RESUMO
The oxidation of the cellular membrane through lipid peroxidation (LPO) is linked to aging and disease. Despite the physiological importance, the chemical mechanisms underlying LPO and oxidative reactions in membranes in general remain incompletely understood, and challenges exist in translating LPO inhibitor efficacies from in vitro to in vivo. The complexity of LPO, including multiple oxidation reactions in complex membrane environments and the difficulty in quantifying reaction kinetics, underlies these difficulties. In this work, we developed a robust and straightforward method for quantifying the oxidation rate kinetics of fluorescent molecules and determined the oxidation kinetics of widely fluorophores used as indicators of membrane LPO, diphenylhexatriene (DPH), BODIPY-C11, and Liperfluo. The measurement is initiated by lipoxygenase, which provides chemical specificity and enables a straightforward interpretation of oxidation kinetics. Our results reveal that the membrane composition significantly impacts the observed kinetics oxidation in DPH and BODIPY-C11 but not Liperfluo. Reaction mechanisms for their lipid peroxide-induced oxidation are proposed. This work provides a foundation for the quantitative analysis of LPO with fluorescence and extricating the complexity of oxidation reactions within membranes.
Assuntos
Compostos de Boro , Corantes Fluorescentes , Peroxidação de Lipídeos , Oxirredução , Cinética , Corantes Fluorescentes/química , Compostos de Boro/química , Membrana Celular/metabolismo , Difenilexatrieno/química , Difenilexatrieno/análogos & derivados , Lipídeos de Membrana/metabolismo , Lipídeos de Membrana/química , Humanos , Lipoxigenase/metabolismoRESUMO
This work intends to describe the physical properties of red blood cell (RBC) membranes in obese adults. The hypothesis driving this research is that obesity, in addition to increasing the amount of body fat, will also modify the lipid composition of membranes in cells other than adipocytes. Forty-nine control volunteers (16 male, 33 female, BMI 21.8 ± 5.6 and 21.5 ± 4.2 kg/m2, respectively) and 52 obese subjects (16 male and 36 female, BMI 38.2± 11.0 and 40.7 ± 8.7 kg/m2, respectively) were examined. The two physical techniques applied were atomic force microscopy (AFM) in the force spectroscopy mode, which allows the micromechanical measurement of penetration forces, and fluorescence anisotropy of trimethylammonium diphenylhexatriene (TMA-DPH), which provides information on lipid order at the membrane polar-nonpolar interface. These techniques, in combination with lipidomic studies, revealed a decreased rigidity in the interfacial region of the RBC membranes of obese as compared to control patients, related to parallel changes in lipid composition. Lipidomic data show an increase in the cholesterol/phospholipid mole ratio and a decrease in sphingomyelin contents in obese membranes. ω-3 fatty acids (e.g., docosahexaenoic acid) appear to be less prevalent in obese patient RBCs, and this is the case for both the global fatty acid distribution and for the individual major lipids in the membrane phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS). Moreover, some ω-6 fatty acids (e.g., arachidonic acid) are increased in obese patient RBCs. The switch from ω-3 to ω-6 lipids in obese subjects could be a major factor explaining the higher interfacial fluidity in obese patient RBC membranes.
Assuntos
Difenilexatrieno/análogos & derivados , Membrana Eritrocítica/fisiologia , Lipidômica/métodos , Obesidade/diagnóstico por imagem , Adolescente , Adulto , Fenômenos Biomecânicos , Estudos de Casos e Controles , Difenilexatrieno/administração & dosagem , Membrana Eritrocítica/metabolismo , Feminino , Polarização de Fluorescência , Humanos , Masculino , Microscopia de Força Atômica , Pessoa de Meia-Idade , Obesidade/metabolismo , Obesidade/fisiopatologia , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Adulto JovemRESUMO
Amyloid-like structure formation by various metabolites represents a significant extension of the amyloidogenic building block family. Similar to protein amyloids, metabolite amyloids induce apoptotic toxicity, a process that was linked to membrane association. Here, we demonstrate that metabolite amyloids interact with model membranes and study the mechanism by molecular dynamics.
