Development of a novel microperfusion chamber for determination of cell membrane transport properties.

A novel microperfusion chamber was developed to measure kinetic cell volume changes under various extracellular conditions and to quantitatively determine cell membrane transport properties. This device eliminates modeling ambiguities and limitations inherent in the use of the microdiffusion chamber and the micropipette perfusion technique, both of which have been previously validated and are closely related optical technologies using light microscopy and image analysis. The resultant simplicity should prove to be especially valuable for study of the coupled transport of water and permeating solutes through cell membranes. Using the microperfusion chamber, water and dimethylsulfoxide (DMSO) permeability coefficients of mouse oocytes as well as the water permeability coefficient of golden hamster pancreatic islet cells were determined. In these experiments, the individual cells were held in the chamber and perfused at 22 degrees C with hyperosmotic media, with or without DMSO (1.5 M). The cell volume change was videotaped and quantified by image analysis. Based on the experimental data and irreversible thermodynamics theory for the coupled mass transfer across the cell membrane, the water permeability coefficient of the oocytes was determined to be 0.47 micron. min-1. atm-1 in the absence of DMSO and 0.65 microns. min-1. atm-1 in the presence of DMSO. The DMSO permeability coefficient of the oocyte membrane and associated membrane reflection coefficient to DMSO were determined to be 0.23 and 0.85 micron/s, respectively. These values are consistent with those determined using the micropipette perfusion and microdiffusion chamber techniques. The water permeability coefficient of the golden hamster pancreatic islet cells was determined to be 0.27 microns. min-1. atm-1, which agrees well with a value previously determined using an electronic sizing (Coulter counter) technique. The use of the microperfusion chamber has the following major advantages: 1) This method allows the extracellular condition(s) to be readily changed by perfusing a single cell or group of cells with a prepared medium (cells can be reperfused with a different medium to study the response of the same cell to different osmotic conditions). 2) The short mixing time of cells and perfusion medium allows for accurate control of the extracellular osmolality and ensures accuracy of the corresponding mathematical formulation (modeling). 3) This technique has wide applicability in studying the cell osmotic response and in determining cell membrane transport properties.     

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

Thermodynamic computer modeling was used to predict the freezing response of single-celled unfertilized hamster ova. The cell membrane transport characteristics were investigated, using a microscope diffusion chamber system. The mean osmotically inactive cell volume was determined to be 21.6% of the initial cell volume. An overall mean value of 0.8 +/- 0.1 micron3/micron2.min.atm (= 18 +/- 2.5 micron/sec) was determined for the membrane hydraulic coefficient, Lp. The effect of the extracellular solute concentration on Lp was determined at room temperature (approximately 23 degrees C). A thermodynamic computer model was used to predict the cell response to freezing. The predicted response was compared to the actual volumetric response observed during freezing on a temperature-controlled cryomicroscope conduction stage. The effect of the cooling rate on the nucleation temperature of unprotected ova and protected ova suspended in a 1.5 M DMSO solution was investigated. Overall mean nucleation temperatures of -13 and -57.1 degrees C were observed for unprotected and protected ova, respectively, where the mean nucleation temperature for protected ova was strongly cooling rate dependent.     

Freezing-induced cellular and membrane dehydration in the presence of cryoprotective agents

Abstract

FTIR and cryomicroscopy have been used to study mouse embryonic fibroblast cells (3T3) during freezing in the absence and presence of DMSO and glycerol. The results show that cell volume changes as observed by cryomicroscopy typically end at temperatures above ?15°C, whereas membrane phase changes may continue until temperatures as low as ?30°C. This implies that cellular dehydration precedes dehydration of the bound water surrounding the phospholipid head groups. Both DMSO and glycerol increase the membrane hydraulic permeability at subzero temperature and reduce the activation energy for water transport. Cryoprotective agents facilitate dehydration to continue at low subzero temperatures thereby decreasing the incidence of intracellular ice formation. The increased subzero membrane hydraulic permeability likely plays an important role in the cryoprotective action of DMSO and glycerol. In the presence of DMSO water permeability was found to be greater compared to that in the presence of glycerol. Two temperature regimes were identified in an Arrhenius plot of the membrane hydraulic permeability. The activation energy for water transport at temperature ranging from 0 to ?10°C was found to be greater than that below ?10°C. The non-linear Arrhenius behavior of Lp has been implemented in the water transport model to simulate cell volume changes during freezing. At a cooling rate of 1°C min^-1, ～5% of the initial osmotically active water volume is trapped inside the cells at ?30°C.     

