RESUMO
BACKGROUND AND PURPOSE: The high potency antipsychotic drug trifluoperazine (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluoromethyl)-(10)H-phenothiazine dihydrochloride; TFP) may either counteract or promote suicidal cell death or apoptosis. Similar to apoptosis, erythrocytes may enter eryptosis, characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis can be stimulated by an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) and inhibited by nitric oxide (NO). We explored whether TFP treatment of erythrocytes induces phosphatidylserine exposure, cell shrinkage, and calcium influx, whether it impairs S-nitrosylation and whether these effects are inhibited by NO. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and protein nitrosylation from fluorescence switch of the Bodipy-TMR/Sypro Ruby signal. RESULTS: Exposure of human erythrocytes to TFP significantly enhanced the percentage of annexin-V-binding cells, raised [Ca2+]i, and decreased S-nitrosylation. The effect of TFP on annexin-V-binding was not affected by removal of extracellular Ca2+ alone, but was significantly inhibited by pre-treatment with sodium nitroprusside (SNP), an effect significantly augmented by additional removal of extracellular Ca2+. A 3 hours treatment with 0.1 µM Ca2+ ionophore ionomycin triggered annexin-V-binding and cell shrinkage, effects fully reversed by removal of extracellular Ca2+. CONCLUSIONS: TFP induces eryptosis and decreases protein S-nitrosylation, effects blunted by nitroprusside. The effect of nitroprusside is attenuated in the presence of extracellular Ca2+.
Assuntos
Eriptose/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Trifluoperazina/toxicidade , Potenciais de Ação/efeitos dos fármacos , Cálcio/metabolismo , Tamanho Celular/efeitos dos fármacos , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/fisiologia , Hemólise/efeitos dos fármacos , Humanos , Ionomicina/toxicidade , Microscopia de Fluorescência , Óxido Nítrico/metabolismo , Técnicas de Patch-Clamp , Fosfatidilserinas/toxicidade , Processamento de Proteína Pós-Traducional/efeitos dos fármacosRESUMO
The phenomenon of physical contact between red blood cells and artificial surfaces is considered. A fully three-dimensional mathematical model of a bilayer membrane in contact with an artificial surface is presented. Numerical results for the different geometries and adhesion intensities are found to be in agreement with experimentally observed geometries obtained by means of digital holographic microscopy.
Assuntos
Forma Celular , Eritrócitos/citologia , Animais , Adesão Celular , Humanos , Bicamadas Lipídicas/química , Modelos Teóricos , Propriedades de SuperfícieRESUMO
BACKGROUND/AIMS: The exposure of phosphatidylserine (PS) on the outer membrane leaflet of red blood cells (RBCs) serves as a signal for suicidal erythrocyte death or eryptosis, which may be of importance for cell clearance from blood circulation. PS externalisation is realised by the scramblase activated by an increase of intracellular Ca2+ content. It has been described in literature that RBCs show an increased intracellular Ca2+ content as well as PS exposure when becoming aged up to 120 days (which is their life span). However, these investigations were carried out after incubation of the RBCs for 48 h. The aim of this study was to investigate this effect after short-time incubation using a variety of stimulating substances for Ca2+ uptake and PS exposure. METHODS: We separated RBCs by age in five different fractions by centrifugation using Percoll density gradient. The intracellular Ca2+ content and the PS exposure of RBCs with different age has been investigated after treatment with lysophosphatidic acid (LPA) as well as after activation of protein kinase C (PKC) using phorbol-12 myristate-13 acetate (PMA). For positive control RBCs were treated with 4-bromo-A23187. Measurement techniques included flow cytometry and live cell imaging (fluorescence microscopy). RESULTS: The percentage of RBCs showing increased Ca2+ content as well as the PS exposure did not change significantly in dependence on cell age after short-time incubation in control experiments (without stimulating substances) or using LPA or PMA. However, we confirm findings reported that Ca2+ content and the PS exposure of RBCs increased after 48 h incubation. CONCLUSION: No significant differences of intracellular Ca2+ content and PS exposure can be seen for RBCs of different age in resting state or after stimulation of Ca2+ uptake at short-time incubation.
