Induction of Heme Oxygenase 1 by Nitrosative Stress

Nitric oxide and S-nitrosothiols modulate a variety of important physiological activities. In vascular cells, agents that release NO and donate nitrosonium cation (NO+), such as S-nitrosoglutathione, are potent inducers of the antioxidant protein heme oxygenase 1 (HO-1) (Foresti, R., Clark, J. E., Green, C. J., and Motterlini, R. (1997)J. Biol. Chem. 272, 18411–18417; Motterlini, R., Foresti, R., Bassi, R., Calabrese, V., Clark, J. E., and Green, C. J. (2000) J. Biol. Chem. 275, 13613–13620). Here, we report that Angeli's salt (AS) (0.25–2 mm), a compound that releases nitroxyl anion (NO−) at physiological pH, induces HO-1 mRNA and protein expression in a concentration- and time-dependent manner, resulting in increased heme oxygenase activity in rat H9c2 cells. A time course analysis revealed that NO−-mediated HO-1 expression is transient and gradually disappears within 24 h, in accordance with the short half-life of AS at 37 °C (t = 2.3 min). Interestingly, multiple additions of AS at lower concentrations (50 or 100 μm) over a period of time still promoted a significant increase in heme oxygenase activity. Experiments performed using a NO scavenger and the NO electrode confirmed that NO−, not NO, is the species involved in HO-1 induction by AS; however, the effect on heme oxygenase activity can be amplified by accelerating the rate of NO− oxidation.N-Acetylcysteine almost completely abolished AS-mediated induction of HO-1, whereas a glutathione synthesis inhibitor (buthionine sulfoximine) significantly decreased heme oxygenase activation by AS, indicating that sulfydryl groups are crucial targets in the regulation of HO-1 expression by NO−. We conclude that NO−, in analogy with other reactive nitrogen species, is a potent inducer of heme oxygenase activity and HO-1 protein expression. These findings indicate that heme oxygenase can act both as a sensor to and target of redox-based mechanisms involving NO and extend our knowledge on the biological function of HO-1 in response to nitrosative stress.