Transport parameters in the human red cell membrane: solute-membrane interactions of amides and ureas

We have studied the permeability of a series of hydrophilic amides and ureas through the red cell membrane by determining the three phenomenological coefficients which describe solute-membrane interaction: the hydraulic permeability (Lp), the phenomenological permeability coefficient (omega i) and the reflection coefficient (sigma i). In 55 experiments on nine solutes, we have determined that the reflection coefficient (after a small correction for solute permeation by membrane dissolution) is significantly less than 1.0 (P less than 0.003, t-test), which provides very strong evidence that solute and water fluxes are coupled as they cross the red cell membrane. It is proposed that the aqueous channel is a tripartite assembly, comprising H-bond exchange regions at both faces of the membrane, joined by a narrower sieve-specific region which crosses the lipid. The solutes bind to the H-bond exchange regions to exchange their solvation shell with the H-bonds of the channel; the existence of these regions is confirmed by the finding that the permeation of all the amides and ureas requires binding to well-characterized sites with Km values of 0.1-0.5 M. The sieve-specific regions provide the steric restraints which govern the passage of the solutes according to their size; their existence is shown by the findings that: (1) the reflection coefficient (actually the function [1-corrected sigma i]) is linearly dependent upon the solute molecular diameter; and (2) the permeability coefficient is linearly dependent upon solute molar volume. These several observations, taken together, provide strong arguments which lead to the conclusion that the amides and urea cross the red cell membrane in an aqueous pore.     

Freezing response and optimal cooling rates for cryopreserving sperm cells of striped bass, Morone saxatilis

This study explored the optimization of techniques for sperm cryopreservation of an economically important fish species, the striped bass Morone saxatilis. The volumetric shrinkage or the water transport response during freezing of sperm cells was obtained using a differential scanning calorimeter (DSC) technique. Water transport was obtained in the presence of extracellular ice at a cooling rate of 20 degrees C/min in two different media: (1) without cryoprotective agents (CPAs), and (2) with 5% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder of length of 22.8 microm and diameter 0.288 microm and was assumed to have an osmotically inactive cell volume (V(b)) of 0.6 V(0), where V(0) is the isotonic or initial cell volume. By fitting a model of water transport to the experimentally determined water transport data, the best fit membrane permeability parameters (reference membrane permeability to water, L(pg) or L(pg)[cpa] and the activation energy, E(Lp) or E(Lp)[cpa]) were determined and ranged from L(pg)=0.011-0.001 microm/min-atm, and E(Lp)=40.2-9.2 kcal/mol). The parameters obtained in this study suggested that the optimal rate of cooling for striped bass sperm cells in the presence and absence of DMSO range from 14 to 20 degrees C/min. These theoretically predicted rates of optimally freezing M. saxatilis sperm compared quite closely with independent and experimentally determined optimal rates of cooling striped bass sperm.     

The effect of dimethylsulfoxide on the water transport response of rat hepatocytes during freezing.