Assuntos
Adenina/metabolismo , Bicamadas Lipídicas/metabolismo , Substâncias Macromoleculares/metabolismo , Triptofano/metabolismo , Tirosina/metabolismo , Adenina/química , Adenina/toxicidade , Alanina/química , Anisotropia , Difenilexatrieno/análogos & derivados , Difenilexatrieno/química , Fluorescência , Células HEK293 , Humanos , Ligação de Hidrogênio , Bicamadas Lipídicas/química , Substâncias Macromoleculares/química , Substâncias Macromoleculares/toxicidade , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Polímero Poliacetilênico , Polímeros/química , Polímeros/metabolismo , Poli-Inos/química , Poli-Inos/metabolismo , Triptofano/química , Triptofano/toxicidade , Tirosina/química , Tirosina/toxicidadeRESUMO
The present study aimed to evaluate the anti-melanogenic activity of 1,6-diphenyl-1,3,5-hexatriene and its derivatives in B16F10 murine melanoma cells and zebrafish embryos. Twenty five (1E,3E,5E)-1,6-bis(substituted phenyl)hexa-1,3,5-triene analogs were synthesized and their non-cytotoxic effects were predictively analyzed using three-dimensional quantitative structure-activity relationship approach. Inhibitory activities of these synthetic compounds against melanin synthesis were determined by evaluating melanin content and melanogenic regulatory enzyme expression in B16F10 cells. The anti-melanogenic activity was verified by observing body pigmentation in zebrafishes treated with these compounds. Compound #2, #4, and #6 effectively decreased melanogenesis induced by α-melanocyte-stimulating hormone. In particular, compound #2 remarkably lowered the mRNA and protein expression levels of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and TYRP2 in B16F10 cells and substantially reduced skin pigmentation in the developed larvae of zebrafish. These findings suggest that compound #2 may be used as an anti-melanogenic agent for cosmetic purpose.
Assuntos
Difenilexatrieno/análogos & derivados , Melaninas/biossíntese , Melanócitos/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Difenilexatrieno/farmacologia , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Hormônios Estimuladores de Melanócitos/farmacologia , Melanócitos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Pigmentação da Pele/efeitos dos fármacos , Peixe-ZebraRESUMO
Quercetin (Q) was used as substrate for regioselective glycosylation at the C-7 position catalyzed by Beauveria bassiana AM278 strain. As a result the glycoside quercetin 7-O-ß-d-(4â³-O-methyl)glucopyranoside (Q 7-MeGlu) was formed. The goal of the studies was to determine the anti-oxidative (liposome membrane protection against free radicals IC50Q 7-MeGlu = 5.47 and IC50Q = 4.49 µM) and anti-inflammatory (COX-1 and COX-2 enzymes activity inhibition) properties of Q 7-MeGlu as compared to Q. Every attempt was made to clarify the antioxidant activity of these molecules, which are able to interact with egg phosphatidylcholine liposomes, using a fluorometric method (by applying the probes MC540, TMA-DPH and DPH). The results indicated that Q 7-MeGlu and Q are responsible for increasing the packing order, mainly in the hydrophilic but also in hydrophobic regions of the membrane (Q > Q 7-MeGlu). These observations, confirmed by a ¹H-NMR method, are key to understanding their antioxidant activity which is probably caused by the stabilizing effect on the lipid membranes. The results showed that Q 7-MeGlu and Q have ability to quench the human serum albumin (HSA) intrinsic fluorescence through a static quenching mechanism. The results of thermodynamic parameters indicated that the process of formation complexes between studied molecules and HSA was spontaneous and caused through Van der Waals interactions and hydrogen bonding.
