J. Stadler

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.

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.

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.

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

ObjectiveThis study determined the mechanism used by neutrophils (PMNs) to induce hepatocellular injury. Summary Background DataNeutrophils 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. MethodsNeutrophils 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. ResultsPMA 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. ConclusionsPMNs 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.

Tumor Necrosis Factor Alpha Inhibits Hepatocyte Mitochondrial Respiration and Induces Release of Cytoplasmic Enzymes

Tumor necrosis factor alpha (TNFα) has been identified as a major mediator of physiologic and pathophysiologic responses to infections and traumatic insults. While TNFα enhances tissue repair and host defense against microbials [1] at low concentrations, high concentrations lead to tissue damage and to a typical shock syndrome [2]. As endotoxin was shown to be one of the most important stimuli for TNFα release, it was subsequently demonstrated that many features of the septic shock syndrome are induced by TNFα [3]. In sepsis, Kupffer cells, the largest population of fixed macrophages in the body, are thought to be a major source of TNFα [4]. Due to their close relationship to Kupffer cells, hepatocytes are exposed to TNFα released from Kupffer cells in addition to TNFα coming from the intenstine into the portal bloodstream. Therefore, it may be relevant to investigate whether TNFα contributes to hepatocellular dysfunction, which is a serious problem in sepsis [5]. The mechanisms of TNFα-mediated cytotoxicity are not very well understood. One of the established effects of TNFα at the subcellular level is mitochondrial swelling [6] and a decrease of mitochondrial respiration in tumor cell lines [7]. Because endotoxemia also results in impaired mitochondrial function of hepatocytes [8] we undertook the following experiments to demonstrate whether this effect is also mediated by TNFα.

Nitric Oxide Synthesis in Sponge Matrix Allografts Coincides with the Initiation of the Allogeneic Response

The investigation of the biological effects of nitric oxide (∙N = O) has been of increasing interest in the recent past. Nitric oxide is a product of oxidative L-arginine metabolism, in which L-arginine is metabolized to L-citrulline and ∙N = O by the yet not fully characterized enzyme ∙N = O synthase.

Nitric Oxide Regulates Prostaglandin E2 Release by Rat Kupffer Cells

Nitric oxide has recently been identified as an intermediate of mammalian nitrogen metabolism [1, 2]. This highly reactive radical is derived from the amino acid L-arginine and degrades within seconds into its stable end products nitrite and nitrate. Important biological functions of nitric oxide include regulation of vascular tone [1], inhibition of thrombocyte adherence and aggregation [3], as well as enhancement of macrophage cytotoxicity [4]. Macrophage nitric oxide biosynthesis is inducible, namely by inflammatory mediators such as lipopolysaccharide (LPS) [5]. Kupffer cells, the fixed macrophages of the liver, also produce nitric oxide in response to LPS [6]. In addition, LPS-stimulated Kupffer cells release cytokines, which induce nitric oxide synthesis in hepatocytes [7]. The role of nitric oxide in the septic liver is not very well understood. Experiments were undertaken to determine whether nitric oxide synthesis influences other functions of Kupffer cells, specifically the release of eicosanoids.