Reaction of peroxynitrite with HEPES or MOPS results in the formation of nitric oxide donors.
We investigated the effects of organic buffers on the NO-like biological activities of ONOO-. In HEPES buffer (50 mM), ONOO- (1 mM) induced a 20-fold increase in endothelial cGMP accumulation and the effect was comparable to that elicited by a maximally active concentration of the NO donor DEA/NO. ONOO- produced a 12-fold increase of cGMP in MOPS buffer (50 mM), but was virtually inactive in phosphate buffer (50 mM). Electrochemical detection of NO showed that the biological effects of ONOO- in HEPES or MOPS were due to accumulation of compounds that released NO in the presence of copper ions. CuCl2-induced formation of NO was completely blocked by the Cu(I) chelator neocuproine but unaffected by the Cu(II) chelator cuprizone, pointing to a Cu(I)-catalyzed decomposition pathway. Formation of NO from ONOO- was not detectable in phosphate buffer, in agreement with the lack of effect of ONOO- on cGMP accumulation in this buffer. These data demonstrate that certain buffer components present in cell culture media may yield artificial results in experiments with authentic ONOO-.
Schmidt
K
K
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Austria. kurt.schmidt@kfunigraz.ac.at
Pfeiffer
S
S
Mayer
B
B
D000818:Animals; D002021:Buffers; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D006531:HEPES; D009566:Nitrates / Q000494:pharmacology*; D009569:Nitric Oxide / Q000494:pharmacology*; D016877:Oxidants / Q000494:pharmacology*; D013552:Swine
D002021:Buffers; D009566:Nitrates; D016877:Oxidants; C039642:peroxynitric acid; D009569:Nitric Oxide; D006531:HEPES