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Biomedical subjects

T R Billiar

Publications and source records attributed to T R Billiar.

At least 217 records · Page 12Linked to original sources
PubMed1993

Endogenous nitric oxide inhibits the synthesis of cyclooxygenase products and interleukin-6 by rat Kupffer cells.

Macrophage production of nitric oxide (.N = O) leads to considerable alterations of vital metabolic pathways in various target cells. The present study tested whether .N = O synthesis by Kupffer cells (KCs), the resident macrophages of the liver, interferes with the secretory function of these cells. As in other macrophage-type cells, the combination of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) was a potent stimulus of .N = O synthesis by KC. Treatment with LPS and IFN-gamma also induced significant production of prostaglandin E2 (PGE2), thromboxane B2 (TBX2), tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6. Inhibition of .N = O synthesis by KC. Treatment with LPS and IFN-gamma also induced significant production of prostaglandin E2 (PGE2), thromboxane B2 (TBX2), tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6. Inhibition of .N = O synthesis by the L-arginine analogue of NG-monomethyl-L-arginine (NMA) resulted in a further increase of PGE2, TXB2, and IL-6 but not IL-1 and TNF-alpha production, indicating specific inhibitory effects of endogenous .N = O synthesis on the secretory activity of KCs. PGE2 production was most sensitive to the suppressive effect of .N = O and increased 24 h after stimulation with LPS and IFN-gamma from 16.3 +/- 4.9 ng/10(6) KCs without NMA to 94.3 +/- 17.9 ng/10(6) KCs with NMA. This effect of NMA was reversed by a 10-fold increase of the L-arginine concentration. No recovery of PGE2 production was seen when .N = O synthesis was blocked after 24 h. NMA treatment increased cyclooxygenase activity more than threefold, suggesting that .N = O inhibits PGE2 and TXB2 production through diminished PGH2 availability. .N = O synthesis did not significantly affect total protein synthesis or viability of the KCs. These results show that .N = O influences the production of specific inflammatory mediators by KCs.

Animals↗
PubMed1993

Nonheme iron-nitrosyl complex formation in rat hepatocytes: detection by electron paramagnetic resonance spectroscopy.

Isolated rat hepatocytes were examined by EPR spectroscopy after exposure to inflammatory stimuli (interferon-gamma [IFN-gamma], tumor necrosis factor-alpha [TNF-alpha], interleukin-1 beta [IL-1 beta], and lipopolysaccharide [LPS]) in vitro, after in vivo immune activation by Corynebacterium parvum, and after exposure to .N = O and to nitroprusside (nitroferricyanide), an NO-donating nitrovasodilator. Hepatocytes exposed to IFN-gamma, TNF-alpha, IL-1 beta, and LPS demonstrated the appearance of a g = 2.04 axial EPR signal indicative of the formation of nonheme iron-nitrosyl complexes. Concurrent incubation with L-NG-monomethylarginine (L-NMMA), a competitive inhibitor of .N = O synthase, prevented the appearance of the signal. The g = 2.04 signal was localized in the cytosolic fraction of hepatocyte extracts. Hepatocytes freshly isolated from C. parvum-treated rats exhibited a modest g = 2.04 signal, which was increased by a factor of approximately 2.5-fold upon subsequent 24-h culture in media without additional stimuli. This increase was prevented by L-NMMA in the culture medium and also by the presence of rat erythrocytes added to the culture. In the presence of erythrocytes, virtually all of the .N = O produced was oxidized by reaction with intracellular hemoglobin within the erythrocyte, as judged by the relative amounts of nitrite and nitrate detected. These results suggest that in this model system .N = O is sufficiently stable and diffusible to escape from the hepatocyte and diffuse into the erythrocyte without first reacting with oxygen or with intracellular iron at the site of its formation within the hepatocyte. Treatment of hepatocytes with exogenous .N = O or nitroprusside generated an identical g = 2.04 signal of much greater intensity than with cytokines plus LPS. Treatment with nitroprusside also caused the appearance of a signal from pentacyanonitrosylferrate ion, verifying the previously reported metabolism of this nitrovasodilator by reduction and liberation of cyanide ion and .N = O. These results indicate significant differences in intracellular nonheme iron nitrosylation in hepatocytes compared to cytotoxic activated macrophages, which may correlate with the differences in physiological function of .N = O in these two systems.

Animals↗
PubMed1993

Nitric oxide synthase expression is induced in hepatocytes in vivo during hepatic inflammation.

