The water permeability of the monoolein/triolein bilayer membrane

The water permeability of the lipid bilayer can be used as a probe of membrane structure. A simple model of the bilayer, the liquid hydrocarbon model, views the membrane as a thin slice of bulk hydrocarbon liquid. A previous study (Petersen, D. (1980) Biochim. Biophys. Acta 600, 666–677) showed that this model does not accurately predict the water permeability of the monoolein/n-hexadecane bilayer: the measured activation energy for water permeation is 50% above the predicted value. From this it was inferred that the hydrocarbon chains in the lipid bilayer are more ordered than in the bulk hydrocarbon liquid. The present study tests the liquid hydrocarbon model for the monoolein/triolein bilayer, which has been shown to contain very little triolein in the plane of the membrane (Waldbillig, R.C. and Szabo, G. (1979) Biochim. Biophys. Acta 557, 295–305). Measurements of the water permeability coefficient of the bilayer are compared with predictions of the liquid hydrocarbon model based on measurements of the water permeability coefficient of bulk 8-heptadecene. The predicted and measured values agree quite closely over the temperature range studied (15–35°C): the predicted activation energy is 11.1±0.2 kcal/mol, whereas the measured activation energy for the bilayer is 9.8±0.7 kcal/mol. This close agreement is in contrast with the monoolein/n-hexadecane results and suggests that, insofar as water permeation is concerned, the liquid hydrocarbon model quite closely represents the monoolein/triolein bilayer.     

The effects of cholesterol inclusion on the molecular organisation of bimolecular lipid membranes

Dielectric measurements on planar egg phosphatidylcholine bilayers formed from n-hexadecane solutions indicate that these bilayers contain very low equilibrium concentrations of alkane. In 100 mM KCl the capacitance of the hydrophobic region was found to be 7.0 ±0.2 mF/m². The addition of cholesterol (at 2:1 mole ratio) was found to affect only marginally the capacitance of the hydrophobic region of such bilayers. Precise measurements of the frequency dependence of the bilayer impedance at very low frequencies now allow the resolution of several electrically distinct substructural regions within the bilayer. Examination of the effects of cholesterol inclusion upon the electrical parameters of these substructural regions indicate that cholesterol spans the acetyl region (i.e. the region containing the glycerol bridge of the phosphatidylcholine molecules in the bilayer) with the hydroxyl group of the cholesterol molecules located inbetween the phosphate group and the glycerol oxygens of the phosphatidylcholine molecules. The capacitance of the hydrophobic region of both phosphatidylcholine and phosphatidylcholine/cholesterol bilayers formed from n-hexadecane solutions was found to decrease slightly as the external KCl concentration was decreased.     

The thickness of monoolein lipid bilayers as determined from reflectance measurements

Measurements of the reflectance of monoolein $\text{n-}\text{alkane}$ and monoolein/squalene lipid bilayers have been made. The total thickness of the bilayer was calculated from the dependence of reflectance on the refractive index of the aqueous salt or sucrose solution surrounding the bilayer. The total thickness was then compared to the thickness of the hydrocarbon chain region as determined from capacitance measurements. From this comparison, we found that the thickness of each polar region of the bilayers in salt solutions was $\text{0.5 ± 0.1}\text{nm}$, independent of the hydrocarbon solvent used. When the aqueous solutions contained sucrose, each polar region was approx. 0.9 nm thick. When $\text{n-}\text{tetradecane}$ and $\text{n-}\text{hexadecane}$ were used as solvents, microlenses of solvent trapped in the monoolein bilayer increased the reflectance. After about one hour, the coalescence of microlenses into larger lenses allowed the reflectance of the bilayer alone to be measured. The use of reflectance to measure the thickness of monoolein bilayers appears to be consistent with other methods and to give useful information about the structure of lipid bilayers.     

NMR studies of glycerol permeability in lipid vesicles, erythrocytes and the alga Dunaliella

Glycerol diffusional permeabilities through the cytoplasmic cell membrane of Dunaliella salina, the cell envelope of pig erythrocyte and egg phosphattidylcholine vesicles were measured by NMR spectroscopy employing the spin-echo method and nuclear T₁ relaxation. The following permeability coefficients (P) and corresponding enthalpies of activation (ΔH^‡) were determined for glycerol at 25°C: for phosphatidylcholine vesicles 5·10⁻⁶ cm/s and 11±2 kcal/mol; for pig erythrocytes 7·10⁻⁸ cm/s and 18±3 kcal/mol, respectively; for the cytoplasmic membrane of D. salina the permeability at 17°C was found to be exceptionally low and only a lower limit (P<5·10⁻¹¹cm/s) could be calculated. At temperatures above 50°C a change in membrane permeability occurred leading to rapid leakage of glycerol accompanied by cell death. The data reinforce the notion that the cytoplasmic membrane of Dunaliella represents a genuine anomaly in its exceptional low permeability to glycerol.     

