RESUMO
Hypoxia plays a role in many diseases and can have a wide range of effects on cardiac metabolism depending on the extent of the hypoxic insult. Noninvasive imaging methods could shed valuable light on the metabolic effects of hypoxia on the heart in vivo. Hyperpolarized carbon-13 magnetic resonance spectroscopy (HP 13 C MRS) in particular is an exciting technique for imaging metabolism that could provide such information. The aim of our work was, therefore, to establish whether hyperpolarized 13 C MRS can be used to assess the in vivo heart's metabolism of pyruvate in response to systemic acute and chronic hypoxic exposure. Groups of healthy male Wistar rats were exposed to either acute (30 minutes), 1 week or 3 weeks of hypoxia. In vivo MRS of hyperpolarized [1-13 C] pyruvate was carried out along with assessments of physiological parameters and ejection fraction. Hematocrit was elevated after 1 week and 3 weeks of hypoxia. 30 minutes of hypoxia resulted in a significant reduction in pyruvate dehydrogenase (PDH) flux, whereas 1 or 3 weeks of hypoxia resulted in a PDH flux that was not different to normoxic animals. Conversion of hyperpolarized [1-13 C] pyruvate into [1-13 C] lactate was elevated following acute hypoxia, suggestive of enhanced anaerobic glycolysis. Elevated HP pyruvate to lactate conversion was also seen at the one week timepoint, in concert with an increase in lactate dehydrogenase (LDH) expression. Following three weeks of hypoxic exposure, cardiac metabolism of pyruvate was comparable with that observed in normoxia. We have successfully visualized the effects of systemic hypoxia on cardiac metabolism of pyruvate using hyperpolarized 13 C MRS, with differences observed following 30 minutes and 1 week of hypoxia. This demonstrates the potential of in vivo hyperpolarized 13 C MRS data for assessing the cardiometabolic effects of hypoxia in disease.
Assuntos
Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Hipóxia/metabolismo , Miocárdio/metabolismo , Animais , Hipóxia/sangue , Masculino , Oxigênio/sangue , Ratos WistarRESUMO
Evaluation of: Araki H, Tazawa H, Kanaya N, et al. Oncolytic virus-mediated p53 overexpression promotes immunogenic cell death and efficacy of PD-1 blockade in pancreatic cancer. Mol Ther Oncolytics. 2022;27:3-13.Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with poor prognosis. PDAC has a dense, desmoplastic stroma and immunosuppressive microenvironment, which impedes current treatment options. Immunotherapy delivered via oncolytic virotherapy is one potential solution to these barriers. Immune checkpoint inhibitors may facilitate immunogenic cell death by improving immune cell infiltration in cancer cells. PD-1 blockade shows better clinical outcomes for certain cancers. The addition of p53 to stimulate cell cycle arrest remains a novel field of research. The evaluated article by Araki et al. explores the efficacy of PD-1 blockade with oncolytic adenovirus platforms on immunogenic cell death and the possibility of combining PD-1 blockade and p53-activation. In vitro analysis showed increased cell death in multiple cell lines infected with AdV mediating p53 expression. The underlying process may attribute to apoptosis and autophagy, with evidence of increased immunogenic cell death. In vivo models demonstrated improved efficacy of p53-expressing AdV, particularly with the addition of PD-1 blockade which appears to be related to CD8+ cell infiltration.
