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PubMed1991

An ultrarapid filtration method adapted to the measurements of water and solute permeability of synthetic and biological vesicles.

An ultrarapid filtration method was adapted to the determination of water and solute permeability of membrane vesicles. This method consisted of measuring substance washout from vesicles first loaded with 3H2O or labeled solutes, placed on filters, and rinsed at high rates for short periods. The retention of the vesicles on the filters was analyzed and was found to be a function of the nature and porosity of the filters as well as of the vesicle origin. Washing buffer flow rate and washing duration did not affect vesicle retention. The diffusional water permeability of cholesterol-free liposomes was determined at 16 degrees C. Its value was reduced by a factor of 2.5 when the liposomes were prepared with 20% cholesterol and a threefold increase was noted when the liposomes were preincubated with gramicidin (6 mg/g lipid). Water permeability of liposomes was strongly temperature-dependent: Ea = 15.3 kcal/mol. Diffusional water permeability of pink ghosts was also measured: a value of (4.4 +/- 0.2) X 10(-3) cm/s (n = 3) was obtained at 13 degrees C. This permeability was reduced by 45.2% with 0.4 mM HgCl2. The urea permeability of intestinal and renal brush-border membrane vesicles was (1.15 +/- 0.18) X 10(-6) cm/s (n = 7) and (1.67 +/- 0.08) X 10(-6) cm/s (n = 9), respectively. The renal value was reduced by a factor of 4.4 by 100 mM thiourea. This ultrarapid filtration technique provides an accurate method of transport measurement in sealed membranes such as liposomes and plasma membrane vesicles.

Animals↗
PubMed1984

Permeability of the rat oral mucosa to organic solutes measured in vivo.

The permeability of the oral mucosa to organic solutes of different molecular volumes, oil-to-water distribution coefficients, and pKa was studied in an in-vivo system. The calculated permeability coefficients were independent of time of application and the concentration of the solutes, a finding consistent with transport by simple diffusion. The permeability of organic acids was pH dependent whereas permeability to compounds with a degree of ionization that did not change over the pH range was not affected by pH. Increasing the chain length from two to eight carbons in an organic acid series resulted in an increase of both the oil-to-water distribution coefficient and the permeability coefficient; the permeability coefficient was decreased by the addition of oxygen in the form of hydroxyl groups. These results indicate that there is similarity in the way that lipid-soluble substances diffuse across oral mucosa and other lipid membranes. Results using compounds with oil-to-water distribution coefficients less than that of water suggest that these compounds traverse the oral mucosa by two additional routes. One, for compounds with molar volumes less than 80 cm3/ml, is via pores; whereas, the second, utilized by larger compounds, is probably by an intercellular route.

Animals↗
PubMed1996

DCCD-sensitive proton permeability of bacterial photosynthetic membranes. Cross-reconstitution studies with purified bovine heart Fo subunits.

The DCCD-sensitive proton permeability of chromatophores, from a green strain of Rhodobacter Capsulatus is potentiometrically detected following the proton release induced by a transmembrane diffusion potential imposed by a valinomycin-mediated potassium influx with a procedure already used for bovine heart submitochondrial particles (ESMP) and vesicles from Escherichia coli (Zanotti et al. (1994) Eur. J. Biochem. 222, 733-741). In the photosynthetic system, addition of increasing amounts of DCCD inhibits, with a similar titre, both proton permeability and MgATP-dependent ATPase activity as detected in the dark. The titre for 50% inhibition coincides with that obtained measuring proton permeability and ATP hydrolysis in ESMP. Upon removal of F1, the passive proton permeability is much less sensitive to DCCD in chromatophores than in USMP, suggesting that in chromatophores the F1-Fo interaction shapes the DCCD-sensitive proton conducting pathway. Addition of the purified mitochondrial FoI-PVP and oligomycin sensitivity-conferring (OSCP) proteins to the F1 stripped chromatophores restored the sensitivity of proton permeability to DCCD detected in untreated chromatophores. Analysis of the binding of 14C[DCCD] on F1 stripped chromatophores shows that the increase of DCCD sensitivity of proton permeability, caused by addition of mitochondrial Fo proteins, is related to an increase of the binding of the inhibitor to subunit c of Fo sector of ATP synthase complex.

