Immunosuppressant FK506 enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity.

Immunosuppressants FK506 and cyclosporin A inhibit neurotoxicity of N-methyl-D-aspartate in primary cortical cultures, while having no effect on quisqualate- and kainate-mediated neurotoxicity. Rapamycin completely reverses the neuroprotective effect of FK506. Both FK506 and cyclosporin A inhibit NMDA-elicited/nitric oxide-mediated increases in cGMP levels in cortical cultures. FK506 has no effect on sodium nitroprusside-induced increases in cGMP. In a stably transfected human kidney 293 cell line overexpressing the gene encoding nitric oxide synthase [L-arginine, NADPH:oxygen oxidoreductase (nitric oxide-forming), EC 1.14.13.39], FK506 inhibits the calcium ionophore A23187, stimulated increases in nitrite (a breakdown product of nitric oxide), and potentiates phorbol ester-mediated inhibition of nitrite formation. FK506-mediated inhibition of nitric oxide formation is completely reversed by rapamycin. Calcineurin dephosphorylates protein kinase C-mediated phosphorylation of nitric oxide synthase. FK506 prevents the calcineurin-mediated dephosphorylation of nitric oxide synthase and thereby diminishes the enzyme''s catalytic activity. These data establish nitric oxide synthase as a calcineurin substrate. Nitric oxide synthase catalytic activity is regulated by the phosphorylation state of the enzyme. Enhanced phosphorylation of nitric oxide synthase diminishes catalytic activity, and dephosphorylation (through activation of calcineurin) enhances catalytic activity. The neuroprotective effect of FK506 and cyclosporin A presumably involves the inhibition of calcineurin, preventing the dephosphorylation of nitric oxide synthase and its subsequent activation.     

beta-Lapachone reduces endotoxin-induced macrophage activation and lung edema and mortality

beta-Lapachone, a 1,2-naphthoquinone, is a novel chemotherapeutic agent. It has been shown to be capable of suppressing inducible nitric oxide synthase expression and function in rat alveolar macrophages. The authors further performed experiments to examine the molecular mechanism of beta-lapachone on LPS-induced responses in rat alveolar macrophages and to evaluate its in vivo antiinflammatory effect. A significant increase in nitrite production and inducible nitric oxide synthase expression was elicited in macrophages treated with LPS that was inhibited by coincubation with beta-lapachone. beta-Lapachone could also inhibit the production of tumor necrosis factor-alpha induced by LPS. LPS induces protein tyrosine phosphorylation and nuclear factor-kappaB binding activity by gel mobility shift assay in macrophages. These events were significantly inhibited by beta-lapachone. Furthermore, beta-lapachone in vivo protected against the induction of lung edema, lung-inducible nitric oxide synthase protein expression and nuclear factor-kappaB activation, lethality, and increased plasma nitrite and serum tumor necrosis factor-alpha levels induced by LPS. These results indicate that beta-lapachone suppresses inducible nitric oxide synthase induction and tumor necrosis factor-alpha production mediated by the inhibition of protein tyrosine phosphorylation and nuclear factor-kappaB activation caused by LPS. This results in a beneficial effect in an animal model of sepsis.     

N-Acetyl-cysteine modulates inducible nitric oxide synthase gene expression in human hepatocytes

BACKGROUND/AIMS: A major role has been described for inducible nitric oxide (NO) synthase in several chronic inflammatory liver diseases. N-Acetyl-cysteine (NAC) is a sulfhydryl donor molecule with antioxidant and antiinflammatory effects. It attenuates NO generation following lipopolysaccharide injection in rats. Our goal was to study the effect of NAC on NO synthase induction in hepatocytes in response to proinflammatory cytokines. METHODS: The effect of NAC on NO synthase induction was studied in the human hepatocyte cell lines HepG2 and 2.2.15 treated with a mixture of proinflammatory cytokines. Interactions between NAC and cytokines on nuclear factor-kappaB (NF-kappaB) activation and NO synthase promoter transactivation were investigated. RESULTS: NAC dose-dependently modulated the induction of NO synthase mRNA expression, the release of nitrites and the formation of NF-kappaB binding complexes in cytokine-treated hepatocytes. NAC also reduced the transactivation of the NO synthase promoter. CONCLUSIONS: Our data show that exposure of hepatocytes to NAC modulated NO synthase expression and NF-kappaB activity, the key responses of the hepatocyte to inflammatory mediators. These data constitute preliminary evidence that NAC might have hepatoprotective actions of potential relevance in chronic inflammatory liver diseases, mediated partially through the modulation of NO production.     

