Membrane hydration correlates to cellular biophysics during freezing in mammalian cells

Cell survival during freezing applications in biomedicine is highly correlated to the temperature history and its dependent cellular biophysical events of dehydration and intracellular ice formation (IIF). Although cell membranes are known to play a significant role in cell injury, a clear correlation between the membrane state and the surrounding intracellular and extracellular water is still lacking. We previously showed that lipid hydration in LNCaP tumor cells is related to cellular dehydration. The goal of this study is to build upon this work by correlating both the phase state of the membrane and the surrounding water to cellular biophysical events in three different mammalian cell types: human prostate tumor cells (LNCaP), human dermal fibroblasts (HDF), and porcine smooth muscle cells (SMC) using Fourier Transform Infrared spectroscopy (FTIR). Variable cooling rates were achieved by controlling the degree of supercooling prior to ice nucleation (− 3 °C and − 10 °C) while the sample was cooled at a set rate of 2 °C/min. Membranes displayed a highly cooperative phase transition under dehydrating conditions (i.e. NT = − 3 °C), which was not observed under IIF conditions (NT = − 10 °C). Spectral analysis showed a consistently greater amount of ice formation during dehydrating vs. IIF conditions in all cell types. This is hypothesized to be due to the extreme loss of membrane hydration in dehydrating cells that is manifested as excess water available for phase change. Interestingly, changes in residual membrane conformational disorder correlate strongly with cellular volumetric decreases as assessed by cryomicroscopy. A strong correlation was also found between the activation energies for freezing induced lyotropic membrane phase change determined using FTIR and the water transport measured by cryomicroscopy. Reduced lipid hydration under dehydration freezing conditions is suggested as one of the likely causes of what has been termed as “solution effects” injury in cryobiology.     

Membrane permeability changes induce hyperpolarization in transformed lymphoid cells under high-density culture conditions

BACKGROUND: Membrane potential changes in cells from the human lymphoid B cell line, JY, evoked by increasing cell density in culture were investigated, as data published on other cell types are controversial. An attempt was also made to clear the underlying mechanism. METHODS: Nonadherent JY cells were isolated from high-density plateau-phase cultures (type A cells), medium-density log-phase cultures (type B cells), and low-density lag-phase cultures (type C cells). They were analyzed for transmembrane potential, intracellular free concentration of potassium and sodium, membrane permeability for monovalent cations, cell cycle distribution by measuring DNA content, and glucose uptake. RESULTS: C type cells proved to be relatively depolarized (-41 +/- 3 mV) and cells obtained from the highest density cultures hyperpolarized (-60 +/- 3 mV). Intracellular concentrations ([K](i) = 92-97 mM and [Na](i) = 34-35 mM) were almost identical for each type of cell. The sodium/potassium permeability constant ratio in the A and C type of cells was 0.047 and 0.094, respectively. High-density culture conditions resulted in a pronounced G(1)-phase arrest. CONCLUSIONS: Differences in the membrane potential values induced by high-density culture conditions were maintained by changes in the membrane permeability for the monovalent cations.     

Membrane permeability changes in amphibian eggs at ovulation

Electropotential differences between the cytoplasm and external medium have been compared in the mature R. pipiens occyte and the ovulated unfertilized egg as a function of [Na]_o, [K]_o, [Ca]_o and [Cl]_o. In solutions containing 1.0 mM Ca⁺⁺ the oocyte behaved as though it were predominantly permeable to K⁺ and Cl^?, i.e., like a KCl electrode. However, the steady potential decreased with decreasing [Ca]_o and in 5 × 10^?4 mM [Ca]_o the oocyte membrane behaved like a NaCl electrode. Studies on the steady potential as a function of [Na]_o, [K]_o and [Cl]_o in 1.0 mM Ca⁺⁺ or Ca-free solutions suggest that Ca⁺⁺ controls the passive permeability of the oocyte membrane to Na⁺ and Cl^?. In the ovulated unfertilized egg the K⁺ selectivity of the cell membrane disappeared and the system behaved like a NaCl electrode. No effect of external Ca⁺⁺ or K⁺ concentration changes on the steady potential was observed. These results indicate that the ion permeability properties of the ovulated egg are similar to that of the ovarian oocyte in Ca-deficient medium, and suggests that the mechanism of ovulation may involve the removal of Ca⁺⁺ regulation of ion permeability of the egg cell membrane.     

