Role of Glutaredoxin-Mediated Protein S-Glutathionylation in Cellular Nitroglycerin Tolerance

Abstract

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.

Broad regulation of matrix and adhesion molecules in THP-1 human macrophages by nitroglycerin

“…We have shown recently that NTG can cause significant cellular protein thiol oxidation, including S-glutathionylation [23]. Because of this pro-oxidant property, we hypothesize that NTG may alter the expression of MMPs and other ECM proteases and adhesion molecules.…”

Anti-Oxidative Stress and Beyond: Multiple Functions of the Protein Glutathionylation

“…Deglutathionylation may be realized through direct thiol/disulfide exchange reactions with GSH [45]. However, in most cases, this reversible process is dependent on thiol-disulfide oxidoreductases, mainly including glutaredoxin, or glutaredoxin reductase [46,47], thioredoxin, or thioredoxin reductase (Trx/ TrxR system) [48,49], and sulfiredoxin [44,50]. Glutaredoxin, a family of small molecular weight proteins [51], is the first and the most extensively studied oxidoreductase that catalyzes the glutathione-mixed disulfide [52,53].…”

A fluorometric method to quantify protein glutathionylation using glutathione derivatization with 2,3-naphthalenedicarboxaldehyde

“…The quantification of glutathionylation in tissues has also been attempted using a variety of techniques. The same antibodies used to detect glutathionylated proteins on Western blots have also been used in an enzymelinked immunosorbent assay format to measure the level of glutathionylation [13]. Other methods have eluted the disulfidebound glutathione for subsequent measurement [17,18].…”

“…Because of the increasing recognition of the biological and clinical importance of glutathionylation, a range of techniques has been developed to identify and locate glutathionylated proteins and to determine the level of glutathionylation [7][8][9][10][11][12][13][14][15][16][17]. Antibodies that recognize glutathione specifically bound to protein have been used to identify glutathionylated proteins after Western blotting of cell extracts and in tissue sections [9,15,16].…”