Peroxynitrite-mediated inactivation of heme oxygenases

BACKGROUND: Endogenous 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. RESULTS: Exposure 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 IC(50 )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. CONCLUSION: These 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

The effects of azole-based heme oxygenase inhibitors on rat cytochromes

ABSTRACT Heme oxygenases (HOs) catalyze the degradation of heme to biliverdin, carbon monoxide (CO), and free iron. The two major isoforms, HO-1 (inducible) and HO-2 (constitutive), are involved in a variety of physiological functions, including inflammation, apoptosis, neuromodulation, and vascular regulation. Major tools used in exploring these actions have been metalloporphyrin analogs of heme that inhibit the HOs. However, these tools are limited by their lack of selectivity; they affect other hemedependent enzymes, such as cytochromes P450 (P450s), soluble guanylyl cyclase (sGC), and nitric-oxide synthase (NOS). Our laboratory has successfully synthesized a number of nonporphyrin azole-based HO inhibitors (QC-xx) that had little or no effect on sGC and NOS activity. However, their effects on various P450 isoforms have yet to be fully elucidated. To determine the effects of the QC-xx inhibitors on P450 enzyme activity, microsomal preparations of two rat P450 isoforms (2E1 and 3A1/3A2) and two human P450 supersome isoforms (3A4 and 2D6) were incubated with varying concentrations of HO inhibitor, and the activity was determined by spectrophotometric or fluorometric analysis. Results indicated that some QC compounds demonstrated little to no inhibition of the P450s, whereas others did inhibit these P450 isoforms. Four structural regions of QC-xx were analyzed, leading to the identification of structures that confer a decreased effect on both rat and human P450 isoforms studied while maintaining an inhibitory effect on the HOs

Glyceryl trinitrate-induced vasodilation is inhibited by ultraviolet irradiation despite enhanced nitric oxide generation: evidence for formation of a nitric oxide conjugate

ABSTRACT Our objective was to determine whether a stabilized form of nitric oxide (NO) such as an S-nitrosothiol, rather than NO itself, is the vasoactive metabolite produced when glyceryl trinitrate (GTN) interacts with vascular smooth muscle. In a control study, NO formation was measured by a chemiluminescenceheadspace gas method during the incubation of a prototype S-nitrosothiol, namely, S-nitroso-N-acetylpenicillamine (SNAP), in Krebs' solution. NO formation from SNAP was increased when the incubation was carried out in the presence of UV light, indicating that homolytic photolysis of the S-nitrosothiol had occurred. When GTN was incubated with bovine pulmonary artery (BPA) in the absence of UV light, NO was not measurable until 5 min of incubation. By contrast, in the presence of UV light, NO was measurable as early as 0.5 min, and by 5 min, it was higher than that observed in the absence of UV light. BPA rings were relaxed with SNAP and GTN in the absence of UV light, and EC 50 values of 0.24 Ϯ 0.28 M and 10 Ϯ 6 nM, respectively, were observed. In the presence of UV light, the vasodilator response of BPA to SNAP and GTN was attenuated, and EC 50 values of 2.7 Ϯ 3.0 M and 49 Ϯ 23 nM, respectively, were observed. Our results are consistent with the idea that GTN biotransformation by vascular smooth muscle results in the production of a stabilized form of NO, possibly an S-nitrosothiol, and that degradation of this metabolite by UV light results in NO formation accompanied by decreased vasodilation

A Novel, “Double-Clamp” Binding Mode for Human Heme Oxygenase-1 Inhibition

The development of heme oxygenase (HO) inhibitors is critical in dissecting and understanding the HO system and for potential therapeutic applications. We have established a program to design and optimize HO inhibitors using structure-activity relationships in conjunction with X-ray crystallographic analyses. One of our previous complex crystal structures revealed a putative secondary hydrophobic binding pocket which could be exploited for a new design strategy by introducing a functional group that would fit into this potential site. To test this hypothesis and gain further insights into the structural basis of inhibitor binding, we have synthesized and characterized 1-(1H-imidazol-1-yl)-4,4-diphenyl-2-butanone (QC-308). Using a carbon monoxide (CO) formation assay on rat spleen microsomes, the compound was found to be ∼15 times more potent (IC50 = 0.27±0.07 µM) than its monophenyl analogue, which is already a potent compound in its own right (QC-65; IC50 = 4.0±1.8 µM). The crystal structure of hHO-1 with QC-308 revealed that the second phenyl group in the western region of the compound is indeed accommodated by a definitive secondary proximal hydrophobic pocket. Thus, the two phenyl moieties are each stabilized by distinct hydrophobic pockets. This “double-clamp” binding offers additional inhibitor stabilization and provides a new route for improvement of human heme oxygenase inhibitors

