RESUMO
Efficient mechanisms of H(+) ion extrusion are crucial for normal NADPH oxidase function. However, whether the NADPH oxidase-in analogy with mitochondrial cytochromes-has an inherent H(+) channel activity remains uncertain: electrophysiological studies did not find altered H(+) currents in cells from patients with chronic granulomatous disease (CGD), challenging earlier reports in intact cells. In this study, we describe the presence of two different types of H(+) currents in human eosinophils. The "classical" H(+) current had properties similar to previously described H(+) conductances and was present in CGD cells. In contrast, the "novel" type of H(+) current had not been described previously and displayed unique properties: (a) it was absent in cells from gp91- or p47-deficient CGD patients; (b) it was only observed under experimental conditions that allowed NADPH oxidase activation; (c) because of its low threshold of voltage activation, it allowed proton influx and cytosolic acidification; (d) it activated faster and deactivated with slower and distinct kinetics than the classical H(+) currents; and (e) it was approximately 20-fold more sensitive to Zn(2+) and was blocked by the histidine-reactive agent, diethylpyrocarbonate (DEPC). In summary, our results demonstrate that the NADPH oxidase or a closely associated protein provides a novel type of H(+) conductance during phagocyte activation. The unique properties of this conductance suggest that its physiological function is not restricted to H(+) extrusion and repolarization, but might include depolarization, pH-dependent signal termination, and determination of the phagosomal pH set point.
Assuntos
Eosinófilos/metabolismo , Doença Granulomatosa Crônica/metabolismo , Adulto , Criança , Transporte de Elétrons , Ativação Enzimática , Feminino , Doença Granulomatosa Crônica/genética , Humanos , Concentração de Íons de Hidrogênio , Cinética , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Potenciais da Membrana , NADPH Oxidase 2 , NADPH Oxidases/deficiência , NADPH Oxidases/genética , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Explosão RespiratóriaRESUMO
The role of the cytosolic free calcium concentration ([Ca2+]i) and of protein kinase C on the internalization of transferrin and insulin in the human promyelocytic cell line HL60 was investigated. [Ca2+]i was selectively monitored and manipulated by the use of the fluorescent Ca2+ indicator and buffer quin2, while receptor-ligand internalization was studied directly by quantitative electron microscope autoradiography. Decreasing the [Ca2+]i up to 10-fold below resting level had no effect on the internalization of transferrin or insulin. Similarly, a 10-fold elevation of the [Ca2+]i using the calcium ionophore ionomycin caused little or no change in the endocytosis of the two ligands. In contrast, activation of protein kinase C by phorbol myristate acetate markedly stimulated the internalization of both occupied and unoccupied transferrin receptors, even in cells with very low [Ca2+]i. The insulin receptor was found to behave differently in response to phorbol myristate acetate, however, in that only the occupied receptors were stimulated to internalize. We conclude that the [Ca2+]i plays only a minor role in regulating receptor-mediated endocytosis, whereas protein kinase C can selectively modulate receptor internalization depending on receptor type and occupancy.
