Mechanisms and kinetic profiles of superoxide-stimulated nitrosative processes in cells using a diaminofluorescein probe

Author(s)

Damasceno, Fernando Cruvinel ^[1] ; Facci, Romulo Rodrigues ^[1] ; da Silva, Thalita Marques ^[1] ; Toledo, Jr., Jose Carlos ^[1]

Affiliation

^[1] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, BR-14040901 Ribeirao Preto, SP - Brazil

• Document type: Journal article
• Source: Free Radical Biology and Medicine; v. 77, p. 270-280, DEC 2014.
• Web of Science Citations: 4

Abstract

In this study, we examined the mechanisms and kinetic profiles of intracellular nitrosative processes using diaminofluorescein (DAF-2) as a target in RAW 264.7 cells. The intracellular formation of the fluorescent, nitrosated product diaminofluorescein triazol (DAFT) from both endogenous and exogenous nitric oxide (NO center dot) was prevented by deoxygenation and by cell membrane-permeable superoxide (C:1) scavengers but not by extracellular bovine Cu,Zn-SOD. In addition, the DAFT formation rate decreased in the presence of cell membrane-permeable Mn porphyrins that are known to scavenge peroxynitrite (ONOO-) but was enhanced by HCO3-/CO2. Together, these results indicate that nitrosative processes in RAW 264.7 cells depend on endogenous intracellular O-2(center dot-) and are stimulated by ONOO-/CO2-derived radical oxidants. The N2O3 scavenger sodium azide (NaN3) only partially attenuated the DAFT formation rate and only with high NO center dot (> 120 nM), suggesting that DAFT formation occurs by nitrosation (azide-susceptible DAFT formation) and predominantly by oxidative nitrosylation (azide-resistant DAFT formation). Interestingly, the DAFT formation rate increased linearly with NO center dot concentrations of up to 120-140 nM but thereafter underwent a sharp transition and became insensitive to NO center dot. This behavior indicates the sudden exhaustion of an endogenous cell substrate that reacts rapidly with NO center dot and induces nitrosative processes, consistent with the involvement of intracellular O-2(center dot-). On the other hand, intracellular DAFT formation stimulated by a fixed flux of xanthine oxidase-derived extracellular O-2(center dot-) that also occurs by nitrosation and oxidative nitrosylation increased, peaked, and then decreased with increasing NO center dot, as previously observed. Thus, our findings complementarily show that intra- and extracellular O-2(center dot-)-dependent nitrosative processes occurring by the same chemical mechanisms do not necessarily depend on NO center dot concentration and exhibit different unusual kinetic profiles with NO center dot dynamics, depending on the biological compartment in which NO center dot and O-2(center dot-) interact. (C) 2014 Elsevier Inc. All rights reserved. (AU)

• FAPESP's process: 10/00550-2 - Mechanisms of cellular s-nitrosation from free nitric oxide. Involviment of transition metals and reactive species
• Grantee: José Carlos Toledo Junior
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 13/07937-8 - Redoxome - Redox Processes in Biomedicine
• Grantee: Ohara Augusto
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC