Biomedical subjects

T R Billiar

Publications and source records attributed to T R Billiar.

At least 145 records · Page 8Linked to original sources

PubMed1997

Lipopolysaccharide activates the muscularis macrophage network and suppresses circular smooth muscle activity.

Bacterial lipopolysaccharide (LPS) is a causative agent of sepsis-induced ileus. Although it is known that LPS activates macrophages and initiates inflammation, the consequences of LPS on the macrophage network and a potential inflammatory response within the intestinal muscularis have not been investigated. This study was designed to identify cellular and functional changes in rat intestinal muscularis after intraperitoneal LPS. Histo- and immunohistochemistry were used to phenotype leukocytes. Functional alterations were determined using an organ bath. Compared with controls, LPS caused a 21-fold increase in staining for the lymphocyte activation marker-1 (LFA-1) localized to the ED2+ macrophage network 1 h after injection. This was followed by a significant infiltration of neutrophils, mast cells, and monocytes into the muscularis. LPS also caused a 62% reduction in spontaneous circular muscle activity and a 91% suppression of bethanechol-stimulated contractions 12 h after injection. These results demonstrate that endotoxemia 1) acutely activates the muscularis macrophage network, 2) causes the extravasation of leukocytes, and 3) results in circular muscle impairment.

Animals↗

PubMed1997

Dexamethasone suppresses iNOS gene expression by upregulating I-kappa B alpha and inhibiting NF-kappa B.

Cytokine-stimulated inducible nitric oxide synthase (iNOS) gene expression is dependent on nuclear factor-kappa B (NF-kappa B) activation and is suppressed by glucocorticoids (GC). In this study we examined the molecular mechanisms of GC inhibition of iNOS expression in rat hepatocytes. Combinations of tumor necrosis factor-alpha, interleukin-1 beta, and interferon-gamma (cytokine mixture CM) induced high levels of iNOS mRNA and NO synthesis. The synthetic GC dexamethasone markedly repressed iNOS mRNA and protein expression, and nuclear run-on assays showed that this inhibition was occurring at the level of transcription. In addition, transfection studies showed that CM-stimulated activity of a 1.6-kb murine iNOS promoter fragment linked upstream of luciferase was suppressed by dexamethasone. Electromobility shift assays demonstrated that CM induced the appearance of an NF-kappa B complex composed of p50 and p65 subunits; the addition of dexamethasone markedly decreased this band shift. I-kappa B alpha expression was decreased by CM and upregulated in the presence of dexamethasone. Subsequently, nuclear p65 levels were decreased by dexamethasone compared with CM-treated cells. Thus GC repress NF-kappa B DNA-binding activity in rat hepatocytes in part through the upregulation of its inhibitor I-kappa B alpha. These data indicate that one mechanism by which GC block iNOS expression is through the inhibition of NF-kappa B activation resulting in decreased iNOS transcription.

Animals↗

PubMed1997

Hemorrhagic shock induces G-CSF expression in bronchial epithelium.

Hemorrhagic shock (HS) initiates a series of inflammatory processes that includes the activation of polymorphonuclear granulocytic neutrophils (PMN). We tested the hypothesis that HS induces granulocyte colony-stimulating factor (G-CSF), a cytokine that augments PMN effector functions, in the lungs of rats. Sprague-Dawley rats were subjected to compensated or decompensated HS followed by resuscitation and death at 4 or 8 h. Animals subjected to HS demonstrated acute lung injury with PMN infiltration, edema, and hypoxia. Using semiquantitative reverse transcriptase-polymerase chain reaction, we detected a 1.9- to 7.1-fold increase in G-CSF mRNA levels in the lung of animals subjected to HS compared with sham controls. Levels of G-CSF mRNA increased with increased duration of the ischemic phase of resuscitated shock. In situ hybridization revealed that bronchoepithelial cells were the major cellular site of G-CSF mRNA. Thus production of G-CSF mRNA by bronchoepithelial cells is dramatically increased in a rat model of HS that also demonstrated lung injury. Increased local G-CSF levels may contribute to PMN recruitment and activation and resultant lung injury in HS.