Assuntos
Cálcio/fisiologia , Endocitose , Insulina/metabolismo , Proteína Quinase C/fisiologia , Transferrina/metabolismo , Linhagem Celular , Humanos , Leucemia Mieloide Aguda , Receptor de Insulina/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores da Transferrina , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Calciosomes are small cytoplasmic vacuoles identified in various nonmuscle cell types by their content of protein(s) similar to calsequestrin (CS), the Ca2+ storage protein of the muscle sarcoplasmic reticulum (SR). These entities have been interpreted as the "primitive" counterpart of the SR, and suggested to be the organelle target of inositol-1,4,5-triphosphate action (Volpe, P., K. H. Krause, S. Hashimoto, F. Zorzato, T. Pozzan, J. Meldolesi, and D. P. Lew. Proc. Natl. Acad. Sci. USA. 85:1091-1095). Immunoperoxidase and immunogold experiments carried out in both thick and ultrathin cryosections of rat hepatocytes and pancreatic acinar cells by using antimuscle CS antibodies revealed a specific labeling widely distributed in the entire cytoplasm, while nuclei were negative. Individual calciosomes appeared as small (105 nm) membrane-bound vacuoles intermingled with, and often apposed to ER cisternae and mitochondria. Other calciosomes were scattered in the Golgi area, in between zymogen granules and beneath the plasma membrane. The cumulative volume of the CS-positive organelles was measured to account for the 0.8 and 0.45% of the cytoplasm in liver and pancreas cells, respectively. The real total volume of the calciosome compartment is expected to be approximately twice as large. In hepatocytes, structures similar to CS-positive calciosomes were decorated by antibodies against the Ca2+ ATPase of muscle SR, while ER cisternae were not. By dual labeling, colocalization was revealed in 53.6% of the organelles, with 37.6% positive for the ATPase only. CS appeared preferentially confined to the content, and the Ca2+ ATPase to the contour of the organelle. The results suggested a partial segregation of the two antigens, reminiscent of their well-known segregation in muscle SR. Additional dual-label experiments demonstrated that hepatic calciosomes express neither two ER markers (cytochrome-P450 and NADH-cytochrome b5 reductase) nor the endolysosome marker, luminal acidity (revealed by 3-[2,4-dinitroanilino]-3'-amino-N-methyl dipropylamine). Calciosomes appear as unique cytological entities, ideally equipped to play a role in the rapid-scale control of the cytosolic-free Ca2+ in nonmuscle cells.
Assuntos
Calsequestrina/análise , Fígado/análise , Proteínas Musculares/análise , Pâncreas/análise , Vacúolos/análise , Animais , ATPases Transportadoras de Cálcio/análise , Retículo Endoplasmático/análise , Secções Congeladas , Complexo de Golgi/análise , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Fígado/ultraestrutura , Masculino , Mitocôndrias/análise , Pâncreas/ultraestrutura , Ratos , Ratos EndogâmicosRESUMO
Cytosolic free calcium ([Ca2+]i) and fusion of secondary granules with the phagosomal membrane (phagosome-lysosome fusion, P-L fusion) were assessed in single adherent human neutrophils during phagocytosis of C3bi-opsonized yeast particles. Neutrophils were loaded with the fluorescent dye fura2/AM and [Ca2+]i was assessed by dual excitation microfluorimetry. Discharge of lactoferrin, a secondary granule marker into the phagosome was verified by immunostaining using standard epifluorescence, confocal laser scanning and electron microscopy. In Ca2(+)-containing medium, upon contact with a yeast particle, a rapid rise in [Ca2+]i was observed, followed by one or more Ca2+ peaks (maximal value 1,586 nM and median duration 145 s): P-L fusion was detected in 80% of the cells after 5-10 min. In Ca2(+)-free medium the amplitude, frequency and duration of the [Ca2+]i transients were decreased (maximal value 368 nM, mostly one single Ca2+ peak and median duration 75 s): P-L fusion was decreased to 52%. Increasing the cytosolic Ca2+ buffering capacity by loading the cells with MAPT/AM led to a dose-dependent inhibition both of [Ca2+]i elevations and P-L fusion. Under conditions where basal [Ca2+]i was reduced to less than 20 nM and intracellular Ca2+ stores were depleted, P-L fusion was drastically inhibited while the cells ingested yeast particles normally. P-L fusion could be restored in Ca2(+)-buffered cells containing ingested particles by elevating [Ca2+]i with the Ca2(+)-ionophore ionomycin. The present findings directly indicate that although the ingestion step of phagocytosis is a Ca2(+)-independent event, [Ca2+]i transients triggered upon contact with opsonized particles are necessary to control the subsequent fusion of secondary granules with the phagosomal membrane.
