An NMR study of the temperature dependence of the coefficient of water self-diffusion through lipid bilayer membranes

The temperature dependence of the coefficient of water self-diffusion through plane-parallel lipid multilayers of the phospholipid dioleoylphosphatidylcholine oriented on a glass support has been studied in the temperature range of 20-60 degrees C by the method of NMR with magnetic field pulse gradient. The values of the coefficients of transbilayer water diffusion are by four orders of magnitude less than for bulky water and ten times less than the coefficients of lateral diffusion of the lipid under the same conditions. The temperature dependence of the coefficient of water diffusion is described by the Arrhenius law with an apparent activation energy of about 41 kJ/mol, which far exceeds the activation energy for the diffusion of bulky water (18 kJ/mol). The experimental data were analyzed using a "dissolving-diffusion" model, by simulating the passage of water through membrane channels, and by analyzing the exchange of water molecules in states with different modes of translation mobility, including pore channels and bilayer "defects". Each of the approaches used made it possible to take the significance of bilayer permeability for the apparent energy of activation of water diffusion into account and estimate the energies of activation of water diffusion in the hydrophobic moiety of the bilayer, which were found to be close to the values for bulky water. The coefficients of water diffusion in the system under examination and the coefficients of permeation of water through the bilayer were estimated, and the effect of bilayer "defects" on the coefficients of water diffusion along and across bilayers was studied.     

Lateral diffusion of cholesterol and dimyristoylphosphatidylcholine in a lipid bilayer measured by pulsed field gradient NMR spectroscopy

The pulsed field gradient NMR method for measuring self-diffusion has been used for a direct determination of the lateral diffusion coefficient of cholesterol, fluorine labeled at the 6-position, for an oriented lamellar liquid-crystalline phase of dimyristoylphosphatidylcholine (DMPC)/cholesterol/water. It is found that the diffusion coefficients of DMPC and cholesterol are equal over a large temperature interval. The apparent energy of activation for the diffusion process (58 kJ/mol) is about the same as for a lamellar phase of DMPC/water, whereas the phospholipid lateral diffusion coefficient is approximately four times smaller in the presence of cholesterol.     

The effect of cholesterol on the lateral diffusion of phospholipids in oriented bilayers

Pulsed field gradient NMR was utilized to directly determine the lipid lateral diffusion coefficient for the following macroscopically aligned bilayers: dimyristoylphosphatidylcholine (DMPC), sphingomyelin (SM), palmitoyloleoylphosphatidylcholine (POPC), and dioleoylphosphatidylcholine (DOPC) with addition of cholesterol (CHOL) up to approximately 40 mol %. The observed effect of cholesterol on the lipid lateral diffusion is interpreted in terms of the different diffusion coefficients obtained in the liquid ordered (l(o)) and the liquid disordered (l(d)) phases occurring in the phase diagrams. Generally, the lipid lateral diffusion coefficient decreases linearly with increasing CHOL concentration in the l(d) phase for the PC-systems, while it is almost independent of CHOL for the SM-system. In this region the temperature dependence of the diffusion was always of the Arrhenius type with apparent activation energies (E(A)) in the range of 28-40 kJ/mol. The l(o) phase was characterized by smaller diffusion coefficients and weak or no dependence on the CHOL content. The E(A) for this phase was significantly larger (55-65 kJ/mol) than for the l(d) phase. The diffusion coefficients in the two-phase regions were compatible with a fast exchange between the l(d) and l(o) regions in the bilayer on the timescale of the NMR experiment (100 ms). Thus, strong evidence has been obtained that fluid domains (with size of micro m or less) with high molecular ordering are formed within a single lipid bilayer. These domains may play an important role for proteins involved in membrane functioning frequently discussed in the recent literature. The phase diagrams obtained from the analysis of the diffusion data are in qualitative agreement with earlier published ones for the SM/CHOL and DMPC/CHOL systems. For the DOPC/CHOL and the POPC/CHOL systems no two-phase behavior were observed, and the obtained E(A):s indicate that these systems are in the l(d) phase at all CHOL contents for temperatures above 25 degrees C.     