Amino Acid Sequence↗

PubMed1997

Decreased activity of inducible nitric oxide synthase type 2 and modulation of the expression of glutathione S-transferase alpha, bcl-2, and metallothioneins during the differentiation of CaCo-2 cells.

Reactive oxygen species modulate the cell growth of a wide variety of mammalian cells. To determine whether oxidative metabolism is altered during the differentiation process, we studied the expression of pro- and antioxidant proteins in proliferating and differentiated CaCo-2 cells, a human colon adenocarcinoma cell line. Nitric oxide synthase type 2 (iNOS) produces nitric oxide (NO). Depending on its rate of synthesis, NO may either promote cellular and DNA damage or reduce the ability of other free radicals to induce cell injury. Using Western and Northern blot analysis and arginine conversion assay, we demonstrate that the expression of iNOS decreases when cells undergo differentiation. This biological event entails a diminished production of NO metabolites and correlates with the loss of activation of soluble guanylate cyclase activity. In differentiated cells, a 2-fold down-regulation of the nuclear factor kappa B activity was observed, suggesting that nuclear factor kappa B could be one of the iNOS gene regulatory factors in the CaCo-2 model. In parallel, we studied the expression of other antioxidant proteins including glutathione S-transferase alpha (GST alpha), bcl-2, and the metallothioneins (MTs). We show that the protein levels of GST alpha and MT increase during the differentiation of CaCo-2 cells, whereas bcl-2 levels decrease. Our investigation indicates that the expression of iNOS, GST alpha, bcl-2, and MT is associated with the enterocytic differentiation. The shift in the expression of specific antioxidant genes during CaCo-2 cell differentiation may occur to avoid alterations in the cell redox potential.

Actins↗

PubMed1997

Inhibition of nitric oxide synthesis during severe shock but not after resuscitation increases hepatic injury and neutrophil accumulation in hemorrhaged rats.

Hemorrhagic shock results in hepatocellular dysfunction and hepatic injury that may contribute to the development of liver failure and multiple organ dysfunction in trauma patients. The specific mediators involved in this process remain incompletely defined. We have previously demonstrated that inhibition of nitric oxide (NO) synthesis in a rat model of moderately severe hemorrhagic shock increases hepatic injury, suggesting that NO synthesis is beneficial after hemorrhage. To further define the role of NO in hepatic function during hemorrhagic shock, rats were subjected to a severe hemorrhagic shock insult in which they were bled to a mean arterial pressure of 40 mmHg until 40% of their shed blood had been returned and then were resuscitated. Rats were treated with the NO synthase inhibitor L-nitroarginine methyl ester (L-NAME) or the NO donor S-nitroso-N-acetylpenicillamine beginning either during the hypotensive period or after resuscitation. When instituted during the hypotensive period, low dose L-NAME infusion significantly increased hepatic injury. When L-NAME was infused after resuscitation, no increase in hepatic injury was detected even when the L-NAME dose was increased by a factor of four. The increased hepatic injury produced by L-NAME was associated with increased myeloperoxidase content in the lung, suggesting that L-NAME led to a greater accumulation of neutrophils during shock. Administration of the NO donor S-nitroso-N-acetylpenicillamine reduced hepatocellular enzyme release. Our results suggest that ongoing NO synthesis during the hypotensive phase of hemorrhagic shock is essential in preventing shock-induced hepatic injury and this may be due, in part, to the interaction between NO and circulating neutrophils.

Alanine Transaminase↗

PubMed1996

Stable expression of human inducible nitric oxide synthase in V79 Chinese hamster cells.

