Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes.

Abstract

Nitric oxide (NO-) is a short-lived mediator which can be induced in a variety of cell types and produces many physiologic and metabolic changes in target cells. The inducible or high-output NO' synthase (NOS) pathway was first characterized in macrophages activated by lipopolysaccharide (LPS) and interferon v (IFN-y). Hepatocytes also express an inducible NOS following exposure to the combination of endotoxin (LPS) and tumor necrosis factor (TNF), interleukin 1 Following the discovery of the nitric oxide (NO-) pathway and its identification as endothelium-derived relaxing factor, a variety of cell types such as macrophages (1, 2), endothelial cells (3, 4), smooth muscle cells (5), and neurons (6, 7) have been shown to produce NO' from L-arginine. Constitutive and inducible isoforms of the NO' synthase (NOS) enzyme exist, and they differ in structure and regulation (8). The neuronal constitutive NOS is a 150-kDa protein whose activity is dependent upon calcium and calmodulin (7); the inducible macrophage NOS is a 130-kDa protein which is thought to function independently of calcium/calmodulin (9, 10). The constitutive NOS cDNA has been cloned from rat cerebellum and identifies an ==10-kilobase (kb) mRNA on Northern blot analysis (11), while the inducible murine macrophage NOS has been cloned from RAW264.7 cells by three groups and identifies an -4.4-kb mRNA (12-14). The physiologic importance of NON as a vasodilator, neurotransmitter, and antimicrobial/antitumor agent is rapidly becoming apparent. Previous work showed that rat hepatocyte/Kupffer cell cocultures stimulated with lipopolysaccharide (LPS) produce large amounts of nitrite (NO-) and nitrate (NO-), the stable end products of the NO-pathway (15). Further, it was demonstrated that hepatocytes also produce NO-in vivo during chronic hepatic inflammation (16,17) and in vitro in response to conditioned Kupffer cell supernatant (18) or to a mixture of LPS and the cytokines tumor necrosis factor (TNF), interleukin 1 (IL-1), and interferon y (IFN-y) (19). Human hepatocytes were also stimulated to produce NO' by the same combination of endotoxin and cytokines as rat hepatocytes (20). However, essentially nothing has been reported about the direct signals required for inducible NOS gene activation. Therefore, the present study was undertaken to characterize the molecular regulation of the inducible rat hepatocyte NOS by endotoxin and cytokines known to up-regulate hepatocyte NO' synthesis. MATERIALS AND METHODSIsolation of Hepatocytes. Hepatocytes were isolated from male rats (200-250 g, Harlan-Sprague-Dawley) by a modification of the in situ collagenase (Sigma) perfusion technique of Seglen (21). Hepatocytes were separated from nonparenchymal cells by differential centrifugation at 50 x g and then passed over a 30%o Percoll gradient to obtain a highly purified cell population. Hepatocyte purity assessed by microscopy was >98% and viability consistently exceeded 95% by trypan blue exclusion. For the in vivo studies, hepatocytes were harvested from rats 3...

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

“…We previously showed that DEX inhibits inducible nitric oxide synthase gene expression and nitric oxide production in cultured primary hepatocytes stimulated with TNF-a and interleukin-1b by suppressing NF-kB activation. 34,35 Furthermore, DEX can inhibit the production of proinflammatory cytokines in immune cells by the suppression of NF-kB activation. 5 We found that pretreatment of hepatocytes with DEX for 2-12 h inhibited NF-kB activation, as determined by gel-shift assay, in cultured rat primary hepatocytes stimulated with TNF-a (data not shown), suggesting that DEX-mediated upregulation of cFLIP is not due to NF-kB activation.…”

The hepatic circulation in health and disease: Report of a single-topic symposium

“…NO production by inducible NOS (iNOS) in the liver, in contradistinction to eNOS generation of NO, is independent of calcium and largely dependent on the level of iNOS gene expression. 41 Although the liver is not thought to express iNOS basally, every major cell type in the liver expresses iNOS under appropriate circumstances, particularly the hepatocyte. 42 TNF-␣, interleukin 1␤, and interferon gamma all have the capability to induce iNOS transcription in hepatocytes, although IL-1 is the most potent in human and rat hepatocytes.…”

Inflammatory Modulation of Hepatocyte Apoptosis by Nitric Oxide: In Vivo, In Vitro, and In Silico Studies

“…In vitro, interleukin-1 (IL-1), tumor necrosis factor (TNF), interferon-γ (IFN-γ), and bacterial lipopolysaccharide (LPS) act synergistically to induce iNOS expression maximally in hepatocytes. [15] Interleukin-1 is the most potent single inducer of iNOS, [16] while redox stress further activates iNOS gene transcription. [11] In vivo, hepatocyte iNOS expression has been identified in response to endotoxemia, [17] hemorrhagic shock, [18,19] hepatic and intestinal ischemia/reperfusion, [20] and during hepatic regeneration [21] or hepatitis.…”