Role of glutaredoxin-mediated protein S-glutathionylation in cellular nitroglycerin tolerance.
We hypothesize that nitroglycerin (NTG) causes direct oxidation of multiple cellular sulfhydryl (SH) proteins and that manipulation of SH redox status affects NTG tolerance. In LLC-PK1 cells, we found that nitrate tolerance, as indicated by cGMP accumulation toward NTG, was accompanied by increased protein [(35)S]cysteine incorporation, significant S-glutathionylation of multiple proteins, and decreased metabolic activity of several SH-sensitive enzymes, including creatine kinase, xanthine oxidoreductase, and glutaredoxin (GRX). Cells overexpressing GRX exhibited reduced cellular protein S-glutathionylation (PSSG) and absence of NTG tolerance, whereas those with silenced GRX showed increased extent of NTG-induced tolerance. Incubation of LLC-PK1 cells with oxidized glutathione led to several major observations associated with nitrate tolerance, namely, reduced cGMP accumulation, PSSG formation, superoxide accumulation, and the attenuation of these events by vitamin C. Aortic S-glutathionylated proteins increased approximately 3-fold in rats made tolerant in vivo to NTG and showed significant negative correlation with vascular responsiveness ex vivo. NTG incubation in EA.hy926 endothelial cells and LLC-PK1 cells led to increased S-glutathionylation and activity of p21(ras), a known mediator of cellular signaling. These results indicate that the hallmark events of NTG tolerance, such as reduced bioactivation and redox signaling, are associated with GRX-dependent protein deglutathionylation.
Tsou
Pei-Suen
PS
Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, NY 14260-1200, USA.
Addanki
Vamsi
V
Haas
Jessica A
JA
Page
Nathaniel A
NA
Fung
Ho-Leung
HL
D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western; D003402:Creatine Kinase / Q000378:metabolism; D006152:Cyclic GMP / Q000096:biosynthesis; D004361:Drug Tolerance; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D054477:Glutaredoxins / Q000235:genetics / Q000502:physiology*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D018374:LLC-PK1 Cells; D005996:Nitroglycerin / Q000494:pharmacology*; D010084:Oxidation-Reduction; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013552:Swine; D014162:Transfection; D014664:Vasodilation / Q000187:drug effects; D014665:Vasodilator Agents / Q000494:pharmacology*
D054477:Glutaredoxins; D013438:Sulfhydryl Compounds; D014665:Vasodilator Agents; D003402:Creatine Kinase; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D005996:Nitroglycerin; D005978:Glutathione; D006152:Cyclic GMP