RESUMO
To study the proliferative response of hematopoietic cells to growth factors at the molecular level, we developed a cell-free system for growth factor-dependent initiation of genomic DNA replication. Nuclei were isolated from the IL-3-dependent cell line NFS/N1-H7 after a 10-h period of IL-3 deprivation. Cytosolic and membrane-containing subcellular fractions were prepared from proliferating NFS/N1-H7 cells. Nuclei from the nonproliferating cells (+/- IL-3) showed essentially no incorporation of [3H]thymidine during a 16-h incubation with a mixture of unlabeled GTP, ATP, UTP, CTP, dGTP, dATP, dCTP, and [3H]dTTP. When the combination of IL-3, a cytosolic fraction, and a membrane-containing fraction from proliferating cells was added to nuclei from nonproliferating cells, a burst of [3H]thymidine incorporation into DNA began after a 12-h lag period, attained a maximal rate at 16 h, and reached a level of 860 pmol thymidine/10(6) nuclei at 24 h (corresponding to replication of approximately 56% total mouse genomic DNA). This DNA synthesis was inhibited approximately 90% by the specific DNA polymerase alpha inhibitor aphidicolin. Deletion of a single cellular component or IL-3 from the system resulted in a marked reduction of DNA replication (-membrane, 80 +/- 4%; -cytosol, 90% +/- 4%; -IL-3, 74 +/- 7% inhibition). This model requires a growth factor (IL-3), a sedimentable cell fraction containing its receptor and possibly additional membrane-associated components, and a cytosolic fraction. It appears to recapitulate the molecular events required for progression from early G1 to S phase of the cell cycle induced by IL-3 binding to its receptor.
Assuntos
Núcleo Celular/metabolismo , Replicação do DNA/efeitos dos fármacos , Interleucina-3/farmacologia , Animais , Afidicolina , Bromodesoxiuridina/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Sistema Livre de Células , Diterpenos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Cinética , Camundongos , Moldes GenéticosRESUMO
The NADPH-dependent O2-.-generating oxidase in subcellular fractions from the neutrophils of three male patients with chronic granulomatous disease was compared with the corresponding preparations from normal neutrophils. The oxidase from normal neutrophils contained flavin adenine dinucleotide in an approximately 0.9:1 molar ratio with cytochrome b559. Each of the three chronic granulomatous disease patients had decreased amounts of the flavoprotein component of the oxidase fraction. The oxidase from two chronic granulomatous disease patients had undetectable amounts of cytochrome b559 whereas the third patient had a normal content of cytochrome b559, which was spectrally indistinguishable from the normal. The intrinsic cytochrome b559 in the oxidase fraction from stimulated neutrophils of the latter chronic granulomatous disease patient was not reduced by NADPH under anaerobic conditions, in distinction with the previously reported reduction of the normal cytochrome b559 under identical conditions. We conclude that the flavoprotein component of the oxidase may mediate transfer of electrons from NADPH to the cytochrome b559 in normal neutrophils, and that deficiency of this flavoprotein is associated with the chronic granulomatous disease phenotype in the three patients studied.
Assuntos
Flavina-Adenina Dinucleotídeo/deficiência , Doença Granulomatosa Crônica/enzimologia , NADH NADPH Oxirredutases/sangue , NADPH Oxidases , NADP/farmacologia , Neutrófilos/enzimologia , Complexo de Proteína do Fotossistema II , Adolescente , Adulto , Grupo dos Citocromos b/sangue , Flavina-Adenina Dinucleotídeo/farmacologia , Humanos , Lactente , Masculino , Oxirredução , Espectrometria de Fluorescência , Frações Subcelulares/enzimologiaRESUMO
Sodium fluoride (20 mM) effected rapid hydrolysis of phosphatidylinositol bisphosphate (PIP2) in human neutrophils. Intracellular free Ca2+ levels increased after PIP2 hydrolysis but before respiratory burst activation. Both the increase in intracellular free Ca2+ levels and the extent of functional activation were dependent on the availability of extracellular Ca2+. The rate of F(-)-stimulated PIP2 hydrolysis, however, was not affected when the rise in cytosolic Ca2+ was severely limited by depletion of extracellular Ca2+. Fluoride caused the specific hydrolysis of PIP2 in isolated neutrophil plasma membranes. This effect occurred in the presence of low levels of available Ca2+ and was accompanied by the release of inositol phosphates. We conclude that PIP2 hydrolysis is an early event in the response of neutrophils to F-. This response is not Ca2+-regulated but may lead to an influx of Ca2+ from the extracellular medium. Activation of a PIP2-specific phospholipase independent of a change in cytosolic free Ca2+ levels may be the initial event in the stimulus-response pathway triggered by fluoride.
