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 muM). 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 muM), 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.