Proton pump activity is required for active uptake of chloride in isolated amphibian skin exposed to freshwater

Net proton secretion and unidirectional chloride fluxes were measured in isolated skin of toads ( Bufo bufo) and frogs ( Rana esculenta) mounted in an Ussing chamber and exposed to a Ringer's solution on the serosal side and a freshwater-like solution (1-3 mM Cl(-)) on the external side. Active proton secretion was 34.2+/-2.0 pmol.cm(-2).s(-1) ( n=18) in frog skin, and 16.7+/-1.7 pmol.cm(-2).s(-1) ( n=10) in toad skin. Proton secretion by toad skin was dependent on the transepithelial potential ( V(T)), and an amiloride-insensitive short-circuit current was stimulated by exogenous CO(2)/HCO(3)(-), indicating the presence of a rheogenic proton pump. Cl(-) influx was 37.4+/-7.5 pmol.cm(-2).s(-1) ( n=14) in frog skin and 19.5+/-3.5 pmol.cm(-2).s(-1) ( n=11) in toad skin. In toad skin, the mean Cl(-) flux ratio was larger than expected for simple electro-diffusion. In 8 of 11 sets of paired skins, influx was greater than the efflux indicating active uptake of Cl(-). Cl(-) influx in toad skin was unaffected by large perturbations (100-150 mV) of V(T), which was accomplished by adding amiloride to the outer bath under open circuit conditions. A component of the Cl(-) efflux seemed to be dependent on V(T). 4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS; 0.3 mM or 1.3 mM) inhibited Cl(-) influx and, surprisingly, increased Cl(-) efflux in toad skin. Influx and efflux of Cl(-) in toad skin were highly dependent on the external [Cl(-)] in the freshwater range (0.1-4 mM). (36)Cl(-) influx decreased whereas the total Cl(-) efflux increased as a function of external [Cl(-)]. These data indicate the presence of a DIDS-sensitive, electroneutral carrier mechanism with an external binding site for Cl(-). Ethoxzolamide (100 micro M), an inhibitor of carbonic anhydrase, reduced proton secretion and Cl(-) influx in frog skin. Concanamycin A (0.1-10 micro M), a specific vacuolar-type proton pump (V-ATPase) inhibitor, significantly reduced proton secretion in frog skin. In addition, concanamycin A (1 micro M) significantly reduced Cl(-) influx in frog skin. We suggest that the active proton secretion and Cl(-) influx are coupled. We hypothesise that an apical V-ATPase is capable of energising active Cl(-) uptake in fresh water by creating a favourable gradient for an apical HCO(3)(-) exit in exchange for external Cl(-). The data also suggest that a carbonic anhydrase activity provides H(+) and HCO(3)(-) for apically co-expressed proton pumps and Cl(-)/HCO(3)(-) exchangers.     

Water, proton, and urea transport in toad bladder endosomes that contain the vasopressin-sensitive water channel

Vasopressin (VP) increases the water permeability of the toad urinary bladder epithelium by inducing the cycling of vesicles containing water channels to and from the apical membrane of granular cells. In this study, we have measured several functional characteristics of the endosomal vesicles that participate in this biological response to hormonal stimulation. The water, proton, and urea permeabilities of endosomes labeled in the intact bladder with fluorescent fluid-phase markers were measured. The diameter of isolated endosomes labeled with horse-radish peroxidase was 90-120 nm. Osmotic water permeability (Pf) was measured by a stopped-flow fluorescence quenching assay (Shi, L.-B., and A. S. Verkman. 1989. J. Gen. Physiol. 94:1101-1115). The number of endosomes formed when bladders were labeled in the absence of a transepithelial osmotic gradient increased with serosal [VP] (0-50 mU/ml), and endosome Pf was very high and constant (0.08-0.10 cm/s, 18 degrees C). When bladders were labeled in the presence of serosal-to-mucosal osmotic gradient, the number of functional water channels per endosome decreased (at [VP] = 0.5 mU/ml, Pf = 0.09 cm/s, 0 osmotic gradient; Pf = 0.02 cm/s, 180 mosmol gradient). Passive proton permeability was measured from the rate of pH decrease in voltage-clamped endosomes in response to a 1 pH unit gradient (pHin = 7.5, pHout = 6.5). The proton permeability coefficient (PH) was 0.051 cm/s at 18 degrees C in endosomes containing the VP-sensitive water channel; PH was not different from that measured in vesicles not containing water channels. Measurement of urea transport by the fluorescence quenching assay gave a urea reflection coefficient of 0.97 and a permeability coefficient of less than 10(-6) cm/s. These results demonstrate: (a) VP-induced endosomes from toad urinary bladder have extremely high Pf. (b) In states of submaximal bladder Pf, the density of functional water channels in endosomes in constant in the absence of an osmotic gradient, but decreases in the presence of a serosal-to-mucosal gradient, suggesting that the gradient has a direct effect on the efficiency of packaging of water channels into endosomes. (c) The VP-sensitive water channel does not have a high proton permeability. (d) Endosomes that cycle the water channel do not contain urea transporters. These results establish a labeling procedure in which greater than 85% of labeled vesicles from toad urinary bladder are endosomes that contain the VP-sensitive water channel in a functional form.     