Assuntos
Carcinoma Ductal Pancreático , Imunoterapia , Terapia Viral Oncolítica , Neoplasias Pancreáticas , Proteína Supressora de Tumor p53 , Humanos , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/imunologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Imunoterapia/métodos , Animais , Adenoviridae/genética , Vírus Oncolíticos/genética , Vírus Oncolíticos/imunologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Morte Celular Imunogênica , Microambiente Tumoral , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismoRESUMO
Chronic hypoxia decreases cardiomyocyte respiration, yet the mitochondrial mechanisms remain largely unknown. We investigated the mitochondrial metabolic pathways and enzymes that were decreased following in vivo hypoxia, and questioned whether hypoxic adaptation was protective for the mitochondria. Wistar rats were housed in hypoxia (7 days acclimatisation and 14 days at 11% oxygen), while control rats were housed in normoxia. Chronic exposure to physiological hypoxia increased haematocrit and cardiac vascular endothelial growth factor, in the absence of weight loss and changes in cardiac mass. In both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria isolated from hypoxic hearts, state 3 respiration rates with fatty acid were decreased by 17-18%, and with pyruvate were decreased by 29-15%, respectively. State 3 respiration rates with electron transport chain (ETC) substrates were decreased only in hypoxic SSM, not in hypoxic IFM. SSM from hypoxic hearts had decreased activities of ETC complexes I, II and IV, which were associated with decreased reactive oxygen species generation and protection against mitochondrial permeability transition pore (MPTP) opening. In contrast, IFM from hypoxic hearts had decreased activity of the Krebs cycle enzyme, aconitase, which did not modify ROS production or MPTP opening. In conclusion, cardiac mitochondrial respiration was decreased following chronic hypoxia, associated with downregulation of different pathways in the two mitochondrial populations, determined by their subcellular location. Hypoxic adaptation was not deleterious for the mitochondria, in fact, SSM acquired increased protection against oxidative damage under the oxygen-limited conditions.
Assuntos
Metabolismo Energético , Hipóxia/metabolismo , Mitocôndrias Cardíacas/metabolismo , Aconitato Hidratase/metabolismo , Adaptação Fisiológica , Animais , Respiração Celular , Doença Crônica , Modelos Animais de Doenças , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Ácidos Graxos/metabolismo , Hematócrito , Masculino , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Estresse Oxidativo , Ácido Pirúvico , Ratos , Ratos Wistar , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Rat dermal fibroblasts were grown as monolayers, and changes in the fine structure of the cells that occurred during 12 hr incubation in a medium containing protein and excess of retinol (vitamin A alcohol) were studied by electron microscopy. There is little change during the first 6 hr, although some of the nuclei have highly convoluted membranes. During the subsequent 3 hr, there is some disorganization of the mitochondrial cristae; the cisternae of the rough-surfaced endoplasmic reticulum diminish in number; and the amount of smooth membranous material and free ribosomes increases. There is a rapid decline in the respiratory activity of the cells after 6 hr exposure to the vitamin. It is concluded that the primary action of excess of retinol is to cause alterations in the membranes of the cells and that these alterations affect the functions of the mitochondria and endoplasmic reticulum.
Assuntos
Álcoois/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Animais , Membrana Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Meios de Cultura , Retículo Endoplasmático/fisiologia , Complexo de Golgi/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Membranas/efeitos dos fármacos , Microscopia Eletrônica , Mitocôndrias/fisiologia , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ribossomos/efeitos dos fármacos , Vitamina A/farmacologiaRESUMO
The importance of cell swelling in the fusion of erythrocytes by three different chemical treatments has been investigated with cells that were cytoplasmically labelled with 6-carboxyfluorescein. Hen erythrocytes, which had been pre-incubated with ionophore A23187 and 5 mM Ca2+ to cause a proteolytic breakdown of the membrane skeleton, were induced to fuse by applying an osmotic shock. Human erythrocytes that had been incubated in an isotonic salt/buffer solution, which was progressively diluted and which contained 0.5 mM La3+ to minimise cell lysis, were also fused. In addition, the fusion of human erythrocytes by 40% poly(ethylene glycol) began only when the poly(ethylene glycol) was diluted, and it mostly occurred when the diluted polymer solution was subsequently replaced by isotonic buffer. In related experiments, the effect of an osmotic gradient on electrically induced cell fusion has been studied. Human erythrocytes in 150 mM erythritol fused more readily than less swollen cells in 200-400 mM erythritol when subjected to a 20 microseconds pulse of 3.5 kV X cm-1, indicating that the extent of cell fusion induced by the breakdown pulse is governed by the combined electrical-compressive and osmotic forces. Since osmotic phenomena are already known to be important in exocytosis, we suggest that these observations on cell fusion indicate that osmotic forces may provide the driving force for many membrane fusion reactions in biological systems.