Animals↗
PubMed1977

Modulation of drug permeability in Chinese hamster ovary cells. Possible role for phosphorylation of surface glycoproteins.

We have previously reported that the uptake of colchicine and other drugs in Chinese hamster ovary (CHO) cells can be greatly enhanced by the addition of metabolic inhibitors such as cyanide (See, Y.P., Carlsen, S.A., Till, J.E. and Ling, V. (1974) Biochim. Biophys. Acta 373, 242-252). This has led us to postulate the presence of an active drug permeability barrier in these cells. In this paper we provide evidence for the dependence of this permeability barrier on intracellular ATP levels. Colchicine-resistant mutants of CHO cells exhibiting a reduced drug permeability, however, can maintain this drug permeability barrier at much lower ATP levels, suggesting that they possess an altered active drug permeability barrier. We have also observed a membrane-associated protein kinase-phosphoprotein phosphatase system in the isolated membranes of mutant and wild-type cells. Differences in the intrinsic protein phosphorylation patterns between the membranes of these cells have led us to conclude that the control of the drug permeability barrier may be mediated via the phosphorylation of at least two high molecular weight surface glycoproteins.

Adenosine Triphosphate↗
PubMed1978

The influence of the lipid on the water permeability of artificial membranes.

The water permeabilities of artificial membranes formed formed from various monoglycerides or phospholipids in alkane solvents have been measured using an osmotic method, and it has been shown that the permeability depends upon the type of lipid used. For monoglycerides, the permeability was found to increase with the unsaturation and decrease with the length of the acyl chain. Membranes formed from either egg phosphatidylcholine or dioleyl phosphatidylcholine had an osmotic permeability coefficient of approx. 35--40 micrometer/s at 25 degrees C; with sphingomyelin as the membrane lipid, the permeability was an order of magnitude lower than that for phosphatidylcholine. It is suggested that the water permeabilities of biological membranes might be partly controlled by the types of lipid present.

Glycerides↗
PubMed1979

Transepithelial permeability in the rabbit pancreas.

1. The transepithelial permeability in the isolated rabbit pancreas has been studied with the aid of radioactive markers added to the bathing medium. 2. After addition of these compounds in 2 mM concentration to the medium, they equilibrate within 30 min to a steady-state concentration in the secreted fluid. The latter concentrations, expressed as percent of those in the bathing medium, are: urea 100%, glycerol 90%, erythritol 95%, mannitol 60%, lactose 5%, sucrose 4% and inulin 3%. 3. Addition of 10(-5) M carbachol to the bathing medium after 60 or 90 min of incubation results in an increase of the concentrations of mannitol, lactose sucrose and inulin in the secreted fluid. Maximal concentrations, reached about 35 min after addition of the stimulant, are: mannitol 65%, lactose 31%, sucrose 23%, inulin 8%. 4. No change in the concentration of urea is observed, while the concentrations of glycerol and erythritol increase always to 100% after addition of 10(-5) M carbachol. 5. For sucrose and lactose the increase in permeability appears to be dependent on the concentration of carbachol. 6. There is no increase in the extracellular space for lactose, sucrose and inulin after incubating fragments of the rabbit pancreas with 10(-5) M carbachol. 7. Addition of atropine 5 min or more after carbachol stimulation has no effect on enzyme secretion, but markedly inhibits the increase in sucrose permeability. 8. These results indicate that: (a) the permeability of the transcellular transport route in the isolated rabbit pancreas is determined by the size of the permeating molecules, (b) this route is probably extracellular, (c) its permeability is increased by a cholinergic agent in dose-dependent fashion, (d) the increase in permeability is not caused by the enzyme secretion as such.

Animals↗
PubMed1981

Permeability of axon membranes to local anesthetics.

