RESUMO
OBJECTIVE: To evaluate the anti-inflammatory effects of exogenous surfactants and surfactant phospholipid without surfactant proteins (SP-A and SP-D) on the lipopolysaccharide- (LPS) stimulated rat alveolar macrophage (AM) cell line NR8383. METHODS: Exogenous surfactants (beractant, calfactant or colfosceril) and surfactant phospholipid (dipalmitoyl phosphatidylcholine, DPPC), standardized to phospholipid content of 25-1,000 microg/ml were incubated with LPS- (1 microg/ml) stimulated NR8383 AMs. RESULTS: TNF-alpha and IL-1beta secretion and nitric oxide (NO) formation following LPS stimulation were inhibited by treatment with surfactants or DPPC. Furthermore, LPS-dependent NO production and iNOS protein levels were significantly suppressed in cells pretreated for one hour with beractant compared to beractant added simultaneously with or following LPS. Additionally, LPS-stimulated oxidative burst, measured by flow cytometry, was significantly decreased by beractant. Finally, beractant inhibited the translocation of NF-kappaB from cytoplasmic into nuclear extract in LPS-stimulated NR8383 AMs. CONCLUSIONS: Exogenous surfactants and surfactant phospholipid inhibit secretion of proinflammatory cytokines and NO in NR8383 AMs. The inhibitory effects of beractant on oxygen radical and LPS-induced NO formation may result from unique mechanisms of decreasing cell signaling. The anti-inflammatory activity of surfactant products used in the treatment of neonatal respiratory distress syndrome (RDS) may depend upon the specific preparation or dose used.
Assuntos
Imunossupressores/imunologia , Macrófagos Alveolares/imunologia , Surfactantes Pulmonares/imunologia , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Animais , Produtos Biológicos/metabolismo , Linhagem Celular , Combinação de Medicamentos , Álcoois Graxos/metabolismo , Humanos , Interleucina-1beta/imunologia , Macrófagos Alveolares/citologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Fosfolipídeos/metabolismo , Fosforilcolina/metabolismo , Polietilenoglicóis/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Ratos , Explosão Respiratória , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Excessive nitric oxide (NO) generated by hepatic cells in response to lipopolysaccharide (LPS) and inflammatory substances (e.g., platelet-activating factor [PAF]) is a key contributor to the pathophysiological outcomes observed in the liver during sepsis. In rats subjected to liver-focused endotoxemia, inducible nitric oxide synthase (iNOS) levels in the intact liver were elevated by 6 hours; cell-specific expression of iNOS messenger RNA (mRNA) was Kupffer cells (KCs), endothelial cells, and hepatocytes. Elevated serum alanine transaminase (ALT) levels at 6 hours confirmed hepatic damage. Pretreatment of endotoxemic rats with PAF receptor antagonists BN 50739 or WEB 2170 reduced serum ALT and iNOS mRNA levels in the intact liver. Pretreatment of cultured KCs with BN 50739 or WEB 2170 inhibited both LPS and PAF-induced iNOS mRNA formation. In addition, LPS-induced iNOS protein levels in KCs pretreated with BN 50739 or WEB 2170 were decreased. Exposure of KCs to either LPS or PAF caused the translocation of the p65 subunit of nuclear factor kappa B (NF-kappaB) into the nucleus and this process was attenuated by BN 50739 and WEB 2170. There was concomitant inhibition of LPS-dependent degradation of the inhibitory protein IkappaBalpha and increase in intracellular Ca(2+) in KC treated with BN 50739 or WEB 2170. Also, in KCs, LPS was able to induce iNOS mRNA expression independent of CD14. This response was inhibited by pretreatment of KCs with either BN 50739 or WEB 2170. Our findings indicate that PAF receptor antagonists convey protection against hepatocellular injury accompanied by a decrease in nitric oxide (NO) formation in the livers of endotoxemic rats.