Assuntos
Fusão Celular , Membrana Eritrocítica , Animais , Cálcio/farmacologia , Galinhas , Citoesqueleto/efeitos dos fármacos , Eletricidade , Fluoresceínas , Humanos , Microscopia Eletrônica , Polietilenoglicóis , Equilíbrio HidroeletrolíticoRESUMO
A comparative study has been made of the abilities of retinol and its derivatives to induce cell fusion and haemolysis of hen erythrocytes and to cause swelling of rat liver mitochondria. Retinol, retinaldehyde, alpha-retinoic acid, iso-13-retinol and to a lesser extent retinyl acetate were active in all three systems. Iso-13-retinoic acid was extremely membranolytic but did not produce stable fused cells. By contrast retinoic acid, its cyclopentyl derivative RO8-7699, and the long chain fatty acid esters of retinol, viz. the oleate, linoleate and palmitate esters, were neither fusogenic nor haemolytic, nor did they affect mitochondria.
Assuntos
Fusão Celular/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Dilatação Mitocondrial/efeitos dos fármacos , Vitamina A/farmacologia , Animais , Galinhas , Eritrócitos/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Ratos , Retinaldeído/farmacologia , Relação Estrutura-Atividade , Tretinoína/análogos & derivados , Tretinoína/farmacologia , Vitamina A/análogos & derivadosRESUMO
Preparations of poly(ethylene glycol) 6000 (PEG) from five different commercial sources have been purified, and their ability to fuse hen erythrocytes has been investigated. Quantitative assessments of cell fusion showed that before purification one of the preparations (PEG Wako), was able to induce limited fusion (5-6%) of erythrocytes with conditions (1 min incubation with 50% w/w PEG) under which the other four unpurified preparations of PEG were inactive. On purification, PEG (Wako) became inactive. By contrast, when erythrocytes were incubated with 45% w/w PEG for 15 min, extensive fusion (23-27%) occurred with all five unpurified preparations of PEG. Under these conditions, the fusogenic properties of four of the preparations of PEG were unaffected by purification: fusion induced by PEG (Wako) was, however, decreased on purification from 27% to 19%. It appears that polymeric poly(ethylene glycol) is itself able to fuse cells, but that some commercial preparations, e.g. PEG (Wako), have enhanced fusogenic properties resulting from the presence of contaminating substances. No relationship between the absorbance at 290 nm of PEG and its fusogenic properties was found in this study. The addition of small quantities of fusogenic lipid-soluble compounds to PEG was, however, observed to enhance cell fusion by up to 50%.
Assuntos
Fusão Celular/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Animais , Antioxidantes/farmacologia , Galinhas , Sinergismo Farmacológico , Eritrócitos/efeitos dos fármacos , Feminino , Glicerídeos/farmacologia , Técnicas In Vitro , Espectrofotometria Ultravioleta , Vitamina A/farmacologiaRESUMO
There are many reports of hemi-fusion in phospholipid vesicles but few published studies on hemi-fusion in cells. We report evidence from both fluorescence microscopy and freeze-fracture electron microscopy for hemi-fusion in the electrofusion of human erythrocytes. We have also characterised the conditions that favour hemi-fusion as opposed to complete fusion, and discuss the possibility that hemi-fusion might precede complete electrically-induced cell fusion. A membrane probe (DiIC16) and a cytoplasmic probe (6-carboxyfluorescein) were used to investigate the behaviour of doubly-labelled human erythrocytes which were aligned in chains by dielectrophoresis and then exposed to high voltage breakdown pulses. Some of the cells were fused by the pulses, as shown by diffusion of both membrane and cytoplasmic probes from labelled to unlabelled cells. With other cells, the membrane probe diffused into unlabelled cells after the breakdown pulses, without the cytoplasmic probe diffusing into unlabelled cells or leaking into the medium. Membrane fusion (hemi-fusion) thus occurred without cytoplasmic fusion in these erythrocytes. Such cells were irreversibly, but fragilely, attached to their neighbours by the breakdown pulses. There was an inverse relationship between conditions that permit complete fusion and those that favour hemi-fusion, with respect to breakdown pulse length, breakdown voltage and, in particular, osmolarity and temperature. The incidence of hemi-fusion in 250 mM erythritol was twice that in 150 mM erythritol, and hemi-fusion was 5-fold greater at 25 degrees C than at 20 degrees C. Hemi-fused erythrocytes occasionally fused completely on heating to 50 degrees C, demonstrating that hemi-fusion can proceed to complete cell fusion. Freeze fracture electron micrographs of preparations of hemi-fused cells revealed long-lived, complementary depressions and protrusions on the E- and P-fracture faces, respectively, of tightly apposed cells that may mediate hemi-fusion. The possibility that the fusion of closely adjacent human erythrocytes by electrical breakdown pulses may involve an intermediate, shared bilayer structure, which is stable in certain conditions but which can be ruptured by osmotic swelling of the permeabilised cells, is discussed.
