Original contribution
Dioxygen-dependent metabolism of nitric oxide in mammalian cells

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Abstract

Steady-state gradients of NO within tissues and cells are controlled by rates of NO synthesis, diffusion, and decomposition. Mammalian cells and tissues actively decompose NO. Of several cell lines examined, the human colon CaCo-2 cell produces the most robust NO consumption activity. Cellular NO metabolism is mostly O2-dependent, produces near stoichiometric NO3, and is inhibited by the heme poisons CN, CO (KI ≈ 3 μM), phenylhydrazine, and NO and the flavoenzyme inhibitor diphenylene iodonium. NO consumption is saturable by O2 and NO and shows apparent KM values for O2 and NO of 17 and 0.2 μM, respectively. Mitochondrial respiration, O2, and H2O2 are neither sufficient nor necessary for O2-dependent NO metabolism by cells. The existence of an efficient mammalian heme and flavin-dependent NO dioxygenase is suggested. NO dioxygenation protects the NO-sensitive aconitases, cytochrome c oxidase, and cellular respiration from inhibition, and may serve a dual function in cells by limiting NO toxicity and by spatially coupling NO and O2 gradients.

Introduction

NO modulates arteriole pressure and regulates tissue O2 concentrations. NO is also a natural antibiotic belonging to the arsenal of free radicals and oxidants deployed by leukocytes and other host cells to combat invading microorganisms, viruses, and neoplastic tissue [1], [2]. Other important physiological functions of NO include the transduction of neuronal messages [3]. NO production is tightly regulated by calmodulin and caveolin, and by the complex controls on the expression of various isoforms of NO synthase [4], [5].

Multiple pathways of NO removal can impact NO signaling and toxicity. Hb is an important sink for NO within the vasculature [6], [7]. NO reacts rapidly with oxygenated Hb to form NO3 [8], [9] and reacts equally rapidly with deoxyHb to form a stable nitrosylated Hb [10], [11], [12]. The removal of NO by circulating Hb is controlled by the spatial distribution and movement of erythrocytes within capillaries and by the high density of Hb within membranes [13], [14], [15], [16]. Within red muscle tissue, the abundant oxygenated Mb dioxygenates NO to form NO3 [6], and this pathway appears important for NO function [17].

In rat lungs, NO is consumed by an active mechanism that is only partly affected by blood flow, and Hb-dependent scavenging, suggesting the presence of alternate pathways for NO metabolism in tissues [18]. In addition, recent mathematical models for NO diffusion and decomposition in tissues suggest an important role for cellular NO metabolism in the maintenance of NO gradients [19]. The diffusion-limited bimolecular reaction of NO with O2 provides a potential route for NO decomposition in cells and tissues and produces toxic ONOO [20]. However, the ONOO pathway is dependent upon both NO and O2 concentrations. In cells, efficient and abundant O2 scavengers [21], [22] in concert with NO scavengers [23], [24] can synergistically inhibit the ONOO pathway. NO also reacts with O2 in a termolecular reaction to form NO2, and ultimately NO2. The reaction of NO with O2 is accelerated in the hydrophobic interior of biological membranes [25], however, this reaction is still slow at physiological NO and O2 concentrations. In addition, NO reacts directly with a myriad of target biomolecules that contribute to NO decomposition including the labile [4Fe-4S]-containing aconitases [26], [27], [28]. In this regard, cytochrome c oxidase [29], [30], [31], [32], cytochrome c [33], cytochrome P450 [34], 15-lipoxygenase [35], prostaglandin H synthase [36], and peroxidases [37] are targets for NO inhibition and are invariably thought to serve important roles in NO metabolism.

During our investigations of NO-mediated inactivation of aconitase and its repair, we have observed O2-dependent activities for NO metabolism and aconitase protection in Escherichia coli [23], [38] and mammalian cells [39]. A flavoHb confers a protective NO dioxygenase activity in E. coli. The flavoHb, and NOD activity, are expressed in prokaryotes, yeast, and fungi [23], [24], [38], [40], [41], [42], [43], [44], [45], [46], [47]. Our investigations of the substrate requirements, product, and inhibitor sensitivities of the NO metabolic activity in mammalian cells have revealed a similar flavohemoprotein-dependent catalytic mechanism for NO conversion to NO3. However, the mammalian cell activity is largely lost upon cell disruption, suggesting a loss of necessary components or an instability of the activity during cell fractionation. The properties and possible functions of the activity are discussed.

Section snippets

Reagents

Porcine heart isocitrate dehydrogenase, cis-aconitate, Aspergillus niger glucose oxidase, heparin sodium salt, endothelial cell growth supplement, NADP+, glucose-6-phosphate, glucose-6-phosphate dehydrogenase from baker’s yeast, FCCP, L-amino acids, NaCN, rotenone, oligomycin, antimycin A, human Hb A0, human heart Mb, phenylhydrazine-HCl, H2O2, and DPI were purchased from Sigma (St. Louis, MO, USA). Myxothiazole, bovine erythrocyte Cu,Zn-containing SOD (5,000 U per mg), bovine liver catalase

Effect of dioxygen and cyanide on NO-mediated aconitase inactivation

[4Fe-4S]-containing aconitases have been shown to be targets for NO-mediated damage in bacteria and mammalian cells exposed to macrophages, NO donor compounds, or pure NO [26], [27], [28], [38], [55]. We were interested in determining the sensitivity of the aconitases to various NO concentrations, measuring the repair of inactive aconitase, and using aconitase to identify protective NO detoxification activities in mammalian cells.

Aconitase activity is slowly but progressively lost during

Discussion

NO is a highly diffusible gaseous molecule showing selective and specialized functions throughout the biosphere [70]. NO is important for denitrification, energy production, niche maintenance, and inter- and intracellular communication in various life forms. In mammals, the rate of NO degradation as well as its formation may serve as a very specific means to influence vascular tone, tissue oxygenation, and other NO-controlled functions in localized regions [6], [19]. The half-life of NO infused

Acknowledgements

This work was supported in part by an American Heart Association Grant (9730193N) to P.R.G.

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