RESUMO
The size increase of small unilamellar vesicles composed of binary mixtures either of saturated fatty acid phosphatidylcholines with different chain lengths or of saturated and unsaturated phosphatidylcholines was found to depend on the miscibility properties of the lipid components. No size increase was detected in vesicles formed by two miscible phosphatidylcholines. In vesicles composed of two lipids which are partially immiscible in the gel state, a size increase was observed at temperatures which mainly overlapped the range of temperatures of the lipid phase transition. The rate of size increase of vesicles composed of two lipids which are immiscible in the gel state was faster than that of vesicles composed of two partially immiscible phosphatidylcholines, and the process occurred not only at the temperature ranges of the lipid phase transition, but also when both lipids were in the gel state. The vesicle size increase process occurred without the mixing of the internal content of the vesicles. A model is proposed in which the presence of 'fractures' between membrane regions of different fluidity and/or lipid composition controls the rate of this process.
Assuntos
Lipossomos , Fosfatidilcolinas , Fenômenos Químicos , Físico-Química , Ácidos Graxos , Ácidos Graxos Insaturados , Fluoresceínas , Polarização de Fluorescência , Fluidez de Membrana , Fusão de Membrana , Microscopia Eletrônica , Tamanho da Partícula , TemperaturaRESUMO
The surface density of the fluorescent probe N-(lissamine Rhodamine B sulfonyl)dipalmitoylphosphatidylcholine is the same in the two lipid leaflets of phosphatidylcholine bilayers containing the probe. In the liquid-crystalline state, the probe molecules aggregate above a threshold amount, approximately 0.2 mol/mol phospholipids. Above this threshold value, the surface density of the free probe molecules is constant, and all probe molecules added are incorporated in the aggregated form. The aggregation of the probe increases by approximately 20% when the medium pH is lowered to 4. In the gel state, the probe aggregation is higher than that in the liquid-crystalline state, and the free probe molecules distribute unevenly in the bilayer surface. Even though the results obtained in our model system cannot be directly extrapolated to all model systems, we point out that care is to be taken in the use of the probe. In fact, only in membranes in the liquid-crystalline state in which the amount of probe molecules to phospholipid molecules is lower than 1:7 the fluorescence response of the probe is independent of the pH changes and of the molecular aggregation.
Assuntos
Corantes Fluorescentes , Bicamadas Lipídicas , Fosfatidilcolinas , Fosfatidiletanolaminas , Rodaminas , Xantenos , Cloreto de Cálcio , Concentração de Íons de Hidrogênio , Cinética , Espectrometria de Fluorescência , Propriedades de Superfície , TermodinâmicaRESUMO
Addition to energized rat liver mitochondria of low micromolar concentrations of the thiol oxidant, copper-o-phenanthroline [Cu(OP)2], causes opening of the permeability transition pore, a cyclosporin A-sensitive channel. The effects of Cu(OP)2 can be reversed by reduction with dithiothreitol (DTT), suggesting that a dithiol-disulfide interconversion is involved. However, at variance with all pore inducers known to act through dithiol oxidation, the effects of Cu(OP)2 are not prevented by treatment of mitochondria with low (10-20 microM) concentrations of N-ethylmaleimide (NEM). Rather, these concentrations of NEM potentiate the inducing effects of Cu(OP)2. We show that this enhancing effect of NEM is blocked by the subsequent addition of DTT, indicating that potentiation by NEM is mediated by an oxidative event rather than by substitution as such. We find that also pore induction by high (0.5-1.0 mM) concentrations of NEM in the absence of oxidants is completely blocked by reduction with DTT or beta-mercaptoethanol. These results underscore the unexpected importance of oxidative events in pore opening by substituting agents. Since we find that pore opening by Cu(OP)2 or by high concentrations of NEM is not accompanied by dimerization of the adenine nucleotide translocase, we conclude that the translocase itself is not the target of the pore-inducing oxidative events triggered by Cu(OP)2 and NEM.