Assuntos
Eritrócitos/efeitos dos fármacos , Fosfatidilserinas/farmacologia , Fatores Etários , Cálcio/metabolismo , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Eritrócitos/citologia , Eritrócitos/metabolismo , Citometria de Fluxo , Humanos , Lisofosfolipídeos/farmacologia , Microscopia de Fluorescência , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
BACKGROUND/AIMS: The increase of the intracellular Ca2+ content as well as the exposure of phosphatidylserine (PS) on the outer cell membrane surface after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA) has been investigated by a variety of research groups. Carrying out experiments, which we described in several previous publications, we observed some discrepancies when comparing data obtained by different investigators within our research group and also between batches of LPA. In addition, we found differences comparing the results of double and single labelling experiments (for Ca2+ and PS). Furthermore, the results of PS exposure depended on the fluorescent dye used (annexin V-FITC versus annexin V alexa fluor® 647). Therefore, it seems necessary to investigate these methodological approaches in more detail to be able to quantify results and to compare data obtained by different research groups. METHODS: The intracellular Ca2+ content and the PS exposure of RBCs separated from whole blood have been investigated after treatment with LPA (2.5 µM) obtained from three different companies (Sigma-Aldrich, Cayman Chemical Company, and Santa Cruz Biotechnology Inc.). Fluo-4 and x-rhod-1 have been used to detect intracellular Ca2+ content, annexin V alexa fluor® 647 and annexin V-FITC have been used for PS exposure measurements. Both parameters (Ca2+ content, PS exposure) were studied using flow cytometry and fluorescence microscopy. RESULTS: The percentage of RBCs showing increased intracellular Ca2+ content as well as PS exposure changes significantly between different LPA manufacturers as well as on the condition of mixing of LPA with the RBC suspension. Furthermore, the percentage of RBCs showing PS exposure is reduced in double labelling compared to single labelling experiments and depends also on the fluorescent dye used. Finally, data on Ca2+ content are slightly affected whereas PS exposure data are not affected significantly by the measuring method (flow cytometry, fluorescence microscopy). CONCLUSION: The LPA batch used and the mixing procedure of LPA and the RBC suspension has to be taken into consideration when comparing results of intracellular Ca2+ content and PS exposure of RBCs after LPA activation. In addition, one should consider that the results of single and double labelling experiments might be different depending on the fluorescent dyes used.
Assuntos
Cálcio/metabolismo , Eritrócitos/metabolismo , Lisofosfolipídeos/metabolismo , Fosfatidilserinas/metabolismo , Cálcio/análise , Eritrócitos/citologia , Citometria de Fluxo , Corantes Fluorescentes/análise , Humanos , Microscopia de Fluorescência , Imagem Óptica , Fosfatidilserinas/análiseRESUMO
BACKGROUND/AIMS: In previous publications we were able to demonstrate the exposure of phosphatidylserine (PS) in the outer membrane leaflet after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA), phorbol-12 myristate-13acetate (PMA), or 4-bromo-A23187 (A23187). It has been concluded that three different mechanisms are responsible for the PS exposure in human RBCs: (i) Ca2+-stimulated scramblase activation (and flippase inhibition) by A23187, LPA, and PMA; (ii) PKCα activation by LPA and PMA; and (iii) enhanced lipid flip flop caused by LPA. Further studies aimed to elucidate interconnections between the increased Ca2+ content, scramblase- and PKCα-activation. In addition, the role of the Ca2+-activated K+ channel (Gardos channel) activity in the process of PS exposure needs to be investigated. METHODS: The intracellular Ca2+ content and the PS exposure of RBCs have been investigated after treatment with LPA (2.5 µM), PMA (6 µM), or A23187 (2 µM). Fluo-4 and annexin V-FITC has been used to detect intracellular Ca2+ content and PS exposure, respectively. Both parameters (Ca2+ content, PS exposure) were studied using flow cytometry. Inhibitors of the scramblase, the PKCα, and the Gardos channel have been applied. RESULTS: The percentage of RBCs showing PS exposure after activation with LPA, PMA, or A23187 is significantly reduced after inhibition of the scramblase using the specific inhibitor R5421 as well as after the inhibition of the PKCα using chelerythrine chloride or calphostin C. The inhibitory effect is more pronounced when the scramblase and the PKCα are inhibited simultaneously. Additionally, the inhibition of the Gardos channel using charybdotoxin resulted in a significant reduction of the percentage of RBCs showing PS exposure under all conditions measured. Similar results were obtained when the Gardos channel activity was suppressed by increased extracellular K+ content. CONCLUSION: PS exposure is mediated by the Ca2+-dependent scramblase but also by PKCα activated by LPA and PMA in a Ca2+-dependent and a Ca2+-independent manner. Furthermore, we hypothesize that a hyperpolarisation of RBCs caused by the opening of the Gardos channel is essential for the scramblase activity as well as for a fraction of the LPA-induced Ca2+ entry.