Assuntos
Cálcio/fisiologia , Fura-2/análogos & derivados , Lisossomos/fisiologia , Neutrófilos/metabolismo , Fagocitose/fisiologia , Fagossomos/fisiologia , Benzofuranos , Cálcio/metabolismo , Citosol/metabolismo , Ácido Egtázico , Imunofluorescência , Corantes Fluorescentes , Humanos , Imuno-Histoquímica , Lactoferrina/metabolismo , Lisossomos/metabolismo , Fusão de Membrana/fisiologia , Microscopia Eletrônica , Fagossomos/metabolismoRESUMO
The sequence of events leading to clathrin-coated pit (CCP) nucleation on the cell surface and to the incorporation of receptors into these endocytic structures is still imperfectly understood. In particular, the question remains as to whether receptor tails initiate the assembly of the coat proteins or whether receptors migrate into preformed CCP. This question was approached through a dissection of the mechanisms implemented by Nef, an early protein of human and simian immunodeficiency virus (HIV and SIV, respectively), to accelerate the endocytosis of cluster of differentiation antigen type 4 (CD4), the major receptor for these viruses. Results collected showed that: (a) Nef promotes CD4 internalization via an increased association of CD4 with CCP; (b) the Nef-mediated increase of CD4 association with CCP is related to a doubling of the plasma membrane area occupied by clathrin-coated structures; (c) this increased CCP number at the plasma membrane has functional consequences preferentially on CD4 uptake and does not significantly affect transferrin receptor internalization or fluid-phase endocytosis; (d) the presence of a CD4 cytoplasmic tail including a critical dileucine motif is required to induce CCP formation via Nef; and (e) when directly anchored to the cytoplasmic side of the plasma membrane, Nef itself can promote CCP formation. Taken together, these observations lead us to propose that CD4 can promote CCP generation via the connector molecule Nef. In this model, Nef interacts on one side with CD4 through a dileucine-based motif present on CD4 cytoplasmic tail and on the other side with components of clathrin-coated surface domain (i.e., adaptins). These Nef-generated complexes would then initiate the nucleation of CCP.
Assuntos
Invaginações Revestidas da Membrana Celular/fisiologia , Endocitose , Produtos do Gene nef/fisiologia , Antígenos CD4/metabolismo , Invaginações Revestidas da Membrana Celular/efeitos dos fármacos , Invaginações Revestidas da Membrana Celular/metabolismo , Citoplasma/fisiologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , HIV-1/fisiologia , Humanos , Leucina/fisiologia , Células Tumorais Cultivadas , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Bradicinina/farmacologia , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Calreticulina , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Citometria de Fluxo , Homeostase , Immunoblotting , Receptores de Inositol 1,4,5-Trifosfato , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Receptores da Bradicinina/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Ribonucleoproteínas/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Tapsigargina/farmacologia , TransfecçãoRESUMO
The concentration of cytosolic ionized calcium, [Ca2+]i, was measured in intact neutrophils by use of a fluorescent indicator trapped in the icytoplasm. A given rise of [Ca2+]i elicited by the chemotactic peptide formylmethionylleucylphenylalanine (FMLP) was associated with a much greater degree of superoxide generation and myeloperoxidase secretion than was the same or larger [Ca2+]i produced by a specific calcium ionophore, ionomycin, which bypasses cell surface receptors. Thus, FMLP appears to generate some important excitatory signal in addition to a rise in [Ca2+]i and exocytosis and superoxide generation in neutrophils may not be simply dependent on [Ca2+]i as is widely supposed.
Assuntos
Cálcio/fisiologia , Exocitose , Neutrófilos/fisiologia , Oxigênio/metabolismo , Superóxidos/metabolismo , Citoplasma/fisiologia , Éteres/farmacologia , Humanos , Ionomicina , Ionóforos/farmacologia , Lisossomos/enzimologia , N-Formilmetionina/análogos & derivados , N-Formilmetionina/farmacologia , N-Formilmetionina Leucil-Fenilalanina , Oligopeptídeos/farmacologiaRESUMO
Subcellular gradients of cytosolic free Ca2+ concentration, [Ca2+]i, are thought to be critical for the localization of functional responses within a cell. A potential but previously unexplored mechanism for the generation of gradients of [Ca2+]i is the accumulation of Ca2+ stores at the site of Ca2+ action. The distribution of the Ca2+ store markers Ca(2+)-dependent adenosine triphosphatase and calreticulin was investigated in resting and phagocytosing human neutrophils. Both proteins showed an evenly distributed fine granular pattern in nonphagocytosing cells, but became markedly concentrated in the filamentous actin-rich cytoplasmic area around the ingested particle during phagocytosis. This redistribution began at early stages of phagocytosis and did not depend on an increase in [Ca2+]i. Thus, accumulation of Ca2+ stores in a restricted area of the cell may contribute to the generation of localized increases in [Ca2+]i.
