RESUMO
Lipopolysaccharide (LPS) treatment results in widespread expression of the inducible isoform of nitric oxide (NO) synthase (iNOS). Although there is evidence for the expression of iNOS in heart tissue, regulation of myocardial iNOS expression is not known. To determine the time course and degree of iNOS induction in the adult heart, we examined iNOS mRNA expression and enzyme activity in (1) rat left ventricular tissue after LPS treatment in vivo, and (2) cultured, long-term rat cardiac myocytes maintained in serum and exposed to interleukin-1 beta, tumor necrosis factor-alpha, interferon-gamma, and/or LPS. iNOS mRNA was detected by Northern blot analysis and in situ hybridization. iNOS enzyme activity was measured in extracts of whole heart, and nitrate and nitrite (the stable end-products of NO) accumulation was quantified in cardiomyocyte culture media. iNOS mRNA was not detected in untreated hearts or cultured myocytes but was apparent within 3 h in both hearts obtained from LPS-treated animals and in cytokine-treated myocytes. In whole heart, iNOS mRNA expression peaked by 6 h after LPS and declined by 12 and 24 h. In situ hybridization demonstrated perinuclear localization of iNOS mRNA in both cardiac vascular smooth muscle and myocytes with maximal expression at 6 h after LPS injection. In cardiac myocytes, iNOS expression was maximal at 12 to 24 h, persisted through 48 h, and was partially inhibited by dexamethasone. Interferon-gamma was the most potent single cytokine with regards to myocyte iNOS induction. Nitric oxide release in cytokine-stimulated cardiac myocytes was largely in the form of nitrate and was associated with increased glucose uptake and lactate release; the former finding indicates that NO interacts with myocardial heme proteins and/or oxyradicals, while the latter suggests inhibition of oxidative metabolism. Although non-myocardial cells may significantly contribute to iNOS expression in whole heart tissue, significant iNOS expression and NO production also take place within the myocyte. Induced NO production may regulate myocardial perfusion and impair myocardial function and metabolism.
Assuntos
Ventrículos do Coração/enzimologia , Interferon gama/farmacologia , Interleucina-1/farmacologia , Óxido Nítrico Sintase/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Animais , Células Cultivadas , Ventrículos do Coração/efeitos dos fármacos , Hibridização In Situ , Lipopolissacarídeos , Masculino , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
Lipopolysaccharide (LPS)-binding protein (LBP) has been reported to be an acute-phase protein. LBP binds to LPS with a high affinity; LPS-LBP complexes then interact with the receptor CD14, resulting in increased expression of LPS-inducible genes. Hepatocytes represent a major source of LBP, but little is known about the regulation of rodent hepatocyte LBP synthesis. In these studies, undertaken to characterize hepatocyte LBP expression, we show that greater-than-20-fold increases in LBP mRNA levels in hepatocytes occurred following injection of LPS or turpentine in rats. In primary cultures of rat hepatocytes, the addition of interleukin-6 (IL-6) and LPS led to 4.5- and 3.2-fold stimulation in LBP mRNA levels, respectively. The induction of LBP by IL-6 or LPS was attenuated by dexamethasone. In contrast to IL-6 and LPS, in the presence of 10(-6) M dexamethasone, IL-1 and tumor necrosis factor (TNF) led to maximal LBP mRNA induction levels, 4.7- and 3.8-fold, respectively, suggesting that IL-6 and LPS stimulate LBP expression by mechanisms different from those of IL-1 and TNF. Similar induction levels of LBP mRNA were seen in rat H35 hepatoma cells for all four stimuli, and dexamethasone inhibited these responses. Dexamethasone alone increased the spontaneous induction in primary hepatocytes at early time points but suppressed induction at later time points. Furthermore, hepatocytes from rats treated with LPS in vivo exhibited a > 10-fold increase in mRNA expression in response to LPS and enhanced responses to TNF and IL-1. As with the normal hepatocytes, dexamethasone inhibited the LPS-dependent induction in the LPS-treated rat hepatocytes. These data suggest that LBP synthesis by hepatocytes is under the control of LPS, IL-1, TNF, IL-6, and glucocorticoids and that the LPS treatment primes hepatocytes for subsequent responses to LPS, TNF, and IL-1 for LBP synthesis.
