Modifying effect of concanavalin A and diamide on erythrocyte sensitivity to cold shock]

The temperature (0 degrees C and 37 degrees C) and the medium tonicity (0.15-1.20 M NaCl) were shown to affect erythrocyte agglutination by concanavalin A. Treatment of cells with lectin caused no significant decrease in the erythrocyte hemolysis upon cooling. Diamide, unlike concanavalin A used at concentrations above 2.0 M decreases the cell sensitivity to the cold shock. The changes in the erythrocyte susceptibility to cooling within the temperature range of 37-0 degrees C correlate with changes in the electrophoretic spectrum of membrane proteins. The progressive decrease in the spectrin bands intensity with a simultaneous formation of high molecular weight protein aggregates not included in the gel composition was observed after diamide treatment. The diamide effect depends on the medium tonicity, at which the treatment was performed, being especially well pronounced in hypertonic media with 0.8-1.2 M NaCl concentrations, the maximal spectrin aggregation being observed under these conditions. It is suggested that the main factor of the mechanism underlying the erythrocyte hypertonic cold shock is the increase in the association of peripheral cytoskeleton proteins with plasma membrane in osmotically dehydrated cells which limits the ability of lipids to adapt during cooling and results in the stabilization of defects in the membrane structure at low temperatures. Diamide eliminates these unfavourable changes eventually resulting in the dissociation of peripheral proteins from the cytoplasmic surface of the membrane on the protein aggregation.     

Mechanism of erythrocyte cryohemolysis, induced by cationic amphipaths: synergism of induction of the "discocyte-stomatocyte III" transition due to chlorpromazine and medium tonicity]

It is determined that chlorpromazine, a cationic amphipath usually protecting erythrocytes under conditions of hypertonic cryohemolysis is an efficient inductor of the cell lysis in case of cooling in media with tonicity close to the physiological (the isotonic cryohemolysis). Both chlorpromazine and tonicity of the medium influence the alterations in the state of cells, which is confirmed by synergy of the "discocyte-stomatocyte III" transition induction. The above process may be considered as a critical stage of structural modification of erythrocytes. Transition through this stage coincides with appearance of sensitivity to cooling in cells.     

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.     

Recovery of the viability of Chinese hamster V79-4 cells after combined exposure to hyperthermia and radiation

The survival of cells overheated (42 degrees C) before gamma irradiation is increased by holding them in the growth medium at 37 degrees C before treatment with hypertonic NaCl solution. The substantial synergistic effect of hyperthermia and radiation takes place when the cells are treated with a 1.5 M NaCl solution immediately after the combined action of these inactivating factors. The synergistic effect is decreased by holding the cells in the nutrient medium at 37 degrees C for 4 hours before hypertonic treatment.     

Factors influencing survival of mammalian cells exposed to hypothermia. II. Effects of various hypertonic media

Survival of Chinese hamster lung (V79) cells, exposed as a function of time to hypothermia in tissue culture, in isosmotic and various hypertonic media was measured using a colony assay. The mechanism of hypothermic cell killing is different above and below 7 degrees C in this cell line. Addition of NaCl or mannitol to increase the tonicity to 400 mOsm greatly decreased the survival at 10 degrees C while addition of KCl had no significant effect. When these experiments were repeated at 5 degrees C, addition of either NaCl, KCl, or mannitol was detrimental to long-term cell survival. Furthermore, addition of mannitol to the medium did not improve survival when cells were stored at 7 degrees C. Addition of KCl at 5 or 10 degrees C or NaCl at 5 degrees C only affected the cells' ability to accumulate sublethal damage, while addition of mannitol at 5 or 10 degrees C affected both of the above and the cold sensitivity of the cells. Addition of NaCl at 10 degrees C only affected the latter. These experiments suggest that prevention of cell swelling by these conditions, while possibly necessary during clinical hypothermic organ storage, is detrimental to single cell survival at these temperatures.     

Recovery from thermal damage of mammalian cells fixed by treatment with a hypertonic sodium chloride solution

The survival of Chinese hamster cell V79-4 after hyperthermic treatment (42 degrees C, 40 minutes) in the exponential growth phase considerably increases with the duration of holding them in the growth medium at 37 degrees C before hypertonic salt treatment (1.5 M NaCl, 15 minutes). The experimental data are interpreted as a recovery of mammalian cells from thermal lesions, whose lethal action manifests itself at high salt concentrations.     

Apoptosis of murine BW 5147 thymoma cells induced by cold shock.