Assuntos
Antioxidantes/química , Beauveria/metabolismo , Glicosídeos/química , Lipossomos/química , Quercetina/química , Antioxidantes/isolamento & purificação , Beauveria/química , Beauveria/crescimento & desenvolvimento , Meios de Cultura/química , Ciclo-Oxigenase 1/química , Ciclo-Oxigenase 2/química , Di-Hidropiridinas/química , Difenilexatrieno/análogos & derivados , Difenilexatrieno/química , Corantes Fluorescentes/química , Glicosídeos/isolamento & purificação , Glicosilação , Humanos , Fosfatidilcolinas/química , Quercetina/isolamento & purificação , Albumina Sérica Humana/química , Soluções , EstereoisomerismoRESUMO
The production of pork with moderate amounts of intramuscular fat (IMF) without an increase in subcutaneous fat is highly desirable for the meat industry. Several studies indicate that dietary protein reduction during the growing-finishing period of pigs enhances IMF content, but its consequence on carcass fat deposition is still contradictory. In this study, we hypothesized that the effects of reduced protein diets (RPD), corrected or not with the limiting amino acid lysine, on subcutaneous fat deposition from pigs with distinct genotypes are mediated by adipose membranes biophysical properties. In total, 36 crossbred (Large White×Landrace×Pietrain - a lean genotype) and purebred (Alentejana breed - a fatty genotype) male pigs were randomly assigned to the control group, the RPD group or the reduced protein diet equilibrated for lysine (RPDL) group, allowing a 2×3 factorial arrangement (n=6). Backfat thickness and total fatty acid content were higher in Alentejana relative to crossbred pigs. Although dietary treatments did not change backfat thickness, RPD and RPDL increased total fatty acids content of subcutaneous fat. In order to understand this effect, adipose tissue membranes isolated from pig's subcutaneous fat were assayed for glycerol permeability and fluidity, using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-(trimethylamino)-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) probes. The glycerol transport across adipose membranes was not mediated by aquaglyceroporins and remained unchanged across dietary groups. Regardless of lysine correction, RPD increased membrane fluidity at the hydrocarbon region (lower DPH fluorescence anisotropy) in both genotypes of pigs. This result was associated with a lower ratio between oleic acid and linoleic acid on membrane's fatty acid composition. Adipose membrane's cholesterol content was independent from genotype and diet. Taken together, the present study shows that dietary protein reduction is successful in maintaining backfat thickness, although a negative side effect was observed on total fatty acids in subcutaneous fat, which may be due to changes in the fluidity of adipose membranes.
Assuntos
Dieta com Restrição de Proteínas/veterinária , Ácidos Graxos/análise , Fluidez de Membrana , Carne Vermelha/normas , Gordura Subcutânea/química , Suínos/fisiologia , Tecido Adiposo/metabolismo , Animais , Composição Corporal , Cruzamento , Proteínas Alimentares/administração & dosagem , Proteínas Alimentares/metabolismo , Difenilexatrieno/análogos & derivados , Lisina/metabolismo , Masculino , Obesidade/metabolismo , Gordura Subcutânea/metabolismoRESUMO
Fluorescence spectroscopy and microscopy have been utilized as tools in membrane biophysics for decades now. Because phospholipids are non-fluorescent, the use of extrinsic membrane probes in this context is commonplace. Among the latter, 1,6-diphenylhexatriene (DPH) and its trimethylammonium derivative (TMA-DPH) have been extensively used. It is widely believed that, owing to its additional charged group, TMA-DPH is anchored at the lipid/water interface and reports on a bilayer region that is distinct from that of the hydrophobic DPH. In this study, we employ atomistic MD simulations to characterize the behavior of DPH and TMA-DPH in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and POPC/cholesterol (4:1) bilayers. We show that although the dynamics of TMA-DPH in these membranes is noticeably more hindered than that of DPH, the location of the average fluorophore of TMA-DPH is only ~3-4Å more shallow than that of DPH. The hindrance observed in the translational and rotational motions of TMA-DPH compared to DPH is mainly not due to significant differences in depth, but to the favorable electrostatic interactions of the former with electronegative lipid atoms instead. By revealing detailed insights on the behavior of these two probes, our results are useful both in the interpretation of past work and in the planning of future experiments using them as membrane reporters.
Assuntos
Colesterol/química , Difenilexatrieno/análogos & derivados , Difenilexatrieno/química , Corantes Fluorescentes/química , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Fluorescência , Polarização de Fluorescência , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Fluidez de Membrana , Eletricidade Estática , Termodinâmica , Água/químicaRESUMO
The small intestinal brush border is a specialized cell membrane that needs to withstand the solubilizing effect of bile salts during assimilation of dietary nutrients and to achieve detergent resistance; it is highly enriched in glycolipids organized in lipid raft microdomains. In the present work, the fluorescent lipophilic probes FM 1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide), FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl)hexatrienyl)pyridinium dibromide), TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate), and CellMask Orange plasma membrane stain were used to study endocytosis from the enterocyte brush border of organ-cultured porcine mucosal explants. All the dyes readily incorporated into the brush border but were not detectably endocytosed by 5 min, indicating a slow uptake compared with other cell types. At later time points, FM 1-43 clearly appeared in distinct punctae in the terminal web region, previously shown to represent early endosomes (TWEEs). In contrast, the other dyes were relatively "endocytosis resistant" to varying degrees for periods up to 2 h, indicating an active sorting of lipids in the brush border prior to internalization. For some of the dyes, a diphenylhexatriene motif in the lipophilic tail seemed to confer the relative endocytosis resistance. Lipid sorting by selective endocytosis therefore may be a process in the enterocytes aimed to generate and maintain a unique lipid composition in the brush border.
