Hyperosmotic injury in mammalian cells. Survival of CHO cells in unprotected and DMSO-treated cultures

Cultures of CHO cells growing as monolayers or held in suspension were exposed to increased concentrations of NaCl in Eagle's MEM. Analysis of posthypertonic survival indicated that the cells' ability to withstand hyperosmolal (596–7550 mosm) environment is anchorage-dependent, cells in suspension being extremely sensitive to hyperosmotic conditions. By trypsinizing monolayers at various intervals during hypertonic exposure (3300 or 7550 mosm), the existence of a repair mechanism has been revealed. The repair is abolished by inhibitors of RNA or DNA synthesis. Media made hyperosmolal with nonpenetrating sugars (sucrose, sorbitol) do not elicit a repair process. DMSO at 5 or 10% levels is able to prevent the hypertonic killing of cells growing as monolayers. Cells in suspension which have been allowed to recover from the trypsin action, but not the freshly trypsinized cells, were also protected by DMSO. The DMSO effect seems to be mediated by the presence, probably at the membrane level, of a trypsin-removable material (glycoprotein?).     

Parallel effects of freezing and osmotic stress on the ATPase activity and protein composition of the plasma membrane of winter rye seedlings

The objective of this study was to determine the influence of freezing versus hypertonic stress on the ATPase activity and polypeptide profile of the plasma membrane of nonacclimated winter rye leaves (Secale cereale L. cv Puma). Exposure of leaves to hypertonic sorbitol solutions resulted in a similar extent of injury as did freezing to subzero temperatures that resulted in equivalent osmotic stresses. When isolated with a two-phase partition system of aqueous polymers, the plasma membrane fractions of control, frozen, or hypertonically stressed leaves were of similar purity as judged by the distribution of marker enzyme activities. When assayed in the presence of Triton X-100 (0.05% w/w), ATPase activity was decreased only slightly in plasma membrane fractions isolated from either frozen or hypertonically stressed leaves. In contrast, the specific ATPase activity of the plasma membrane fractions assayed in the absence of Triton X-100 increased following freezing or hypertonic stress. As a result, the Triton X-100 stimulation of the ATPase activity decreased significantly from sixfold in control leaves to threefold in lethally stressed leaves and reflects an increase in the permeability of the plasma membrane vesicles. The increased permeability was also manifested as a decrease in H⁺-transport following exposure to freezing or hypertonic stress. Both freezing and hypertonic exposure at subzero temperatures altered the polypeptide profile of the plasma membrane, but with the exception of one polypeptide, there was no difference between the two treatments.     

Effect of hypertonic stress on mammalian cell lines and its relevance to freeze-thaw injury

The effect of subjecting the mammalian cell lines MRC-5 and CHO to hypertonic salt concentrations (0.16 to 2.4 m) and returning them to isotonic conditions was investigated. Parameters for measuring cell size, viability and release of radiochemical markers were used to determine the relative susceptibilities of the two cell lines to hypertonic stress and the relative effects of increasing and decreasing hypertonicity. The aim of this study was to determine how great a role hypertonic stress plays in freeze-thaw damage of mamalian cells. This type of study has been extensively used for erythrocytes but not for nucleated mamamlian cell lines.The findings were that considerable cell shrinkage occurred, with a minimum size at 0.6 m NaCl, but that this caused no cell injury or death. Injury, measured by cation leakage and release of membrane and cytoplasmic labels occurred whilst the cell was swelling after reaching its minimum volume. MRC-5 cells succumbed at relatively low salt concentrations and became denatured. CHO cells withstood far high salt concentrations but were then damaged during dilution back to isotonic conditions. Comparison of the data obtained from hypertonic stress experiments and freeze-thaw experiments showed many similarities for CHO cells and indicated that the cell membrane could withstand high salt concentrations both at constant and changing temperatures but were prone to injury on dilution back to isotonic conditions. MRC-5 cells were shown to be very prone to cold shock and the results indicated that they probably succumb to damage and death during the hypertonic phase of cooling rather than thawing thus explaining their much lower survival from freeze-thaw experiments than CHO cells. The influence of DMSO in delaying cell damage to higher salt concentrations and lessening disruptive swelling during dilution were also demonstrated.     

Cold shock hemolysis in human erythrocytes studied by spin probe method and freeze-fracture electron microscopy.

