Redox biological reactions are now accepted to bear the Janus faceted feature of promoting both physiological signaling responses and pathophysiological cues. Endogenous antioxidant molecules participate in both scenarios. This review focuses on the role of crucial cellular nucleophiles, such as glutathione, and their capacity to interact with oxidants and to establish networks with other critical enzymes such as peroxiredoxins. We discuss the importance of the Nrf2-Keap1 pathway as an example of a transcriptional antioxidant response and we summarize transcriptional routes related to redox activation. As examples of pathophysiological cellular and tissular settings where antioxidant responses are major players we highlight endoplasmic reticulum stress and ischemia reperfusion. Topologically confined redox-mediated post-translational modifications of thiols are considered important molecular mechanisms mediating many antioxidant responses, whereas redox-sensitive microRNAs have emerged as key players in the posttranscriptional regulation of redox-mediated gene expression. Understanding such mechanisms may provide the basis for antioxidant-based therapeutic interventions in redox-related diseases.

Nitric oxide (NO) is a ubiquitous intercellular messenger molecule synthesized from the amino acid L-arginine by NO synthases in diverse cells and tissues. NO is synthesized in vascular endothelial cells and appears to play an important role in the control of blood pressure and platelet aggregation. A detailed understanding of the regulation of NO synthesis by endothelial cells has been hampered by the lack of molecular clones for endothelial NO synthase; the isolation and characterization of such clones is reported herein. The constitutive NO synthases present in endothelial cells and in brain share common biochemical and pharmacologic features. We purified NO synthase from bovine brain and determined the amino acid sequence of several tryptic peptides. The sequence of the bovine brain peptides is nearly identical to the deduced amino acid sequence previously determined for the rat brain NO synthase. These sequence data were utilized to design PCR-generated NO synthase cDNA probes, which were used to isolate clones encoding NO synthase from a bovine aortic endothelial cell (BAEC) cDNA library. A full-length NO synthase cDNA clone was isolated, representing a protein of 1205 amino acids with a molecular mass of 133 kDa; transfection of this clone in a heterologous expression system demonstrated the expected enzymatic activity. The deduced amino acid sequence of the BAEC NO synthase cDNA differs at numerous residues from the sequence determined for the purified bovine brain protein and shows 50-60% sequence identity with recently isolated molecular clones for murine macrophage and rat brain NO synthase isoforms. Bovine genomic Southern blots probed with bovine brain and BAEC NO synthase cDNA probes identify distinct bands, indicating that these cDNAs are the products of different genes. Prolonged treatment of BAECs with the cytokine tumor necrosis factor alpha, which we have previously shown to result in a marked increase in NO synthase activity, is associated with a decrease in the abundance of the 4.8-kilobase BAEC NO synthase transcript. The increase in BAEC NO synthase activity induced by tumor necrosis factor alpha is thus likely to involve posttranscriptional mechanisms or the induction of a distinct endothelial NO synthase isoform.

Protein S‐glutathiolation, the reversible covalent addition of glutathione to cysteine residues on target proteins, is emerging as a candidate mechanism by which both changes in the intracellular redox state and the generation of reactive oxygen and nitrogen species may be transduced into a functional response. This review will provide an introduction to the concepts of oxidative and nitrosative stress and outline the molecular mechanisms of protein regulation by oxidative and nitrosative thiol‐group modifications. Special attention will be paid to recently published work supporting a role for S‐glutathiolation in stress signalling pathways and in the adaptive cellular response to oxidative and nitrosative stress. Finally, novel insights into the molecular mechanisms of S‐glutathiolation as well as methodological problems related to the interpretation of the biological relevance of this post‐translational protein modification will be discussed.

Mitochondrial production of oxidants contributes to a variety of pathological conditions including the vascular complications of diabetes, neurodegenerative diseases, and cellular senescence. We postulated that a transcriptional coactivator, peroxisome proliferator activated receptor-gamma coactivator 1alpha (PGC-1alpha), a major regulator of oxidative metabolism and mitochondrial biogenesis, could be involved in the transcriptional regulation of the mitochondrial antioxidant defense system in vascular endothelial cells. We show that PGC-1alpha is present in human, bovine, and mouse endothelial cells and positively modulates the expression of the mitochondrial detoxification system. Endothelial cells that overexpress PGC-1alpha show reduced accumulation of reactive oxygen species (ROS), increased mitochondrial membrane potential, and reduced apoptotic cell death both in basal and oxidative stress conditions. Downregulation of PGC-1alpha levels by siRNA reduces the expression of mitochondrial detoxification proteins. These results unveil a novel regulatory pathway that links mitochondrial activity and mitochondrial oxidative stress protective systems. In addition, they suggest that PGC-1alpha could play a crucial protective role in vascular complications of diabetes, where the mitochondrial metabolism of glucose has been shown to result in oxidative stress and vascular endothelial cell dysfunction.