Oxygen radicals alter the cell membrane potential in a renal cell line (LLC-PK1) with differentiated characteristics of proximal tubular cells.
We employed a carbocyanine dye (1,1',3,3,3',3'-hexamethylindocarbocyanine iodide) to measure the plasma membrane potential of LLC-PK1 renal epithelial cells exposed to either xanthine oxidase-generated oxygen radicals or to hydrogen peroxide. Measurements were performed using a fluorescent-activated cell sorter to record fluorescence on a cell by cell basis. Initial exposure of cells to low concentrations of either H2O2 or xanthine oxidase resulted in a transient increase in membrane potential relative to control cells (P less than 0.001), followed by an exponential decline in potential (P less than 0.001). The addition of extracellular catalase diminished the H2O2-related decline in potential, consistent with a role for hydrogen peroxide in producing this effect. Pretreatment of cells with inhibitors of intracellular catalase and superoxide dismutase prior to exposure to xanthine oxidase caused an even larger decline in potential (P less than 0.001). Cells could be partially protected from the radical-mediated loss of potential by incubating them in a hypertonic (400 mosmolal) environment during radical exposure. Similarly, the loss of membrane potential was increased after incubation of cells in a hypotonic (200 mosmolal) environment during radical exposure. These observations are consistent with a reduction in membrane potential effected by exposure to oxygen radicals (including superoxide anion and hydrogen peroxide). This reduction may be prevented, in part, by radical scavenging enzymes and by reducing the degree of cellular swelling in response to oxygen radical exposure.
D002232:Carbocyanines; D002374:Catalase / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D005456:Fluorescent Dyes; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006982:Hypertonic Solutions / Q000494:pharmacology; D007038:Hypotonic Solutions / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects*; D010100:Oxygen / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism
D002232:Carbocyanines; D005456:Fluorescent Dyes; D005609:Free Radicals; D006982:Hypertonic Solutions; D007038:Hypotonic Solutions; C051385:1,1',3,3,3',3'-hexamethylindocarbocyanine; D006861:Hydrogen Peroxide; D002374:Catalase; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D010100:Oxygen