When human erythrocytes are osmotically stressed or chemically treated, they hemolyze on cooling below 10 degrees C (called cold shock). We have studied the effects of osmotic stress and cooling on the state of membrane by the spin-probe method and freeze-fracture electron microscopy. At room temperature, the membrane fluidity detected by 12-doxyl stearate spin probe showed a steady decrease with osmolality in hypertonic NaCl solutions up to 900 mOsm/kg, above which it remained unchanged. In hypertonic sucrose solutions, the electron paramagnetic resonance spectra showed an additional pair of absorptions, indicating development of regions, in the membrane, further immobilized than in NaCl solutions. Mobility of a cholesterol analogue probe, androstane, did not show change by hypertonicity, but the spectral intensity dropped at 1,200 mOsm/kg, probably due to formation of loose aggregates in the cholesterol phase. On cooling the osmotically stressed cells in NaCl solution, the isotropic rotational correlation time vs. inverse temperature plot of 12-doxyl stearate probe exhibited a step-wise discontinuity at approximately 10 degrees C, suggestive of a drastic transition in the state of the membrane. At about the same temperature, the freeze-fracture pattern of osmotically stressed cells revealed the development of large wrinkles and aggregation of membrane particles, in contrast to the case of the cells in isotonicity. Significance of these findings in understanding cold shock hemolysis is discussed.     

Hypertonic cryohemolysis of human red blood cells

Summary Hypertonic cryohemolysis of human erythrocytes is caused by incubation of the cells in hypertonic medium at a temperature of 20–50°C (stage 1), with subsequent cooling to 0°C (stage 2). In 0.86m sucrose hemolysis increases, with increasing stage 1 temperature, whereas in 1m NaCl cryohemolysis has a temperature optimum at a stage 1 temperature of about 30°C.Cryohemolysis is inhibited by preceding ATP depletion of the cells and bypreincubation of the cells in hypertonic medium at 0°C. In general, anesthetics inhibit cryohemolysis strongly. Only in 1m NaCl at stage 1 temperatures in the range of 40–50°C is cryohemolysis stimulated by these drugs, if present during the entire incubation period. This effect is abolished, however, when the anesthetic is added after piror incubation of the cells at 40–50°C in 1m NaCl.Ghost-bound ANS fluorescence indicates complicated conformation changes in the membrane structure during the various experimental stages leading to cryohemolysis.Some of the experimental results can be considered as examples of molecular hysteresis, thus indicating several different metastable structures of the membrane, under various experimental conditions.The described results support the working hypothesis of Green and Jung that the experimental procedure results in membrane protein damage, preventing normal adaptation of the membrane during cooling.     

Induction of aldose reductase expression in rat kidney mesangial cells and Chinese hamster ovary cells under hypertonic conditions

Rat kidney cortex mesangial cells (MES) and Chinese hamster ovary cells (CHO) responded to hypertonicity (600 mosmol/kg) in culture by accumulating sorbitol. The accumulation of sorbitol was due to increased aldose reductase (AR) activity, apparently brought about by increased levels of AR mRNA and protein. The levels of AR mRNA increased approximately 60-fold in MES cells and 30-fold in CHO cells by 24 h in culture media (300 mosmol/kg supplemented with 150 mM NaCl, 600 mosmol/kg total). AR activity also markedly increased (14- to 16-fold above control), but MES took 4 days and CHO 6 days to reach this maximum. Other osmolytes, raffinose and sorbitol (at concentrations of 250 to 300 mM) elicited the same response as that of 150 mM NaCl. These data show that AR expression is induced in MES and CHO cells under hypertonic conditions. Of special interest is the induction of large amounts of AR in rat kidney cortex mesangial cells, a target tissue of diabetes and a site where excessive accumulation of sorbitol is suspected to be a critical factor in diabetic nephropathy.     