Assuntos
Cálcio/sangue , Neutrófilos/metabolismo , Fosfatos de Fosfatidilinositol , Fosfatidilinositóis/sangue , Fluoreto de Sódio/farmacologia , Membrana Celular/metabolismo , Citosol/metabolismo , Humanos , Hidrólise , Cinética , Neutrófilos/efeitos dos fármacos , Oxirredução , Fosfatidilinositol 4,5-Difosfato , Fosfolipídeos/sangueRESUMO
The mRNA for Rvp.1 (rat ventral prostate) increases in abundance before gland involution after androgen deprivation. Rvp.1 is homologous to CPE-R, the high-affinity intestinal epithelial receptor for Clostridium perfringens enterotoxin (CPE), and is sufficient to mediate CPE binding and trigger subsequent toxin-mediated cytolysis. Rvp.1 (claudin-3) and CPE-R (claudin-4) are members of a larger family of transmembrane tissue-specific claudin proteins that are essential components of intercellular tight junction structures regulating paracellular ion flux. However, claudin-3 and claudin-4 are the only family members capable of mediating CPE binding and cytolysis. The present study was designed to study the expression of claudin-3 and claudin-4 in human prostate tissue as potential targets for CPE toxin-mediated therapy for prostate cancer. On human multiple-tissue Northern blot analysis, mRNAs for both claudin-3 and claudin-4 were expressed at high levels in prostate tissue. In normal prostate tissue, expression of claudin-3 was localized exclusively within acinar epithelial cells by in situ mRNA hybridization. Compared with expression within prostate epithelial cells in surrounding normal glandular tissue, expression of claudin-3 mRNA remained high in the epithelium of prostate adenocarcinoma (10 of 10) and prostatic intraepithelial neoplasia (five of five). Prostate adenocarcinoma cells metastatic to bone were obtained from a patient with disease progression during antiandrogen therapy. These metastatic cells were prostate-specific antigen-positive by immunohistochemical staining and also expressed functional CPE receptors as measured by sensitivity to CPE-induced cell lysis. The persistent high level of claudin-3 expression in prostate adenocarcinoma and functional cytotoxicity of CPE in metastatic androgen-independent prostate adenocarcinoma suggests a new potential therapeutic strategy for prostate cancer.
Assuntos
Adenocarcinoma/metabolismo , Enterotoxinas/toxicidade , Proteínas de Membrana/biossíntese , Neoplasias da Próstata/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/secundário , Androgênios/fisiologia , Animais , Neoplasias da Medula Óssea/tratamento farmacológico , Neoplasias da Medula Óssea/metabolismo , Neoplasias da Medula Óssea/secundário , Chlorocebus aethiops , Claudina-3 , Claudina-4 , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Enterotoxinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Masculino , Proteínas de Membrana/genética , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias da Próstata/tratamento farmacológico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Células VeroRESUMO
We previously showed that the proliferative response of a serum- and interleukin-3 (IL-3)-dependent murine myeloid cell line, NFS/N1-H7, was partially inhibited by pertussis toxin as a result of toxin-induced increased adenylate cyclase activity. In the present studies, we examined the role of the phosphoinositide cycle in the proliferative response of these cells and demonstrated that there was no change in PIP (phosphatidylinositol bisphosphate)-specific phospholipase C activity in response to IL-3 alone. However, serum caused a pertussis toxin-insensitive increase in PIP2-specific phospholipase C activity as reflected by decreased cellular levels of 32P-labelled PIP2. Proliferation of a subline selected from val-12-mutant H-ras-transfected NFS-H7 cells, clone E5, was insensitive to pertussis toxin, occurred in the absence of serum but remained serum-stimulatable and absolutely dependent on IL-3. This val-12 mutant ras-expressing cell line showed an increase in 32P-labelled PIP (phosphatidylinositol phosphate) in response to serum whereas the parent cell line did not. Membrane fractions from 32P-labelled ras-transfected cells displayed higher GTP gamma S-, GTP-, or F(-)-stimulated PIP2-specific phospholipase C activity compared to membranes from the parent cell line. Thus serum-dependence and adenylate cyclase-mediated pertussis toxin-sensitivity of the parent cell line was bypassed by val-12 mutant ras p21, possibly as a result of increased PIP2-specific phospholipase C activity.
