Inhibition of ornithine decarboxylase potentiates nitric oxide production in LPS-activated J774 cells

We have examined whether modulation of the polyamine biosynthetic pathway, through inhibition by alpha-difluoromethylornithine (DFMO) of the rate limiting enzyme, ornithine decarboxylase (ODC), modulates NO synthesis in J774 macrophages. DFMO potentiated LPS-stimulated nitrite production in both a concentration- and time-dependent manner, increasing nitrite levels by 48+/-5% at 10 mM. This effect was observed in cells pre-treated with DFMO for 24 h prior to stimulation with LPS. Addition of DFMO 12 h after LPS failed to potentiate LPS-induced nitrite production. Supplementation of the culture medium with horse serum (10%) in place of foetal calf serum (10%) caused no significant change in either LPS-induced nitrite production or in the ability of DFMO (10 mM) to potentiate LPS-induced NO synthesis. Metabolism of L-[3H]arginine to L-[3H]citrulline by partially purified inducible nitric oxide synthase (iNOS) was not significantly altered by either DFMO (1-10 mM) or by putrescine (0.001-1 mM), spermidine (0.001-1 mM) or spermine (0.001-1 mM). iNOS activity was also unaffected by 1 mM EGTA but was markedly attenuated (70+/-0.07%) by L-NMMA (100 microM). Pre-incubation of cells with DFMO (10 mM; 24 h) prior to activation with LPS resulted in enhanced (approximately 2 fold) iNOS protein expression. These results show that DFMO potentiates LPS-induced nitrite production in the murine macrophage cell line J774. Since the only known mechanism of action of DFMO is inhibition of ODC, and thus polyamine biosynthesis, we conclude that expression of iNOS can be critically regulated by endogenous polyamines.     

The utilization of dipeptides containing L-arginine by chicken macrophages

L-Arginine is the precursor of NO, a cytotoxic agent of macrophages. Studies were carried out to determine whether dipeptides containing arginine can be utilized by lipopolysaccharide (LPS)-activated avian macrophages for NO production. A chicken macrophage cell line, the HD11 cell, was used in all experiments. Peptidase activities were observed in fetal bovine serum (FBS) and macrophage serum free medium (Mac-SFM). Therefore, the utilization of dipeptides by macrophages was examined using Dulbecco's modified Eagle medium (D-MEM), a chemically defined medium, in short-term culture without FBS. Nitrite accumulation in the culture medium was used as the indicator of NO production. At concentrations of 0.15 mM in the culture media, L-leucinyl-L-arginine was 89% as effective as L-arginine in providing substrate for NO production. L-Argininyl-L-leucine was 38% as effective as L-arginine. The effectiveness increased to 93 and 58%, respectively, when the concentrations of dipeptides and arginine were 1.0 mM. Both values were slightly higher in a second experiment (97 and 70%, respectively). L-Lysine (10 mM) inhibited nitrite formation from all three sources of L-arginine. In studies of initial rates of transport by HD11 cells in Hanks Balanced Salts solution (HBSS), both L-argininyl-L-leucine and L-leucinyl-L-arginine inhibited arginine uptake. As lysine and arginine share a common transporter for cationic amino acids and are known to compete for transport, these studies suggest that the peptides were hydrolyzed extracellularly, yielding arginine that was transported into the cell where it served as a substrate for NO synthesis.     

Induction of manganese superoxide dismutase in BV-2 microglial cells

The regulation of the manganese-dependent superoxide dismutase (Mn-SOD) was studied in immortalized microglial cells (line BV-2). BV-2 cells, activated with lipopolysaccharide (LPS), exhibited an increase in Mn-SOD-like immunoreactivity, that was associated with an accumulation of nitrite in the culture medium and an increase in immunoreactivity for the inducible type of nitric oxide synthase (i-NOS). The i-NOS inhibitor L-N6-(1-iminoethyl)-lysine (NIL, 600 microM) suppressed the nitrite accumulation and the increase in Mn-SOD-like immunoreactivity in activated cells without significant effect on the level of i-NOS-like immunoreactivity. The NO donor sodium nitroprusside dose-dependently increased Mn-SOD-like immunoreactivity in NIL-pretreated BV-2 cells. These results indicate that the induction of Mn-SOD in activated BV-2 cells is mediated in part by NO, or its metabolites.     

