Stopped-flow study of anesthetic effect on water-transport kinetics through phospholipid membranes. Interfacial versus lipid core ligands

We have compared ligand effects between polar and apolar anesthetic molecules upon water transport across phospholipid membranes by kinetic analysis of the osmotic swelling rate, using a stopped-flow technique. Chloroform and 1-hexanol were used as interfacial ligands, and carbon tetrachloride and n-hexane were used as their counterparts, representing lipid core action. Because anesthetics transform the solid-gel membrane into a liquid-crystalline state, and because phospholipid membranes display an anomaly in permeability at the phase transition, dimyristoylphosphatidylcholine vesicles were studied at temperatures above the main phase transition to avoid this anomaly. All these molecules increased the osmotic swelling rate. However, a significant difference was observed in the activation energy, delta Ep, between polar and apolar molecules; delta Ep was almost unaltered by the addition of polar molecules (chloroform and 1-hexanol), whereas it was decreased by apolar molecules (carbon tetrachloride and n-hexane). The obtained results were analyzed in terms of the dissolution-diffusion mechanism for water permeation across the lipid membrane. It is suggested that polar molecules affect water permeability by altering the partition of water between the membrane interior and water phase, and apolar molecules affect it by altering both the partition and the diffusion of water within the membrane interior.     

Molecular origin of biphasic response of main phase-transition temperature of phospholipid membranes to long-chain alcohols

A statistical mechanical theory is proposed which explains the molecular mechanism of the nonlinear response of the phase-transition temperature of phospholipid vesicle membranes to added 1-alkanols. By assuming that the free energy of transfer of 1-alkanols from the aqueous phase to the membrane and the interaction energy between 1-alkanol molecules are linear functions of alkanol alkyl chain-length, the nonlinear behavior is explained in the Bragg-Williams approximation. For dipalmitoylphosphatidylcholine vesicle membranes, the theory reveals a larger free energy of transfer of 1-alkanols from the aqueous phase to the solid-gel membrane than to the liquid-crystalline membrane when the number of carbon atoms of 1-alkanol exceeds 12. When the intermolecular interaction force between 1-alkanol molecules residing in the gel phase is stronger than the interaction force between those residing in the liquid-crystalline phase, the ligand effect is to tighten the lipid matrix structure, causing the transition temperature to rise. The interaction force is a quadratic function of 1-alkanol concentration; hence, the response of the transition temperature to the 1-alkanol concentration is nonlinear. At low concentrations of the long-chain 1-alkanols that predominantly elevate the transition temperature, this intermolecular interaction force is negligible. In this case, the entropic effect of the incorporated ligand molecules, which loosens the lipid matrix, predominates, and the transition temperature decreases. The biphasic action of long-chain 1-alkanols originates from the balance of these two opposing effects: entropy and intermolecular interaction.     

Activation of phospholipase D by osmotic cell swelling

In response to osmotic cell swelling, Intestine 407 cells react with a rapid and transient activation of phospholipase D (PLD). To investigate the role of PLD during the regulatory volume decrease, cells were treated with 1-butanol resulting in a depletion of PLD substrates. Activation of volume-regulated anion channels, but not the cell swelling-induced release of taurine, was largely inhibited in the presence of low concentrations of 1-butanol. In addition, hypotonicity-induced exocytosis, ATP release and subsequent endocytosis were found to be largely abrogated. The results support a model of cell volume regulation in which PLD plays an essential role in the cell swelling-induced vesicle cycling and in the activation of volume-sensitive anion channels.     

Osmotic gradient-induced water permeation across the sarcolemma of rabbit ventricular myocytes

