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T R Billiar

Publications and source records attributed to T R Billiar.

At least 235 records · Page 13Linked to original sources
PubMed1992

Cytokines and lipopolysaccharide induce nitric oxide synthase in cultured rat pulmonary artery smooth muscle.

In the current study, we describe cytokine and Escherichia coli lipopolysaccharide (LPS) induction of nitric oxide (NO) synthase mRNA levels in cultured smooth muscle from rat pulmonary artery (RPASM). Exposure of RPASM to interleukin-1 beta, interferon-gamma, or LPS alone did not significantly affect NO synthesis, as determined by nitrite concentrations in media. Exposure to tumor necrosis factor-alpha caused a modest (2x) increase in nitrite production. In contrast, exposure to a combination of the above three cytokines and LPS caused a large increase in NO synthesis. Exposure of RPASM to this combination caused an increase in mRNA levels of NO synthase (as described by Northern blot analysis with 32P-cDNA probe to an inducible form of NO synthase present in murine macrophages) that was apparent as early as 4 h. Expression of the induced gene product after exposure to the cytokine and LPS mixture was evident by significant increases in nitrite production at 12 h. Production of nitrite was completely abolished in the presence of NG-monomethyl-L-arginine (NMA), and this inhibition was reversible by the addition of excess L-arginine. NO synthase mRNA levels were not affected by NMA. The nitrite production induced by the combination of cytokines and LPS was abolished by pretreating cells with cycloheximide. These data indicate that a combination of cytokines and LPS affect expression of the gene for the inducible form of NO synthase in cultured RPASM.

Amino Acid Oxidoreductases↗
PubMed1991

Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide.

Although IL-1 is an important modulator of chondrocyte metabolism, the postreceptor events triggered by IL-1 remain obscure. The present study shows that IL-1 induces the biosynthesis of nitric oxide (.N = O) by articular chondrocytes. Synthesis of .N = O is also induced by LPS. Other inflammatory mediators such as IFN-gamma, fibroblast growth factor, and TNF-alpha fail to provoke the production of .N = O, but they increase the potency of IL-1. A combination of IL-1, LPS, and TNF-alpha was shown to induce maximal production of 355 +/- 51 nmol/10(6) cells/72 h of nitrite (NO2-), which was measured as a stable end-product of .N = O generation. The biosynthesis of .N = O requires an induction period of approximately 6 h and continues for at least 72 h. Inhibition of .N = O production with the competitive inhibitor NG-monomethyl-L-arginine (NMA) leads to a suppression of gelatinase and PGE2 synthesis by chondrocytes activated with IL-1 alone. In contrast, NMA enhances the synthesis of both gelatinase and PGE2 after activation with a combination of IL-1, LPS, and TNF-alpha. An increase of PGE2 synthesis from 42.0 +/- 21.0 to 174.0 +/- 33.5 ng/10(6) cells/72 h resulted from the addition of NMA when these stimulatory agents were combined. Exposure of IL-1 and fibroblast growth factor-stimulated chondrocytes to authentic, exogenous .N = O led to an increase of PGE2 synthesis from 5.6 +/- 1.7 of untreated cells to 15.8 +/- 6.8 ng/10(6) of .N = O treated cells within the 1st h. This was followed by a suppression of PGE2 synthesis within the next 2 h.

Animals↗
PubMed1991

Effect of endogenous nitric oxide on mitochondrial respiration of rat hepatocytes in vitro and in vivo.

Nitric oxide, a highly reactive radical, was recently identified as an intermediate of L-arginine metabolism in mammalian cells. We have shown that nitric oxide synthesis is induced in vitro in cultured hepatocytes by supernatants from activated Kupffer cells or in vivo by injecting rats with nonviable Corynebacterium parvum. In both cases, nitric oxide biosynthesis in hepatocytes was associated with suppression of total protein synthesis. This study attempts to determine the effect of nitric oxide biosynthesis on the activity of specific hepatocytic mitochondrial enzymes and to determine whether inhibition of protein synthesis is caused by suppression of energy metabolism. Exposure of hepatocytes to supernatants from activated Kupffer cells led to a 30% decrease of aconitase (Krebs cycle) and complex I (mitochondrial electron transport chain) activity. Using NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthesis, we demonstrated that the inhibition of mitochondrial aconitase activity was due, in part, to the action of nitric oxide. In contrast, in vivo nitric oxide synthesis of hepatocytes from Corynebacterium parvum-treated animals had no effect on mitochondrial respiration. This suggests that inhibition of protein synthesis by nitric oxide is not likely to be mediated by inhibition of energy metabolism.