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Etilmaleimida/farmacologia , Membranas Intracelulares/fisiologia , Mitocôndrias Hepáticas/química , Compostos de Sulfidrila/química , Reagentes de Sulfidrila/farmacologia , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Ciclosporina/farmacologia , Dimerização , Dissulfetos/química , Ditiotreitol/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Mercaptoetanol , Translocases Mitocondriais de ADP e ATP , Oxirredução , Permeabilidade/efeitos dos fármacos , Fenantrolinas/farmacologia , Ratos , Ratos Wistar , Substâncias Redutoras/farmacologiaRESUMO
Cation translocation across the membrane of cytochrome oxidase reconstituted vesicles may be followed with a simple spectrophotometric method. Cytochrome oxidase reconstituted vesicles, supplemented with ascorbate and cytochrome c. induce large spectral changes of the positive dye safranine, reversed by uncouplers and inhibitors of respiration. The dye is probably accumulated in the inner space of the vesicles, where it reaches high concentrations and aggregates. The spectral shifts and the absorbance changes, due to aggregation, are proportional to the amount of the dye taken up and depend on the respiratory control. In the presence of potassium, valinomycin causes an inhibition, whereas nigericin stimulates the dye uptake. The data are discussed in terms of electrical potential dependent fluxes.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona , Corantes , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Membranas Artificiais , Modelos Biológicos , Nigericina , Consumo de Oxigênio , Fenazinas , Fosfolipídeos , Cloreto de Potássio , Ligação Proteica , Glycine max , Espectrofotometria , ValinomicinaRESUMO
Dipalmitoyl phosphatidylcholine vesicles incubated in the presence of increasing amounts of myristic acid showed a progressive translocation of phospholipid molecules across a dialysis membrane. The rate of phospholipid translocation increased abruptly at a 'critical' value of myristic acid concentrations. The translocation rate of mixed dipalmitoyl phosphatidylcholine/myristic acid vesicles obtained by cosonicating the two components was also dependent on a 'critical' fatty acid concentration. A marked release of K+ and different responses of fluorescent probes to the fatty acid addition were observed at this concentration.
Assuntos
Ácidos Graxos/farmacologia , Bicamadas Lipídicas/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolipídeos/metabolismo , Cromatografia em Gel , Diálise , Cinética , Potássio/metabolismoRESUMO
Size enlargement of dipalmitoyl phosphatidylcholine vesicles was greatly accelerated in the range of the phase-transition temperatures, when fatty acid concentration was above a threshold level ('critical' concentration). This 'critical' concentration varied with the length of the fatty acid chain. The size enlargement process had second-order kinetics dependent on the vesicle concentration. Alkaline pH and low ionic strength inhibited the rate of size enlargement. Phospholipid exchange between dimyristoyl and dipalmitoyl phosphatidyl-choline vesicles increased abruptly above a 'critical' fatty acid concentration. The donor vesicles were those vesicles in which fatty acids reached the 'critical' concentration. The phospholipid exchange occurred both in fluid- and in solidstate vesicles. The 'critical' fatty acid concentration accelerating the phospholipid exchange process was lower than that accelerating the size enlargement process. The phospholipid exchange process explained in terms of a diminished hydrophobic attraction among the phospholipid molecules of the bilayer occurs via a free phospholipid molecule transfer through the aqueous phase. The size enlargement process is interpreted in terms of high fatty acid concentration in the membrane fluid domains. The membrane structure is locally perturbed inducing vesicle sticking after collision.