Assuntos
Membrana Celular/metabolismo , Endocitose , Enterócitos/metabolismo , Corantes Fluorescentes/metabolismo , Jejuno/metabolismo , Metabolismo dos Lipídeos , Microscopia de Fluorescência , Animais , Membrana Celular/ultraestrutura , Difenilexatrieno/análogos & derivados , Difenilexatrieno/metabolismo , Enterócitos/ultraestrutura , Isoquinolinas/metabolismo , Jejuno/ultraestrutura , Cinética , Microvilosidades , Técnicas de Cultura de Órgãos , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , SuínosRESUMO
Secretory phospholipase A(2) exhibits much greater activity toward apoptotic versus healthy cells. Various plasma membrane changes responsible for this phenomenon have been proposed, including biophysical alterations described as "membrane fluidity" and "order." Understanding of these membrane perturbations was refined by applying studies with model membranes to fluorescence measurements during thapsigargin-induced apoptosis of S49 cells using probes specific for the plasma membrane: Patman and trimethylammonium-diphenylhexatriene. Alterations in emission properties of these probes corresponded with enhanced susceptibility of the cells to hydrolysis by secretory phospholipase A(2). By applying a quantitative model, additional information was extracted from the kinetics of Patman equilibration with the membrane. Taken together, these data suggested that the phospholipids of apoptotic membranes display greater spacing between adjacent headgroups, reduced interactions between neighboring lipid tails, and increased penetration of water among the heads. The phase transition of artificial bilayers was used to calibrate quantitatively the relationship between probe fluorescence and the energy of interlipid interactions. This analysis was applied to results from apoptotic cells to estimate the frequency with which phospholipids protrude sufficiently at the membrane surface to enter the enzyme's active site. The data suggested that this frequency increases 50-100-fold as membranes become susceptible to hydrolysis during apoptosis.
Assuntos
Apoptose , Fluidez de Membrana , Fosfolipases A2/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Biofísica/métodos , Calibragem , Domínio Catalítico , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Difenilexatrieno/análogos & derivados , Difenilexatrieno/química , Citometria de Fluxo/métodos , Humanos , Hidrólise , Lipídeos/química , Ácidos Palmíticos/química , Espectrometria de Fluorescência/métodos , Tapsigargina/química , Fatores de Tempo , Água/químicaRESUMO
Aluminum belongs to a group of potential toxic elements capable of penetrating the human body. In this paper, the effect of aluminum concentrations on red blood cell membranes using different fluorescent probes able to localize in various parts of the phospholipid bilayer (TMA-DPH, laurdan and pyrene) were studied. Our results confirm that human erythrocytes exposed to aluminum undergo physico-chemical modifications at the membrane level. A decrease in fluorescence anisotropy of TMA-DPH and in the polarity of the lipid bilayer with a concomitant shift toward a gel phase was observed, and the pyrene excimerization coefficient (kex) increased. Furthermore, the presence of aluminum induced lipid peroxidation and reduced the activity of erythrocyte antioxidant enzymes (SOD, CAT and GSHPx). Al-induced morphological changes on the erythrocyte membrane surface were monitored using atomic force microscopy. These results provide further information on the target of action of different aluminum amounts.
