Ionophore-mediated uptake of ciprofloxacin and vincristine into large unilamellar vesicles exhibiting transmembrane ion gradients

A new method, based on the ion-translocating properties of the ionophores nigericin and A23187, is described for loading large unilamellar vesicles (LUVs) with the drugs vincristine and ciprofloxacin. LUVs composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) (55:45 mol/mol) or sphingomyelin (SPM)/Chol (55:45 mol/mol) exhibiting a transmembrane salt gradient (for example, internal solution 300 mM MnSO4 or K2SO4; external solution 300 mM sucrose) are incubated in the presence of drug and, for experiments involving divalent cations, the chelator EDTA. The addition of ionophore couples the outward movement of the entrapped cation to the inward movement of protons, thus acidifying the vesicle interior. External drugs that are weak bases can be taken up in response to this induced transmembrane pH gradient. It is shown that both nigericin and A23187 facilitate the rapid uptake of vincristine and ciprofloxacin, with entrapment levels approaching 100% and excellent retention in vitro. Following drug loading, the ionophores can be removed by gel exclusion chromatography, dialysis, or treatment with biobeads. In vitro leakage assays (addition of 50% mouse serum) and in vivo pharmacokinetic studies (in mice) reveal that the A23187/Mn2+ system exhibits superior drug retention over the nigericin/K+ system, and compares favorably with vesicles loaded by the standard DeltapH or amine methods. The unique features of this methodology and possible benefits are discussed.     

Accumulation of doxorubicin and other lipophilic amines into large unilamellar vesicles in response to transmembrane pH gradients

The uptake of the anticancer agent doxorubicin into large unilamellar vesicles (LUVs) exhibiting a transmembrane pH gradient (inside acidic) has been investigated using both kinetic and equilibrium approaches. It is shown that doxorubicin uptake into the vesicles proceeds via permeation of the neutral form and that uptake of the drug into LUVs with an acidic interior is associated with high activation energies (Ea) which are markedly sensitive to lipid composition. Doxorubicin uptake into egg-yolk phosphatidylcholine (EPC) LUVs exhibited an activation energy of 28 kcal/mol, whereas for uptake into EPC/cholesterol (55:45, mol/mol) LUVs Ea = 38 kcal/mol. The equilibrium uptake results obtained are analyzed in terms of a model which includes the buffering capacity of the interior medium and the effects of drug partitioning into the interior monolayer. From the equilibrium uptake behaviour, a doxorubicin partition coefficient of 70 can be estimated for EPC/cholesterol bilayers. For a 100 nm diameter LUV, this indicates that more than 95% of encapsulated doxorubicin is partitioned into the inner monolayer, presumably located at the lipid/water interface. This is consistent with 13C-NMR behaviour as a large proportion of the drug appears membrane associated after accumulation as reflected by a broadening beyond detection of the 13C-NMR spectrum. The equilibrium accumulation behaviour of a variety of other lipophilic amines is also examined in terms of the partitioning model.     

Factors influencing uptake and retention of amino-containing drugs in large unilamellar vesicles exhibiting transmembrane pH gradients

The level of uptake and retention of amino-containing drugs in large unilamellar vesicles (LUVs) following uptake in response to a transmembrane pH gradient (DeltapH) can vary dramatically depending on the drug. For example, the anticancer drugs doxorubicin and epirubicin can be readily retained, whereas the anticancer drug vincristine and the antibiotic ciprofloxacin tend to leak out rapidly. In this investigation, we examine the influence of the hydrophobicity of the entrapped amines (that induce the DeltapH) and the anionic lipid content of the LUV on drug retention. It is shown that entrapment of increasingly hydrophobic monoamines (methylamine to amylamine) all lead to an induced DeltapH of 3 units and essentially complete drug uptake under the conditions employed, but do not lead to improved retention of vincristine and ciprofloxacin. However, significantly improved retention could be achieved by substitution of the anionic lipid distearoylphosphatidylglycerol (DSPG) for distearoylphosphatidylcholine (DSPC) in the LUV bilayer. Further, it is shown that if the induced DeltapH is reduced to 1.4 units (driven by entrapped diamine) nearly 100% accumulation of doxorubicin and epirubicin could be achieved, whereas only 25% loading for vincristine and ciprofloxacin was observed. Taken together these results provide methodology for improving (weak base) drug retention in liposomes and indicate that drugs that can partition into the lipid bilayer exhibit improved uptake and retention characteristics.     