Assuntos
Cálcio/metabolismo , Neutrófilos/metabolismo , Fagocitose , Actinas/análise , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Calreticulina , Imunofluorescência , Humanos , Neutrófilos/imunologia , Neutrófilos/ultraestrutura , Organelas/metabolismo , Organelas/ultraestrutura , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Ribonucleoproteínas/metabolismoRESUMO
Phagocytosis of C3bi- or IgG-opsonized yeast particles in human neutrophils was found to be associated with an increased formation of inositol phosphates and diacylglycerol. Pertussis toxin only marginally affected phagocytosis of IgG- and C3bi-opsonized particles and the associated formation of second messengers. Forskolin, which induced a threefold rise of cellular cAMP, however, markedly inhibited both C3bi- and IgG-mediated phagocytosis as well as the particle-induced formation of inositol phosphates and diacylglycerol. These observations are in contrast to what was found to occur with chemotactic factors and indicate that chemotactic and phagocytic signaling can be regulated independently in human neutrophils. Since C3bi-mediated phagocytosis has been shown to occur at vanishingly low cytosolic free calcium levels, calcium-depleted cells were used to study the importance of the inositol cycle for the engulfment of C3bi-opsonized particles. Despite a total lack of receptor-induced formation of inositol phosphates, a significantly increased accumulation of diacylglycerol accompanied the ingestion of C3bi-opsonized particles. These data show that the engulfment of C3bi-opsonized particles can occur independently of both a calcium transient and an increased inositol phosphate production. However, the observed accumulation of diacylglycerol, not derived from phosphoinositides, suggests that this second messenger play a role in the control of the engulfment process.
Assuntos
Antígenos de Diferenciação/fisiologia , Cálcio/metabolismo , Diglicerídeos/biossíntese , Glicerídeos/biossíntese , Fosfatos de Inositol/biossíntese , Neutrófilos/fisiologia , Fagocitose , Receptores de Complemento/fisiologia , Receptores Fc/fisiologia , Fosfatos Açúcares/biossíntese , Antígenos de Diferenciação/metabolismo , Colforsina/farmacologia , Complemento C3b/metabolismo , Citosol/metabolismo , Diglicerídeos/metabolismo , Humanos , Imunoglobulina G/metabolismo , Fosfatos de Inositol/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Proteínas Opsonizantes , Toxina Pertussis , Fagocitose/efeitos dos fármacos , Receptores de Complemento/efeitos dos fármacos , Receptores de Complemento 3b , Receptores Fc/metabolismo , Receptores de IgG , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The activation patterns of surface adherent neutrophils are modulated via interaction of extracellular matrix proteins with neutrophil integrins. To evaluate neutrophil bactericidal activity, Staphylococcus aureus adherent to biological surfaces were incubated with neutrophils and serum, and the survival of surface bacteria was determined. When compared to albumin-coated surfaces, the bactericidal activity of neutrophils adherent to purified human extracellular matrix was markedly enhanced (mean survival: 34.2% +/- 9.0% of albumin, P less than 0.0001) despite similar efficient ingestion of extracellular bacteria. Enhancement of killing was observed when surfaces were coated with purified constituents of extracellular matrix, i.e., fibronectin, fibrinogen, laminin, vitronectin, or type IV collagen. In addition to matrix proteins, the tetrapeptide RGDS (the sequence recognized by integrins) crosslinked to surface bound albumin was also active (survival: 74.5% +/- 5.5% of albumin, P less than 0.02), and fibronectin-increased killing was inhibited by soluble RGDS. Chemiluminescence measurements and experiments with CGD neutrophils revealed that both oxygen-dependent and -independent bactericidal mechanisms are involved. In conclusion, matrix proteins enhance intracellular bactericidal activity of adherent neutrophils, presumably by integrin recognition of RGDS-containing ligands. These results indicate a role for extracellular matrix proteins in the enhancement of the host defense against pyogenic infections.