The permeability of the neutral form of tertiary amine local anesthetics across squid axon membranes was studied by utilizing three different experimental methods: (1) narcotic action of axon excitability was measured by monitoring the time derivative of action potential and the results were analyzed in terms of a diffusion reaction equation of local anesthetics to obtain their permeabilities; (2) the influx of local anesthetic into the axon was measured by use of the radioisotope tracer technique; and (3) the desorption rates of the neutral form of local anesthetics from lipid monolayers were measured and the desorption rate was correlated with permeability. The relative permeabilities obtained for procaine, lidocaine and tetracaine by the above three methods were comparable. The order of relative permeabilities was procaine greater than lidocaine greater than tetracaine, and had an inverse correlation with the partition coefficients of anesthetics at oil/water phases. Some discussion concerning the concept of permeability is made when the partition coefficient of a permeant molecule is high.

Action Potentials↗
PubMed1984

The mechanism of vanadium action on selective K+-permeability in human erythrocytes.

Low concentrations of chelating agents such as EDTA prevent the air oxidation of vanadyl (VO2+, +4 oxidation state) to vanadate (VO3-, +5 oxidation state). Under these conditions, the ionophore A23187 mediates the rapid entry of vanadyl into human erythrocytes. In the presence of A23187, vanadyl at concentrations in excess of EDTA gives rise to a dramatic increase in K+ permeability, which is very similar to the Gardos Ca2+-induced K+ permeability increase with respect to ion selectivity, response to inhibitors, effects of pH, and stimulation by external K+. In ultrapure media with very low Ca2+, however, vanadyl has no effect on K+ permeability. These experiments suggest that Ca2+ is displaced from EDTA by vanadyl and then enters the cell via A23187 where it triggers the increase in K+ permeability. This hypothesis is confirmed by experiments demonstrating that vanadyl does displace Ca2+ from EDTA. Vanadate, an inhibitor of Ca2+-ATPase, causes a selective increase in K+ permeability in metabolically depleted cells, but the increase is abolished by low concentrations of EDTA, indicating that this effect is also due to entry of extracellular Ca2+. Earlier observations of effects of vanadyl and vanadate on erythrocyte K+ permeability can thus be explained on the basis of inhibition of the Ca2+ pump by vanadium, leading to an increase in intracellular Ca2+ concentration.

Biological Transport↗
PubMed1984

25-Hydroxysterols increase the permeability of liposomes to Ca2+ and other cations.

25-Hydroxycholesterol and 25-hydroxy vitamin D-3 increased the permeability of liposomes to Ca2+ measured by the arsenazo III encapsulation technique. This effect was sensitive to the lipid composition of the membrane, with changes that decreased the motional freedom of phospholipid acyl chains decreasing Ca2+ permeability. The greatest permeability was observed with the zwitter-ionic phospholipids, phosphatidylcholine and phosphatidylethanolamine, whereas the acidic phospholipids, phosphatidylinositol and phosphatidylserine, depressed Ca2+ permeability. The effect was not specific for Ca2+. Other divalent cations were translocated in the order Mn2+ greater than Mg2+ = Ca2+ much greater than Sr2+ = Ba2+. The permeability of liposomes to the monovalent cation, Na+, was also substantially increased. The effect did not appear to be due to ionophoretic properties of the sterols, and it is suggested that perturbation of the membranes by the polar 25-hydroxyl group may play a role in increasing membrane permeability.

Arsenazo III↗
PubMed1985

Modification of vesicle surfaces with amphiphilic sterols. Effect on permeability and in vivo tissue distribution.

In this paper, we describe the permeability of vesicles prepared with various synthetic cholesterol derivatives. Cholesterol derivatives with side-chains ending in hydroxyl groups reduced the permeability of unilamellar vesicles. However, addition of cholesterol derivatives with terminal amino groups makes the vesicles more permeable. Vesicles prepared with a short-chain amino-cholesterol derivative were found to be less permeable in phosphate-buffered saline, but not in bovine serum, while long-chain amino-cholesterol-containing vesicles were very permeable in both media. Studies in vivo indicate a rapid clearance rate for intravenously administered amino-cholesterol-containing vesicles with a concomitant increase in liver uptake. However, no difference was found in either the clearance or tissue distribution of control vesicles and the less permeable hydroxyl-cholesterol-containing vesicles.