Role of NF-kappaB activation and nitric oxide expression during PGE protection against d-galactosamine-induced cell death in cultured rat hepatocytes.

Prostaglandin E1 (PGE1) reduces cell death in experimental and clinical liver dysfunction. Nitric oxide (NO) mediates PGE1 protection against D-galactosamine (D-GalN)-induced cell death. Nuclear factor kappa-B (NF-kappaB) plays a protective role in different experimental models of cell death. We investigated if NF-kappaB was responsible for inducible nitric oxide synthase (iNOS) expression and cytoprotection induced by PGE1 against D-GalN cell death in cultured hepatocytes. Rat hepatocytes were isolated following the classical method of collagenase perfusion of liver. A kinetic study of cell death, NF-kappaB activation, mRNA and protein iNOS expression, and NO production was carried in hepatocytes treated with D-GalN (5 mM) in the presence or absence of PGE1 (1 microM) administered 2 h before the hepatotoxin. A proteasome inhibitor was used to evaluate the role of NF-kappaB activation in our experimental conditions. PGE1 protection against D-GalN-induced cell death was associated with its capacity to rapidly enhance NF-kappaB activation, mRNA and protein iNOS expression, and NO production in D-GalN-treated hepatocytes. The inhibition of NF-kappaB activation abolished iNOS expression and cell protection by PGE1 in hepatocytes treated with the hepatotoxin. The present study shows that the cytoprotection by PGE1 against D-GalN-induced apoptosis was related to NF-kappaB-dependent iNOS expression.     

Peroxynitrite formation during rat hepatic allograft rejection

The role of nitric oxide (NO) on tissue injury of hepatic allografts during rejection remains controversial. We investigated inducible nitric oxide synthase (iNOS) expression and formation of peroxynitrite in ACI rat liver grafts implanted in recipients. Animals were divided into four experimental groups: group I, isografts; group II, untreated hepatic allografts; group III, allografts treated with FK506; and group IV, allografts pretreated with donor-specific blood transfusion (DST). Serum nitrite/nitrate, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) concentrations increased significantly in group II rats after transplantation but were significantly lower in groups I, III, and IV. The numbers of macrophages that reacted with an antimacrophage iNOS monoclonal antibody as well as iNOS messenger RNA (mRNA) levels in liver specimens were also much lower in groups I, III, and IV as compared with group II. Immunostaining and Western blot analysis showed prominent tissue nitrotyrosine expression in untreated hepatic allografts, but not in allografts treated with FK506 or donor-specific blood. These results suggest that one of the mechanisms by which production of NO results in injury in rat hepatic allografts may be because of its reaction with superoxide to form peroxynitrite.     

An enhancement of nitric oxide production regulates energy metabolism in rat hepatocytes after a partial hepatectomy.