Assuntos
Proteínas de Fase Aguda , Proteínas de Transporte/metabolismo , Dexametasona/farmacologia , Interleucina-1/fisiologia , Interleucina-6/fisiologia , Lipopolissacarídeos/farmacologia , Fígado/efeitos dos fármacos , Glicoproteínas de Membrana , Fator de Necrose Tumoral alfa/fisiologia , Animais , Proteínas de Transporte/genética , Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Fígado/metabolismo , Masculino , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Marked differences in induced nitric oxide (NO) synthesis occur between species. We have previously shown that both human and rat hepatocytes express an inducible NO synthase in response to cytokines and lipopolysaccharide. In this study, we compare the expression and regulation of cytokine-induced NO synthase in hepatocytes isolated from three species, human, rat, and mouse. On stimulation with tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interferon gamma (IFN gamma), and lipopolysaccharide (LPS), it was found that hepatocytes from all three species produce high levels of NO with levels of production exhibiting the following hierarchy: rat hepatocytes > mouse hepatocytes > human hepatocytes. Whereas rat and mouse hepatocytes express inducible NO synthase messenger RNA (mRNA) in response to TNF alpha, IL-1 beta, or IFN gamma as a single stimulus, human hepatocytes respond to LPS alone. Inhibition of NO generation through transforming growth factor (TGF-beta 1) was seen in mouse (77% +/- 5.9) and rat hepatocytes (17% +/- 2.6) whereas only about 10% was seen in human hepatocytes. Epidermal growth factor (EGF) was shown to inhibit NO synthesis in human and mouse hepatocytes but not rat. A marked NO-dependent inhibition of total protein synthesis was seen in rat and human hepatocytes, whereas mouse hepatocytes showed almost no inhibition in protein synthesis when stimulated. NO-dependent cyclic guanosine monophosphate (cGMP) release was found in all three species.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Aminoácido Oxirredutases/biossíntese , Expressão Gênica/efeitos dos fármacos , Fígado/enzimologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Células Cultivadas , GMP Cíclico/metabolismo , Dexametasona/farmacologia , Indução Enzimática , Humanos , Interferon gama/farmacologia , Interleucina-1/farmacologia , Interleucinas/farmacologia , Isoenzimas/biossíntese , Lipopolissacarídeos/farmacologia , Fígado/efeitos dos fármacos , Camundongos , Nitratos/análise , Óxido Nítrico Sintase , Nitritos/análise , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes/farmacologia , Especificidade da Espécie , Fator de Necrose Tumoral alfa/farmacologia , ômega-N-MetilargininaRESUMO
Hepatocyte plating density is known to affect cell function. Human and rat hepatocytes have been shown to express the inducible nitric oxide synthase (INOS) in response to cytokines plus lipopolysaccharide (LPS). The following studies were performed to determine the effects of hepatocyte plating density on the regulation of INOS. Rat hepatocytes were plated at densities from 10(4) to 20 x 10(4) hepatocytes/cm2 and stimulated with a combination of LPS, interferon-gamma, interleukin-1, and tumor necrosis factor. We found that NO2- plus NO3- released from stimulated hepatocytes declines with increasing hepatocyte density. Similar effects were seen for 3',5'-cyclic monophosphate release into supernatants and in the amount of nonheme iron-nitrosyl signals measured by electron paramagnetic resonance spectroscopy. Limitations of substrate (L-arginine) and 5,6,7,8-tetrahydrobiopterin were excluded as cause of the reduced nitric oxide generation at higher densities. Although mRNA levels for INOS were not influenced when measured at 24 h, there was a marked reduction in INOS enzyme activity and INOS protein detectable by Western blotting at higher cell density. Total protein synthesis decreased as hepatocyte density increased in both nonstimulated and stimulated hepatocytes at higher cell densities. These data suggest that reduced INOS translation may account for the density-dependent reduction in INOS activity in cultured hepatocytes. The importance of this phenomenon remains to be determined in vivo but has important implications for the in vitro study of INOS expression.