Pyrimidinoceptor-mediated potentiation of inducible nitric-oxide synthase induction in J774 macrophages. Role of intracellular calcium

We have shown that, in murine J774 macrophages, binding of UTP to pyrimidinoceptors stimulates phosphoinositide (PI) breakdown and an increase in [Ca2+]i. In this study, UTP modulation of the expression of inducible nitric-oxide synthase (iNOS) was investigated. Although UTP alone had no effect, stimulation of J774 cells with a combination of UTP (10-300 microM) and LPS (0.1-3 microgram/ml) resulted in a potentiated increase in nitrite levels. In parallel, the amount of iNOS protein induced by LPS was also potentiated by UTP treatment. The UTP potentiating effect was attenuated by U73122, suggesting involvement of the downstream signaling pathways of phosphatidylinositide turnover. The tyrosine kinase inhibitor genistein inhibited both the LPS-induced nitrite response and the UTP potentiation. Conversely, two protein kinase C inhibitors, Ro 31-8220 and Go 6976, and a phosphatidylcholine-specific phospholipase C inhibitor, D609, inhibited LPS-stimulated nitrite induction, but did not affect the potentiating effect of UTP, which was also unaffected by pretreatment with phorbol 12-myristate 13-acetate for 8 h. Furthermore, the UTP-induced potentiation was abolished by BAPTA/AM or KN-93 (a selective inhibitor of Ca2+/calmodulin-dependent protein kinase (CaMK)). Nitrite potentiation and iNOS induction were prominent when UTP was added simultaneously with LPS, with the potentiating effect being lost when UTP was added 3 h after treatment with LPS. Pyrrolidinedithiocarbamate (3-30 microM), an inhibitor of NF-kappaB, caused a concentration-dependent reduction in the nitrite response to LPS and UTP. In electrophoretic mobility shift assays, LPS produced marked activation of NF-kappaB and AP-1, which was potentiated by UTP. LPS-induced degradation of IkappaB-alpha as well as the phosphorylation of IkappaB-alpha were also increased by UTP. Moreover, the UTP-potentiated activation of NF-kappaB and AP-1 and the degradation and phosphorylation of IkappaB-alpha were inhibited by KN-93. Taken together, these data demonstrate that nucleotides, especially UTP, can potentiate the LPS-induced activation of NF-kappaB and AP-1 and of iNOS induction via a CaMK -dependent pathway and suggest that the UTP-dependent up-regulation of iNOS may constitute a novel element in the inflammatory process.     

Interleukin-13 is a more potent inhibitor of the expression of inducible nitric oxide synthase in smooth muscle cells than in macrophages: a comparison with interleukin-4 and interleukin-10

This study compares the effects of interleukin (IL)-13, a cytokine with anti-inflammatory properties, with those of IL-4 or IL-10 on the expression of inducible nitric oxide synthase (iNOS) protein and activity in 1) a murine macrophage cell line (J774.2) activated with lipopolysaccharide (LPS) and 2) rat aortic smooth muscle cells (RASM) activated with LPS plus interferon-gamma. Pretreatment of macrophages with IL-4 or IL-13 caused a similar, concentration-dependent inhibition of the formation of nitrite and the expression of iNOS protein elicited by LPS. In contrast, IL-13 was a much more potent inhibitor of the formation of nitrite and the expression of iNOS protein in activated RASM than IL-4. IL-10 caused only a small, but significant, inhibition of the nitrite formation induced by LPS in macrophages and RASM. Pretreatment of J774.2 macrophages, but not of RASM, with the phosphatidylinositol-3-kinase inhibitor, wortmannin (10-100 nM), attenuated the inhibition by either IL-13 or IL-4 of the LPS-induced increase in nitrite in a dose-related fashion. Thus, IL-13 is more potent than IL-4 in preventing the expression of iNOS protein and activity in activated RASM, whereas IL-13 and IL-4 are equipotent in inhibiting the expression of iNOS protein and activity in J774.2 macrophages.     