Suprazero cooling conditions significantly influence subzero permeability parameters of mammalian ovarian tissue

To model the cryobiological responses of cells and tissues, permeability characteristics are often measured at suprazero temperatures and the measured values are used to predict the responses at subzero temperatures. The purpose of the present study was to determine whether the rate of cooling from +25 to +4 degrees C influenced the measured water transport response of ovarian tissue at subzero temperatures in the presence or absence of cryoprotective agents (CPAs). Sections of freshly collected equine ovarian tissue were first cooled either at 40 degrees C/min or at 0.5 degrees C/min from 25 to 4 degrees C, and then cooled to subzero temperatures. A shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine ovarian tissue sections. After ice was induced to form in the extracellular fluid within the specimen, the sample was frozen from the phase change temperature to -50 degrees C at 5 degrees C/min. Replicate samples were frozen in isotonic medium alone or in medium containing 0.85 M glycerol or 0.85 M dimethylsulfoxide. The water transport response of ovarian tissue samples cooled at 40 degrees C/min from 25 to 4 degrees C was significantly different (confidence level >95%) from that of tissue samples cooled at 0.5 degrees C/min, whether in the presence or absence of CPAs. We fitted a model of water transport to the experimentally-derived volumetric shrinkage data and determined the best-fit membrane permeability parameters (L(pg) and E(Lp)) of equine ovarian tissue during freezing. Subzero water transport parameters of ovarian tissue samples cooled at 0.5 degrees C/min from 25 to 4 degrees C ranged from: L(pg) = 0.06 to 0.73 microm/min.atm and E(Lp) = 6.1 to 20.5 kcal/mol. The corresponding parameters of samples cooled at 40 degrees C/min from 25 to 4 degrees C ranged from: L(pg) = 0.04 to 0.61 microm/min.atm and E(Lp) = 8.2 to 54.2 kcal/mol. Calculations made of the theoretical response of tissue at subzero temperatures suggest that the optimal cooling rates to cryopreserve ovarian tissue are significantly dependent upon suprazero cooling conditions.     

Osmotic response of individual cells during freezing. II. Membrane permeability analysis

An analytical model is presented to simulate the freezing of individual yeast cells. In addition the model is solved numerically on a digital computer to obtain values for cell volume as a function of temperature, based on the thermal protocol during freezing, and the transport parameters of the cell membrane. The numerical procedure was modified to enable values for the membrane hydraulic permeability reference coefficient, Lpg, and activation energy, ELp, to be deduced by nonlinear analysis of complementary experimental data (10). It was observed that the apparent values of both Lpg and ELp increase with cooling rate, from Lpg = 0.0116 micrometer 3 micrometers-2 atm-1 min-1 and ELp = 19.4 kJ mol-1 for 9 degrees K/min to Lpg = 2.11 micrometers 3 micrometer-2 atm-1 min-1 and ELp = 101 kJ mol-1 for 35 degrees K/min. The deduced permeabilities fall within the range of values determined in a prior study by Levin (6). Analysis with the model also indicates that the turgor pressure exerts a negligible effect on yeast exposed to freezing stress.     

Relative effects of cooling and warming rates on mammalian cells during the freeze-thaw cycle

The relative roles of cooling and warming rates on cell survival during a freeze-thaw cycle were investigated. Basically the faster the warming rate, the better the cells survive. One of the factors influencing this is the extended phase transition period at the slower thawing rates. The warming rate had a significant effect on cell damage and recovery, but this was not as great as comparative changes in the cooling rate were. This investigation also showed that under certain freeze-thaw conditions there was a lack of correlation between the two methods used for quantifying cell recovery (RI) and cell damage (PCR) as measured by radiochromate release. The analysis of the relationship between RI and PCR showed that PCR could be used to measure both lethal and nonlethal damage and enabled a clearer interpretation of cellular damage during cooling and thawing to be made.     