Assuntos
Calcimicina/farmacologia , Cálcio/metabolismo , Lisofosfolipídeos/farmacologia , Fosfatidilserinas/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Anexina A5/genética , Anexina A5/metabolismo , Benzofenantridinas/farmacologia , Células Cultivadas , Charibdotoxina/farmacologia , Contagem de Eritrócitos , Eritrócitos , Regulação da Expressão Gênica , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Metomil/análogos & derivados , Metomil/farmacologia , Naftalenos/farmacologia , Fosfatidilserinas/química , Proteínas de Transferência de Fosfolipídeos/antagonistas & inibidores , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Transdução de SinaisRESUMO
BACKGROUND/AIMS: Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. METHODS: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. RESULTS: Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. CONCLUSION: An increase of intracellular Ca2+ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs.
Assuntos
Cálcio/farmacologia , Micropartículas Derivadas de Células/ultraestrutura , Eritrócitos/citologia , Proteína Quinase C/metabolismo , Calcimicina/análogos & derivados , Calcimicina/farmacologia , Tamanho Celular/efeitos dos fármacos , Micropartículas Derivadas de Células/efeitos dos fármacos , Micropartículas Derivadas de Células/metabolismo , Contagem de Eritrócitos , Humanos , Lisofosfolipídeos/farmacologia , Microscopia de Força Atômica/métodos , Tamanho da Partícula , Ésteres de Forbol/farmacologia , Fosfatidilserinas/farmacologia , Análise de Célula Única/métodosRESUMO
BACKGROUND: Blood collected in an anticoagulant can be kept refrigerated in an unmodified state within 5 - 6 weeks. Oxidative damage is considered to be a one of the major factors contributing to the development of storage lesions. Lipid and membrane proteins oxidation results in changes in cation gradients that affect the cell survival. AIM: In the present study we used the natural antioxidants and ion channels blockers (L-carnosine, spermine, phloretin and their mixtures) to prolong "survival" of red blood cells (RBCs), measured as the lack of PS exposure and cell hemolysis, in the Alsever's preservative solution upon hypothermic storage. RESULTS: We show that the mixture of carnosine (20 mM), spermine (20 µM) and phloretin (100 µM) effectively blunted phosphatidylserine (PS) exposure, Ca(2+) accumulation and RBCs hemolysis in non-leukoreduced low (â¼ 2%) hematocrit samples after 36 days of storage as well as after 1 day of post-storage incubation of the stored cells in physiological saline solution. In addition, a slight but significant decrease in PS exposure was observed in non-leukoreduced high (â¼ 20%) hematocrit samples after 36 days of storage with the mixture of substances. CONCLUSION: We conclude that the use of the mixture of natural antioxidants (carnosine, spermine, and phloretin) as an additive to blood preservative solution provides better RBCs storage and "survival".
Assuntos
Antioxidantes/farmacologia , Eritrócitos/efeitos dos fármacos , Compostos de Anilina/química , Cálcio/metabolismo , Carnosina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Eritrócitos/citologia , Eritrócitos/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Floretina/farmacologia , Fosfatidilserinas/toxicidade , Espermina/farmacologia , Xantenos/químicaRESUMO
Dipalmitoylphosphatidylcholine (DPPC) and 1,2-palmitoyl-phosphatidic acid (DPPA) liposomes, prepared by conventional rotary evaporation method, have similar structural organization, though they have significant differences. The similarity is that both types of lipids create standard bilayer liposomes with strong hydrophobic forces between lipids tails and with homogeneous bonds of hydrogen and electrostatic nature between hydrophilic lipids heads. By the calorimetric method, it has been shown that hydrophobic bonds break but liposomes' destruction does not occur by heating till 150 °C. As for bonds between lipid heads in liposomes, their cooperative destruction takes place at 41 °C for DPPC and 66 °C for DPPA liposomes. In the case of thermal distraction of DPPC liposomes, two so-called pre transitions peaks were observed before the main transition peak, which indicates that DPPC liposomes' structure is multilamellar. DPPA liposomes have one cooperative heat absorption peak, which points to a unilamellar structure of such liposomes. Substances of hydrophobic/hydrophilic nature, incorporated into the liposomes, are placed in hydrophobic or hydrophilic parts of liposomes, which lead to a change in calorimetric peak shapes and thermodynamic parameters. It has been shown that gold nanoparticles, incorporated into the DPPC liposomes, are able to enter Caco-2 cells. In contrast, these nanoparticles do not enter red blood cells.