Ronald D. Curran

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).

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.

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.

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.

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‐γ (IFN‐γ) was a potent stimulus of N=O synthesis by KC. Treatment with LPS and IFN‐γ also induced significant production of prostaglandin E2 (PGE2), thromboxane B2 (TBX2), tumor necrosis factor a (TNF‐ α), interleukin‐1 (IL‐1), and IL‐6. Inhibition of N=O synthesis by the L‐arginine analogue NG‐monomethyl‐L‐ar‐ ginine (NMA) resulted in a further increase of PGEs, TXBs, and EL‐6 but not BL‐l and TNF‐α 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‐γ from 16.3 ± 4.9 ng/106 KCs without NMA to 94.3 ± 17.9 ng/106 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./

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.—Curran, R. D.; Ferrari, F. K.; Kispert, P. H.; Stadler, J.; Stuehr, D. J.; Simmons, R. L.; Billiar, T. R. Nitric oxide and nitric oxide‐generating compounds inhibit hepatocyte protein synthesis. FASEB J. 5: 2085–2092; 1991.

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

Background and MethodsNitric 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, Kupfler 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. ResultsInjection 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. ConclusionsNitric 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 livers response to a systemic inflammatory stimulus.

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.

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.

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.