be determined by a quantitative ratio of osmotic pressure values in the cell-suspension medium system in particular temperature and tonic environmental conditions.     

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.     

Oxytocinergic and serotonergic systems involvement in sodium intake regulation: satiety or hypertonicity markers?

Previous studies demonstrated the inhibitory participation of serotonergic (5-HT) and oxytocinergic (OT) neurons on sodium appetite induced by peritoneal dialysis (PD) in rats. The activity of 5-HT neurons increases after PD-induced 2% NaCl intake and decreases after sodium depletion; however, the activity of the OT neurons appears only after PD-induced 2% NaCl intake. To discriminate whether the differential activations of the 5-HT and OT neurons in this model are a consequence of the sodium satiation process or are the result of stimulation caused by the entry to the body of a hypertonic sodium solution during sodium access, we analyzed the number of Fos-5-HT- and Fos-OT-immunoreactive neurons in the dorsal raphe nucleus and the paraventricular nucleus of the hypothalamus-supraoptic nucleus, respectively, after isotonic vs. hypertonic NaCl intake induced by PD. We also studied the OT plasma levels after PD-induced isotonic or hypertonic NaCl intake. Sodium intake induced by PD significantly increased the number of Fos-5-HT cells, independently of the concentration of NaCl consumed. In contrast, the number of Fos-OT neurons increased after hypertonic NaCl intake, in both depleted and non-depleted animals. The OT plasma levels significantly increased only in the PD-induced 2% NaCl intake group in relation to others, showing a synergic effect of both factors. In summary, 5-HT neurons were activated after body sodium status was reestablished, suggesting that this system is activated under conditions of satiety. In terms of the OT system, both OT neural activity and OT plasma levels were increased by the entry of hypertonic NaCl solution during sodium consumption, suggesting that this system is involved in the processing of hyperosmotic signals.     

Effect of osmotic shock on the viability, optical density and permeability of heated Escherichia coli cells

The object of this work was to study the effect of a short incubation in 0.01 M tris buffer, pH 7.0, with a different NaCl content (0-10%) on the viability, optic density and permeability of intact and heated at 52 degrees C Escherichia coli B/r cells. In contrast to the intact cells, the viability of the heated cells depended on osmotic pressure in the medium into which they were transferred after heating. The survival rate was highest when the cells were transferred into an isotonic buffer. In the case of hypotonic and hypertonic media, the survival rate of the cells decreased owing to the death of cells which were responsible for the formation of small colonies under the isotonic conditions. This was accompanied with a more intensive drop in the optic density of bacterial suspensions while their permeability increased (when the cells were transferred into the hypotonic conditions). The role of membranes in the processes of bacterial heat inactivation is discussed on the basis of the results obtained.     

A new transfection method of mouse FM3A mammary carcinoma cells with plasmid DNA

High-frequency transfection of mouse FM3A cells with plasmid pSV2neo DNA was achieved by incubation of the cells with DNA plus polybrene for 6 hours followed by an osmotic shock with hypertonic NaCl solution. When incubated for 20 min at 34 degrees C, FM3A cells showed resistance to the osmolarity change from 0.1 to 9.0% NaCl in the medium. Within this range of NaCl concentration, 5-7% gave the highest efficiency of transfection. Both linear and circular forms of plasmid DNA produced transformants with equal efficiency. This method was simple, reproducible and carrier DNA was not required. The efficiency was about 100 times higher than that of the widely used method with DNA-calcium phosphate precipitates. Transformed cells were stable and different numbers of plasmid DNA copies were detected with different restriction sites.     

The chemical composition and the osmotic pressure of the aqueous humor and plasma of the rabbit

Measurements were made of the osmotic pressure of plasma, and of aqueous humor taken from the anterior chamber of the right and left eyes and from the posterior chamber of unanesthetized rabbits. Aqueous humor from the anterior chamber was found to be hypertonic to the plasma by approximately 3 mM/liter equivalent of sodium chloride. The aqueous humor from the anterior and posterior chambers of the right and left eyes was isotonic. The concentration of chloride in the anterior and posterior chambers was the same. The concentration of all the major components of the aqueous humor and plasma has been determined by chemical analysis on fluid samples obtained from unanesthetized rabbits at approximately the same time. The calculated osmotic pressure of the total of these substances in terms of sodium chloride equivalent agrees to within better than 1 per cent of the total osmotic pressure as measured experimentally. The distribution of some individual anions and cations of the aqueous humor and plasma was determined. This distribution is widely different from that which would obtain at a state of equilibrium. The positive and negative charges carried by the ions in the aqueous humor were approximately equal. Sources of error in the experiments are discussed.     