A recombinant expression vector containing the full-length cDNA for human inducible nitric oxide (NO) synthase was constructed for constitutive expression in V79 Chinese hamster cells. Expression was followed by Western analyses using three different NO synthase antisera. Activity remained stable during 4 months of continued cultivation. Activities were 25 pmol min-1 mg-1 cytosolic protein with L-arginine and 47 pmol min-1 mg-1 cytosolic protein with NG-hydroxy-L-arginine as substrates. Activity was concentration-dependently inhibited by inhibitors such as NG-methyl-L-arginine, NG-nitro-L-arginine, NG-nitro-L-arginine methyl ester, aminoguanidine, and S-methyl-isothiourea. The rank order of inhibitor potencies was different from published results obtained with rodent inducible NOS. Parental V79 cells do not express and cannot be induced for NO synthase activity. Therefore, the genetically engineered V79 cell line is defined for the cDNA-encoded human inducible NO synthase. The new cell line may serve as a useful tool to study human inducible NO synthase.

Animals↗

PubMed1996

Expression and immunoaffinity purification of human inducible nitric-oxide synthase. Inhibition studies with 2-amino-5,6-dihydro-4H-1,3-thiazine.

Recombinant human inducible nitric-oxide synthase (rH-iNOS) was expressed in the baculovirus system and purified by a novel immunoaffinity column. rH-iNOS and its native counterpart from cytokine-stimulated primary hepatocytes exhibited similar molecular mass of 130 kDa on SDS-polyacrylamide gel electrophoresis, recognition by antipeptide antibodies, specific activities, and IC50 values for inhibitors. The active dimeric form exhibited a specific activity range of 114-260 nmol/min/mg at 37 degrees C and contained 1.15 +/- 0.04 mol of calmodulin/monomer. The enzyme exhibited a Soret lambdamax at 396 nm with a shoulder at 460 nm and contained 0. 28-0.64 mol of heme/monomer. Dithionite reduction under CO yielded an absorbance maximum at 446 nm, indicating a P450-type heme. Imidazole induced a type II difference spectrum, reversible by L-Arg. 2-Amino-5,6-dihydro-4H-1,3-thiazine (ADT) was competitive versus L-Arg (Ki = 22.6 +/- 1.9 nM), reversed the type II difference spectrum induced by imidazole (Kd = 17.7 nM), and altered the CO-ferrous absorbance of rH-iNOS. L-Arg did not perturb the CO-ferrous adduct directly, but it partially reversed the ADT-induced absorbance shift, indicating that both bind similarly to the protein but interact differently with the heme.

Chromatography, High Pressure Liquid↗

PubMed1996

Competition for tetrahydrobiopterin between phenylalanine hydroxylase and nitric oxide synthase in rat liver.

Tetrahydrobiopterin (BH4) is an important cofactor for two hepatic enzymes, inducible nitric oxide synthase (iNOS) and phenylalanine hydroxylase (PAH), and competition for BH4 between the two enzymes might limit hepatic iNOS or PAH activity. To test this hypothesis, we determined whether conversion of phenylalanine to tyrosine was modified by changes in NO synthase activity, and conversely whether NO synthesis was limited by the rate of phenylalanine conversion to tyrosine in rat hepatocytes and perfused livers. NO production was decreased only slightly, when flux through PAH was maximized in isolated perfused livers, and in isolated hepatocytes only when BH4 synthesis was inhibited. Increases in NO synthesis did not reduce tyrosine formation from phenylalanine. Phenylalanine markedly increased biopterin synthesis, whereas arginine had no effect. Thus, basal BH4 synthesis appears to be adequate to support iNOS activity, whereas BH4 synthesis is increased to support PAH activity.

Animals↗

PubMed1996

A cohort of supporting metabolic enzymes is coinduced with nitric oxide synthase in human tumor cell lines.