Effect of hypertonicity on hexose transporter regulation in chicken embryo fibroblasts

The regulation of hexose transporters of cultured fibroblasts was investigated by exposing chicken embryo fibroblasts (CEF) to hypertonic culture medium, a condition known to enhance hexose transport activity. The effects of hypertonicity and the role of protein synthesis were examined with CEF in the basal (glucose fed) and transport enhanced (glucose starved) states. Glucose-fed CEF exposed to hypertonic conditions developed four-fold enhancement of hexose transport activity within 4 hrs; this declined in the following 20 hrs to a level slightly higher than the fed control. Protein synthesis was required in part for this effect, since the presence of cycloheximide during hypertonic exposure of fed CEF blocked the increase in of transport by almost 50%. Although the increased transport produced by glucose starvation was not further enhanced by hypertonicity, hypertonic treatment of starved CEF during glucose refeeding largely prevented the loss of transport activity to the basal, fed state. The hypertonic effects were concentration dependent (240mOsm optimal) and could be elicited with NaCl, KCl, or sucrose. Hypertonic treatment typically led to a greater than 50% decline in the incorporation of [3H]leucine into acid-insoluble fractions. The changes in transport were evident at the plasma membrane level, and studies of membrane vesicles prepared from hypertonically treated fed CEF showed a doubling of both [3H]cytochalasin B binding and the Vmax of D-glucose transport. These findings indicate that exposure of CEF to hypertonic conditions has some effects similar to those produced by glucose starvation and suggest that protein synthesis is to some extent involved in the regulation of hexose transporters in CEF.     

Postfreeze viability of human renal epithelial carcinoma cells related to phase transformations in ternary solutions

The postfreeze viability of human renal epithelial carcinoma cells frozen in solutions based on a complex physiologic support medium to which additions of NaCl and a cryoprotective agent, either glycerol or dimethyl sulfoxide (DMSO) were made, have been determined by a dye exclusion technique. The support medium consisted of either Eagle's Minimum Essential Medium with Hanks' salts added (MEM) or this same medium supplemented with 20 vol% heat-inactivated fetal calf serum (MEM + FCS). Glycerol was found to be an ineffective cryoprotective agent for these cells, while DMSO was highly effective. Addition of NaCl along with the DMSO further improved the viability of cells frozen at −196 °C. Freezing and thawing rates were found to be important with a slow freezing rate, 2.5 °C/min, and a rapid thawing rate, 240°C/min, yielding the best results.Maximum viability occurred in solutions containing 80 to 95 wt.% (MEM + FCS) with the balance being DMSO and NaCl in the weight ratio of 9:1. In addition to primary ice formation, two nonequilibrium glassy phases were observed during DTA studies of these solutions (10). The exintence of these vitreous states reduces the chances thet cells will be exposed to hypertonic concentrations of salt in the extracellur fluids during freezing-out of primary ice.     

Morphological alterations in mammalian neurons in vitro in response to hypertonic solutions

Summary Neurons in cultures of central nervous tissue exhibited marked structural changes when exposed to hypertonic solutions. Cellular reactions were described in living neurons as well as after fixation and staining in preparations observed with both the light and electron microscope. The structures involved in these changes were mainly the nucleolus, the nucleus and the Nissl substance.Nucleolus In living neurons, observed with phase contrast optics, the nucleolus became invisible in hypertonic medium. This change occurred within a few seconds, and it was reversible when the cells were brought back to isotonic solutions. Fixation of the cells while exposed to hypertonic solution caused the nucleolus to reappear as a granular body. In stained preparations it appeared as a more irregular body in contrast to the smoothly outlined nucleolus in normal cells. In electron microscopic preparations of neurons which were fixed while exposed to hypertonic solutions the nucleolus was visible only as nucleolar shadow, overlaid by a few small irregular bodies of higher electron density than other nuclear contents.Nucleus The nuclear membrane of living neurons exposed to hypertonic media lost much of its sharp definition and became rather hazy in outline. The nuclear diameter increased about 10% in hypertonic medium, and the nuclear space became somewhat denser when observed with the phase contrast microscope. In Nissl stained preparations the nuclear space was filled with many small granular or rod-shaped bodies in contrast to the clear vesicular appearance of the nuclei of untreated cells. In electron microscopic preparations the nuclear space exhibited a spotty appearance due to the presence of electron dense and light areas.Nissl Substance In living neurons immersed in hypertonic solutions the Nissl substance showed a slight increase in phase density, especially after repeated changes between hypertonic and isotonic solutions. Sometimes a distinct striation in the Nissl substance appeared. In Nissl stained preparations there was no marked change observed in comparison with normal cells. However, in the electron microscope, the Nissl substance of hypertonically treated cells exhibited a marked structural change. The membrane-bound spaces of the endoplasmic reticulum assumed a rather precise orientation parallel to the cell membrane so that in extreme cases a concentric arrangement of endoplasmic cisternae was observed. The normal arrangement of ribosomal granules in rosettes and clusters became disturbed and the granules were more uniformly distributed.The cells as whole units showed a distinct shrinkage in hypertonic solution which may account for the more crowded appearance of various organelles such as mitochondria and Golgi complexes. There was also a marked increase in agranular reticulum profiles and small membrane bound vesicles in treated cells. Vacuoles appeared frequently in the cytoplasm of treated cells; they disappeared upon re-immersion in isotonic medium.This investigation was supported by USPHS Grants NB 03114-04, NB 00690-11 and 5 T 1 GM 495 from the National Institutes of Health, Bethesda, Maryland.Acknowledgement. Mrs. Eleanor W. Morris and Mr. Edwin E. Pitsinger, Jr. gave indispensible aid with the management of the cultures and with photographic procedures.     

