Free radical-mediated membrane depolarization in renal and cardiac cells

Cell membrane potential was measured with a flow cytometer by quantitating the intracellular accumulation of a fluorescent cationic carbocyanine dye. We used this system to demonstrate depolarization upon the addition of hydrogen peroxide (10-1,000 microM) and ferrous chloride (25-100 microM) to cultures of either neonatal rat myocardial or LLC-PK1 renal epithelial cells. Ferrous chloride-induced depolarization was prevented by superoxide dismutase, catalase and dimethyl sulfoxide, suggesting roles for the superoxide anion, hydrogen peroxide and the hydroxyl radical in effecting this depolarization, possibly through a Fenton-type reaction mechanism. Supplementation of either cell type with 2 microM tocopherol acid succinate during growth in tissue culture, prior to exposure to the oxidizing agent, decreased the magnitude of the depolarization in both cell types. The results are consistent with a role for tocopherols in scavenging free radical species responsible for the depolarization of the cell membrane.