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5TH INTERNATIONAL CONFERENCE ON METALLOTHIONEIN SYMPOSIUM PAPERS

Cell-Surface Protein Disulfide Isomerase Is Required for Transnitrosation of Metallothionein by S-Nitroso-Albumin in Intact Rat Pulmonary Vascular Endothelial Cells

Li-Ming Zhang^*, Claudette St. Croix, Rong Cao, Karla Wasserloos, Simon C. Watkins, Troy Stevens, Song Li^||, Vladimir Tyurin, Valerian E. Kagan and Bruce R. Pitt^,1

^* Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261; Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15261; Department of Pharmacology, University of South Alabama School of Medicine, Mobile, Alabama 36688; and ^|| Center for Pharmacogenetics and Department of Pharmaceutical Science, University of Pittsburgh School of Pharmacy, Pittsburgh, Pennsylvania 15261

To whom requests for reprints should be addressed at ¹ Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, 100 Technology Drive, Cellomics Building, Pittsburgh, PA 15219. E-mail: brucep{at}pitt.edu

Abstract

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 U S A 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).

Key Words: metallothionein • fluorescence resonance energy transfer • S-nitroso-albumin • transnitrosation • cell surface protein disulfide isomerase • pulmonary endothelium