The mechanism of water permeation across the sarcolemma was characterized by examining the kinetics and temperature dependence of osmotic swelling and shrinkage of rabbit ventricular myocytes. The magnitude of swelling and the kinetics of swelling and shrinkage were temperature dependent, but the magnitude of shrinkage was very similar at 6 degrees, 22 degrees, and 37 degrees C. Membrane hydraulic conductivity, Lp, was approximately 1.2 x 10(-10) liter.N-1.s-1 at 22 degrees C, corresponding to an osmotic permeability coefficient, Pf, of 16 microns.s-1, and was independent of the direction of water flux, the magnitude of the imposed osmotic gradient (35-165 mosm/liter), and the initial cell volume. This value of Lp represents an upper limit because the membrane was assumed to be a smooth surface. Based on capacitive membrane area, Lp was 0.7 to 0.9 x 10(-10) liter.N-1.s-1. Nevertheless, estimates of Lp in ventricle are 15 to 25 times lower than those in human erythrocytes and are in the range of values reported for protein- free lipid bilayers and biological membranes without functioning water channels (aquaporin). Evaluation of the effect of unstirred layers showed that in the worst case they decrease Lp by < or = 2.3%. Analysis of the temperature dependence of Lp indicated that its apparent Arrhenius activation energy, Ea', was 11.7 +/- 0.9 kcal/mol between 6 degrees and 22 degrees C and 9.2 +/- 0.9 kcal/mol between 22 degrees and 37 degrees C. These values are significantly greater than that typically found for water flow through water-filled pores, approximately 4 kcal/mol, and are in the range reported for artificial and natural membranes without functioning water channels. Taken together, these data strongly argue that the vast majority of osmotic water flux in ventricular myocytes penetrates the lipid bilayer itself rather than passing through water-filled pores.     

Anesthetic-protein interaction Random versus helix polylysine monolayers and interaction with 1-alkanols

Penetration of 1-alkanols into monolayers of hydrophobic polypeptides, poly(ε-benzyloxycarbonyl-l-lysine) and poly(ε-benzyloxycarbonyl-dl-lysine), was compared with their adsorption on the air/water interface in the absence of monolayers. The polypeptide prepared from l-lysine is generally considered to be in the α-helical form whereas dl-copolymer polypeptide contains random-coiled portions due to the structural incompatibility between the two isomers. The free energy of adsorption of 1-alkanols on the air/water interface at dilute concentrations was ?0.68 kcal·mol^?1 per methylene group and 0.15 kcal·mol^?1 for the hydroxyl group at 25°C. In the close-packed state, the surface area occupied by each molecule of 1-alkanols of varying carbon chain-lengths showed nearly a constant value of about 27.2 Ų, indicating perpendicular orientation of the alkanol molecules at the interface. About 75% of the water surface was covered by 1-butanol in this close-packed state. The mode of adsorption of 1-alkanols on the vacant air/water interface followed the Gibbs surface excess while the mode on the polypeptide membranes followed the Langmuir adsorption isotherm, indicating that the latter is characterized by the presence of a finite number of binding sites. The free energies of adsorption of 1-alkanols on the l-polymer monolayers were more negative than those on the vacant air/water interface and less negative than those on the dl-copolymer monolayers. Thus, the affinity of 1-alkanols to the interface was in the order of vacant air/water interface <l-polymer <dl-copolymer. The difference between the air/water interface and l-polymer was about 0.54 kcal·mol^?1 and that between l-polymer and dl-copolymer was 0.17 kcal·mol^?1 at 25°C: the adsorption of 1-alkanols to the dl-copolymer was favored compared to the l-polymer. The polar moieties of the backbone of the dl-copolymer may be exposed to the aqueous phase at the disordered portion. Dipole interaction between this portion and 1-alkanol molecules may account for the enhanced adsorption of the alkanols to the dl-copolymer.     

Osmotic swelling of hepatocytes increases membrane conductance but not membrane capacitance.

We have used the whole-cell patch-clamp technique to study changes in membrane conductance and membrane capacitance after osmotic swelling in rat hepatocytes. Hypoosmotic solutions induced an instantaneous increase in the volume of patch-clamped cells that was followed by a slow decline reminiscent of regulatory volume decrease as seen in intact cells. These morphological changes were associated with a transient increase in membrane conductance. The rise in conductance was not correlated with changes in capacitance, neither in time after the initiation of cell swelling nor in magnitude. Therefore we conclude that an osmotically induced increase in conductance is probably a result of the activation of existent channels in the plasmalemma and not a result of the fusion of vesicle membrane containing ionic channels.     

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.     

Permeability of lipid membranes revised in relation to freeze-thaw processes.

Water and solute activity gradients created during freeze-thaw processes produce water and solute fluxes across the cell membrane resulting in volume changes. Under these conditions, osmotic and thermal stresses affect the curvature, the phase behavior, and the surface properties of the lipid bilayer. These structural changes are not considered by the classical formalisms describing permeability of lipid membranes to water and nonelectrolytes such as the Nernst-Planck equation, Eyring's absolute rate theory, and Kedem-Katchalsky's thermodynamic of irreversible processes approach. In this paper, the influence of such changes on the glycerol permeation kinetics are reported. The results indicate that osmotic and chemical effects of the cryoprotectant on the membrane properties affect the rate of volume swelling depending on whether the membrane is in the gel or in the liquid crystalline state.     