Electroinsertion of glycophorin A in interdigitation-fusion giant unilamellar lipid vesicles

Previously we demonstrated that transmembrane back insertion of glycophorin A, a solubilizable intrinsic protein, can be obtained in dipalmitoylphosphatidylcholine multilamellar vesicles, MLVs, by electropulsation (Raffy, S., and Teissié, J. (1995) Eur. J. Biochem. 230, 722-732). Here we report that transmembrane back insertion of protein is obtained by electropulsion of unilamellar giant vesicles, termed interdigitation-fusion vesicles (IFVs), which are better membrane models than MLVs due to their unilamellarity. Electropulsation promotes a field-dependent local permeabilization of the lipid layer, as shown by the associated leakage of entrapped calcein. Glycophorin insertion is assayed by immunofluorescence. Electroinsertion is obtained by pulsing the vesicle/protein mixture. Glycophorin insertion is observed under more drastic electrical conditions than needed for permeabilization. Direct observation of glycophorin insertion in the vesicles under a microscope shows a localized process in agreement with the theoretical prediction. A quantitative evaluation of the immunofluorescence pattern shows that insertion was higher on one side of the vesicle than on the other. This suggests that an electrophoretic movement of the solubilized glycophorin could take place during electropulsation. Insertion of glycophorin, a prefolded intrinsic protein, is then obtained in the lipid bilayer brought transiently to the electropermeabilized state.     

Cholesterol mobilization and regression of atheroma in cholesterol-fed rabbits induced by large unilamellar vesicles

The antiatherogenic properties of repeated injections of egg phosphatidylcholine large unilamellar vesicles (LUVs) of 100 nm diameter were tested in an experimental model for atherosclerosis. Forty eight rabbits were divided into two diet groups fed standard rabbit chow or fed a cholesterol-enriched diet (0.5% by weight) to induce the formation of atherosclerotic lesions. Prior to the initiation of LUV therapy, the cholesterol diet was ceased and all animals were returned to standard rabbit chow. The treatment protocol consisted of a total of 10 bolus injections of vesicles, at a phospholipid dose of 300 mg/kg body weight or the equivalent volume of saline, with one injection given to each animal every 10 days. LUV injections brought about a large movement of cholesterol into the blood pool and resulted in a significant reduction in the cholesterol content as well as the degree of surface plaque involvement of aortic tissue in atherosclerotic animals. Most notably, the thoracic aorta of LUV-treated animals exhibited a 48% reduction in tissue cholesterol content per gram of protein compared to saline-treated controls. Histochemical analyses revealed that aortas from animals receiving the repeated injections of LUVs displayed less cholesterol deposits in lesions, and a moderate reduction in intimal-to-medial thickness. This regression of atheroma, induced by LUV therapy, was observed even though animals possessed persistent elevated plasma cholesterol levels after the cholesterol-enriched diet was ceased. These results suggest that repeated injections of LUVs, working with endogenous HDL, may be a useful therapy in the management of atherosclerosis.     

Effect of membrane surface potential on the uptake and the inhibition of cationic compounds in rat intestinal brush-border membrane vesicles and liposomes

The effect of membrane surface potential on the uptake of tryptamine, an organic cation, by rat intestinal brush-border membrane vesicles was investigated. In the presence of an inside-negative K(+)-diffusion potential, the manner of initial uptake of tryptamine appeared to be pH-dependent and the uptake in the acidic medium was lower than that in the neutral medium. Changes in surface potential of brush-border membrane vesicles were monitored using 8-anilino-1-naphthalenesulfonic acid (ANS) and the results suggested that the membrane surface potential (negative charge on the membrane surface) decreased in the acidic medium. A good correlation was observed between the K(+)-diffusion potential-dependent uptake of tryptamine and membrane surface potential monitored by ANS at various pH levels. The uptake of tryptamine by liposomes (large unilamellar vesicles), which contained various amounts of dipalmitoylphosphatidylserine (DPPS), was also examined. The uptake of tryptamine decreased with a decrease of DPPS content in the liposomes, and was correlated with the membrane surface potential monitored by ANS. Moreover, the effect of organic cations on the uptake of tryptamine by intestinal brush-border membrane vesicles was examined. The uptake of tryptamine was inhibited by tetracaine and imipramine. The inhibitory effect of these cations was well correlated with changes in the membrane surface potential in the presence of tetracaine or imipramine. These results suggest that the K(+)-diffusion potential-dependent uptake of tryptamine by intestinal brush-border membrane vesicles is affected by membrane surface potential, and the inhibition of tryptamine uptake originates in changes in the membrane surface potential caused by the organic cations.     