The diffusional water permeability in the halotolerant alga Dunaliella as measured by nuclear magnetic resonance

T₁ nuclear relaxation measurements of ¹H and ¹⁷O of water have been applied to study the kinetics of the diffusional transport of water across the cytoplasmic cell membrane of Dunaliella salina and Dunaliella bardawil. The water permeability coefficients at 25°C were found to be 1.5·10⁻³ cm/s and 1.8·10⁻³ cm/s, respectively, with an activation energy of 3.7 kcal/mol. The results indicate that the cell membrane of Dunaliella exhibits high diffusional permeability to water, similar in magnitude to that found for other cells and model membranes, and a relatively low activation energy. This regularity is in contrast to the exceptionally low glycerol permeability of the membrane (Brown, F.F., Sussman, I., Avron, M. and Degani, H. (1982) Biochim. Biophys. Acta 690, 165–173).     

Electrical capacity of black lipid films and of lipid bilayers made from monolayers

Planar bilayer membranes were formed from monolayers of a series of monounsaturated monoglycerides and lecithins. The hydrocarbon thickness of these membranes, as calculated from the electrical capacity, increases with the length of the fatty acid chain. The specific capacity of monoolein bilayers was found to be 0.745 μF/cm² which is nearly twice that of a monoolein black film made in the presence of decane, but is close to that obtained after freezing out the solvent from the black film. The hydrocarbon thickness of the bilayer, as calculated with a dielectric constant of 2.1, is considerably less than twice the length of the extended hydrocarbon chain of the monoglyceride.The specific capacity ($\text{C}{}_{\mathrm{<mtext>m</mtext>}}$) of bilayers made from monoolein monolayers showed a negligible voltage dependence, whereas the $\text{C}{}_{\mathrm{<mtext>m</mtext>}}$ increased significantly at a voltage of 150 mV in the case of Mueller-Rudin-type monoolein films with $\text{n-}\text{decane}$ as a solvent.     

A structural transition in egg lecithin-cholesterol bilayers at 12 °C

The thermal coefficient of expansion of egg lecithin bilayer thickness, αd₁, was measured as a function of its cholesterol content up to mole ratio lecithin/cholesterol of 1:1, and over the temperature range 0–40 °C. At all cholesterol contents αd₁ changes abruptly at approximately 12 °C indicating a structural transition at this temperature. Above 12 °C, αd₁ decreases monotonically from −2·10⁻³ for pure egg lecithin to −1·10^–3 at mole ratio 1:1. Below 12 °C αd₁ is walways higher than above 12 °C and shows a sharp, anomalously high value of −6·10⁻³ at the mole ratio 2:1. The results have been interpreted as the movement of cholesterol into the bilayer or the formation of lecithin-cholesterol “complexes” at temperatures below 12 °C. Similar studies with phosphatidylinositol containing cholesterol showed no structural transition and lysolecithin containing cholesterol behaved differently giving two lamellar phases in equilibrium.     

Transport methods for probing the barrier domain of lipid bilayer membranes.