Assuntos
Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Fosfolipases Tipo C/metabolismo , Toxina Adenilato Ciclase , Animais , Sangue , Divisão Celular/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Meios de Cultura , Interleucina-3/fisiologia , Cinética , Camundongos , Mutação , Toxina Pertussis , Fosfatidilinositol 4,5-Difosfato , Fosfatidilinositóis/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Timidina/metabolismo , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The functional role of a mutant RAS gene in immortal myeloid cell proliferation was examined in a fastidious interleukin-3 (IL-3) dependent cell line (NFS/N1.H7) formed by forced proliferation in IL-3 of marrow cells of the NFS/N mouse. The NFS/N1.H7 cell line was strictly dependent upon IL-3 for growth, and the cell line could be activated by phorbol esters (PMA) to augment IL-3 dependent proliferation, but when pKC was downregulated, diminished IL-3 proliferative response resulted. Transfection (electroporation) of the T24 RAS-containing vector pAL8 to NFS/N1.H7 led to clones (H7 NeoRas.F3, H7 NeoRas.E2) that had incorporated the entire 6.6 Kb human mutant H-RAS genome. The mutant RAS-containing clones demonstrated greater proliferation than parent cells or cells containing a control (neo-resistance) vector over a range of suboptimal IL-3 does and in optimal IL-3 concentrations had a faster doubling rate than parent cells. The clone H7 NeoRas.F3 was studied biochemically, and found to constitutively form 3-fold more 3H-diacylglycerol than the parent cell line upon exposure to 3H-glycerol. PMA could partially repair the proliferative defect of NFS/N1.H7 compared to the RAS-expressor. These studies affirm a secondary, accelerating role for a mutant RAS gene product acting through pKC to promote clonal expansion of immortal myeloid cells stimulated by IL-3.
Assuntos
Células da Medula Óssea , Proteínas de Caenorhabditis elegans , Divisão Celular/efeitos dos fármacos , Genes ras , Interleucina-3/farmacologia , Proteína Quinase C/fisiologia , Animais , Proteínas de Transporte , Linhagem Celular , Diglicerídeos/fisiologia , Ativação Enzimática , Camundongos , Mutação , Receptores de Droga/metabolismo , TransfecçãoRESUMO
A relationship was proved between constitutive activity of leukemic cell c-jun-N-terminal kinase (JNK) and treatment failure in AML. Specifically, early treatment failure was predicted by the presence of constitutive JNK activity. The mechanistic origins of this association was sought. A multidrug resistant leukemic cell line, HL-60/ADR, characterized by hyperexpression of c-jun and JNK activity, was transfected with a mutant c-jun vector, whose substrate N-terminal c-jun serines were mutated. Down-regulated expression occurred of c-jun/AP-1-dependent genes, catalase and glutathione-S-transferase (GST) pi, which participate in cellular homeostasis to oxidative stress and xenobiotic exposure. MRP-efflux was abrogated in HL-60/ADR cells with dominant-negative c-jun, perhaps because MRP1 protein expression was also lost. Heightened sensitivity to daunorubicin resulted in cells subjected to this change. Biochemical analysis in 67 primary adult AML samples established a statistical correlation between cellular expression of c-jun and JNK activity, JNK activity with hyperleukocytosis at presentation of disease, and with exuberant MRP efflux. These findings reflect the survival role for c-jun/AP-1 and its regulatory kinase previously demonstrated for yeast in homeostatic response to oxidative stress and in operation of ATP-binding cassette efflux pumps, and may support evolutionary conservation of such function. Thus, JNK and c-jun may be salient drug targets in multidrug resistant AML.
Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Leucemia Mieloide/enzimologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Doença Aguda , Células da Medula Óssea/patologia , Divisão Celular , Daunorrubicina , Resistência a Múltiplos Medicamentos/fisiologia , Células HL-60 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de SinaisRESUMO
The factor-dependent cell line FDC-P1 has been utilized as a model of interleukin 3 (IL-3)-dependent myeloid cell proliferation. However, it has been recently observed that active phorbol esters (e.g., phorbol 12-myristate 13-acetate) may entirely replace IL-3 to promote its proliferation. These observations reveal abnormal regulation of protein kinase C (pkC) (absence of downregulation or overexpression). This property allowed a test of the hypothesis that the T24 RAS (codon 12) oncogene acts by constitutive and persistent pkC activation, driving proliferation. FDC-P1 cells were transfected by electroporation with the T24 RAS-containing vector pAL 8, or with a control vector pSVX Zip Neo, and neomycin-resistant clones were selected. Multiple RAS-transfectant clones were categorized for their growth factor requirement and incorporation of the 6.6-kb human mutant H-RAS genome. IL-3-independent clones had incorporated multiple (more than two) copies of the entire 6.6-kb RAS genome. The incorporation of multiple 6.6-kb RAS genomes was correlated with high-level p21 RAS expression. No evidence for autostimulatory growth factor production by clones containing the RAS oncogene was observed. Thus, acquisition of growth factor independence in myeloid cells by abundant expression of a RAS oncogene is linked, in part, to abnormal regulation of pkC, which acts as a collaborating oncogene.
Assuntos
Transformação Celular Neoplásica , Genes ras , Granulócitos/citologia , Proteína Quinase C/metabolismo , Divisão Celular , Linhagem Celular , Regulação para Baixo , Resistência a Medicamentos/genética , Ativação Enzimática , Expressão Gênica , Vetores Genéticos , Granulócitos/enzimologia , Humanos , Interleucina-3/farmacologia , Mutação , Neomicina , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
Certain qualitative abnormalities in neutrophils and blood monocytes are associated with frequent, severe, and recurrent bacterial infections leading to fatal sepsis, while other qualitative defects demonstrated in vitro may have few or no clinical sequelae. These qualitative defects are discussed in terms of the specific functions of locomotion, phagocytosis, degranulation, and bacterial killing.
Assuntos
Leucócitos/fisiopatologia , Actinas/biossíntese , Atividade Bactericida do Sangue , Movimento Celular , Síndrome de Chediak-Higashi/sangue , Fatores Quimiotáticos/biossíntese , Proteínas do Sistema Complemento/deficiência , Grânulos Citoplasmáticos/fisiopatologia , Deficiência de Glucosefosfato Desidrogenase/sangue , Doença Granulomatosa Crônica/sangue , Humanos , Imunoglobulina E , Recém-Nascido , Monócitos/fisiopatologia , Neutrófilos/fisiopatologia , Oxigênio/sangue , Fagócitos/fisiologia , Fagocitose , SíndromeRESUMO
A subcellular particulate fraction from normal neutrophils that was enriched in NADPH-dependent O-.2-generating activity (Gabig, T. G., Schervish, E. W., and Santinga, J. T. (1982) J. Biol. Chem. 257, 4114-4119) has been further characterized. This preparation contained 0.25 +/- 0.02 nmol of flavin adenine dinucleotide/mg of protein and 0.28 +/- 0.01 nmol of cytochrome b/mg of protein. Measurable amounts of riboflavin or flavin mononucleotide were not present. The flavoprotein was completely resolved from the cytochrome b by selective bile salt extraction of the particulate oxidase fraction. The identical subcellular particulate fraction was studied in the neutrophils from two male patients with chronic granulomatous disease. The neutrophil oxidase fraction from one of the chronic granulomatous disease patients had a cytochrome b component that was spectrally abnormal, but a normal content of flavin adenine dinucleotide. The fraction from this patient's neutrophils corresponding to the resolved flavoprotein from normal cells had fluorescence excitation and emission spectra that were identical to the normal flavoprotein. The neutrophil oxidase fraction from the second chronic granulomatous disease patient had a quantitatively and spectrally normal cytochrome b but less than 8% of the normal amount of flavin adenine dinucleotide. The fraction from the latter patient's neutrophils corresponding to the resolved flavoprotein from normal cells had no detectable flavoprotein by fluorescence excitation and emission spectroscopy. It is postulated that these two patients represent distinct mutants in two separate components of the neutrophil NADPH-dependent O-.2-generating oxidase system, flavoprotein and cytochrome b.
