RESUMO
Phosphatidylinositol (PtdIns) synthesis and polyphosphoinositide (PPI) formation were measured as the incorporation of [32P]orthophosphate ([32P]Pi) or [3H]inositol into non-stimulated intact human neutrophil membrane phospholipids. The rate of PtdIns "de novo" synthesis appeared to be a slow mechanism when compared to the rapid incorporation of [32P]Pi into PPIs. Of the "de novo" synthesized [3H]PtdIns, 70% was further phosphorylated to PPI. Nevertheless, this PPI pool represented less than 0.01% of the total nmols of PPIs formed evaluated as [32P]Pi labeling, indicating that PPI formation mainly involves a no "de novo" synthesized phosphatidylinositol pool. When evaluated at short incubation times, oscillations in the formation of PPIs were detected. A rapid phase was characterized after 30 s of incubation with [32P]Pi Phosphorylation levels returned to an equilibrium state within a minute, and the second phase peaked at 5 min., returning to equilibrium at 15 min. The fluctuant kinetics though not the equilibrium level of PPI formation, could be abolished by neomycin. On the other hand, a selective inhibition of the rapid phase of PPI synthesis occurred in the presence of the tyrosine kinase inhibitor genistein. When the incorporations of [gamma-32P]-adenosine triphosphate (ATP) or [32P]Pi into human neutrophil particulate fraction membranes were evaluated, PPIs synthesis showed fluctuations independently of the precursor used. Noticeably, [32P]from [32P]Pi was incorporated more efficiently into PPIs than that from [gamma-32P]ATP, when evaluated in parallel using equal specific activities for both radiolabeled precursors and under non-ATP synthesizing conditions. Moreover, the incorporation of [32P]Pi into particulate fraction PPIs was not abolished by high concentrations of non-radiolabeled ATP, and metabolically inhibited PMNs showed high rates of PPI synthesis. These data suggest that PPI formation is not necessarily a futile cycle in PMNs.
Assuntos
Lipídeos de Membrana/biossíntese , Neutrófilos/metabolismo , Fosfatos de Fosfatidilinositol/biossíntese , Trifosfato de Adenosina/metabolismo , Adulto , Antimetabólitos/farmacologia , Desoxiglucose/farmacologia , Difosfatos/metabolismo , Metabolismo Energético/efeitos dos fármacos , Genisteína/farmacologia , Humanos , Cinética , Magnésio/farmacologia , Neomicina/farmacologia , Neutrófilos/efeitos dos fármacos , Oligomicinas/farmacologia , Fosfatidilinositóis/biossíntese , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidoresRESUMO
In response to formyl-Met-Leu-Phe (fMLP), human neutrophils (PMN) generate superoxide anion (O2-) by the enzyme complex NADPH oxidase. The modulation of phosphoinositide (PPI) turnover and the activation of phospholipases C (PLC) and D (PLD) have been shown to be early steps in the oxidative response of fMLP-stimulated PMN. Although the physiological nonapeptide bradykinin (BK) is involved in inflammation, its participation in PMN activation has not been properly studied. In this work, activation of signal transduction pathways that mediate the oxidative response, and the modulation of the NADPH oxidase activity by BK, are analyzed. A direct comparison between the signal transduction pathway induced by BK and fMLP is also made. BK was not able to elicit O2- production by PMN. Nevertheless, several signal transduction pathways associated with PMN activation were triggered by BK. The nonapeptide induced the phosphorylation of prelabeled membrane PPI. This phenomenon was imitated by PMA and inhibited by H7 and staurosporine, thus suggesting the participation of protein kinase c (PKC). A loss of labeled [32P]PPI was triggered by fMLP. The fact that both PMA and fMLP stimulated O2- production but modulated PPI turnover in different ways, indicates that PPI labeling does not correlate with the oxidative response. Because PKC activation seemed to be a prerequisite for BK-induced modulation of PPI turnover, PLC activation could act as an intermediate step in this mechanism. Our results show that BK activated a PIP2-PLC measured as the release of [3H]IP3. On the contrary, a PC-PLD was highly stimulated by fMLP but not by BK. The fact that BK induced PLC activity but neither that of PLD nor NADPH oxidase, whereas fMLP triggered the activation of both phospholipases and evoked the PMN respiratory burst, suggests that diacylglycerol (DAG) from PIP2 as well as PA or PA-derived DAG, synergize to trigger the PMN oxidative response. Finally, BK inhibited O2- production by fMLP-activated PMN in a time-dependent manner. Since BK did not induce NO production by PMN, the inhibitory effect on the oxidative function was not due to ONOO- formation. These data show that BK plays an important role in inflammation by modulating the PMN function.
