期刊
EXPERIMENTAL BIOLOGY AND MEDICINE
卷 231, 期 9, 页码 1507-1515出版社
SAGE PUBLICATIONS LTD
DOI: 10.1177/153537020623100909
关键词
metallothionein; fluorescence resonance energy transfer; S-nitroso-albumin; transnitrosation; cell surface protein disulfide isomerase; pulmonary endothelium
资金
- NHLBI NIH HHS [R37 HL65697, K08 HL79456, HL70807] Funding Source: Medline
- NIGMS NIH HHS [GM53789] Funding Source: Medline
S-nitrosation of the metal binding protein, metallothionein (MT) appears to be a critical link in affecting endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)derived nitric oxide (NO)-induced changes in cytoplasmic and nuclear labile zinc, respectively. Although low molecular weight S-nitrosothiols also appear to affect this signaling system, less is known about the ability of extracellular protein nitrosothiols to transnitrosate MT. Accordingly, we synthesized fluorescently labeled S-nitroso-albumin (SNO-albumin, a major protein S-nitrosothiol in plasma) and determined, via confocal microscopy in fixed tissue, that it is transported into cultured rat pulmonary vascular endothelial cells in a temperature sensitive fashion. The cells were transfected with an expression vector that encodes human MT-IIa cDNA sandwiched between enhanced cyan (donor) and yellow (acceptor) fluorescent proteins (FRET-MT) that can detect conformational changes in MT through fluorescence resonance energy transfer (FRET). SNO-albumin and the membrane-permeant low molecular weight S-nitroso-L-cysteine ethyl ester (L-SNCEE) caused a conformational change in FRET-MT as ascertained by full spectral laser scanning confocal microscopy in live rat pulmonary vascular endothelial cells, a result which is consistent with transnitrosation of the reporter molecule. Transnitrosation of FRET-MT by SNO-albumin, but not L-SNCEE, was sensitive to antisense oligonucleotide-mediated inhibition of the expression of cell surface protein disulfide isomerase (csPDI). These results extend the original observations of Ramachandran et aL (Ramachandran N, Root P, Jiang XM, Hogg PJ, Mutus B. Proc Natl Acad Sci USA 98: 9539-9544, 2001) and suggest that csPDI-mediated denitrosation helps to regulate the ability of the major plasma NO carrier (SNO-albumin) to transnitrosate endothelial cell molecular targets (e.g. MT).
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