Although nitric oxide (NO) has cytotoxic activity against certain tumor cell lines, some human tumor cell lines can themselves produce NO by expressing the inducible isoform of NO synthase (iNOS). As rates of cellular NO synthesis play a major role in determining whether NO has cytotoxic or cytoprotective effects at anatomic sites of NO production, identification of cellular processes which regulate rates of NO synthesis by iNOS is important for understanding the role of NO in tumor cell biology. This study demonstrates that argininosuccinate synthetase and GTP-cyclohydrolase-I, which catalyze rate-limiting steps in the synthesis of iNOS substrate (arginine) and cofactor (tetrahydrobiopterin), respectively, are coinduced with iNOS expression in two human tumor cell lines. These results indicate that coinduction of these supporting metabolic pathways helps to maximize cellular NO synthesis by iNOS in human tumor cells, suggesting that these pathways might be useful targets for pharmacologic intervention in NO-producing human tumor cells.

Adenocarcinoma↗

PubMed1996

Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.

Animals↗

PubMed1996

Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter.

The expression of inducible nitric oxide synthase (NOS2) is complex and is regulated in part by gene transcription. In this investigation we studied the regulation of NOS2 in a human liver epithelial cell line (AKN-1) which expresses high levels of NOS2 mRNA and protein in response to tumor necrosis factor alpha, interleukin 1 beta, and interferon gamma (cytokine mix, CM). Nuclear run-on analysis revealed that CM transcriptionally activated the human NOS2 gene. To delineate the cytokine-responsive regions of the human NOS2 promoter, we stimulated AKN-1 cells with CM following transfection of NOS2 luciferase constructs. Analysis of the first 3.8 kb upstream of the NOS2 gene demonstrated basal promoter activity but failed to show any cytokine-inducible activity. However, 3- to 5-fold inductions of luciferase activity were seen in constructs extending up to -5.8 and -7.0 kg, and a 10-fold increase was seen upon transfection of a -16 kb construct. Further analysis of various NOS2 luciferase constructs ligated upstream of the thymidine kinase promoter identified three regions containing cytokine-responsive elements in the human NOS2 gene: -3.8 to -5.8, -5.8 to -7.0, and -7.0 to -16 kb. These results are in marked contrast with the murine macrophage NOS2 promoter in which only 1 kb of the proximal 5' flanking region is necessary to confer inducibility to lipopolysaccharide and interferon gamma. These data demonstrate that the human NOS2 gene is transcriptionally regulated by cytokines and identify multiple cytokine-responsive regions in the 5' flanking region of the human NOS2 gene.

Blotting, Northern↗

PubMed1996

Glucagon inhibits hepatocyte nitric oxide synthesis.

OBJECTIVE: To investigate the effects of glucagon on nitric oxide (NO) synthesis in cultured rat hepatocytes. SETTING: Laboratory. MATERIALS: Male Sprague-Dawley rats (weight, 200-250 g). INTERVENTIONS: Isolated rat hepatocytes were cultured with interleukin-1 to stimulate NO synthesis. Glucagon was added at increasing concentrations (from 10(-9) to 2 x 10(-5) mol/L) at the time of interleukin-1 stimulation. Selected cultures were treated with the adenylate cyclase inhibitor, SQ 22536 (from 10(-5) to 10(-3) mol/L). MAIN OUTCOME MEASURES: Nitric oxide synthesis was assessed by measuring the concentrations of culture supernatant nitrite and nitrite plus nitrate, hepatocyte nitric oxide synthase-2 (NOS-2) messenger RNA (mRNA), and NOS-2 protein. RESULTS: Interleukin-1 stimulated hepatocyte NO synthesis, and this synthesis was inhibited by glucagon in a dose-dependent manner. Glucagon inhibited the accumulation of supernatant nitrite and the expression of NOS-2 mRNA and NOS-2 protein. SQ 22536 restored glucagon-induced decreases in NO synthesis. CONCLUSIONS: Glucagon inhibits NO synthesis in interleukin-1-stimulated hepatocytes in vitro. This inhibition seems to be mediated by glucagon-induced changes in cyclic adenosine monophosphate.

Animals↗

PubMed1996

Heat shock response inhibits cytokine-inducible nitric oxide synthase expression in rat hepatocytes.

