RESUMO
We studied biological effects of mannan, a polysaccharide immunomodulator from C. albicans, that interacts with mannose receptor in vivo. It is shown that preliminary administration of mannan (5 times in a dose of 50 mg/kg or 2 times in a dose of 100 mg/kg) to mice with acute lipemia induced by lipase inhibitor poloxamer 407 (300 mg/kg) reduces the serum concentrations of atherogenic LDL, cholesterol, and triglycerides. Administration of mannan to intact mice and animals with acute lipemia reduces triglyceride concentration and causes labilization of lysosomal membranes in the liver. Serum activity of chitotriosidase, a marker of macrophage activation, was elevated in mice with acute lipemia treated with mannan. Thus, mannan from C. albicans is a promising hypolipidemic polysaccharide compound, similar by its activity to ß-glycan, a component of LPS.
Assuntos
Polissacarídeos Fúngicos/farmacologia , Hiperlipidemias/tratamento farmacológico , Fígado/efeitos dos fármacos , Mananas/farmacologia , Poloxâmero/farmacologia , Animais , Candida albicans/química , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Polissacarídeos Fúngicos/isolamento & purificação , Hexosaminidases/sangue , Hiperlipidemias/sangue , Hiperlipidemias/induzido quimicamente , Hiperlipidemias/patologia , Injeções Intraperitoneais , Membranas Intracelulares/química , Membranas Intracelulares/efeitos dos fármacos , Fígado/metabolismo , Lisossomos/química , Lisossomos/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Mananas/isolamento & purificação , Camundongos , Camundongos Endogâmicos CBA , Triglicerídeos/sangueRESUMO
Recent studies demonstrated that lipids influence the assembly and efficiency of membrane-embedded macromolecular complexes. Similarly, lipids have been found to influence chloroplast precursor protein binding to the membrane surface and to be associated with the Translocon of the Outer membrane of Chloroplasts (TOC). We used a system based on chloroplast outer envelope vesicles from Pisum sativum to obtain an initial understanding of the influence of lipids on precursor protein translocation across the outer envelope. The ability of the model precursor proteins p(OE33)titin and pSSU to be recognized and translocated in this simplified system was investigated. We demonstrate that transport across the outer membrane can be observed in the absence of the inner envelope translocon. The translocation, however, was significantly slower than that observed for chloroplasts. Enrichment of outer envelope vesicles with different lipids natively found in chloroplast membranes altered the binding and transport behavior. Further, the results obtained using outer envelope vesicles were consistent with the results observed for the reconstituted isolated TOC complex. Based on both approaches we concluded that the lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylinositol (PI) increased TOC-mediated binding and import for both precursor proteins. In contrast, enrichment in digalactosyldiacylglycerol (DGDG) improved TOC-mediated binding for pSSU, but decreased import for both precursor proteins. Optimal import occurred only in a narrow concentration range of DGDG.
Assuntos
Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Membranas Intracelulares/metabolismo , Lipídeos/farmacologia , Precursores de Proteínas/metabolismo , Cloroplastos/efeitos dos fármacos , Corantes Fluorescentes/metabolismo , Galactolipídeos/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Pisum sativum/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ribulose-Bifosfato Carboxilase/metabolismo , Lipossomas Unilamelares/metabolismoRESUMO
BACKGROUND: Data from environmental exposure to nanoparticles (NPs) suggest that chronic exposure may increase the incidence of lung, cardiovascular and neurodegenerative diseases. Impairment of cell function by intracellular accumulation of NPs is also suspected. Many types of NPs have been detected in the endosomal-lysosomal system and, upon repeated exposure, alterations of the endosomal-lysosomal system may occur. To identify such effects we compared the effect of carboxyl polystyrene particles (CPS) of different sizes (20-500 nm) on lysosomes of the endothelial cell line EAhy926 after short (24h) and long (72h-96h) exposure times. Lysosomal localization of CPS, as well as lysosomal pH, lysosomal membrane integrity, morphology of the endosomal-lysosomal system and activities of the lysosomal enzymes,cathepsin B and sulfatases, upon exposure to CPS were recorded. RESULTS: CPS in sizes ≤100 nm showed high co-localization with lysosomes already after 4h, larger CPS after 24h. None of the particles at non-cytotoxic concentrations caused marked changes in lysosomal pH or destroyed lysosomal membrane integrity. At 24h of exposure, 20 nm CPS induced significant dilatation of the endosomal-lysosomal system and reduced activity of lysosomal sulfatases. After 72h, these alterations were less pronounced. CONCLUSIONS: Despite accumulation in lysosomes CPS induced only small changes in lysosomes. Upon longer contact, these changes are even less pronounced. The presented panel of assays may serve to identify effects on lysosomes also for other NPs.