Exposure of thymoma BW 5147 cells to cold (0-2 degrees C) followed by rewarming at 37 degrees C (cold shock) resulted in internucleosomal DNA cleavage. Sensitivity to cold shock-induced cell death was critically dependent on the serum concentration in the medium and limited to serum-deficient medium (2% serum concentration), whereas cells in the complete growth medium (10%) were completely resistant. RNA/protein-synthesis inhibitors (cycloheximide and actinomycin D) had no effect on cold shock-induced DNA cleavage in BW 5147 cells. The DNA fragmentation seems to be independent of increase in the cytosolic Ca2+ level. Moreover, reduction in the calcium content of the external medium by EGTA induced DNA cleavage. Incubation of BW 5147 cells in the presence of colchicine and cytochalasin B led to the apoptosis. The latter suggests that the internucleosomal DNA cleavage induced by cold shock may be concerned with the disruption of some cytoskeletal network caused by cooling. The results are discussed in relation to cell proliferation.     

The influx of calcium ions into human erythrocytes during cold storage

1. When human erythrocytes, suspended in iso-osmotic sucrose containing CaCl(2), are stored at 3 degrees C, Ca(2+) influx into the cells occurs. Simultaneously, efflux of K(+), Na(+), Cl(-) and water takes place and cell volume diminishes. 2. The extent of Ca(2+) influx increases with duration of cold storage and with increasing concentration of Ca(2+) in the suspending medium. 3. Erythrocytes that have been thus loaded with Ca(2+) exhibit Ca(2+) efflux against a concentration gradient when subsequently incubated at 37 degrees C. 4. Ca(2+) influx likewise occurs when the sucrose of the medium is replaced by iso-osmotic solutions of other non-ionized compounds. 5. Replacement of sucrose by iso-osmotic KCl or NaCl greatly diminishes the rate of Ca(2+) influx during cold storage; however, in iso-osmotic choline chloride, Ca(2+) influx is as rapid as in sucrose. 6. Preincubation of erythrocytes in iso-osmotic sucrose at 37 degrees C causes rapid efflux of K(+) and Na(+) and renders the cell membranes highly permeable to Ca(2+) during subsequent cold storage. 7. Preincubation of erythrocytes in iso-osmotic NaCl at 37 degrees C with trypsin or neuraminidase is without effect on the permeability of the membrane towards Ca(2+). 8. The experimental results lead to the conclusion that the main prerequisite for Ca(2+) influx into erythrocytes is the partial depletion of the cells of their univalent cations.     

Hypertonic cryohemolysis: Ionophore and pH effects

Human erythrocytes suspended at 37 degrees C in hypertonic solution of either electrolytes or nonelectrolytes undergo hemolysis when the temperature is lowered toward 0 degrees C (Green, F.A., Jung, C.Y. 1977 J. Membrane Biol. 33:249). In the present studies this hypertonic cryohemolysis was profoundly affected by the pH of incubation, and was completely abolished at ph 5. In hypertonic NaCl, there was an apparent pH optimum at 6--6.5. In hypertonic sucrose, on the other hand, hemolysis increased progressively with increasing pH between 6 and 9. Amphotericin B inhibited hypertonic cryohemolysis in NaCl or KCl solution. No inhibiting effect of amphotericin B was observed when hypertonicity was due to sodium sulfate or sucrose. Valinomycin also inhibited hypertonic cryohemolysis in KCl, but did not affect the process in NaCl or sucrose solution. SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate) and phloretin interfered with this valinomycin effect, whereas phlorizin did not. These results indicate that dissipation of an osmotic gradient across membranes may be responsible for the inhibition of the hemolysis by these inophores. Iso-osmotic cell shrinkage induced by valinomycin in 150 mM NaCl solution did not result in cryohemolysis.     

Multifaceted freezing injury in human polymorphonuclear cells at high subfreezing temperatures

Extracellular freezing injury at high subzero temperatures in human polymorphonuclear cells (PMNs) was studied with a cryomicroscope, electron microscope, and functional assays (phagocytosis, microbicidal activity, and chemotaxis). There are at least four major factors in freezing injury: osmotic stress, chilling, cold shock, and dilution shock. Extracellularly frozen PMNs lose functions when cooled to -2 degrees C without a cryoprotectant. Cells lose volume on freezing to the same degree as in hypertonic exposure. PMNs have a minimum volume to which they can shrink without injury. Greater dehydration produces irreversible injury to cellular functions, and cells eventually collapse under high osmotic stress. Chilling sensitivity is seen in slowly chilled, supercooled PMNs below -5 degrees C; at -7 degrees C, functions are lost in 1 h. This injury can be prevented by the addition of Me2SO but not glycerol. Me2SO does not, however, prevent cold shock (injury due to rapid cooling), which is seen during cooling at 10 degrees C/min to -14 degrees C, but not during slow cooling at 0.5 degrees C/min. One of the problems of using glycerol as a cryoprotectant stems from the high sensitivity of PMNs to dilution shock during the dilution or removal of glycerol.     