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.     

SNAT2 silencing prevents the osmotic induction of transport system A and hinders cell recovery from hypertonic stress

Under hypertonic conditions the induction of SLC38A2/SNAT2 leads to the stimulation of transport system A and to the increase in the cell content of amino acids. In hypertonically stressed human fibroblasts transfection with two siRNAs for SNAT2 suppressed the increase in SNAT2 mRNA and the stimulation of system A transport activity. Under the same condition, the expansion of the intracellular amino acid pool was significantly lowered and cell volume recovery markedly delayed. It is concluded that the up-regulation of SNAT2 is essential for the rapid restoration of cell volume after hypertonic stress.     

Internalization-competent influenza hemagglutinin mutants form complexes with clathrin-deficient multivalent AP-2 oligomers in live cells

Most membrane proteins are endocytosed through clathrin-coated pits via AP-2 adaptor complexes. However, little is known about the interaction of internalization signals with AP-2 in live cells in the absence of clathrin lattices. To investigate this issue, we employed cells cotransfected with pairs of antigenically distinct influenza hemagglutinin (HA) mutants containing different internalization signals of the YXXZ family. To enable studies on the possible association of the naturally trimeric HAs into higher order complexes via binding to AP-2, we exploited the inability of HAs from different influenza strains to form mutual trimers. Thus, we coexpressed HA pairs from different strains (Japan and X:31) bearing similar cytoplasmic tails mutated to include internalization signals. Using antibody-mediated immunofluorescence co-patching on live cells, we demonstrate that internalization-competent HA mutants form higher order complexes and that this clustering depends on the strength of the internalization signal. The clustering persisted in cells treated with hypertonic medium to disperse the clathrin lattices, as validated by co-immunoprecipitation experiments. The clustering of HAs bearing strong internalization signals appears to be mediated via binding to AP-2, as indicated by (i) the coprecipitation of alpha-adaptin with these HAs, even in hypertonically treated cells; (ii) the co-localization (after hypertonic treatment) of AP-2 with antibody-mediated patches of these mutants; and (iii) the dispersal of the higher order HA complexes following chlorpromazine treatment, which removes AP-2 from the plasma membrane. These results suggest that even in the absence of clathrin lattices, AP-2 exists in multivalent complexes capable of simultaneously binding several internalization signals from the same family.     

Differential repair of potentially lethal damage in exponentially growing and quiescent 9L cells

The alteration of potentially lethal damage repair by postirradiation treatment with hypertonic saline (0.5 M PBS) was investigated in exponentially growing and quiescent 9L cells in vitro. A single dose of X rays (8.5 Gy) immediately followed by a 30-min treatment with hypertonic PBS at 37 degrees C reduced the survival of exponentially growing 9L cells by a factor of 13-18 compared to survival of irradiated immediately and delayed-plated cells, while the survival of quiescent cells was reduced by only a factor of 5-8. Survival curves confirmed the relative resistance of the quiescent 9L cells versus exponentially growing 9L cells to X rays plus hypertonic treatment. Both the slope and the shoulder of the survival curve were reduced to a greater extent in exponentially growing cells than in the quiescent cells by hypertonic treatment. The response of quiescent cells cannot be explained by either the duration of hypertonic treatment or the redistribution of the cells into G1 phase. We show that quiescent 9L cells can recover from hypertonically induced potentially lethal damage when incubated under conditions which have been found to delay progression through the cell cycle, and postulate that an altered chromatin structure or an enhanced repair capacity of quiescent 9L cells may be responsible for their resistance.     