Assuntos
Bradicinina/farmacologia , NADH NADPH Oxirredutases/sangue , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fosfatidilinositóis/sangue , Fosfolipase D/sangue , Fosfolipases Tipo C/sangue , Sequência de Aminoácidos , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Dados de Sequência Molecular , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADH NADPH Oxirredutases/efeitos dos fármacos , NADPH Oxidases , Neutrófilos/enzimologia , Óxido Nítrico/biossíntese , Óxido Nítrico/sangue , Fosfolipase D/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Estimulação Química , Superóxidos/sangue , Acetato de Tetradecanoilforbol/farmacologia , Fosfolipases Tipo C/efeitos dos fármacosRESUMO
Nitric oxide synthase (NOS) inhibitors have been reported to modulate luminol-dependent chemiluminescence (CL) in rat macrophages, whereas the potent oxidant peroxynitrite (ONOO-) was shown to react with luminol to yield CL in a cell-free system. We evaluated the role of the L-arginine/NOS pathway in luminol CL by phorbol ester-activated human polymorphonuclear (PMN) leukocytes using the NOS inhibitors NG-monomethyl-L-arginine (L-NMMA) and N-iminoethyl-L-ornithine (L-NIO). Nitric oxide (.NO) release was determined by oxidation of oxymyoglobin. In addition, the effect of NOS inhibitors on superoxide anion O2.-) production was measured. Luminol CL was notably diminished by L-NMMA in a dose-dependent manner. Superoxide dismutase (SOD) also decreased luminol CL and L-NMMA potentiated light emission decrease produced by SOD. Nitric oxide and O2.- production was significantly decreased by L-NMMA; moreover, luminol-dependent CL but not O2.- production was attenuated by L-NIO. These data suggest that products of catalytic activity of both .NO synthase and NADPH oxidase are required to elicit maximal luminol CL in this system. These studies demonstrate that the NOS synthase pathway is involved in luminol CL by human PMN, and they suggest that ONOO- would be an unrecognized mediator in this phenomenon.
Assuntos
Inibidores Enzimáticos/farmacologia , Luminol/farmacologia , Neutrófilos/efeitos dos fármacos , Óxido Nítrico Sintase/antagonistas & inibidores , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Humanos , Medições Luminescentes , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidases , Neutrófilos/metabolismo , Nitratos/farmacologia , Óxido Nítrico/metabolismo , Ornitina/análogos & derivados , Ornitina/farmacologia , Oxirredução , Oxiemoglobinas/metabolismo , Ratos , Superóxido Dismutase/farmacologia , Superóxidos/metabolismo , ômega-N-MetilargininaRESUMO
We have studied microsomal phospholipid, cholesterol and protein concentration in rat renal papilla, medulla and cortex during postnatal development, and the relationship between these membranes biochemical parameters and morphological changes. We also determined DNA concentration in each kidney zone. No changes were observed either in papillary microsomal phospholipids, proteins and cholesterol or in DNA concentration from 10-to 70-day-old rats. Medullary microsomal proteins and cholesterol did not change but a significant increase was observed in the microsomal phospholipid concentration during development; in this case, medullary DNA was significantly lower at 70 than at 10 days. In contrast, all biochemical parameters in renal cortex were significantly higher during development except for DNA concentration which suffered a great decrease. These biochemical findings demonstrate that the developmental pattern is different in each zone of the kidney and confirm the fact that the papilla, in newborn rats, is almost fully developed whereas the renal cortex and medulla are immature.
Assuntos
Envelhecimento/metabolismo , Rim/crescimento & desenvolvimento , Rim/metabolismo , Animais , Animais Recém-Nascidos , Colesterol/metabolismo , DNA/metabolismo , Rim/citologia , Microssomos/metabolismo , Índice Mitótico , Fosfolipídeos/metabolismo , Proteínas/metabolismo , Ratos , Ratos Wistar , Distribuição TecidualRESUMO
The objective of this study was to determine nitric oxide (NO) and superoxide anion release (O-2) by neutrophils (PMNs) in the septic multiple organ dysfunction syndrome (MODS) and to compare them with the response of normal cells to lipopolysaccharide (LPS) and cytokines. NO production was measured by the release of nitrites in the medium, its maximal production rate by a modified oxyhemoglobin assay and O-2 by standard methods. Normal cells were incubated with LPS, gamma interferon (IFN-gamma), or tumor necrosis factor (TNF-alpha) alone or in combination. Results showed that PMN release of both NO and O-2 was reduced in septic samples; in contrast, an association of LPS, IFN-gamma, and TNF-alpha promoted maximal NO release by normal cells (40-50%). We conclude that while interaction of normal PMNs with cytokines increases NO and O-2 release, progression of sepsis to a multiple organ dysfunction impairs these responses in both functions.