Membrane leakage of solutes after thermal shock or freezing

Washed human erythrocytes were cooled at different rates from +37 °C to 0 °C in hypertonic solutions of either NaCl (1.2 m) or of a mixture of sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). Thermal shock hemolysis was measured and the surviving cells were examined for their mass and cell water content and also for net movements of sodium, potassium, and ¹⁴C-sucrose. The results were compared with those obtained from cells in sucrose (40% $\text{w}\text{v}$) initially, cooled at different rates to ?196 °C and rapidly thawed.The cells cooled to 0 °C in NaCl (1.2 m) showed maximal hemolysis at the fastest cooling rate studied (39 °C/min). In addition in the surviving cells this cooling rate induced the greatest uptake of ¹⁴C-sucrose and increase in cell water and cell mass and also entry of sodium and loss of cell potassium. A different dependence on cooling rate was seen with the cells cooled from +37 °C to 0 °C in sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). In this solution, survival decreased both at slow and fast cooling rates correlating with the greatest uptake of cell sucrose and increase in cell water. There was extensive loss of cell potassium and uptake of sodium at all cooling rates, the cation concentrations across the cell membrane approaching unity.The cells frozen to ?196 °C at different cooling rates in sucrose (40% $\text{w}\text{v}$) initially, also showed sucrose and water entry on thawing together with a loss of cell potassium and an uptake of cell sodium. More sucrose entered the cells cooled slowly (1.8 ° C/min) than those cooled rapidly (318 ° C/min).These results show that cooling to 0 °C in hypertonic solutions (thermal shock) and freezing to ?196 °C both induce membrane leaks to sucrose as well as to sodium and potassium. These leaks are not induced by the hypertonic solutions themselves but are due to the effects of the added stress of the temperature reduction on the membranes modified by the hypertonic solutions. The effects of cooling rate are explicable in terms of the different times of exposure to the hypertonic solutions. These results indicate that the damage observed after thermal shock or slow freezing is of a similar nature.     

Effect of cooling on the motility and function of human spermatozoa

Human spermatozoa were cooled from 37 to 0 degrees C at 10 degrees C min(-1) in 5 degrees C steps with 1 min equilibration at each step, the temperature control was +/- 0.1 degrees C. Spermatozoa were held at 0 degrees C for 5 min and then rewarmed at the same rate. No significant effect of cooling on the straight-line velocity was found using computer-aided semen analysis. The physiological function of spermatozoa was also examined before and after cooling using hypoosmotic swelling, ionophore-provoked acrosome reaction, and binding to fragments of human zonae pellucidae. Spermatozoa were cooled either in seminal plasma or in conventional IVF medium with or without fractionation by centrifugation through a discontinuous Percoll gradient. When spermatozoa were cooled and rewarmed in seminal plasma there was no significant change in either the ionophore-induced acrosome reaction or the binding to zona pellucida fragments. When spermatozoa were fractionated by centrifugation through Percoll an increased response in both was seen. However, following cooling and rewarming, a significant decline in the response of both occurred. We suggest that motility alone is not a reliable predictor of changes in other physiological functions of spermatozoa following cooling. Furthermore, short-term cooling appears to have no significant detrimental effect on normozoospermic samples and cold shock may be avoided in the clinical context by controlled cooling and warming.     

Dynamic light scattering studies on hydrodynamic properties of fibrinogen-fibronectin complex.