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Ácidos Fosfatídicos/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Células CACO-2 , Cálcio/metabolismo , Permeabilidade da Membrana Celular , Colesterol/química , Colesterol/metabolismo , Eritrócitos/metabolismo , Fluoresceína/química , Fluoresceína/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Ouro/química , Humanos , Ligantes , Lipossomos , Nanopartículas/química , Tamanho da Partícula , Ácidos Fosfatídicos/metabolismo , TermodinâmicaRESUMO
BACKGROUND/AIMS: Protein kinase Cα (PKCα) is activated by an increase in cytosolic Ca(2+) in red blood cells (RBCs). Previous work has suggested that PKCα directly stimulates the CaV2.1 channel, whereas other studies revealed that CaV2.1 is insensitive to activation by PKC. The aim of this study was to resolve this discrepancy. METHODS: We performed experiments based on a single cell read-out of the intracellular Ca(2+) concentration in terms of Fluo-4 fluorescence intensity and phosphatidylserine exposure to the external membrane leaflet. Measurement modalities included flow cytometry and live cell imaging. RESULTS: Treatment of RBCs with phorbol 12-myristate 13-acetate (PMA) led to two distinct populations of cells with an increase in intracellular Ca(2+): a weak-responding and a strong-responding population. The EC50 of PMA for the number of cells with Ca(2+) elevation was 2.7±1.2 µM; for phosphatidylserine exposure to the external membrane surface, it was 2.8±0.5 µM; and for RBC haemolysis, it was 2.9±0.5 µM. Using pharmacological manipulation with the CaV2.1 inhibitor ω-agatoxin TK and the broad protein kinase C inhibitor Gö6983, we are able to show that there are two independent PMA-activated Ca(2+) entry processes: the first is independent of CaV2.1 and directly PKCα-activated, while the second is associated with a likely indirect activation of CaV2.1. Further studies using lysophosphatidic acid (LPA) as a stimulation agent have provided additional evidence that PKCα and CaV2.1 are not directly interconnected in a signalling chain. CONCLUSION: Although we provide evidence for a lack of interaction between PKCα and CaV2.1 in RBCs, further studies are required to decipher the signalling relationship between LPA, PKCα and CaV2.1.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Eritrócitos/metabolismo , Proteína Quinase C-alfa/metabolismo , Agatoxinas/farmacologia , Compostos de Anilina/química , Cálcio/metabolismo , Canais de Cálcio Tipo N/química , Sinalização do Cálcio/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Citometria de Fluxo , Hemólise , Humanos , Indóis/farmacologia , Cinética , Lisofosfolipídeos/farmacologia , Maleimidas/farmacologia , Fosfatidilserinas/farmacologia , Proteína Quinase C-alfa/antagonistas & inibidores , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia , Xantenos/químicaRESUMO
BACKGROUND/AIMS: In a variety of investigations described in the literature it was not clear to what extent the transmembrane potential red blood cells (RBCs) was changed after the cells have been transferred into low ionic strength (LIS) solutions. Another open question was to find out how fast the transmembrane potential of RBCs in LIS solution will change and which final new equilibrium value will be reached. METHODS: The transmembrane potential of human and bovine RBCs was investigated using the potential-sensitive fluorescent dye DIBAC4(3) (bis(1,3-dibutylbarbituric acid) trimethine oxonol) as well as the CCCP (carbonylcyanide-m-chlorophenylhydrazone) method. RESULTS: Under physiological conditions the transmembrane potential was about -10 mV in agreement with literature data. However, when the RBCs were transferred into an isosmotic low ionic strength medium containing sucrose the transmembrane potential increased to +73 mV and +81 mV for human and bovine RBCs, respectively. In case of human RBCs it continuously decreased reaching finally an equilibrium state of -10 mV again after 30 - 60 min. For bovine RBCs the transmembrane potential declined more slowly reaching a value of +72 mV after 30 min. CONCLUSIONS: Investigations of parameters of RBCs depending on transmembrane potential cannot be performed with human RBCs in LIS media.