The mechanism of a P-450 enzyme-aromatase; a molecular modelling perspective for the removal of the C(19) methyl and aromatisation of the steroid A ring

1 Here we compared the effects of various inhibitors of the activity of protein tyrosine kinase on (i) the expression of the activity of the inducible isoform of nitric oxide (NO) synthase (iNOS) caused by endotoxin (lipopolysaccharide, LPS) in cultured macrophages, (ii) the induction of iNOS and cyclooxygenase 2 (COX-2) protein and activity in rats with endotoxaemia, and (iii) the circulatory failure and organ dysfunction caused by LPS in the anesthetized rat. 2 Activation of murine cultured macrophages with LPS (1 microgram ml-1) resulted, within 24 h, in a significant increase in nitrite (an indicator of the formation of NO) in the cell supernatant. This increase in nitrate was attenuated by the tyrphostins AG126, AG556, AG490 or AG1641 or by genistein in a dose-dependent fashion (IC50: approximately 15 microM). In contrast, tyrphostin A1 (an analogue of tyrphostin AG126) or daidzein (an analogue of genistein) had no effect on the rise in nitrite caused by LPS. 3 Administration of LPS (E. coli, 10 mg kg-1, i.v.) caused hypotension and a reduction of the pressor responses elicited by noradrenaline (NA, 1 microgram kg-1, i.v.). Pretreatment of rats with the tyrphostins AG126, AG490, AG556, AG1641 or A1 attenuated the circulatory failure caused by LPS. Although genistein attenuated the vascular hyporeactivity to NA, it did not affect the hypotension caused by LPS. Daidzein did not affect the circulatory failure caused by LPS. 4 Endotoxaemia for 360 min resulted in rises in the serum levels of (i) urea and creatinine (indicators of renal failure), (ii) alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin and gamma-glutamyl transferase (gamma GT) (indicators of liver injury/dysfunction), lipase (an indicator of pancreatic injury) as well as lactate (an indicator of tissue hypoxia). None of the tyrosine kinase inhibitors tested had a significant effect on the rise i the serum levels of urea, but the tyrphostins AG126, AG556 or A1 significantly attenuated the rises in the serum level of creatinine caused by LPS. In addition, all tyrphostins and genistein attenuated the liver injury/failure, the pancreatic injury, the hypoglycaemia and the lactic acidosis caused by LPS. In contrast, daidzein did not reduce the organ injury/dysfunction or the lactic acidosis caused by LPS. 5 Injection of LPS resulted (within 90 min) in a substantial increase in the serum level of tumor necrosis factor alpha (TNF alpha), which was attenuated by pretreatment of LPS-rats with any of the tyrphostins used. Genistein, but not daidzein, also reduced the rise in the serum levels of TNF alpha caused by LPS. Endotoxaemia for 6 h also resulted in a substantial increase in the expression of iNOS and COX-2 protein and activity in the lung, which was attenuated by pretreatment of LPS-rats with the tyrphostins AG126, AG556 or genistein, but not by daidzein. 6 Thus, tyrphostins (AG126, AG556, AG1641 or A1) and genistein, but not daidzein (inactive analogue of genistein), prevent the (i) circulatory failure, (ii) the multiple organ dysfunction (liver and pancreatic dysfunction/injury lactacidosis, hypoglycaemia), as well as (iii) the induction of iNOS and COX-2 protein and activity in rats with endotoxic shock.     

