S-Nitrosation of proteins during D-galactosamine-induced cell death in human hepatocytes

Nitric oxide (NO) participates in the cell death induced by d-Galactosamine (d-GalN) in hepatocytes, and NO-derived reactive oxygen intermediates are critical contributors to protein modification and hepatocellular injury. It is anticipated that S-nitrosation of proteins will participate in the mechanisms leading to cell death in d-GalN-treated human hepatocytes. In the present study, d-GalN-induced cell death was related to augmented levels of NO production and S-nitrosothiol (SNO) content. The biotin switch assay confirmed that d-GalN increased the levels of S-nitrosated proteins in human hepatocytes. S-nitrosocysteine (CSNO) enhanced protein S-nitrosation and altered cell death parameters that were related to S-nitrosation of the executioner caspase-3. Fifteen S-nitrosated proteins participating in metabolism, antioxidative defense and cellular homeostasis were identified in human hepatocytes treated with CSNO. Among them, seven were also identified in d-GalN-treated hepatocytes. The results here reported underline the importance of the alteration of SNO homeostasis during d-GalN-induced cell death in human hepatocytes.     

Role of nitric oxide in actin depolymerization and programmed cell death induced by fusicoccin in sycamore (Acer pseudoplatanus) cultured cells

Programmed cell death (PCD) plays a vital role in plant development and is involved in defence mechanisms against biotic and abiotic stresses. Different forms of PCD have been described in plants on the basis of the cell organelle first involved. In sycamore ( Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin (FC) induces cell death. However, only a fraction of the dead cells shows the typical hallmarks of animal apoptosis, including cell shrinkage, chromatin condensation, DNA fragmentation and release of cytochrome c from the mitochondrion. In this work, we show that the scavenging of nitric oxide (NO), produced in the presence of FC, by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and rutin inhibits cell death without affecting DNA fragmentation and cytochrome c release. In addition, we show that FC induces a massive depolymerization of actin filaments that is prevented by the NO scavengers. Finally, the addition of actin-depolymerizing drugs induces PCD in control cells and overcomes the inhibiting effect of cPTIO on FC-induced cell death. Vice versa, the addition of actin-stabilizing drugs to FC-treated cells partially inhibits the phytotoxin-induced PCD. These results suggest that besides an apoptotic-like form of PCD involving the release of cytochrome c , FC induces at least another form of cell death, likely mediated by NO and independent of cytochrome c release, and they make it tempting to speculate that changes in actin cytoskeleton are involved in this form of PCD.     

Inhibition of nitric oxide synthesis in primary cultured mouse hepatocytes by alpha-lipoic acid

Recent work shows that septic or endotoxic shock is associated with lipopolysaccharide and cytokine mixture-induced nitric oxide (NO) synthesis in liver. Here we found that DL-alpha-lipoic acid inhibited but other thiol-containing antioxidants such as glutathione and N-acetylcysteine enhanced lipopolysaccharide and cytokine mixture (referred as LPS/CM)-induced NO synthesis in hepatocytes. The inhibitory action of alpha-lipoic acid on hepatocyte NO synthesis was as potent as that of NG-monomethyl-L-arginine without obvious cytotoxicity. Deletion by diethylmaleate or inhibition by buthionine sulfoximine of intracellular glutathione caused a significant decrease in hepatocyte NO synthesis, implying that increased intracellular reduced glutathione levels could not be the reason for alpha-lipoic acid inhibited NO synthesis. alpha-Lipoic acid inhibition of NO synthesis seems to be from alpha-lipoic acid improved carbohydrate metabolism in hepatocytes. Since alpha-lipoic acid is an essential compound existing naturally in physiological systems, it may serve as both a research and therapeutic agent for sepsis.     

Induction of nitric oxide production by natural killer cells: its role in tumor cell death.

It has been recognized that natural killer (NK) cells destroy AK-5 tumor cells, largely by cytolysis and apoptosis. The objective of this study was to elucidate the existence and the role of nitric oxide (NO) during this killing. The target cell killing ability of NK cells was associated with an increased production of NO with higher expression of inducible nitric oxide synthase. In part, the production of NO was confirmed by significant increase in cell lysis in the presence of l-arginine and attenuation of cell lysis, DNA fragmentation, and apoptosis by N(omega)-nitro-l-arginine methyl ester (L-NAME). An increased oxidation of intracellularly trapped dichlorofluorescein was observed in NK cells, which was effectively prevented by L-NAME. Exposure of AK-5 cells to chemically generated NO also induced DNA fragmentation in AK-5 cells. Further evidence for the involvement of NO in apoptosis was provided by the inhibition of specific cleavage of PARP and activation of CPP32 by L-NAME. Increased production of NO with simultaneous enhancement of the cytotoxic activity of NK cells from sc tumor-transplanted animals has been implicated in tumor regression when compared to the ip tumor-bearing animals. Overall, these observations suggest an important role for NO during NK cell-mediated apoptosis and lysis of AK-5 cells.     