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 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.     

Water permeation through the lipid bilayer membrane. Test of the liquid hydrocarbon model

According to the liquid hydrocarbon model, the lipid bilayer is viewed simply as a thin slice of bulk hydrocarbon liquid. This allows the water permeability of the bilayer to be calculated from bulk properties. In this paper the prediction of the liquid hydrocarbon model is compared with the known water permeability coefficient of the glycerol monoolein/n-hexadecane bilayer (Fettiplace, R. (1978) Biochim. Biophys. Acta 513, 1–10). As the alkyl chain of glycerol monoolein is equivalent to 8-heptadecene, the water permeability coefficient of 8-heptadecene/n-hexadecane mixtures was measured for temperatures between 20 and 35°C. The mole fraction of n-hexadecane in the bulk liquid was chosen at each temperature to match the known mole fraction of n-hexadecane in the bilayer (White, S. (1976) Nature 262, 421–422). The predicted water permeability coefficient agrees with the measured value at 32°C but is 40% above the measured value at 20°C. The apparent activation energy predicted by the liquid hydrocarbon model is 9.0 ± 0.3 kcal/mol, while the measured value is 14.2 ± 1.0 kcal/mol. The failure of the liquid hydrocarbon model probably results from a different molecular organization of the hydrocarbon chains in the bilayer and in the bulk liquid.     

Roles of Aquaporin-3 Water Channels in Volume-Regulatory Water Flow in a Human Epithelial Cell Line

Membrane water transport is an essential event not only in the osmotic cell volume change but also in the subsequent cell volume regulation. Here we investigated the route of water transport involved in the regulatory volume decrease (RVD) that occurs after osmotic swelling in human epithelial Intestine 407 cells. The diffusion water permeability coefficient (Pd) measured by NMR under isotonic conditions was much smaller than the osmotic water permeability coefficient (Pf) measured under an osmotic gradient. Temperature dependence of Pf showed the Arrhenius activation energy (Ea) of a low value (1.6 kcal/mol). These results indicate an involvement of a facilitated diffusion mechanism in osmotic water transport. A mercurial water channel blocker (HgCl₂) diminished the Pf value. A non-mercurial sulfhydryl reagent (MMTS) was also effective. These blockers of water channels suppressed the RVD. RT-PCR and immunocytochemistry demonstrated predominant expression of AQP3 water channel in this cell line. Downregulation of AQP3 expression induced by treatment with antisense oligodeoxynucleotides was found to suppress the RVD response. Thus, it is concluded that AQP3 water channels serve as an essential pathway for volume-regulatory water transport in, human epithelial cells.     

Effect of grafted polyethylene glycol (PEG) on the size, encapsulation efficiency and permeability of vesicles

Liposomes have been prepared by the vesicle extrusion method (VETs) from mixtures of dipalmitoylphosphatidylcholine (DPPC), phosphatidylinositol (PI) and dipalmitoylphosphatidylethanolamine with covalently linked poly(ethylene glycol) molecular mass 5000 and 2000 (DPPE-PEG 5000 and DPPE-PEG 2000) covering a range of 0-7.5 mole%. The encapsulation of D-glucose has been studied and found to be markedly dependent on the mole% DPPE-PEG. The permeability of the liposomes to D-glucose has been measured both as a function of temperature and liposome composition. The permeability coefficients for D-glucose increase with mole% DPPE-PEG 5000 and with temperature over the range 25-50 degrees C. The activation energies for glucose permeability range from 90 to 23 kJ mol(-1). The decrease in activation energy with increasing temperature is attributed to an increasing number of bilayer defects as the liposome content of PEG-grafted lipid is increased. The dependence of D-glucose encapsulation as a function of PEG-grafted lipid content is discussed in terms of the conformation of the PEG molecules on the inner surface of the bilayer. For liposomes containing DPPE-PEG 5000 the relative percentage encapsulation of glucose, assuming that the PEG surface layer excludes glucose, is comparable to that predicted from the mushroom and brush conformational models.     

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.     

Lateral diffusion of small compounds in human stratum corneum and model lipid bilayer systems.

An image-based technique of fluorescence recovery after photobleaching (video-FRAP) was used to measure the lateral diffusion coefficients of a series of nine fluorescent probes in two model lipid bilayer systems, dimyristoylphosphatidylcholine (DMPC) and DMPC/cholesterol (40 mol%), as well as in human stratum corneum-extracted lipids. The probes were all lipophilic, varied in molecular weight from 223 to 854 Da, and were chosen to characterize the lateral diffusion of small compounds in these bilayer systems. A clear molecular weight dependence of the lateral diffusion coefficients in DMPC bilayers was observed. Values ranged from 6.72 x 10(-8) to 16.2 x 10(-8) cm2/s, with the smaller probes diffusing faster than the larger ones. Measurements in DMPC/cholesterol bilayers, which represent the most thorough characterization of small-solute diffusion in this system, exhibited a similar molecular weight dependence, although the diffusion coefficients were lower, ranging from 1.62 x 10(-8) to 5.60 x 10(-8) cm2/s. Lateral diffusion measurements in stratum corneum-extracted lipids, which represent a novel examination of diffusion in this unique lipid system, also exhibited a molecular weight dependence, with values ranging from 0.306 x 10(-8) to 2.34 x 10(-8) cm2/s. Literature data showed that these strong molecular weight dependencies extend to even smaller compounds than those examined in this study. A two-parameter empirical expression is presented that describes the lateral diffusion coefficient in terms of the solute's molecular weight and captures the size dependence over the range examined. This study illustrates the degree to which small-molecule lateral diffusion in stratum corneum-extracted lipids can be represented by diffusion in DMPC and DMPC/cholesterol bilayer systems, and may lead to a better understanding of small-solute transport across human stratum corneum.     

Effect of atrial natriuretic factor on the water permeability of endothelial cells.

Atrial natriuretic factor increases the water permeability of the whole endothelium. This study investigates how it would affect the transcellular osmotic water permeability of bovine artery endothelial cells. The cyclic-GMP production by the isolated cells was maximal for 10(-6)M atrial natriuretic factor within 30 minutes at 37 degrees C. The cyclic-GMP protein kinase cell concentration was 1.87 +/- 0.15 ng/mg protein. The control apparent water permeability of the cells measured by light scattering was 195 +/- 11 microns/sec (n = 5). Membrane folding revealed by light and scanning electron microscopy indicated that their true water permeability values would be close to 20-40 microns/sec, similar to the values for lipid membranes. The energy activation calculated from the temperature dependence of water permeability between 15 degrees C and 37 degrees C was 9.3 kcal/mol. This value suggests water movement through the lipid bilayer and not through water channels. Atrial natriuretic factor 10(-6)M did not significantly increase the water permeability of the cells. Hence, atrial natriuretic factor-stimulated increase in water permeability of the endothelium is more related to changes in paracellular water pathways than in transcellular water flux.     

Effect of temperature on water transport through aquaporins

The mean effective water self-diffusion coefficient in maize root segments under the effect of aquaporin blocker (mercuric chloride, 0.1 mM) was measured using the spin-echo NMR method with pulsed magnetic field gradient within the temperature range from 10 to 35 °C. HgCl₂ caused the reduction in water diffusion by 30 % as compared to the control samples. Temperature dependences of water self-diffusion coefficients showed two linear regions with different values of Q₁₀ and activation energy, E_a. As the temperature reduced from 20 to 10 °C, E_a values calculated from the Arrhenius plots were close to those of bulk water (20 ± 3 kJ mol⁻¹) and slightly changed for the sample pretreated HgCl₂. Within the temperature range from 25 to 35 °C the slope of temperature dependences became steeper and E_a values were 31 ± 3 kJ mol⁻¹ for the control and 40 ± 4 kJ mol⁻¹ for the treated sample. In the vicinity of 20 °C, the temperature dependence of water diffusion via the mercury-sensitive water channels showed extreme value. In the region, the specific area of the mercury-sensitive aquaporins was 0.004 % of the total cell surface area. The data indicate that water transfer via aquaporins is sensitive to temperature, and the contributions of the transmembrane pathways (aquaporins, lipid bilayer) differ in different temperature ranges.     

