Nitric oxide donation and nitrite assays in the presence of thiols and albumin as determined by Griess' and Werringloer's methods.
ABSTRACT Nitric oxide (NO) or nitrite (NO2-) were assayed using the Werringloer's method or the Griess' method, respectively, in the presence or absence of various thiols, amino acids, or albumin. This has been done because both methods are used to determine the generation of endogenous NO from L-arginine or exogenous NO from drugs in vivo, paying little attention to biological constituents which may affect results of these assays. Albumin, reduced glutathione (GSH), cysteine and N-acetylcysteine, but not other amino acids lowered the amount of NO2- as detected by Griess' method no matter whether sodium nitrite or 3-morpholinosydnonimine (SIN-1) were used as a source of NO2-. This happened probably because at low pH of the reaction mixture the corresponding nitrosothiols were formed and thus NO2- was not accessible for detection. However, this phenomenon was not seen when instead of SIN-1 another NO donor--S-nitroso-N-acetylpenicillamine (SNAP) was used. SNAP is a nitrosothiol itself and physiological low molecular thiols (e.g. GSH or cysteine) displaced NO from SNAP. An increase in the amount of released NO was detectable by both Werringloer's and Griess' methods. Only the presence of 700 microns of albumin steadily suppressed the detection of NO or NO2- no matter what was the source of these species. It is concluded that low molecular thiols and albumin may differently influence the detection of both NO and NO2- which derive from various NO donors or sodium nitrite.
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ABSTRACT: Nitric oxide (NO) produced in inflammatory lesions may play a major role in the destruction of oligodendrocytes in multiple sclerosis and experimental allergic encephalomyelitis. The transformed murine oligodendroglial line N20.1 is much more resistant than primary oligodendrocytes to killing by the NO generator S-nitroso-N-acetyl-DL-penicillamine (SNAP). This observation prompted investigation of the mechanisms leading to cell death in the N20.1 cells and comparison of SNAP with another NO donor, sodium nitroprusside (SNP). We observed that N20.1 cells were 30 times more sensitive to SNP than to SNAP. The specific NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) protected against SNP only, not against SNAP. However, dithiothreitol protected against both SNAP and SNP, indicating that S-nitrosylation of cysteines plays a major role in the cytotoxicity of both NO donors. We did not observe any formation of peroxynitrite or increase of Ca2+ concentration with either SNAP or SNP, thus excluding their involvement in the mechanisms leading to N20.1 cell death. Based on two observations, (a) potentiation of the cytotoxic effect of SNP when coincubated with ferricyanide or ferrocyanide, but not sodium cyanide, and (b) protection by deferoxamine, an iron cyanide chelator, we conclude that the greater sensitivity of N20.1 cells to SNP compared with SNAP is due to synergism between NO released and the iron cyanide portion of SNP, with the cyanide accounting for very little of the cytotoxicity. Finally, SNP but not SNAP induces some apoptosis, as shown by DNA laddering and protection by a caspase-3 inhibitor. These results suggest that low levels of NO in combination with increased iron content lead to apoptotic cell death rather than the necrotic cell death seen with higher levels of NO generated by SNAP.Journal of Neurochemistry 04/1999; 72(3):1050-60. · 3.97 Impact Factor
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ABSTRACT: Non-receptor protein tyrosine kinases (NRPTKs)-dependent inflammatory signal transduction cascades play key roles in immuneregulation. However, drug intervention through NRPTKs-involved immunoregulation mechanism in microglia (the major immune cells of the central nervous system) has not been widely investigated. A main aim of the present study is to elucidate the contribution of two major NRPTKs (Syk and Jak2) in neuroinflammation suppression by a bioactive sesquiterpene dimmer (DSF-27). We found that LPS-stimulated BV-2 cells activated Syk and further initiated Akt/NF-κB inflammatory pathway. This Syk-dependent Akt/NF-κB inflammatory pathway can be effectively ameliorated by DSF-27. Moreover, Jak2 was activated by LPS, which was followed by transcriptional factor Stat3 activation. The Jak2/Stat3 signal was suppressed by DSF-27 through inhibition of Jak2 and Stat3 phosphorylation, promotion of Jak/Stat3 inhibitory factors PIAS3 expression, and down-regulation of ERK and p38 MAPK phosphorylation. Furthermore, DSF-27 protected cortical and mesencephalic dopaminergic neurons against neuroinflammatory injury. Taken together, our findings indicate NRPTK signaling pathways including Syk/NF-κB and Jak2/Stat3 cascades are potential anti-neuroinflammatory targets in microglia, and may also set the basis for the use of sesquiterpene dimmer as a therapeutic approach for neuroinflammation via interruption of these pathways.Toxicology and Applied Pharmacology 01/2014; · 3.98 Impact Factor