Aconitate Hydratase↗
PubMed1991

Nitric oxide and nitric oxide-generating compounds inhibit hepatocyte protein synthesis.

Hepatocytes are stimulated to produce nitric oxide (NO.) from L-arginine in response to conditioned Kupffer cell medium or a combination of cytokines. Associated with the production of NO.in hepatocytes, there is a profound decrease in total protein synthesis ([3H]leucine incorporation). This report demonstrates that authentic NO.and the NO.-generating compound S-nitroso-N-acetylpenicillamine inhibit hepatocyte total protein synthesis in a reversible and concentration-dependent fashion. In parallel with the suppression of hepatocyte total protein synthesis, authentic NO.inhibits the production of two specific hepatocyte proteins, albumin and fibrinogen, without influencing the quantity of albumin mRNA. Although authentic NO.induces a rapid increase in cGMP levels in hepatocytes, the addition of the cGMP analog 8-bromoguanosine 3':5' cyclic monophosphate to unstimulated HC cultures does not reproduce the inhibition of total protein synthesis. These data show that NO.is the hepatocyte L-arginine metabolite that inhibits protein synthesis. Furthermore, these findings indicate that NO.does not inhibit hepatocyte protein synthesis solely through the activation of soluble guanylate cyclase but appears to affect a translational or posttranslational process.

Albumins↗
PubMed1991

Nitrogen oxide levels in patients after trauma and during sepsis.

The mediators responsible for maintenance of the hyperdynamic state and the low systemic vascular resistance (SVR) observed in sepsis have not been elucidated. Nitric oxide (.N = O) is a mediator with numerous functions, including regulation of vascular tone and a role in macrophage-mediated cytostasis and microbiostasis. Thirty-nine critically ill trauma and septic patients were studied to determine the relationship between .N = O production and the hyperdynamic state. high plasma levels of NO2-/NO3- (the stable end products of .N = O) were observed in septic patients (p less than 0.02). Low SVR and high endotoxin levels were associated with high NO2-/NO3- values (p = 0.029, p = 0.002). Changes in .N = O levels may mediate the vasodilation seen in sepsis. Low NO2-/NO3- levels were observed in trauma patients (p less than 0.001) and remained low even in the presence of sepsis (p = 0.001).

Adult↗
PubMed1991

Effect of exogenous and endogenous nitric oxide on mitochondrial respiration of rat hepatocytes.

Although nitric oxide (.N = O) biosynthesis is inducible in rat hepatocytes (HC), the physiological significance of .N = O production by these cells is unknown. Short exposure of HC to authentic .N = O led to a concentration-dependent inhibition of mitochondrial aconitase, NADH-ubiquinone oxidoreductase, and succinate-ubiquinone oxidoreductase (complexes I and II of the mitochondrial electron transport chain). Most susceptible to .N = O inhibition was mitochondrial aconitase, in which a reduction in enzyme activity to 20.2 +/- 1.6% of control was observed. In contrast to mitochondrial aconitase, cytosolic aconitase activity was not inhibited by .N = O. After exposure to a maximal inhibitory concentration of .N = O, mitochondrial aconitase activity recovered completely within 6 h. Complex I did not fully recover within this incubation period. Endogenous .N = O biosynthesis was induced in HC by a specific combination of cytokines and lipopolysaccharide. After 18 h of incubation with these stimuli, a significant inhibition of mitochondrial aconitase activity to 70.8 +/- 2.4% of controls was detected. However, this was due only in part to the action of .N = O. A non- .N = O-dependent inhibition of mitochondrial function appeared to be mediated by tumor necrosis factor.

Animals↗
PubMed1991

Nitric oxide synthesis in the in vivo allograft response: a possible regulatory mechanism.