Assuntos
Células Endoteliais/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Nanopartículas/toxicidade , Poliestirenos/toxicidade , Catepsina B/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/ultraestrutura , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Nanopartículas/química , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Poliestirenos/química , Fatores de TempoRESUMO
Fabry disease is an X-linked lysosomal storage disorder that leads to abnormal accumulation of glycosphingolipids due to a deficiency of alpha-galactosidase A (AGAL). The consequences of these alterations on the targeting of membrane proteins are poorly understood. Glycosphingolipids are enriched in Triton-X-100- resistant lipid rafts [detergent-resistant membranes (DRMs)] and play an important role in the transport of several membrane-associated proteins. Here, we show that In fibroblasts of patients suffering from Fabry disease, the colocalization of AGAL with the lysosomal marker LAMP2 is decreased compared with wild-type fibroblasts concomitant with a reduced transport of AGAL to lysosomes. Furthermore, overall composition of membrane lipids in the patients' fibroblasts as well as in DRMs reveals a substantial increase in the concentration of glycolipids and a slight reduction of phosphatidylethanolamine (PE). The altered glycolipid composition in Fabry fibroblasts is associated with an intracellular accumulation and impaired trafficking of the Triton-X-100 DRM-associated membrane glycoprotein dipeptidyl peptidase IV (DPPIV) in transfected Fabry cells, whereas no effect could be observed on the targeting of aminopeptidase N (ApN) that is not associated with this type of DRM. We propose that changes in the lipid composition of cell membranes in Fabry disease disturb the ordered Triton X-100 DRMs and have implications on the trafficking and sorting of DRM-associated proteins and the overall protein-lipid interaction at the cell membrane. Possible consequences could be altered signalling at the cell surface triggered by DRM-associated proteins, with implications on gene regulation and subsequent protein expression.
Assuntos
Detergentes/farmacologia , Dipeptidil Peptidase 4/metabolismo , Doença de Fabry/metabolismo , Proteínas de Membrana/metabolismo , Octoxinol/farmacologia , Técnicas de Cultura de Células , Dipeptidil Peptidase 4/genética , Doença de Fabry/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Complexo de Golgi/metabolismo , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/enzimologia , Proteínas de Membrana/genética , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Transporte Proteico/fisiologia , Esfingolipídeos/metabolismo , alfa-Galactosidase/genéticaRESUMO
Two different methods, stimulation of transport by fatty acyl-coenzyme A (CoA) and inhibition of transport by a nonhydrolyzable analogue of palmitoyl-CoA, reveal that fatty acylation is required to promote fusion of transport vesicles with Golgi cisternae. Specifically, fatty acyl-CoA is needed after the attachment of coated vesicles and subsequent uncoating of the vesicles, and after the binding of the NEM-sensitive fusion protein (NSF) to the membranes, but before the actual fusion event. We therefore suggest that an acylated transport component participates, directly or indirectly, in membrane fusion.
Assuntos
Acil Coenzima A/farmacologia , Proteínas de Transporte/metabolismo , Complexo de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Fusão de Membrana/efeitos dos fármacos , Organelas/metabolismo , Proteínas de Transporte Vesicular , Transporte Biológico , Sistema Livre de Células , Coenzima A/farmacologia , Detergentes/farmacologia , Etanol/farmacologia , Complexo de Golgi/efeitos dos fármacos , Membranas Intracelulares/efeitos dos fármacos , Cinética , Proteínas Sensíveis a N-Etilmaleimida , Octoxinol , Organelas/efeitos dos fármacos , Polietilenoglicóis/farmacologiaRESUMO
Lysosome-related organelles are cell type-specific intracellular compartments with distinct morphologies and functions. The molecular mechanisms governing the formation of their unique structural features are not known. Melanosomes and their precursors are lysosome-related organelles that are characterized morphologically by intralumenal fibrous striations upon which melanins are polymerized. The integral membrane protein Pmel17 is a component of the fibrils and can nucleate their formation in the absence of other pigment cell-specific proteins. Here, we show that formation of intralumenal fibrils requires cleavage of Pmel17 by a furin-like proprotein convertase (PC). As in the generation of amyloid, proper cleavage of Pmel17 liberates a lumenal domain fragment that becomes incorporated into the fibrils; longer Pmel17 fragments generated in the absence of PC activity are unable to form organized fibrils. Our results demonstrate that PC-dependent cleavage regulates melanosome biogenesis by controlling the fibrillogenic activity of a resident protein. Like the pathologic process of amyloidogenesis, the formation of other tissue-specific organelle structures may be similarly dependent on proteolytic activation of physiological fibrillogenic substrates.