Successful improvement of cryopreservation protocols for cells in suspension requires knowledge of how such cells respond to the biophysical stresses of freezing (intracellular ice formation, water transport) while in the presence of a cryoprotective agent (CPA). This work investigates the biophysical water transport response in a clinically important cell type--isolated hepatocytes--during freezing in the presence of dimethylsulfoxide (DMSO). Sprague-Dawley rat liver hepatocytes were frozen in Williams E media supplemented with 0, 1, and 2 M DMSO, at rates of 5, 10, and 50 degrees C/min. The water transport was measured by cell volumetric changes as assessed by cryomicroscopy and image analysis. Assuming that water is the only species transported under these conditions, a water transport model of the form dV/dT = f(Lpg([CPA]), ELp([CPA]), T(t)) was curve-fit to the experimental data to obtain the biophysical parameters of water transport--the reference hydraulic permeability (Lpg) and activation energy of water transport (ELp)--for each DMSO concentration. These parameters were estimated two ways: (1) by curve-fitting the model to the average volume of the pooled cell data, and (2) by curve-fitting individual cell volume data and averaging the resulting parameters. The experimental data showed that less dehydration occurs during freezing at a given rate in the presence of DMSO at temperatures between 0 and -10 degrees C. However, dehydration was able to continue at lower temperatures (< -10 degrees C) in the presence of DMSO. The values of Lpg and ELp obtained using the individual cell volume data both decreased from their non-CPA values--4.33 x 10(-13) m3/N-s (2.69 microns/min-atm) and 317 kJ/mol (75.9 kcal/mol), respectively--to 0.873 x 10(-13) m3/N-s (0.542 micron/min-atm) and 137 kJ/mol (32.8 kcal/mol), respectively, in 1 M DMSO and 0.715 x 10(-13) m3/N-s (0.444 micron/min-atm) and 107 kJ/mol (25.7 kcal/mol), respectively, in 2 M DMSO. The trends in the pooled volume values for Lpg and ELp were very similar, but the overall fit was considered worse than for the individual volume parameters. A unique way of presenting the curve-fitting results supports a clear trend of reduction of both biophysical parameters in the presence of DMSO, and no clear trend in cooling rate dependence of the biophysical parameters. In addition, these results suggest that close proximity of the experimental cell volume data to the equilibrium volume curve may significantly reduce the efficiency of the curve-fitting process.     

Measurement and simulation of water and methanol transport in algal cells

BACKGROUND: Experimental data and a complementary biophysical model are presented to describe the dynamic response of a unicellular microalga to osmotic processes encountered during cryopreservation. METHOD OF APPROACH: Chlorococcum texanum (C. texanum) were mounted on a cryoperfusion microscope stage and exposed sequentially to various solutions of sucrose and methanol. Transient volumetric excursions were determined by capturing images of cells in real time and utilizing image analysis software to calculate cell volumes. A biophysical model was applied to the data via inverse analysis in order to determine the plasma membrane permeability to water and to methanol. The data were also used to determine the elastic modulus of the cell wall and its effect on cell volume. A three-parameter (hydraulic conductivity (Lp), solute permeability; (omega), and reflection coefficient, (sigma)) membrane transport model was fit to data obtained during methanol perfusion to obtain constitutive property values. These results were compared with the property values obtained for a two coefficient (Lp and omega) model. RESULTS: The three-parameter model gave a value for sigma not consistent with practical physical interpretation. Thus, the two-coefficient model is the preferred approach for describing simultaneous water and methanol transport. The rate of both water and methanol transport were strongly dependent on temperature over the measured temperature range (25 degrees C to -5 degrees C) and cells were appreciably more permeable to methanol than to water at all measured temperatures. CONCLUSION: These results may explain in part why methanol is an effective cryoprotective agent for microalgae.     

Studies on membrane permeability of zebrafish (Danio rerio) oocytes in the presence of different cryoprotectants