Hyperosmotic injury in mammalian cells 2. Surface alterations of CHO cells in unprotected and DMSO-treated cultures

Chinese hamster ovary (CHO) cells exposed for up to 2 hr to hypertonic Eagle's MEM were examined for surface changes by scanning electron microscopy. Cells hypertonically stressed in the presence of DMSO were identically monitored. In media supplemented with NaCl, little changes were observed below 2600 mosm. Above this level there is a concentration-and time-dependent disappearance of surface microvilli and blebs as well as a reduction in the plasma membrane ruffling activity. At hyperosmolalities of about 3000 mosM the cells became devoid of any surface projections and demonstrated multiple blister-like patches. In 20 × isotonic solution, the cells are flattened, the usual surface projections are absent, and the membrane appears to be full of pits and holes, giving the cells the appearance of an overcooked fried egg. In hypertonic sucrose the surface of CHO cells developed a gradual thickening and agglutination of surface components at tonicities of about 5000 mosm.DMSO at 5 or 10% (w/v) concentration considerably mitigated the surface alterations of hypertonically NaCl stressed cells. Enzymatic digestions with pronase simulated some of the changes seen with hyperosmotic salt. Neuraminidase treatment produced no demonstrable change in surface topography.     

High-frequency transfection of mouse FM3A mammary carcinoma cells in suspension culture with plasmid DNA

High-frequency transfection of mouse FM3A cells, grown in suspension, with plasmid pSV2neo DNA was achieved by incubation of the cells with DNA plus polybrene for 6 h followed by an osmotic shock with a hypertonic NaCl solution. When incubated for 20 min at 34 degrees C, FM3A cells showed resistance to the osmolarity change from 0.1 to 9.0% NaCl in the medium. Within this concentration range, 5-7% gave the highest efficiency of transfection. Both linear and circular forms of plasmid DNA produced transformants with equal efficiency. This method was simple, reproducible, and carrier DNA was not required. The efficiency was about 100 times higher than that of the method with DNA-calcium phosphate precipitates. Transformed cells were stable and different numbers of plasmid DNA copies were detected.     

Deglycerolization of red blood cells: A new dilution-filtration system

In this work, we present a new version of the dilution-filtration system for rapidly deglycerolizing a large volume of cryopreserved blood. In our earlier system, one of the major problems was the damage induced to the red blood cells (RBCs) due to high osmolality change at the dilution point. Therefore, we devised a new system to solve this problem. First, we theoretically simulated the osmolality variation in the new system and the variation of the maximum and minimum volumes of the RBCs at the dilution point to examine the effects of operating parameters/conditions. Next, we experimentally validated the effects of these operating parameters by deglycerolizing porcine blood. The results show that when the initial NaCl concentration in the hypertonic solution is 18%, the volume of the hypertonic solution is 200 mL, and the flow rate of the filtrate is 50 mL/min, the system can effectively remove glycerin from 200 mL of porcine blood in 30 min, with ∼87% RBC survival rate and ∼73% RBC recovery rate. Our results indicated that in the new system the concentration and the volume of the hypertonic solution used to dilute the blood are the important parameters that need to be adjusted to reduce osmotic damage to the RBCs. In addition, a fast filtrate flow rate is highly recommended. This work can significantly contribute to the development of a more efficient and effective system for deglycerolizing large volumes of cryopreserved blood in clinic.     

Effect of hypertonicity on survival of unprotected human cultured cells following freezing and thawing

An investigation was carried out on the post-thaw survival of unprotected human heteroploid EUE cells, either maintained in isotonic medium (0.137 M NaCl) or adapted to hypertonicity (0.356 M NaCl) and frozen in medium with an increased concentration of NaCl. A fivefold increase in the survival fraction of the adapted cells in comparison with the unadapted ones was observed when cells were frozen in isotonic medium. When cells were frozen in hypertonic medium (0.356 M NaCl), the two cell types exhibit comparable survival values. The results are discussed, with special attention to cell defense mechanisms against freezing injury.     

The effects of various salt and sucrose solutions on the U.V.L. sensitivity of CHO cells.

The response of cultured CHO cells to U.V.L. irradiation during treatment with anisotonic solutions shows that treatment with hypotonic sucrose, NaCl or KCl solutions causes an increase in the cellular U.V.L. sensitivity, while exposure to hypertonic solutions causes a large decrease in U.V.L. sensitivity. Cells exposed to 1.8 M sucrose, NaCl or KCl solutions and given a U.V.L. dose of 252 erg/mm2 towards the end of the 20 min solution exposure time have survival levels which are respectively 228,26, and 23 times higher than the controls, i.e. cells irradiated in phosphate buffered saline. Cell volume data obtained using a Coulter counter, and nuclear area data of attached cells obtained using an optical microscope with a micrometer reticle, show that cell and nuclear size are related to U.V.L. sensitivity. That is, as cells shrink and the nuclear area decreases, the cells become more U.V.L.-resistant. During hypotonic treatment with 0.1 M NaCl, the cell volume, nuclear area and U.V.L. sensitivity increased in the first 2 to 4 min of exposure time, but at longer exposure times (greater than 3 to 4 min), cell volume, nuclear area and cellular U.V.L. sensitivity decreased. For 0.1 M KCl treatment the cells initially displayed a rapid increase in volume, nuclear area and U.V.L. sensitivity, but at the longer exposure times no decrease in cell and nuclear size were observed, and a slight increase in U.V.L. sensitivity occurred. Changes in U.V.L. sensitivity were related to changes in nuclear size and cell volume; however, calculations showed that during hypertonic treatment there is an ionic effect as well as an osmotic effect. That is, the cellular U.V.L. survival in equal hypertonic concentrations of NaCl or KCl was lower than in the same concentration of sucrose.