Assuntos
Flavoproteínas/sangue , Doença Granulomatosa Crônica/sangue , NADP/farmacologia , Neutrófilos/enzimologia , Oxirredutases/sangue , Grupo dos Citocromos b/sangue , Flavina-Adenina Dinucleotídeo/sangue , Humanos , Masculino , Espectrometria de Fluorescência , Superóxidos/sangueRESUMO
Intact neutrophils possess a cellular mechanism that efficiently deactivates the microbicidal O2-generating NADPH oxidase during the respiratory burst (Akard, L. P., English, D., and Gabig, T. G. (1988) Blood 72, 322-327). The present studies directed at identifying the molecular mechanism(s) involved in NADPH oxidase deactivation showed that a heat- and trypsin-insensitive species in the cytosolic fraction from normal unstimulated neutrophils was capable of deactivating the membrane-associated NADPH oxidase isolated from opsonized zymosan- or phorbol 12-myristate 13-acetate-stimulated neutrophils. This cytosolic species also deactivated the cell-free-activated oxidase. Deactivation by this cytosolic species occurred in the absence of NADPH-dependent catalytic turnover and was reversible, since NADPH oxidase activity could be subsequently reactivated in the cell-free system. The sedimentable particulate fraction from unstimulated neutrophils did not demonstrate deactivator activity. Deactivator activity was demonstrated in the neutral lipid fraction of neutrophil cytosol extracted with chloroform:methanol. Following complete purification of cytosolic deactivator activity by thin layer chromatography and reversed phase high performance liquid chromatography, the deactivator species was shown to be a lipid thiobis ester compound by mass spectroscopy. Cellular metabolism of this compound in human neutrophils may reveal a unique mechanism for enzymatic control of the NADPH oxidase system and thereby play an important role in regulation of the inflammatory response.
Assuntos
NADH NADPH Oxirredutases/sangue , NADPH Oxidases , Neutrófilos/metabolismo , Propionatos/sangue , Sulfetos/sangue , Superóxidos/sangue , Membrana Celular/enzimologia , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Citosol/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Cinética , NADH NADPH Oxirredutases/antagonistas & inibidores , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Propionatos/isolamento & purificação , Sulfetos/isolamento & purificação , Acetato de Tetradecanoilforbol/farmacologia , Zimosan/farmacologiaRESUMO
The article reviews the enzymatic and electron transfer properties of a low-potential FAD-dependent flavoprotein that is a component of the NADPH-dependent O2-.-generating respiratory burst oxidase of phagocytes. Current methods available for isolation of the respiratory burst oxidase and the flavoprotein component of the complex are also reviewed. These studies and data obtained from affinity-labeling of respiratory burst oxidase components, suggest that the flavoprotein has a molecular weight of 65-67 kD. The prevailing evidence suggests that the flavoprotein functions as a dehydrogenase/electron transferase and can directly catalyse NADPH-dependent O2-.formation when isolated. However, in neutrophil plasma membranes, the prevailing evidence suggests that the flavoprotein functions primarily to transfer electrons from NADPH to cytochrome b-245 and that this latter redox component is the catalytic side of O2-.formation. A working model for the arrangement of the flavoprotein and cytochrome b-245 components of the respiratory burst oxidase in neutrophil membranes is proposed.
Assuntos
Flavoproteínas/isolamento & purificação , NADH NADPH Oxirredutases/isolamento & purificação , NADPH Oxidases , Flavoproteínas/metabolismo , Humanos , NADH NADPH Oxirredutases/metabolismo , Neutrófilos/enzimologia , Superóxidos/metabolismoRESUMO
NADPH-oxidase of stimulated human neutrophil membranes was solubilized in Triton X-100 and activity reconstituted with FAD, 8-F-FAD, 8-phenyl-S-FAD, and 8-S-FAD. The enzyme had similar affinities for all the flavins with Km values in the 60-80 nM range. Vmax was found to increase 4-fold with increasing redox midpoint potential of the flavin. 8-F-FAD reconstituted with the enzyme was reactive toward thiophenol, suggesting exposure of the 8-position to solvent, a finding supported by unsuccessful attempts to label the enzyme with the photoaffinity probe 8-N3-[32P]FAD. Solubilized oxidase stabilized the red thiolate form of 8-S-FAD, a characteristic of flavoproteins of the dehydrogenase/electron transferase classes which stabilize the blue neutral form of the flavin semiquinone radical.