Assuntos
Eritrócitos/fisiologia , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/química , Bovinos , Citometria de Fluxo , Corantes Fluorescentes/química , Humanos , Potenciais da Membrana , Concentração OsmolarRESUMO
DIDS, NPPB, tannic acid (TA) and AO1 are widely used inhibitors of Cl(-) channels. Some Cl(-) channel inhibitors (NPPB, DIDS, niflumic acid) were shown to affect phosphatidylserine (PS) scrambling and, thus, the life span of human red blood cells (hRBCs). Since a number of publications suggest Ca(2+) dependence of PS scrambling, we explored whether inhibitors of Cl(-) channels (DIDS, NPPB) or of Ca(2+)-activated Cl(-) channels (DIDS, NPPB, TA, AO1) modified intracellular free Ca(2+) concentration ([Ca(2+)]i) and activity of Ca(2+)-activated K(+) (Gardos) channel in hRBCs. According to Fluo-3 fluorescence in flow cytometry, a short treatment (15 min, +37 °C) with Cl(-) channels inhibitors decreased [Ca(2+)]i in the following order: TA > AO1 > DIDS > NPPB. According to forward scatter, the decrease of [Ca(2+)]i was accompanied by a slight but significant increase in cell volume following DIDS, NPPB and AO1 treatments. TA treatment resulted in cell shrinkage. According to whole-cell patch-clamp experiments, TA activated and NPPB and AO1 inhibited Gardos channels. The Cl(-) channel blockers further modified the alterations of [Ca(2+)]i following ATP depletion (glucose deprivation, iodoacetic acid, 6-inosine), oxidative stress (1 mM t-BHP) and treatment with Ca(2+) ionophore ionomycin (1 µM). The ability of the Cl(-) channel inhibitors to modulate PS scrambling did not correlate with their influence on [Ca(2+)]i as TA and AO1 had a particularly strong decreasing effect on [Ca(2+)]i but at the same time enhanced PS exposure. In conclusion, Cl(-) channel inhibitors affect Gardos channels, influence Ca(2+) homeostasis and induce PS exposure of hRBCs by Ca(2+)-independent mechanisms.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Células Cultivadas , Eletrofisiologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/efeitos dos fármacos , Nitrobenzoatos/farmacologia , Taninos/farmacologiaRESUMO
Extracellular vesicles (EVs) are cell-derived membrane particles that include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies, and other EV subsets. EVs are involved in intercellular communication and the transport of macromolecules between cells. Here, we propose and test the ability of red blood cell (RBC)-derived EVs (RBC-EVs) as putative drug carriers. EVs were produced by treating RBCs with Phorbol-12-myristate-13-acetate (PMA) and separating from the cells by differential centrifugation steps. RBC-EVs were characterized by size determination, flow cytometry, and scanning electron microscopy (SEM). EVs were loaded with DNA plasmids coding for the green fluorescent protein (GFP) by electroporation. The DNA-loaded EVs (DNA-EVs) were used to transfect THP-1-derived macrophages and analyzed by fluorescence microscopy and flow cytometry. The results showed that RBC-EVs had an almost spherical shape and a polydispersity in their size with an average of 197 ± 44 nm and with a zeta potential of -36 ± 8 mV. RBC-EVs were successfully loaded with DNA but associated with an increase of the polydispersity index (PdI) and showed a positive signal with Picogreen. DNA-EVs were almost completely taken up by macrophages within 24 h, however, resulting in the expression of the GFP in a subpopulation of macrophages. As the way, we designed that RBC-EVs could be potential nucleic acid carriers when the immune system was addressed. This study may contribute to the understanding of the role of EVs in the development of microvesicle-based vehicles.
RESUMO
The exposure of phosphatidylserine (PS) on the outer membrane leaflet of red blood cells (RBCs) serves as a signal for eryptosis, a mechanism for the RBC clearance from blood circulation. The process of PS exposure was investigated as function of the intracellular Ca(2+) content and the activation of PKCα in human and sheep RBCs. Cells were treated with lysophosphatidic acid (LPA), 4-bromo-A23187, or phorbol-12 myristate-13 acetate (PMA) and analysed by flow cytometry, single cell fluorescence video imaging, or confocal microscopy. For human RBCs, no clear correlation existed between the number of cells with an elevated Ca(2+) content and PS exposure. Results are explained by three different mechanisms responsible for the PS exposure in human RBCs: (i) Ca(2+)-stimulated scramblase activation (and flippase inhibition) by LPA, 4-bromo-A23187, and PMA; (ii) PKC activation by LPA and PMA; and (iii) enhanced lipid flop caused by LPA. In sheep RBCs, only the latter mechanism occurs suggesting absence of scramblase activity.