Nitric oxide (NO.) is a short-lived biologic mediator produced by the enzyme NO. synthase (NOS) which exists in constitutive and inducible isoforms. Previously, we have shown that hepatocytes express an inducible NOS in vitro following exposure to the combination of lipopolysaccharide and inflammatory cytokines. The purpose of the present study is to characterize the induction of NOS in vivo in rat hepatocytes during chronic hepatic inflammation triggered by Corynebacterium parvum injection and to correlate NO. synthesis with the timing of liver injury. Using Northern blot hybridization, hepatocyte-inducible NOS mRNA was detected 3 days after C. parvum administration and was not found in normal hepatocytes. Hepatocyte NOS activity was significantly increased 3 to 7 days after C. parvum. Plasma concentrations of nitrite and nitrate (NO2- + NO3-), the stable end products of NO. oxidation, increased from a basal concentration of 21.0 +/- 2.5 to 2439.6 +/- 364.2 microM 3 days after injection. Urinary excretion of NO2- + NO3- also increased in a parallel manner. Plasma liver injury enzymes were elevated three to sixfold in vivo at 3 to 5 days following C. parvum and coincided with the period of maximal NO production. The results show that NO. is produced directly by hepatocytes in vivo during hepatic inflammation and suggest a role for NO. in mediating the hepatic response to inflammatory stimuli.

Amino Acid Oxidoreductases↗
PubMed1993

Hepatocyte injury by activated neutrophils in vitro is mediated by proteases.

OBJECTIVE: This study determined the mechanism used by neutrophils (PMNs) to induce hepatocellular injury. SUMMARY BACKGROUND DATA: Neutrophils have been shown to be potent mediators of cell and tissue injury and have been hypothesized to contribute to the hepatic injury that occurs after trauma and infection. Oxygen radical scavengers protect the liver in vivo from inflammatory injury and it has been suggested that PMNs are the source of these toxic oxygen radicals. The specific mechanism used by PMNs to produce hepatocellular damage, however, has not been determined. METHODS: Neutrophils were cultured in vitro with hepatocytes (HCs) and stimulated with phorbol 12-myristate 13-acetate (PMA) to induce HC injury in the presence of oxygen radical scavengers and protease inhibitors. RESULTS: PMA induced a PMN-mediated HC injury that was dependent on the number of PMNs present and the concentration of PMA. Protease inhibitors reduced the extent of HC injury, while oxygen radical scavengers had no effect. Hydrogen peroxide, directly applied, was able to injure HCs, but only at concentrations greater than those that could be produced by PMA-stimulated PMNs. CONCLUSIONS: PMNs are cytotoxic to cultured HCs, predominantly due to the release of proteolytic enzymes, while HCs appear relatively resistant to oxidative injury. Involvement of neutrophil toxic oxygen radicals in hepatic damage in vivo may require impairment of HC antioxidant defenses or may involve injury to nonparenchymal liver cells with secondary effects on HCs.

Animals↗
PubMed1993

Nitric oxide production is inhibited in trauma patients.

Elevated levels of nitrates/nitrites, the stable endproducts of nitric oxide (NO), were recently observed in septic patients. In this setting, NO maintains blood flow by vasodilation and inhibition of platelet aggregation. Trauma patients were found to have low plasma levels of nitrates/nitrites, even when they developed sepsis. The current study substantiated that trauma patients have suppressed production of NO; reductions in plasma nitrate/nitrite levels correlated with low urinary excretion of these endproducts. Nitric oxide production was upregulated in trauma patients with clinical infection compared with trauma patients without infection, but was still significantly suppressed compared with nitric oxide production in normal controls. The inability of trauma patients to produce NO may be an important component of the susceptibility of these patients to infection.

Adolescent↗
PubMed1993

Cytokine induction of interferon regulatory factor-1 in hepatocytes.

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.

Animals↗
PubMed1993

Antisense oligodeoxynucleotide to inducible nitric oxide synthase inhibits nitric oxide synthesis in rat pulmonary artery smooth muscle cells in culture.

BACKGROUND: We recently demonstrated the induced expression of the inducible nitric oxide synthase (iNOS) gene in cultured rat pulmonary artery smooth muscle (RPASM) in response to lipopolysaccharide and cytokines, using a complementary DNA probe to murine macrophage iNOS. Because nitric oxide (NO) can be cytotoxic, iNOS in the pulmonary vasculature may contribute to lung injury in sepsis. We designed an antisense oligodeoxynucleotide complementary to the iNOS messenger RNA (mRNA) sequence to determine whether the probe prevented iNOS translation. METHODS: RPASM, preincubated in the presence of antisense and sense oligodeoxynucleotide to the first 18 bases after the initiation codon of iNOS mRNA, was exposed to interferon-gamma and tumor necrosis factor-alpha to induce NO production (as measured by NO2-, the stable end product of NO formation). RESULTS: Interferon-gamma and tumor necrosis factor-alpha induced NO production in RPASM: The antisense probe caused up to a 36% decrease in cytokine-induced NO2- production in a concentration-dependent manner (1 to 10 mumol/L). The sense probe had no effect. CONCLUSIONS: Increased transcription of iNOS mRNA is an essential step in the induced production of NO by RPASM: Antisense probes partially inhibit iNOS expression in vitro, suggesting its use to inhibit iNOS expression under pathologic conditions.