Animals↗
PubMed1986

Concentration- and time-dependence of amphotericin-B induced permeability changes across ergosterol-containing liposomes.

The effect of amphotericin B on the permeability properties of liposomes prepared by reverse-phase evaporation was examined by using an osmotic method. This study has revealed that the magnitude and type of the alterations in permeability induced by amphotericin B in liposomes made of egg phosphatidylcholine and ergosterol depend not only on the amphotericin B concentration in the external aqueous solution but also on the time elapsed after mixing. Thus, low amphotericin B concentrations (from 0.2 to 1.2 microM) led to, (1) an small increment of the total extent of shrinkage of liposomes suspended in non-electrolytes such as urea or salts like KNO3, (2) an enhancement of urea and salt permeabilities at the same time scale at which volume changes were measured (ms to s), (3) a maximal blocking by tetraethylammonium of amphotericin B-induced urea permeability and (4) an enhancement of glucose permeability but only after liposomes were incubated with amphotericin B for some minutes before mixing. The high amphotericin B concentration regime (beyond 1.2 microM) led to, (1) a decrease of the total extent of shrinkage of liposomes immediately after rapid mixing of liposomes with urea solutions containing amphotericin B and (2) a 50% reduction of the tetraethylammonium blocking of amphotericin B-induced urea permeability. These results are explained by assuming that amphotericin B may form in ergosterol-containing liposomes two types of active channel differing in internal diameter.

Amphotericin B↗
PubMed1986

The water and ionic permeability induced by polyene antibiotics across plasma membrane vesicles from Leishmania sp.

An osmotic method has been used to study the effect of the polyene antibiotics amphotericin B, nystatin and candicidin on the water permeability of plasma membranes prepared from Leishmania sp. The effect of amphotericin B on the permeability of Leishmania membranes to a salt such as potassium nitrate was also investigated. A non-linear and saturable enhancement of water and salt permeability was measured with increasing polyene concentrations, which could be adjusted to Hill cooperativity equation. The antibiotic concentrations that induce at 30 degrees C half-maximal effects on the water permeability of Leishmania vesicles were 0.021 microM for candicidin, 0.21 microM for amphotericin B and 1.4 microM for nystatin. At 30 degrees C, the concentration of amphotericin B required to induce half of the maximal effect on the permeability of Leishmania vesicles to potassium nitrate was 1.8 microM. The temperature dependence for amphotericin B, nystatin and candicidin enhancement of the water permeability of Leishmania vesicles was determined by using Q10 data at 20 and 30 degrees C. The estimated activation energies at increasing polyene concentrations display the same general pattern for all three polyene antibiotics investigated, that is, a maximal positive value at about the polyene concentrations required for half-maximal effect. The significance of these results for understanding the mechanisms of action of polyene antibiotics on natural membranes is discussed.

Amphotericin B↗
PubMed1987

Effects of local anesthetics on the passive permeability of sarcoplasmic reticulum vesicles to Ca2+ and Mg2+.