D-galactosamine-induced cell damage enhances specific binding of prostaglandin E1 to rat hepatocytes.

Specific bindings of [3H]prostaglandin E1 ([3H]PGE1), 125I-glucagon and [3H]norepinephrine to D-galactosamine (GalN)-treated rat hepatocytes in primary culture were investigated. After a two hour-treatment with GalN (1 and 10mg/ml), hepatocytes showed an enhanced specific binding to [3H]PGE1, whereas 125I-glucagon binding was little affected and [3H]norepinephrine binding was strongly diminished. Scatchard plot analysis indicated an increase of binding sites of [3H]PGE1. This unusual manner of [3H]PGE1 binding is suggested to indicate a special property of PGE1 receptor and may be associated with the cytoprotective effect of prostaglandins.     

Iron induces protection and necrosis in cultured cardiomyocytes: Role of reactive oxygen species and nitric oxide

We investigate here the role of reactive oxygen species and nitric oxide in iron-induced cardiomyocyte hypertrophy or cell death. Cultured rat cardiomyocytes incubated with 20 μM iron (added as FeCl₃–Na nitrilotriacetate, Fe–NTA) displayed hypertrophy features that included increased protein synthesis and cell size, plus realignment of F-actin filaments along with sarcomeres and activation of the atrial natriuretic factor gene promoter. Incubation with higher Fe–NTA concentrations (100 μM) produced cardiomyocyte death by necrosis. Incubation for 24 h with Fe–NTA (20–40 μM) or the nitric oxide donor Δ-nonoate increased iNOS mRNA but decreased iNOS protein levels; under these conditions, iron stimulated the activity and the dimerization of iNOS. Fe–NTA (20 μM) promoted short- and long-term generation of reactive oxygen species, whereas preincubation with l-arginine suppressed this response. Preincubation with 20 μM Fe–NTA also attenuated the necrotic cell death triggered by 100 μM Fe–NTA, suggesting that these preincubation conditions have cardioprotective effects. Inhibition of iNOS activity with 1400 W enhanced iron-induced ROS generation and prevented both iron-dependent cardiomyocyte hypertrophy and cardioprotection. In conclusion, we propose that Fe–NTA (20 μM) stimulates iNOS activity and that the enhanced NO production, by promoting hypertrophy and enhancing survival mechanisms through ROS reduction, is beneficial to cardiomyocytes. At higher concentrations, however, iron triggers cardiomyocyte death by necrosis.     

Dynamic compression inhibits the synthesis of nitric oxide and PGE(2) by IL-1beta-stimulated chondrocytes cultured in agarose constructs

Both mechanical loading and interleukin-1beta (IL-1beta) are known to regulate metabolic processes in articular cartilage through pathways mediated by nitric oxide ((*)NO) and PGE(2). This study uses a well-characterized model system involving isolated chondrocytes cultured in agarose constructs to test the hypothesis that dynamic compression alters the synthesis of (*)NO and PGE(2) by IL-1beta-stimulated articular chondrocytes. The data presented demonstrate for the first time that dynamic compression counteracts the effects of IL-1beta on articular chondrocytes by suppressing both (*)NO and PGE(2) synthesis. Inhibitor experiments indicated that the dynamic compression-induced inhibition of PGE(2) synthesis and stimulation of proteoglycan synthesis were (*)NO mediated, while compression-induced stimulation of cell proliferation was (*)NO independent. The inhibition of (*)NO and PGE(2) by dynamic compression is a finding of major significance that could contribute to the development of novel strategies for the treatment of cartilage-degenerative disorders.     