Water permeability in different epithelial barriers

The water permeability properties of a series of epithelial barriers (the toad urinary bladder [TUB], the rat caecum [RC], the distal human colon [DHC], and the human amnion [HA] were studied in different experimental conditions. Three parameters were simultaneously determined: the water permeability coefficient in the presence of a transepithelial hydrostatic gradient (Phydr); the water permeability coefficient in the presence of an osmotic gradient (Posm); and the transepithelial potential difference (dV). All experiments were performed with the same experimental device, allowing comparison of the permeability properties of the barriers tested. The results obtained were: (1) TUB (N = 8): Phydr = 0.079 +/- 0.008 cm/s; Posm = 0.0004 +/- 0.0002 cm/s; dV = 31 +/- 5 mV; (2) TUB after ADH (N = 8): Phydr = 0.093 +/- 0.012 cm/s; Posm = 0.0065 +/- 0.0011 cm/s; dV = 52 +/- 8; (3) RC (N = 10): Phydr = 0.18 +/- 0.02 cm/s; Posm = 0.0019 +/- 0.0004 cm/s; dV = 3.9 +/- 0.1 mV; (4) RC adapted to a high K diet (N = 10): Phydr = 0.21 +/- 0.02 cm/s; Posm = 0.0018 +/- 0.0006 cm/s; dV = 4.5 +/- 0.5 mV; (5) DHC (N = 6): Phydr = 0.22 +/- 0.03 cm/s; Posm = 0.002 +/- 0.05 cm/s; dV = 15 +/- 3 mV; (6) HA (N = 10): Phydr = 0.32 +/- 0.05 cm/s; Posm = 0.0154 +/- 0.0015; dV = 0. The results show a good correlation between Phydr and dV, but not between dV and Posm or between Posm and Phydr.     

The water permeability of toad urinary bladder. I. Permeability of barriers in series with the luminal membrane

Antidiuretic hormone (ADH) induces a large increase in the water permeability of the luminal membrane of toad urinary bladder. Measured values of the diffusional water permeability coefficient, Pd(w), are spuriously low, however, because of barriers within the tissue, in series with the luminal membrane, that impede diffusion. We have now determined the water permeability coefficient of these series barriers in fully stretched bladders and find it to be approximately 6.3 X 10(- 4) cm/s. This is equivalent to an unstirred aqueous layer of approximately 400 microns. On the other hand, the permeability coefficient of the bladder to a lipophilic molecule, hexanol, is approximately 9.0 X 10(-4) cm/s. This is equivalent to an unstirred aqueous layer of only 100 microns. The much smaller hindrance to hexanol diffusion than to water diffusion by the series barriers implies a lipophilic component to the barriers. We suggest that membrane-enclosed organelles may be so tightly packed within the cytoplasm of granular epithelial cells that they offer a substantial impediment to diffusion of water through the cell. Alternatively, the lipophilic component of the barrier could be the plasma membranes of the basal cells, which cover most of the basement membrane and thereby may restrict water transport to the narrow spaces between basal and granular cells.     