1-Alkanols and membranes: A story of attraction

Although 1-alkanols have long been known to act as penetration enhancers and anesthetics, the mode of operation is not yet understood. In this study, long-time molecular dynamics simulations have been performed to investigate the effect of 1-alkanols of various carbon chain lengths onto the structure and dynamics of dimyristoylphosphatidylcholine bilayers. The simulations were complemented by microcalorimetry, continuous bleaching and film balance experiments. In the simulations, all investigated 1-alkanols assembled inside the lipid bilayer within tens of nanoseconds. Their hydroxyl groups bound preferentially to the lipid carbonyl group and the hydrocarbon chains stretched into the hydrophobic core of the bilayer. Both molecular dynamics simulations and experiments showed that all 1-alkanols drastically affected the bilayer properties. Insertion of long-chain 1-alkanols decreased the area per lipid while increasing the thickness of the bilayer and the order of the lipids. The bilayer elasticity was reduced and the diffusive motion of the lipids within the bilayer plane was suppressed. On the other hand, integration of ethanol into the bilayer enlarged the area per lipid. The bilayer became softer and lipid diffusion was enhanced.     

Influence of the growth at high osmolality on the lipid composition, water permeability and osmotic response of Lactobacillus bulgaricus

Changes in water permeability and membrane packing were measured in cells of Lactobacillus bulgaricus and in vesicles prepared with lipids extracted from them. The osmotic response of whole cells and vesicles is compared with the one of bacteria grown in a high osmolal medium. Both bacteria and vesicles, behave as osmometers. This means that the volume decrease is promoted by the outflow of water, driven by the NaCl concentration difference, arguing that neither Na+ nor Cl- permeates the cell or the lipid membrane in these conditions. Therefore, the volume changes can be correlated with the rate of water permeation across the cell or the vesicle membranes. The permeation of water was analyzed as a function of the lipid species by measuring the volume changes and the saturation ratio of the lipids. To put into relevance the membrane processes, the permeation properties of lipid vesicles prepared with lipids extracted from bacteria grown in normal and high osmolality conditions were also analyzed. The permeation response was correlated with the physical properties of the membrane of whole cells and vesicles, by means of fluorescence anisotropy of diphenyl hexatriene (DPH). The modifications in membrane properties are related with the changes in the membrane composition triggered by the growth in a high osmolal medium. The changes appear related to an increase in the sugar content of the whole pool of lipids and in the saturated fatty acid residues.     

1-Alkanols and membranes: a story of attraction

Although 1-alkanols have long been known to act as penetration enhancers and anesthetics, the mode of operation is not yet understood. In this study, long-time molecular dynamics simulations have been performed to investigate the effect of 1-alkanols of various carbon chain lengths onto the structure and dynamics of dimyristoylphosphatidylcholine bilayers. The simulations were complemented by microcalorimetry, continuous bleaching and film balance experiments. In the simulations, all investigated 1-alkanols assembled inside the lipid bilayer within tens of nanoseconds. Their hydroxyl groups bound preferentially to the lipid carbonyl group and the hydrocarbon chains stretched into the hydrophobic core of the bilayer. Both molecular dynamics simulations and experiments showed that all 1-alkanols drastically affected the bilayer properties. Insertion of long-chain 1-alkanols decreased the area per lipid while increasing the thickness of the bilayer and the order of the lipids. The bilayer elasticity was reduced and the diffusive motion of the lipids within the bilayer plane was suppressed. On the other hand, integration of ethanol into the bilayer enlarged the area per lipid. The bilayer became softer and lipid diffusion was enhanced.     