Effect of anisotonic NaCl treatment on cellular ultrastructure of V79 Chinese hamster cells. Part 1. Mitotic cells

The ultrastructural modifications produced by anisotonic NaCl treatment of Chinese hamster mitotic cells were observed at three NaCl concentrations which have been frequently used in radiosensitization studies: 0.05, 0.5 and 1.5 M. After exposure to 0.05 M NaCl, many well-spread chromosomes are visible. The chromatin fibres are well dispersed and membraneous material is associated with the chromosomes. After hypertonic treatment with 0.5 M NaCl, the chromosomes have a uniform, structureless appearance with some coalescing into larger anaphase-like masses. At 1.5 M NaCl, large scale cellular dehydration is apparent, and filamentous structures such as microfilaments are tightly constricted. The degree of chromosome staining is also reduced below the level of the cytoplasm. After both hypo- and hypertonic NaCl treatment the chromosomes appear swollen relative to untreated cells, but hypertonic treatment causes chromosome clumping and dissociates chromatin. Conformational changes in the chromatin may restrict the capacity for DNA repair and be related to cellular radiosensitivity.     

Hyperosmotic relaxation lysis of chromaffin granules is caused by interactions between the granular membrane and intragranular vesicles

Bovine chromaffin granules undergo irreversible structural changes during osmotic shrinkage in hypertonic sucrose and salt solutions, such that, on reexposure to isoosmotic conditions they do not regain their original morphology, but undergo lysis ('hyperosmotic relaxation lysis'). Irreversible alterations of granules were induced by hypertonic incubations lasting for as little as 1 min. Fluorescence and EPR membrane labelling experiments showed that hypertonicity did not induce membrane loss for instance by inwardly or outwardly directed pinching off of membrane material. The mean sizes of chromaffin granules as a function of increasing and subsequently decreasing osmotic pressure were measured by photon correlation spectroscopy; there was no significant difference in sizes of hyperosmotically pretreated granules as compared with controls. Freeze-fracture electron micrographs showed the formation of 'twins' and 'triplets' under hypertonic conditions. They also revealed intragranular vesicles of 50-200 nm in diameter in both hypertonically and isotonically suspended granules. 'Twin' and 'triplet' granules were formed by the attachment of intragranular vesicles to the granule membranes. We suggest that hyperosmotic relaxation lysis is caused by the fact that this adhesion partly prevents the granule membrane from reexpanding, thus, leading to its rupture.     

二甲基亚砜作用下重组CHO细胞胞内乙型肝炎表面抗原的定位

在培养环境中添加二甲基亚砜 (DMSO) 能分别提高重组 CHO 细胞乙型肝炎表面抗原（HBsAg）的产量和比生产速率70%和3.2倍以上, 但同时发现胞内HBsAg 的积累量是对照组的7.2倍。为了分析胞内HBsAg 积累的区域,采用电镜技术分析后发现,经DMSO处理后的CHO细胞胞内出现了很多的扩张区域,这些扩张区域分布整个胞浆,有的扩张区域已经侵蚀到细胞核上,而对照组未发现明显的扩张区域。进一步利用免疫电镜技术分析后发现,经DMSO处理后的细胞胞内大量积累的HBsAg主要分布在这些扩张区域中,同时发现在细胞核膜上也有分布,这可能是由于扩张区域侵蚀细胞核造成的。以上工作有助于揭示在DMSO作用下重组CHO细胞胞内HBsAg大量积累的机制。     

Effects of osmolarity on taste receptor cell size and function

Osmotic effects onsalt taste were studied by recording from the rat chorda tympani (CT)nerve and by measuring changes in cell volume of isolated rat fungiformtaste receptor cells (TRCs). Mannitol, cellobiose, urea, or DMSO didnot induce CT responses. However, the steady-state CT responses to 150 mM NaCl were significantly increased when the stimulus solutions alsocontained 300 mM mannitol or cellobiose, but not 600 mM urea or DMSO.The enhanced CT responses to NaCl were reversed when the saccharideswere removed and were completely blocked by addition of 100 µMamiloride to the stimulus solution. Exposure of TRCs to hyperosmoticsolutions of mannitol or cellobiose induced a rapid and sustaineddecrease in cell volume that was completely reversible, whereasexposure to hypertonic urea or DMSO did not induce sustained reductionsin cell volume. These data suggest that the osmolyte-induced increasein the CT response to NaCl involves a sustained decrease in TRC volumeand the activation of amiloride-sensitive apicalNa⁺ channels.

     

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.