Assuntos
Azepinas/farmacologia , Endotoxemia/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteínas I-kappa B , Células de Kupffer/enzimologia , Lipopolissacarídeos/toxicidade , Fígado/enzimologia , Óxido Nítrico Sintase/genética , Fator de Ativação de Plaquetas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Triazóis/farmacologia , Alanina Transaminase/sangue , Alanina Transaminase/genética , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/enzimologia , Proteínas de Ligação a DNA/metabolismo , Células de Kupffer/citologia , Células de Kupffer/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Fator de Ativação de Plaquetas/antagonistas & inibidores , RNA Mensageiro/genética , Ratos , Ratos Sprague-DawleyRESUMO
The onset of liver injury is a pivotal event during endotoxemia. Lipopolysaccharide (LPS) activates the Kupffer cells (KC), the resident macrophages of the liver, to generate an abundance of inflammatory substances, including nitric oxide (NO). Elevated levels of NO are thought to contribute to the propagation of liver injury during sepsis. Calcium, a major second messenger in several cellular signaling events, is required by the KC for the generation of inducible nitric oxide synthase (iNOS). The purpose of this study was to determine whether calcium channel antagonists limit hepatic injury and iNOS expression in vivo following LPS exposure and to evaluate their effects on the regulation of iNOS expression in cultured KC. In rats subjected to LPS for 6 h, the serum alanine aminotransferase (ALT) level was elevated significantly; this response was accompanied by an increase in iNOS mRNA formation in the intact liver. Pretreatment of rats with calcium channel antagonists (i.e., diltiazem, nifedipine, or verapamil) before LPS exposure attenuated the serum ALT level and iNOS mRNA expression in the liver. Pretreatment of cultured KC with calcium channel antagonists for 1 h followed by the addition of LPS markedly repressed iNOS protein and mRNA expression. Time-course studies revealed that calcium channel antagonists were most effective at inhibiting LPS-induced iNOS mRNA formation by KC when added before LPS. Treatment of KC with calcium channel antagonists prior to the addition of LPS decreased nuclear levels of the p65 subunit of nuclear factor-kappaB and prevented the LPS-dependent degradation of the inhibitory protein IkappaBalpha. Thus our findings indicate that under endotoxemic conditions calcium channel antagonists limit hepatocellular injury that is accompanied by an inhibition of LPS-mediated iNOS expression in rat liver KC.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Proteínas de Ligação ao Cálcio , Lipopolissacarídeos/farmacologia , Fígado/enzimologia , Óxido Nítrico Sintase/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Diltiazem/farmacologia , Proteínas I-kappa B , Cinética , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/enzimologia , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nifedipino/farmacologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Sinaptotagmina I , Sinaptotagminas , Verapamil/farmacologiaRESUMO
The liver plays a major role in metabolism and elimination of leukotrienes (LT). It produces cysteinyl leukotrienes (cLT), and cLT have been implicated in hepatocellular toxicity in several models of lipopolysaccharide (LPS)-associated liver injury. However, the liver cell types responsible for cLT production are poorly defined, and the expression of the LT-synthesis enzymes, 5-lipoxygenase (5-LO) and LTC4 synthase (LTC4-S), in liver cells has never been demonstrated. The aim of the present study was to examine the ability of rat liver cells to produce cLT by determining whether hepatocytes, Kupffer cells, and sinusoidal endothelial cells express mRNA and enzyme activities of the LT-synthesis enzymes and whether expression is altered by LPS. 5-LO mRNA was expressed in whole liver, and expression was enhanced by LPS. Cell fractionation studies demonstrated that expression was present in Kupffer cells and sinusoidal endothelial cells, but not in hepatocytes. LTC4-S mRNA was detected in whole liver, hepatocytes, and sinusoidal endothelial cells, but not in Kupffer cells. Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) showed that LPS increased LTC4-S expression in hepatocytes by a factor of 3 (n = 3; P < .03). LTC4-S enzyme activity in the microsomal fraction of hepatocytes was also increased from 0.52 +/- 0.13 to 1.90 +/- 0.66 nmol . mg protein-1 . 5 min-1 (n = 6; P < .015) after LPS treatment. These results indicate that hepatocytes do not possess the ability for de novo synthesis of cLT from arachidonic acid, but they may actively participate in cLT production by conjugation of LTA4 with glutathione to produce LTC4. LPS enhances LTC4-S expression in hepatocytes. This intrinsic cLT production may contribute to hepatocellular injury during inflammation.
