Interference of Carboxy-PTIO with Nitric Oxide- and Peroxynitrite-Mediated Reactions

doi:10.1016/S0891-5849(96)00407-8

Free Radical Biology and Medicine

Volume 22, Issue 5, 1997, Pages 787-794

https://doi.org/10.1016/S0891-5849(96)00407-8 Get rights and content

Abstract

Carboxy-PTIO reacts rapidly with NO to yield NO₂ and has been used as a scavenger to test the importance of nitric oxide (NO) in various physiological conditions. This study investigated the effects of carboxy-PTIO on several NO- and peroxynitrite-mediated reactions. The scavenger potently inhibited NO-induced accumulation of cGMP in endothelial cells but potentiated the effect of the putative peroxynitrite donor SIN-1. Carboxy-PTIO completely inhibited peroxynitrite-induced formation of 3-nitrotyrosine from free tyrosine (EC₅₀ = 36 ± 5 μM) as well as nitration of bovine serum albumin. Peroxynitrite-mediated nitrosation of GSH was stimulated by the drug with an EC₅₀ of 0.12 ± 0.03 mM, whereas S-nitrosation induced by the NO donor DEA/NO (0.1 mM) was inhibited by the scavenger with an IC₅₀ of 0.11 ± 0.03 mM. Oxidation of NO with carboxy-PTIO resulted in formation of nitrite without concomitant production of nitrate. Our results demonstrate that the effects of carboxy-PTIO are diverse and question its claimed specificity as NO scavenger. Copyright © 1997 by Elsevier Science Inc.

Introduction

NO has several important biological functions, including the regulation of vascular tone, inhibition of platelet aggregation, modulation of synaptic transmission in the brain, and neurotransmission in the peripheral nervous system.[1] NO can undergo a variety of reactions within cells, and to understand NO-mediated regulatory effects it is necessary to study the rates of these reactions and the activity of their products.[2] The reactions with molecular oxygen, superoxide, and iron proteins are already known to be physiologically significant.[3] The reaction with oxygen is second order with respect to NO,4, 5, 6 and yields intermediates with potential cytotoxic and mutagenic properties.7, 8 It is not yet clear which of the possible nitrogen oxides are intermediates in this reaction.4, 9

One of the fastest reactions of NO is the nearly diffusion-controlled combination with superoxide to form peroxynitrite,10, 11 a potent oxidant of many biological molecules.[12] In a metal-catalyzed reaction, peroxynitrite nitrates several phenolic compounds including free and peptide tyrosine.13, 14, 15 Peroxynitrite is protonated with a pKa of 6.8, and the corresponding peroxynitrous acid rapidly decomposes to form a potent oxidant with hydroxyl radical-like properties.[10] The precise mechanism of peroxynitrite decomposition is not clear yet, but it appears that free radical intermediates are not involved.[12]

Several reports indicate that peroxynitrite has similar physiological effects to NO. It induces NO-like relaxation of vascular smooth muscle,16, 17 inhibits platelet aggregation,[17] and stimulates purified soluble guanylyl cyclase.[18] These effects may be due to formation of small amounts of NO during peroxynitrite decomposition[16] or result from S-nitrosation of thiols followed by release of free NO from the corresponding thionitrites.18, 19 Thus, selective NO scavengers are required to discriminate between free NO and other species exhibiting NO-like biological activity. Reduced hemoglobin was described as a potent and specific scavenger of NO,[20] but authentic peroxynitrite reacts rapidly with hemoglobin in a manner indistinguishable from NO-induced formation of methemoglobin.[21] Another class of NO scavengers are nitronyl nitroxides.[22] The prototypes of such stable free radicals are PTIO and its water-soluble derivative carboxy-PTIO, which react rapidly with NO to yield the corresponding imidazolineoxyl and free NO₂ radical.[23] Based on susceptibility to carboxy-PTIO, it was reported that EDRF was identical with NO, whereas the nitrergic neurotransmitter producing relaxation of certain smooth muscles was not.23, 24 To find out whether carboxy-PTIO can be regarded as a specific NO scavenger, we have studied the effect of this drug on several NO- and peroxynitrite-mediated reactions.

Section snippets

Materials

Solutions of peroxynitrite (80–100 mM) were prepared as described[21] and diluted to 10 mM with H₂O prior to experiments. GSNO and carboxy-PTIO were purchased from ALEXIS Corp., Switzerland. DEA/NO was from NCI Chemical Carcinogen Repository, Kansas City, MO; 10-fold concentrated stock solutions of the NO donor were prepared daily in 10 mM NaOH. SIN-1, a generous gift from Cassella-Riedel, Frankfurt, Germany, was dissolved at pH 5.0 prior to use. The monoclonal nitrotyrosine antibody was a

Results

The ability of carboxy-PTIO to scavenge NO was assessed by determination of NO-induced accumulation of cGMP in cultured porcine aortic endothelial cells in the absence and presence of the drug. Basal cGMP levels of unstimulated cells were 2.2 ± 0.7 pmol/10⁶ cells. As shown in Fig. 1, 1 μM DEA/NO induced a 20-fold increase in cGMP (44 ± 12.5 pmol/10⁶ cells) and this effect was inhibited by 0.1 mM carboxy-PTIO to 16.3 ± 4.8% of the control. In the presence of the Ca²⁺ ionophore A 23187 (0.3 μM),

Discussion

Our results on inhibition of NO-induced cGMP accumulation in cultured cells and inhibition of NO-stimulated soluble guanylyl cylcase confirm that carboxy-PTIO is a potent scavenger of NO, but the action profile of this compound may be more complex than hitherto assumed. Unexpectedly, carboxy-PTIO did not inhibit but potentiated the effect of SIN-1 on cGMP accumulation in endothelial cells. Decomposition of SIN-1 yields NO and superoxide,[36] suggesting that the sydnonimine may be a donor of

Acknowledgements

We thank an anonymous referee for his comments on oxidation of GSH by NO₂ and Margit Rehn for excellent technical assistance. This work was supported by Grants 10655, 10859, 11478 (to B.M.), F712 (K.S.), and 11301 (to E.R.W.) of the Fonds zur Förderung der Wissenschaftlichen Forschung in Österreich.

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Original ContributionInterference of Carboxy-PTIO with Nitric Oxide- and Peroxynitrite-Mediated Reactions

Abstract

Introduction

Section snippets

Materials

Results

Discussion

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

J. Biol. Chem.

Arch. Biochem. Biophys.

Eur. J. Pharmacol.

Biochem. Pharmacol.

Anal. Biochem.

FEBS Lett.

FEBS Lett.

J. Inorg. Biochem.

Arch. Biochem. Biophys.

Eur. J. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

Nitric oxidePhysiology, pathophysiology, and pharmacology

Pharmacol. Rev.

EPR characterization of molecular targets for NO in mammalian cells and organelles

FASEB J.

Reactions of the bioregulatory agent nitric oxide in oxygenated aqueous media: Determination of the kinetics for oxidation and nitrosation by intermediates generated in the NO/O2 reaction

Chem. Res. Toxicol.

DNA deaminating ability and genotoxicity of nitric oxide and its progenitors

Science

Original Contribution
Interference of Carboxy-PTIO with Nitric Oxide- and Peroxynitrite-Mediated Reactions

Reactions of the bioregulatory agent nitric oxide in oxygenated aqueous media: Determination of the kinetics for oxidation and nitrosation by intermediates generated in the NO/O₂ reaction