A high molecular weight 'cryogel' was obtained as insoluble complexes by cold incubation at near-freezing temperatures from heparinized plasma of patients with rheumatoid arthritis. After the cryogel was solubilized at 37 degrees C, 1:1 complex of fibrinogen and fibronectin was purified at room temperature by affinity chromatography on a gelatin-Sepharose 4B. Hydrodynamic properties of the complex were investigated as a function of temperature and NaCl concentration using a dynamic light scattering. The diffusion coefficients of the complex at 20 degrees C decreased with increasing of NaCl concentration as free fibronectin. The complex appears to be a more compact form at low ionic concentration, which is associated with conformational changes of fibronectin. The diffusion coefficient of the complex at 20 degrees C in 0.05 M TrisHCl(pII7.4) containing 0.5 M NaCl was estimated as 8.5 x 10(-8) cm2s-1. The complex did not dissociate over the temperature range from 20 to 37 degrees C. The diffusion coefficients of the complex decreased significantly at 12 degrees C and 40 degrees C. The thermal denaturation of fibrinogen molecule in the complex was observed at 40 degrees C. The CONTIN analysis of the light scattering data showed that the complex associated to form higher aggregates at 15 degrees C, but not at near-freezing temperature. The equilibrium between the complex and higher aggregates appeared reversible.     

Prostaglandin E1 binding protein on the surface of primary cultured hepatocytes

Prostaglandin E1 (PGE1) was bound to primary cultured rat hepatocytes in a receptor-dependent manner in serum-free medium at 4 degrees C. When added at a concentration of 2 X 10(-9) M, maximal specific binding occurred within 60-90 min. Trypsin treatment of the cells reduced the binding capacity to about 50% of that of untreated cells. Scatchard-analysis of the binding data showed that the cells had an apparent dissociation constant of 1.2 X 10(-8) M and a binding capacity of 580 fmol (approximately 3.5 X 10(11) PGE1 receptors)/mg of protein. In experiments at 37 degrees C, maximal specific binding occurred within 5 min and was 6-7 times that at 4 degrees C, but the amount of bound PGE1 decreased rapidly after 5 min due to metabolism of PGE1 in the hepatocytes. Thin-layer chromatographic analysis showed that the material bound to the cell surface consisted of intact PGE1 and its metabolites at 37 degrees C, but PGE1 only at 4 degrees C.     

Determination of temperature and cooling rate which induce cold shock in stallion spermatozoa

Experiments were conducted to determine temperatures between 24 and 4 degrees C at which stallion spermatozoa are most susceptible to cold shock damage. Semen was diluted to 25 x 10(6) spermatozoa/ml in a milk-based extender. Aliquots of extended semen were then cooled in programmable semen coolers. Semen was evaluated by computerized semen analysis initially and after 6, 12, 24, 36 and 48 hours of cooling. In Experiment 1A, semen was cooled rapidly (-0.7 degrees C/minute) from 24 degrees C to either 22, 20, 18 or 16 degrees C; then it was cooled slowly (-0.05 degrees C/minute) to a storage temperature of 4 degrees C. In Experiment 1B, rapid cooling proceeded from 24 degrees C to either 22, 19, 16, or 13 degrees C, and then slow cooling occurred to 4 degrees C. Initiating slow cooling at 22 or 20 degrees C resulted in higher (P<0.05) total and progressive motility over the first 24 hours of cooling than initiating slow cooling at 16 degrees C. Initiation of slow cooling at 22 or 19 degrees C resulted in higher (P<0.05) total and progressive motility over 48 hours of cooled storage than initiation of slow cooling at 16 or 13 degrees C. In Experiment 2A, semen was cooled rapidly from 24 to 19 degrees C, and then cooled slowly to either 13, 10, 7 or 4 degrees C, at which point rapid cooling was resumed to 4 degrees C. Resuming the fast rate of cooling at 7 degrees C resulted in higher (P<0.05) total and progressive motility at 36 and 48 hours of cooled storage than resuming fast cooling at 10 or 13 degrees C. In Experiment 2B, slow cooling proceeded to either 10, 8, 6 or 4 degrees C before fast cooling resumed to 4 degrees C. There was no significant difference (P>0.05) at most storage times in total or progressive motility for spermatozoa when fast cooling was resumed at 8, 6 or 4 degrees C. In Experiment 3, cooling units were programmed to cool rapidly from 24 to 19 degrees C, then cool slowly from 19 to 8 degrees C, and then resume rapid cooling to storage temperatures of either 6, 4, 2 or 0 degrees C. Storage at 6 or 4 degrees C resulted in higher (P<0.05) total and progressive motility over 48 hours of storage than 0 or 2 degrees C.     

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.     