Assuntos
Compostos de Alumínio/farmacologia , Cloretos/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Adulto , Cloreto de Alumínio , Antioxidantes/metabolismo , Transporte Biológico/efeitos dos fármacos , Difenilexatrieno/análogos & derivados , Difenilexatrieno/metabolismo , Membrana Eritrocítica/ultraestrutura , Polarização de Fluorescência , Glutationa/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Microscopia de Força Atômica , Pessoa de Meia-IdadeRESUMO
Polyhexamethylene biguanide (PHMB) is a cationic biocide. The antibacterial mode of action of PHMB (at concentrations not exceeding its minimal inhibitory concentration) upon Listeria innocua LRGIA 01 was investigated by Fourier transformed infrared spectroscopy and fluorescence anisotropy analysis. Fourier transformed infrared spectra of bacteria treated with or without PHMB presented some differences in the lipids region: the CH(2)/CH(3) (2924 cm(-1)/2960 cm(-1)) band areas ratio significantly increased in the presence of PHMB. Since this ratio generally reflects membrane phospholipids and membrane microenvironment of the cells, these results suggest that PHMB molecules interact with membrane phospholipids and, thus, affect membrane fluidity and conformation. To assess the hypothesis of PHMB interaction with L. innocua membrane phospholipids and to clarify the PHMB mode of action, we performed fluorescence anisotropy experiments. Two probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and its derivative 1-[4-(trimethyl-amino)-phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), were used. DPH and TMA-DPH incorporate inside and at the surface of the cytoplasmic membrane, respectively. When PHMB was added, an increase of TMA-DPH fluorescence anisotropy was observed, but no changes of DPH fluorescence anisotropy occurred. These results are consistent with the hypothesis that PHMB molecules perturb L. innocua LRGIA 01 cytoplasmic membrane by interacting with the first layer of the membrane lipid bilayer.
Assuntos
Biguanidas/farmacologia , Membrana Celular/efeitos dos fármacos , Desinfetantes/farmacologia , Listeria/efeitos dos fármacos , Fluidez de Membrana/efeitos dos fármacos , Membrana Celular/química , Difenilexatrieno/análogos & derivados , Difenilexatrieno/química , Polarização de Fluorescência , Bicamadas Lipídicas/análise , Bicamadas Lipídicas/química , Fosfolipídeos/análise , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The interaction of positively-charged antibiotic gentamicin with cell membranes was studied to determine if any changes in membrane organization were induced by the drug. Opossum kidney epithelia (OK) cells were used as models of eukaryotic cells. Two methods were used: laurdan fluorescence spectroscopy and fluorescence anisotropy recordings on 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) labeled cell suspensions. Both methods showed an altered membrane hydration and fluidity of gentamicin treated cells. Liposomes prepared from dimyristoyl-phosphatidylcholine (DMPC) mixed with cardiolipin, which mimics the heterogeneous charge composition of the natural cell membrane, were used to determine the effect of gentamicin on artificial bilayers. The membrane lipid packing as revealed by generalized polarization (GP) and fluorescence anizotropy variation with increasing temperature was studied. It was found that the generalized polarization of liposomal membranes containing a negatively charged lipid (cardiolipin) is higher in the presence of gentamicin; in the membrane of living cell (OK), gentamicin induces, on the contrary, a decrease of general polarization. Considering the role of membrane organization in the function of transmembrane channels and receptors, our findings suggest hypotheses that may explain the permeation of gentamicin through the living cell membrane by using these channels.
Assuntos
Membrana Celular/química , Células Epiteliais/química , Gentamicinas/química , Rim/química , Lipossomos/química , Lipídeos de Membrana/química , 2-Naftilamina/análogos & derivados , Animais , Transporte Biológico , Cardiolipinas/química , Membrana Celular/metabolismo , Dimiristoilfosfatidilcolina/química , Difenilexatrieno/análogos & derivados , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Polarização de Fluorescência , Corantes Fluorescentes , Rim/citologia , Rim/metabolismo , Lauratos , Membranas Artificiais , Gambás , Poliaminas/química , Polieletrólitos , Espectrometria de Fluorescência , Eletricidade EstáticaRESUMO
Human-ß-defensins HBD-1-3 are important components of the innate immune system. Synthetic peptides Phd-1-3 with a single disulphide bond, spanning the cationic C-terminal region of HBD-1-3, have antimicrobial activity. The interaction of Phd-1-3 with model membranes was investigated using isothermal titration calorimetry (ITC) and steady-state fluorescence polarization to understand the biophysical basis for the mechanism of antimicrobial action. Calorimetric titration of POPE:POPG (7:3) vesicles with peptides at 25°C and 37°C showed complex profiles with two distinct regions of heat changes. The data indicate binding of Phd-1-3 at 37°C to both negative and zwitterionic lipid vesicles is exothermic with low enthalpy values (ΔH~-1.3 to -2.8kcal/mol) as compared to amphipathic helical antibacterial peptides. The adsorption of peptides to negatively charged lipid membranes is modulated by electrostatic interactions that are described by surface partition equilibrium model using Gouy-Chapman theory. However, this model could not explain the isotherms of peptide binding to zwitterionic lipid vesicles. Fluorescence polarization of TMA-DPH (1-[4-(trimethylammonio) phenyl]-6-phenyl-1,3,5-hexatriene) and DPH (1,6-diphenyl-1,3,5-hexatriene) located in the head group and acyl chain region respectively, indicates that the peptides interact with interfacial region of negatively charged membranes. Based on the results obtained, we conclude that adsorption of cationic peptides Phd-1-3 on lipid surface do not result in conformational change or pore formation. It is proposed that interaction of Phd-1-3 with the negatively charged lipid head group causes membrane destabilization, which in turn affects the efficient functioning of cytoplasmic membrane proteins in bacteria, resulting in cell death.