Assuntos
Células Eucarióticas/enzimologia , Glicoproteínas/metabolismo , Melanossomas/enzimologia , Microfibrilas/enzimologia , Proteínas/metabolismo , Subtilisinas/metabolismo , Compartimento Celular/fisiologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Detergentes/farmacologia , Endossomos/metabolismo , Endossomos/ultraestrutura , Células Eucarióticas/ultraestrutura , Células HeLa , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Melaninas/metabolismo , Melanossomas/ultraestrutura , Glicoproteínas de Membrana , Microfibrilas/ultraestrutura , Microscopia Eletrônica , Octoxinol/farmacologia , Peptídeo Hidrolases/metabolismo , Pró-Proteína Convertases , Estrutura Terciária de Proteína/fisiologia , Solubilidade/efeitos dos fármacos , Antígeno gp100 de MelanomaRESUMO
Enhanced formation of reactive oxygen species (ROS), superoxide (O2*-), and hydrogen peroxide (H2O2) may result in either apoptosis or other forms of cell death. Here, we studied the mechanisms underlying activation of the apoptotic machinery by ROS. Exposure of permeabilized HepG2 cells to O2*- elicited rapid and massive cytochrome c release (CCR), whereas H2O2 failed to induce any release. Both O2*- and H2O2 promoted activation of the mitochondrial permeability transition pore by Ca2+, but Ca2+-dependent pore opening was not required for O2*--induced CCR. Furthermore, O2*- alone evoked CCR without damage of the inner mitochondrial membrane barrier, as mitochondrial membrane potential was sustained in the presence of extramitochondrial ATP. Strikingly, pretreatment of the cells with drugs or an antibody, which block the voltage-dependent anion channel (VDAC), prevented O2*--induced CCR. Furthermore, VDAC-reconstituted liposomes permeated cytochrome c after O2*- exposure, and this release was prevented by VDAC blocker. The proapoptotic protein, Bak, was not detected in HepG2 cells and O2*--induced CCR did not depend on Bax translocation to mitochondria. O2*--induced CCR was followed by caspase activation and execution of apoptosis. Thus, O2*- triggers apoptosis via VDAC-dependent permeabilization of the mitochondrial outer membrane without apparent contribution of proapoptotic Bcl-2 family proteins.