Cardiac adenylate metabolism : possible relationship to autoreguation of coronary blood flow

The metabolism of 5'-AMP by 5'-nucleotidase, adenylate deaminase and adenylate kinase was examined in heart homogenates of rat, rabbit, dog, pigeon and turtle. The study was conducted in consideration of the possibility that adenosine, a catabolic product of 5'-AMP, may control vasotone for the autoregulation of coronary blood flow. The relative activities of homogenates of hearts from various species to form adenosine by the action of 5'-nucleotidase generally supported such a role for this nucleoside. Those species anticipated to have the largest potential requirements for coronary vasodilation, i.e. those whose oxygen consumption is known to increase significantly during physical exertion, had the highest levels of cardiac 5'-nucleotidase. An exception to this was the pigeon which had no detectable cardiac 5'-nucleotidase; the order of levels of this enzyme in hearts of the other species tested was: rat > dog > rabbit > turtle. The turtle ventricle, by virtue of its high content of adenylate deaminase and low content of 5'-nucleotidase appeared to catabolize 5'-AMP largely by deamination to IMP. Homogenates of pigeon ventricle contained the greatest activity of adenylate kinase, indicating that the heart of this species is equipped for preservation of ATP by resynthesis from ADP. Enzyme histochemistry revealed that most 5'-nucleotidase of mammalian hearts was localized in the endothelial cells of capillaries. Therefore, if adenosine is involved in regulation of coronary perfusion, its source may be capillary endothelial cells rather than cardiac muscle cells. 5'-Nucleotidase was partially purified from an acetone powder of rat heart. It was active over a broad range of pH with an optimum at pH 8.5. The enzyme was stimulated up to 5-fold by Mg(++) [formula omitted]; Mn(++) and Ni(++) also stimulated activity. The K for 5'-AMP was 2.1 x 10(-5)M in the absence of 16Mg and 2.3 x 10 M in the presence of 16 mM MgCl(2). Certain of its properties indicated that the production of adenosine might be favoured under conditions in which coronary vasodilation would be required and vice-versa. For example, the enzyme was inhibited by ATP, whose levels are greatest in well oxygenated hearts in which energy charge is high. Not all properties of 5'-nucleotidase were consistent with enhanced adenosine formation at reduced energy charge. Both ADP and orthophosphate, the levels of which increase when energy charge decreases, inhibited the enzyme; in fact ADP was a more powerful inhibitor than ATP. In addition, the enzyme was not specific for 5'-AMP but hydrolyzed a variety of nucleoside 5'-monophosphates; and the hydrolysis of 5'-AMP was competitively inhibited by UMP. In the absence of Mg(++) , inhibition by ADP was of the mixed (competitive- non-competitive) type. In the presence of 16 mM MgCl(2), inhibition was non-competitive. On the basis of these data and Dixon plots of inhibition as a function of ADP concentration, it is suggested that two conformations of the enzyme are possible; one which is competitively inhibited by ADP. The simple non-competitive inhibition by ADP, observed in the presence of 16 mM MgCl(2), is attributed to Mg(++) -induced preference for the latter conformation.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat

Cysteine-independent activation/inhibition of heme oxygenase-2

<b>Figure 1: Menadione activation of recombinant FL-hHO-2 (open circles) was similar to the same enzyme without thiols-FL-hHO-2 Cys265,282-Ala (closed triangles).</b><div><br></div><div><b>Figure 2: QC-2350 inhibition of recombinant and microsomal HO-2.</b> </div><div><br></div><div><b>How to cite this article:<i> </i></b><i>Vukomanovic D, Rahman MN, Maines MD, Ozolinš TR, Szarek WA, Jia Z, Nakatsu K. Cysteine-independent activation/inhibition of heme oxygenase-2. Med Gas Res 2016;6:10-13.</i></div