Diffusional water permeability of mammalian red blood cells

An extensive programme of comparative nuclear magnetic resonance measurements of the membrane diffusional permeability for water (P_d) and of the activation energy (E_a,d) of this process in red blood cells (RBCs) from 21 mammalian species was carried out. On the basis of P_d, these species could be divided into three groups. First, the RBC's from humans, cow, sheep and “large” kangaroos (Macropus giganteus and Macropus rufus) had P_d values 5 × 10⁻³ cm/s at 25°C and 7 × 10⁻³ cm/s at 37°C. The RBCs from other marsupial species, mouse, rat, guinea pig and rabbit, had P_d values roughly twice higher, whereas echidna RBCs were twice lower than human RBCs. The value of E_a,d was in most cases correlated with the values of P_d. A value of E_a,d -26 kJ/mol was found for the RBCs from humans and the species having similar P_d values. Low values of E_a,d (ranging from 15 to 22 kJ/mol) appeared to be associated with relatively high values of P_d. The highest value of E_a,d (33 kJ/mol) was found in echidna RBCs. This points to specialized channels for water diffusion incorporated in membrane proteins; a relatively high water permeability of the RBC membrane could be due to a greater number of channel proteins. There are, however, situations where a very high water permeability of RBCs is associated with a high value of E_a,d (above 25 kJ/mol) as in the case of RBCs from mouse, rat and tree kangaroo. Moreover, it was found that P_d in different species was positively correlated to the RBC membrane phosphatidylcholine and negatively correlated to the sphingomyelin content. This suggests that in addition to the number of channel proteins, other factors are involved in the water permeability of the RBC membrane.     

Membrane water permeability and aquaporin expression increase during growth of maize suspension cultured cells

Aquaporins (AQPs) are water channels that allow cells to rapidly alter their membrane water permeability. A convenient model for studying AQP expression and activity regulation is Black Mexican Sweet (BMS) maize cultured cells. In an attempt to correlate membrane osmotic water permeability coefficient (P_f) with AQP gene expression, we first examined the expression pattern of 33 AQP genes using macro-array hybridization. We detected the expression of 18 different isoforms representing the four AQP subfamilies, i.e. eight plasma membrane (PIP), five tonoplast (TIP), three small basic (SIP) and two NOD26-like (NIP) AQPs. While the expression of most of these genes was constant throughout all growth phases, mRNA levels of ZmPIP1;3 , ZmPIP2;1 , ZmPIP2;2, ZmPIP2;4 and ZmPIP2;6 increased significantly during the logarithmic growth phase and the beginning of the stationary phase. The use of specific anti-ZmPIP antisera showed that the protein expression pattern correlated well with mRNA levels. Cell pressure probe and protoplast swelling measurements were then performed to determine the P_f. Interestingly, we found that the P_f were significantly increased at the end of the logarithmic growth phase and during the steady-state phase compared to the lag phase, demonstrating a positive correlation between AQP abundance in the plasma membrane and the cell P_f.     

Membrane permeability changes at early stages of influenza hemagglutinin-mediated fusion

While biological membrane fusion is classically defined as the leak-free merger of membranes and contents, leakage is a finding in both experimental and theoretical studies. The fusion stages, if any, that allow membrane permeation are uncharted. In this study we monitored membrane ionic permeability at early stages of fusion mediated by the fusogenic protein influenza hemagglutinin (HA). HAb2 cells, expressing HA on their plasma membrane, fused with human red blood cells, cultured liver cells PLC/PRF/5, or planar phospholipid bilayer membranes. With a probability that depended upon the target membrane, an increase of the electrical conductance of the fusing membranes (leakage) by up to several nS was generally detected. This leakage was recorded at the initial stages of fusion, when fusion pores formed. This leakage usually accompanied the "flickering" stage of the early fusion pore development. As the pore widened, the leakage reduced; concomitantly, the lipid exchange between the fusing membranes accelerated. We conclude that during fusion pore formation, HA locally and temporarily increases the permeability of fusing membranes. Subsequent rearrangement in the fusion complex leads to the resealing of the leaky membranes and enlargement of the pore.     

Methods for rapidly altering the permeability of mammalian cells

Various agents alter mammalian cells so that they rapidly become nonspecifically permeable to substances that ordinarily do not penetrate intact cells. Thus, toluene renders liver cells permeable to nucleotides and macromolecules. Tween 80 and Tween 60 act in similar fashion, and the effect is reversible. Dextran sulfate reversibly alters the permeability of Ehrlich ascites tumor cells, which offers a tool for studying the control of macromolecular syntheses and other processes. Brief exposure to external ATP alters the permeability of certain transformed mouse cells but not of untransformed cells. The effect of ATP is rapidly reversible.     