Assuntos
Araquidonato 5-Lipoxigenase/genética , Expressão Gênica , Glutationa Transferase/genética , Leucotrienos/biossíntese , Fígado/enzimologia , Animais , Endotélio Vascular/enzimologia , Células de Kupffer/enzimologia , Lipopolissacarídeos/farmacologia , Masculino , Microssomos Hepáticos/enzimologia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Treatment of cultured rat Kupffer cells with lipopolysaccharide (LPS) resulted in a time-dependent increase in the expression of the inducible isoform of nitric-oxide synthase (iNOS). Agents that elevated intracellular cAMP levels (e.g. forskolin, dibutyryl cAMP, cholera toxin, and isoproterenol) markedly decreased nitrite production and iNOS protein formation by LPS-stimulated Kupffer cells. Furthermore, inhibition of LPS-induced nitrite formation and iNOS protein levels by these agents was enhanced in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Forskolin, the most potent inhibitor of LPS-induced nitrite formation by Kupffer cells, decreased iNOS mRNA levels in a time-dependent manner. Time course studies indicated that forskolin was most effective at inhibiting LPS-induced nitrite formation and iNOS mRNA levels by Kupffer cells when added before LPS. Message stability studies established that forskolin did not enhance the rate of decay of LPS-induced iNOS mRNA. Nuclear run-on assays revealed that forskolin decreased LPS-induced transcription of the iNOS gene. Treatment of Kupffer cells with LPS induced the translocation of the p65 subunit of nuclear factor kappaB (NF-kappaB) into the nucleus, and this process was abolished by forskolin. In addition, the LPS-dependent degradation of IkappaBalpha was not observed in forskolin-treated cells; the levels of the p65 subunit of NF-kappaB were minimal in the nucleus at the same time. Also, we observed that forskolin induced transcription of the IkappaBalpha gene in a time-dependent manner and in addition up-regulated LPS-induced IkappaBalpha mRNA levels. Taken together, this study indicates that the attenuation of LPS-induced iNOS formation in Kupffer cells by elevated intracellular cAMP levels occurs by preventing the degradation of IkappaBalpha which suppresses the activation of NF-kappaB and inhibits the onset of transcription of the iNOS gene.
Assuntos
AMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas I-kappa B , Células de Kupffer/enzimologia , Fígado/enzimologia , Óxido Nítrico Sintase/biossíntese , Animais , Transporte Biológico , Núcleo Celular/metabolismo , Células Cultivadas , Colforsina/farmacologia , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células de Kupffer/citologia , Lipopolissacarídeos/farmacologia , Fígado/citologia , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Nitritos/metabolismo , RNA Mensageiro/análise , Ratos , Choque Séptico , Transcrição Gênica/efeitos dos fármacosRESUMO
Endothelin (ET) is a potent peptide mediator exhibiting a wide variety of effects in both the parenchymal and nonparenchymal hepatic cells. In the Kupffer cell, ET activates several transmembrane signaling pathways to generate numerous second messengers including the phospholipase C-generated products inositol-1,4,5-trisphosphate and diacylglycerol and the cyclooxygenase product prostaglandin E2 via specific ETB-type receptors. In addition to these findings, we have now demonstrated that endothelin stimulates the production of nitric oxide (NO) in the Kupffer cell in a time- and concentration-dependent manner. Western blot analysis indicates that ET-stimulated NO production occurs though activation of the inducible form of the nitric oxide synthase enzyme. These findings have important implications as the stimulation of NO production by ET may be part of the physiological response to inflammation or infection. Elevated levels of ET and NO have been found to be associated with numerous hepatic pathophysiological conditions that may contribute to derangements in the vascular system seen in these conditions.
Assuntos
Endotelina-3/farmacologia , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/metabolismo , Óxido Nítrico/biossíntese , Animais , Cálcio/farmacologia , Quelantes/farmacologia , Ácido Egtázico/farmacologia , Técnicas In Vitro , Cinética , Masculino , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , ômega-N-Metilarginina/farmacologiaRESUMO
Acute endotoxic shock is accompanied by an increase in the production of nitric oxide (NO) by several different hepatic cell types. Platelet-activating factor (PAF) is a potent proinflammatory mediator with many pathophysiological actions and, in fact, elevated plasma and tissue levels of PAF are observed in animal models of endotoxic shock. The current study demonstrates that PAF induced nitrite formation, the end product of nitric oxide synthesis, by Kupffer cells in a dose- and time-dependent manner. Moreover, PAF was seen to initiate NO synthase gene expression and protein synthesis. PAF augmented lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase messenger RNA (mRNA), protein, nitrite and cyclic guanosine monophosphate (cGMP) levels in Kupffer cells. Treatment of Kupffer cells with actinomycin D or cycloheximide inhibited PAF- and LPS-stimulated nitrite and nitric oxide synthase protein formation confirming that de novo synthesis of the enzyme occurred. In Kupffer cells, the presence of an arginine analog, NG-methyl-L-arginine, attenuated nitrite formation induced by PAF and LPS alone or in combination. L-arginine is the principal substrate for nitric oxide synthase. PAF and LPS individually and in combination induced a time-dependent uptake of L-[3H]-arginine into the Kupffer cell, and this response was sensitive to cycloheximide. The current study indicates that exogenous PAF contributes to the induction of nitric oxide synthase by LPS in cultured rat Kupffer cells.
