Nitric oxide physiological responses and delivery mechanisms probed by water-soluble Roussin's red ester and {Fe(NO)2}10 DNIC.
Dinitrosyl-iron complexes (DNICs) are stable carriers for nitric oxide (NO), an important biological signaling molecule and regulator. However, the insolubility of synthetic DNICs, such as Roussin's red ester (RRE), in water has impaired efforts to unravel their biological functions. Here, we report a water-soluble and structurally well-characterized RRE [Fe(mu-SC2H4COOH)(NO)2]2 (DNIC-1) and a {Fe(NO)2}(10) DNIC [(PPh2(Ph-3-SO3Na))2Fe(NO)2] (DNIC-2), their NO-induced protein regulation, and their cellular uptake mechanism using immortalized vascular endothelial cells as a model. Compared with the most common NO donor, S-nitroso-N-acetyl-penicillamine (SNAP), the in vitro NO release assay showed that both DNICs acted as much slower yet higher stoichiometric NO-release agents with low cytotoxicity (IC50 > 1 mM). Furthermore, L-cysteine facilitated NO release from SNAP and DNIC-1, but not DNIC-2, in a dose- and time-dependent manner. EPR spectroscopic analysis showed, for the first time, that intact DNIC-1 can either diffuse or be transported into cells independently and can transform to either paramagnetic protein bound DNIC in the presence of serum or [DNIC-(Cys)2] with excess L-cysteine under serum-free conditions. Both DNICs subsequently induced NO-dependent upregulation of cellular heat shock protein 70 and in vivo protein S-nitrosylation. We conclude that both novel water-soluble DNICs have potential to release physiologically relevant quantities of NO and can be a good model for deciphering how iron-sulfur-nitrosyl compounds permeate into the cell membrane and for elucidating their physiological significance.
Chen
Yi-Ju
YJ
Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan.
Ku
Wei-Chi
WC
Feng
Li-Ting
LT
Tsai
Ming-Li
ML
Hsieh
Chung-Hung
CH
Hsu
Wen-Hwei
WH
Liaw
Wen-Feng
WF
Hung
Chen-Hsiung
CH
Chen
Yu-Ju
YJ
D002460:Cell Line; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004337:Drug Carriers / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D016503:Drug Delivery Systems / Q000379:methods*; D004353:Drug Evaluation, Preclinical; D042783:Endothelial Cells / Q000187:drug effects / Q000473:pathology; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000737:chemistry* / Q000494:pharmacology; D008954:Models, Biological; D008958:Models, Molecular; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D009589:Nitrogen Oxides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D009603:Nitroso Compounds / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility; D013997:Time Factors; D015854:Up-Regulation / Q000187:drug effects; D014867:Water / Q000737:chemistry
D004337:Drug Carriers; D018840:HSP70 Heat-Shock Proteins; D009589:Nitrogen Oxides; D009603:Nitroso Compounds; D014867:Water; C028357:Roussin red methyl ester; D009569:Nitric Oxide; C073661:dinitrosyl iron complex; D007501:Iron