Assuntos
Grupo dos Citocromos c/metabolismo , Mitocôndrias/enzimologia , Porinas/metabolismo , Superóxidos/metabolismo , Trifosfato de Adenosina/metabolismo , Apoptose/fisiologia , Carcinoma Hepatocelular , Caspase 3 , Caspases/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Grupo dos Citocromos c/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/enzimologia , Lipossomos/metabolismo , Neoplasias Hepáticas , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Oxidantes/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transfecção , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/enzimologia , Canais de Ânion Dependentes de VoltagemRESUMO
Nosocomial infections have become a serious threat in our times and are getting more difficult to handle due to increasing development of resistances in bacteria. In this light, cold atmospheric plasma (CAP), which is known to effectively inactivate microorganisms, may be a promising alternative for application in the fields of dentistry and dermatology. CAPs are partly ionised gases, which operate at low temperature and are composed of electrons, ions, excited atoms and molecules, reactive oxygen and nitrogen species. In this study, the effect of CAP generated from ambient air was investigated against Enterococcus faecalis, grown on agar plates or as biofilms cultured for up to 72 h. CAP reduced the colony forming units (CFU) on agar plates by > 7 log10 steps. Treatment of 24 h old biofilms of E. faecalis resulted in CFU-reductions by ≥ 3 log10 steps after CAP treatment for 5 min and by ≥ 5 log10 steps after CAP treatment for 10 min. In biofilm experiments, chlorhexidine (CHX) and UVC radiation served as positive controls and were only slightly more effective than CAP. There was no damage of cytoplasmic membranes upon CAP treatment as shown by spectrometric measurements for release of nucleic acids. Thus, membrane damage seems not to be the primary mechanism of action for CAP towards E. faecalis. Overall, CAP showed pronounced antimicrobial efficacy against E. faecalis on agar plates as well as in biofilms similar to positive controls CHX or UVC.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/fisiologia , Plâncton/microbiologia , Gases em Plasma/farmacologia , Biofilmes/crescimento & desenvolvimento , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Enterococcus faecalis/citologia , Enterococcus faecalis/crescimento & desenvolvimento , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismoRESUMO
Recent developments in the field of fullerene C60 and its derivatives suggest its suitability in a wide range of applications ranging from photovoltaic instruments, development of solar based cells, cosmetics to enzyme inhibition treatment, and so on. These innovative applications raised possibilities of intentional or oblivious human-particle contact leading to possible deleterious effects on human health. The current study deals with the interaction of dextran functionalized fullerene C60 (Dex-C60) on Chinese Hamster Ovary cells. The results showed that the cell viability was not affected by Dex-C60 treatment even at higher concentrations. Treatment of Dex-C60 did not affect mitochondrial membrane potential and the integrity of lysosomal and cytoskeletal membrane. DNA ladder assay and nuclear staining showed that the DNA remains intact, and no fragmentation or nuclear condensation was visible. From flow cytometry analysis, the viable population of treated cells was seemed to be remaining similar to that of untreated cells. Hence, from the current result, it is concluded that Dex-C60 can be a potential candidate for various biomedical applications.
Assuntos
Células CHO/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Fulerenos/toxicidade , Polímeros/toxicidade , Animais , Cricetulus , DNA/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Membranas Intracelulares/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacosRESUMO
PURPOSE: This study examined the state of mitochondrial physiology subsequent to exposing lens epithelium to high ambient galactose (Gal), which upon conversion to galactitol (GalOH) and resultant intracellular accumulation thereof, leads to profound destabilization of mitochondrial membrane potential (Deltapsim). Further, we determined whether the aldose reductase (AR) inhibitor, Sorbinil, or estrogen (17beta-E2, and its isomer, 17alpha-E2, which exhibits marginal binding affinity for estrogen receptor), administered prior to and concomitant with Gal exposure might prevent or delay mitochondrial membrane depolarization. METHODS: Secondary cultures of bovine lens epithelial cells (BLECs), as well as a virally-transformed human lens epithelial cell line (HLE-B3), were maintained in 40 mM galactose (Gal) for up to seven days in the presence and absence of Sorbinil, 17beta-E2 or 17alpha-E2. Endogenous accumulation of reactive oxygen species (ROS) was assessed by loading cells with H2DCF-DA, which upon oxidation in the presence of ROS transitions to the fluorescent compound, DCF. To assess Deltapsim, confocal microscopy was employed in conjunction with the potentiometric dye, JC-1. Intracellular polyol content was determined by gas chromatography. Cells were monitored for apoptosis and necrosis as determined by annexin V-propidium iodide staining and visualized by confocal fluorescence microscopy. RESULTS: BLECs, more so than HLE-B3 cells, accumulate high intracellular levels of GalOH upon exposure to high ambient Gal. BLECs were significantly depolarized while HLE-B3 cells showed little depolarization over the same course of Gal exposure. The addition of either 17alpha-E2 or 17beta-E2 to BLECs, over a dose range of 0.01 microM to 1.0 microM, prevented mitochondrial membrane depolarization as did the addition of 0.1 mM Sorbinil. The polyol content in BLECs after 3 days of exposure to Gal was 282 nmol/mg