Assuntos
Membranas Artificiais , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , beta-Defensinas/química , Adsorção , Bactérias/química , Calorimetria Indireta/métodos , Di-Hidropiridinas/química , Difenilexatrieno/análogos & derivados , Difenilexatrieno/química , Polarização de Fluorescência/métodos , HumanosRESUMO
The main function of background two-pore potassium (K(2P)) channels KCNK3/9/15 is to stabilize the cell membrane potential. We previously observed that membrane potential depolarization enhances the release of HIV-1 viruses. Because membrane polarization affects the biomembrane directly, here we examined the effects of KCNK3/9/15 on the budding of nonviral vesicles. We found that depolarization by knocking down endogenous KCNK3/9/15 promoted secretion of cell-derived vesicles. We further used Vpu (an antagonist of KCNK3) as a model for the in vivo study of depolarization-stimulated secretion. Vpu is a HIV-1-encoded, ion channel-like protein (viroporin) capable of enhancing virus release and depolarizing the cell membrane potential. We found that Vpu could also promote nonviral vesicle release, perhaps through a similar mechanism that Vpu utilizes to promote viral particle release. Notably, T cells expressing Vpu alone became pathologically low in intracellular K(+) and insensitive to extracellular K(+) or membrane potential stimulation. In contrast, heterologous expression of KCNK3 in T cells stabilized the cell potentials by maintaining intracellular K(+). We thus concluded that KCNK3/9/15 expression limits membrane depolarization and depolarization-induced secretion at least in part by maintaining intracellular K(+).
Assuntos
Membrana Celular/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/virologia , Difenilexatrieno/análogos & derivados , Difenilexatrieno/metabolismo , Exocitose/efeitos dos fármacos , HIV-1/fisiologia , Células HeLa , Proteínas do Vírus da Imunodeficiência Humana/farmacologia , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Espaço Intracelular/virologia , Células K562 , Potenciais da Membrana/efeitos dos fármacos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Potássio/metabolismo , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidoresRESUMO
INTRODUCTION TMA-DPH (trimethylamine-diphenylhexatriene) is a fluorescent membrane probe that has classically been used to label the outer leaflet of a membrane bilayer, to label the outer leaflet of the plasma membrane in cells, and to report on membrane dynamics using the techniques of fluorescence polarization and/or fluorescence lifetime. This probe has also been used to follow exocytosis and endocytosis of labeled plasma membranes. The interaction of the aqueous environment with mitochondrial inner membrane dynamics has also been studied following the fluorescence polarization and the lifetime of TMA-DPH. This protocol describes the use of TMA-DPH to label the plasma membrane.
