GEA 3162 decomposes to co-generate nitric oxide and superoxide and induces apoptosis in human neutrophils via a peroxynitrite-dependent mechanism.
1. GEA 3162 (1,2,3,4,-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-chloride), has powerful effects on neutrophil function and apoptosis, but the underlying mechanisms are unclear, particularly with respect to the possible roles of nitric oxide (NO) and/or peroxynitrite (ONOO(-)). 2. Our hypothesis was that GEA 3162 is a generator of ONOO(-) and that its biological effects on neutrophil apoptosis differ from those of a conventional NO donor. The effects of GEA 3162 were compared to those of the established ONOO(-) donor, 3-morpholinosydnonimine (SIN-1), and the NO donor, diethylamine diazeniumdiolate (DEA/NO) in neutrophils from healthy volunteers. Electrochemical detection and electron paramagnetic resonance were used to define the NO-related species generated from these agents. 3. GEA 3162 and SIN-1 influence neutrophil apoptosis differently from DEA/NO. All three compounds induced morphological neutrophil apoptosis. However, both GEA 3162 and SIN-1 paradoxically inhibited internucleosomal DNA fragmentation, whereas DEA/NO induced fragmentation compared to control. 4. In contrast to DEA/NO, generation of free NO was not detectable in solutions of GEA 3162 or SIN-1 (100 microm). However, Cu/Zn superoxide dismutase (SOD; 50-750 U ml(-1)) unmasked NO generated from these compounds in a concentration-dependent manner. GEA 3162 and SIN-1 oxidised the O(2)(-)- and ONOO(-)-sensitive dye, dihydrorhodamine 123 (DHR 123; 1 microm), suggesting that ONOO(-) released from these compounds is responsible for oxidation of DHR 123. 5. We conclude that GEA 3162 is an ONOO(-) donor with pro-apoptotic properties that more closely resemble SIN-1 than the NO donor, DEA/NO. Moreover, unlike NO, ONOO(-) induces apoptosis in neutrophils via a mechanism that does not require DNA fragmentation.
Taylor
Emma L
EL
Centre for Inflammation Research, Rayne Laboratory, Medical School, University of Edinburgh, Teviot Place, Edinburgh EH8 9AG.
Rossi
Adriano G
AG
Shaw
Catherine A
CA
Dal Rio
Francesco P
FP
Haslett
Christopher
C
Megson
Ian L
IL
D017209:Apoptosis / Q000187:drug effects*; D002469:Cell Separation; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects*; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D012235:Rhodamines; D013481:Superoxides / Q000378:metabolism*; D014230:Triazoles / Q000378:metabolism* / Q000494:pharmacology*
D005456:Fluorescent Dyes; D020030:Nitric Oxide Donors; D012235:Rhodamines; D014230:Triazoles; C058319:dihydrorhodamine 123; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002385:linsidomine; C082874:GEA 3162; D008981:Molsidomine