Investigation into fish oocyte membrane permeability is essential for developing successful protocols for their cryopreservation. The aim of the present work was to study the permeability of the zebrafish (Danio rerio) oocyte membrane to water and cryoprotectants before cryopreservation protocol design. The study was conducted on stage III and stage V zebrafish oocytes. Volumetric changes of stage III oocytes in different concentrations of sucrose were measured after 20 min exposure at 22 degrees C and the osmotically inactive volume of the oocytes (Vb) was determined using the Boyle-van't Hoff relationship. Volumetric changes of oocytes during exposure to different cryoprotectant solutions were also measured. Oocytes were exposed to 2 M dimethyl sulphoxide (DMSO), propylene glycol (PG), and methanol for 40 min at 22 degrees C. Stage III oocytes were also exposed to 2 M DMSO at 0 degrees C. Oocyte images were captured on an Olympus BX51 cryomicroscope using Linkham software for image recording. Scion Image was used for image analysis and diameter measurement. The experimental data were fitted to a two-parameter model using Berkeley Madonna 8.0.1 software. Hydraulic conductivity (L(p)) and solute (cryoprotectant) permeability (Ps) were estimated using the model. The osmotically inactive volume of stage III zebrafish oocytes was found to be 69.5%. The mean values+/-SE of Lp were found to be 0.169+/-0.02 and 0.196+/-0.01 microm/min/atm in the presence of DMSO and PG, respectively, at 22 degrees C, assuming an internal isosmotic value for the oocyte of 272 mOsm. The Ps values were 0.000948+/-0.00015 and 0.000933+/-0.00005 cm/min for DMSO and PG, respectively. It was also shown that the membrane permeability of stage III oocytes decreased significantly with temperature. No significant changes in cell volume during methanol treatment were observed. Fish oocyte membrane permeability parameters are reported here for the first time. The Lp and Ps values obtained for stage III zebrafish oocytes are generally lower than those obtained from successfully cryopreserved mammalian oocytes and higher than those obtained with fish embryos and sea urchin eggs. It was not possible to estimate membrane permeability parameters for stage V oocytes using the methods employed in this study because stage V oocytes experienced the separation of outer oolemma membrane from inner vitelline during exposure to cryoprotectants.     

An experimental study of some indigenous drugs with special reference to hydraulic permeability

The effect of commonly used indigenous drugs for hepatic disorders i.e. Tinospora cordifolia, (Guduchi/Amrita), Andrographis paniculata (Kalmegha), Picrorhiza kurroa (Kutki), Phyllantnus niruri (Bhoomyamalaki) and Berberis aristata (Daruharidra) was tested on the hydraulic permeability of water in the presence of bile salt through a transport cell model. The data on hydraulic permeability were calculated as t (time). JV = Lp x AP, where Lp = hydraulic conductivity and AP is the pressure difference. It was observed that the value of controlled hydraulic permeability (0.49 x 10(-8) M3 S(-1) N(-1)) decreased in the presence of indigenous drugs and bile salt. The results suggest that these drugs might have the cell membrane stabilizing property which may lead to prevention of the toxic effect of bile salts in various hepatic disorders.     

Cryoprotectant permeability parameters for cells used in a bioengineered human corneal equivalent and applications for cryopreservation

A human corneal equivalent is being developed with applications in pharmaceutical testing and biomedical research, but the distribution of this engineered tissue, depends on successful cryopreservation. Cryopreservation of tissues depends on the presence of cryoprotectants, their addition and removal, and exposure to conditions during freezing and thawing, all of which depend on cellular membrane permeabilities to water and cryoprotectant. This study defines the permeability properties that define the rate of water and cryoprotectant movement across the plasma membrane of isolated human corneal endothelial, keratocyte, and epithelial cells. Cells were transferred from isotonic conditions (300 mosm/kg) to 0.5, 1, or 2 M dimethyl sulfoxide and propylene glycol solutions at constant temperature, and cell volumes monitored using an electronic particle counter. Histograms describing cell volume changes over time after cryoprotectant exposure allowed calculation of hydraulic conductivity (Lp), cryoprotectant permeability (Ps), and the reflection coefficient (sigma). Experimental values for Lp and Ps at 4, 13, 22, and 37 degrees C were used to determine the Arrhenius activation energy (Ea). Defining the permeability parameters and temperature dependencies allows simulation of responses of human corneal cells to addition and removal of cryoprotectants and to freezing conditions, allowing amount of supercooling, intracellular electrolyte concentration, and intracellular cryoprotectant concentration to be calculated. Simulations also show that the constituent cells in the bioengineered cornea respond differently to addition and removal of cryoprotectants and to freezing. This study has defined the requirements during cryopreservation for the corneal cells; future work will define the matrix requirements which will allow the development of a cryopreservation protocol.     