Assuntos
Eritrócitos/efeitos dos fármacos , Fosfatidilserinas/farmacologia , Animais , Calcimicina/análogos & derivados , Calcimicina/farmacologia , Cálcio/metabolismo , Eritrócitos/metabolismo , Citometria de Fluxo , Humanos , Lisofosfolipídeos/farmacologia , Ésteres de Forbol/farmacologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteína Quinase C-alfa/metabolismo , OvinosRESUMO
In patients with Gárdos channelopathy (p.R352H), an increased concentration of intracellular Ca2+ was previously reported. This is a surprising finding because the Gárdos channel (KCa3.1) is a K+ channel. Here, we confirm the increased intracellular Ca2+ for patients with the KCa3.1 mutation p.S314P. Furthermore, we provide the concept of KCa3.1 activity resulting in a flickering of red blood cell (RBC) membranepotential, which activates the CaV2.1 channel allowing Ca2+ to enter the RBC. Activity of the nonselective cation channel Piezo1 modulates the aforementioned interplay in away that a closed Piezo1 is in favor of the KCa3.1-CaV2.1 interaction. In contrast, Piezo1 openings compromise the membrane potential flickering, thus limiting the activity of CaV2.1. With the compound NS309, we mimic a gain-of-function mutation of KCa3.1. Assessing the RBC Ca2+ response by Fluo-4-based flow cytometry and by measuring the membrane potential using the Macey-Bennekou-Egée method, we provide data that support the concept of the KCa3.1/CaV2.1/Piezo1 interplay as a partial explanation for an increased number of high Ca2+ RBCs. With the pharmacological inhibition of KCa3.1 (TRAM34 and Senicapoc), CaV2.1 (ω-agatoxin TK), and Piezo1 (GsMTx-4), we could project the NS309 behavior of healthy RBCs to the RBCs of Gárdos channelopathy patients.
Assuntos
Canalopatias , Agatoxinas , Cálcio/metabolismo , Eritrócitos/metabolismo , Humanos , Canais Iônicos/genéticaRESUMO
BACKGROUND: Cryohydrocytosis is an inherited dominant hemolytic anemia characterized by mutations in a transmembrane segment of the anion exchanger (band 3 protein). Transfection experiments performed in Xenopus oocytes suggested that these mutations may convert the anion exchanger into a non-selective cation channel. The present study was performed to characterize so far unexplored ion transport pathways that may render erythrocytes of a single cryohydrocytosis patient cation-leaky. DESIGN AND METHODS: Cold-induced changes in cell volume were monitored using ektacytometry and density gradient centrifugation. Kinetics, temperature and inhibitor-dependence of the cation and water movements in the cryohydrocytosis patient's erythrocytes were studied using radioactive tracers and flame photometry. Response of the membrane potential of the patient's erythrocyte membrane to the presence of ionophores and blockers of anion and cation channels was assessed. RESULTS: In the cold, the cryohydrocytosis patient's erythrocytes swelled in KCl-containing, but not in NaCl-containing or KNO(3)-containing media indicating that volume changes were mediated by an anion-coupled cation transporter. In NaCl-containing medium the net HOE-642-sensitive Na(+)/K(+) exchange prevailed, whereas in KCl-containing medium swelling was mediated by a chloride-dependent K(+) uptake. Unidirectional K(+) influx measurements showed that the patient's cells have abnormally high activities of the cation-proton exchanger and the K(+),Cl(-) co-transporter, which can account for the observed net movements of cations. Finally, neither chloride nor cation conductance in the patient's erythrocytes differed from that of healthy donors. Conclusions These results suggest that cross-talk between the mutated band 3 and other transporters might increase the cation permeability in cryohydrocytosis.