BACKGROUND/AIMS: Infection after a liver resection often results in hepatic failure. Nitric oxide is one of the candidates which has been suspected to cause cellular dysfunction during infection in the liver. We have previously reported that the inflammatory cytokine interleukin-1beta (IL-1beta) induced the expression of the inducible nitric oxide synthase gene in primary cultured rat hepatocytes. We hypothesized that an enhancement of nitric oxide production after the resection was implicated in a change in liver energy metabolism, thus resulting in liver dysfunction. METHODS: In this study, we performed a 70% hepatectomy or a sham operation in rats, and then isolated hepatocytes from the remnant liver by collagenase perfusion. The cultured hepatocytes were treated with cytokines including IL-1beta. The effects on nitric oxide induction, the ATP content and ketone body ratio (acetoacetate/beta-hydroxybutyrate) were then compared between the partial hepatectomized (PH) and sham-operated (control) rats. RESULTS: IL-1beta augmented the induction of nitric oxide production two-fold in hepatocytes from the PH rats as compared to the control rats. IL-1beta markedly decreased the ATP content in the PH rats, although IL-1beta also decreased the ATP content in the control rats, but to a lesser extent. IL-1beta also decreased the ketone body ratio in both groups. The addition of L-arginine further stimulated the inhibition of the ATP levels and the ketone body ratio concomitantly with increased nitric oxide production in the PH rats. N(G)-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, abolished the effects of IL-1beta on the ATP levels and ketone body ratio, as well as on the nitric oxide production. CONCLUSIONS: These results demonstrate that the decreased ATP content observed in PH rats resulted from an increase in nitric oxide production. The decrease in ketone body ratio indicates that nitric oxide-induced mitochondrial dysfunction contributes significantly to ATP attenuation in hepatocytes. Therefore, the regulation of nitric oxide induction may be crucial for preventing liver failure after a hepatic resection.     

Effects of cyclosporin A and FK506 on Fc epsilon receptor type I-initiated increases in cytokine mRNA in mouse bone marrow-derived progenitor mast cells: resistance to FK506 is associated with a deficiency in FK506-binding protein FKBP12.

The inhibitory effects of cyclosporin A (CsA) and FK506 on Fc epsilon receptor type I-initiated increases in cytokine mRNA and the expression of their intracellular binding proteins were studied in interleukin 3 (IL-3)-dependent, mouse bone marrow-derived mast cells (BMMCs). In BMMCs sensitized with IgE anti-trinitrophenyl, CsA inhibited trinitrophenylated bovine serum albumin-induced increases in mRNA for IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 in a dose-related manner (IC50 values of 4, 65, and 130 nM, respectively). FK506 did not inhibit hapten-specific increases of mRNA for TNF-alpha or IL-6, and for IL-1 beta the IC50 was greater than 50-fold higher than that of CsA. Neither agent inhibited exocytosis of the endogenous secretory granule mediators beta-hexosaminidase and histamine at the IC50 values for inhibition of increases in cytokine mRNA. BMMCs expressed cyclophilin, and CsA inhibited the phosphatase activity of cellular calcineurin with an IC50 of approximately 8 nM. That CsA inhibited IL-1 beta mRNA accumulation in IgE-activated BMMCs with an IC50 similar to that for inhibition of calcineurin activity, whereas the IC50 values were approximately 20-fold higher for the inhibition of TNF-alpha and IL-6 mRNA, suggests that the induction of TNF-alpha and IL-6 is less dependent upon calcineurin activity than is the induction of IL-1 beta. BMMCs were deficient in the 12-kDa FK506-binding protein FKBP12, but not FKBP13, as assessed by RNA and protein blot analyses. FK506 did not inhibit calcineurin phosphatase activity in BMMCs, even at drug concentrations of 1000 nM. The resistance of BMMCs to inhibition of Fc epsilon receptor type I-mediated increases in cytokine mRNA by FK506 is most likely due to their deficiency of FKBP12 and the related inability to inhibit the activity of calcineurin.     

