Abstract
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 degrees C (t(12) = 2.3 min). Interestingly, multiple additions of AS at lower concentrations (50 or 100 microm) 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.
Highlights
Nitric oxide and S-nitrosothiols modulate a variety of important physiological activities
We utilized rat H9c2 cells to examine the effect of a NOϪ generator (Angeli’s salt) on heme oxygenase 1 (HO-1) protein expression as well as heme oxygenase activity in an attempt to discern the contribution of NO and its redox forms in the cellular adaptation to the stress inflicted by nitrosative reactions
Angeli’s Salt Increases Heme Oxygenase Activity, HO-1 Protein, and mRNA Expression in Rat H9c2 Cells—A concentrationdependent increase in heme oxygenase activity was observed in cells exposed for 6 h to 0.25–1 mM Angeli’s salt (AS); higher concentrations of the drug (1.5 and 2 mM) did not further enhance heme oxygenase activity but rather promoted a reduction in the activation of the enzyme (Fig. 1A)
Summary
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. The biological response(s) mediated by NO cannot be confined solely to the ability of this free radical to interact with important intracellular targets but must be extended to the reactivity of the nitrosonium cation (NOϩ) and the nitroxyl anion (NOϪ), respectively, the one-electron oxidation and reduction products of NO [33] Each of these redox forms can evoke a variety of biological responses depending upon their concentration and location, the presence of thiols, and the composition of the cellular microenvironment [33, 34]. We utilized rat H9c2 cells to examine the effect of a NOϪ generator (Angeli’s salt) on HO-1 protein expression as well as heme oxygenase activity in an attempt to discern the contribution of NO and its redox forms in the cellular adaptation to the stress inflicted by nitrosative reactions (i.e. nitrosative stress)
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