Assuntos
Atividade Bactericida do Sangue , Matriz Extracelular/fisiologia , Neutrófilos/imunologia , Sequência de Aminoácidos , Adesão Celular , Fibrinogênio/fisiologia , Fibronectinas/fisiologia , Doença Granulomatosa Crônica/fisiopatologia , Humanos , Peróxido de Hidrogênio/metabolismo , Técnicas In Vitro , Integrinas/fisiologia , Dados de Sequência Molecular , Oligopeptídeos , Oxigênio/toxicidade , Fagocitose , Staphylococcus aureus/imunologia , Relação Estrutura-AtividadeRESUMO
To study the mediation of Ca2+ influx by second messengers in myeloid cells, we have combined the whole-cell patch clamp technique with microfluorimetric measurements of [Ca2+]i. Me2SO-differentiated HL-60 cells were loaded with the fluorescent Ca2+ indicator Indo-1, allowed to adhere to glass slides, and patch-clamped. Receptor agonists and Ca(2+)-ATPase inhibitors were applied by superfusion and inositol phosphates by microperfusion through the patch pipette. In voltage-clamped cells, [Ca2+]i elevations with a sustained phase could be induced by (a) the chemoattractant receptor agonist FMLP, (b) the Ca(2+)-releasing second messenger myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P3], as well as its nonmetabolizable analogues, and (c) the Ca(2+)-ATPase inhibitor cyclopiazonic acid, which depletes intracellular Ca2+ stores. In the absence of extracellular Ca2+, responses to all stimuli were short-lasting, monophasic transients; however, subsequent addition of Ca2+ to the extracellular medium led to an immediate [Ca2+]i increase. In all cases, the sustained phase of the [Ca2+]i elevations could be inhibited by millimolar concentrations of extracellular Ni2+, and its amplitude could be decreased by depolarization of the plasma membrane. Thus, the sustained phase of the Ca2+ elevations was due to Ca2+ influx through a pathway sensitive to the electrical driving force and to Ni2+. No Ca2+ influx could be observed after (a) plasma membrane depolarization in resting cells, (b) an imposed [Ca2+]i transient independent of receptor activation, or (c) microperfusion of myo-inositol(1,3,4,5)tetrahisphosphate (Ins(1,3,4,5)P4). Also, Ins(1,3,4,5)P4 did not have additive effects when co-perfused with a submaximal concentration of Ins(1,4,5)P3. Our results suggest that, in myeloid cells, activation of chemoattractant receptors induces an electrogenic, Ni(2+)-sensitive Ca2+ influx via generation of Ins(1,4,5)P3. Ins(1,4,5)P3 might activate Ca2+ influx directly, or by depletion of intracellular Ca2+ stores, but not via [Ca2+]i increase or Ins(1,3,4,5)P4 generation.
Assuntos
Cálcio/metabolismo , Fosfatos de Inositol/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Citosol/metabolismo , Humanos , Indóis/farmacologia , Potenciais da Membrana , Células Tumorais CultivadasRESUMO
CD4, a member of the immunoglobulin superfamily, is not only expressed in T4 helper lymphocytes but also in myeloid cells. Receptor-mediated endocytosis plays a crucial role in the regulation of surface expression of adhesion molecules such as CD4. In T lymphocytes p56lck, a CD4-associated tyrosine kinase, prevents CD4 internalization, but in myeloid cells p56lck is not expressed and CD4 is constitutively internalized. In this study, we have investigated the role of cyclic AMP (cAMP) in the regulation of CD4 endocytosis in the myeloid cell line HL-60. Elevations of cellular cAMP were elicited by 1) cholera toxin, 2) pertussis toxin, 3) forskolin and IBMX, 4) NaF, or 5) the physiological receptor agonist prostaglandin E1. All five interventions led to an inhibition of CD4 internalization. Increased cAMP levels did not inhibit endocytosis per se, because internalization of insulin receptors and transferrin receptors and fluid phase endocytosis were either unchanged or slightly enhanced. The mechanism of cAMP inhibition was further analyzed at the ultrastructural level. CD4 internalization, followed either by quantitative electron microscopy autoradiography or by immunogold labeling, showed a rapid and temperature-dependent association of CD4 with clathrin-coated pits in control cells. This association was markedly inhibited in cells with elevated cAMP levels. Thus these findings suggest a second-messenger regulation of CD4 internalization through an inhibition of CD4 association with clathrin-coated pits in p56lck-negative cells.