Subzero water permeability parameters and optimal freezing rates for sperm cells of the southern platyfish, Xiphophorus maculatus

This study reports the subzero water transport characteristics (and empirically determined optimal rates for freezing) of sperm cells of live-bearing fishes of the genus Xiphophorus, specifically those of the southern platyfish Xiphophorus maculatus. These fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. Water transport during freezing of X. maculatus sperm cell suspensions was obtained using a shape-independent differential scanning calorimeter technique in the presence of extracellular ice at a cooling rate of 20 degrees C/min in three different media: (1) Hanks' balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol, and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder with a length of 52.35 microm and a diameter of 0.66 microm with an osmotically inactive cell volume (Vb) of 0.6 V0, where V0 is the isotonic or initial cell volume. This translates to a surface area, SA to initial water volume, WV ratio of 15.15 microm(-1). By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best fit membrane permeability parameters (reference membrane permeability to water at 0 degrees C, Lpg or Lpg [cpa] and the activation energy, E(Lp) or E(Lp) [cpa]) were found to range from: Lpg or Lpg [cpa] = 0.0053-0.0093 microm/minatm; E(Lp) or E(Lp) [cpa] = 9.79-29.00 kcal/mol. By incorporating these membrane permeability parameters in a recently developed generic optimal cooling rate equation (optimal cooling rate, [Formula: see text] where the units of B(opt) are degrees C/min, E(Lp) or E(Lp) [cpa] are kcal/mol, L(pg) or L(pg) [cpa] are microm/minatm and SA/WV are microm(-1)), we determined the optimal rates of freezing X. maculatus sperm cells to be 28 degrees C/min (in HBSS), 47 degrees C/min (in HBSS+14% glycerol) and 36 degrees C/min (in HBSS+10% DMSO). Preliminary empirical experiments suggest that the optimal rate of freezing X. maculatus sperm in the presence of 14% glycerol to be approximately 25 degrees C/min. Possible reasons for the observed discrepancy between the theoretically predicted and experimentally determined optimal rates of freezing X. maculatus sperm cells are discussed.     

Cryomicroscopic determination of the membrane osmotic properties of human monocytes at subfreezing temperatures

Monocytes were isolated from fresh whole human blood and resuspended in Hanks balanced salt solution; a portion of the cells was mixed with an equal volume of 2M dimethyl sulfoxide (DMSO) to form a 1 M solution. Microliter volumes of cell suspension were placed directly onto a computer-controlled cryostage and cooled to a predetermined subzero temperature. Ice was nucleated in the extracellular medium and a continuous video record was made of the subsequent osmotically induced volume changes of individual cells owing to exposure to the concentrated extracellular solutes. Selected micrographs emphasizing the initial transient data were digitized for computer analysis with an interactive boundary tracing algorithm to determine metric parameters of specific cells, and apparent volume changes were measured as a function of elapsed time after nucleation. The Kedem-Katchalsky-coupled transport equations were fit to the data using a network thermodynamic model implemented on a microcomputer to determine values for the permeability properties Lp, omega, and sigma. Experiments were performed over the temperature range from -7 degrees to -10 degrees C. Cells pre-equilibrated with DMSO had a lower Lp and a higher activation energy, delta E, than without additive, although the statistical significance of the difference could not be substantiated. It was found that the movement of DMSO across the plasma membrane in response to extracellular freezing was apparently so much smaller than the water flux that values for omega and sigma could not be determined from the data base.     

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.     