Activated macrophages are known to oxidatively metabolize L-arginine to nitric oxide and citrulline. We have recently shown that nitric oxide is a potent inhibitory molecule in the in vitro rat mixed-splenocyte culture, resulting in inhibition of proliferation and cytolytic T-cell induction. We undertook this study using the sponge matrix allograft model in the rat to determine whether nitric oxide plays a role in an in vivo allograft response. Our experiments showed that on day 6 after grafting, when cytolytic activity of allograft-infiltrating cells is first detected, allogeneic graft fluid contains higher levels of NO2-/NO3- (the stable endproducts of nitric oxide metabolism) than syngeneic graft fluid. Furthermore, evaluation of the supernatants of cultured graft-infiltrating cells revealed that allogeneic graft-infiltrating cells spontaneously produce higher amounts of nitric oxide than syngeneic graft-infiltrating cells. The nitric oxide production was inhibited in the presence of NG-monomethyl-L-arginine (NMA), the competitive inhibitor of nitric oxide production. Most of the nitric oxide production was observed in the adherent macrophage fraction of the allograft-infiltrating cells. When allograft-infiltrating cells were cultured in the presence of NMA, donor-specific cytolytic activity was observed, whereas allograft-infiltrating cells cultured in the absence of NMA showed no cytolytic activity. These data show that nitric oxide production may play an important regulatory role in the allograft response.

Animals↗
PubMed1990

Alloantigen-induced activation of rat splenocytes is regulated by the oxidative metabolism of L-arginine.

Activated macrophages have been demonstrated to metabolize the amino acid L-arginine by the oxidative pathway to produce nitric oxide, citrulline, and NO2-/NO3-. Nitric oxide has been shown to be cytostatic for tumor targets and to inhibit the mitochondrial respiration and other functions of the macrophages that produce it. Addition of NG monomethyl-L-arginine (NMA), a competitive inhibitor of oxidative L-arginine metabolism, to rat splenocyte (SPL) MLC results in allospecific lymphocyte proliferation and CTL induction. In the absence of NMA, neither proliferation nor CTL induction is observed. Citrulline and NO2-/NO3- levels in the supernatants of rat SPL MLC are decreased in the presence of NMA compared with cultures without NMA. NMA also augments the proliferation and CTL induction in mouse SPL MLC. Detectable levels of cytokines able to induce T cell proliferation were present in supernatants of rat SPL MLC without NMA on days 1 to 5 of culture. Supernatants of cultures with NMA contained detectable levels of cytokines on days 1 to 3 and undetectable levels by days 4 and 5 of culture, concomitant with the observed lymphocyte proliferation and presumed depletion of cytokines. Thus, inhibition of rat SPL proliferation to alloantigen seems not to be caused by the lack of production of cytokines able to induce T cell proliferation. The inhibition of proliferation and CTL induction in rat SPL cultures may be caused by a direct effect of the cytostatic products of oxidative L-arginine metabolism on lymphocyte proliferation, or by an indirect deleterious effect on the mitochondrial respiration and viability of macrophages that oxidatively metabolize L-arginine. Alternatively, diversion of L-arginine to the oxidative pathway may affect production of polyamines that are necessary for cell growth and proliferation.

Animals↗
PubMed1990

Inducible cytosolic enzyme activity for the production of nitrogen oxides from L-arginine in hepatocytes.

The in vivo conditions needed for the induction of nitrogen oxide synthesis by hepatocytes were determined. Hepatocytes obtained from rats injected with killed Corynebacterium parvum spontaneously produced NO2(-)+NO3- in culture and were found to contain cytosolic enzyme activity for nitrogen oxide synthesis. The enzyme activity required both L-arginine and NADPH, and was not found in hepatocytes obtained from normal rats or rats injected with lipopolysaccharide (LPS) alone. In contrast, nonparenchymal cells were stimulated to synthesize NO2(-)+NO3- by LPS. These results show the presence of inducible cytosolic enzyme activity for nitrogen oxide synthesis in hepatocytes, which is distinct from nonparenchymal cell NO. synthesis.

Animals↗
PubMed1990

Modulation of nitrogen oxide synthesis in vivo: NG-monomethyl-L-arginine inhibits endotoxin-induced nitrate/nitrate biosynthesis while promoting hepatic damage.

Attempts were made to promote or inhibit nitric oxide (. N = O) synthesis in a murine model of hepatic damage (Corynebacterium parvum followed by lipopolysaccharide; LPS) to determine the role of . N = O in the liver injury. Moderate hepatic damage and increases in circulating NO2-/NO3- levels were detectable after C. parvum alone. Administration of LPS to these mice resulted in severe hepatic damage and acute elevations in circulating nitrogen oxide levels. L-arg had no influence on the C. parvum or LPS-induced changes. NG-monomethyl-L-arginine (NMA) had no effect in the absence of LPS, but when given with LPS, a dose-dependent suppression in plasma NO2-/NO3- levels and an increase in liver injury were seen. The NMA-induced changes were partially reversed by the simultaneous administration of L-arg. These findings suggest a protective role for . N = O in this model.