Two experimental techniques have been utilized to explore the barrier properties of lecithin/decane bilayer membranes with the aim of determining the contributions of various domains within the bilayer to the overall barrier. The thickness of lecithin/decane bilayers was systematically varied by modulating the chemical potential of decane in the annulus surrounding the bilayer using different mole fractions of squalene in decane. The dependence of permeability of a model permeant (acetamide) on the thickness of the solvent-filled region of the bilayer was assessed in these bilayers to determine the contribution of this region to the overall barrier. The flux of acetamide was found to vary linearly with bilayer area with Pm = (2.9 +/- 0.3) x 10(-4) cm s-1, after correcting for diffusion through unstirred water layers. The ratio between the overall membrane permeability coefficient and that calculated for diffusion through the hydrocarbon core in membranes having maximum thickness was 0.24, suggesting that the solvent domain contributes only slightly to the overall barrier properties. Consistent with these results, the permeability of acetamide was found to be independent of bilayer thickness. The relative contributions of the bilayer interface and ordered hydrocarbon regions to the transport barrier may be evaluated qualitatively by exploring the effective chemical nature of the barrier microenvironment. This may be probed by comparing functional group contributions to transport with those obtained for partitioning between water and various model bulk solvents ranging in polarity or hydrogen-bonding potential. A novel approach is described for obtaining group contributions to transport using ionizable permeants and pH adjustment. Using this approach, bilayer permeability coefficients of p-toluic acid and p-hydroxymethyl benzoic acid were determined to be 1.1 +/- 0.2 cm s-1 and (1.6 +/- 0.4) x 10(-3) cm s-1, respectively. From these values, the -OH group contribution to bilayer transport [delta(delta G0-OH)] was found to be 3.9 kcal/mol. This result suggests that the barrier region of the bilayer does not resemble the hydrogen-bonding environment found in octanol, but is somewhat less selective (more polar) than a hydrocarbon solvent.     

Phospholipid surface density determines the partitioning and permeability of acetic acid in DMPC:cholesterol bilayers

Relationships between the permeability coefficient (P_HA) and partition coefficient (K _m/w) of acetic acid and the surface density of DMPC:cholesterol bilayers have been investigated. Permeability coefficients were measured in large unilamellar vesicles by NMR line broadening. Bilayer surface density, , was varied over a range of 0.5–0.9 by changing cholesterol concentration and temperature. The temperature dependence of P_HA for acetic acid exhibits Arrhenius behavior with an average apparent activation energy (E _a ) of 22±3 kcal/mole over a cholesterol mole fraction range of 0.00–0.40. This value is much greater than the enthalpy change for acetic acid partitioning between bulk decane and water (H° = 4.8±0.8 kcal/mole) and the calculated E _a (= 8.0 kcal/mole) assuming a bulk phase permeability model which includes the enthalpy of transfer from water to decane and the temperature dependence of acetic acid's diffusion coefficient in decane. These results suggest that dehydration, previously considered to be a dominant component, is a minor factor in determining E _a . Values of 1n P_HA decrease linearly with the normalized phospholipid surface density with a slope of = -12.4±1.1 (r = 0.90). Correction of P_HA for those temperature effects considered to be independent of lipid chain order (i.e., enthalpy of transfer from water to decane and activation energy for diffusion in bulk hydrocarbon) yielded an improved correlation ( = -11.7±0.5 (r = 0.96)). The temperature dependence of K_m/w is substantially smaller than that for P_HA and dependent on cholesterol composition. Values of 1n K_m/w decrease linearly with the surface density with a slope of = -4.6±0.3 (r = 0.95), which is 2.7-fold smaller than the slope of the plot of 1n P_HA vs. . Thus, chain ordering is a major determinant for molecular partitioning into and transport across lipid bilayers, regardless of whether it is varied by lipid composition or temperature.This work was supported by grants from Glaxo, INTERx/Merck, and University of Utah Research Committee.     

Liver Freezing Response of the Freeze-Tolerant Wood Frog, Rana sylvatica, in the Presence and Absence of Glucose. II. Mathematical Modeling

The “two-step” low-temperature microscopy (equilibrium and dynamic) freezing methods and a differential scanning calorimetry (DSC) technique were used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing, in Part I of this study. In this study, the experimentally determined dynamic water transport data are curve fit to a model of water transport using a standard Krogh cylinder geometry (Model 1) to predict the biophysical parameters of water transport: L_pg = 1.76 μm/min-atm and E_Lp = 75.5 kcal/mol for control liver cells and L_pg[cpa] = 1.18 μm/min-atm and E_Lp[cpa] = 69.0 kcal/mol for liver cells equilibrated with 0.4 M glucose. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5°C/min but do so when cooled at 2°C/min. Cells also retained twice as much intracellular fluid in the presence of 0.4 M glucose than in control tissue when cooled at 5°C/min. The ability of R. sylvatica liver cells to retain water during fast cooling (≥5°C/min) appears to be primarily due to its liver tissue architecture and not to a dramatically lower permeability to water, in comparison to mammalian (rat) liver cells which do dehydrate completely when cooled at 5°C/min. A modified Krogh model (Model 2) was constructed to account for the cell–cell contact in frog liver architecture. Using the same biophysical permeability parameters obtained with Model 1, the modified Krogh model (Model 2) is used in this study to qualitatively explain the experimentally measured water retention in some cells during freezing on the basis of different volumetric responses by cells directly adjacent to vascular space versus cells at least one cell removed from the vascular space. However, at much slower cooling rates (1–2°C/h) experienced by the frog in nature, the deciding factor in water retention is the presence of glucose and the maintenance of a sufficiently high subzero temperature (≥−8°C).     

