Abstract:
The effects of non-lethal oxidative stress on cytosolic calcium regulation in hepatocytes are inadequately understood. It was hypothesized that non-lethal oxidative stress and calcium mobilizing agonists are co-toxic and that non-lethal oxidative injury would result in impaired responsiveness to receptor mediated-calcium mobilization. 'Methods'. Oxidative injury was induced in a dose-dependent manner in cultured hepatocytes using the prooxidant chemical, menadione. A lethal (100[mu]M) and a non-lethal (10[mu]M) dose of menadione were defined. Co-toxicity of the calcium-mobilizing purinergic receptor agonist, ATP, and the effects of menadione on ATP-related calcium signaling were determined by incubating hepatocytes for 2 hours with menadione and then exposing hepatocytes to normally non-toxic concentrations of ATP. A propidium iodide exclusion assay was used to assess for cell necrosis. Nuclear stains with Hoechst 33258 and a fluorescein-based TUNEL assay were used to assess apoptosis. Cytosolic free calcium was evaluated using Fura-2-AM. (Abstract shortened by UMI.)