Assuntos
Ácidos Graxos/metabolismo , Bicamadas Lipídicas/metabolismo , Fosfolipídeos/metabolismo , Cinética , TemperaturaRESUMO
Mitochondria from a variety of sources possess a regulated inner membrane channel, the permeability transition pore (MTP), which is responsible for the 'permeability transition', a sudden permeability increase to solutes with molecular masses < or = 1500 Da, most easily observed after Ca2+ accumulation. The MTP is a voltage-dependent channel blocked by cyclosporin A with Ki in the nanomolar range. The MTP open probability is regulated by both the membrane potential and matrix pH. The probability of pore opening increases as the membrane is depolarized, while it decreases as matrix pH is decreased below 7.3 through reversible protonation of histidine residues. Many physiological and pathological effectors, including Ca2+ and ADP, modulate MTP operation directly through changes of the gating potential rather than indirectly through changes of the membrane potential (Petronilli, V., Cola, C., Massari, S., Colonna, R. and Bernardi, P. (1993) J. Biol. Chem. 268, 21939-21945). Here we present recent work from our laboratory indicating that (i) the voltage sensor comprises at least two vicinal thiols whose oxidation-reduction state affects the MTP gating potential; as the couple becomes more oxidized the gating potential increases; conversely, as it becomes more reduced the gating potential decreases; (ii) that MTP opening is fully reversible, as mitochondria maintain volume homeostasis through several cycles of pore opening/closure; and (iii) that the mechanism of MTP inhibition by cyclosporin A presumably involves a mitochondrial cyclophilin but does not utilize a calcineurin-dependent pathway.
Assuntos
Ciclosporina/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Isomerases de Aminoácido/fisiologia , Animais , Arsenitos/farmacologia , Cálcio , Proteínas de Transporte/fisiologia , Ácido Egtázico , Canais Iônicos/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Peptidilprolil Isomerase , Permeabilidade/efeitos dos fármacos , Ratos , Transdução de SinaisRESUMO
We have studied the rate of membrane fusion, the lipid dynamics and order and the membrane permeability of phosphatidylcholine vesicles as a function of pH. Acidification induced very different effects depending on the state of the bilayer. In liquid-crystalline bilayers, acidification decreased the rate of membrane fusion, the acyl chain motion and disorder and the rate of K+ release, whereas in solid bilayers acidification increased the rate of membrane fusion, the lipid acyl chain disorder and the rate of K+ release. These pH-dependent modifications are interpreted in terms of conformational and/or packing changes of the phosphatidylcholine head group in the membrane. In solid bilayers, these changes are not easily accommodated by the rigid structure, and the resulting stress leads to an unstable bilayer.
Assuntos
Fusão Celular , Permeabilidade da Membrana Celular , Metabolismo dos Lipídeos , Fosfatidilcolinas/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Polarização de Fluorescência , Concentração de Íons de Hidrogênio , Potássio/metabolismo , Temperatura , Valinomicina/farmacologiaRESUMO
The interaction of diphtheria toxin and its enzymatically deficient mutants crm 176 and crm 197 with liposomes has been studied by turbidity measurement and hydrophobic photolabelling with photoactivatable phosphatidylcholines. Diphtheria toxin and crm 176 at neutral pH bind to the surface of lipid bilayers while crm 197 also appears to interact with the fatty acid chains of phospholipids. All proteins undergo a change in conformation over the same range of acidic pH and become able to insert in the lipid bilayer. The tighter lipid interaction of crm 197 may account for its higher cell association constant. The possibility is discussed that the binding of diphtheria toxin to cells is mediated by both a protein receptor and an interaction with the head group of phospholipids.
Assuntos
Toxina Diftérica/genética , Metabolismo dos Lipídeos , Receptores de Superfície Celular , Receptores Colinérgicos/metabolismo , Toxina Diftérica/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Concentração de Íons de Hidrogênio , Peptídeos e Proteínas de Sinalização Intercelular , Lipossomos , Nefelometria e Turbidimetria , Fosfolipídeos/metabolismoRESUMO
This paper reports an investigation on the effects of the bipyridylium herbicide, paraquat, on rat liver mitochondria in vitro. We show that paraquat induces a Ca(2+)-dependent permeability increase of the inner mitochondrial membrane leading to membrane depolarization, uncoupling and matrix swelling. The permeability increase is not observed in the absence of Ca2+ accumulation, and is not due to a direct effect of paraquat on the membrane energy level, as assessed by measurements of membrane potential, respiration and mitochondrial permeability to solutes at high concentrations of paraquat in the presence of excess ethylene-bis(oxoethylenenitrilo)tetraacetic acid (EGTA), a Ca2+ chelator. The Ca(2+)-dependent permeability increase is due to inappropriate opening of the endogenous permeability transition pore (MTP), a regulated, voltage-dependent channel of the inner mitochondrial membrane. The pore is primarily affected by paraquat through a shift of the gating potential to more negative values, allowing pore opening at physiological membrane potential. This effect apparently involves oxidation of a critical dithiol in the pore voltage sensor, while other regulatory aspects of the MTP (matrix pH and Ca2+) are unaffected by paraquat, which is not transported inside the mitochondrial matrix. The effects of paraquat on MTP opening depend on inhibition of electron transfer at Site I by rotenone, or by respiratory chain inhibition by nitric oxide, one of the proposed endogenous mediators of paraquat toxicity to the lung (Berisha, H.I., Hedayatollah, P., Absood, A., and Said, S.I. (1994) Proc. Natl. Acad. Sci. USA 91, 7445-7449). Taken together, these data provide an additional biochemical mechanism by which paraquat may affect cell function, and support the idea that mitochondrial damage is an important determinant in paraquat toxicity (Hirai, K.-I., Ikeda, K., and Wang, G.-Y. (1992) Toxicology 72, 1-16).