Assuntos
Antígenos CD4/metabolismo , Invaginações Revestidas da Membrana Celular/fisiologia , Endocitose/imunologia , Receptores de Superfície Celular/fisiologia , Sistemas do Segundo Mensageiro/fisiologia , Antígenos CD4/efeitos dos fármacos , Invaginações Revestidas da Membrana Celular/efeitos dos fármacos , Invaginações Revestidas da Membrana Celular/ultraestrutura , AMP Cíclico/farmacologia , Endocitose/efeitos dos fármacos , Células HL-60 , Humanos , Insulina/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Transferrina/efeitos dos fármacos , Transferrina/metabolismo , Quinases da Família src/metabolismoRESUMO
Rat liver was homogenized and subjected to differential centrifugation. When the low speed nuclear pellet was processed on a Percoll gradient, plasma membrane markers and Ins(1,4,5)P3 binding activity purified together. The high speed (microsomal) fraction was subfractionated by sucrose density gradient centrifugation, resulting in 10-fold enrichment of [32P]-Ins(1,4,5)P3 binding. In the sucrose density gradient fractions there was an inverse relationship between the enrichment of plasma membrane markers and Ins(1,4,5)P3 binding sites. Endoplasmic reticulum markers showed a moderate enrichment in the fractions displaying high Ins(1,4,5)P3 binding activity. Calcium binding proteins in the homogenate and in the microsomal subfractions were separated by SDS/PAGE. A 60 kD protein, stained metachromatically with Stains-All was identified as calreticulin with immunoblotting. Its enrichment pattern was similar to that of Ins(1,4,5)P3 binding sites, indicating the co-existence of these two elements of Ca(2+)-metabolism in the same intracellular compartment in the liver.
Assuntos
Canais de Cálcio , Proteínas de Ligação ao Cálcio/isolamento & purificação , Inositol 1,4,5-Trifosfato , Fígado/química , Receptores de Superfície Celular/isolamento & purificação , Receptores Citoplasmáticos e Nucleares , Ribonucleoproteínas/isolamento & purificação , Frações Subcelulares/química , Animais , Biomarcadores , Calreticulina , Compartimento Celular , Fracionamento Celular , Membrana Celular/química , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Poliacrilamida , Retículo Endoplasmático/química , Feminino , Immunoblotting , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Fígado/ultraestrutura , Microssomos Hepáticos/química , Peso Molecular , Ratos , Ratos WistarRESUMO
Calciosomes are intracellular organelles in HL-60 cells, neutrophils and various other cell types, characterized by their content of a Ca2+-binding protein that is biochemically and immunologically similar to calsequestrin (CS) from muscle cells. In subcellular fractionation studies the CS-like protein copurifies with functional markers of the inositol 1,4,5-trisphosphate (IP3) releasable Ca2+-store. These markers (ATP-dependent Ca2+-uptake and IP3-induced Ca2+-release) show a subcellular distribution which is clearly distinct from the endoplasmic reticulum and other organelles. In morphological studies, antibodies against rabbit skeletal muscle CS protein specifically stained hitherto unrecognized vesicles with a diameter between 50 and 250 nm. Thus both, biochemical and morphological studies indicate that the calsequestrin containing intracellular Ca2+-store, now referred to as the calciosome, is distinct from other known organelles such as endoplasmic reticulum. Calciosomes are likely to play an important role in intracellular Ca2+-homeostasis. They are possibly the intracellular target of inositol 1,4,5-trisphosphate and thus the source of Ca2+ that is redistributed into the cytosol following surface receptor activation in non-muscle cells.