Assuntos
Infecções Bacterianas/metabolismo , Citocinas/farmacologia , Endotoxinas/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Óxido Nítrico/biossíntese , Adulto , Idoso , Ânions/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Consumo de Oxigênio , Superóxidos/metabolismoRESUMO
Nitric oxide (.NO) release, oxygen uptake and hydrogen peroxide (H2O2) production elicited by increasing phorbol 12-myristate 13-acetate (PMA) concentrations were measured in human neutrophils. Half-maximal activities were sequentially elicited at about 0.0001-0.001 micrograms PMA/ml (.NO) and 0.001-0.01 micrograms PMA/ml (H2O2). At saturated PMA concentrations, .NO production, oxygen uptake and H2O2 release were 0.56 +/- 0.04, 3.32 +/- 0.52 and 1.19 +/- 0.17 nmol.min-1.10(6) cells-1. .NO production accounts for about 30% of the total oxygen uptake. Luminol-dependent chemiluminescence, reported to detect NO reactions in other inflammatory cells, was also half-maximally activated at about 0.001-0.01 micrograms PMA/ml. Preincubation with NG-monomethyl-L-arginine (L-NMMA) decreased O2 uptake and .NO release but increased H2O2 production, while superoxide dismutase (SOD) increased .NO detection by 30%. Chemiluminescence was also reduced by preincubation with L-NMMA and/or SOD. The results indicate that .NO release is part of the integrated response of stimulated human neutrophils and that, in these cells, kinetics of .NO and O2.- release favour the formation of other oxidants like peroxynitrite.
Assuntos
Peróxido de Hidrogênio/metabolismo , Neutrófilos/metabolismo , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Explosão Respiratória , Humanos , Técnicas In Vitro , Cinética , Luminol/farmacologia , Consumo de Oxigênio , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Con el objeto de establecer la eficacia predictiva de enfermedad cardíaca coronaria (ECC) que poseen determinados componentes sanguíneos, se investigó su concentración en 249 pacientes 2 días antes de ser sometidos a cirugía cardiovascular. Los analitos evaliados fueron: Glucosa (glucosa-oxidasa), hemoglobina-glucosada (resina de intercambio catiónico), Calcio iónico (electrodo específico), Acido úrico (uricasa), Colesterol (enzimático), HDL-Colesterol (Gordon) y Triglicéridos (enzimático), empleándose técnicas automatizadas. El LDLColesterol fue calculado por la fórmula de Friedewald. Los resultados obtenidos fueron analizados agrupando a los enfermos por sexo y edad y comparados con una población control. En los varones con ECC el colesterol total se encontró significativamente aumentado al compararse con los controles (241,9 ñ 44,7 vs 223,6 ñ 43,0 mg/dl (p < 0,01) entre los 25 y 49 años, perdiendo luego carácter predictivo a medida que la edad avanza. Los triglicéridos fueron más altos (197 ñ 107,3 vs 161,6 ñ 97,7, p < 0,05), en la población de varones enferma entre los 25 y 69 años. Las mujeres con ECC no mostraron diferencias estadísticamente significativas en su colesterol total y sus triblicéricos fueron mayores sólo en la población enferma entre los 30 y 49 años. El ácido úrico fue mayor en los varones con ECC con edades entre los 25 y 49 años (p < 0,05). El HDL-Colesterol mostró valores francamente inferiores en ambos sexos y a todas las edades estudiadas (p < 0,001). Los valores de hemoglobina-glucosada, glucosa y calcio iónico no se diferenciaron de los controles, en los diferentes grupos estudiados. El análisis de los datos aquí presentados revela que sólo el HDL-Colesterol y la relación Colesterol/HDL-Colesterol alertan sobre el riesgo de enfermedad coronaria en ambos sexos y a cualquier edad. Es posible que los otros analitos estudiados se comporten como mejores indicadores de ECC al asociarlos con la presencia de otros factores de riesgo
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Doença das Coronárias/etiologia , Lipídeos/sangue , Ácido Úrico/sangue , Fatores Etários , Argentina , Colesterol/sangue , Glucose Oxidase/sangue , Fatores Sexuais , Triglicerídeos/sangue , Estados UnidosRESUMO
