Research

Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis

Catherine A Shaw¹ , David J Webb¹ , Adriano G Rossi² and Ian L Megson³

¹  Centre for Cardiovascular Science, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK

²  MRC Centre for Inflammation Research, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK

³  Free Radical Research Facility, Department of Diabetes and Cardiovascular Science, UHI Millennium Institute, Inverness, UK

author email corresponding author email

Journal of Inflammation 2009, 6:14doi:10.1186/1476-9255-6-14

Published:	7 May 2009

Abstract

Background

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^-). 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.

Methods

Characterisation 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.

Results

Electrochemistry 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.

Conclusion

These 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.