ascorbic-acid and 2-oxothiazolidine-4-carboxylic-acid

Vitamin C and thiol agents improve vasomotor function. To determine whether these compounds directly affect endothelial function, nitric oxide (NO) synthesis was measured in human aortic endothelial cells treated with ascorbic acid or the thiol modulating agents lipoic acid or L-2-oxothiazolidine-4-carboxylic acid (OTC). A dose-dependent increase in A23187-stimulated NO synthesis and elevated cGMP levels were observed in all cases except for OTC. Cellular GSH levels were not significantly increased, and the GSH/GSSG ratio was not significantly affected by treatment of the cells with lipoic acid, OTC, or ascorbic acid. Thus, vitamin C and lipoic acid potentiate endothelial NO synthesis and bioactivity by mechanisms that appear to be independent of cellular GSH levels and redox environment.

Topics: Antioxidants; Aorta; Ascorbic Acid; Endothelium, Vascular; Glutathione; Humans; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Pyrrolidonecarboxylic Acid; Thiazoles; Thiazolidines; Thioctic Acid

Cellular glutathione is released during apoptosis and may play a role in the regulation of the mitochondrial permeability transition pore. The question of whether only cytosolic glutathione is important in apoptosis, or whether mitochondrial glutathione also plays a role, was investigated using gamma-glutamyltranspeptidase-deficient knockout mice. Thymocytes from these mice were found to have both glutathione pools diminished and they were more susceptible to dexamethasone (DEX)-induced apoptosis. Supplementation with N-acetylcysteine (NAC) and L-2-oxothiazolidine-4-carboxylic acid replenished both glutathione pools and provided protection from apoptosis. Ascorbate supplementation was beneficial to the mitochondrial glutathione pool, but apoptosis was not prevented. NAC supplementation caused an increase in reactive oxygen species formation and cardiolipin oxidation, but had no adverse affect on the amount of apoptotic cells. Our results suggest that the glutathione status is an important factor in apoptosis and indirect evidence indicates that the cytosolic pool of glutathione may be important in DEX-induced apoptosis, with mitochondrial events being secondary, and may reflect the execution phase.

Topics: Acetylcysteine; Animals; Apoptosis; Ascorbic Acid; Dexamethasone; gamma-Glutamyltransferase; Glutathione; Mice; Mice, Knockout; Pyrrolidonecarboxylic Acid; Thiazoles; Thiazolidines; Thymus Gland

Antioxidants are often added to culture media as cytoprotective agents. We examined the effects of antioxidants on the results and interpretation of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay for cell viability. Without cells, the thiol-containing antioxidant compounds beta-mercaptoethanol, dithiothreitol, pyrrolidine-dithiocarbamate, and N-acetyl-L-cysteine (acetylcysteine) reduced MTT tetrazolium salts to a blue formazan product in a dose-dependent manner. Addition of the compounds L-ascorbic acid and (+)-alpha-tocopherol acid succinate had different effects. In contrast, addition of the antioxidants N-acetyl-5-methoxytryptamine and (-)-2-oxo-4-thiazolidine carboxylic acid, which do not contain reactive thiol groups, did not result in the development of blue formazan product. These results showed that antioxidants, and potentially other chemotherapeutic compounds that contain free thiol groups or other reducing equivalents, readily reduce MTT to produce the blue formazan, irrespective of the viability of the cells present. This undescribed reaction can, therefore, significantly influence the results and interpretation of cell-viability experiments.

Topics: Acetylcysteine; Antioxidants; Ascorbic Acid; Colorimetry; Coloring Agents; Culture Media; Dithiothreitol; Kinetics; Melatonin; Mercaptoethanol; Oxidation-Reduction; Pyrrolidines; Pyrrolidonecarboxylic Acid; Reagent Kits, Diagnostic; Sulfhydryl Compounds; Tetrazolium Salts; Thiazoles; Thiazolidines; Thiocarbamates; Vitamin E