FK506 attenuates developing and established joint inflammation and suppresses interleukin 6 and nitric oxide expression in bacterial cell wall induced polyarthritis

OBJECTIVE: To determine the efficacy of therapeutic administration of FK506 (Tacrolimus) in suppressing developing and established joint inflammation, proinflammatory cytokine expression, and nitric oxide (NO) production in peptidoglycan-polysaccharide (PG/PS) induced experimental polyarthritis in rats. METHODS: Chronic joint inflammation was induced by intraperitoneal injection of PG/PS, and joint inflammation was quantified using arthritis index and paw volume. Serum and joint levels of interleukin 6 (IL-6) were measured by bioassay and Western blot analysis respectively, and serum levels of NO production were determined by the Griess procedure and the expression of the inducible isoform of nitric oxide synthase (i-NOS) in the joints was determined by Western blot analysis. RESULTS: Arthritis induced by PG/PS is biphasic, progressing through an initial acute phase and a remission phase, which is followed by a persistent chronic phase. Daily administration of FK506 initiated during the remission phase significantly attenuated the onset and development of chronic joint inflammation. We observed a significant reduction in joint inflammation and swelling, an apparent suppression of pannus development, and minimal erosive damage to the articular cartilage and subchondral bone. Fully established chronic joint inflammation was also ameliorated by daily administration of FK506. Joint swelling and inflammation was significantly reduced by 5 days post-treatment with FK506 and the erosive activity associated with the pannus appeared diminished. The elevated expression of IL-6 and NO characteristic of chronic joint inflammation in the serum and in joint tissue was significantly reduced by FK506 treatment. CONCLUSION: Therapeutic administration of FK506 has a profound antiinflammatory effect on the development of the chronic, erosive arthritis induced by PG/PS. This attenuation in joint inflammation was associated with suppression of IL-6 and NO production systemically and locally in the joints. Our data suggest that FK506 may be effective in the treatment of chronic joint inflammation associated with rheumatoid arthritis.     

Peroxisome proliferator-activated receptor-gamma ligands inhibit nitric oxide synthesis in vascular smooth muscle cells

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a key player in glucose metabolism. If PPARgamma ligands modulate nitric oxide (NO) synthesis in the vascular tissue, they may affect the process of plaque formation and postangioplasty restenosis. We investigated the effects of PPARgamma ligands on NO synthesis in vascular smooth muscle cells. Incubation of cultures with interleukin-1beta (10 ng/mL) for 24 hours caused a significant increase in the production of nitrite, a stable metabolite of NO, in cultured rat vascular smooth muscle cells. The PPARgamma agonists troglitazone and 15-deoxy-triangle up(12,14)-prostaglandin J(2) (15d-PG J(2)) dose-dependently inhibited nitrite production by interleukin-1beta-stimulated vascular smooth muscle cells. Decreased interleukin-1beta-induced nitrite production by the PPARgamma agonists was accompanied by decreased inducible NO synthase mRNA and protein accumulation. Interleukin-1beta induced nuclear factor-kappaB activation in vascular smooth muscle cells, and both troglitazone and 15d-PG J(2) markedly suppressed this nuclear factor-kappaB activation. PPARgamma ligands inhibit NO synthesis in cytokine-stimulated vascular smooth muscle cells, suggesting that these agonists may act directly on the vascular smooth muscle and influence the process of atherosclerosis and restenosis.     

Lipopolysaccharide and interleukin 1 augment the effects of hypoxia and inflammation in human pulmonary arterial tissue.

The combined effects of hypoxia and interleukin 1, lipopolysaccharide, or tumor necrosis factor alpha on the expression of genes encoding endothelial constitutive and inducible nitric oxide synthases, endothelin 1, interleukin 6, and interleukin 8 were investigated in human primary pulmonary endothelial cells and whole pulmonary artery organoid cultures. Hypoxia decreased the expression of constitutive endothelial nitric oxide synthase (NOS-3) mRNA and NOS-3 protein as compared with normoxic conditions. The inhibition of expression of NOS-3 corresponded with a reduced production of NO. A combination of hypoxia with bacterial lipopolysaccharide, interleukin 1 beta, or tumor necrosis factor alpha augmented both effects. In contrast, the combination of hypoxia and the inflammatory mediators superinduced the expression of endothelin 1, interleukin 6, and interleukin 8. Here, we have shown that inflammatory mediators aggravate the effect of hypoxia on the down-regulation of NOS-3 and increase the expression of proinflammatory cytokines in human pulmonary endothelial cells and whole pulmonary artery organoid cultures.