The State of Water in Human and Dog Red Cell Membranes

The apparent activation energy for the water diffusion permeability coefficient, P_d, across the red cell membrane has been found to be 4.9 ± 0.3 kcal/mole in the dog and 6.0 ± 0.2 kcal/mole in the human being over the temperature range, 7° to 37°C. The apparent activation energy for the hydraulic conductivity, L_p, in dog red cells has been found to be 3.7 ± 0.4 kcal/mole and in human red cells, 3.3 ± 0.4 kcal/mole over the same temperature range. The product of L_p and the bulk viscosity of water, η, was independent of temperature for both dog and man which indicates that the geometry of the red cell membrane is not temperature-sensitive over our experimental temperature range in either species. In the case of the dog, the apparent activation energy for diffusion is the same as that for self-diffusion of water, 4.6–4.8 kcal/mole, which indicates that the process of water diffusion across the dog red cell membrane is the same as that in free solution. The slightly, but significantly, higher activation energy for water diffusion in human red cells is consonant with water-membrane interaction in the narrower equivalent pores characteristic of these cells. The observation that the apparent activation energy for hydraulic conductivity is less than that for water diffusion across the red cell membrane is characteristic of viscous flow and suggests that the flow of water across the membranes of these red cells under an osmotic pressure gradient is a viscous process.     

Vasopressin and water permeability of artificial lipid membranes

Vasopressin markedly stimulated the water permeability of bilayer lipid membranes: a two-fold increase was measured at 25° in presence of 1.7·10⁻⁹ M (50 μunits/ml) vasopressin. Oxytocin and a mixture of the amino acids comprising the vasopressin molecule could not substitute for vasopressin at comparable concentration. The experimental activation energy of water transport was reduced in the presence of vasopressin from 14 to 4 kcal/mole, in agreement with the effect of the hormone on water permeability of toad bladder.     

The nonelectrolyte permeability of planar lipid bilayer membranes

The permeability of lecithin bilayer membranes to nonelectrolytes is in reasonable agreement with Overton's rule. The is, Pd alpha DKhc, where/Pd is the permeability coefficient of a solute through the bilayer, Khc is its hydrocarbon:water partition coefficient, and D is its diffusion coefficient in bulk hydrocarbon. The partition coefficients are by far the major determinants of the relative magnitudes of the permeability coefficients; the diffusion coefficients make only a minor contribution. We note that the recent emphasis on theoretically calculated intramembranous diffusion coefficients (Dm'S) has diverted attention from the experimentally measurable and physiologically relevant permeability coefficients (Pd'S) and has obscured the simplicity and usefulness of Overton's rule.     

The State of Water in the Isolated Toad Bladder in the Presence and Absence of Vasopressin

An attempt has been made to assess the validity of applying the frictional and viscous coefficients of bulk water to the movement of water and solutes through the urinary bladder of the toad. The temperature dependence of diffusion of THO, C¹⁴-urea, C¹⁴-thiourea, and net water transfer across the bladder was determined in the presence and absence of vasopressin. The activation energy for diffusion of THO was 9.8 kcal per mole in the absence of vasopressin and 4.1 kcal per mole with the hormone present. Activation energies simultaneously determined following vasopressin for diffusion and net transfers of water were similar, and in the same range as known activation energies for diffusion and viscous flow in water. Urea had activation energies for diffusion of 4.1 and 3.9 kcal per mole in the absence and presence of vasopressin, respectively. Thiourea had a high activation energy for diffusion of 6.3 kcal per mole, which was unchanged, 6.6 kcal per mole, following hormone. These findings suggest that in its rate-limiting permeability barrier, water is present in a structured state, offering a high resistance to penetration by water. Vasopressin enlarges the aqueous channels so that the core of water they contain possesses the physical properties of ordinary bulk water. Urea penetrates the tissue via these aqueous channels while thiourea is limited by some other permeability barrier.     

