Natural compounds derived from foods modulate nitric oxide production and oxidative status in epithelial lung cells.

The effects of natural antioxidants on nitric oxide (NO) modulation and oxidative status were determined in rat epithelial lung cells (L-2). Cells were stimulated with cytokines and treated with one of the following: resveratrol, soybean saponin group B (SSB), quercetin, genistein, olive leaf polyphenol concentrate (OLPC), or N-acetyl-L-cystein (NAC). NAC had no effect on NO levels, whereas resveratrol and OLPC were found to be effective in reducing nitrite levels, modifying iNOS mRNA, and decreasing free radical production. OLPC affected the levels of MnSOD while resveratrol did not, indicating that they act via different pathways. Quercetin and genistein reduced nitrite levels without affecting iNOS levels, presumably by scavenging NO. SSB did not affect nitrite levels, but exposure did reduce iNOS mRNA expression and protein levels, possibly due to antioxidant activity. Naturally occurring antioxidants, in particular resveratrol and OLPC, may have therapeutic potential in the treatment of inflammatory diseases.

Negative feedback between secretory and cytosolic phospholipase A2 and their opposing roles in ovalbumin-induced bronchoconstriction in rats.

Phospholipase A2 (PLA2) hydrolyzes cell membrane phospholipids (PL) to produce arachidonic acid and lyso-PL. The PLA2 enzymes include the secretory (sPLA2) and cytosolic (cPLA2) isoforms, which are assumed to act synergistically in production of eicosanoids that are involved in inflammatory processes. However, growing evidence raises the possibility that in airways and asthma-related inflammatory cells (eosinophils, basophils), the production of the bronchoconstrictor cysteinyl leukotrienes (CysLT) is linked exclusively to sPLA2, whereas the bronchodilator prostaglandin PGE2 is produced by cPLA2. It has been further reported that the capacity of airway epithelial cells to produce CysLT is inversely proportional to PGE2 production. This seems to suggest that sPLA2 and cPLA2 play opposing roles in asthma pathophysiology and the possibility of a negative feedback between the two isoenzymes. To test this hypothesis, we examined the effect of a cell-impermeable extracellular sPLA2 inhibitor on bronchoconstriction and PLA2 expression in rats with ovalbumin (OVA)-induced asthma. It was found that OVA-induced bronchoconstriction was associated with elevation of lung sPLA2 expression and CysLT production, concomitantly with suppression of cPLA2 expression and PGE2 production. These were reversed by treatment with the sPLA2 inhibitor, resulting in amelioration of bronchoconstriction and reduced CysLT production and sPLA2 expression, concomitantly with enhanced PGE2 production and cPLA2 expression. This study demonstrates, for the first time in vivo, a negative feedback between sPLA2 and cPLA2 and assigns opposing roles for these enzymes in asthma pathophysiology: sPLA2 activation induces production of the bronchoconstrictor CysLT and suppresses cPLA2 expression and the subsequent production of the bronchodilator PGE2.

Interactions between nitric oxide and arachidonic acid in lung epithelial cells: possible roles for peroxynitrite and superoxide.

This study investigated interactions between nitric oxide synthesis and phospholipase A2 (PLA2) activation in lung epithelial cells. Nitrite formation, inducible nitric oxide synthase expression, and [3H]arachidonic acid (AA) release were determined following treatment with: (1) the nitric oxide synthase inhibitors N(G)-nitro-L-arginine methyl esther (L-NAME) and aminoguanidine; (2) arachidonyl trifluoromethyl ketone (AACOCF3), a specific cytosolic PLA2 inhibitor; (3) S-morpholinosydnonimine (SIN-1), a nitric oxide donor which provokes peroxynitrite formation; (4) trolox, a free radical scavenger, and (5) the AA release agonists calcium ionophore, phorbol 12-myristate 13-acetate, and sodium vanadate. The results demonstrated that (1) L-NAME and aminoguanidine inhibited agonist-induced AA release by 40 and 65%, respectively; (2) AACOCF3 inhibited nitrite formation and inducible nitric oxide synthase expression in a dose-dependent manner; (3) SIN-1, together with AA release agonists, significantly increased the AA output, and (4) trolox counteracted the SIN-1 effects. Our results demonstrate cross talk between nitric oxide synthase and PLA(2) pathways, with a possible intermediary role for peroxynitrite and superoxide.

A leukotriene receptor antagonist modulates iNOS in the lung and in a leukotriene-free cell model.

Nitric oxide (NO), an important cell signaling molecule, is considered a marker of inflammatory response and is elevated in asthmatics. This study investigated the effects of montelukast (a leukotriene receptor antagonist) on iNOS expression and activity in a Brown Norway (BN) rat allergic inflammation model and in L2 lung epithelial cells. Allergic inflammation was induced by ovalbumin (OVA) injection in BN rats followed by treatment with either montelukast or dexamethasone (DX). Allergen inhalation was performed, and post-allergen Penh was measured 5 min after the challenge. Cysteinyl leukotriene levels were measured in bronchoalveolar lavage (BAL) fluid and lung iNOS expression and activity determined. These parameters were also measured in cytokine stimulated L2 lung epithelial cells. iNOS expression was significantly higher in OVA challenged rats compared to the naive, DX, and montelukast treated groups, as confirmed by immunohistochemistry and Western blot analysis. However, no significant differences in NOS activity were found. Cysteinyl leukotriene measured in BAL was significantly higher in all OVA challenged rats compared to naive controls. Incubation of L2 cells with a mixture of interferon gamma (IFNgamma), lipopolysaccharide (LPS), and tumor necrosis factor (TNFalpha) resulted in high levels of nitrite formation resulting from iNOS induction. Treatment of cytokine stimulated cells with DX or montelukast significantly decreased iNOS expression and activity. No detectable cysteinyl leukotrienes were found in the supernatant fluid of L2 cells. This study confirms the ability of montelukast to modulate iNOS function and raises the possibility that changes in iNOS expression and activity may occur via pathways independent of cysteinyl leukotrienes.