Assuntos
Membrana Celular/metabolismo , Técnicas Citológicas/métodos , Difenilexatrieno/análogos & derivados , Coloração e Rotulagem/métodos , Difenilexatrieno/metabolismo , FluorescênciaRESUMO
BACKGROUND: The involvement of platelets in the pathogenesis of diabetic vascular complications is supported by several studies. Type 1 diabetic (T1D) platelets show increased adhesiveness and aggregation related to a modification of nitric oxide synthase activity. Moreover, different cell types from diabetic patients showed a decreased membrane Na(+) /K(+) -ATPase activity, which might be involved in diabetic complications. The aim of this study was to investigate whether T1D at onset is able to induce alterations of platelet physicochemical and functional properties and whether these changes are affected by hyperglycaemia. METHODS: The study was performed on 50 young subjects: 30 patients (1-14 years) affected by T1D and 20 age- and gender-matched healthy subjects. We analyzed platelet membrane fluidity by fluorescent anisotropy of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene and 1,6-diphenyl-1,3,5-hexatriene, Na(+) /K(+) -ATPase, nitric oxide, and peroxynitrite production. RESULTS: In T1D subjects, we observed an increased fluidity in the plasma membrane outer part and greater rigidity in the internal part compared with that in controls. Na(+) /K(+) -ATPase activity and nitric oxide levels were significantly reduced, while peroxynitrite production was increased compared with that in controls. Moreover, correlations found between the above parameters were correlated with fasting glycaemia and haemoglobin A(1c). CONCLUSIONS: T1D patients exhibit structural and functional modifications of platelet membrane properties and alterations of nitric oxide metabolism due to diabetes per se and not to chronic hyperglycaemia, insulin therapy, or ageing. These results support the hypothesis that oxidative attack could be an important early event in the pathogenesis of diabetic complications.
Assuntos
Plaquetas/fisiologia , Doenças Cardiovasculares/etiologia , Diabetes Mellitus Tipo 1/sangue , Adolescente , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/complicações , Difenilexatrieno/análogos & derivados , Feminino , Polarização de Fluorescência , Humanos , Lactente , Masculino , Fluidez de Membrana , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/farmacologia , Risco , ATPase Trocadora de Sódio-Potássio/sangueRESUMO
In this report, we describe an effect of photodynamic therapy (PDT) on membrane trafficking in murine 1c1c7 hepatoma cells. A brief exposure of 1c1c7 cells to a 20 nM concentration of the phosphatidylinositol kinase class-3 antagonist wortmannin led to the rapid appearance of cytoplasmic vacuoles. Fluorescence monitoring of plasma membrane-associated 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TDPH) over time demonstrated that the wortmannin-induced vacuoles were derived from endocytosed plasma membrane. Low-dose photodamage catalyzed by the lysosomal photosensitizer NPe6, prior to the addition of wortmannin, prevented formation of these vacuoles. NPe6 was found to suppress for several hours the normal trafficking of TDPH-labeled plasma membrane to the cytosol, and the formation of punctate TDPH-labeled cytoplasmic vesicles. The ability of NPe6-induced photodamage to suppress wortmannin-induced vacuolization occurred under conditions that did not disrupt lysosomes and were at or below the threshold of cytostatic/cytotoxic effects. Furthermore, the suppressive effects of NPe6-PDT were not prevented by inclusion of an agent that stabilized lysosomal membranes, or by E64d, an inhibitor of lysosomal cathepsin proteases. Mitochondrial photodamage was less effective at preventing wortmannin-induced vacuole formation and PDT directed against the ER had no effect. The role of photodamage to the endocytic pathway may be a hitherto unexplored effect on cells that selectively accumulate photosensitizing agents. These results indicate that photodamage directed against endosomes/lysosomes has effects independent of the release of lysosomal proteases.
Assuntos
Endocitose/efeitos da radiação , Fármacos Fotossensibilizantes/farmacologia , Androstadienos/farmacologia , Animais , Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Difenilexatrieno/análogos & derivados , Difenilexatrieno/química , Endossomos/efeitos dos fármacos , Endossomos/efeitos da radiação , Corantes Fluorescentes/química , Leucina/análogos & derivados , Leucina/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Lisossomos/efeitos da radiação , Camundongos , Fotoquimioterapia , Porfirinas/toxicidade , Fatores de Tempo , Vacúolos/metabolismo , WortmaninaRESUMO
Herbal remedies containing root extracts of Panax ginseng are commonly used for complementary or alternative therapies. Ginsenosides, the major components of root extracts, are responsible for ginseng's pharmacological and biological effects; however, their mechanisms of action are unclear. We examined whether membrane cholesterol was involved in the mechanism of action of ginsenoside Rh2 in cultured cells. In B16 melanoma cells, Rh2 (18.5 µM) induced dendrite formation within 2 h. Depletion of cholesterol by pretreatment with 10 mM methyl-ß-cyclodextrin suppressed this effect of Rh2. Rh2 did not change the cellular cholesterol content and the immunofluorescence staining pattern of the lipid-raft-associated molecules, ganglioside GM3, Caveolin-1, Flotillin-1, and Flotillin-2, for up to 3 or 6 h. However, within 2 min of addition, Rh2 changed the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) but not of 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). DPH is more sensitive than TMA-DPH to changes in the physical properties of membrane lipid bilayers regulated by cholesterol. These results suggest that Rh2 affects the physical properties of cholesterol-regulated membrane lipid bilayers and could lead to changes in cellular functions.