Sarcoplasmic reticulum vesicles are used here as model membrane system to question the hypothesis of enhancement of permeability of cations by anesthetics, particularly that of Ca2+ and of Mg2+. The effects of dibucaine (up to 800 microM), tetracaine (up to 2 mM), lidocaine (up to 10 mM) and procaine (up to 10 mM) on the permeability of these membranes to Ca2+ and Mg2+ have been measured. We have used an experimental approach based on the light scattering method (Kometani, T. and Kasai, M. (1978) J. Membrane Biol. 41, 295-308). It has been found that all the local anesthetics cited above markedly increase the permeability of sarcoplasmic reticulum vesicles to Mg2+ and, in the concentration range tested herein, only dibucaine and tetracaine increase the permeability to Ca2+. The kinetic analysis of the time dependence of the light-scattering data after the osmotic shock shows that, in the absence of local anesthetics, the Mg2+ influx can be described as proceeding through a unique type of channel. However, Ca2+ influx appears to involve two channel of different kinetic properties. Because the relative fraction of both types of Ca2+ channel is similar to the average ratio between light and heavy vesicles in unfractionated sarcoplasmic reticulum, we suggest that each type of channel can be preferentially located in one of these fractions. The determined rate constants for Ca2+ permeability through both types of channel are 0.77 +/- 0.08 min-1 (fast channels) and 0.025 +/- 0.005 min-1 (slow channels) and that for Mg2+ is 0.08 +/- 0.02 min-1. These results agree with data obtained by other groups using different experimental approaches. Dibucaine and tetracaine significantly alter the rate of Mg2+ and Ca2+ influx through the slow channels. In addition, these two local anesthetics also produce the effect that the Mg2+ influx cannot be described with only one exponential process, thus suggesting a differential effect on vesicles of different density. The increase of Ca2+ and Mg2+ permeability by dibucaine and by tetracaine is found at concentrations of these drugs that do not produce a noticeable inhibition of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles.

Anesthetics, Local↗
PubMed1987

Effects of halothane, caffeine, dantrolene and tetracaine on the calcium permeability of skeletal sarcoplasmic reticulum of malignant hyperthermic pigs.

Preparing skeletal sarcoplasmic reticulum from both normal and malignant hyperthermia susceptible pigs, the effects of various drugs on the passive calcium permeability of these sarcoplasmic reticulum preparations were studied. It was found that, in the absence of halothane, the permeability of heavy sarcoplasmic reticulum prepared from malignant hyperthermia susceptible pigs was much higher than that of normal pigs. It was observed that halothane, at concentrations above 10 microM (well below anesthetic concentrations, which are on the order of 1 mM), increased the permeability of sarcoplasmic reticulum. The Hill coefficient of the effect of halothane ranged from 1.96 to 2.25, suggesting that some kind of cooperativity was involved in this reaction. The effects of caffeine were similar to those of halothane. Inhibitors, such as tetracaine and ruthenium red inhibited both the calcium permeability and the halothane-induced increment. The Hill coefficient of the effect of tetracaine was 1.75. The mode of inhibition suggests that tetracaine directly binds with the calcium channel to inhibit the calcium efflux. On the contrary, dantrolene did not affect the calcium permeability of the sarcoplasmic reticulum. However, it inhibited the halothane-induced and caffeine-induced increments of the permeability. The Hill coefficient of inhibition by dantrolene ranged from 2.3 to 3.9, suggesting that several molecules of dantrolene may interact cooperatively with one calcium release channel to inhibit the effect of halothane. These results suggest that dantrolene has a unique inhibitory action, which may be related to its efficacy in ameliorating the syndrome of malignant hyperthermia.

Animals↗
PubMed1988

Permeability studies of lipid vesicles from alkalophilic Bacillus firmus showing opposing effects of membrane isoprenoid and diacylglycerol fractions and suggesting a possible basis for obligate alkalophily.

Previous studies of the membrane lipids of extremely alkalophilic bacilli had indicated that both facultative and obligate alkalophiles contained a substantial fraction of isoprenoid lipid as well as high concentrations of cardiolipin. Facultative alkalophiles differed from obligate strains in having a phospholipid fatty acid composition that would be expected to result in a more ordered membrane structure. Current studies of ion permeability in vesicles prepared from lipids from obligately alkalophilic Bacillus firmus RAB and its facultatively alkalophilic strain, OF4, support the suggestion that membranes of the latter strain form a tighter barrier structure, with the difference especially pronounced at near neutral pH values. The water permeability of whole cells and the reflection coefficients for acetamide in vesicles were also consistent with a tighter membrane in the facultatively alkalophilic strain than in the obligately alkalophilic strain. The permeability properties of vesicles prepared from phospholipids from these organisms were studied as a function of the addition of either homologous membrane isoprenoid or diacylglycerol. For each permeability parameter that was assayed, in lipids from both strains, the isoprenoid fraction decreased the permeability, whereas the diacylglycerol fraction increased the permeability of the vesicles to solute.