protein. Co-addition of Sorbinil during the 3-day exposure period prevented any significant accumulation of GalOH. Co-administration of either isoform of estrogen did not block GalOH synthesis and the level of attained intracellular accumulation was similar to that of Gal alone. The observed accumulation of ROS from HLE-B3 cells subsequent to 3 days of Gal exposure was negligible and consistent with that of control cells maintained in physiological medium. Intracellular accumulation of ROS with 3-day, Gal-maintained BLECs, exhibited a marginal but statistically significant increase over control cells maintained in physiological medium (5.5 mM glucose) and similar levels of ROS were generated irrespective of the presence of estrogen with Gal. Bolus addition of 100 microM hydrogen peroxide to 3-day, Gal plus Sorbinil-maintained BLECs failed to induce a change in mitochondrial membrane potential. Evidence of apoptosis or necrosis was negligible through 7 days of sustained exposure to high ambient Gal. CONCLUSIONS: Polyol accumulation promotes mitochondrial membrane depolarization and the decrease in Deltapsim is prevented by prior addition and co-administration of Sorbinil or estrogen with Gal. Unlike Sorbinil, estrogens' mode of action is not via the inhibition of aldose reductase activity. The data supports the theory that with Gal plus estradiol-treated cells, at a given intracellular polyol load, a larger portion of the mitochondrial population retains Deltapsim, and hence continues to function relative to Gal-treated cells. Results with 17alpha-E2 indicate that maintaining Deltapsim, in the face of chronic polyol accumulation, is likely to be mediated via a nuclear estrogen receptor-independent mechanism. The failure of supraphysiological levels of hydrogen peroxide added to Gal plus Sorbinil-maintained BLECs to depolarize mitochondria indicates that polyol accumulation, not ROS generation, is the causative factor responsible for the loss of mitochondrial membrane potential.
Assuntos
Estradiol/farmacologia , Cristalino/efeitos dos fármacos , Cristalino/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Polímeros/metabolismo , Aldeído Redutase/antagonistas & inibidores , Animais , Apoptose , Benzimidazóis , Carbocianinas , Bovinos , Células Cultivadas , Sinergismo Farmacológico , Eletrofisiologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Corantes Fluorescentes , Galactose/farmacologia , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Cristalino/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Microscopia Confocal , Espécies Reativas de Oxigênio/metabolismoRESUMO
Paraformaldehyde fixation of platelets stabilizes surface antigens while altering some that are associated with cellular activation. Present experiments show that the asymmetric distribution of phosphatidylserine in platelets was especially sensitive to paraformaldehyde treatment. It was found that this reagent induced a dose- and time-dependent translocation of phosphatidylserine to the membrane surface as measured by annexin V binding and flow cytometry. The percent phosphatidylserine-positive cells increased from about 5% to >90%. Chelation of extracellular Ca(2+) with EGTA partially blocked this translocation. Spectrofluorimetric analysis of fluo-3 loaded platelets indicates that paraformaldehyde caused a concomitant elevation of intracellular Ca(2+) concentrations, [Ca(2+)](i). ATP levels also declined in paraformaldehyde-treated cells, suggesting that the rise in [Ca(2+)](i) ensued in part from decreased activity of calcium pumps. Previous studies indicate that phosphatidylserine externalization arises from Ca(2+)-activated randomization of membrane phospholipids and decreased transport of phosphatidylserine from the outer to the inner leaflet of the plasma membrane. In light of present results, paraformaldehyde fixation is best avoided particularly in studies involving platelet apoptosis or activation.
Assuntos
Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Formaldeído/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Polímeros/farmacologia , Trifosfato de Adenosina/metabolismo , Plaquetas/metabolismo , Relação Dose-Resposta a Droga , Humanos , Membranas Intracelulares/metabolismo , Valores de Referência , Propriedades de Superfície , Fatores de TempoRESUMO
Peptides corresponding to the BH3 domains of Bax (BaxBH3) or Bcl-2 (Bcl2BH3) are potent inducers of apoptosis when fused to the Atennapedia plasma membrane translocation domain (Ant). BaxBH3Ant and Bcl2BH3Ant caused a mitochondrial membrane permeabilization (MMP) and apoptosis, via a mechanism that was not inhibited by overexpressed Bcl-2 or Bcl-X(L), yet partially inhibited by cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore. When added to isolated mitochondria, BaxBH3 and Bcl2BH3 induced MMP, which was inhibited by CsA. However, Bcl-2 or Bcl-X(L) failed to inhibit MMP induced by BaxBH3 and Bc2BH3 in vitro, while they efficiently suppressed the induction of MMP by the Vpr protein (from human immunodeficiency virus-1), a ligand of the adenine nucleotide translocator (ANT). BaxBH3 but not Bcl2BH3 was found to interact with ANT, and only BaxBH3 (not Bcl2BH3) permeabilized ANT proteoliposomes and induced ANT to form non-specific channels in electrophysiological experiments. In contrast, both BaxBH3 and Bcl2BH3 were able to stimulate channel formation by recombinant Bax protein. Thus, BaxBH3 might induce MMP via an action on at least two targets, ANT and Bax-like proteins. In contrast, Bcl2BH3 would elicit MMP in an ANT-independent fashion. In purified mitochondria, two ligands of ANT, bongkrekic acid and the protein vMIA from cytomegalovirus, failed to prevent MMP induced by BaxBH3 or Bcl2BH3. In conclusion, BaxBH3 and Bcl2BH3 induce MMP and apoptosis through a mechanism which overcomes cytoprotection by Bcl-2 and Bcl-X(L).
