ponceau-s and tin-mesoporphyrin

Heme oxygenase (HO)-1, which is a rate-limiting enzyme involved in the catabolism of heme, is upregulated by a variety of stresses including oxidative stresses and inflammatory cytokines, in many cell types. Recent studies have suggested that upregulation of HO-1 might provide cytoprotection and immunomodulatory functions in addition to its obvious role in heme metabolism. In this study, we examined whether HO-1 was upregulated following degranulation in mast cells that initiate vigorous immunity reactions. To trigger degranulation, rat basophilic leukemia (RBL)-2H3 cells were passively sensitized using an antiserum collected from ovalbumin (OA) immunized-Brown Norway rats, and the cells were stimulated by treatment with OA. Degranulation was confirmed by measuring the release of beta-hexosaminidase. HO-1 mRNA and presence of HO-1 protein were detected using Northern blot and Western blot analyses, respectively. The effect of the antioxidant N-acetyl-L-cysteine (NAC) on HO-1 expression was also tested. HO-1 mRNA transiently increased at 1--2 h after RBL-2H3 cells were stimulated to degranulate. Its mRNA increases were dependent on the extent of degranulation. Following the upregulation of HO-1 mRNA, HO-1 protein was also increased. We also detected intracellular production of reactive oxygen species following degranulation in RBL-2H3 cells. NAC attenuated the HO-1 expression in a dose-dependent manner. This is the first report to reveal induction of both HO-1 mRNA and protein by degranulation in RBL-2H3 cells. We showed that NAC inhibited HO-1 upregulation. These results suggest that oxidative stress in activated RBL-2H3 cells results in the upregulation of HO-1.

Topics: Acetylcysteine; Animals; Azo Compounds; Blotting, Northern; Blotting, Western; Cell Degranulation; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fluoresceins; Fluorescence; Free Radical Scavengers; Heme Oxygenase-1; Leukemia, Basophilic, Acute; Metalloporphyrins; Ovalbumin; Rats; Rats, Inbred BN; Reactive Oxygen Species; RNA, Messenger; Up-Regulation