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

Abstract

SummaryNitric 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-% and endotoxin. Increased levels of nitrite (NO2-) and nitrate (N03-) in culture supernatants were associated with NADPH-dependent NOS activity in the cell lysates. The production of NO2-and N03-was inhibited by N6-monomethyl I.-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.A variety of cell types have been shown to produce nitric oxide (NO) from t-arginine by either a constitutive and/or an inducible enzyme (1). Recent reports provide strong evidence that vasorelaxation, induced through the constitutive and inducible NO pathway, might play an important role in the regulation of vascular tone in humans (2, 3). Little evidence for the existence of inducible NO enzyme activity in specific human cell types has been documented, despite its clear potential role in the elimination of tumor cells (4) and intra-and extracellular pathogens (recently reviewed in reference 5), as well as in the induction of sustained hypotension (6), as shown in animal models.We have shown that rat hepatocytes (HC) cocultured with Kupffer ceils and stimulated with LPS can produce large amounts of nitrite (NO2-) and nitrate (N03-), the stable end products of NO (7). Furthermore, we have shown that HC alone are capable of biosynthesis of NO when exposed to various immunostimuli (8-10). The expression of the inducible nitric oxide synthase (NOS) alters various HC functions in vitro. These include a substantial decrease in total protein synthesis (7, 11), the inhibition of mitochondrial aconitase (12), and the stimulation of cyclic guanylate monophosphate (cGMP) synthesis and release (13). In vivo studies suggest that hepatic NO can protect the liver from damage in sepsis (14). In this communication, we report that nitrogen oxides are also produced in large amounts by human HC in a reproducible manner, demonstrating that a human call type can, indeed, express an inducible NOS. Materials and MethodsCulture Medium. HC cultures were performed in Williams medium E (Gibco Laboratories, Grand Island, NY) supplemented with 10 .6 M insulin, 15 mM Hepes, t-glutamine, penicillin, streptomycin, and 10% dialyzed calf serum. Additional culture reagents included t-arginine hydrochloride (Gibco Laboratories) and N ~-monomethyl-r-arginine acetate (NMA), prepared by a modification of the method previously described (15).Hepatocyte Isolation. In acc...

Nitric oxide localized to spinal cords of mice with experimental allergic encephalomyelitis: an electron paramagnetic resonance study.

“…No EPR signals corresponding to nitrosyl-heme proteins within the peripheral blood could be identified. Although hepatocytes are known to be induced by cytokines (IFN-% TNF) to produce NO (29), an iron-nitrosyl signal was not observed in the liver, an organ unaffected by EAE. Perhaps any NO released from the CNS reacted locally with oxygen or superoxides before binding to heme proteins in the blood or distal organs.…”

TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer

“…In contrast, NOS2 (also called iNOS) requires induction generally, but is able to produce NO concentrations in the micromolar range (reviewed by Ambs et al, 1997). NOS2 gene expression can be induced by bacterial endotoxins, proinflammatory cytokines or hypoxia (Wild et al, 1986;Nussler et al, 1992;Lombard and Guarente, 2000) in many cell types, including macrophages (Xie et al, 1992) and hepatocytes (Mowat et al, 1990;Lombard and Guarente, 2000;Vodovotz et al, 2004) as well as in a variety of human tumors (reviewed by Ambs et al, 1997). During chronic viral hepatitis, the upregulation of certain proinflammatory cytokines, like tumor necrosis factor-a (TNF-a) and interferon-g (IFN-g), has been repeatedly demonstrated (Gonzalez-Amaro et al, 1994;Mihm et al, 1996).…”

Dexamethasone protects primary cultured hepatocytes from death receptor-mediated apoptosis by upregulation of cFLIP

“…15 In accordance with institutional review guidelines of the University of Pittsburgh, human hepatocytes were obtained by collagenase digestion of human donor livers not used for transplantation. 45 Highly purified hepatocytes (498% purity and 498% viability by trypan blue exclusion) were suspended in Williams medium E supplemented with 10% calf serum, 1 mM insulin, 2 mM L-glutamine, 15 mM HEPES (pH 7.4), 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were plated in 100 mm Petri dishes (5 ml/dish) and 12-well plates (1 ml/well) at a concentration of 1 Â 10 6 and 2 Â 10 5 cells/ml, respectively, and cultured in a CO 2 incubator (5% CO 2 /95% air) at 371C for 16 h. Cells were pretreated with or without DEX and incubated with 2000 U/ml TNF-a plus 0.2 mg/ml actinomycin D (ActD) (TNF-a þ ActD) or 0.5 mg/ml anti-Fas antibody (Jo2) plus 0.2 mg/ml ActD.…”