Assuntos
Apoptose/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/farmacologia , Animais , Eletrofisiologia , Citometria de Fluxo , Células HeLa , Hepatócitos , Humanos , Membranas Intracelulares/efeitos dos fármacos , Canais Iônicos/metabolismo , Células Jurkat , Lipossomos/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fragmentos de Peptídeos/síntese química , Permeabilidade/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
Activation of the mitochondrial permeability transition may contribute to excitotoxic neuronal death (Ankarcrona et al., 1996; Dubinsky and Levi, 1998). However, cyclosporin A (CsA), a potent inhibitor of the permeability transition in liver mitochondria, only protects against neuronal injury by limited doses of glutamate and selected ischemic paradigms. The lack of consistent CsA inhibition of the mitochondrial permeability transition was analyzed with the use of isolated brain mitochondria. Changes in the permeability of the inner mitochondrial membrane were evaluated by monitoring mitochondrial membrane potential (Deltapsi), using the distribution of tetraphenylphosphonium, and by monitoring mitochondrial swelling, using light absorbance measurements. Metabolic impairments, large Ca(2+) loads, omission of external Mg(2+), or low doses of palmitic acid or the protonophore FCCP exacerbated Ca(2+)-induced sustained depolarizations and swelling and eliminated CsA inhibition. BSA restored CsA inhibition in mitochondria challenged with 50 microm Ca(2+), but not with 100 microm Ca(2+). CsA failed to prevent Ca(2+)-induced depolarization or to repolarize mitochondria when mitochondria were depolarized excessively. Similarly, CsA failed to prevent mitochondrial swelling or PEG-induced shrinkage after swelling when the Ca(2+) challenge produced a strong, sustained depolarization. Thus in brain mitochondria CsA may be effective only as an inhibitor of the permeability transition and the Ca(2+)-activated low permeability state under conditions of partial depolarization. In contrast, ADP plus oligomycin inhibited both permeabilities under all of the conditions that were tested. In situ, the neuroprotective action of CsA may be limited to glutamate challenges sufficiently toxic to induce the permeability transition but not so severe that mitochondrial depolarization exceeds threshold.
Assuntos
Encéfalo/metabolismo , Ciclosporina/farmacologia , Inibidores Enzimáticos/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Animais , Química Encefálica , Cálcio/metabolismo , Cálcio/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Ciclofilinas/farmacologia , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Membranas Intracelulares/metabolismo , Magnésio/metabolismo , Magnésio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Oligomicinas/farmacologia , Oniocompostos/farmacocinética , Compostos Organofosforados/farmacocinética , Ácido Palmítico/farmacologia , Permeabilidade/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Ratos , Soroalbumina Bovina/farmacologiaRESUMO
Location and dynamics of the voltage-dependent pore-forming icosapeptide alamethicin have been studied using spin labels which were linked directly and via spacers to the C-terminus of the amphiphilic alpha-helix. Ion-transport activities of these derivatives were found to be very similar to those of natural alamethicin in green plant thylakoids chosen as a model system. The shape of the electron spin resonance spectra indicates segmental motion of the nitroxide rather than rotation of the whole peptide. A population of spins showing narrow lines in the presence of thylakoids or lipid vesicles is attributed to alamethicin in the aqueous solution. A second population shows rotational correlation times greater than 10(-9) s and is bound to the membranes, the C-termini residing in an environment with a polarity close to that of water. This population is inaccessible to the hydrophilic, charged line broadening agent chromium oxalate. Since spectral shapes and amplitudes of spectra are unchanged by additions of unlabelled peptide, it is concluded that the ESR detectable spins are bound to peptides essentially in the monomeric state. Alamethicin induced pore formation under flash illumination is demonstrated by measurement of kinetics of proton deposition in the thylakoid interior. When pores are opened by illuminating thylakoids and thus applying a membrane potential, mainly the bound population is affected by a process reversibly suppressing the signal, whereas only limited disappearance of label from the external medium is detected. Apparently, the potential causes a change in the conformation of the peptide which leads to a further immobilisation of the label, possibly due to a deeper insertion of the alpha-helices into the lipid membrane. However, evidence has been presented experimentally that there is no detectable change of potential prior to the opening of the pore.