Characterization of Water Channels in Wheat Root Membrane Vesicles

The functional significance of water channels in wheat (Triticum aestivum L.) root membranes was assessed using light scattering to measure vesicle shrinking in response to osmotic gradients rapidly imposed in a stopped flow apparatus. Vesicles were obtained from both a plasma membrane fraction and a plasma membrane-depleted endomembrane fraction including tonoplast vesicles. Osmotic water permeability (Pos) in the endomembrane fraction was high (Pos= 86.0 [mu]m s-1) with a low activation energy (EA= 23.32 kJ mol-1 [plus or minus] 3.88 SE), and was inhibited by mercurials (K1= 40 [mu]M HgCl2, where K1 is the inhibition constant for half-maximal inhibition), suggesting participation of water channels. A high ratio of osmotic to diffusional permeability (Pd) (using D2O as a tracer, Pos/Pd = 7 [plus or minus] 0.5 SE) also supported this view. For the endomembrane fraction there was a marked decrease in Pos with increasing osmotic gradient that was not observed in the plasma membrane fraction. Osmotic water permeability in the plasma membrane fraction was lower (Pos= 12.5 [mu]m s-1) with a high activation energy (EA= 48.07 kJ mol-1 [plus or minus] 3.63 SE) and no mercury inhibition. Nevertheless, Pos/Pd was found to be substantially higher than one (Pos= 3 [plus or minus] 0.2 SE), indicating that water channels mediated water flow in this fraction, too. Possible distortion of the Pos/Pd value by unstirred layer effects was shown to be unlikely.     

Dimensions of polar pathways through rabbit gallbladder epithelium

Summary The permeability of rabbit gallbladder to hydrophilic nonelectrolytes, with molecular weights from 20 to 60,000, has been studied. Restriction in the diffusion of the small electrolytes is very significant up to glycerol, which suggests permeation through aqueous pores with equivalent radii of 4 Å. An extracellular pathway is responsible for the permeation of the larger solutes. This extracellular pathway shows no restriction in diffusion of molecules up to the size of inulin. Dextran (15,000 to 17,000 mol wt) is significantly restricted. Albumin permeability is <10^–8 cm sec^–1. These observations can be equated with equivalent, pore radii of 40 Å for the shunt pathway.Increasing osmolarities of the incubation medium cause decreased cell-membrane permeability and increased shunt permeability. 0.5mm phloretin induces a 60% reduction in urea permeability and a 168% increase in antipyrine permeability. No effect on the osmotic water permeability or on the shunt permeability is observed in the presence of phloretin. The apparent activation energy of urea permeation changes from values consistent with diffusion in bulk water, to values consistent with diffusion through hydrocarbon regions. This suggests that the polar route for urea permeation is blocked by phloretin.The contribution of the shunt pathway to osmotic flow induced by sucrose or NaCl gradients is smaller than 16% according to Poiseuille's flow calculations. Tetraethylammoniumchloride and albumin have been shown to be osmotically more effective than sucrose, suggesting a greater shunt contribution to the total water flow.     

Effects of divalent cations,temperature, osmotic pressure gradient,and vesicle curvature on phosphatidylserine vesicle fusion

Summary Fusion of phosphatidylserine vesicles induced by divalent cations, temperature and osmotic pressure gradients across the membrane was studied with respect to variations in vesicle size. Vesicle fusion was followed by two different methods: 1) the Tb/DPA fusion assay, whereby the fluorescent intensity upon mixing of the internal aqueous contents of fused lipid vesicles was monitored, and 2) measurement of the changes in turbidity of the vesicle suspension due to vesicle fusion. It was found that the threshold concentration of divalent cations necessary to induce vesicle fusion depended on the size of vesicles; as the diameter of the vesicle increased, the threshold value increased and the extent of fusion became less. For the osmotic pressure-induced vesicle fusion, the larger the diameter of vesicles, the smaller was the osmotic pressure gradient required to induce membrane fusion. Divalent cations, temperature increase and vesicle membrane expansion by osmotic pressure gradient all resulted in increase in surface energy (tension) of the membrane. The degree of membrane fusion correlated with the corresponding surface energy changes of vesicle membranes due to the above fusion-inducing agents. The increase in surface energy of 9.5 dyn/cm from the reference state corresponded to the threshold point of phosphatidylserine membrane fusion. An attempt was made to explain the factors influencing fusion phenomena on the basis of a single unifying theory.     

The influence of 1-alkanols and external pressure on the lateral pressure profiles of lipid bilayers

The suggestion by Robert Cantor, that drug-induced pressure changes in lipid bilayers can change the conformational equilibrium between open and closed states of membrane proteins and thereby cause anesthesia, attracted much attention lately. Here, we studied the effect of both large external pressure and of 1-alkanols of different chain lengths—some of them anesthetics, others not—on the lateral pressure profiles across dimyristoylphosphatidylcholine (DMPC) bilayers by molecular dynamics simulations. For a pure DMPC bilayer, high pressure both reduced and broadened the tension at the interface hydrophobic/hydrophilic and diminished the repulsion between the phospholipid headgroups. Whereas the effect of ethanol on the lateral pressure profile was similar to the effect of a large external pressure on a DMPC bilayer, long-chain 1-alkanols significantly amplified local maxima and minima in the lateral pressure profile. For most 1-alkanols, external pressure had moderate effects and did not reverse the changes 1-alkanols exerted on the pressure profile. Nevertheless, assuming the bent helix model as a simple geometric model for the transmembrane region of a membrane protein, protein conformational equilibria were shifted in opposite directions by addition of 1-alkanols and additional application of external pressure.     

