Non-protein thiols flux to S-nitrosothiols in endothelial cells: an LPS redox signal.
Lipopolysaccharide (LPS) injures blood vessels by activating pathways in the endothelium that lead either to cell survival and proliferation or apoptosis. It has been suggested that these outcomes are determined when reactive oxygen and nitrogen intermediates oxidize low molecular weight non-protein thiols (NPSHs) such as glutathione (GSH) and cysteine (Cys), which serve as major intracellular reducing agents. The oxidoreduction of NPSHs could be an important redox signal if it were shown to occur rapidly following injury. Towards that end, cultured bovine aortic endothelial cells were stained with the thiol fluorescent probe, monobromobimane (MBB). Most of the acid extractable MBB-reactive adducts are GSH (approximately 90%) and Cys (approximately 90%). Within 1 min of LPS exposure, 50-70% of the MBB-reactive NPSHs are consumed without evidence for concomitant net generation of superoxide, hydrogen peroxide, singlet oxygen, or glutathione disulfide (GSSG). Although LPS induces an increased rate of thiol-disulfide exchange, the slight increase does not explain the magnitude of NPSH consumption. Within the first 10 min of recovery from LPS exposure, the MBB-reactive NPSH fluorescence returns at or slightly above baseline values. When HgCl2 was added to the acid extract, one mole of S-nitrosothiol oxidizing equivalent was found for every mole of MBB-reactive NPSH consumed. It is suspected that the rapid flux of MBB-reactive NPSHs and Hg2+-inducible oxidants reflects transition of GSH to GSNO (S-nitrosoglutathione) and could be an important redox signal in endothelial cells exposed to LPS.
Rubin
D B
DB
Department of Medicine, Rush University Medical College, Chicago, Illinois 60612, USA.
Reznik
G
G
Weiss
E A
EA
Young
P R
PR
D000818:Animals; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D017669:Mercury Compounds / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D009603:Nitroso Compounds / Q000378:metabolism*; D009952:Ornithine / Q000031:analogs & derivatives* / Q000494:pharmacology; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D017382:Reactive Oxygen Species; D026422:S-Nitrosoglutathione; D026082:Singlet Oxygen; D013481:Superoxides / Q000378:metabolism
D001643:Bridged Bicyclo Compounds; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D017669:Mercury Compounds; D009603:Nitroso Compounds; D016877:Oxidants; D017382:Reactive Oxygen Species; D013481:Superoxides; D026082:Singlet Oxygen; D009569:Nitric Oxide; C065027:N(G)-iminoethylornithine; D026422:S-Nitrosoglutathione; D009952:Ornithine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D005978:Glutathione; C015728:calomel; D003545:Cysteine; D010100:Oxygen; C028381:monobromobimane