Assuntos
Cálcio/metabolismo , Neutrófilos/metabolismo , Organelas/metabolismo , Retículo Sarcoplasmático/metabolismo , Transporte Biológico Ativo , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Calsequestrina/metabolismo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Humanos , Inositol 1,4,5-Trifosfato , Fosfatos de Inositol/fisiologia , Neutrófilos/ultraestrutura , Organelas/classificaçãoRESUMO
The role of changes in cytosolic free calcium concentration ([Ca2+]i) in the assembly and disassembly of actin during adhesion and phagocytosis was evaluated. Rhodamine-phalloidin staining combined with quantitative fluorescence and confocal laser scanning microscopy was used to measure local F-actin changes in single adherent human neutrophils phagocytosing yeast particles on different surfaces and under different calcium conditions. Cells were suspended in a) calcium-containing medium (CCM) or b) calcium-free medium (CFM) or c) were first depleted of calcium (i.e., MAPT/AM-loaded in CFM) and then suspended in CFM (MAPT). In parallel, local [Ca2+]i changes were monitored using a fura-2 ratio imaging system. In CCM or CFM, attachment to the substrate and formation of pseudopods around a yeast particle generated, within a few seconds, rises in [Ca2+]i, both around the phagosome and in the cell body. During continued phagocytosis, [Ca2+]i was more elevated around the phagosome compared to the rest of the cell. No [Ca2+]i fluctuations were observed in MAPT cells. Adhesion and phagocytosis led to a several-fold increase in F-actin. The increase was transient in cells in CCM and CFM, but remained high in Ca-depleted neutrophils. A distinct ring of F-actin was formed around a phagosome with a yeast particle. Twenty min after ingestion the amount of this actin decreased more than 50% in CCM and CFM cells but increased by 40 to 100% in MAPT cells. The accumulation of F-actin in MAPT cells was reduced to resting levels by adding Ca2+ and ionomycin after ingestion. This treatment reestablished the periphagosomal [Ca2+]i rises, as observed in CCM cells. In conclusion, the present study shows that the actin polymerization, occurring in human neutrophils during adhesion and phagocytosis, is not influenced by changes in [Ca2+]i, whereas the subsequent depolymerization is. The accumulation of actin filaments around the phagosome in calcium-depleted cells could be involved in the inhibition of phagolysosome fusion seen in the absence of [Ca2+]i changes (Jaconi et al., J. Cell Biol. 110, 1555-1564 (1990)). This suggests that the actin network, controlled by [Ca2+]i, regulates the movement of granules during phagocytosis.
Assuntos
Actinas/sangue , Cálcio/metabolismo , Neutrófilos/metabolismo , Fagocitose , Adesão Celular , Humanos , Neutrófilos/imunologia , Neutrófilos/fisiologia , Fagossomos/metabolismoRESUMO
It was previously demonstrated that C3bi- but not IgG-mediated phagocytosis in human neutrophils is a Ca2+-independent process [(1985) Nature 315, 509-511]. The objective of this study was to elucidate the nature of an additional messenger signal(s) that may be involved in receptor-mediated phagocytosis in these cells. The C3bi- and IgG-mediated phagocytic ability of neutrophils was inhibited by forskolin, a potent activator of adenylate cyclase. It was found that forskolin induced a 3-fold increase in cAMP levels and simultaneously reduced the uptake of both C3bi- and IgG-opsonized particles by 65%. This inhibition was reversed by exposure to either phorbol myristate acetate (PMA) or diacylglycerol (OAG), which are both activators of protein kinase C, but not by the inactive analog 4 alpha-PMA. PMA could also restore the phagocytic ability of neutrophils with an experimentally impaired calcium response. These results suggest that activation of protein kinase C might be an important transducing signal controlling receptor-mediated phagocytosis in human neutrophils.
Assuntos
Neutrófilos/fisiologia , Fagocitose , Proteína Quinase C/sangue , Receptores Imunológicos/fisiologia , AMP Cíclico/sangue , Diglicerídeos/farmacologia , Humanos , Técnicas In Vitro , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Proteína Quinase C/fisiologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The present review provides a critical quantitative analysis of the use of quinolones in osteomyelitis. Only papers published in peer-reviewed journals and related to the following areas were selected: experimental osteomyelitis, penetration of quinolones into human bone and clinical use in comparative and non-comparative studies. Cumulated results show clinical success rates of more than 90% in osteomyelitis caused by Enterobacteriaceae, after prolonged oral use of ciprofloxacin. However, further comparative studies using oral quinolones as single agents or in combination (versus standard parenteral therapy) are required in osteomyelitis due to S. aureus or P. aeruginosa, or in more complicated situations such as diabetic osteomyelitis or foreign body infection.