Galactosamine decreases nitric oxide formation in cultured rat hepatocytes: mechanism of suppression

We have shown that nitric oxide production is dramatically decreased in rat primary hepatocyte cultures exposed to galactosamine. Cotreatment of the cells with uridine, which is known to prevent cytotoxicity, was found to also attenuate NO loss. In the present study, two possible mechanisms for the decreased nitric oxide production were examined. First, we examined the possibility that galactosamine could interfere with the uptake of extracellular arginine by the cultured hepatocytes. Cellular uptake of arginine was determined after addition of 14C-arginine at the time of hepatocyte attachment. Uptake of arginine was rapid in control cultures, and both the rate and level of uptake were unchanged by the addition of a cytotoxic concentration of galactosamine (4 mM). In addition, increased concentrations of arginine in the cell culture medium did not ameliorate the galactosamine-induced decrease in production of nitric oxide. Second, we determined whether the synthesis of inducible nitric oxide synthase in the hepatocyte cultures was inhibited by addition of galactosamine. Hepatocyte levels of inducible nitric oxide synthase were determined immunochemically at various times after the addition of galactosamine (4 mM). In control cultures, inducible nitric oxide synthase was detectable at 7 and 24 hours after attachment. In contrast, no nitric oxide synthase protein was detectable at any time in the galactosamine-treated cultures. Furthermore, addition of galactosamine after inducible nitric oxide synthase had already been synthesized (6.5 h after attachment) did not result in suppression of nitric oxide production in the hepatocyte cultures. The present studies suggest that galactosamine suppresses nitric oxide production in hepatocyte cultures by inhibiting synthesis of inducible nitric oxide synthase, rather than by interference in cellular uptake of arginine.     

Lipopolysaccharide-induced dopaminergic cell death in rat midbrain slice cultures: role of inducible nitric oxide synthase and protection by indomethacin

Glial cell activation associated with inflammatory reaction may contribute to pathogenic processes of neurodegenerative disorders, through production of several cytotoxic molecules. We investigated the consequences of glial activation by interferon-gamma (IFN-gamma)/lipopolysaccharide (LPS) in rat midbrain slice cultures. Application of IFN-gamma followed by LPS caused dopaminergic cell death and accompanying increases in nitrite production and lactate dehydrogenase release. Aminoguanidine, an inhibitor of inducible nitric oxide synthase (iNOS), or SB203580, an inhibitor of p38 mitogen-activated protein kinase, prevented dopaminergic cell loss as well as nitrite production. SB203580 also suppressed expression of iNOS and cyclooxygenase-2 (COX-2) induced by IFN-gamma/LPS. A COX inhibitor indomethacin protected dopaminergic neurons from IFN-gamma/LPS-induced injury, whereas selective COX-2 inhibitors such as NS-398 and nimesulide did not. Notably, indomethacin was able to attenuate neurotoxicity of a nitric oxide (NO) donor. Neutralizing antibodies against tumour necrosis factor-alpha and interleukin-1beta did not inhibit dopaminergic cell death caused by IFN-gamma/LPS, although combined application of these antibodies blocked lactate dehydrogenase release and decrease in the number of non-dopaminergic neurons. These results indicate that iNOS-derived NO plays a crucial role in IFN-gamma/LPS-induced dopaminergic cell death, and that indomethacin exerts protective effect by mechanisms probably related to NO neurotoxicity rather than through COX inhibition.     

The morphological and immunocytochemical evaluation of primary rat hepatocytes undergoing spontaneous cell death: modulation by the nitric oxide donor S-nitroso-N-acetylpenicillamine

Nitric oxide (NO) is one of the smallest molecules synthesised in the human body. It is produced by three distinct nitric oxide synthase isoenzymes (NOS) and plays a number of physiological functions in many organs and tissues. Among its numerous properties is the ability to influence programmed cell death. NO can either inhibit or induce apoptosis depending on the context of its production. In the liver, NO is produced in greater amounts especially during inflammation. The effect of NO in liver physiology and pathophysiology can be both beneficial and detrimental. Therefore, the aim of our study was to examine NO effect on cell viability and cell death in primary rat hepatocyte culture. By using NO donor, S-nitroso-N-acetylpenicillamine (SNAP), the potential of exogenously delivered NO to influence spontaneous cell death in culture was examined. The morphological approach was used in order to discriminate between apoptotic and necrotic cell death. The nitrite level, urea production and alanine aminotransferase leakage were determined in the culture medium. The immunocytochemical detection of three apoptotic markers: cleaved caspase-3, cleaved caspase-9 and lamin A, was performed. Immunocytochemical analysis of hepatocyte apoptosis revealed different labelling pattern for each method, while the detection of cleaved caspase-3 best correlated with defined phenotypical criteria. Our data showed that under present conditions NO improved the viability of primary rat hepatocytes compared to untreated cells. This was manifested by the increase of viable hepatocytes in contrast to the decrease of necrotic and apoptotic hepatocytes as assessed by the morphological examination of cell culture. The NO effect was dose-dependent in the range of SNAP concentration between 200-800 microM.     