Assuntos
Estimulação Elétrica , Membrana Eritrocítica/fisiologia , Eritrócitos/fisiologia , Fusão de Membrana , Citoplasma/fisiologia , Membrana Eritrocítica/ultraestrutura , Eritrócitos/citologia , Eritrócitos/ultraestrutura , Corantes Fluorescentes , Técnica de Fratura por Congelamento , Humanos , Técnicas In Vitro , Microscopia Eletrônica , Microscopia de FluorescênciaRESUMO
An assay for procoagulant activity has been used to investigate the Ca2(+)-dependent exposure of phosphatidylserine at the surface of human erythrocytes that were induced to swell and to fuse osmotically. Since the phosphatidylserine of human erythrocytes is located in the inner leaflet of the plasma membrane, it is inaccessible in intact cells which therefore had no procoagulant activity in an isotonic solution of sucrose. The procoagulant activity of erythrocytes incubated in increasingly hypotonic, sucrose solutions containing Mg2+ paralleled the percentage haemolysis, reflecting the accessibility of phosphatidylserine in an increasing number of lysed cells. However, cells in mildly hypotonic sucrose solutions containing Ca2+ had an abnormally high procoagulant activity indicating that phosphatidylserine was exposed in intact cells under these conditions. Erythrocytes that were subjected to continuous swelling at 37 degrees C, which was induced by entry of the permeant molecule poly(ethylene glycol) 400 (PEG 400) developed procoagulant activity in the presence of Ca2+ prior to extensive lysis. Cells treated in this way also fused. With Mg2+, PEG 400-treated erythrocytes lysed without fusing, and the development of procoagulant activity paralleled the rate of lysis. Erythrocytes incubated with ionophore A23187, subtilisin, and Ca2+ developed procoagulant activity (with less than 20% lysis), and they fused on subsequent exposure to a hypotonic medium. The procoagulant activity reached its maximum before fusion could be induced in the hypotonic medium. It is concluded that the entry of Ca2+ facilitates a translocation of phosphatidylserine to the outer leaflet of the erythrocyte plasma membrane that plays an important role in fusion protocols that involve cell swelling. It is also suggested that transbilayer movements of phosphatidylserine could be an important control factor in the cell biology of membrane fusion phenomena.