Assuntos
Anemia Hemolítica/genética , Anemia Hemolítica/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/genética , Transporte de Íons , Mutação , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Adulto , Anemia Hemolítica/sangue , Cátions/sangue , Cátions/metabolismo , Temperatura Baixa , Deformação Eritrocítica , Eritrócitos/metabolismo , Humanos , Masculino , Potássio/sangue , Potássio/metabolismoRESUMO
Digital holographic microscopy (DHM) enables a quantitative multifocus phase contrast imaging that has been found suitable for technical inspection and quantitative live cell imaging. The combination of DHM with fast and robust autofocus algorithms enables subsequent automated focus realignment by numerical propagation of the digital holographically reconstructed object wave. In combination with a calibrated optical imaging system, the obtained propagation data quantify axial displacements of the investigated sample. The evaluation of quantitative DHM phase contrast images also enables an effective determination of lateral cell displacements. Thus, 3-D displacement data are provided. Results from investigations on sedimenting red blood cells and HT-1080 fibrosarcoma cells in a collagen tissue model demonstrate that DHM enables marker-free automated quantitative dynamic 3-D cell tracking without mechanical focus adjustment.
Assuntos
Eritrócitos/citologia , Eritrócitos/fisiologia , Holografia/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia/métodos , Reconhecimento Automatizado de Padrão/métodos , Algoritmos , Inteligência Artificial , Movimento Celular/fisiologia , Células Cultivadas , Humanos , Aumento da Imagem/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Processamento de Sinais Assistido por ComputadorRESUMO
Mature human erythrocytes are dependent on anerobic glycolysis, i.e. catabolism (oxidation) of one glucose molecule to produce two ATP and two lactate molecules. Proliferating tumor cells mimick mature human erythrocytes to glycolytically generate two ATP molecules. They deliberately avoid or switch off their respiration, i.e. tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) machinery and consequently dispense with the production of additional 36 ATP molecules from one glucose molecule. This phenomenon is named aerobic glycolysis or Warburg effect. The present review deals with the fate of a glucose molecule after entering a mature human erythrocyte or a proliferating tumor cell and describes why it is useful for a proliferating tumor cell to imitate a mature erythrocyte. Blood consisting of plasma and cellular components (99% of the cells are erythrocytes) may be regarded as a mobile organ, constantly exercising a direct interaction with other organs. Therefore, the use of drugs, which influences the biological activity of erythrocytes, has an immediate effect on the entire organism. Abbreviations: TCA: tricarboxylic acid cycle; OXPHOS: oxidative phosphorylation; GSH: reduced state of glutathione; NFκB: Nuclear factor of kappa B; PKB (Akt): protein kinase B; NOS: nitric oxide synthase; IgG: immune globulin G; H2S: hydrogen sulfide; slanDCs: Human 6-sulfo LacNAc-expressing dendritic cells; IL-8: interleukin-8; LPS: lipopolysaccharide; ROS: reactive oxygen species; PPP: pentose phosphate pathway; NADPH: nicotinamide adenine dinucleotide phosphate hydrogen; R5P: ribose-5-phophate; NAD: nicotinamide adenine dinucleotide; FAD: flavin adenine dinucleotide; O2â-: superoxide anion; G6P: glucose 6-phosphate; HbO2: Oxyhemoglobin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GAP: glyceraldehyde-3-phosphate; 1,3-BPG: 1,3-bis-phosphoglycerate; 2,3-BPG: 2,3-bisphosphoglycerte; PGAM1: phosphoglycerate mutase 1; 3-PG: 3-phosphoglycerate; 2-PG: 2-phosphoglycerate; MIPP1: Multiple inositol polyphosphate phosphatase; mTORC1: mammalian target of rapamycin complex 1; Ru5P: ribulose 5-phosphate; ox-PPP: oxidative branch of pentose phosphate pathway; PGK: phosphoglycerate kinase; IFN-γ: interferon-γ; LDH: lactate dehydrogenase; STAT3: signal transducer and activator of transcription 3; Rheb: Ras homolog enriched in Brain; H2O2: hydrogen peroxide; ROOH: lipid peroxide; SOD: superoxide dismutase; MRC: mitochondrial respiratory chain; MbFe2+-O2: methmyoglobin; RNR: ribonucleotide reductase; PRPP: phosphoribosylpyrophosphate; PPi: pyrophosphate; GSSG: oxidized state of glutathione; non-ox-PPP: non-oxidative branch of pentose phosphate pathway; RPI: ribose-5-phosphate isomerase; RPE: ribulose 5-phosphate 3-epimerase; X5P: xylulose 5-phosphate; TK: transketolase; TA: transaldolase; F6P: fructose-6-phosphate; AR2: aldose reductase 2; SD: sorbitol dehydrogenase; HK: hexokinase; MG: mehtylglyoxal; DHAP: dihydroxyacetone phosphate; TILs: tumor-infiltrating lymphocytes; MCTs: monocarboxylate transporters; pHi: intracellular pH; Hif-1α: hypoxia-induced factor 1; NHE1: sodium/H+ (Na+/H+) antiporter 1; V-ATPase: vacuolar-type proton ATPase; CAIX: carbonic anhydrase; CO2: carbon dioxide; HCO3-: bicarbonate; NBC: sodium/bicarbonate (Na+/HCO3-) symporter; pHe: extracellular pH; GLUT-1: glucose transporter 1; PGK-1: phosphoglycerate kinase 1.