Nitric oxide production by peritoneal macrophages of cirrhotic rats: a host response against bacterial peritonitis

BACKGROUND & AIMS: Patients and rats with cirrhosis and ascites are prone to develop peritonitis. The aim of this study was to assess whether peritoneal macrophages of cirrhotic rats without peritoneal infection produce nitric oxide and express inducible NO synthase (iNOS). METHODS: NO2- accumulation produced by macrophages from control rats and cirrhotic rats with ascites was determined. iNOS messenger RNA and protein expression were analyzed by Northern and Western blot and immunocytochemical analysis. The in vivo effects of inhibiting iNOS were investigated by giving the specific iNOS inhibitor L-N-(1-iminoethyl)-lysine (L-NIL) or sterile saline to 9 and 7 cirrhotic rats with ascites, respectively. RESULTS: Cirrhotic macrophages produced NO2- that was around fourfold greater than that of control macrophages after 30 hours in culture. Northern and Western blot and immunocytochemical analysis showed the presence of iNOS messenger RNA and protein in macrophages of cirrhotic rats. Ascites cultures were positive in all rats administered L-NIL and negative in those administered saline. CONCLUSIONS: Macrophages of cirrhotic rats produce NO and express iNOS messenger RNA and protein, and these changes are not a consequence of overt bacterial infection. Because iNOS inhibition results in peritoneal infection, these results suggest that iNOS induction in macrophages of cirrhotic rats is a host defense response to prevent bacterial peritonitis.     

Rapid estimation of regurgitant volume by the proximal isovelocity surface area method in mitral regurgitation: Can continuous-wave Doppler echocardiography be omitted?

To determine whether heme oxygenase-1 (HO-1) protein is induced by endogenous nitric oxide (NO) in rat glial cultures, we examined the effects of lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), and NO donors such as S-nitroso-N-acetylpenicillamine (SNAP), in mixed glial cells and in vivo rat hippocampus. In cultured glial cells, treatment with LPS induced the expression of 130-kd inducible NO synthase (iNOS) after 6 h, and NO2- accumulation and enhancement of the protein level of 33-kd HO-1 after 12 h. In addition, treatment with SNAP induced HO-1 expression after 6 h. Although NOS inhibitors such as NG-nitro-L-arginine (NNA) and NG-methyl-L-arginine did not change LPS-induced iNOS expression, these inhibitors suppressed both NO2- accumulation and the enhancement of HO-1. Immunocytochemistry showed that treatment with LPS for 24 h induced iNOS immunoreactivity predominantly in ameboid microglia, while this treatment induced HO-1-immunoreactivity in both microglia and astrocytes. In in vivo rat hippocampus, microinjection of LPS plus IFN-gamma, or SNAP after 24 h also induced HO-1 immunoreactivity in reactive microglia and astrocytes. In addition, intraperitoneal administration of NNA inhibited HO-1 immunoreactivity induced by the microinjection of LPS plus IFN-gamma. These results suggest that endogenous NO production by iNOS in microglia causes autocrine and paracrine induction of HO-1 protein in microglia and astrocytes in vitro and in rat brain.     