Con el objeto de establecer la eficacia predictiva de enfermedad cardíaca coronaria (ECC) que poseen determinados componentes sanguíneos, se investigó su concentración en 249 pacientes 2 días antes de ser sometidos a cirugía cardiovascular. Los analitos evaliados fueron: Glucosa (glucosa-oxidasa), hemoglobina-glucosada (resina de intercambio catiónico), Calcio iónico (electrodo específico), Acido úrico (uricasa), Colesterol (enzimático), HDL-Colesterol (Gordon) y Triglicéridos (enzimático), empleándose técnicas automatizadas. El LDLColesterol fue calculado por la fórmula de Friedewald. Los resultados obtenidos fueron analizados agrupando a los enfermos por sexo y edad y comparados con una población control. En los varones con ECC el colesterol total se encontró significativamente aumentado al compararse con los controles (241,9 ñ 44,7 vs 223,6 ñ 43,0 mg/dl (p < 0,01) entre los 25 y 49 años, perdiendo luego carácter predictivo a medida que la edad avanza. Los triglicéridos fueron más altos (197 ñ 107,3 vs 161,6 ñ 97,7, p < 0,05), en la población de varones enferma entre los 25 y 69 años. Las mujeres con ECC no mostraron diferencias estadísticamente significativas en su colesterol total y sus triblicéricos fueron mayores sólo en la población enferma entre los 30 y 49 años. El ácido úrico fue mayor en los varones con ECC con edades entre los 25 y 49 años (p < 0,05). El HDL-Colesterol mostró valores francamente inferiores en ambos sexos y a todas las edades estudiadas (p < 0,001). Los valores de hemoglobina-glucosada, glucosa y calcio iónico no se diferenciaron de los controles, en los diferentes grupos estudiados. El análisis de los datos aquí presentados revela que sólo el HDL-Colesterol y la relación Colesterol/HDL-Colesterol alertan sobre el riesgo de enfermedad coronaria en ambos sexos y a cualquier edad. Es posible que los otros analitos estudiados se comporten como mejores indicadores de ECC al asociarlos con la presencia de otros factores de riesgo (AU)
Assuntos
Estudo Comparativo , Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Doença das Coronárias/etiologia , Lipídeos/sangue , Glucose Oxidase/sangue , Colesterol/sangue , Triglicerídeos/sangue , Ácido Úrico/sangue , Fatores Sexuais , Fatores Etários , Argentina , Estados UnidosRESUMO
The study included 249 patients two days before cardiovascular surgery and 73,915 control subjects. Results obtained were analyzed by grouping the individuals according to sex and age. In coronary heart disease (CHD) in males, total cholesterol was found higher than in controls (mean +/- D.S.: 241.9 +/- 44.7 vs 223.6 +/- 43.0 mg/dl, p < 0.01) between 25 and 49 years of age, this significance being lost with age. Triglycerides were also higher (197 +/- 107.3 vs 161.6 +/- 97.7 mg/dl, p < 0.01) in the CHD male population between ages 25 and 69. In CHD females, triglycerides were higher (116.9 +/- 56.2 vs 91.5 +/- 43.3 mg/dl, p < 0.05) between ages 25 and 49; cholesterol showed no difference at any of the ages studied. HDL-C was much lower in both sexes of CHD patients at all ages studied (p < 0.001). Uric acid was higher in CHD males between ages 25 and 49 (p < 0.05), this significance being lost in the older age CHD group. Other components such as glycated hemoglobin, glucose and ionized calcium, were not different from those of the control group.
Assuntos
Doença das Coronárias/sangue , Lipídeos/sangue , Adulto , Fatores Etários , Idoso , Argentina , Colesterol/sangue , Doença das Coronárias/etiologia , Feminino , Glucose Oxidase/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Fatores Sexuais , Triglicerídeos/sangue , Estados Unidos , Ácido Úrico/sangueRESUMO
The study included 249 patients two days before cardiovascular surgery and 73,915 control subjects. Results obtained were analyzed by grouping the individuals according to sex and age. In coronary heart disease (CHD) in males, total cholesterol was found higher than in controls (mean +/- D.S.: 241.9 +/- 44.7 vs 223.6 +/- 43.0 mg/dl, p < 0.01) between 25 and 49 years of age, this significance being lost with age. Triglycerides were also higher (197 +/- 107.3 vs 161.6 +/- 97.7 mg/dl, p < 0.01) in the CHD male population between ages 25 and 69. In CHD females, triglycerides were higher (116.9 +/- 56.2 vs 91.5 +/- 43.3 mg/dl, p < 0.05) between ages 25 and 49; cholesterol showed no difference at any of the ages studied. HDL-C was much lower in both sexes of CHD patients at all ages studied (p < 0.001). Uric acid was higher in CHD males between ages 25 and 49 (p < 0.05), this significance being lost in the older age CHD group. Other components such as glycated hemoglobin, glucose and ionized calcium, were not different from those of the control group.