Animals↗
PubMed1990

Kupffer cell:hepatocyte cocultures release nitric oxide in response to bacterial endotoxin.

Nitric oxide (NO.) is a short-lived intermediate in a biochemical pathway where L-arginine is converted to L-citrulline and nitrite/nitrate (NO2-/NO3-). This highly reactive molecule is the biologically active component of this inducible pathway in macrophages. Using a rat Kupffer cell:hepatocyte (KC:HC) coculture model, we have previously shown that this combination of cells produces large quantities of both citrulline and NO2-/NO3- if exposed to lipopolysaccharides (LPS) but we did not determine whether nitric oxide was produced or released. We had also shown that this L-arginine metabolism was associated with a profound decrease in total protein synthesis. In these experiments, we show that KC:HC cocultures release nitric oxide into the culture supernatant if exposed to LPS. NO. production by these cells requires L-arginine and is inhibited by NG-mono-methyl-L-arginine. In addition, the time course for NO. release by KC:HC cocultures parallels the previously reported time course for NO2-/NO3- synthesis and the decrease in protein synthesis, supporting the hypothesis that NO. is the reactive nitrogen intermediate of the pathway responsible for this inhibition of protein synthesis. Finally, we show that KC:HC cocultures release more NO. than KC alone in response to LPS, and we propose that the combination of KC and HC acts as a functional unit capable of generating large amounts of NO. from L-arginine in gram-negative sepsis.

Animals↗
PubMed1990

Kupffer cell-hepatocyte interactions and the changes in 1-14C-arachidonate incorporation in response to endotoxin in vitro.

The present experiments were undertaken to elucidate the effect of either the hepatocyte (HC) or hepatocyte supernatant on prelabeled endotoxin (LPS)-stimulated Kupffer cell (KC) arachidonic acid utilization. HC, KC, or their coculture were incubated for 18 hours with labeled 1-14C- arachidonic acid followed by a 24 hour incubation with 10 micrograms/ml LPS. LPS had no effect on the percent distribution of labeled arachidonate into the HC phospholipid or neutral lipid. KC showed a decreased percent neutral lipid labeled arachidonic acid distribution with generally no effect on the phospholipid. However, KC:HC cocultures or the addition of HC supernatant to KC exposed to LPS dramatically reversed the labeled arachidonate distribution into the KC with an increased incorporation into neutral lipid. Labeled PGE2 and PGD2 were increased in the KC following incubation with HC supernatant while only labeled PGE2 levels were elevated in the cocultures. The changes in the distribution of cell's labeled arachidonate required the addition of LPS. These results suggest that the HC can promote changes in the lipid fraction during sepsis by elaborating a substance that can modulate labeled arachidonate distribution in the KC lipids as well as stimulate prostaglandin production.

Animals↗
PubMed1990

Multiple cytokines are required to induce hepatocyte nitric oxide production and inhibit total protein synthesis.

The etiology and mechanisms by which severe trauma or sepsis induce hepatic failure are unknown. Previously we showed that Kupffer cells (KC), the fixed macrophages of the liver, induce a profound decrease in hepatocyte (HC) total-protein synthesis when exposed to endotoxin. Furthermore we demonstrated that endotoxin-activated KCs induce these changes in HC protein synthesis through the induction of a novel L-arginine-dependent biochemical pathway within the HC. In this pathway, the guanido nitrogen of L-arginine is converted to the highly reactive molecule nitric oxide (NO.). To identify the KC factors that act as signals for induction of HC NO. biosynthesis, recombinant cytokines were added to HC cultures and HC nitrogen oxide production and protein synthesis levels were determined. We found that no single cytokine, but rather a specific combination of tumor necrosis factor, interleukin-1, interferon-gamma, and endotoxin, were required for maximal induction of HC nitrogen oxide production. This specific combination of cytokines induced a 248.8 +/- 26.0 mumol/L (micromolar) increase in HC nitrogen oxide production and simultaneously inhibited HC total protein synthesis by 36.1% +/- 3.1%. These data demonstrate that multiple cytokines, produced by endotoxin-activated KC, induce the production of NO. within HC, which in turn leads to the inhibition of HC total-protein synthesis.

Animals↗