Amino Acid Oxidoreductases↗
PubMed1993

Inflammation, immunoregulation, and inducible nitric oxide synthase.

Nitric oxide (NO), the molecule of the year 1992, is gaining recognition as an important biological mediator. Its multitude of physiologic and pathophysiologic functions result from both a wide distribution of synthesis and diverse mechanisms of action. Besides its functions as a potent vasodilator and neurotransmitter, NO is important in inflammation and immunity. Both beneficial and detrimental consequences of induced synthesis have been discovered. Information is now accumulating on the regulation and function of induced NO. The recent cloning of human inducible NO cDNA should assist in defining the role of inducible NO in human physiology and pathophysiology.

Amino Acid Oxidoreductases↗
PubMed1992

Nitric oxide-induced S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase inhibits enzymatic activity and increases endogenous ADP-ribosylation.

Using conditions that produced chronic inflammation in rat liver, we were able to find a correlation between induction of nitric oxide production and inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). This enzyme is a tetramer composed of identical M(r) 37,000 subunits. The tetramer contains 16 thiol groups, four of which are essential for enzymatic activity. Our information indicates that four thiol groups are S-nitrosylated by exposure to authentic nitric oxide (NO) gas. Furthermore, NO decreased GAPDH activity while increasing its auto-ADP-ribosylation. Reduced nicotinamide adenine dinucleotide and dithiothreitol are required for the S-nitrosylation of GAPDH caused by the NO-generating compound sodium nitroprusside. Our results suggests that a new and important action of nitric oxide on cells is the S-nitrosylation and inactivation of GAPDH. S-Nitrosylation of GAPDH may be a key covalent modification of multiple regulatory consequences in chronic liver inflammation.

Adenosine Diphosphate Ribose↗
PubMed1992

Stimulation of the nitric oxide synthase pathway in human hepatocytes by cytokines and endotoxin.

Nitric oxide (NO) is a short-lived biologic mediator that is shown to be induced in various cell types and to cause many metabolic changes in target cells. Inhibition of tumor cell growth and antimicrobial activity has been attributed to the stimulation of the inducible type of the NO synthase (NOS). However, there is limited evidence for the existence of such inducible NOS in a human cell type. We show here the induction of NO biosynthesis in freshly isolated human hepatocytes (HC) after stimulation with interleukin 1, tumor necrosis factor (TNF), IFN-gamma, and endotoxin. Increased levels of nitrite (NO2-) and nitrate (NO3-) in culture supernatants were associated with NADPH-dependent NOS activity in the cell lysates. The production of NO2- and NO3- was inhibited by NG-monomethyl L-arginine and was associated with an increase in cyclic guanylate monophosphate release. The data presented here provide evidence for the existence of typical inducible NO biosynthesis in a human cell type.

Amino Acid Oxidoreductases↗
PubMed1992

Increased circulating nitrogen oxides after human tumor immunotherapy: correlation with toxic hemodynamic changes.

BACKGROUND: Toxicity to interleukin-2 (IL-2) tumor immunotherapy is manifested principally by the vascular leak syndrome, hypotension, and a hyperdynamic response with low systemic vascular resistance. Nitric oxide (.N = O), a recently discovered biological mediator of vascular smooth muscle relaxation, is produced in increased amounts by numerous cell types exposed to a number of inflammatory cytokines. PURPOSE: Our purpose was to determine if there is an increased production of .N = O in patients receiving IL-2 tumor immunotherapy, and, if so, whether increases in .N = O production correlate with hemodynamic instability. METHODS: Twelve patients undergoing immunotherapy trials with IL-2 and anti-CD3 monoclonal antibody-activated lymphocytes (T-AK cells) were studied. Plasma levels of nitrate (NO3-), the stable end metabolic product of .N = O synthesis, were measured before and at the end of IL-2 treatment cycles. RESULTS: We observed a ninefold increase in plasma levels of NO3- in patients after 7 days of treatment (P less than .0001). A significant decrease in both systolic and diastolic blood pressures was observed in all patients (P less than .001). CONCLUSIONS: We propose that mediated induction of .N = O synthase enzyme leads to progressive increases in .N = O production which, in turn, produces clinically significant hypotension. IMPLICATIONS: Since .N = O synthesis can be competitively inhibited by L-arginine analogues, a possible pharmacologic modulation of .N = O production could potentially contribute to better management of toxic side effects seen in IL-2 cancer therapies.