Assuntos
Cálcio/farmacologia , Agregação Eritrocítica , Eritrócitos/metabolismo , Fusão de Membrana , Fosfatidilserinas/metabolismo , Transporte Biológico , Calcimicina/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Humanos , Soluções Hipotônicas , Pressão Osmótica , Polietilenoglicóis/farmacologia , SacaroseRESUMO
The procoagulant activity of human erythrocytes, which provides a measure of the translocation of acidic phospholipids from the inner to the outer monolayer of the plasma membrane, has been compared with the percentage cell fusion in experiments on the effects of electrical breakdown pulses under differing experimental conditions. After treatment with breakdown pulses of 20 microseconds or longer (5 kV cm-1), the plasma membranes of erythrocytes in 250 mM sucrose exhibited an almost complete loss of asymmetry with respect to acidic phospholipids. As the breakdown voltage was increased from 2 to 5 kV cm-1 (with breakdown pulses of 99 microseconds), the surface exposure of acidic phospholipids and cell fusion increased approximately in parallel. Furthermore, with 99 microseconds pulses and a voltage of 3 kV cm-1, a decrease in the osmolarity from 250 to 150 mM of the sucrose medium was accompanied by an increase in both the surface exposure of acidic phospholipids and the extent of cell fusion. Breakdown pulses of 2-5 microseconds were sufficient to cause a marked loss of asymmetry, but no cell fusion was observed unless the pulse length was at least 20 microseconds. Kinetic experiments indicated that exposure of the acidic phospholipids at the cell surface was more likely to be due to a direct effect of the electric field pulses on plasma membrane structure than to secondary effects, such as the action of endogenous proteinases on the membrane skeleton. It seems possible that a localised, surface exposure of acidic phospholipids may contribute to the 'long-lived fusogenic state' (Sowers, A.E. (1986) J. Cell Biol. 102, 1358-1362) and the 'transient permeant structures' (Teissié, J. and Rols, M.P. (1986) Biochem. Biophys. Res. Commun. 140, 258-266) that enable cell fusion to occur when contact between cells is established after they have been subjected to field pulses. Our observations also provide circumstantial support for the concept that changes in the phospholipid asymmetry of membranes may be important in physiologically-occurring instances of biomembrane fusion.
Assuntos
Fusão Celular , Eritrócitos/metabolismo , Fosfolipídeos/metabolismo , Animais , Bovinos , Estimulação Elétrica , Hemólise , Humanos , Concentração de Íons de Hidrogênio , Soluções Hipotônicas , Cinética , Osmose , Sacarose , Propriedades de Superfície , Tromboplastina/metabolismoRESUMO
The interaction which occurs between an anti-lipid antibody (TEPC 15) and two phospholipids, phosphatidylcholine and phosphatidylethanolamine, when they are arranged in a lipid monolayer system has been studied. It is shown that the antibody is stabilised under the influence of a high lateral pressure when it is mixed with a lipid monolayer and that the behaviour of the antibody depends upon the lipid used. Measurements of the surface pressure and surface potential parameters of the lipid monolayers indicate that the antibody interacts differently with phosphatidylcholine compared with phosphatidylethanolamine. The antibody also exhibits a different interaction when it is pretreated with phosphorylcholine prior to being spread with a phosphatidylcholine monolayer. The interaction of the antibody with phosphatidylcholine-cholesterol monolayers has also been studied.
Assuntos
Anticorpos/imunologia , Lipídeos de Membrana/imunologia , Membranas Artificiais , Fosfatidilcolinas/imunologia , Fosfatidiletanolaminas/imunologia , Fosforilcolina/farmacologia , Propriedades de SuperfícieRESUMO
Poly(ethylene glycol) 6000 induced a concentration-dependent, time-dependent decrease in the latency of the reaction between Arsenazo III sequestered in liposomes and extraliposomal Ca2+. This was mediated by a gross change in liposomal permeability, i.e. by a release of Arsenazo III from liposomes rather than simply by an entry of Ca2+. The loss of latency was strongly temperature-dependent, and it was markedly diminished on increasing the cholesterol content of the liposomes. It was apparently not due to an osmotic stress of the polymer. The high activation energy found (63 kJ . mol-1) is thought to indicate that the loss of latency resulted from local discontinuities in the lipid bilayers, caused by dehydration, rather than from partial or total lysis. Related microscopy experiments indicated that the polymer also caused the liposomes to fuse, and it is suggested that membrane fusion may have occurred at the sites of dehydration-induced discontinuities in adjacent bilayers. In addition, the polymer was found to enhance the permeability of hen erythrocytes of Ca2+ in a manner that was comparable to its effect on liposomal latency, and it is proposed that gel fusion induced by poly(ethylene glycol) may occur at the sites of similarly induced discontinuities in the phospholipid bilayers of two closely adjacent cells.