Comparison of the permeability properties and post-thaw motility of ejaculated and epididymal bovine spermatozoa

There are very few experimental reports on the comparative water transport (membrane permeability) characteristics of ejaculated and epididymal mammalian spermatozoa during freezing. In the present study, we report the effects of cooling ejaculated and epididymal bovine sperm from the same males with and without the presence of a cryoprotective agent, glycerol. Water transport data during freezing of ejaculated and epididymal bovine sperm suspensions were obtained at a cooling rate of 20 °C/min under two different conditions: (1) in the absence of any cryoprotective agents, CPAs and, (2) in the presence of 0.7 M glycerol. Using values published in the literature, we modeled the spermatozoa as a cylinder of length 39.8 μm and a radius of 0.4 μm with an osmotically inactive cell volume, V_b, of 0.61V_o, where V_o is the isotonic cell volume. The subzero water transport response is analyzed to determine the variables governing the rate of water loss during cooling of bovine spermatozoa, i.e. the membrane permeability parameters (reference membrane permeability, L_pg and activation energy, E_Lp). The predicted best-fit permeability parameters ranged from, L_pg = 0.021–0.038 μm/min-atm and E_Lp = 27.8–41.1 kcal/mol. The subzero water transport response and consequently the subzero water transport parameters are not significantly different between the ejaculated and epididymal bovine spermatozoa under corresponding cooling conditions. If this observation is found to be more generally valid for other mammalian species as well, then in the future the sperm extracted from the testes of a postmortem male could be optimally cryopreserved using procedures similar to those derived for ejaculated sperm.     

Temperature Dependence of Vasopressin Action on the Toad Bladder

Toad bladders were challenged with vasopressin at one temperature, fixed on the mucosa with 1% glutaraldehyde, and then subjected to an osmotic gradient at another temperature. Thus, the temperature dependence of vasopressin action on membrane permeability was distinguished from the temperature dependence of osmotic water flux. As the temperature was raised from 20° to 38°C, there was a substantial increase in the velocity of vasopressin action, but osmotic flux was hardly affected. In this range of temperature the apparent energy of activation for net water movement across the bladder amounted to only 1.2 kcal/mole, a value well below the activation energy for bulk water viscosity. It is suggested that osmotic water flux takes place through narrow, nonpolar channels in the membrane. When the temperature was raised from 4° to 20°C, both vasopressin action as well as osmotic water flux were markedly enhanced. Activation energies for net water movement were now 8.5 kcal/mole (4°–9°C) and 4.1 kcal/mole (9°–20°C), indicating that the components of the aqueous channel undergo conformational changes as the temperature is lowered from 20°C. At 43°C bladder reactivity to vasopressin was lost, and irreversible changes in selective permeability were observed. The apparent energy of activation for net water movement across the denatured membrane was 6.6 kcal/mole. Approximately 1 µosmol of NaCl was exchanged for 1 µl of H₂O across the denatured membrane.     

Osmotic water permeability of small intestinal brush-border membranes

Summary A stopped-flow nephelometric technique was used to examine osmotic water flow across small intestinal brush-border membranes. Brush-border membrane vesicles (BBMV) were prepared from rat small intestine by calcium precipitation. Scattered 500 nm light intensity at 90° to incident was a linear function of the number of vesicles in suspension, and of the reciprocal of the suspending medium osmolality. When BBMV were mixed with hyperosmotic mannitol solutions there was a rapid increase in the intensity of scattered light that could be fit to a single exponential function. The rate constant for vesicle shrinking varied with temperature and the size of the imposed osmotic gradient. At 25°C and an initial osmotic gradient of 50 mOsm, the rate constant was 1.43±0.044 sec^–1. An Arrhenius plot of the temperature dependence of vesicle shrinking showed a break at about 25°C with an activation energy of 9.75±1.04 kcal/mole from 11 to 25°C and 17.2±0.55 kcal/mole from 25 to 37°C. The pore-forming antibiotic gramicidin increased the rate of osmotically driven water efflux and decreased the activation energy of the process to 4.51±0.25 kcal/mole. Gramicidin also increased the sodium permeability of these membranes as measured by the rate of vesicle reswelling in hyperosmotic NaSCN medium. Gramicidin had no effect on mannitol permeability. Assuming spherical vesicles of 0.1 m radius, an osmotic permeability coefficient of 1.2×10^–3 cm/sec can be estimated for the native brush-border membranes at 25°C. These fesults are consistent with the solubility-diffusion model for water flow across small intestinal BBMV but are inconsistent with the existence there of large aqueous pores.