Assuntos
Células de Kupffer/efeitos dos fármacos , Células de Kupffer/metabolismo , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/toxicidade , Óxido Nítrico/biossíntese , Fator de Ativação de Plaquetas/administração & dosagem , Animais , Arginina/análogos & derivados , Arginina/metabolismo , Arginina/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Células Cultivadas , GMP Cíclico/biossíntese , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Sinergismo Farmacológico , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Cinética , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase/genética , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Choque Séptico/metabolismo , ômega-N-MetilargininaRESUMO
Circulating endothelin (ET) levels are elevated in conditions such as endotoxemia, hepatic ischemia-reperfusion injury, or orthotopic liver transplantation, and this potent peptide may contribute to hepatic pathophysiology. We measured the surface binding of [125I]ET-1 to rat Kupffer cells in primary culture at 4 degrees C; the dissociation constant (Kd) was 270 pmol/L, and the apparent Bmax was 3,000 receptors/cell. At 37 degrees C, total association (surface binding plus internalization) was much greater than at 4 degrees C, indicating that internalization of the receptor-ligand complex is rapid; the apparent Kd was 30 pmol/L, comparable with other reports for hepatic-derived cells. Studies using [125I]ET-1, [125I]ET-3, and specific ET (ant)agonists showed that Kupffer cells possess predominantly ET(B) type receptors. Prior treatment with 500 pmol/L unlabeled endothelin rapidly ( < 15 minutes) occluded 60% of subsequent [125I]ET association; using 5 nmol/L unlabeled ET, this occlusion occurred within 1 minute. [125I]ET association with Kupffer cells was unaffected by short-term (approximately 1 hour) treatment with cyclic adenosine monophosphate (cAMP), but long-term (20 hour) treatment resulted in a twofold increase in [125I]ET association with no change in the apparent Kd. Stimulation of protein kinase C in Kupffer cells by phorbol 12-myristate acetate had a dual regulatory effect on [125I]ET association. Short-term ( < 1 hour) treatment with phorbol 12-myristate acetate decreased [125I]ET-3 association by 50%, whereas prolonged treatment (20 hour) increased association twofold. In both cases, the apparent Kd for [125I]-endothelin was unaltered.
Assuntos
Endotelinas/metabolismo , Células de Kupffer/metabolismo , Animais , Células Cultivadas , AMP Cíclico/fisiologia , Dimetil Sulfóxido/farmacologia , Regulação para Baixo , Células de Kupffer/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Endotelina/efeitos dos fármacos , Receptores de Endotelina/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Endothelins are potent peptide mediators that elicit glycogenolytic and vasoconstrictor actions in the liver. Endothelins were found to stimulate the synthesis and release of the lipid mediator platelet-activating factor in cultured rat Kupffer cells. Endothelin-mediated synthesis of platelet-activating factor required extracellular calcium in that the calcium chelator, EGTA and nifedipine, a calcium ion channel blocker, inhibited platelet-activating factor synthesis. The phospholipase A2 inhibitor, 4-bromophenacyl bromide, strongly inhibited endothelin-induced platelet activating factor synthesis. Endothelin-stimulated platelet activating factor synthesis was inhibited after treatment of Kupffer cells with cholera toxin, whereas pertussis toxin inhibited only this response to endothelin-1. Agents that elevate intracellular cyclic AMP levels were found to inhibit endothelin-induced platelet-activating factor synthesis in Kupffer cells. Staurosporine, a protein kinase C inhibitor minimized endothelin-induced platelet-activating factor synthesis but phorbol myristate acetate, an activator of protein kinase C, did not affect endothelin-induced platelet activating factor synthesis. Thus, the current study demonstrates that activation of an endothelin receptor in cultured rat Kupffer cells results in the synthesis and release of platelet-activating factor. The importance of endothelin-mediated platelet-activating factor synthesis relates to the mechanism of intercellular signaling occurring between endothelial cells (i.e., the site of endothelin synthesis) and Kupffer cells (i.e., the site of formation of secondary mediators such as platelet-activating factor and eicosanoids) within the rat liver exposed to various types of pathophysiological episodes.