Abstract
BackgroundEndogenous nitric oxide (NO) and carbon monoxide (CO) are generated by nitric oxide synthase and heme oxygenase, respectively. Like NO, CO has been accepted as an important cellular signaling molecule in biological systems. An up-regulation in both gene and protein expression of heme oxygenase-1 (HO-1) under oxidative/nitrosative stress has been well documented, and the protective role of HO-1 and HO-2 against oxidative damage is proposed. However, data on the direct effect of reactive oxygen/nitrogen species (ROS/RNS) on HO function is incomplete. Using gas chromatography to quantify carbon monoxide (CO) formation from heme oxidation, we investigated the effects of peroxynitrite (ONOO-) on the in vitro catalytic activity of rat spleen (HO-1) and brain (HO-2) microsomal heme oxygenases.ResultsExposure to ONOO- led to concentration-dependent but reversible decreases in the activity of microsomal rat spleen and brain HO activity. Spleen HO activity was 100-fold more sensitive to ONOO--dependent inactivation compared to that of the brain, with IC50 values of 0.015 ± 0.005 mM and 1.25 ± 0.25 mM respectively. Inhibition of both rat spleen and brain microsomal HO activity was also observed with tetra-nitromethane, a tyrosine nitrating agent, as well as two NO donors, S-nitrosoglutathione (GSNO) and diethylamine NONOate (DEA-NONOate). However, no additive effect was found following the application of NO donors and ONOO- together.ConclusionThese results indicate that ONOO- may regulate HO-1 and HO-2 activities by mechanisms that involve different interactions with these proteins. It is suggested that while nitration of tyrosine residues and oxidation of sulfhydryl groups may be involved, consideration should be given to other facets of ONOO- chemistry. This inhibition of HO activity offers a mechanism for cross talk between the nitric oxide synthase and HO systems.
Highlights
Endogenous nitric oxide (NO) and carbon monoxide (CO) are generated by nitric oxide synthase and heme oxygenase, respectively
Effect of ONOO- and TNM on Microsomal heme oxygenases (HO) activity Exposure of either rat spleen or brain microsomes to ONOO- resulted in decreases of HO activity in a concentration dependent manner (Figure 1A and Figure 1B)
heme oxygenase-1 (HO-1) was more sensitive towards ONOO--mediated inactivation compared to HO-2 (Figure 1A and Figure 1B)
Summary
Endogenous nitric oxide (NO) and carbon monoxide (CO) are generated by nitric oxide synthase and heme oxygenase, respectively. An up-regulation in both gene and protein expression of heme oxygenase-1 (HO-1) under oxidative/nitrosative stress has been well documented, and the protective role of HO-1 and HO-2 against oxidative damage is proposed. Using gas chromatography to quantify carbon monoxide (CO) formation from heme oxidation, we investigated the effects of peroxynitrite (ONOO-) on the in vitro catalytic activity of rat spleen (HO-1) and brain (HO-2) microsomal heme oxygenases. Heme oxygenases (HO, EC 1.14.99.3) are a highly conserved family of proteins that catalyse the oxidative cleavage of heme at the α-meso carbon to yield equimolar amounts of iron, carbon monoxide (CO) and biliverdin. HO-1 (the inducible isoform) is predominantly expressed in the spleen, the primary site of heme catabolism but has been detected in many different tissues including the liver and the kidney. The involvement of HO and products of heme catabolism have been studied extensively with (page number not for citation purposes)
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