Assuntos
Fígado/citologia , Fígado/metabolismo , Óxido Nítrico/biossíntese , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Biopterinas/análogos & derivados , Biopterinas/farmacologia , Contagem de Células , Células Cultivadas , GMP Cíclico/metabolismo , Citocinas/farmacologia , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Óxido Nítrico Sintase , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , ômega-N-MetilargininaRESUMO
BACKGROUND: Many of the physiologic derangements resulting in septic shock are caused by inflammatory mediators such as nitric oxide (NO) and cytokines produced in response to bacterial endotoxin or, more specifically, lipopolysaccharide. The recent development of a novel class of lipopolysaccharide antagonists offers the opportunity to block this response selectively. In this article we investigated the ability of one of these antagonists, B464 (Eisai), to block lipopolysaccharide-induced release of macrophage NO and cytokines. METHODS: The mouse macrophage cell line RAW264.7 was grown in vitro and exposed to (1) media control, (2) B464 alone, (3) lipopolysaccharide alone, or (4) lipopolysaccharide plus graded concentrations of B464. Supernatants were assayed for nitrite plus nitrate, the stable end products of NO, as well as tumor necrosis factor-alpha and interleukin-6. Total cellular RNA was examined for inducible NO synthase and interleukin-6 mRNA. RESULTS: Lipopolysaccharide-stimulated increases in NO, tumor necrosis factor, and interleukin-6 production were blocked by B464. Reduction of NO was also seen at the level of inducible NO synthase mRNA. Induction of interleukin-6 mRNA was also suppressed. CONCLUSION: B464 is a novel potent specific antagonist of lipopolysaccharide-induced macrophage NO and cytokine production.
Assuntos
Interleucina-6/biossíntese , Lipídeo A/análogos & derivados , Lipopolissacarídeos/antagonistas & inibidores , Óxido Nítrico/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Aminoácido Oxirredutases/genética , Animais , Sequência de Carboidratos , Linhagem Celular , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Interleucina-6/genética , Lipídeo A/análise , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Óxido Nítrico Sintase , RNA Mensageiro/análiseRESUMO
Following trauma and tissue injury, patients frequently suffer infections and septic complications. Tissue injury is associated with the induction of the hepatic acute-phase response, but how this phenotypic expression by hepatocytes influences their subsequent response to endotoxin (lipopolysaccharide, LPS) or inflammatory cytokines is unknown. We have shown that both rat and human hepatocytes maximally express the enzyme-inducible nitric oxide synthase (iNOS) in response to a combination of LPS and the cytokines tumor necrosis factor (TNF), interferon-gamma (IFN-gamma), and interleukin-1. Furthermore, we have shown that the in vivo induction of the acute-phase response following tissue injury (hind limb turpentine injection) is not associated with hepatocyte iNOS expression. In this study, we show that the phenotypic change associated with the acute-phase response following tissue injury primes the hepatocyte to subsequently express iNOS in vitro in response to LPS alone as well as TNF and IFN-gamma. This expression of iNOS can be seen as early as 3 hr following the initial injury and lasts up to 24 hr. Early postinjury changes result in maximal expression following stimulation with TNF or IFN-gamma. Later (24 hr post-injury) changes reveal LPS to be the most potent inducer with as little as 0.01 microgram/ml LPS being required for iNOS mRNA expression. The in vivo correlate of tissue injury (turpentine injection) followed by sepsis (intraperitoneal LPS injection) resulted in a three- to fourfold rise in plasma levels of the stable end-products of nitric oxide production, nitrite, and nitrate (NO2- + NO3-), over levels seen in cases of sepsis alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Queimaduras Químicas/metabolismo , Fígado/metabolismo , Óxido Nítrico/biossíntese , Ferimentos e Lesões/metabolismo , Animais , Células Cultivadas , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Fígado/patologia , Masculino , Nitratos/sangue , Nitritos/sangue , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/farmacologia , TerebintinaRESUMO
