Identification of N-iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells.

1. The synthesis of nitric oxide (NO) from L-arginine by rat peritoneal neutrophils (PMN) and the murine macrophage cell-line J774 and the inhibition of this synthesis by N-iminoethyl-L-ornithine (L-NIO), NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine (L-NNA) and its methyl ester (L-NAME) were investigated. 2. L-NIO was the most potent inhibitor in both types of cells while L-NMMA was less active. L-NNA and L-NAME had no significant effect in PMN and L-NNA produced only approximately 40% inhibition of the generation of NO in the J774 cells at the highest concentration tested (300 microM). 3. The inhibitory effect of L-NIO was rapid in onset, requiring 10 min pre-incubation to achieve its full inhibitory activity, while the other compounds required 20-60 min pre-incubation to achieve their full effect. 4. The inhibitory effect of L-NIO (10 microM) on intact cells could not be reversed by L-arginine (300 microM) but could be prevented by concomitant incubation with this compound (300 microM), while the effect of the other inhibitors could be reversed by a 3-5 fold molar excess of L-arginine. 5. The NO synthase from both PMN and J774 cells was cytosolic and NADPH- but not Ca2(+)-dependent, with Km values for L-arginine of 3.3 +/- 0.8 and 4.2 +/- 1.1 microM respectively. 6. L-NIO was the most potent inhibitor of the neutrophil and J774 enzymes with IC50 values of 0.8 +/- 0.1 and 3 +/- 0.5 microM respectively. Furthermore, the effect of L-NIO was irreversible. The other three compounds were less potent, reversible inhibitors. 7. The inhibitory effects of all these compounds were enantiomerically specific. 8. These data indicate that L-NIO is a novel, potent, rapid in onset and irreversible inhibitor of NO synthase in phagocytic cells. The rapid uptake of L-NIO compared with the other compounds indicates that phagocytic cells have different uptake mechanisms for L-arginine analogues.

Therapeutic potential of fish oil in the treatment of ulcerative colitis.

In a pilot study six patients with active ulcerative colitis and six healthy controls were given fish oil (MaxEPA) containing 3-4 g of eicosapentaenoic acid daily for a period of 12 weeks. There was a significant improvement in the patients' symptoms and histological appearance of the rectal mucosa by the end of the treatment period. There was significant fall in neutrophil chemiluminescence during treatment in patients, whereas no change was observed in the control group. Neutrophil leukotriene B4 levels fell significantly during treatment. Serum from patients receiving fish oil was significantly less chemotactic for neutrophils compared with control serum. Eicosapentaenoic acid inhibited neutrophil chemotaxis and chemiluminescence in vitro. The omega-3 fatty acids, which occur naturally in fish oils, may exert a beneficial effect by decreasing the production of inflammatory mediators.

Interleukin-8 inhibits the induction of nitric oxide synthase in rat peritoneal neutrophils.

The effect of interleukin(IL)-8 and leukotriene B4 (LTB4) on the induction of nitric oxide (NO) synthase in rat peritoneal neutrophils (PMN) ex vivo was studied. IL-8, but not LTB4, caused concentration-dependent inhibition of the induction of a Ca(2+)-independent NO synthase ex vivo. The effect of IL-8 was not attributable to the synthesis of an inhibitor of this enzyme. These findings suggest complex regulatory control of the induction of NO synthase by cytokines.

Induction of nitric oxide synthase in rat peritoneal neutrophils and its inhibition by dexamethasone.

Rat peritoneal polymorphonuclear leukocytes (PMN) elicited with oyster glycogen contain a Ca(2+)-independent nitric oxide (NO) synthase which is induced in vivo in a time-dependent manner. When washed PMN containing low levels of enzyme activity were cultured ex vivo further expression of NO synthase was observed. This was inhibited by cycloheximide indicating that de novo synthesis of the enzyme occurred during the ex vivo incubation. Enzyme activity was enhanced by interferon (IFN)-gamma, but not by tumor necrosis factor (TNF)-alpha when added ex vivo. However, IFN-gamma and TNF-alpha synergized to increase further the expression of NO synthase. Treatment of rats with dexamethasone inhibited the induction of NO synthase in elicited PMN. This treatment reduced the accumulation of PMN by approximately 30%, without affecting cell viability. Dexamethasone also inhibited the induction of the NO synthase ex vivo in a concentration-dependent manner. Furthermore, the enhanced enzyme activity following treatment of PMN with cytokines was also inhibited by dexamethasone. Once induced, dexamethasone did not affect enzyme activity. These data indicate that PMN elicited in the rat peritoneum with oyster glycogen express an NO synthase in vivo and ex vivo. The induction of the enzyme can be further stimulated ex vivo with IFN-gamma and TNF-alpha and inhibited by dexamethasone. The inhibition of the induction of NO synthase in the PMN by dexamethasone may contribute to the anti-inflammatory activity of this and other glucocorticoids.

Synthesis of nitric oxide from L-arginine by neutrophils. Release and interaction with superoxide anion.

Stimulated rat peritoneal neutrophils release a platelet inhibitory factor with the pharmacological properties of NO. This release is inhibited by NG-monomethyl-L-arginine and L-canavanine, indicating that it occurs through a mechanism similar to that in vascular endothelial cells and macrophages. As the degree of stimulation increases, the factor released is progressively inactivated by concomitant release of superoxide anions.