Assuntos
Flavina-Adenina Dinucleotídeo/análogos & derivados , Flavina-Adenina Dinucleotídeo/farmacologia , NADH NADPH Oxirredutases/sangue , Neutrófilos/enzimologia , Membrana Celular/enzimologia , Detergentes , Humanos , Cinética , NADH NADPH Oxirredutases/isolamento & purificação , NADPH Oxidases , Octoxinol , Fagócitos/enzimologia , Polietilenoglicóis , Ligação Proteica , SolubilidadeRESUMO
Neutrophil adherence stimulated by phorbol myristate acetate (PMA) was investigated by quantitating the attachment of 51Cr-labeled neutrophils to plastic surfaces and to the endothelium of umbilical veins mounted in compartmentalized Lucite chambers. PMA-induced adherence could be functionally separated into an induction phase requiring cellular metabolism and a Mg++ dependent maintenance phase that was independent of cellular metabolism. Thus, metabolic inhibitors (N-ethylmaleimide, 2-deoxyglucose) blocked adherence when added to neutrophils prior to PMA, but did not cause detachment of cells adhering as a consequence of prior exposure to PMA. PMA failed to induce adherence of neutrophils incubated at low (0.4 degree C) temperature, but temperature reduction, even for prolonged periods, did not cause detachment of adherent cells. Thus, the attractive forces that mediate stimulated adherence persist independently of any sustained metabolic response to the inducing stimulus. However, removal of Mg++ from the media above adherent cells resulted in immediate detachment, indicating that the cation was required for the persistent expression or maintenance of the attractive forces involved. The extent of stimulated adherence correlated well with the extent of degranulation when rates were varied by limiting the incubation time or stimulus concentration. This correlation was not absolute; in the absence of Mg++, PMA induced degranulation normally but failed to enhance adherence. To explain these findings, we investigated the possibility that PMA-stimulated adherence was maintained by Mg++-dependent cellular adherence molecules released during exocytosis. Supernatants of stimulated neutrophils were devoid of adherence-promoting activity, and only weak activity was recovered in supernatants of mechanically disrupted neutrophils. PMA effectively stimulated the tight adherence of degranulated neutrophil cytoplasts to plastic surfaces and did so in the absence of stimulated granule enzyme release. Thus, conditions have been identified under which degranulation occurs in the absence of adherence (removal of Mg++) and adherence occurs without concurrent degranulation. Since neutrophil cytoplasts do contain some granule products and granule material can be identified on cytoplast membranes, it is possible that degranulation or granule products may be involved in the adherent response. However, hyperadherence was shown to develop in the absence of de novo degranulation.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Neutrófilos/citologia , Adesão Celular/efeitos dos fármacos , Diferenciação Celular , Citocalasina B/farmacologia , Etilmaleimida/farmacologia , Humanos , Magnésio/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Zimosan/metabolismoRESUMO
Chronic granulomatous disease (CGD) is a genetically transmitted disorder thought to result from defect(s) in the activation or turnover of the NADPH dependent O2- generating oxidase enzyme system of human neutrophils and monocytes. The normal oxidase may be a flavoprotein-cytochrome b559 complex; therefore, these components of the oxidase were quantitated in the neutrophils from patients and family members of two unrelated CGD kindreds. The male propositus from an X-linked recessive kindred had a neutrophil oxidase fraction with low FAD content (26 pmol/mg protein) and undetectable cytochrome b559 (less than 5 pmol/mg protein). The male propositus from an autosomal recessive kindred had a neutrophil oxidase fraction with low FAD content (34 pmol FAD/mg protein), but normal cytochrome b559 content (170 pmol cytochrome b559/mg protein). Both parents of this latter CGD patient had normal FAD and cytochrome b559 content in their neutrophil oxidase fraction. We conclude that the carrier state in certain X-linked recessive female carriers of CGD can be detected by partial deficiencies of both flavoprotein and cytochrome b559 components of the oxidase, whereas presumed heterozygous carriers of certain autosomal recessive CGD kindreds cannot be detected by this means.