Assuntos
Alameticina , Antibacterianos , Membranas Intracelulares/metabolismo , Lipossomos , Modelos Biológicos , Plantas/metabolismo , Alameticina/farmacologia , Antibacterianos/farmacologia , Cloroplastos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Membranas Intracelulares/efeitos dos fármacos , Nigericina/farmacologia , Oxigênio/análise , Espectrometria de Fluorescência , Marcadores de Spin/síntese química , Valinomicina/farmacologiaRESUMO
Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to study the interaction of lindane with model and native membranes. Lindane disorders the gel phase of liposomes reconstituted with dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC), since it broadens and shifts the main phase transition, but no apparent effect is detected in the fluid phase. These effects of lindane are more pronounced in bilayers of short-chain lipids, e.g., DMPC. In equimolar mixtures containing DMPC and DSPC, lindane preferentially interacts with the more fluid lipid species inducing lateral phase separations. However, in mixtures of DMPC and DPPC, the insecticide only broadens and shifts the main phase transition, i.e., an effect similar to that observed in bilayers of pure lipids. Lindane has no apparent effect in DMPC bilayers enriched with high cholesterol content (greater than or equal to 30 mol%), whereas disordering effects can still be detected in bilayers with low cholesterol (less than 30 mol%). Apparently, lindane does not perturb the fluid phase of representative native membranes, namely, mitochondria, sarcoplasmic reticulum, myelin, brain microsomes and erythrocytes in agreement with the results obtained in fluid phospholipid bilayers, despite the reasonable incorporation of the insecticide in these membranes, as previously reported (Antunes-Madeira, M.C. and Madeira, V.M.C. (1985) Biochim. Biophys. Acta 820, 165-172).
Assuntos
Hexaclorocicloexano/farmacologia , Fluidez de Membrana/efeitos dos fármacos , Animais , Membrana Celular/efeitos dos fármacos , Colesterol , Difenilexatrieno , Relação Dose-Resposta a Droga , Polarização de Fluorescência , Técnicas In Vitro , Membranas Intracelulares/efeitos dos fármacos , Lipossomos , Lipídeos de Membrana/fisiologia , TemperaturaRESUMO
The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.
Assuntos
Congelamento , Galactolipídeos , Fusão de Membrana , Lipídeos de Membrana/análise , Fenilalanina/farmacologia , Tilacoides/efeitos dos fármacos , Triptofano/farmacologia , Daunorrubicina/farmacologia , Diglicerídeos , Glicolipídeos , Membranas Intracelulares/efeitos dos fármacos , Lipossomos/química , Permeabilidade/efeitos dos fármacos , FosfatidilcolinasRESUMO
The outer mitochondrial membrane pore at a voltage above 20 to 30 mV can adopt a state of low conductance which may restrict free permeability of mitochondrial substrates. In order to obtain insight into the physiological meaning of this property we took advantage of the fact that the low conductance pore state could be induced by a polyanion in lipid bilayer membranes as well as in intact mitochondria. Upon reconstitution in artificial bilayers the pore in this substate became exclusively cation selective when the polarity of the applied voltage was negative on the cis-side. This behaviour of the pore would explain why induction of the low conductance pore state in intact mitochondria led to a complete inhibition of mitochondrial intermembranous kinases, such as creatine kinase and adenylate kinase, but not of peripheral kinases, for example hexokinase, when utilizing external ATP. The possibility that the inner membrane potential might be transduced to the outer membrane in the contact sites, suggests the existence of cation selective pores in these sites. This aspect may be important in the regulation of peripheral kinases like creatine kinase, nucleoside diphosphate kinase and adenylate kinase which are located behind the outer mitochondrial membrane.