Assuntos
Anti-Infecciosos/uso terapêutico , Osteomielite/tratamento farmacológico , 4-Quinolonas , Animais , Anti-Infecciosos/farmacocinética , Osso e Ossos/metabolismo , Humanos , Osteomielite/metabolismoRESUMO
The present review provides an updated critical analysis of the use of quinolones in osteomyelitis and orthopaedic prosthetic infections. Only papers published in peer-reviewed journals and related to the following areas were selected: experimental osteomyelitis, penetration of quinolones into human bone, and clinical use in comparative and noncomparative studies. Local drug carriers impregnated with quinolones allow high local antibiotic concentrations to be achieved in experimental systems. Considerable clinical experience has been gained mostly with ciprofloxacin and ofloxacin. Cumulated results in clinical trials show clinical success rates of more than 90% in osteomyelitis caused by Enterobacteriaceae. The combination of quinolones and rifampicin for the treatment of staphylococcal osteomyelitis as well as orthopaedic prosthetic infections appears very promising in clinical studies with a small number of patients. However, further comparative studies using quinolones as single agents or in combination (versus standard parenteral therapy) remain necessary in osteomyelitis due to Staphylococcus aureus or Pseudomonas aeruginosa. In particular, studies with the newer quinolones should be strongly encouraged in acute or chronic osteomyelitis and in more complicated situations such as diabetic osteomyelitis or foreign-body infection.
Assuntos
Anti-Infecciosos/uso terapêutico , Osteomielite/tratamento farmacológico , Infecções Relacionadas à Prótese/tratamento farmacológico , 4-Quinolonas , Animais , HumanosRESUMO
Central venous catheters are widely used in clinical practice; however, complications such as venous thrombosis or infection are frequent. The physical and biological effects of a coating procedure designed to improve the blood-contacting properties of polyurethane central venous catheters (CVCs) were studied. The surface atomic composition of poly(vinyl pyrrolidone) (PVP)-coated or uncoated Pellethane single lumen CVCs was characterized by electron spectroscopy for chemical analysis (ESCA), which confirmed the presence of an oxygen-rich PVP layer on the former material. Topological analysis of both single and triple lumen CVCs by scanning force microscopy (SFM) revealed a very smooth surface in PVP-coated catheters compared to the more frequent surface irregularities found either in uncoated Pellethane or in four additional randomly selected, commercially available triple lumen polyurethane CVCs. The PVP-coated Pellethane showed a strong reduction in either fibrinogen or fibronectin adsorption compared to all other PVP-free polyurethane CVCs. This decreased protein adsorption led to a proportional reduction in protein-mediated adhesion of either Staphylococcus aureus or Staphylococcus epidermidis and in the binding of a monoclonal antibody directed against the cell-binding domain of fibronectin. Increased surface smoothness and hydrophilic properties of polyurethane CVCs might decrease the risk of bacterial colonization and infection.
Assuntos
Cateterismo Venoso Central/normas , Cateterismo/normas , Poliuretanos/química , Povidona/química , Adsorção , Anticorpos Monoclonais , Aderência Bacteriana , Microanálise por Sonda Eletrônica , Ensaio de Imunoadsorção Enzimática , Fibronectinas/imunologia , Fibronectinas/metabolismo , Humanos , Microscopia de Força Atômica , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/metabolismo , Staphylococcus epidermidis/metabolismo , Propriedades de SuperfícieRESUMO
Persistent staphylococcal infections are a major medical problem, especially when they occur on implanted materials or intravascular catheters. This review describes some of the recently discovered molecular mechanisms of Staphylococcus aureus attachment to host proteins coating biomedical implants. These interactions involve specific surface proteins, called bacterial adhesins, that recognize specific domains of host proteins deposited on indwelling devices, such as fibronectin, fibrinogen, or fibrin. Elucidation of molecular mechanisms of S aureus adhesion to the different host proteins may lead to the development of specific inhibitors blocking attachment of S aureus, which may decrease the risk of bacterial colonization of indwelling devices.