During sepsis or inflammation, the liver expresses various protective phenotypes such as the acute phase response or the heat shock response (HSR). Inducible nitric oxide synthase (NOS2) is also expressed in the liver in these conditions and may protect the liver under some circumstances and promote injury in others. We have previously reported that the acute phase response and NOS2 expression are differentially regulated, though both can be expressed simultaneously. The HSR is known to prevent expression of other genes, but its effects on NOS2 expression in the liver is unknown. Therefore, we examined how the HSR influences NOS2 expression in primary rat hepatocytes. Sodium arsenite (Ars) or hyperthermia (43 degrees C) induced the synthesis of hsp72 messenger RNA (mRNA) and protein in hepatocytes, indicating activation of the HSR. In the absence of the HSR, combinations of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma) stimulated high levels of NOS2 mRNA and nitric oxide (NO) synthesis. However, treatment with Ars or heat shock significantly attenuated cytokine-induced NOS2 mRNA and NO production. The addition of the nuclear factor kappaB (NF-kappaB) inhibitor pyrrolidine dithiocarbamate also inhibited NOS2 expression, suggesting a role for NF-kappaB in the cytokine induction of NOS2 in hepatocytes. Cytokines induced the appearance of an NF-kappaB complex as shown in gel retardation assays; however, induction of the HSR by Ars partially prevented cytokine-induced formation of this band while hyperthermia had a more complete inhibition. Furthermore, preinduction of the HSR prevented the activation of the NOS2 promoter construct in hepatocytes transfected with a 1.6 kilobase NOS2 promoter linked to luciferase. These findings show that NO production in stressed cells can be modulated by the HSR, possibly through repression of NOS2 gene transcription via the inhibition of NF-kappaB.

Animals↗

PubMed1996

Effects of hepatocellular mitogens on cytokine-induced nitric oxide synthesis in human hepatocytes.

The synthesis of induced nitric oxide (NO) is regulated by several cytokines, including growth factors produced following hepatic injury and inflammation. However, little information is available on the role of growth factors in regulating the inducible NO synthase in human hepatocytes. The capacity of hepatocellular mitogens (HGF, EGF, and TGF-alpha) to regulate the inducible NO synthase (iNOS) was studied in human hepatocytes incubated with inflammatory cytokines and lipopolysaccharide (LPS). Furthermore, the effects of hepatic mitogens on NO-induced changes in DNA and protein synthesis was studied. It was found that NO-mediated decrease of protein and DNA synthesis were partially reversed by the mitogens. This was associated with a down-regulation in cytokine-mediated hepatocyte NO formation, iNOS mRNA expression, and NOS enzyme activity. Cytokine-induced NO formation or SNAP, an NO donor, added with cytokines increased hepatocyte chromatin condensation but no DNA fragmentation was observed. The increase in chromatin condensation was partially reversed by hepatic mitogens and corresponded with the inhibition of NO production. Thus, the hepatic mitogens, HGF, EGF, and TGF-alpha, all suppress iNOS expression and it is the suppression of iNOS that appears to be responsible for the mitogen-reduced preservation of DNA and protein synthesis and prevention of chromatin condensation.

Cells, Cultured↗

PubMed1996

PGE2 and LTB4 inhibit cytokine-stimulated nitric oxide synthase type 2 expression in isolated rat hepatocytes.

Prostaglandins have been shown to have a wide range of effects on nitric oxide synthesis when studied in different cell populations. The proximity of hepatocytes to eicosanoid-producing endothelial cells and Kupffer cells prompted us to determine the effects of PGE2 and LTB4 on hepatocyte NO production by the inducible nitric oxide synthase (iNOS, NOS-2) in vitro. PGE2 decreased hepatocyte NO synthesis in a concentration-dependent manner when the cells were stimulated with a combination of cytokines or IL-1 alone. LTB4 had a similar effect. PGE2 had to be present at the time of cytokine exposure to produce maximal inhibition of NO synthesis. Reduced synthesis of NO2- was associated with reduced NOS-2 mRNA levels suggesting that the induction of NOS-2 was inhibited. These findings demonstrate that eicosanoids can regulate hepatocyte NO synthesis in vitro.

