Morphologic and functional correlates of plasma membrane injury during oxidant exposure

Plasma membrane injury by exposure to hydrogen peroxide was examined in a renal epithelial cell line (LLC-PK1). Morphologic and functional parameters of plasma membrane integrity were studied in an attempt to eludicate the sequence of membrane alterations during the evolution of hydrogen peroxide-mediated injury. These parameters included plasma membrane potential and permeability, plasma membrane bleb formation, cellular size, and plating efficiency. Plasma membrane potential was the earliest parameter affected by hydrogen peroxide exposure. Half maximal depolarization occurred within 15-30 min of exposure to 1 mM, after 10-15 min exposure to 100 mM and after over 150 min exposure to 10 microM hydrogen peroxide. After exposure to 1 mM hydrogen peroxide, the following sequence of events was seen; increased plasma membrane blebbing (30 min), cell swelling (90-125 min) and increased plasma membrane permeability (150-240 min). After a 30 min exposure to 1 mM hydrogen peroxide, cellular plating efficiency, measured at 24 h, was reduced by 50% (P less than .001). These changes were accelerated, although their order of appearance was unchanged, at higher concentrations of hydrogen peroxide. We conclude that functional and morphologic expressions of cellular injury in this model occur in a defined sequence with plasma membrane depolarization representing the earliest marker of membrane injury during hydrogen peroxide exposure.