Regulation of the inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide

1. In airway epithelium, nitric oxide (NO) is synthesized in the setting of inflammation by inducible nitric oxide synthase (iNOS). Although the role of epithelial derived NO in the regulation of human airways is unknown, prostaglandin E2 (PGE2) is recognised as an important inhibitory mediator in human airways. Cyclo-oxygenase (COX) is the rate limiting enzyme in the production of prostanoids and since inflammatory pathways enhance the expression of an inducible COX (COX-2), both COX-2 and iNOS may be co-expressed in response to an inflammatory stimulus. Although regulation of the COX-2 pathway by NO has been demonstrated in animal models, its potential importance in human airway epithelium has not been investigated. 2. The effect of endogenous and exogenous NO on the COX-2 pathway was investigated in the A549 human airway epithelial cell culture model. Activity of the COX-2 pathway was assessed by PGE2 EIA, and iNOS pathway activity by nitrite assay. A combination cytokine stimulus of interferon gamma (IFNgamma) 100 u ml(-1), interleukin-1beta (IL-1beta) 1 u ml(-1) and lipopolysaccharide (LPS) 10 microg ml(-1) induced nitrite formation which could be inhibited by the competitive NOS inhibitor N(G)-nitro-L-arginine-methyl-ester (L-NAME). IL-1beta alone (1-50 u ml(-1) induced PGE2 formation without significant nitrite formation, a response which was inhibited by the COX-2 specific inhibitor nimesulide. Submaximal stimuli used for further experiments were IFNgamma 100 u ml(-1), IL-1beta 1 u ml(-1) and LPS 10 microg ml(-1) to induce both the iNOS and COX-2 pathways, and IL-1beta 3 u ml(-1) to induce COX-2 without iNOS activity. 3. Cells treated with IFNgamma 100 u ml(-1), IL-1beta I u ml(-1) and LPS 10 microg ml(-1) for 48 h either alone, or with the addition of L-NAME (0 to 10(-2) M), demonstrated inhibition by L-NAME of PGE2 (3.61 +/- 0.55 to 0.51 +/- 0.04 pg/l0(4) cells; P<0.001) and nitrite (34.33 +/- 8.07 to 0 pmol/10(4) cells; P<0.001) production. Restoration of the PGE2 response (0.187 +/- 0.053 to 15.46 +/- 2.59 pg/10(4) cells; P<0.001) was observed after treating cells with the same cytokine stimulus and L-NAME 10(-6) M, but with the addition of the NOS substrate L-arginine (0 to 10(-5) M). 4. Cells incubated with IL-1beta 3 u ml(-1) for 6 h, either alone or with addition of the NO donor S-nitroso-acetyl-penicillamine (SNAP) (0 to 10(-4) M), demonstrated increased PGE2 formation (1.23 +/- 0.03 to 2.92 +/- 0.19 pg/10(4) cells; P< 0.05). No increase in PGE2 formation was seen when the experiment was repeated in the presence of the guanylate cyclase inhibitor methylene blue (50 microM). Cells treated with SNAP alone did not demonstrate an increased PGE2 formation. Cells incubated with IL-1beta 3 u ml(-1) for 6 h in the presence of dibutyryl cyclic guanylate monophosphate (0 to 10(-3) M) also demonstrated an increased PGE2 response (2.56 +/- 0.21 to 4.53 +/- 0.64 pg/10(4) cells; P<0.05). 5. These data demonstrate that in a human airway epithelial cell culture system, both exogenous and endogenous NO increase the activity of the COX-2 pathway in the setting of inflammatory cytokine stimulation, and that this effect is likely to be mediated by guanylate cyclase. This suggests a role for NO in the regulation of human airway inflammation.     

5,6-Dihydroxyindole-2-carboxylic acid, a diffusible melanin precursor, is a potent stimulator of lipopolysaccharide-induced production of nitric oxide by J774 macrophages

Pre-incubation of J774 murine macrophages with 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a diffusible intermediate in the biosynthesis of eumelanins, leads to a marked increase in the levels of nitric oxide (NO) produced by lipopolysaccharide (LPS)-induced NO-synthase (iNOS). The effect varies with DHICA concentration being maximum at a concentration of 1 x 10(-6)M, and is suppressed by the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). No stimulation is observed when macrophages are exposed to DHICA after activation with LPS, indicating that the indole does not affect the catalytic activity of iNOS. These results point to a hitherto unrecognized role of DHICA as a chemical messenger mediating interaction between active melanocytes and macrophages in epidermal inflammatory and immune responses.     

Effect of cycloheximide on the expression of LPS-inducible iNOS, IFN-beta, and IRF-1 genes in J774 macrophages

The effect of cycloheximide (CHX) on the gene expression for inducible NO synthase (iNOS), interferon (IFN)-beta, and IFN regulatory factor (IRF)-1 was examined in LPS-stimulated J774 macrophages. LPS caused increased expression of mRNAs specific for iNOS, IFN-beta, and IRF-1 with different kinetics. Addition of CHX resulted in inhibition of the LPS-induced iNOS gene expression and parallel decrease in NO production. In contrast, expression of IFN-beta and IRF-1 genes in response to LPS was potentiated in the presence of CHX. These results indicate that de novo protein synthesis is not required for IFN-beta and IRF-1 gene expression and that ongoing protein synthesis including IFN-beta and IRF-1 may be involved in the induction process of iNOS in mouse macrophages.