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.     

Effects of diffusion limitation on immobilized nitrifying microorganisms at low temperatures

Activation energies of suspended and immobilized nitrifying bacteria were determined and compared to determine if diffusion limitation results in decreased sensitivity for temperature. The activation energy for the respiration activity of suspended Nitrosomonas europaea and Nitrobacter agilis was found to be 86.4 and 58.4 kJ mol(-1), respectively. The activation energy for oxygen diffusion in the support material, kappa-carrageenan, determined from the effect of temperature on the effective diffusion coefficient (D), was 17.2 kJ mol(-1). Consequently, the apparent actvation energy of diffusion limited cells should be lower. It was indeed shown that due to the effect of diffusion limitation and to temperature effects on the Monod constant K(s), the immobilized-cell activity was less sensitive to temperature. The apparent activation energy for immobilized Ns. europaea was between 28.6 and 94.2 kJ mol(-1) and for immobilized Nb. agilis between 1.4 and 72.9 kJ mol(-1), depending on the oxygen concentration and temperature. (c) 1995 John Wiley & Sons, Inc.     

Lateral diffusion of saturated phosphatidylcholines in cholesterol-containing bilayers

A pulsed field gradient NMR was used to study lateral diffusion in the cholesterol-containing oriented bilayers of saturated (dipalmitoyl- and dimyristoyl-) phosphatidylcholines, upon their limiting hydration. Similar dependences of lateral diffusion coefficients on temperature and cholesterol concentration were observed, which agree with phase diagram showing the presence of the regions of disordered and ordered liquid-crystalline phases and a two-phase region. Under the same conditions, the lateral diffusion coefficient of dipalmitoylphosphatidylcholine is lower, which agrees qualitatively with its larger molecular weight. The comparison of data for dipalmitoylphosphatidylcholine with previous results for dipalmitoylsphingomyelin-cholesterol bilayers under the same conditions, in spite of similarity of phase diagrams, shows large (two–three times) differences in the lateral diffusion coefficient and a different profile of its dependence on cholesterol concentration. The comparison of data for dimyristoylphosphatidylcholine with previous results shows that the values of lateral diffusion coefficient and the shape of its dependence on cholesterol concentration coincide at high concentrations (>15 mol%) but differ at lower concentrations The revealed disagreement may be caused by the fact that the measurements were carried out at different water content in the system. At limiting hydration (more than 35% of water), the lateral diffusion coefficient for lipids decreases when cholesterol concentration rises, while at water content about 25% (as a result of equilibrium hydration from vapors) the lateral diffusion coefficient of phosphatidylcholine may be independent of cholesterol concentration. This is the consequence of the denser packing of molecules in the bilayer at reduced water content, an effect that competes with the ordering effect of cholesterol.     

On the Problem of Diffusivity in Heterogeneous Biological Materials with Random Structure

Biological tissues are multicompartmental heterogeneous media composed of cellular and subcellular domains. Randomly walking water molecules may have different diffusion coefficients and densities (concentrations) in different domains, namely within cells and within the outer medium. Results of the proposed effective media scale-averaging iterative scheme are used to explore the effects of a large range of microstructural and compositional parameters on the apparent (effective) diffusion coefficient. A self-consistent modelling framework for predicting the steady-state effective diffusion coefficient is presented; the framework reveals the strong dependence of the apparent diffusion coefficient on the ratio of the microscopic diffusion coefficients of the comprising phases, permeability of the cells, and their volume fractions.     