Assuntos
Cátions , Membranas Intracelulares/fisiologia , Proteínas de Membrana/fisiologia , Mitocôndrias/ultraestrutura , Porinas , Adenilato Quinase/antagonistas & inibidores , Adenilato Quinase/metabolismo , Animais , Encéfalo/ultraestrutura , Permeabilidade da Membrana Celular , Creatina Quinase/metabolismo , Condutividade Elétrica , Hexoquinase/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/ultraestrutura , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Bicamadas Lipídicas , Potenciais da Membrana , Mitocôndrias Hepáticas/ultraestrutura , Polieletrólitos , Polímeros/farmacologia , Ratos , Canais de Ânion Dependentes de VoltagemRESUMO
Saponin permeabilization of rough microsomes in the presence of high salt revealed a novel pool of prothrombin associated by ionic interactions to the microsomal membrane. The lumenal content was obtained by treating rough microsomes with 0.32% saponin in a low salt (0.05 M KCl) buffer. By a subsequent treatment with 0.32% saponin in a slightly alkaline high salt buffer a fraction of peripherally associated membrane prothrombin was released from rough microsomes. Finally, the membrane-bound fraction was solubilized with 2.5% Triton X-100. The lumenal content fraction, the peripherally membrane-associated and the membrane-bound fraction from normal rats contained 55%, 29% and 16% of the total rough microsomal prothrombin, respectively. The corresponding fractions from warfarin-treated rats contained 86%, 5% and 9% of the total prothrombin. Following (14)C-gamma-carboxylation of intact microsomes for 30 min, the novel membrane-associated and the membrane-bound pool contained 42% and 33%, respectively, of labeled prothrombin. A similar distribution was found with warfarin-treated rats.
Assuntos
Microssomos Hepáticos/efeitos dos fármacos , Saponinas/farmacologia , Animais , Anticoagulantes , Soluções Tampão , Dióxido de Carbono/química , Radioisótopos de Carbono , Fracionamento Químico , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Masculino , Microssomos Hepáticos/metabolismo , Octoxinol , Permeabilidade , Polietilenoglicóis , Precursores de Proteínas/metabolismo , Protrombina/química , Protrombina/metabolismo , Ratos , Ratos Wistar , Vitamina K/metabolismo , VarfarinaRESUMO
Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca(2+)-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-beta-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-beta-cyclodextrin complexes restored the Ca(2+)-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca(2+), annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca(2+) concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca(2+) concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.
Assuntos
Anexina A2/metabolismo , Cálcio/análise , Colesterol/farmacologia , Membranas Intracelulares/efeitos dos fármacos , beta-Ciclodextrinas , Anexina A2/química , Cálcio/farmacologia , Grânulos Cromafim/metabolismo , Ciclodextrinas/farmacologia , Relação Dose-Resposta a Droga , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Lipossomos/química , Lipídeos de Membrana/metabolismo , Ligação Proteica/efeitos dos fármacosRESUMO
The membrane disordering efficiency of four local anesthetics, including lidocaine, tetracaine, dibucaine and heptacaine (piperidinoethyl ester of 2-heptyloxyphenylcarbamic acid) has been studied by spin-labeling methods. The disordering efficiency of the drugs in rat total brain lipid liposomes was quantitated with the initial slope value of the order parameter versus drug concentration curve, the so-called change-in-order parameter value. Using the positional isomers of m-doxyl stearic acids (m = 5, 12 and 16), it has been demonstrated that the tested drugs reveal quite different disordering efficiency. There is a clear tendency of increasing disordering efficiency towards the methyl terminal of the lipid acyl chains. By a comparison of order parameter versus drug concentration and temperature at three depths of rat brain total lipid liposomes and synaptosomes, it is shown that the 'fluidizing effect' of local anesthetics does not correspond to fluidization of membrane by temperature and that tetracaine and dibucaine do not have equal disordering efficiency as judged by their solubility in the membrane. The disordering efficiency of these drugs on the hydrocarbone core of a membrane qualitatively corresponds to their anesthetic potency. Similar results were obtained in liposomes and synaptosomes. It is assumed that there is a similar incorporation of the local anesthetics in the liposomes and in the lipid part of synaptosomes.