Melatonin desensitizing effects on the in vitro responses to MCH,alpha-MSH,isoproterenol and melatonin in pigment cells of a fish (S. marmoratus), a toad (B. ictericus), a frog (R. pipiens), and a lizard (A. carolinensis), exposed to varying photoperiodic regimens

Melatonin is a weak dose-independent lightening agonist in fish skin, a moderate dose-dependent lightening agonist in toad skin and a potent lightening agent in frog and lizard skins (reversing in a dose-dependent manner the darkening caused by alpha-melanocyte-stimulating hormone). In frog skins, previous exposure to melatonin reduced further lightening actions of the indoleamine, and in toad skins, increasing concentrations of melatonin elicited decreasing lightening responses, suggesting an autodesensitizing action of the hormone. Various concentrations of melatonin diminished the responses to the lightening agonist melanin-concentrating hormone (MCH) in fish skins and to the darkening agonists alpha-MSH in toad, frog and lizard skins and isoproterenol in frog skins. In vitro inhibitory actions of melatonin are mimicked in the absence of the hormone in skin preparations from toads kept in continuous darkness for 48 hr. The lipophylic nature of the indoleamine associated with the results herein described suggests intracellular actions of melatonin on vertebrate pigment cells.     

Very high water permeability in vasopressin-induced endocytic vesicles from toad urinary bladder

The regulation of transepithelial water permeability in toad urinary bladder is believed to involve a cycling of endocytic vesicles containing water transporters between an intracellular compartment and the cell luminal membrane. Endocytic vesicles arising from luminal membrane were labeled selectively in the intact toad bladder with the impermeant fluid-phase markers 6-carboxyfluorescein (6CF) or fluorescein-dextran. A microsomal preparation containing labeled endocytic vesicles was prepared by cell scraping, homogenization, and differential centrifugation. Osmotic water permeability was measured by a stopped-flow fluorescence technique in which microsomes containing 50 mM mannitol, 5 mM K phosphate, pH 8.5 were subject to a 60-mM inwardly directed gradient of sucrose; the time course of endosome volume, representing osmotic water transport, was inferred from the time course of fluorescence self-quenching. Endocytic vesicles were prepared from toad bladders with hypoosmotic lumen solution treated with (group A) or without (group B) serosal vasopressin at 23 degrees C, and bladders in which endocytosis was inhibited by treatment with vasopressin at 0-2 degrees C (group C), or with vasopressin plus sodium azide at 23 degrees C (group D). Stopped-flow results in all four groups showed a slow rate of 6CF fluorescence decrease (time constants 1.0-1.7 s for exponential fit) indicating a component of nonendocytic 6CF entrapment into sealed vesicles. However, in vesicles from group A only, there was a very rapid 6CF fluorescence decrease (time constant 9.6 +/- 0.2 ms, SEM, 18 separate preparations) with an osmotic water permeability coefficient (Pf) of greater than 0.1 cm/s (18 degrees C) and activation energy of 3.9 +/- 0.8 kcal/mol (16 kJ/mol). Pf was inhibited reversibly by greater than 60% by 1 mM HgCl2. The rapid fluorescence decrease was absent in vesicles in groups B, C, and D. These results demonstrate the presence of functional water transporters in vasopressin-induced endocytic vesicles from toad bladder, supporting the hypothesis that water channels are cycled to and from the luminal membrane and providing a functional marker for the vasopressin-sensitive water channel. The calculated Pf in the vasopressin-induced endocytic vesicles is the highest Pf reported for any biological or artificial membrane.     

The single file hypothesis and the water channels induced by antidiuretic hormone

Unidirectional and net water movements were determined at minute intervals in frog urinary bladders. The changes in both parameters were followed, during the action of antidiuretic hormone (ADH), at different temperatures and stirring conditions. After correction for external unstirred layer effects, the ratio of the osmotic (Pf) and diffusional (Pd) permeability coefficients was remarkably constant, at different times and in different experimental conditions. In the presence of ADH the delta Pf/delta Pd ratio in the mucosal border was probably greater than 9. On the other hand, in nonstimulated preparations the ratio was smaller, and probably not different from 1. These results, together with previous observations indicating that other small molecules (like urea) are excluded from the ADH-induced channel, might indicate that single-file water movement can occur through this structure. Alternatively, the delta Pf/delta Pd ratio could result from a complex geometric arrangement in series with the aqueous pore.     

