Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis.

Catherine A Shaw,Ian L Megson,David J Webb,Adriano G Rossi

doi:10.1186/1476-9255-6-14

Abstract

BackgroundNitric oxide (NO) can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-). In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDMϕ), and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP) is able to limit apoptosis in this cell type.MethodsCharacterisation of the NO-related species generated by (Z)-1- [2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-, chloride (GEA-3162) was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR) spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMϕ. Resultant MDMϕ were treated for 24 h with DETA/NO (100 – 1000 μM) or GEA-3162 (10 – 300 μM) in the presence or absence of BAY 41–2272 (1 μM), isobutylmethylxanthine (IBMX; 1 μM), 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 μM) or 8-bromo-cGMP (1 mM). Apoptosis in MDMϕ was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining.ResultsElectrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO) had no effect on cell viability, but ONOO- (GEA-3162) caused a concentration-dependent induction of apoptosis in MDMϕ. Preconditioning of MDMϕ with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX), or the NO-independent stimulator of soluble guanylate cyclase, BAY 41–2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner.ConclusionThese results demonstrate disparities between the ability of NO and ONOO- to induce apoptosis in human MDMϕ. Furthermore, this study provides evidence for a novel cGMP-dependent pre-conditioning mechanism to limit ONOO--induced apoptosis in human MDMϕ.

Highlights

Nitric oxide (NO) can be both pro- and anti-apoptotic in various cell types, including macrophages
The generally accepted paradigm is that lower NO concentrations produced constitutively by endothelial NO synthase and neuronal NO synthase are cytoprotective via primarily cyclic guanosine monophosphate (cGMP)-dependent mechanisms, whilst higher, supraphysiological concentrations generated in some pathologies by the inducible form of NOS mediate apoptosis via mechanisms independent of cGMP signalling [16]
NO Release from (Z)1- [2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1 (DETA/NO) and GEA-3162 DETA/NO (300 μM) generated a slow and prolonged NO release in Iscove's modified Dulbecco's tissue culture medium (IMDM) that remained steady throughout a 30 min recording period

Summary

Introduction

Nitric oxide (NO) can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-). Failure of inflammatory cells to undergo apoptosis, or failure of subsequent phagocytic removal of apoptotic cells is believed to result in incomplete resolution and an exacerbation of the inflammatory response [1,2,3,4]. The signalling molecule, nitric oxide (NO), has previously been reported to induce apoptosis in various cell types, including macrophages [8,9,10,11,12]. The generally accepted paradigm is that lower NO concentrations produced constitutively by endothelial NO synthase (eNOS) and neuronal NO synthase (nNOS) are cytoprotective via primarily cGMP-dependent mechanisms, whilst higher, supraphysiological concentrations generated in some pathologies by the inducible form of NOS (iNOS) mediate apoptosis via mechanisms independent of cGMP signalling [16]

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Conclusion