Osmotic water permeability of plasma and vacuolar membranes in protoplasts I. High osmotic water permeability in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>) root cells as measured by a new method

Intra- and transcellular water movements in plants are regulated by the water permeability of the plasma membrane (PM) and vacuolar membrane (VM) in plant cells. In the present study, we investigated the osmotic water permeability of both PM (P ( f1)) and VM (P ( f2)), as well as the bulk osmotic water permeability of a protoplast (P ( f(bulk))) isolated from radish (Raphanus sativus) roots. The values of P ( f(bulk)) and P ( f2) were determined from the swelling/shrinking rate of protoplasts and isolated vacuoles under hypo- or hypertonic conditions. In order to minimize the effect of unstirred layer, we monitored dropping or rising protoplasts (vacuoles) in sorbitol solutions as they swelled or shrunk. P ( f1) was calculated from P ( f(bulk)) and P ( f2) by using the 'three-compartment model', which describes the theoretical relationship between P ( f1), P ( f2) and P ( f(bulk)) (Kuwagata and Murai-Hatano in J Plant Res, 2007). The time-dependent changes in the volume of protoplasts and isolated vacuoles fitted well to the theoretical curves, and solute permeation of PM and VM was able to be neglected for measuring the osmotic water permeability. High osmotic water permeability of more than 500 mum s(-1), indicating high activity of aquaporins (water channels), was observed in both PM and VM in radish root cells. This method has the advantage that P ( f1) and P ( f2) can be measured accurately in individual higher plant cells.     

Kinetics of water penetration into unsonicated liposomes effects ofn-alkanols and cholesterol

Summary The rate of swelling of egg lecithin liposomes under osmotic shock has been studied employing a stopped-flow spectrophotometer. Incorporation of cholesterol and simple alcohols into the liposomal structure elicits a biphasic response in swelling rate: at low concentrations these additives increase but at high concentrations they decrease water permeabilty. For simplen-alkanols, the effects can be correlated with structure. Specifically, the concentration of alcohol required to elicit maximal permeability as well as the maximal permeability decreases with increasing length of the alcohol. These effects are accounted for on the basis of modification of the orientation and packing of lecithin molecules in the bilayer membrane of the liposome.     

The permeability of liposomes to nonelectrolytes. I. Activation energies for permeation.

The effect of temperature on the permeability of nonelectrolytes across liposomal membranes above and below their transition temperature has been studied by using an osmotic method. Below their transition temperature, liposomes are osmotically insensitive structures but, on addition of gramicidin A, the water permeability so increased that the permeability of solutes could be studied. The measured activation energies for permeation of a variety of nonelectrolytes has been found to increase when a) there is an increase in the capability of the solutes to form hydrogen bonds in water, b) the cholesterol concentration in the membranes increases and c) the membranes pass from a liquid-crystalline to a solid-crystalline state. The change in the activation energy for permeation per hydrogen bond is about 1.8 kcal/mole for all the different liposome systems investigated; the only solute tested that deviated from this correlation was urea, whose activation energy for permeation across a gramicidin-containing system was much lower than expected from its hydrogen-bonding capacity. This finding suggests that urea is permeating across the gramicidin pore. Although the literature contains only incomplete data relating the activation energies for permeation of nonelectrolytes across biological membranes to their hydrogen-bonding capacity, the available evidence suggests that there is a similar correlation to that found in liposomes. Thus, the average increase in the activation energy per hydrogen bond for permeation across ox red cell membranes (Jacobs, Glassman & Parpart, J. Cell. Comp. Physiol. 7:197, 1935) is 2.2 plus or minus 0.4 kcal/mole, a value that is similar to that obtained in liposomes. However, the activation energies for water and urea are - in such a system - very much lower than expected, suggesting that they, too, are permeating by some parallel route such as an aqueous pore.