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Lipossomos/metabolismo , Polietilenoglicóis/farmacologia , Animais , Arsenazo III/metabolismo , Cálcio/metabolismo , Fusão Celular/efeitos dos fármacos , Galinhas , Feminino , Técnica de Fratura por Congelamento , Cinética , TermodinâmicaRESUMO
We have previously reported that acidic phospholipids are exposed at the surface of human erythrocytes when the cells are subjected to electrical breakdown. It has now been shown that the prothrombinase assay, which was used previously for the determination of acidic phospholipids, is specific for phosphatidylserine under the conditions of our experiments. In the light of this finding, we have investigated and characterised factors that govern cell lysis, cell fusion, and the formation of giant cells induced by electrical breakdown with human erythrocytes in media of low ionic strength. Divalent cations (1.1 mM) protected the cells against haemolysis, in the order Mn2+ > Ca2+ > Ba2+ > Mg2+ >> Zn2+, whereas about 99% of the cells lysed immediately on breakdown in the presence of Na+ or K+ (2.1 mM), or Al3+ (0.95 mM). The lengths of pearl chains of fused erythrocytes formed was similarly greatest with Mn2+ and decreased progressively with Ba2+, Zn2+, Ca2+ and Mg2+. No cell fusion occurred with Na+, K+, or Al3+. It is suggested that interactions with phosphatidylserine, which is exposed at the cell surface by electrical breakdown, may enable Mn2+, Ba2+ and Ca2+ ions to inhibit cell lysis (via membrane resealing) and facilitate cell fusion. Following electrically-induced cell fusion, erythrocytes round-up into giant cells. It has previously been proposed that Ca2+ ions accelerate the rounding-up process. However, data are presented which show that, as with erythrocytes treated with Sendai virus, the formation of rounded, giant cells following cell fusion depends on the osmotic swelling properties of permeabilised erythrocytes. Osmotic swelling may also have induced any hemi-fused cells present to fuse completely. Zn2+ ions anomalously enabled erythrocytes to round-up very rapidly into giant cells following electrical breakdown. This phenomenon may result from an interaction of Zn2+ ions with cysteine groups in membrane proteins, which decreases the immediate loss of ions that occurs when erythrocytes are subjected to electrical breakdown in low-ionic-strength media.
Assuntos
Cátions Bivalentes/química , Eritrócitos/química , Fosfolipídeos/química , Zinco/química , Cádmio/química , Fusão Celular/efeitos dos fármacos , Tamanho Celular , Cobalto/química , Estimulação Elétrica , Eritrócitos/citologia , Eritrócitos/metabolismo , Células Gigantes , Hemólise/efeitos dos fármacos , Humanos , Concentração Osmolar , Fosfatidilserinas/análise , Tromboplastina , Zinco/metabolismoRESUMO
The effects of competitive inhibitors of transglutaminase on the formation of myotubes by the fusion of myoblasts in vitro has been investigated. Myotube formation was inhibited when myoblasts from 11-day-old chick embryos were cultured in vitro in the presence of 10 mM histamine or 0.2 mM dansyl cadaverine. The inhibitions observed were reversed when the treated cells were subsequently cultured in normal medium. Glycine methyl ester also inhibited myotube formation but sarcosine methyl ester, which is not a competitive inhibitor of transglutaminase, had little if any inhibitory action. The formation of myotubes was not inhibited by cultivation in normal medium adjusted to pH 8.0-8.1, indicating that the observed effects of histamine and of dansyl cadaverine were not mediated by a lysosomotropic effect. Inhibition of myotube formation in the presence of histamine was accompanied by the production of abnormal multinucleated cells, indicating that myoblast fusion occurred in the treated cultures but that the fused cells failed to elongate into normal myotubes. Transglutaminase activity has been found in cell-free lysates of embryonic chick myoblasts and it is concluded that a transglutaminase enzyme, activated by an increase in the concentration of intracellular Ca2+, plays an important role in stabilising the cytoskeletal network of developing myotubes.