Amino Acid Oxidoreductases↗
PubMed1992

Liver nonparenchymal cells are stimulated to provide interleukin 6 for induction of the hepatic acute-phase response in endotoxemia but not in remote localized inflammation.

It has been postulated that Kupffer cells provide signals that regulate hepatocyte responses in sepsis and inflammation. Although in vitro data support such a hypothesis, to our knowledge, no in vivo evidence has been reported. We injected rats with lipopolysaccharide intraperitoneally to simulate sepsis or turpentine intramuscularly to mimic localized inflammation. Both treatments are known to induce the hepatic acute-phase response. Liver nonparenchymal cells and hepatocytes were isolated and placed in culture. Hepatocyte fibrinogen synthesis was measured as an indication of interleukin 6 exposure, while nonparenchymal interleukin 6 production was measured directly. Both lipopolysaccharide and turpentine stimulated a sharp increase in hepatocyte fibrinogen synthesis (turpentine greater than lipopolysaccharide). However, only lipopolysaccharide injection was associated with increased nonparenchymal cell interleukin 6 synthesis. Increased circulating levels of interleukin 6 could be found only after lipopolysaccharide injection. In addition, tumor necrosis factor synthesis was enhanced by lipopolysaccharide but not turpentine. Our data show that nonparenchymal cells are stimulated to provide the interleukin 6 signal to hepatocytes in endotoxemia but not in remote localized inflammation, even though both treatments stimulate the hepatic acute-phase response. Our findings support paracrine functions for liver sinusoidal cells in certain septic states.

Acute-Phase Reaction↗
PubMed1992

Inhibition of nitric oxide synthesis during endotoxemia promotes intrahepatic thrombosis and an oxygen radical-mediated hepatic injury.

Corynebacterium parvum-treated mice produce large amounts of circulating nitrogen oxides and develop a severe liver injury in response to lipopolysaccharide (LPS). Concurrent administration of NG-monomethyl-L-arginine not only suppresses nitric oxide synthesis in these animals but also profoundly increases the hepatic damage following LPS. In this report, we present evidence that the increased hepatic damage from inhibition of nitric oxide synthesis is mediated in part by superoxide and hydroxyl radicals. The hepatic damage induced by suppressing nitric oxide production during endotoxemia could be reduced by treating mice with superoxide dismutase and deferoxamine, scavengers of superoxide and hydroxyl radicals, respectively. This damage could also be prevented by treating mice with the anticoagulant heparin sodium. The results suggest that nitric oxide synthesis during endotoxemia is important in preventing hepatic damage by reducing oxygen radical-mediated hepatic injury and preventing intravascular thrombosis.

Animals↗
PubMed1992

Tumor necrosis factor alpha inhibits hepatocyte mitochondrial respiration.

Although direct cytotoxicity is a well-established phenomenon of tumor necrosis factor alpha (TNF alpha)-induced tissue damage, the intracellular events leading to cell death are still poorly understood. To study the cytotoxic effects of TNF alpha on normal parenchymal cells, rat hepatocytes were purified and incubated with various concentrations of TNF alpha. Mitochondrial respiration, total protein synthesis, and enzyme release were measured to assess metabolic performance and cell integrity. Treatment with TNF alpha suppressed mitochondrial respiration in a concentration-dependent fashion, resulting in a reduction of the activity of complex I of the respiratory chain to 67.0 +/- 3.5% of that of untreated hepatocytes by 2000 U/mL TNF alpha. Under these conditions protein synthesis and the release of intracellular enzymes were significantly increased. Both hepatocellular enzyme release and inhibition of mitochondrial respiration appear to be associated with the generation of reactive oxygen intermediates by the hepatocyte itself, because oxygen radical scavengers prevented these adverse effects of TNF alpha. Inhibition of protein synthesis by cycloheximide as well as addition of cyclic adenosine monophosphate synergistically enhanced the suppression of mitochondrial respiration by TNF alpha, resulting in complex I activity of 6.9 +/- 1.6% and 24.9 +/- 2.9% of that of untreated cells. These data indicate that inhibition of mitochondrial respiration is one of the mechanisms by which TNF alpha induces tissue injury.

Alanine Transaminase↗
PubMed1992

Nitric oxide synthesis serves to reduce hepatic damage during acute murine endotoxemia.