Effect of the lipid phase transition on the kinetics of H+/OH− diffusion across phosphatidic acid bilayers

The kinetics of H⁺/OH^? diffusion across dimyristoyl phosphatidic acid bilayer membranes was measured by following the absorbance of the pH-sensitive indicator Cresol red ($\text{o-}\text{cresolsulfonphthalein}$) entrapped in single lamellar vesicles after rapidly changing the external pH in a stopped-flow apparatus. The H⁺/OH^?-permeability coefficient was found to be in the 10^?5 to 10^?3 cm·s^?1 range. The lipid phase transition has a strong influence on the permeation kinetics as the permeability coefficients in the liquid-crystalline phase are drastically higher. The permeability shows no maximum at the phase transition temperature as is the case for other ions, but displays a similar temperature dependence as water permeation. This is also reflected in the high activation energy of approx. 20 kcal/mol and supports the hypothesis (Nichols, J.W. and Deamer, D.W. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2038–2042) of H⁺/OH^? permeation via hydrogen bonded water molecules. A second slower kinetic phase is also observed, where the permeation is obviously controlled by counterion diffusion. The temperature dependence of this slow process displays the for ion diffusion characteristic maximum in the permeability at the phase-transition temperature.     

Electrochemical measurement of lateral diffusion coefficients of ubiquinones and plastoquinones of various isoprenoid chain lengths incorporated in model bilayers.

The long-range diffusion coefficients of isoprenoid quinones in a model of lipid bilayer were determined by a method avoiding fluorescent probe labeling of the molecules. The quinone electron carriers were incorporated in supported dimyristoylphosphatidylcholine layers at physiological molar fractions (<3 mol%). The elaborate bilayer template contained a built-in gold electrode at which the redox molecules solubilized in the bilayer were reduced or oxidized. The lateral diffusion coefficient of a natural quinone like UQ10 or PQ9 was 2.0 +/- 0.4 x 10(-8) cm2 s(-1) at 30 degrees C, two to three times smaller than the diffusion coefficient of a lipid analog in the same artificial bilayer. The lateral mobilities of the oxidized or reduced forms could be determined separately and were found to be identical in the 4-13 pH range. For a series of isoprenoid quinones, UQ2 or PQ2 to UQ10, the diffusion coefficient exhibited a marked dependence on the length of the isoprenoid chain. The data fit very well the quantitative behavior predicted by a continuum fluid model in which the isoprenoid chains are taken as rigid particles moving in the less viscous part of the bilayer and rubbing against the more viscous layers of lipid heads. The present study supports the concept of a homogeneous pool of quinone located in the less viscous region of the bilayer.     

Urea permeability of human red cells

The rate of unidirectional [14C]urea efflux from human red cells was determined in the self-exchange and net efflux modes with the continuous flow tube method. Self-exchange flux was saturable and followed simple Michaelis-Menten kinetics. At 38 degrees C the maximal self-exchange flux was 1.3 X 10(-7) mol cm-2 s-1, and the urea concentration for half-maximal flux, K1/2, was 396 mM. At 25 degrees C the maximal self-exchange flux decreased to 8.2 X 10(-8) mol cm-2 s-1, and K1/2 to 334 mM. The concentration-dependent urea permeability coefficient was 3 X 10(-4) cm s-1 at 1 mM and 8 X 10(-5) cm s-1 at 800 mM (25 degrees C). The latter value is consonant with previous volumetric determinations of urea permeability. Urea transport was inhibited competitively by thiourea; the half-inhibition constant, Ki, was 17 mM at 38 degrees C and 13 mM at 25 degrees C. Treatment with 1 mM p-chloromercuribenzosulfonate inhibited urea permeability by 92%. Phloretin reduced urea permeability further (greater than 97%) to a "ground" permeability of approximately 10(-6) cm s-1 (25 degrees C). This residual permeability is probably due to urea permeating the hydrophobic core of the membrane by simple diffusion. The apparent activation energy, EA, of urea transport after maximal inhibition was 59 kJ mol-1, whereas in control cells EA was 34 kJ mol-1 at 1 M and 12 kJ mol-1 at 1 mM urea. In net efflux experiments with no extracellular urea, the permeability coefficient remained constantly high, independent of a variation of intracellular urea between 1 and 500 mM, which indicates that the urea transport system is asymmetric. It is concluded that urea permeability above the ground permeability is due to facilitate diffusion and not to diffusion through nonspecific leak pathways as suggested previously.     

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.