Protective effect of keishi-bukuryo-gan and its constituent medicinal plants against nitric oxide donor-induced neuronal death in cultured cerebellar granule cells.

Keishi-bukuryo-gan (Gui-Zhi-Fu-Ling-Wan) (KBG) is a traditional Chinese/Japanese medical (Kampo) formulation that has been administered to patients with "Oketsu" (blood stagnation) syndrome. In the process of neuronal cell death induced by brain ischemia, excessive generation of nitric oxide (NO) free radicals is implicated in the neurotoxicity. In the present study, we examined the protective effects of KBG and its constituent medicinal plants against NO donors, sodium nitroprusside (SNP) and 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (NOC18)-induced neuronal death in cultured rat cerebellar granule cells (CGCs). MTT assay showed cell viability to be significantly increased by the addition of KBG extract (KBGE) (100 microg/ml), Cinnamomi Cortex extract (CCE) (3, 10 and 30 microg/ml), Paeoniae Radix extract (PRE) (100 microg/ml) and Moutan Cortex extract (MCE) (10 and 30 microg/ml) compared with exposure to SNP (30 microM, 24 h) only. Also, cell viability was significantly increased by the addition of KBGE (100 and 300 microg/ml), CCE (30 and 100 microg/ml), PRE (100 and 300 microg/ml) and MCE (30 and 100 microg/ml) compared with exposure to NOC 18 (100 microM, 48 h) only. Persicae Semen extract and Hoelen extract did not protect against NO donor-induced neuronal death. These results suggest that KBG has protective effect against NO-mediated neuronal death in cultured CGCs and that it is derived from Cinnamomi Cortex, Paeoniae Radix and Moutan Cortex.     

Age-related decline of inducible nitric oxide synthase gene expression in primary cultured rat hepatocytes

Hepatocytes exhibit a non-specific immune response by expressing the enzyme inducible nitric oxide (NO) synthase (iNOS, NOS2) through the stimulation of a mixture of cytokines, or a single cytokine such as interleukin-1beta. We examined the age-dependent inducibility of the iNOS gene expression and the capacity of NO production in response to lipopolysaccharide (LPS) or interleukin-1beta (IL-1beta) in primary cultured rat hepatocytes that were isolated from the livers of rats, 3 (young) and 24 (aging) months of age. NO production (NO2-), indicating iNOS activity, was much higher in the young rat hepatocytes following stimulation with LPS or IL-1beta. Likewise, in the young hepatocytes, Western blot analyses showed much higher protein levels in the iNOS expression; it was also a little higher in mRNA levels that were analyzed by RT-PCR. Furthermore, after stimulation with IL-1beta, the levels of transactivation of nuclear factor-KB (NF-kappaB) that were involved in the induction of the iNOS gene were reduced without a significant difference in the aged cells. Therefore, the decrease of NO formation in the aged hepatocytes was due to the belated and incomplete inducibility of the iNOS protein expression, together with a minor contribution of the reduced-transactivation of NF-kappaB. These results suggest that the age-related decline of the iNOS gene expression in primary rat hepatocytes may be associated with the increased incidence of many infective diseases with aging.     

Activation of the cell death program by nitric oxide involves inhibition of the proteasome.

The ubiquitin/proteasome pathway mediates the degradation of many short-lived proteins that are critically involved in the regulation of cell proliferation and cell death, including the tumor suppressor protein p53. Accumulation of p53 and induction of apoptosis in RAW 264.7 macrophages in response to nitric oxide are well established. However, the molecular mechanisms involved in nitric oxide-induced p53 accumulation are unknown. Here we show that, similar to nitric oxide, treatment of macrophages with specific proteasome inhibitors, including clastolactacystin-beta-lactone, induces p53 accumulation and apoptosis, suggesting that nitric oxide may affect the activity of the proteasome. In support of this hypothesis, both exposure of cells to S-nitrosoglutathione and stimulation of endogenous nitric oxide production by lipopolysaccharide/interferon-gamma treatment result in inhibition of proteasome activity as measured in vitro by the degradation of the proteasome-specific substrate succinyl-Leu-Leu-Val-Tyr-4-methylcoumarin-7-amide. Moreover, chemically diverse nitric oxide donors interfere with proteasome-mediated degradation of polyubiquitinated p53 in vitro. These data imply that nitric oxide-induced apoptosis and accumulation of p53 are, at least in part, mediated by inhibition of the proteasome.     