BACKGROUND AND METHODS: Nitric oxide synthesis occurs both in vitro and in vivo in response to inflammatory stimuli and can have profound effects on the local cellular environment. Hepatocytes, Kupffer cells, and endothelial cells produce nitric oxide in vitro, but the in vivo role of this reactive mediator in the liver is unknown. We assessed the role of nitric oxide synthesis during endotoxemia in mice by inhibiting its synthesis with NG-monomethyl-L-arginine after lipopolysaccharide injection and by determining the effects of this inhibition on hepatic damage. RESULTS: Injection of lipopolysaccharide in mice increased plasma nitrite and nitrate concentrations, the stable end products of nitric oxide metabolism, and caused mild hepatic damage as measured by increased circulating hepatocellular enzyme levels. NG-monomethyl-L-arginine decreased plasma nitrite and nitrate values, but increased the lipopolysaccharide-induced hepatic injury. NG-monomethyl-L-arginine caused no hepatic damage when given without lipopolysaccharide. The extent of hepatic damage with NG-monomethyl-L-arginine was proportional to the dose of lipopolysaccharide used and could be reduced with concurrent administration of L-arginine but not D-arginine. CONCLUSIONS: Nitric oxide synthesis provides a protective function against lipopolysaccharide-induced liver injury that increases in importance as the degree of endotoxemia increases. The production of nitric oxide is, therefore, an important part of the liver's response to a systemic inflammatory stimulus.

Acute Disease↗
PubMed1992

Evidence that nitric oxide production by in vivo allosensitized cells inhibits the development of allospecific CTL.

The oxidative metabolism of L-arginine to its bioactive product, nitric oxide (.N = O) has been shown to inhibit rat splenocyte mixed lymphocyte reactions. To determine if alloantigen-induced .N = O production might be operative in vivo, cells that had infiltrated a rat sponge matrix allograft were tested for de novo .N = O production as well as .N = O production upon restimulation with the sensitizing alloantigen. When graft-infiltrating cells were placed in culture, a peak in de novo .N = O production was observed by day 6 graft-infiltrating cells, the time when donor-specific CTL activity by the graft-infiltrating cells was first observed. Upon restimulation with alloantigen, allograft-infiltrating cells produced greatly increased levels of .N = O, and this production was associated with inhibition of lymphocyte cytolytic function. The addition of NG-monomethyl-L-arginine (NMA), the competitive inhibitor of oxidative L-arginine metabolism, inhibited .N = O production and promoted allospecific CTL development. Both observed effects of NMA were reversed by addition of excess L-arginine. Cytokine(s) able to induce proliferation of the IL-2-dependent T cell line CTLL-2 could be detected in alloantigen-stimulated cultures in both the presence and absence of NMA. However, proliferation of the graft-infiltrating cells in response to these cytokines was observed only in the presence of NMA. The immunosuppressive macrolide FK506 was a potent inhibitor of .N = O production in these cultures, presumably acting by inhibiting the production of those cytokines that induce the oxidative L-arginine pathway.

Animals↗
PubMed1992

Association between synthesis and release of cGMP and nitric oxide biosynthesis by hepatocytes.

Hepatocytes are known to synthesize nitric oxide (NO) from L-arginine via an inducible NO synthase. Studies were performed to determine the relationship between hepatocyte NO production and the stimulation of hepatocyte soluble guanylate cyclase. A combination of lipopolysaccharide (LPS), interferon-gamma, tumor necrosis factor, and interleukin-1 stimulates the biosynthesis of large quantities of nitrite and nitrate (NO2- + NO3-). Hepatocyte NO2- + NO3- production was associated with only small increases in intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels but much greater increases in extracellular cGMP release over an 18-h time period. This cGMP synthesis was dependent on the L-arginine concentration and was inhibited in a reversible manner by NG-monomethyl-L-arginine. The cytokines or LPS added alone induced small increases in nitrogen oxide production and concomitant minor elevations in cGMP release. Atrial natriuretic peptide also stimulated the release of cGMP by hepatocytes which appeared to be independent of the cytokine+LPS-induced cGMP release. The addition of probenecid reduced the cGMP release by 66%, while cell damage was excluded as a cause for the extracellular release. Addition of 3-isobutyl-1-methylxanthine, but not M&B 22948, increased hepatocyte intra- and extracellular cGMP levels after cytokine+LPS stimulation. Induction of nitrogen oxide synthesis by hepatocytes in vivo by injecting rats with killed Corynebacterium parvum resulted in increased cGMP levels in freshly isolated hepatocytes and increased cGMP release by the hepatocytes when placed in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