Poly(ethylene glycol)-induced fusion and rupture of dipalmitoylphosphatidylcholine large, unilamellar extruded vesicles

High concentrations (> or = 20 wt %) of poly(ethylene glycol) (PEG) induce large, unilamellar, dipalmitoylphosphatidylcholine model membrane vesicles to fuse when the bilayers contain small amounts of amphipathic peturbant molecules. In addition to fusion, similar concentrations of PEG induce these vesicles to leak their contents. In this paper, we have asked if fusion could occur independently of leakage or if fusion might be described as local bilayer rupture followed by resealing. By following the release of MW 10,000 fluoresceinated dextran trapped inside vesicles, it was determined that PEG-induced leakage was the result of major membrane disruption and not small-pore formation. Fusion of vesicles containing 0.5 mol % palmitic acid was clearly observed at 20 wt % PEG, while 25 wt % was needed to cause rupture. On the other hand, vesicles containing 0.5 mol % lysophosphatidylcholine ruptured at roughly the same concentration needed to induce rupture. Two methods were developed for removing PEG so that fusion products could be characterized. Quasi-elastic light scattering demonstrated that fusing vesicles grew in size and that nonfusing vesicles did not. Moreover, PEG concentrations that induced rupture led to the appearance of species with mean diameters much larger than those of fused vesicles. High-resolution nuclear magnetic resonance showed that the population of large vesicles that correlated with rupture was composed of multilamellar vesicles while the population resulting from fusion alone remained unilamellar. We conclude that, upon incubation with and subsequent removal of PEG, vesicles were either unaffected, or fused to form larger, unilamellar vesicles, or ruptured to form larger, nonunilamellar species.     

Ursodeoxycholate stabilizes phospholipid-rich membranes and mimics the effect of cholesterol: investigations on large unilamellar vesicles

Ichthyophis kohtaoensis, a member of the limbless Gymnophiona, has a specialized subterranean burrowing mode of life and a predominantly olfactory-guided orientation. The only visually guided behavior seems to be negative phototaxis. As these animals possess extremely small eyes (only 540 microm in diameter in adults), functional investigations of single retinal cells by electrophysiological methods have so far failed. Therefore, the content and distribution of retinal transmitters have been investigated as indications for a functioning sense organ in an animal that is supposed to be blind. In this study, the organization and development of the dopaminergic system have been examined in the retinae of embryonic, larval, and adult I. kohtaoensis, by using an antiserum against tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine synthetic pathway, and an antiserum against dopamine itself. Labeled somata are situated in the inner nuclear layer and in the ganglion cell layer. Dopamine-positive fibers form a dense diffuse plexus, that covers the whole inner plexiform layer, whereas tyrosine hydroxylase-immunoreactive processes show a tendency to arborize in a stratified manner. Tyrosine-hydroxylase-immunolabeled fibers can occasionally be observed in the optic nerve head of larval stages. During ontogenesis and larval development, the distribution of transmitter-expressing cells changes and their number decreases, but no general degeneration of the visual system is detectable. Adult Ichthyophis still have retinal transmitters, indicating that the eyes, although obviously playing a minor role in a subterranean ecological niche, retain all the elements of functioning sense organs.     

Modulation of sodium-coupled uptake and membrane fluidity by cisplatin in renal proximal tubular cells in primary culture and brush-border membrane vesicles