Assuntos
Aciltransferases/antagonistas & inibidores , Cálcio/metabolismo , Músculos/embriologia , Aciltransferases/isolamento & purificação , Animais , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Cálcio/fisiologia , Fusão Celular/efeitos dos fármacos , Sistema Livre de Células , Embrião de Galinha , Glicina/análogos & derivados , Glicina/farmacologia , Histamina/farmacologia , Músculos/enzimologia , Sarcosina/análogos & derivados , Sarcosina/farmacologia , TransglutaminasesRESUMO
Cell fusion of embryonic chick myoblasts has been studied in the presence of fat-soluble agents that induce erythrocytes to fuse. Retinol inhibited myoblast fusion but the cells recovered their ability to fuse within 48 h of removal of the retinol from the medium. Myristic acid, oleic acid, glycerol monooleate, linolenic acid and arachidonic acid similarly prevented fusion in myogenic cultures. By contrast, linoleic acid moderately enhanced the fusion of chick skeletal myoblasts. In addition, stearic acid, which does not fuse erythrocytes, inhibited myoblast fusion whereas the saturated, non-fusogenic fatty acid, arachidic acid, was without effect.
Assuntos
Ácidos Graxos/farmacologia , Glicerídeos/farmacologia , Músculos/embriologia , Vitamina A/farmacologia , Animais , Fusão Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Cinética , Músculos/efeitos dos fármacos , Músculos/fisiologia , Relação Estrutura-AtividadeRESUMO
Phosphatidylserine (PS) was exposed at the surface of human umbilical vein endothelial cells (HUVECs) and cultured cell lines by agonists that increase cytosolic Ca(2+), and factors governing the adhesion of T cells to the treated cells were investigated. Thrombin, ionophore A23187 and the Ca(2+)-ATPase inhibitor 2, 5-di-tert-butyl-1,4-benzohydroquinone each induced a PS-dependent adhesion of Jurkat T cells. A23187, which was the most effective agonist in releasing PS-bearing microvesicles, was the least effective in inducing the PS-dependent adhesion of Jurkat cells. Treatment of ECV304 and EA.hy926 cells with EGTA, followed by a return to normal medium, resulted in an influx of Ca(2+) and an increase in adhering Jurkat cells. Oxidised low-density lipoprotein induced a procoagulant response in cultured ECV304 cells and increased the number of adhering Jurkat cells, but adhesion was not inhibited by pretreating ECV304 cells with annexin V. PS was not significantly exposed on untreated Jurkat cells, as determined by flow cytometry with annexin V-FITC. However, after adhesion to thrombin-treated ECV304 cells for 10 min followed by detachment in 1 mM EDTA, there was a marked exposure of PS on the Jurkat cells. Binding of annexin V-FITC to the detached cells was inhibited by pretreating them with unlabelled annexin V. Contact with thrombin-treated ECV304 cells thus induced the exposure of PS on Jurkat cells and, as Jurkat cells were unable to adhere to thrombin-treated ECV304 cells in the presence of EGTA, the adhesion of the two cell types may involve a Ca(2+) bridge between PS on both cell surfaces. The number of T cells from normal, human peripheral blood that adhered to ECV304 cells was not increased by treating the latter with thrombin. However, findings made with several T cell lines were generally, but not completely, consistent with the possibility that adhesion to surface PS on endothelial cells may be a feature of T cells that express both CD4(+) and CD8(+) antigens. Possible implications for PS-dependent adhesion of T cells to endothelial cells in metastasis, and early in atherogenesis, are discussed.