Assuntos
Eritrócitos/metabolismo , Eritrócitos/patologia , Glucose/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Proliferação de Células , Glicólise , HumanosRESUMO
After about 120 days of circulation in the blood stream, erythrocytes are cleared by macrophages in the spleen and the liver. The "eat me" signal of this event is thought to be the translocation of phosphatidylserine from the inner to the outer membrane leaflet due to activation of the scramblase, while the flippase is inactivated. Both processes are triggered by an increased intracellular Ca2+ concentration. Although this is not the only mechanism involved in erythrocyte clearance, in this minireview, we focus on the following questions: Is the intracellular-free Ca2+ concentration and hence phosphatidylserine exposure dependent on the erythrocyte age, i.e. is the Ca2+ concentration, progressively raising during the erythrocyte aging in vivo? Can putative differences in intracellular Ca2+ and exposure of phosphatidylserine to the outer membrane leaflet be measured in age separated cell populations? Literature research revealed less than dozen of such publications with vastly contradicting results for the Ca2+ concentrations but consistency for a lack of change for the phosphatidylserine exposure. Additionally, we performed reanalysis of published data resulting in an ostensive illustration of the situation described above. Relating these results to erythrocyte physiology and biochemistry, we can conclude that the variation of the intracellular free Ca2+ concentration is limited with 10 µM as the upper level of the concentration. Furthermore, we propose the hypothesis that variations in measured Ca2+ concentrations may to a large extent depend on the experimental conditions applied but reflect a putatively changed Ca2+ susceptibility of erythrocytes in dependence of in vivo cell age.
RESUMO
The direct influence of aldosterone on the human and avian red blood cell (RBC) transport systems, Na(+)/K(+) pump, Na(+),K(+),2Cl(-) symporter, and K(+) (Na(+))/H(+) exchanger, was investigated with tracer kinetics. The present work proved that aldosterone has no significant effect on these transport pathways. However, in young human RBCs containing reticulocytes aldosterone showed a significant inhibition of the Na(+),K(+),2Cl(-) symporter. Investigations of the Li(+) efflux via the Na(+)/Li(+) exchanger using atom absorption spectroscopy revealed that aldosterone has no effect on this transporter. Studies of the effect of aldosterone on the Ca(2+) content and the intracellular pH (pH(i)) were carried out on single RBCs with a fluorescence imaging system. Both parameters are affected by aldosterone. The Ca(2+) uptake in the presence of aldosterone, under conditions where the Ca(2+) pump is inhibited, showed marked differences from the control. Since the effect is nifedipine-sensitive, it seems that aldosterone affects a Ca(2+) channel. In addition, aldosterone leads to an acidification of the intracellular medium after an initial alkalisation due to an effect on the Na(+)/H(+) exchanger.
Assuntos
Aldosterona/farmacologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Genoma/genética , Amilorida/farmacologia , Compostos de Anilina/metabolismo , Animais , Cálcio/metabolismo , Galinhas , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Eritrócitos/citologia , Fluorescência , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Transporte de Íons/efeitos dos fármacos , Cinética , Lítio/metabolismo , Potássio/metabolismo , Xantenos/metabolismoRESUMO
Changes of the intracellular Ca2+ content in human red blood cells (RBCs) in glycerol-containing solutions and after freeze-thawing the cells with glycerol and subsequent deglycerolization were investigated with the Ca2+-sensitive fluorescent dye fluo-4 using fluorescence microscopy. In the glycerol-containing solutions the Ca2+ content increased when compared with a physiological medium (Hepes buffered saline solution (HBSS)). This effect was most likely a result of an inhibition of the Ca2+ pump. After inhibiting the Ca2+ pump using o-vanadate, the Ca2+ uptake was not significantly different in the cells in glycerol-containing and physiological medium. Freeze-thawing and deglycerolization of RBCs resulted in a more pronounced increase in the Ca2+ content. Also in this case, the Ca2+ pump seemed to play a major role.