OBJECTIVE: Nitric oxide (NO) is a potent biologic mediator produced by hepatocytes following exposure to cytokines and lipopolysaccharide (LPS). These cytokines are also known to regulate induction of the hepatic acute-phase response. The objective of this study was to determine whether inducible nitric oxide synthase (iNOS), the enzyme that produces NO, is expressed as part of the hepatic acute-phase response. DESIGN: The gene expression for inducible NOS (iNOS) as well as alpha 1-acid glycoprotein (AGP), an established acute-phase reactant, was measured by Northern blot analysis in rat hepatocytes in vivo during endotoxemia (LPS injection) and during the acute-phase response produced by hindlimb turpentine injection. Hepatocyte iNOS messenger RNA (mRNA) levels were correlated with iNOS activity and circulating plasma nitrite and nitrate levels. In vitro, iNOS and AGP mRNA levels were determined in cultured hepatocytes stimulated with interleukin 6 (IL-6), interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), or dexamethasone. RESULTS: The AGP mRNA levels were increased in vivo following both LPS and turpentine injection, while iNOS expression was induced only by LPS injection. Hepatocyte iNOS activity and plasma nitrite and nitrate levels also increased after LPS treatment. In vitro, the cytokine combination IL-6, IL-1 beta, and TNF-alpha induced hepatocyte iNOS expression but had minimal effects on AGP in the absence of dexamethasone. Addition of dexamethasone alone markedly increased AGP mRNA levels, with further increases seen with TNF-alpha or IL-1 beta addition. In contrast, dexamethasone decreased iNOS expression. CONCLUSION: The results show that hepatocyte iNOS expression is not part of the acute-phase response induced by remote inflammation and indicates that iNOS is differentially regulated from the acute-phase reactant, AGP.
Assuntos
Reação de Fase Aguda/enzimologia , Aminoácido Oxirredutases/biossíntese , Endotoxinas/efeitos adversos , Fígado/enzimologia , Orosomucoide/biossíntese , Reação de Fase Aguda/sangue , Aminoácido Oxirredutases/efeitos dos fármacos , Aminoácido Oxirredutases/genética , Animais , Células Cultivadas , Endotoxinas/sangue , Indução Enzimática , Escherichia coli , Regulação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Interleucina-1/farmacologia , Interleucina-6/farmacologia , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/sangue , Fígado/citologia , Masculino , Nitratos/sangue , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase , Nitritos/sangue , Orosomucoide/efeitos dos fármacos , Orosomucoide/genética , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/farmacologia , Terebintina/efeitos adversosRESUMO
BACKGROUND: Interferon regulatory factor-1 (IRF-1) is a transcriptional factor originally cloned from fibroblasts that activates interferons and certain interferon-responsive genes. Because IRF-1 is an "early-immediate" nuclear protein, it can function acutely after trauma or septic stimuli. We have identified IRF-1 expression in hepatocytes in vivo in sepsis. The purpose of this study was to characterize the cytokine signals that up-regulate IRF-1 messenger RNA (mRNA) in cultured hepatocytes. METHODS: Rat hepatocytes were isolated by in situ collagenase perfusion and stimulated in vitro with cytokines. IRF-1 mRNA levels were determined by Northern blot hybridization with a DNA probe for hepatocyte IRF-1 generated with reverse transcription polymerase chain reaction with custom-designed oligonucleotide primers based on the known sequence for T-cell IRF-1. RESULTS: Northern blot of hepatocyte RNA showed a single IRF-1 mRNA band at approximately 2.4 Kb. The mRNA levels were markedly up-regulated (vs control hepatocytes) 2 hours after in vitro stimulation with the cytokines interferon-gamma (17-fold), tumor necrosis factor-alpha (3-fold), and interleukin-1 beta (2-fold). Lipopolysaccharide had no direct effect. CONCLUSIONS: The results showed that IRF-1 is up-regulated in hepatocytes primarily in response to interferon-gamma and to a lesser extent after tumor necrosis factor-alpha or interleukin-1 beta stimulation. This suggests that IRF-1 plays a role in regulating liver gene expression in sepsis; however, the specific genes controlled by IRF-1 remain to be determined.