Diffusion membrane potential in liposomes: setting by ion gradients, absolute calibration and monitoring of fast changes by spectral shifts of diS-C3(3) fluorescence maximum (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Biomembranes, 1993
Liposomes of egg PC/PG (8:2, mol/mol) were multilabelled with PBFI, pyranine and oxonol VI, fluorescent probes for, respectively, K +, H + and membrane potential. Monitoring fluorescence with a multichannel photoncounting spectrofluorometer during K + filling experiments allowed to measure K ÷ influx, the associated H ÷ efflux and the membrane potential, continuously and simultaneously. The proton net efflux quantitatively mirrored the K ÷ net influx. The rate of the K÷/H + exchange diminished progressively as a quasi-equilibrium was reached for both K ÷ and H ÷. In the presence of valinomycin, the measured membrane potential during the K + filling actually corresponded to the Nernst potential calculated from the observed K + gradient. In the absence of valinomyein, it corresponded to the Nernst potential calculated from the observed H ÷ gradient. In the latter case, the permeability coefficient of liposomes to K ÷, calculated from the Goldman-Hodgkin-Katz relation, was 6.10-13 m s-1. The selectivity sequence for alkali cations of liposomes was determined from the measured H ÷ efflux associated to the influx of the different cations. The selectivity sequence corresponded to the series VI of Eisenman, suggesting interaction of the cation with an anionic field of intermediate strength.
The Journal of Membrane Biology, 1982
port have, in the main, utilized artificial membranes, both planar and vesicular. Systems of biological interest, viz., cells and organelles, resemble vesicles in size and geometry. Methods are, therefore, required to extend the results obtained with planar membranes to liposome systems. In this report we present an analysis of a fluorescence technique, using the divalent cation probe chlortetracycline, in small, unilamellar vesicles, for the study of divalent cation fluxes. An ion carrier (X537A) and a pore former (alamethicin) have been studied. The rate of rise of fluorescence signal and the transmembrane ion gradient have been related to transmembrane current and potential, respectively. A second power dependence of ion conduction-including the electrically silent portion thereof-on X537A concentration, has been observed. An exponential dependence of "current" on "transmembrane potential" in the case of alamethicin is also confirmed. Possible errors in the technique are discussed.
Membrane perturbation: studies employing a calcium-sensitive dye, arsenazo III, in liposomes
Proceedings of the National Academy of Sciences, 1976
A metallochromic dye, arsenazo III [2,7-bis-(2-arsonophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid], has been incorporated into the aqueous interspaces of multilamellar liposomes. Addition of Ca produced no shift in the absorbance spectrum of dye captured by liposomes, whereas disruption of liposomes by Triton X-100, followed by Ca, produced the spectrum characteristic of the dye-Ca complex: evidence of latency. Addition of excess ethylenegly-
Colloids and Surfaces B: Biointerfaces, 2007
In order to study mechanisms involved in liposome-cell interaction, this work attempted to assess the influence of vesicle composition on the delivery of liposomal content to Hela cells. In particular, to evaluate pH-sensitive properties and cell interaction of the prepared liposomes, the lipid formulations contained cholesterol (Chol) and they were varied by using phosphatidylcholines with different purity degree: soy lecithin (SL; 80% phosphatidylcholine), a commercial mixture of soy phosphatidylcholine (P90; 90% phosphatidylcholine) or dipalmitoylphosphatidylcholine (DPPC; 99% of purity). A second series of liposomes also contained stearylamine (SA). Dehydration-rehydration vesicles (DRV) were prepared and then sonicated to decrease vesicle size. Vesicle-cell interactions and liposomal uptake were examined by fluorescence microscopy using carboxyfluorescein (CF) and phosphatidylethanolamine-dioleoyl-sulforhodamine B (Rho-PE) as fluorescent markers. Fluorescence dequenching assay was used to study the influence of pH on CF release from the liposomal formulations. Liposome adhesion on the cell surface and internalization were strongly dependent on vesicle bilayer composition. SA vesicles were not endocytosed. DPPC/Chol liposomes were endocytosed but did not release their fluorescent content into the cytosol. SL/Chol and P90/Chol formulations displayed a diffuse cytoplasmic fluorescence of liposomal marker. (A.M. Fadda).
Biochemical and Biophysical Research Communications, 1976
When single cell suspensions of thymic lymphocytes, splenic lymphocytes and platelets are equilibrated with a carbocyanine dye, 3,3'dipentyloxacarbocyanine iodide the fluorescence emission intensity is related to the trans-membrane potential. Addition of valinomycin to these cells provokes a net potassium efflux, and membrane hyperpolarisation, which is reflected as a decrease in fluorescence intensity. Increasing the extracellular potassium concentration, and thus decreasing the concentration gradient of that ion across the membrane, reduces the change in fluorescence intensity induced by valinomycin. The ionophore A23187 induces a calcium influx into thymic lymphocytes and a concomitant increase in fluorescence intensity indicative of hypo-polarisation. The movements of several ionic species may contribute to this altered membrane potential since Na, K, PO4 and Ca are all induced to move across the cell membrane following the application of A23187. Removal of extracellular calcium markedly reduced both the hypopolarisation and ion redistribution elicited by the ionophore. Thus when calcium enters the cell it initiates a chain of events involving ionic rearrangement and altered transmembrane potential which can be monitored using the fluorescent probe system described here.