A theoretically estimated optimal cooling rate for the cryopreservation of sperm cells from a live-bearing fish, the green swordtail Xiphophorus helleri

Sperm cryopreservation of live-bearing fishes, such as those of the genus Xiphophorus is only beginning to be studied, although these fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. To explore optimization of techniques for sperm cryopreservation of these fishes, this study measured the volumetric shrinkage response during freezing of sperm cells of Xiphophorus helleri by use of a shape-independent differential scanning calorimeter (DSC) technique. Volumetric shrinkage during freezing of X. helleri sperm cell suspensions was obtained in the presence of extracellular ice at a cooling rate of 20 degrees C/min in three different media: (1) Hanks' balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol; and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder of 33.3 microm in length and 0.59 microm in diameter with an osmotically inactive cell volume (V(b)) of 0.6V(o), where V(o) is the isotonic or initial cell volume. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best-fit membrane permeability parameters (reference membrane permeability to water, L(pg) or L(pg)[cpa] and the activation energy, E(Lp) or E(Lp)[cpa]) of the Xiphophorus helleri sperm cell membrane were determined. The best-fit membrane permeability parameters at 20 degrees C/min in the absence of CPAs were: L(pg)=0.776 x 10(-15)m3/Ns (0.0046 microm/min atm), and E(Lp)=50.1 kJ/mol (11.97 kcal/mol) (R2=0.997). The corresponding parameters in the presence of 14% glycerol were L(pg)[cpa]=1.063 x 10(-15)m3/Ns (0.0063 microm/min atm), and E(Lp)[cpa]=83.81 kJ/mol (20.04 kcal/mol) (R2=0.997). The parameters in the presence of 10% DMSO were L(pg)[cpa]=1.4 x 10(-15)m3/Ns (0.0083 microm/min atm), and E(Lp)[cpa]=90.96 kJ/mol (21.75 kcal/mol) (R2=0.996). Parameters obtained in this study suggested that the optimal rate of cooling for X. helleri sperm cells in the presence of CPAs ranged from 20 to 35 degrees C/min and were in close agreement with recently published, empirically determined optimal cooling rates.     

Determination of the permeability of human lymphocytes with a microscope diffusion chamber

A diffusion chamber similar to that proposed by J.J. McGrath (J. Microsc., in press) was constructed which allows microscopic observation of osmotically induced volume changes of individual cells in small (microliter) sample volumes. The cells are kept fixed in position in the upper compartment of the chamber by means of a highly permeable membrane and exposed to a step-like change in concentration generated in the lower compartment. An electrical conductivity probe in the upper compartment was used to monitor the temporal change of salt concentration as experienced by the cells. The rise from isotonic to hypertonic can be approximated by an exponential function. Its time constant of tau = 2.08 sec seems to be mainly determined by the change in flushing solution as tau = 1.48 sec was measured with no membrane installed. With human lymphocytes, no loss of cell volume was detected before 5 sec, i.e., when 95% of the final concentration was reached extracellularly. A step change can hence be assumed when modeling exosmosis for determining the lymphocyte membrane permeability. The equations for coupled transport of water and salt were solved numerically and fitted to the experimental data. The results were also compared to various other transport models described in the literature. Human lymphocytes are almost ideally semipermeable with a hydraulic reference permeability of Lp = 4.23 X 10(-4) cm/sec (3.13 X 10(-3) micron X atm-1 X sec-1) at T = 23 degrees C. The temperature and concentration dependence are described by an activation energy Ea = 14.3 kJ/mol and a concentration coefficient alpha 2 = 0.261 osmol/kg. An osmotically inactive volume fraction of 36.9% was determined from the final cell volumes reached asymptotically after shrinkage.     

A re-examination of the minor role of unstirred layers during the measurement of transport coefficients of Chara corallina internodes with the cell pressure probe