The proximal tubule appears to be the main target for the adverse effects of cis-diamminedichloroplatinum (II) (cDDP). We evaluated the early effects of cDDP at concentrations (3 to 67 microM) lower that those which alter cell viability, on three apical transport systems and on the physical state of the brush border membrane (BBM) in rabbit proximal tubule (RPT) cells in primary culture. The maximal effect, corresponding to a 30% decrease in Na(+)-coupled uptake of phosphate (Pi) and alpha-methylglucopyranoside (MGP) and a twofold increase in Na(+)-coupled alanine uptake, was obtained at 17 microM (5 micrograms/ml) cDDP and occurred through a modification of their affinity. At this concentration, cDDP increased BBM fluidity and decreased the BBM cholesterol content by 28%, without increasing the permeability of tight junctions. To clarify the role of cDDP-induced increase in BBM fluidity on alterations of Na(+)-coupled uptake, these parameters were also investigated in BBM vesicles isolated from rabbit renal cortex directly exposed to cDDP. cDDP induced a concentration-dependent inhibition of Na(+)-coupled uptake of MGP, Pi and alanine in BBM vesicles from the renal cortex, associated with a decrease in protein sulfhydryl content, without modifying BBM fluidity. Our findings strongly suggest that the cDDP-induced increase in BBM fluidity in RPT cells results from an indirect mechanism, possibly an alteration of cholesterol metabolism, and did not play a major role in the cDDP-induced inhibition of Na+/Pi and Na+/glucose cotransport systems that may be mainly mediated through a direct chemical interaction with essential sulfhydryl groups of the transporters.     

Role of the membrane potential in serum-stimulated uptake of amino acid in a diploid human fibroblast

The Na+-dependent accumulation of alpha-aminoisobutyric acid (AIB), measured in normal growing and quiescent (serum-deprived) HSWP cells (human diploid fibroblast), was found to be twofold higher (AIB/in/AIBout = 20-25) under the normal growing conditions. Serum stimulation of quiescent cells increases their AIB concentrating capacity by approximately 70% within 1 hr. These observations suggest that the driving forces for AIB accumulation may be reversibly influenced by the serum concentration of the growth medium. Addition of valinomycin (Val) to cells preequilibrated with AIB causes an enhanced accumulation of AIB, suggesting that the membrane potential can serve as a driving force for AIB accumulation. After preequilibration with AIB in 6 mM K+, transfer to 94 mM K+ with Val results in a marked and rapid net loss of AIB. The effect of Val on the accumulation of AIB is greatest in quiescent cells, with the intracellular AIB concentrations reaching those seen both in Val-stimulated normal cells and in Val-stimulated serum-stimulated cells. By adjusting [K+]0, in the presence of Val, the membrane potential of growing cells can be matched to that of quiescent cells or vice versa. When this is done, the two accumulate AIB to the same extent. Hence the AIB accumulating capacity is characteristic of the membrane potential rather than of the growth state. In summary, these data suggest that the accumulation of AIB in HSWP cells is influenced by changes in membrane potential and that a serum-associated membrane hyperpolarization could be responsible for the increased capacity for AIB accumulation in serum-stimulated cells.     

Slow fluorescent indicators of membrane potential: a survey of different approaches to probe response analysis

Basic tenets related to the use of three main classes of potentiometric redistribution fluorescent dyes (carbocyanines, oxonols, and rhodamines) are discussed in detail. They include the structure/function relationship, formation of nonfluorescent (H-type) and fluorescent (J-type) dimers and higher aggregates, probe partitioning between membranes and medium and binding to membranes and intracellular components (with attendant changes in absorption and emission spectra, fluorescence quantum yield and lifetime). The crucial importance of suitable probe-to-cell concentration ratio and selection of optimum monitored fluorescence wavelength is illustrated in schematic diagrams and possible artifacts or puzzling results stemming from faulty experimental protocol are pointed out. Special attention is paid to procedures used for probe-response calibration (potential clamping by potassium in the presence of valinomycin, use of gramicidin D in combination with N-methylglucamine, activation of Ca-dependent K-channels by A23187, the null-point technique). Among other problems treated are dye toxicity, interaction with mitochondria and other organelles, and possible effects of intracellular pH and the quantity of cytosolic proteins and/or RNA on probe response. Individual techniques using redistribution dyes (fluorescence measurements in cuvettes, flow cytometry and microfluorimetry of individual cells including fluorescence confocal microscopy) are discussed in terms of reliability, limitations and drawbacks, and selection of suitable probes. Up-to-date examples of application of slow dyes illustrate the broad range of problems in which these probes can be used.     