Bacillus↗
PubMed1992

A new diffusion chamber system for the determination of drug permeability coefficients across the human intestinal epithelium that are independent of the unstirred water layer.

A new method for determining permeability coefficients, that are independent of the unstirred water layer (UWL), has been developed. The method was used to determine the cellular permeability coefficient of the rapidly absorbed drug testosterone in monolayers of the human intestinal epithelial cell line, Caco-2. Using a new diffusion cell with an effective stirring system based on a gas lift, the cellular permeability coefficient for testosterone was (1.98 +/- 0.13).10(-4) cm/s which is 3.5-times higher than the permeability coefficient obtained in the unstirred system. The thickness of the UWL obtained with the well stirred diffusion cell was 52 +/- 4 microns. This value is much lower than those previously reported in various well stirred in vitro models. The calculated cellular permeability of testosterone was 13-23-times lower than that for an UWL of the same thickness as the epithelial cell (17-30 microns). We conclude that the permeability of the epithelial monolayer must be included in calculations of the thickness of the UWL.

Cell Line↗
PubMed1992

The permeability and the effect of acyl-chain length for phospholipid bilayers containing cholesterol: theory and experiment.

The model of Cruzeiro-Hansson et al. (Biochim. Biophys. Acta (1989) 979, 166-1176) for lipid-cholesterol bilayers at low cholesterol concentrations is used to predict the thermodynamic properties and the passive ion permeability of lipid bilayers as a function of acyl-chain length and cholesterol concentration. Numerical simulations based on the Monte Carlo method are used to determine the equilibrium state of the system near the main gel-fluid phase transition. The permeability is calculated using an ansatz which relates the passive permeability to the amount of interfaces formed in the bilayer when cholesterol is present. The model predicts at low cholesterol contents an increase in the membrane permeability in the transition region both for increasing cholesterol concentration and for decreasing chain length at a given value of the reduced temperature. This is in contrast to the case of lipid bilayers containing high cholesterol concentrations where the cholesterol strongly suppresses the permeability. Experimental results for the Na+ permeability of C15PC and DPPC (C16PC) bilayers containing cholesterol are presented which confirm the theoretical predictions at low cholesterol concentrations.

Algorithms↗
PubMed1994

The solute permeability of thylakoid membranes is reduced by low concentrations of trehalose as a co-solute.

The different efficiencies of sucrose and trehalose in protecting isolated spinach (Spinacia oleracea L.) thylakoids against freeze-thaw damage is quantitatively related to their ability to reduce the solute loading of the vesicles during freezing. In the present paper we show that this effect is based on a reduction of the solute permeability of the membranes. Permeability was measured with 14C-labeled glucose at temperatures between 0 and 10 degrees C. Glucose permeability was reduced by both sucrose and trehalose, with trehalose effective at much lower concentrations than sucrose. An analysis of the temperature dependence of glucose permeability in the presence and absence of trehalose revealed that a 50% reduction in permeability resulted from a 10% increase in activation energy and a 30% decrease in activation entropy. Using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene (DPH), we found that the reduced permeability of the membranes in the presence of trehalose was unaccompanied by a reduction in lipid fluidity. This also excluded the possibility of a solute-induced liquid crystalline to gel phase transition. A reduced partitioning of the hydrophobicity-sensitive dye merocyanine 540 into thylakoids and into membranes containing 50% digalactosyldiacylglycerol in the presence of trehalose as compared to sucrose and glucose showed that the lipid headgroup region of these membranes became less accessible for solutes. No significant difference in merocyanine partitioning in the presence of trehalose as compared to sucrose or glucose was apparent when monogalactosyldiacylglycerol dispersions or phosphatidylcholine vesicles were investigated.

Cell Membrane Permeability↗