The impact of unstirred layers (USLs) during cell pressure probe experiments with Chara corallina internodes has been quantified. The results show that the hydraulic conductivity (Lp) measured in hydrostatic relaxations was not significantly affected by USLs even in the presence of high water flow intensities ('sweep-away effect'). During pressure clamp, there was a reversible reduction in Lp by 20%, which was explained by the constriction of water to aquaporins (AQPs) in the C. corallina membrane and a rapid diffusional equilibration of solutes in arrays where water protruded across AQPs. In osmotic experiments, Lp, and permeability (Ps) and reflection (sigma s) coefficients increased as external flow rate of medium increased, indicating some effects of external USLs. However, the effect was levelling off at 'usual' flow rates of 0.20-0.30 m s(-1) and in the presence of vigorous stirring by air bubbles, suggesting a maximum thickness of external USLs of around 30 microm including the cell wall. Because the diameters of internodes were around 1 mm, internal USLs could have played a significant or even a dominating role, at least in the presence of the rapidly permeating solutes used [acetone, 2-propanol and dimethylformamide (DMF)]. A comparison of calculated (diffusion kinetics) and of measured permeabilities indicated an upper limit of the contribution of USLs for the rapidly moving solute acetone of 29%, and of 15% for the less rapidly permeating DME The results throw some doubt on recent claims that in C. corallina, USLs rather than the cell membrane dominate solute uptake, at least for the most rapidly moving solute acetone.     

Control of water uptake by rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.): role of the outer part of the root

A new pressure-perfusion technique was used to measure hydraulic and osmotic properties of the outer part of roots (OPR) of 30-day-old rice plants (lowland cultivar: IR64, and upland cultivar: Azucena). The OPR comprised rhizodermis, exodermis, sclerenchyma and one cortical cell layer. The technique involved perfusion of aerenchyma of segments from two different root zones (20-50 mm and 50-100 mm from the tip) at precise rates using aerated nutrient solution. The hydraulic conductivity of the OPR (Lp(OPR)=1.2x10(-6) m s(-1) MPa(-1)) was larger by a factor of 30 than the overall hydraulic conductivity (Lp(r)=4x10(-8) m s(-1) MPa(-1)) as measured by pressure chamber and root pressure probe. Low reflection coefficients were obtained for mannitol and NaCl for the OPR (sigma(sOPR)=0.14 and 0.09, respectively). The diffusional water permeability ( P(dOPR)) estimated from isobaric flow of heavy water was smaller by three orders of magnitude than the hydraulic conductivity (Lp(OPR)/ P(fOPR)). Although detailed root anatomy showed well-defined Casparian bands and suberin lamellae in the exodermis, the findings strongly indicate a predominantly apoplastic water flow in the OPR. The Lp(OPR) of heat-killed root segments increased by a factor of only 2, which is in line with the conclusion of a dominating apoplastic water flow. The hydraulic resistance of the OPR was not limiting the passage of water across the root cylinder. Estimations of the hydraulic properties of aerenchyma suggested that the endodermis was rate-limiting the water flow, although the aerenchyma may contribute to the overall resistance. The resistance of the aerenchyma was relatively low, because mono-layered cortical septa crossing the aerenchyma ('spokes') short-circuited the air space between the stele and the OPR. Spokes form hydraulic bridges that act like wicks. Low diffusional water permeabilities of the OPR suggest that radial oxygen losses from aerenchyma to medium are also low. It is concluded that in rice roots, water uptake and oxygen retention are optimized in such a way that hydraulic water flow can be kept high in the presence of a low efflux of oxygen which is diffusional in nature.     

Osmotic water permeability of Necturus gallbladder epithelium

An electrophysiological technique that is sensitive to small changes in cell water content and has good temporal resolution was used to determine the hydraulic permeability (Lp) of Necturus gallbladder epithelium. The epithelial cells were loaded with the impermeant cation tetramethylammonium (TMA+) by transient exposure to the pore-forming ionophore nystatin in the presence of bathing solution TMA+. Upon removal of the nystatin a small amount of TMA+ is trapped within the cell. Changes in cell water content result in changes in intracellular TMA+ activity which are measured with intracellular ion-sensitive microelectrodes. We describe a method that allows us to determine the time course for the increase or decrease in the concentration of osmotic solute at the membrane surface, which allows for continuous monitoring of the difference in osmolality across the apical membrane. We also describe a new method for the determination of transepithelial hydraulic permeability (Ltp). Apical and basolateral membrane Lp's were assessed from the initial rates of change in cell water volume in response to anisosmotic mucosal or serosal bathing solutions, respectively. The corresponding values for apical and basolateral membrane Lp's were 0.66 x 10(-3) and 0.38 x 10(-3) cm/s.osmol/kg, respectively. This method underestimates the true Lp values because the nominal osmotic differences (delta II) cannot be imposed instantaneously, and because it is not possible to measure the true initial rate of volume change. A model was developed that allows for the simultaneous determination of both apical and basal membrane Lp's from a unilateral exposure to an anisosmotic bathing solution (mucosal). The estimates of apical and basal Lp with this method were 1.16 x 10(-3) and 0.84 x 10(-3) cm/s.osmol/kg, respectively. The values of Lp for the apical and basal cell membranes are sufficiently large that only a small (less than 3 mosmol/kg) transepithelial difference in osmolality is required to drive the observed rate of spontaneous fluid absorption by the gallbladder. Furthermore, comparison of membrane and transepithelial Lp's suggests that a large fraction of the transepithelial water flow is across the cells rather than across the tight junctions.     