Effect of vitamin D deficiency on lipid composition and calcium transport in basolateral membrane vesicles from chick intestine

Vitamin D deficiency affects the lipid composition and Ca2+ uptake of intestinal basolateral membranes from chick intestine. The increased cholesterol content causes an increase in the molar ratio cholesterol/phospholipid. Phospholipid classes remain unchanged, but the percentages of arachidonic acid from the from the major phospholipid fractions are increased. After 24 hours of oral administration of 2,000 IU of cholecalciferol to vitamin D-deficient chicks, the cholesterol values do not change, but the amount of arachidonic acid returns to normal values. Ca2+ uptake driven by ATP is diminished in vesicles from intestinal basolateral membranes of vitamin D-deficient chicks. Cholecalciferol treatment returns these values to the controls which might be due mainly to the increased number of Ca2+ pump units. In conclusion, changes in lipid composition and in Ca2+ pump caused by vitamin D deficiency seems to play a role in the decrease of vesicular Ca2+ transport. A single dose of cholecalciferol restores only partially the lipid-protein changes produced by vitamin D deficiency.     

Effect of the dipole potential of a bilayer lipid membrane on gramicidin channel dissociation kinetics

A technique of measuring of the light-induced transients of the gramicidin-mediated electric current across a membrane in the presence of a photosensitizer has been applied for the study of the effect of agents modifying the dipole potential of a bilayer lipid membrane (phloretin, 6-ketocholestanol, and RH421) on the processes of the gramicidin channel dissociation and formation. It is shown that phloretin, known to lower the dipole potential, decelerates the flash-induced decrease in the current, whereas 6-ketocholestanol and RH421, known to raise the dipole potential, accelerate the current decrease. It is revealed that the addition of phloretin leads to a decrease in the dissociation rate constant, whereas addition of either 6-ketocholestanol or RH421 causes an increase in this constant. Single-channel data show that phloretin brings about an increase in the lifetime of the gramicidin channels, whereas RH421 produces a more complicated effect. It is conclude that the dipole potential affects the process of channel dissociation, presumably via the influence on the movement of the dipoles of gramicidin molecules through the layer of the dipole potential drop near the membrane-water interface.     

Kinetics of the interactions of a dicarboxylic porphyrin with unilamellar lipidic vesicles: interplay between bilayer thickness and pH in rate control

The transfer of a dicarboxylic porphyrin from phosphatidylcholine fluid-phase unilamellar vesicles towards albumin is studied focusing on bilayer thickness and pH effects. The kinetics of this process yield the rate constants for the porphyrin flip-flop from the inner to the outer hemileaflet and its exit towards aqueous medium. Phospholipids with monounsaturated 14-22 carbon chains are used. Interplay between bilayer thickness and pH for the control of the rate constants is observed. This results in the amplification, at physiological pH, of the effect of membrane thickness on the flip-flop and exit rates as compared to pH 8.5 and 6.5. These data are explained by the degree of porphyrin burying within the bilayer resulting from a compromise between favorable hydrophobic interactions with the hydrocarbon phase and unfavorable penetration of the polar carboxylic chains. The balance between the two effects depends particularly on the neutralization of one carboxylic chain. Considering the bilayer hydrophobicity profile and the porphyrin size, the optimization of hydrophobic interactions appears dependent on the bilayer thickness. The flip-flop and the exit are governed by neutralization and deprotonation of the carboxylic chains, respectively, the rate of these proton exchanges being dependent on the porphyrin location within the bilayer.     

Ectopic potassium uptake in trk1 trk2 mutants of Saccharomyces cerevisiae correlates with a highly hyperpolarized membrane potential

Null trk1 trk2 mutants of Saccharomyces cerevisiae exhibit a low-affinity uptake of K+ and Rb+. We show that this low-affinity Rb+ uptake is mediated by several independent transporters, and that trk1Delta cells and especially trk1Delta trk2Delta cells are highly hyperpolarized. Differences in the membrane potentials were assessed for sensitivity to hygromycin B and by flow cytometric analyses of cellular DiOC6(3) fluorescence. On the basis of the latter analyses, it is proposed that Trk1p and Trk2p are involved in the control of the membrane potential, preventing excessive hyperpolarizations. K+ starvation and nitrogen starvation hyperpolarize both TRK1 TRK2 and trk1Delta trk2Delta cells, thus suggesting that other proteins, in addition to Trk1p and Trk2p, participate in the control of the membrane potential. The HAK1 K+ transporter from Schwanniomyces occidentalis suppresses the K+-defective transport of trk1Delta trk2Delta cells but not the high hyperpolarization, and the HKT1 K+ transporter from wheat suppresses both defects, in the presence of Na+. We discuss the mechanism involved in the control of the membrane potential by Trk1p and Trk2p and the causal relationship between the high membrane potential (negative inside) of trk1Delta trk2Delta cells and its ectopic transport of alkali cations.     