The Journal of Membrane Biology, 1977
The kinetics of the transport of the 1-anilino-8-naphthalenesulfonate (ANS-, an anionic fluorescent probe of the membrane surface) across phospholipid vesicle membranes have been studied using a stopped-flow rapid kinetic technique. The method has been used to gain detailed information about the mechanism of transport of this probe and to study ionophore-mediated cation transport across the membrane. The technique has also been exploited to study differences between the inside and outside surfaces of vesicles containing pbosphatidyl choline (PC).
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1995
In earlier studies, the fluorescence probe l-(4-(trimethylamino)phenyl)-6-phenylhexa-l,3,5-triene (TMA-DPH) was shown to interact with living cells by instantaneous incorporation into the plasma membrane, according to a water (probe not fluorescent)/membrane (probe highly fluorescent) partition equilibrium. This made it interesting both as a fluorescence anisotropy probe for plasma membrane fluidity determinations and as a quantitative tracer for endocytosis and intracellular membrane traffic. In order to ascertain the limiting concentrations for its use in these applications, we performed a systematic study of its fluorescence properties (intensity, lifetime, anisotropy) in the plasma membrane and in endocytic membranes of intact L929 mouse fibroblasts. Some of the experiments were repeated on mouse-bone-marrow-derived macrophages and on phospholipidic LUV to confirm the results. Rather unexpectedly, it was observed that: (i) the incorporation of TMA-DPH into the membranes, monitored by UV absorption measurements, remained proportional to the probe concentration over the wide range explored (5 -l0 7 M-2.5 • 10 _s M); (ii) however, concerning fluorescence, quenching effects occurred in the membranes above certain critical concentrations. These effects were shown to result from FiSrster-type resonance auto-transfer; (iii) strikingly, the critical concentrations were considerably higher in early-endocytic-vesicle membranes than in the bulk plasma membrane. It was established that membrane fluidity was involved and this was confirmed by the parallel study on phospholipidic vesicles. Potential applications of these properties as a novel approach for evaluating membrane fluidity are suggested.
Fluorescence studies of lipid regular distribution in membranes
Chemistry and Physics of Lipids, 2002
This article reviews the use of fluorescent lipids and free probes in the studies of lipid regular distribution in model membranes. The first part of this article summarizes the evidence and physical properties for lipid regular distribution in pyrene-labeled phosphatidylcholine (PC)/unlabeled PC binary mixtures as revealed by the fluorescence of pyrene-labeled PC. The original and the extended hexagonal superlattice model are discussed. The second part focuses on the fluorescence studies of sterol regular distributions in membranes. The experimental evidence for sterol superlattice formation obtained from the fluorescent sterol (i.e. dehydroergosterol) and non-sterol fluorescent probes (e.g. DPH and Laurdan) are evaluated. Prospects and concerns are given with regard to the sterol regular distribution. The third part deals briefly with the evidence for polar headgroup superlattices. The emphasis of this article is placed on the new concept that membrane properties and activities, including the activities of surface acting enzymes, drug partitioning, and membrane free volume, are fine-tuned by minute changes in the concentration of bulky lipids (e.g. sterols and pyrene-containing acyl chains) in the vicinities of the critical mole fractions for superlattice formation.
A Stability Test of Liposome Preparations Using Steady-State Fluorescent Measurements
Drug Delivery, 2001
The stability of liposome preparations under the action of the nonionic detergent Triton X-100 was measured using the fluorescent molecular probe octadecylrhodamine B (R18). The probe inserted in the lipid bilayer shows a self-quenched fluorescence and the degree of quenching depends both on the probe concentration and the phase state of the lipid membrane. The addition of detergent to the liposomes produces a steep decrease in self-quenching caused by dilution of the probe in the bilayer. The curves of steady-state fluorescence intensity show an abrupt change in slope that corresponds to the point at which liposomes break down into lipid-detergent mixed entities that are different from the earlier liposome-monodisperse population. The lytic process was followed in parallel by dynamic light scattering (DLS), and the analysis of the DLS results agree with the interpretation of the fluorescence measurements. The probe R18 therefore is a useful marker to test the stability of liposome ...
Rapid Transbilayer Movement of the Fluorescent Sterol Dehydroergosterol in Lipid Membranes
Biophysical Journal, 2002
This study establishes a new assay for measuring the transbilayer movement of dehydroergosterol (DHE) in lipid membranes. The assay is based on the rapid extraction of DHE by methyl--cyclodextrin (M-CD) from liposomes. The concentration of DHE in the liposomal membrane was measured by using fluorescence resonance energy transfer (FRET) from DHE to dansyl-phosphatidylethanolamine, which is not extracted from liposomes by M-CD. The method was applied to small (SUV) and large (LUV) unilamellar vesicles of different compositions and at various temperatures. From the kinetics of FRET changes upon extraction of DHE from membranes, rates of M-CD mediated extraction and flip-flop of DHE could be deduced and were found to be dependent on the physical state of the lipid phase. For egg phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in the liquid-crystalline state, halftimes of extraction and transbilayer movement were Ͻ5 s and ϳ20-50 s, respectively, at 10°C. For 1,2-dimyristoyl-sn-glycero-3-phosphocholine-SUV being in the gel state at 10°C, the respective halftimes were 28 s and 5-8 min. Surprisingly, DHE could not be extracted from LUV consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. This might be an indication of specific interactions between DHE molecules in membranes depending on the phospholipid composition of the membrane.