Nitric oxide is a short-lived biologic mediator for diverse cell types. Synthesis of an inducible nitric oxide synthase (NOS) in murine macrophages is stimulated by lipopolysaccharide (LPS) and interferon y. In human hepatocytes, NOS activity is induced by treatment with a combination of tumor necrosis factor, interleukin 1, interferon y, and LPS. We now report the molecular cloning and expression of an inducible human hepatocyte NOS (hep-NOS) cDNA. hep-NOS has 80% amino acid sequence homology to macrophage NOS (mac-NOS Nitric oxide (NO) is a recently recognized messenger molecule mediating diverse functions including vasodilation, neurotransmission, and antimicrobial and antitumor activities. Different cells such as macrophages (1, 2), endothelial cells (3, 4), neurons (5, 6), smooth muscle cells (7,8), and cardiac myocytes (9) produce NO from L-arginine. Constitutive and inducible isoforms of NO synthase (NOS) differ in structure and regulation (reviewed in refs. 10 and 11). Constitutive NOS has been cloned from rat cerebellum (12) and bovine (13,14) and human (15, 16) endothelial cells, whereas inducible murine macrophage NOS (mac-NOS) has been cloned from RAW 264.7 cells (17)(18)(19).Rat hepatocytes make NO in vivo during chronic hepatic inflammation (20,21) and in vitro in response to conditioned Kupffer cell supernatant (22) or to lipopolysaccharide (LPS) and the cytokines tumor necrosis factor (TNF), interleukin 1 (IL-1), and interferon y (IFN-y) (23-25). Since rats treated with LPS manifest inducible NOS in numerous tissues with few macrophages (26), it is possible that more than one isoform of inducible NOS exists. Evidence for inducible human NOS activity has been shown in patients receiving IL-2 cancer therapy (27, 28) and during sepsis (29). Recently, human hepatocytes were stimulated to produce NO by the same combination of LPS and cytokines as rat hepatocytes, providing evidence that a specific human cell expresses inducible NOS (30). We now report the cloning and functional expression of a distinct form of inducible NOS from human hepatocytes. 11 MATERIALS AND METHODSIsolation of Human Hepatocytes. Human hepatocytes were isolated from histologically normal operative wedge resections (in accordance with institutional approval) by using a modification of an in situ collagenase procedure (type IV; Sigma) (30). Briefly, hepatocytes were separated from nonparenchymal cells by differential centrifugation four times at 50 x g. The hepatocytes were then further purified over a 30% Percoll gradient at a concentration of 106 hepatocytes per ml of Percoll to obtain a highly purified cell population (31). Hepatocyte purity by microscopy was >98%, and viability consistently exceeded 95% by trypan blue exclusion.Cell Culture. Hepatocytes (5 x 106) were plated onto 100-mm gelatin-coated Petri dishes (Coming) in 6 ml of culture medium. Medium consisted of Williams media E (GIBCO) with L-arginine (0.50 mM), insulin (1 uM), Hepes (15 mM), L-glutamine, penicillin, streptomycin, and 10%o (vol/vol) low endotox...

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...

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...

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-gamma (IFN-gamma) was a potent stimulus of .N = O synthesis by KC. Treatment with LPS and IFN-gamma also induced significant production of prostaglandin E2 (PGE2), thromboxane B2 (TBX2), tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6. Inhibition of .N = O synthesis by KC. Treatment with LPS and IFN-gamma also induced significant production of prostaglandin E2 (PGE2), thromboxane B2 (TBX2), tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6. Inhibition of .N = O synthesis by the L-arginine analogue of NG-monomethyl-L-arginine (NMA) resulted in a further increase of PGE2, TXB2, and IL-6 but not IL-1 and TNF-alpha 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-gamma from 16.3 +/- 4.9 ng/10(6) KCs without NMA to 94.3 +/- 17.9 ng/10(6) 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.

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