Induction of random microtubule polymerization in cold and drug-treated PtK1 cells following hyperosmotic shock treatment

The effects of hypertonic sucrose on spindle and interphase microtubule (MT) arrays of PtK1 cells were investigated by incubating cells in complete culture medium at 4 degrees or 37 degrees C, with or without hypertonic sucrose, nocodazole or vinblastine (VLB). Results from anti-tubulin immunofluorescence showed that sucrose-induced alterations of spindle morphology seen at 37 degrees C did not occur at cold temperatures, but cold-induced MT loss was diminished. Application of warm hypertonic sucrose following depolymerization of MTs by nocodazole or cold resulted in the formation of a "feltwork" of randomly oriented, short MTs throughout the cytoplasm. These results, and those obtained substituting VLB for nocodazole, suggest that the effects of sucrose depend on the cytoplasmic concentration of soluble tubulin and support the hypothesis that osmotic factors are involved in effects of hypertonic sucrose on MT organization.     

The influence of cyclic heating and cooling on Escherichia coli survival

Repeated heating and cooling in lethal (2-52 degrees C) and nonlethal (2-37 degrees C) temperature ranges resulted in cell death of Escherichia coli B/r and E. coli B(S-1) suspended in 0.01 M phosphate buffer, pH 7.0 at varying osmotic pressure, but not in cow's milk. The lethal effect increased with the rate of heating and with increasing suspension media tonicity; it may be caused by the temperature destabilization of cellular osmotic homeostasis.     

Kinetic data for redox reactions of cytochrome c with Fe(CN)5X complexes and the question of association prior to electron transfer

Use of rigorous equilibration kinetics to evaluate rate constants for the Fe(CN)6 4- reduction of horse-heart cytochrome c in the oxidized form, cyt c (III), has shown that limiting kinetics do not apply with concentrations of Fe(CN)6 4- (the reactant in excess) in the range 2-10 x 10(-4) M, I = 0.10 M (NaCl). The reaction conforms to a first-order rate law in each reactant, and at 25 degrees C, pH 7.2 (Tris), it is concluded that K for association prior to electron transfer is less than 200 M-1. From previous studies at 25 degrees C, ph 7.0 (10(-1) M phosphate), I = 0.242 M (NaCl), a value K = 2.4 x 10(3) M-1 has been reported. Had such a value applied, some or all of the redox inactive complexes Mo(CN)8 4-, Co(CN)6 3-, Cr(CN)6 3-, Zr(C2O4)4 4- present in amounts 5-20 x 10(-4) M would have been expected to associate at the same site and partially block the redox process. No effect on rats was observed. With the reductants Fe(CN)5(4-NH2-py)3- and Fe(CN)5(imid)3-, reactions proceeded to greater than 90% completion and rate laws were again first order in each reactant. Rate constants (M-1 sec-1) at 25 degrees C, pH 7.2 (Tris), I = 0.10 M (NaCl), are Fe(CN)6 4- (3.5 x 10(4)), Fe(CN)5(4-NH2py)3- (6.7 x 10(5), and Fe(CN)5(imid)3- (4.2 x 10(5). Related reactions in which cyt c(II) is oxidized are also first order in each reactant, Fe(CN)6 3- (9.1 x 10(6)), Fe(CN)5(NCS)3- (1.3 x 10(6)), Fe(CN)5(4-NH2py)2- (3.8 x 10(6) at pH 9.4), and Fe(CN)5(NH3)2- (2.75 x 10(6) at ph 8). Redox inactive Co(CN)6 3- (1.0 x 10(-3) M) has no effect on the reaction of Fe(CN)6 3- which suggests that a recent interpretation for the Fe(CN)6 3- oxidation of cyt c(II), I = 0.07 M, may also require reappraisal.     

Rapid cold hardening in the western flower thrips Frankliniella occidentalis

A rapid cold hardening process is reported in first instar larvae of Frankliniella occidentalis. When larvae are transferred directly from 20 degrees C to -11.5 degrees C for 2h there is 78% mortality, whereas exposure to 0 degrees C for 4h prior to transfer to -11.5 degrees C reduces mortality to 10%. The response can also be induced by exposure to 5 degrees C for 4h or by gradual cooling at rates between 0.1 and 0.5 degrees C min(-1.) The acquired cold tolerance is transient and is rapidly lost (after 1h at 20 degrees C). Rapid cold hardening extends survival times at -11.5 degrees C and depresses lethal temperatures in short (2h) exposures. Rearing at 15 degrees C (12L:12D), (a cold acclimation regime for F. occidentalis), does not protect against the cold shock induced by direct transfer to -11.5 degrees C (which rapid cold hardening does) but does extend survival time at -5 degrees C (i.e. increased chill tolerance) whilst rapid cold hardening does not. The rapid and longer term cold hardening responses in F. occidentalis therefore appear to have different underlying mechanisms.