Assuntos
Doença Granulomatosa Crônica/metabolismo , Neutrófilos/metabolismo , Fagócitos/metabolismo , Complexo de Proteína do Fotossistema II , Superóxidos/metabolismo , Criança , Grupo dos Citocromos b/análise , Feminino , Flavina-Adenina Dinucleotídeo/análise , Radicais Livres , Doença Granulomatosa Crônica/genética , Heterozigoto , Humanos , Masculino , Nitroazul de Tetrazólio/metabolismoRESUMO
The enzymatic activity underlying the respiratory burst in human neutrophils was examined in a subcellular fraction with high specific activity and shown to be a membrane-associated complex of a flavoprotein, ubiquinone-10, and cytochrome b559 in an approximate 1.3:1:2 molar ratio. Study of the redox poise of these electron carriers indicated that electron flow in the intact complex from unstimulated cells proceeded: NADPH----E-FAD----ubiquinone-10. Similar studies on the complex prepared from stimulated neutrophils indicated that electron flow proceeded: NADPH----E-FAD----ubiquinone-10----cytochrome b559----oxygen. The active enzyme complex was inhibited by p-chloromercuribenzoate. Inhibition persisted after removal of excess inhibitor, was reversed by dithiothreitol, and could be blocked by prior addition of substrate (NADPH). Inhibition of the active oxidase complex by p-chloromercuribenzoate also inhibited electron flow from NADPH to all purported electron carriers in the chain (i.e. E-FAD, ubiquinone-10, and cytochrome b559). We conclude that activation of the oxidase enzyme complex in the intact neutrophil resulted in linkage of electron carrier function between endogenous ubiquinone-10 and cytochrome b559 and was without demonstrable effect on proximal electron flow. The p-chloromercuribenzoate sensitive site(s) proximal to the initial electron acceptor (E-FAD) did not appear to be altered by the cellular activation process.
Assuntos
Grupo dos Citocromos b/metabolismo , NADH NADPH Oxirredutases/metabolismo , Neutrófilos/enzimologia , Complexo de Proteína do Fotossistema II , Ubiquinona/metabolismo , 4-Cloromercuriobenzenossulfonato/farmacologia , Cromatografia Líquida de Alta Pressão , Ditiotreitol/farmacologia , Ativação Enzimática , Humanos , NADPH Oxidases , OxirreduçãoRESUMO
The resolved flavoprotein and cytochrome b559 components of the NADPH dependent O2- . generating oxidase from human neutrophils were the subject of further study. The resolved flavoprotein, depleted of cytochrome b559, was reduced by NADPH under anaerobic conditions and reoxidized by oxygen. NADPH dependent O2- . generation by the resolved flavoprotein fraction was not detectable, however it was competent in the transfer of electrons from NADPH to artificial electron acceptors. The resolved cytochrome b559, depleted of flavoprotein, demonstrated no measureable NADPH dependent O2- . generating activity and was not reduced by NADPH under anaerobic conditions. The dithionite reduced form of the resolved cytochrome b559 was rapidly oxidized by oxygen, as was the cytochrome b559 in the intact oxidase.
