Thiols are very important antioxidants that protect cells against oxidative insults. Recently, a different and new physiological role has been defined for these compounds because of their involvement in nitric oxide (NO) binding and transport in biological systems. In view of these characteristics, we examined the effect of thiols and NO on the expression of the inducible form of heme oxygenase (HO-1), a stress protein that degrades heme to carbon monoxide and biliverdin. Cultured bovine aortic endothelial cells exposed to the NO donors sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) resulted in increased heme oxygenase activity and HO-1 expression. Co-incubation with N-acetylcysteine, a precursor of glutathione synthesis, significantly attenuated heme oxygenase induction by SNP and SNAP, and a reduction in heme oxygenase activity was also observed when cells were preincubated with N-acetylcysteine for 16 h prior to exposure to NO donors. This effect appears to be associated with NO stabilization by thiols through the formation of S-nitrosothiols. Hydroxocobalamin, a specific NO scavenger, significantly decreased endothelial heme oxygenase activity, indicating a direct involvement of NO released by NO donors to regulate the expression of this stress protein. Moreover, superoxide anion (O 2 . ) and its reaction product with NO, peroxynitrite (ONOO ؊ ), were found to partially contribute to the observed NO-mediated activation of endothelial heme oxygenase. Thus, we suggest the existence of a dynamic equilibrium among free NO, O 2 . , and endogenous glutathione, which might constitute an interactive signaling mechanism modulating stress and adaptive responses in tissues.Heme oxygenase is a widely distributed enzyme in mammalian tissues, and its main function is associated with the degradation of heme to biliverdin, iron, and carbon monoxide (CO).1 Two distinct isoforms of the protein have been characterized revealing that one of the isozymes is constitutive (HO-2), whereas the other is inducible (HO-1) (1). Thus, if the first enzyme is constitutively expressed and is part of the normal cellular metabolism, the second is regarded as a heat shock protein, and its expression is elicited by many conditions and factors that produce an imbalance in the cellular functions. Various agents, including heavy metal ions (2), oxidative stress (3), endotoxins (4), and hemoglobin (5) are capable of inducing HO-1 in different tissues, and recent findings showed that nitric oxide (NO) donors increase HO-1 mRNA in the brain (6) and in cultured hepatocytes (7). Accordingly, we have reported the ability of diverse NO releasing agents to modulate heme oxygenase activity in aortic endothelial cells (8).The physiological importance of HO-1 induction following stress situations is not fully understood, although it has been hypothesized that the expression of this gene is part of the defensive mechanism that cells and tissues are capable of mounting against different stress stimuli. To sustain this idea are the findin...