Membrane fatty acid changes during the cell cycle of CV-1 cells

Monolayers of CV-1 cells were synchronized at the G1/S boundary of the cell cycle by a 24-h 2 mM thymidine blockade. Uptake of tritiated thymidine indicated that the peak DNA synthesis occurred 6-8 h after release from the block and that cell cycle time was 18-20 h. The fatty acid composition of phospholipids extracted from cells at 0, 7, and 18 h postblockade was measured by gas chromatography. The results indicate cyclic changes in membrane fatty acids with a significant increase in long-chain polyunsaturated fatty acids during the DNA synthesis phase (S phase) of the cell cycle.     

Measuring hemodynamic changes during mammalian development

The pathogenesis of many congenital cardiovascular diseases involves abnormal flow within the embryonic vasculature that results either from malformations of the heart or defects in the vasculature itself. Extensive genetic and genomic analysis in mice has led to the identification of an array of mutations that result in cardiovascular defects during embryogenesis. Many of these mutations cause secondary effects within the vasculature that are thought to arise because of altered fluid dynamics. Presumably, cardiac defects disturb or reduce flow and thereby lead to the disruption of the mechanical signals necessary for proper vascular development. Unfortunately, a precise understanding of how flow disruptions lead to secondary vasculature defects has been hampered by the inadequacy of existing analytical tools. Here, we used a fast line-scanning technique for the quantitative analysis of hemodynamics during early organogenesis in mouse embryos, and we present a model system for studying cellular responses during the formation and remodeling of the mammalian cardiovascular system. Flow velocity profiles can be measured as soon as a heart begins to beat even in newly formed vessels. These studies establish a link between the pattern of blood flow within the vasculature and the stage of heart development and also enable analysis of the influence of mechanical forces during development.     

Membrane potential changes during IgE-mediated histamine release from rat basophilic leukemia cells

The membrane potential of rat basophilic leukemia cells (RBL-2H3 cell line) has been determined by monitoring the distribution of the lipophilic [3H] tetraphenylphosphonium cation between the cells and the extracellular medium. By this method, the determined potential of these cells, passively sensitized with IgE, is -93 +/- 5 mV (mean +/- SEM, interior negative). Almost 40% of this membrane potential is rapidly collapsed upon the addition of the proton carrier, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). It is suggested that the FCCP-sensitive fraction of the total membrane potential results from the accumulation of this cation by the mitochondria, which maintains a negative membrane potential. Thus, the resting plasma membrane potential of these cells equals -55 +/- 6 mV. During the process of immunological stimulation by antibodies directed against cell membrane bound IgE, the membrane potential decreases. Moreover, there is a correlation between the extent of degranulation of the cells and the depolarization. It is concluded that in common with other secretory systems, depolarization of the plasma membrane is involved in the stimulus-secretion coupling of the histamine secreting RBL cells.     

Membrane stress increases cation permeability in red cells.

The human red cell is known to increase its cation permeability when deformed by mechanical forces. Light-scattering measurements were used to quantitate the cell deformation, as ellipticity under shear. Permeability to sodium and potassium was not proportional to the cell deformation. An ellipticity of 0.75 was required to increase the permeability of the membrane to cations, and flux thereafter increased rapidly as the limits of cell extension were reached. Induction of membrane curvature by chemical agents also did not increase cation permeability. These results indicate that membrane deformation per se does not increase permeability, and that membrane tension is the effector for increased cation permeability. This may be relevant to some cation permeabilities observed by patch clamping.     

Membrane permeability equations and their solutions for red cells

Summary The mathematical equations for the transport of nonelectrolytes across cell membranes are critically examined and cast in forms suitable for solution which involve fewer approximations than has heretofore been commonly done. For the case of red cells, the equations are developed to include the effect of the variation in apparent nonosmotic water owing to the variation in hemoglobin concentration as the cell swells or shrinks. Two methods of solution of the equations are developed and studied and sample calculations are provided. It is shown that the solutions to the linearized equations commonly found in the literature are insufficiently accurate for some purposes and this inaccuracy is avoided by the methods given here. The importance of retaining the effects of variations in apparent nonosmotic water and in solute volume in the cell is demonstrated.