Potassium ion accumulation at the external surface of the nodal membrane in frog myelinated fibers.

Potassium accumulation associated with outward membrane potassium current was investigated experimentally in myelinated fibers and analyzed in terms of two models-three-compartment and diffusion in an unstirred layer. In the myelinated fibers, as in squid giant axons, the three-compartment model satisfactorily describes potassium accumulation. Within this framework the average space thickness, theta, in frog was 5,900 +/- 700 A, while the permeability coefficient of the external barrier, PK, was (1.5 +/- 0.1) X 10(-2) cm/s. The model of ionic diffusion in an unstirred aqueous layer adjacent to the axolemma, as an alternative explanation for ion accumulation, was also consistent with the experimental data, provided that D, the diffusion constant, was (1.8 +/- 0.2) X 10(-6) cm/s and l, the unstirred layer thickness, was 1.4 +/- 0.1 micron, i.e., similar to the depth of the nodal gap. An empirical equation relating the extent of potassium accumulation to the amplitude and duration of depolarization is given.     

Gram-negative bacteria influence on the ability of the arginine-vasotocin to stimulate osmotic permeability of the frog urinary bladder epithelium

The influence of endogenous gram-negative bacteria colonizing the mucosal epithelium of frog Rana temporaria L. urinary bladders (FUB) on arginine-vasotocin AVT-stimulated osmotic water flow in isolated urinary bladders was investigated. 170 animals were examined and only 40% were contaminated with gram-negative bacteria (about 10(3)-10(6) CFU per hemibladder). Several Enterobacteriaceae species were identified (Hafnia alvei, 36.7%, E. coli, 32.3%, Serratia marcescens, 8.8%, Citrobacter freundii, 4.4% etc.). Basal osmotic water flow level was invariable in "clean" and contaminated FUB, whereas bacterial contamination resulted in considerable decrease in AVT-stimulated water flow ("clean": 2.53 +/- 0.13, n = 59, contaminated: 1.21 +/- 0.17 me/min/cm2, n = 38, p < 0.001, within first 15 min of incubation with 5 x 10(-10)M AVT). Gentamycin protection assay revealed predominantly adhesive forms of bacteria. Thus our data indicated that the presence of gram-negative bacteria colonizing the mucosal epithelium of the urinary bladder results in decreased adility of ADH to rise osmotic water permeability which in turn could impair body osmoregulation.     

Functional water channels and proton pumps are in separate populations of endocytic vesicles in toad bladder granular cells

Functional water channels are retrieved by endocytosis from the apical membrane of toad bladder granular cells in response to vasopressin [Shi, L.-B., & Verkman, A.S. (1989) J. Gen. Physiol. 94, 1101-1115]. To examine whether endocytic vesicles which contain the vasopressin-sensitive water channel fuse with acidic vesicles for entry into a lysosomal pathway, ATP-dependent acidification and osmotic water permeability were measured in endosomes from control bladders and bladders treated with vasopressin (VP) and/or phorbol myristate acetate (PMA). Endosomes were labeled with the fluid-phase markers 6-carboxyfluorescein or fluorescein-dextran. Osmotic water permeability (Pf) was measured by stopped-flow fluorescence quenching and proton ATPase activity by ATP-dependent, N-ethylmaleimide-inhibitable acidification. In a microsomal pellet, Pf was low (less than 0.002 cm/s, 20 degrees C) in labeled endocytic vesicles from control bladders but high (0.05-0.1 cm/s) in a subpopulation (50-70%) of vesicles from VP- and PMA-treated bladders. Following ATP addition, the average drop in pH was 0.1 (control), 0.3 (VP), and 0.2 (PMA) unit. Measurement of pH in individual endocytic vesicles by quantitative image analysis showed that less than 20% of vesicles from VP-treated bladders acidified by greater than 0.5 pH unit. To examine whether water channels and proton pumps were present in the same endocytic vesicles, the pH of endosomes with high and low water permeability was measured from the effect of ATP on the amplitude of the fluorescence quenching signal in response to an osmotic gradient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of microvessel permeability and tissue diffusion coefficient of solutes by laser scanning confocal microscopy