Structure-activity relationships for cocaine congeners in inhibiting dopamine uptake into rat brain synaptic vesicles and bovine chromaffin granule ghosts

Structure-activity relationships of cocaine congeners were determined in inhibiting reserpine-sensitive, Mg++/ATP-dependent uptake of [3H]dopamine into rat brain synaptic vesicles under initial velocity conditions. The C2 carbomethoxy group could be deleted without loss of activity, whereas movement of this group from axial to equatorial orientation increased the potency. There was no need for the ester linkage between the tropane and phenyl rings, nor was there need for the ethylene bridge between C1 and C5 that makes cocaine a tropane instead of a piperidine structure. The structure-activity relationships were different from those for inhibiting neuronal amine transport or blocking voltage-dependent sodium channels. There was no correlation between block of uptake and degree of lipophilicity. The equally lipophilic compounds cocaine and pseudococaine, and WIN 35,065-3 and WIN 35,140, differed in uptake-blocking potency by an order of magnitude (137 vs. 22 microM and 65 vs. 4 microM, respectively). In bovine chromaffin granules, used as a less complex model system for the vesicular uptake system, the rank order of d-amphetamine, cocaine and pseudococaine in perturbing the proton gradient and in changing the membrane potential was different from that in inhibiting uptake. The inhibition of uptake is discussed in terms of the compounds acting as weak bases, transporter substrates or transporter blockers.     

The formation and annealing of structural defects in lipid bilayer vesicles

It is shown that sonication of phospholipid-water dispersions below the crystalline leads to liquid crystalline phase transition temperature (Tc) produces bilayer vesicles with structural defects within the bilayer membrane, which permit rapid permeation of ions and catalyze vesicle-vesicle fusion. These structural defects are annihilated simply by annealing the vesicle suspension above Tc. The rate of annealing was found to be slow, of the order of an hour for T = 3 degrees C above Tc, but annealing is complete within 10 min for T = 10 degrees C above Tc. It is proposed that these structural defects are fault-dislocations in the bilayer structure, which arise from a population defect in the distribution of the lipid molecules between the outer and inner monolayers, when small bilayer fragments reassemble to form the small bilayer vesicles during the sonication procedure. Such a population defect can only be remedied by lipid transport via the inside in equilibrium outside flip-flop mechanism, which would account for the slow kinetics of annealing observed even at 3 degrees C above the phase transition.     

Brij 58, a polyoxyethylene acyl ether, creates membrane vesicles of uniform sidedness. A new tool to obtain inside-out (cytoplasmic side-out) plasma membrane vesicles

Most of the plasma membrane vesicles formed upon homogenization of plant tissue have a right-side-out (cytoplasmic side-in) orientation. Subsequent purification of plasma membrane vesicles using aqueous two-phase partitioning leads to a further enrichment in right-side-out vesicles resulting in preparations with 80-90% of the vesicles in this orientation. Thus, to be able to assay, e.g. the ion-pumping activities of the H(+)-ATPase and the Ca(2+)-ATPase, which expose their active sites towards the cytoplasm, the vesicles have to be inverted. This is very efficiently achieved by including 0.05% of the detergent Brij 58 (C16E20) in the assay medium, which produces 100% sealed, inside-out (cytoplasmic side-out) vesicles from preparations of 80-90% right-side-out vesicles. This was shown by assaying ATP-dependent H+ pumping using the delta pH probe acridine orange and dissipating the H+ gradient with nigericin, and by assaying ATP-dependent Ca2+ transport using 45Ca2+ and dissipating the Ca2+ gradient with the ionophore A23187. The presence of intact vesicles was confirmed by electronmicroscopy. The detergent Brij 58 is a polyoxyethylene acyl ether and a survey among some other members of this series revealed that those with a head group of relatively large size (E20-23) showed this 'non-detergent behavior', whereas those with smaller head groups (E8-10) behaved as normal detergents and permeabilized the membranes. Thus, a very convenient system for studies on ion-pumping activities and other vectorial properties of the plasma membrane is obtained by simply including the detergent Brij 58 in the assay medium.