Assuntos
Metabolismo Energético/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Dilatação Mitocondrial/efeitos dos fármacos , Paraquat/toxicidade , Animais , Ciclosporina/farmacologia , Sinergismo Farmacológico , Homeostase/efeitos dos fármacos , Técnicas In Vitro , Membranas Intracelulares/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/fisiologia , Óxido Nítrico/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Polarografia , Ratos , Ratos WistarRESUMO
To investigate the role of membrane proteins in the fusion process, linear hydrophobic polypeptide gramicidin was used as fusogenic agent in small unilamellar vesicles (SUV) constituted of saturated lecithins. It was found that gramicidin, externally added to a suspension of vesicles, induces a reversible vesicles aggregation. When incorporated into the bilayer, gramicidin induces increase in vesicle size. The vesicle size increase was monitored by column chromatography and transmission electron microscopy. The process of vesicle size increase occurs only when the lipid membrane is in the gel state. A maximum is observed in the kinetics at a temperature of approx. 25 degrees C lower than the phase transition temperature of lipids. Higher rates of vesicle size increase are obtained as the lipid chain length increases. The process is accompanied by a release of internal vesicle content and by membrane lipid mixing.
Assuntos
Gramicidina , Bicamadas Lipídicas , Encéfalo , Dimiristoilfosfatidilcolina , Cinética , Microscopia Eletrônica , Modelos Biológicos , Conformação Molecular , Fosfatidilcolinas , Fosfatidilserinas , Surfactantes Pulmonares , Relação Estrutura-AtividadeRESUMO
The involvement of mitochondrial permeability transition pore (MTP) in cellular processes is generally investigated by indirect means, such as changes in mitochondrial membrane potential or pharmacological inhibition. However, such effects could not be related univocally to MTP. In addition, source of errors could be represented by the increased retention of membrane potential probes induced by cyclosporin A (CsA) and the interactions between fluorescent probes. We developed a direct technique for monitoring MTP. Cells were co-loaded with calcein-AM and CoCl2, resulting in the quenching of the cytosolic signal without affecting the mitochondrial fluorescence. MTP inducers caused a rapid decrease in mitochondrial calcein fluorescence which, however, was not completely prevented by CsA. Besides the large and rapid efflux of calcein induced by MTP agonists, we also observed a constant and spontaneous decrease of mitochondrial calcein which was completely prevented by CsA. Thus, MTP likely fluctuates between open and closed states in intact cells.
Assuntos
Permeabilidade da Membrana Celular , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cobalto/metabolismo , Ciclosporina/farmacologia , Fluoresceínas/metabolismo , Corantes Fluorescentes , Humanos , Microscopia Confocal , Mitocôndrias/efeitos dos fármacosRESUMO
This review summarizes recent work on the regulation of the permeability transition pore, a cyclosporin A-sensitive mitochondrial channel that may play a role in intracellular calcium homeostasis and in a variety of forms of cell death. The basic bioenergetics aspects of pore modulation are discussed, with some emphasis on the links between oxidative stress and pore dysregulation as a potential cause of mitochondrial dysfunction that may be relevant to cell injury.