Interstitium contains a matrix of fibrous molecules that creates considerable resistance to water and solutes in series with the microvessel wall. On the basis of our preliminary studies, by using laser-scanning confocal microscopy and a theoretical model for interstitial transport, we determined both microvessel solute permeability (P) and solute tissue diffusion coefficient (D) of alpha-lactalbumin (Stokes radius 2.01 nm) from the rate of tissue solute accumulation and the radial concentration gradient around individually perfused microvessel in frog mesentery. P(alpha-lactalbumin) is 1.7 +/- 0.7(SD) x 10(-6) cm/s (n = 6). D(t)/D(free) for alpha-lactalbumin is 27% +/- 5% (SD) (n = 6). This value of D(t)/D(free) is comparable to that for small solute sodium fluorescein (Stokes radius 0.45 nm), while p(alpha-lactalbumin) is only 3.4% of p(sodium fluorescein). Our results suggest that frog mesenteric tissue is much less selective to solutes than the microvessel wall.     

Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance

The relationship of tight junction permeability to junction structure and composition was examined using two strains of Madin-Darby canine kidney (MDCK) cells (I and II) which differ greater than 30-fold in transepithelial resistance. This parameter is largely determined by paracellular, and hence junctional, permeability under most conditions. When these two strains of cells were grown on permeable filter supports, they formed monolayers with equivalent linear amounts of junction/area of monolayer. Ultrastructural analysis of these monolayers by thin section EM revealed no differences in overall cellular morphology or in tight junction organization. Morphometric analysis of freeze-fractured preparations indicated that the tight junctions of these two cell strains were similar in both number and density of junctional fibrils. Prediction of transepithelial resistance for the two strains from this freeze-fracture data and a published structure-function formulation (Claude, P. 1978, J. Memb. Biol. 39:219- 232) yielded values (I = 26.5 omega/cm2, II = 35.7 omega/cm2) that were significantly lower than those observed (I = 2,500-5,000 omega/cm2, II = 50-70 omega/cm2). Consistent with these structural studies, a comparison of the distribution and cellular content of ZO-1, a polypeptide localized exclusively to the tight junction, revealed no significant differences in either the localization of ZO-1 or the amount of ZO-1 per micron of junction (I = 1,415 +/- 101 molecules/micron, II = 1,514 +/- 215 molecules/micron).     

Permeability properties of the subepithelial tissues of Necturus gallbladder

The permeability properties of the subepithelial connective tissue of Necturus gallbladder were evaluated by measurement of electrical resistance, dilution potentials and hydraulic water permeability. The gallbladder epithelial cells were removed by scraping and the underlying connective tissue placed in an Ussing chamber. The electrical resistance was 2.2 +/- 0.8 omega X cm2; the tissue was slightly cation selective relative to free solution. The subepithelial tissues restricted the rate of diffusion of small solutes to 50% of the free solution value. The hydraulic water permeability averaged 2.1 X 10(-2) cm/s per atm. We conclude that limitations of the area of subepithelium available for fluid movement are the most important factors in determining the restrictions to solute and water flow offered by the subepithelial tissues.     

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.     

Caco-2 cell permeability and stability of two d-glucopyranuronamide conjugates of thyrotropin-releasing hormone

Caco-2 cell permeability and stability assays were used as an in vitro model to study the intestinal epithelial transport and stability of two analogues of thyrotropin-releasing hormone (TRH; Pyr-His-Pro-NH2). Peptide 1 (Pyr-His-Pro-D-glucopyranuronamide) was more permeable across the Caco-2 cell monolayer compared with the permeability of the parent TRH peptide (Papp=5.10+/-1.89x10(-6) cm/s c.f. Papp=0.147+/-0.0474x10(-6) cm/s respectively). The permeability of peptide 1 was improved threefold by attaching a 2-aminooctanoic acid moiety to the N-terminus to form peptide 2 (2-aminooctanoic acid-Gln-His-Pro-D-glucopyranuronamide) (Papp=16.3+/-2.47x10(-6) cm/s). The half-life for both peptide 1 and peptide 2 was approximately 20 min in a homogenate of Caco-2 cells compared with the half-life of TRH which is approximately 3 min. It was concluded that the permeability of peptides 1 and 2 was enhanced because of their increased stability, while the higher permeability of peptide 2 compared with peptide 1 may be attributed to its increased lipophilicity which results in enhanced passive diffusion.     

