Nitric oxide formation from hydroxylamine by myoglobin and hydrogen peroxide

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Abstract

Hydroxylamine (HA), which is a natural product of mammalian cells, has been shown to possess vasodilatory properties in several model systems. In this study, HA and methyl-substituted hydroxylamines, N-methylhydroxylamine (NMHA) and N,N-dimethylhydroxylamine (NDMHA), have been tested for their ability to generate free diffusible nitric oxide (NO) in the presence of myoglobin (Mb) and hydrogen peroxide. A NO-specific conversion of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) to 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl (carboxy-PTI), measured by electron spin resonance (ESR) spectroscopy, along with nitrite and nitrate production, was observed for HA but not for NMHA and NDMHA. ESR measurements at 77 K showed the formation of the ferrous nitrosyl myoglobin, Mb–NO, in the reaction mixtures containing Mb, H2O2 and HA. Our data also demonstrate that Mb–NO is an end product of the reaction pathway involving Mb, H2O2 and HA, rather than a reaction intermediate in the formation of NO. In summary, our results demonstrate a possible pathway of NO formation from HA, however, the significance of this mechanism for bioactivation of HA in vivo is unknown at the present time.

Introduction

Hydroxylamine (HA) is a natural product of mammalian cells and has been found to possess vasodilatory properties [1]. Studies with blood vessels of rat kidney showed that HA causes dose-dependent vasodilation, presumably by generating nitric oxide (NO) [2]. HA was also found to relax rat aortic rings in a dose-dependent manner, even in the absence of endothelium, which led to the postulation that HA might be endothelium-derived relaxing factor (EDRF) via its decomposition to NO, which causes activation of guanylate cyclase 3, 4. More recently, HA was found to induce vasodilation of isolated rodent lungs with higher potency than nitroglycerin [5]. The generation of NO from HA was suggested to occur by reaction with catalase in the presence of H2O2 [6]. HA was found to generate NO in intact cells as well as in cytosolic preparations, presumably utilizing catalase- and H2O2-mediated pathways [7]. Recent studies by Feelisch et al. [8]have shown that HA can relax endothelium-denuded rabbit aortic strips, although less potently than that which occurs with NO.

In this study, HA and its methyl-substituted derivatives, N-methylhydroxylamine (NMHA) and N,N-dimethylhydroxylamine (NDMHA), were tested for NO generation in incubations containing metmyoglobin and H2O2 and it was shown that only HA is capable of NO delivery under these conditions.

Section snippets

Reagents

Myoglobin (metMb, horse heart), nitrate reductase and NADPH were purchased from Sigma. The concentration of metMb was determined spectroscopically using an extinction coefficient of 1.3×105 M−1 cm−1 at 410 nm. Hydrogen peroxide was obtained from Fisher Scientific and its concentration was determined using an extinction coefficient of 43.6 M−1 cm−1 at 240 nm. HA, NMHA, NDMHA and diethylenetriaminopentaacetic acid (DTPA) were purchased from Aldrich. Sephadex G-25 columns were purchased from

Results

Fig. 1a shows the accumulation of NO2 and NO3 when HA, NMHA and NDMHA, respectively, were incubated with metMb (100 μM) and H2O2 (10 mM). Nitrate was clearly the dominant metabolite; the nitrite contribution to the combined nitrite plus nitrate yield was less than 5% (data not shown). A dose-dependent accumulation of NO2 and NO3 was detected with HA (1.56–12.5 mM), while no detectable levels of NO2 and NO3 were observed for NMHA or NDMHA, even at concentrations as high as 25 mM. The

Discussion

The formation of nitroxide radicals (RR′NO-type) from hydroxylamine and its derivatives, NMHA and NDMHA, in reactions with oxyhemoglobin and oxymyoglobin, was studied previously by ESR. Stolze and Nohl 11, 12found that the stabilities of the nitroxide radicals H2NO from HA, CH3HNO from NMHA and (CH3)2NO from NDMHA varied significantly; the nitroxide radical derived from HA had a lifetime in the range of several seconds and decomposed to uncharacterized products, the nitroxide derived from

Acknowledgements

We wish to thank Drs. Murali C. Krishna, David A. Wink and James B. Mitchell for useful discussion.

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