Assuntos
Adesão Celular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Fosfatidilserinas/farmacologia , Linfócitos T/metabolismo , Anexina A5/farmacologia , Hidroxianisol Butilado/análogos & derivados , Hidroxianisol Butilado/farmacologia , Calcimicina/farmacologia , Cálcio/metabolismo , Linhagem Celular , Ácido Egtázico , Citometria de Fluxo , Humanos , Ionóforos/farmacologia , Cinética , Lipoproteínas LDL/farmacologia , Microscopia de Fluorescência , Trombina/farmacologiaRESUMO
The majority of investigations of the transverse tubules (T-system) of skeletal muscle have been devoted to their role in excitation-contraction coupling, with particular reference to contact with the sarcoplasmic reticulum and the mechanism of Ca2- release. By contrast, this review is concerned with structural and functional aspects of the vacuolation of T-tubules. It covers experimental procedures used in reversible vacuolation induced by the efflux-influx of glycerol and other small nonelectrolytes, sugars, and ions. The characteristics of the phenomenon, associated alterations in muscle function, and the swelling of analogous structures in nonmuscle cells are considered. Possible functions of reversible vacuolation in water balance, transport, membrane repair, muscle pathology, and fatigue are considered, and the potential application of reversible vacuolation in the transfection of skeletal muscle is discussed. In relation to the possible mechanisms involved in reversible vacuolation, particular attention is given to the dynamic and structural aspects of the opening and closing of T-tubules, the origin of vacuolar membranes, and the localized character of tubular swelling.
Assuntos
Membrana Celular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/fisiologia , Músculo Esquelético/ultraestrutura , Vacúolos/metabolismo , Animais , Tamanho Celular , Corantes Fluorescentes/metabolismo , Glicerol/farmacologia , Íons/metabolismo , Microscopia de Fluorescência , Contração Muscular/fisiologia , Músculo Esquelético/efeitos dos fármacos , Compostos de Piridínio/metabolismo , Transfecção , Vacúolos/ultraestruturaRESUMO
The spatio-temporal distribution of intracellular, free calcium ions, [Ca2+]i, induced in human myotubes by electrical stimulation typically showed a relatively large increase of [Ca2+]i in the vicinity of the plasmalemma. The similarity of this distribution, with that observed after the application of caffeine, and the lack of any effect of lanthanum, strongly suggest that the main source of Ca2+ participating in the electrically induced transient is the sarcoplasmic reticulum. Aneurally cultured human myotubes therefore display a 'skeletal muscle type' coupling between membrane depolarization and calcium release. However, the relatively slow time course of the electrically induced transients compared to rat and mouse myotubes, together with the inability of Ca2+ released from the sarcoplasmic reticulum to activate the contractile machinery, implies that aneurally cultured human myotubes achieve only a limited degree of differentiation. The relevance this may have to an apparent delay between the electrically induced rise in intranuclear Ca2+ relative to cytosolic Ca2+ remains to be determined but, at this stage of differentiation, there appears to be some form of barrier to free diffusion between the two cellular compartments.
Assuntos
Cálcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Cafeína/farmacologia , Núcleo Celular/metabolismo , Células Cultivadas/metabolismo , Citoplasma/metabolismo , Condutividade Elétrica , Fura-2 , Humanos , Lantânio/farmacologia , Fatores de TempoRESUMO
The concept that a direct interaction between Ca2+ and phospholipids is a major factor in membrane fusion reactions is questioned. Attention is drawn to a number of findings on associations between fusion and the proteolysis of membrane proteins. It is proposed that hydrophobic polypeptides, which are functionally comparable to the fusogenic proteins of certain viruses but which are produced in cells by the endogenous proteolysis of membrane and cellular proteins, may induce membrane fusion reactions in vivo.
Assuntos
Fusão Celular , Lipídeos de Membrana/fisiologia , Proteínas de Membrana/fisiologia , Peptídeos/fisiologia , Animais , Cálcio/metabolismo , Membrana Celular/fisiologia , Membrana Eritrocítica/fisiologia , Humanos , Substâncias Macromoleculares , Peptídeo Hidrolases/metabolismo , Conformação Proteica , Proteínas Virais/fisiologiaRESUMO
A molecular model for fusion-fission reactions in membranes is proposed that is based on data from studies on artificially induced cell fusion and on the behaviour of phospholipid bilayers: it is put forward as a framework for further investigations into this fundamental property of biological systems.