Assuntos
Grupo dos Citocromos b/sangue , Flavoproteínas/sangue , NADH NADPH Oxirredutases/sangue , NADPH Oxidases , Neutrófilos/enzimologia , Aerobiose , Anaerobiose , Grupo dos Citocromos b/isolamento & purificação , Flavoproteínas/isolamento & purificação , Humanos , Cinética , NADH NADPH Oxirredutases/isolamento & purificação , Espectrometria de FluorescênciaRESUMO
The cell-free system for activation of the neutrophil NADPH oxidase allowed us to examine activation of the oxidase in the absence of its NADPH-dependent turnover. The covalent sulfhydryl-modifying reagent N-ethylmaleimide completely inhibited the activation step (Ki = 40 mumol/L) in the cell-free system but had no effect on turnover of the preactivated particulate NADPH oxidase (up to 1 mmol/L). When N-ethylmaleimide was added to intact neutrophils during the period of maximal O2 generation in response to stimuli that activate the respiratory burst (phorbol myristate acetate, f-Met-Leu-Phe, opsonized zymosan, arachidonic acid), O2- generation ceased within seconds. Study of components of the cell-free activation system indicated that the cytosolic cofactor was irreversibly inhibited by N-ethylmaleimide whereas the N-ethylmaleimide-treated, membrane-associated oxidase could be activated by arachidonate and control cytosolic cofactor. Likewise, the cell-free system prepared from intact neutrophils that had been briefly exposed to N-ethylmaleimide and then washed reflected the effects of N-ethylmaleimide on the isolated cell-free components: cytosolic cofactor activity was absent, but the membrane oxidase remained fully activatable. Thus inhibition of oxidase activation by N-ethylamaleimide unmasked a rapid deactivation step that was operative in intact neutrophils but not in isolated particulate NADPH oxidase preparations. The demonstrated specificity of N-ethylmaleimide for oxidase activation and lack of effect on turnover of the NADPH oxidase suggested that sustained O2- generation by intact neutrophils was a result of continued replenishment of a small pool of active oxidase. The existence of an inactive pool of NADPH oxidase molecules in particulate preparations from stimulated neutrophils was supported more directly by activating these preparations again in the cell-free system.
Assuntos
NADH NADPH Oxirredutases/sangue , Neutrófilos/enzimologia , Consumo de Oxigênio , Sistema Livre de Células/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/enzimologia , Citosol/fisiologia , Ativação Enzimática/efeitos dos fármacos , Etilmaleimida/farmacologia , Humanos , NADH NADPH Oxirredutases/antagonistas & inibidores , NADPH Oxidases , Neutrófilos/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Superóxidos/biossínteseRESUMO
gamma-Hexachlorocyclohexane was found to exert profound effects on the phosphatidylinositol cycle, cytosolic calcium level, and the respiratory burst of human neutrophils. Exposure of neutrophils prelabelled with 32P to 4 X 10(-4) M gamma-hexachlorocyclohexane almost tripled radioactivity in phosphatidic acid and correspondingly decreased radioactivity in phosphatidylinositol 4,5 bisphosphate. Under similar conditions, gamma-hexachlorocyclohexane evoked the generation of superoxide at a rate of over 11 nmol/min/10(6) cells and more than doubled cytosolic-free calcium concentration as monitored by Quin-2 fluorescence. Because intermediates of the phosphatidylinositol cycle, via increases in available calcium levels or activated protein kinase C, are considered potential second messengers for activation of the NADPH-dependent O-2-generating system, we compared neutrophil responses to gamma-hexachlorocyclohexane with responses to phorbol myristate acetate, an activator of protein kinase C with well known effects on neutrophils. Like phorbol myristate acetate, gamma-hexachlorocyclohexane induced neutrophil degranulation but was not an effective chemotactic stimulus. The ability of gamma-hexachlorocyclohexane to induce a pattern of oxidative activation in neutrophil cytoplasts similar to that in intact cells indicated that concurrent degranulation was not required for sustained O-2 generation in response to this agent. When neutrophils or neutrophil cytoplasts exposed to gamma-hexachlorocyclohexane were centrifuged and resuspended in stimulus-free medium, O-2 generation ceased entirely but could be reinitiated by addition of the same stimulus. This finding was in contrast to the continued O-2 production by phorbol myristate acetate-stimulated neutrophils similarly washed and resuspended in stimulus-free medium. Unlike subcellular fractions of phorbol myristate acetate-stimulated neutrophils, corresponding fractions prepared from gamma-hexachlorocyclohexane-stimulated neutrophils contained almost no detectable NADPH-dependent O-2-generating activity. Subcellular oxidase activity was not recovered when cells and membrane fractions were continuously exposed to gamma-hexachlorocyclohexane during disruption and fractionation after cell stimulation, nor could it be induced by the addition of the stimulus to the subcellular fractions. Thus, the stimulus dependence of continuous neutrophil superoxide release evoked by gamma-hexachlorocyclohexane does not merely reflect a physical interaction of the agonist with the enzyme system involved.(ABSTRACT TRUNCATED AT 400 WORDS)