Electrophysiological responses of frog skin to the peptides bombesin, caerulein, and physalaemin

Isolated skins from the frog Rana pipiens were mounted on Ussing-type chambers and bathed with Amphibian Ringer's solution on both sides. Electrical potential difference, resistance, and short-circuit current (SCC) were measured by using calomel electrodes, Ag-AgCl electrodes, Ringer's-agar bridges, and Tektronix digital multimeters. Under the conditions employed, SCC is a measure of net Na+ transport. The frog skin peptides bombesin, caerulein, and physalaemin were administered to the serosal side at concentrations of 0.5, 5.0, and 50 ng/ml. Control electrophysiological parameters were: potential difference, 23 +/- 2 mV; resistance, 738 +/- 59 ohms cm2; and SCC 32 +/- 3 microA/cm2. Although bombesin and caerulein had no significant, reversible effect on potential difference, resistance, or SCC, physalaemin significantly, and reversibly, depressed SCC by 22%. Caerulein did significantly depress SCC, but the response was not reversible.     

Passive transverse mechanical properties as a function of temperature of rat skeletal muscle in vitro

The objective of the current study was to determine the in vitro passive transverse mechanical properties of skeletal muscle with Dynamic Mechanical Thermal Analysis (DMTA) tests. The starting hypotheses was that the time-temperature-superposition principle could be used to expand the DMTA results to a 1 kHz frequency range. Experiments were performed with rat hind leg skeletal muscle tissue samples on a rotational rheometer using a parallel plate geometry. Because of the small size and low modulus of the samples, the standard test geometry was altered and the samples were shifted from the center to the edge of the plates. From strain sweep tests it became clear that for strains smaller than 0.003 the muscle tissue behaves linearly. In the linear region storage moduli ranged between 24 kPa (omega = 1 rad/s) and 42 kPa (omega = 100 rad/s) at T = 4 degrees C and 22 kPa and 33 kPa at 29 degrees C within the experimental frequency range. The loss modulus decreased with increasing frequency and ranged between 7 and 4 kPa at 4 degrees C and 4.5 and 3.5 kPa at 29 degrees C. Although the properties are clearly temperature dependent, a temperature shift in phase angle delta could not be detected, thus Time Temperature Superposition is not allowed for skeletal muscle in vitro.     

Non-ohmic proton conductance of mitochondria and liposomes

Direct measurements of the proton/hydroxyl ion flux across rat liver mitochondria and liposome membranes are reported. H+/OH- fluxes driven by membrane potential (delta psi) showed nonlinear dependence on delta psi both in mitochondria and in liposomes whereas delta pH-driven H+/OH- flux shows linear dependence on delta pH in liposomes. In the presence of low concentrations of a protonophore the H+/OH- flux was linearly dependent on delta psi and showed complex dependence on delta pH. The nonlinearity of H+/OH- permeability without protonophore is described by an integrated Nernst- Plank equation with trapezoidal energy barrier. Permeability coefficients depended on the driving force but were in the range 10(-3) cm/s for mitochondria and 10(-4)-10(-6) cm/s for liposomes. The nonlinear dependence of H+/OH- flux on delta psi explains the nonlinear dependence of electrochemical proton gradient on the rate of electron transport in energy coupling systems.     

Electrogenic H+/OH- movement across phospholipid vesicles measured by spin-labeled hydrophobic ions.

Transmembrane pH gradients created across phospholipid vesicles give rise to time-dependent potentials as determined from the EPR spectra of phosphonium ion spin labels in the system. From the time-dependent spectra, the transmembrane H+/OH- current is obtained and hence the current-voltage curve for the vesicle membrane is obtained. The current-voltage curve is linear with a membrane resistance of 3 +/- 2 X 10(9) omega cm2 corresponding to a membrane permeability of 5 +/- 2 X 10(-7) cm/s. This unusually high permeability is further increased by small amounts of lipid oxidation, CHCl3 or the general anesthetic halothane.     

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.