Animals↗

PubMed1996

Gene therapy.

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Arterial Occlusive Diseases↗

PubMed1996

Nitric oxide produced by cytokine-activated pulmonary artery smooth muscle cells is cytotoxic to cocultured endothelium.

BACKGROUND: We recently demonstrated that rat pulmonary artery smooth muscle (RPASM) generates maximal nitric oxide (NO) when exposed to inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Our hypothesis is that NO produced by cytokine-stimulated RPASM has local cytotoxic effects on endothelium. Accordingly, we designed a pulmonary smooth muscle and endothelial coculture experiment in which the effects of NO on endothelium can be distinguished from the direct effects of cytokines. METHODS: RPASM cells were incubated with a mixture of TNF-alpha (500 units/ml) and IFN-gamma (100 units/ml) for 24 hours. This cytokine mixture was then removed and the NO-producing smooth muscle cells were incubated in a coculture transwell system with rat pulmonary artery endothelial (RPAE) cells. Subsequent NO production (as measured by nitrite concentration in cell supernatants), and the number of viable attached endothelial cells were then measured at 48 hours. RESULTS: RPASM continued to produce large amounts of NO, in the absence of further cytokine stimulation, after a 24-hour exposure to TNF-alpha and IFN-gamma. This RPASM-generated NO decreased the number of viable attached endothelial cells after 24 hour RPASM-RPAE coculture by 57%. The competitive stereospecific inhibitor of inducible NO synthase (iNOS), NG-monomethyl-L-arginine (NMA), returned the inducible NO production to basal levels and reversed the cytotoxic effects on endothelial cells. The number of viable attached endothelial cells returned to control levels. CONCLUSIONS: The NO produced by cytokine-activated RPASM has local cytotoxic effects on RPAE in coculture. Such NO produced in the vasculature may be a factor in the origin of acute lung injury under conditions of trauma and sepsis.

Animals↗

PubMed1996

Cytokine-induced nitric oxide synthase gene transcription is blocked by the heat shock response in human liver cells.

BACKGROUND: Previously we demonstrated that the heat shock response (HSR) inhibits cytokine-stimulated nitric oxide (NO) synthesis and inducible NO synthase (NOS2) expression in hepatocytes. In this study we sought to determine the molecular basis of this inhibition using a human liver cell line. METHODS: After induction of the HSR by sodium arsenite or hyperthermia, the AKN-1 human liver cell line was treated with cytokines to stimulate NOS2 expression and NO production. Western blot analysis for hsp70 was performed, and NOS2 mRNA and 24-hour NO synthesis were quantitated. Cytokine-induced NOS2 promoter activity of AKN-1 cells transfected with a 7.0 kilobase NOS2 promoter luciferase construct and NO production of AKN-1 cells transduced with the NOS2 gene were measured. RESULTS: Sodium arsenite or hyperthermia induced the synthesis of hsp70 protein in AKN-1 cells, indicating activation of the HSR. Cytokines stimulated high levels of NOS2 mRNA and NO production. However, prior induction of the HSR significantly inhibited NOS2 expression and NO synthesis. Cytokine-stimulated NOS2 promoter activity of transfected AKN-1 cells was decreased by 77%, but the HSR did not affect NOS2 enzyme activity in transduced AKN-1 cells. CONCLUSIONS: These findings indicate that the HSR inhibits cytokine-induced NOS2 expression and NO synthesis in AKN-1 cells by preventing NOS2 promoter activation. Effects on NOS2 protein translation or stability were not observed. These data suggest that the HSR, which is expressed in the liver after trauma, shock, or ischemia-reperfusion, blocks NOS2 gene expression at the transcriptional level.

Acute-Phase Reaction↗