Sodium as the major mediator of NO-induced cell death in cultured hepatocytes

NO has been shown to induce cellular injury via inhibition of the mitochondrial respiratory chain and/or oxidative/nitrosative stress. Here, we studied which mechanism and downstream mediator is responsible for NO toxicity to hepatocytes. When cultured rat hepatocytes were incubated with spermineNONOate (0.01-2 mM) at 2, 5, 21 and 95% O(2) in Krebs-Henseleit buffer (37 degrees C), spermineNONOate caused concentration-dependent hepatocyte death (lactate dehydrogenase release, propidium iodide uptake) with morphological features of both apoptosis and necrosis. Increasing O(2) concentrations protected hepatocytes from NO-induced injury. Steady-state NO concentrations were lower at higher O(2) concentrations, suggesting formation of reactive nitrogen oxide species. Despite this, the scavenger ascorbic acid was hardly protective. In contrast, at equal NO concentrations loss of viability was higher at lower O(2) concentrations and inhibitors of hypoxic injury, fructose and glycine (10 mM), strongly decreased NO-induced injury. Upon addition of spermineNONOate, the cytosolic Na(+) concentration rapidly increased. The increase in sodium depended on the NO/O(2) ratio and was paralleled by hepatocyte death. Sodium-free Krebs-Henseleit buffer strongly protected from NO-induced injury. SpermineNONOate also increased cytosolic calcium levels but the Ca(2+) chelator quin-2-AM did not diminish cell injury. These results show that - in analogy to hypoxic injury - a sodium influx largely mediates the NO-induced death of cultured hepatocytes. Oxidative stress and disturbances in calcium homeostasis appear to be of minor importance for NO toxicity to hepatocytes.     

Galactosamine decreases nitric oxide formation in cultured rat hepatocytes: lack of involvement in cytotoxicity

Galactosamine hepatotoxicity in vivo has long been associated with rapid and extensive depletion of hepatic uridine nucleotides. Depletion of uridine nucleotides is considered to be causal in the toxicity, as evidenced by the protective effect of uridine administration. However, the exact mechanism of galactosamine-induced hepatic necrosis is still unclear. We have previously shown that the addition of galactosamine to rat primary hepatocyte cultures dramatically decreases production of nitric oxide, as measured in the 24 hour culture medium. The present study investigates whether decreased nitric oxide production contributes to the toxicity of galactosamine in primary hepatocyte cultures. Similar concentration-response curves were observed for the decrease in nitric oxide production and galactosamine cytotoxicity, raising the possibility that there is a similar mechanism for these effects. Suppression of NO synthesis was a direct effect of galactosamine, rather than an indirect effect due to loss of cells from the cultures. Both cytotoxicity and the decrease in nitric oxide production were attenuated by coaddition of 3 mM uridine. However, galactosamine cytotoxicity was not enhanced by prior inhibition of hepatocellular NO synthesis nor was it attenuated by maintenance of culture NO levels with molsidomine or diethylamine NONOate. These data do not support a role for decreased hepatocyte nitric oxide production in galactosamine hepatocyte toxicity.     

Endotoxin and TNF alpha directly stimulate nitric oxide formation in cultured rat hepatocytes from chronically endotoxemic rats.

We examined the effects of endotoxin on nitric oxide formation in isolated rat hepatocytes in primary culture. Endotoxin was administered either in vivo, by continuous infusion for 30 or 3 h, or in vitro, on cultured cells. The spontaneous production of nitrites in hepatocytes from in vivo ET-infused rats was lower than equivalent saline controls in the absence of added stimuli. However in vitro addition of endotoxin in culture to hepatocytes from 30 h ET-infused rats greatly enhanced production relative to saline controls. This effect was mimicked by TNF alpha, and activators of protein kinase C (PMA and Ca2+ ionophore A23187). The effects of ET were blocked by NMMA, dexamethasone and protein synthesis inhibitors Actinomycin D and cycloheximide. No in vitro effect of ET was observed in the 3 h infusion model. The results show that chronic exposure to sub-lethal levels of ET primes liver parenchymal cells for the production of nitric oxide, when exposed in vitro to ET or TNF alpha.