Determination of bull sperm membrane permeability to water and cryoprotectants using a concentration-dependent self-quenching fluorophore

The objective of this study was to determine the membrane permeability characteristics of bovine spermatozoa. These included the hydraulic conductivity (Lp), the permeability coefficients (Ps) of four common cryoprotective agents (CPAs) and the associated reflection coefficients (sigma). Stopped-flow fluorometry was applied in order to capture rapid cell volume changes under anisosmotic conditions in the absence or presence of permeant solutes (CPAs). This technique utilizes a concentration-dependent self-quenching entrapped fluorophore. The resulting cell volume changes were used in three-parameter fitting calculations to compute Lp in the absence of permeant solutes and Ps and Lp in the presence of permeating solutes (CPAs) at 22 degrees C. The hydraulic conductivity in the absence of permeating solutes was estimated to be 0.68+/-0.05 microm/min/atm (mean+/-SEM). Hydraulic conductivity (Lp) in the presence of CPAs was 0.91+/-0.27 (mean+/-SEM), 0.29+/-0.04, 0.42+/-0.05, and 0.39+/-0.03 microm/min/atm in the presence of dimethylsulfoxide (Me(2)SO), glycerol, propylene glycol (PG), and ethylene glycol (EG), respectively. The values for Ps were estimated to be 1.72+/-0.36, 1.75+/-0.03, 2.47+/-0.24, and 1.49+/-0.33 x 10(-3)cm/min for Me(2)SO, glycerol, PG, and EG, respectively. The data were used to simulate volume excursions during addition and removal of CPA, to predict the different effects of the four CPAs.     

Water channels in Chara corallina

Water relations parameters ofChara corallina inter-nodes weremeasured using the single cell pressure probe. The effect ofmercurials, which are recognized as non-specific water channelinhibitors, was examined. HgCl₂ concentrations greater than5 mmol m^–3 were found to inhibit hydraulic conductivity{Lp) close to 90%, whereas pCMPS was found to have no effecton Lp. The activation energy of water flow was increased significantlyfrom 21.0 kJ mol^–1 to 45.6 kJ mol^–1, following theapplication of HgCl₂. These results are in accordance with evidencefor Hg²⁺sensitive water channels in the plasma membrane of charophytes(Henzler and Steudle, 1995; Tazawa et al., 1996). The metaboliceffects must, however, be considered in view of the rapid inhibitionof respiration and the depolarization of the membrane potentialwith HgCl₂ concentrations lower than those found to affect Lp.It was possible to measure simultaneously water relations andmembrane PD, in order to examine the contribution of potassiumchannels to Lp. Cells were induced into a K⁺ permeable state.The K⁺ channels, assumed to be open, were subsequently blockedby various blockers. No significant difference in Lp was foundfor any of these treatments. Finally, the permeability of C.corallina membranes to ethanol was examined. HgCl₂ was foundto cause a decrease in reflection coefficient, coinciding witha decrease in Lp, but there was no change in the ethanol permeabilitycoefficient. This has been interpreted in terms of both thefrictional model and composite model of non-electrolyte membranetransport. Key words: Water channels, Chara, hydraulic, conductivity, membrane transport models, reflection coefficient