Assuntos
Colesterol/metabolismo , Dendritos/efeitos dos fármacos , Ginsenosídeos/farmacologia , Melanoma/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Dendritos/metabolismo , Difenilexatrieno/análogos & derivados , Difenilexatrieno/química , Imunoensaio de Fluorescência por Polarização , Gangliosídeos/metabolismo , Melanoma/patologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , CamundongosRESUMO
The present study aimed to investigate the role played by the leaflets of the plasma membrane in the uptake of drugs into cells and in their extrusion by P-glycoprotein and multidrug resistance-associated protein 1. Drug accumulation was monitored by fluorescence resonance energy transfer from trimethylammonium-diphenyl-hexatriene (TMA-DPH) located at the outer leaflet to a rhodamine analog. Uptake of dye into cells whose mitochondria had been inactivated was displayed as two phases of TMA-DPH fluorescence quenching. The initial phase comprised a rapid drop in fluorescence that was neither affected by cooling the cells on ice, nor by activity of mitochondria or ABC transporters. This phase reflects the association of dye with the outer leaflet of the plasma membrane. The subsequent phase of TMA-DPH fluorescence quenching occurred in drug-sensitive cell lines with a half-life in the range 20-40 s. The second phase of fluorescence quenching was abolished by incubation of the cells on ice and was transiently inhibited in cells with active mitochondria. Thus, the second phase of fluorescence quenching reflects the accumulation of dye in the cytoplasmic leaflet of the plasma membrane, presumably as a result of flip-flop of dye across the plasma membrane and slow diffusion from the inner leaflet into the cells. Whereas activity of P-glycoprotein prevented the second phase of fluorescence quenching, the activity of multidrug resistance-associated protein 1 had no effect on this phase. Thus, P-glycoprotein appears to pump rhodamines from the cytoplasmic leaflet either to the outer leaflet or to the outer medium.
Assuntos
Antineoplásicos/farmacologia , Membrana Celular , Difenilexatrieno/análogos & derivados , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Antineoplásicos/uso terapêutico , Transporte Biológico , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Difenilexatrieno/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Leucemia/tratamento farmacológico , Rodaminas/farmacologia , Espectrometria de FluorescênciaRESUMO
The fertilizing potential of human spermatozoa relies on their ability to capacitate as they travel through the female reproductive tract. During this process, cholesterol is released from the plasma membrane, altering its architecture and dynamics. Using ISolate gradients, we obtained high (L90)- and low (L45)-quality spermatozoa from asthenozoospermic human semen samples. We tested the hypothesis that the lower fertilizing ability of asthenozoospermic L90 cells could be related to a lower ability to increase their membrane fluidity during capacitation. We assessed two sets of fluorescent probes: (i) DPH, TMA-DPH and PA-DPH which senses the hydrophobic core, cytosolic and exofacial leaflets of the bilayer, respectively and (ii) Laurdan, sensitive to the amount of water molecules intercalated between lipid moieties of the membrane (membrane hydration). Before capacitation, membrane fluidity of asthenozoospermic sperm populations was similar to the corresponding fractions of normozoospermic cells when evaluated with DPH, TMA-DPH or PA-DPH. Asthenozoospermic whole samples displayed lower plasma membrane hydration than normozoospermic cells as evidenced with Laurdan. After capacitation, asthenozoospermic L45 and L90 cells failed to increase their membrane fluidity in opposition to normozoospermic cells. Interestingly, membrane hydration significantly correlated with the main sperm motion parameters analysed, being a low membrane hydration associated with poor sperm movement. These results show that low-motility spermatozoa are unable to respond to capacitation with the necessary changes in membrane fluidity. This defect in sperm plasma membrane rheology may be responsible for their poor functional quality and low fertilizing ability.