Use of a vibrating electrode to measure changes in calcium fluxes across the cell membranes of oxidatively challenged Aplysia nerve cells.

Abstract

A self-referencing and non-invasive Ca(2+)-sensitive vibrating electrode was used to assess the effects of hydrogen peroxide-induced oxidative challenges on the efflux and influx of calcium across the plasma membrane of single nerve cells cultured from abdominal ganglion of Aplysia californica. A reduced net efflux of Ca2+ from the cell soma occurred immediately after the addition of hydrogen peroxide (0.0025 mM, 0.005 mM or 0.01 mM) to the culture medium, indicating damage to the cell membrane or Ca2+ transport mechanism. There then followed a marked efflux, the extent and duration of which was related to the concentration of hydrogen peroxide used and which may reflect compensatory activity by the Ca2+ regulatory mechanisms in the plasmalemma. No morphological changes were observed in cells challenged with 0.0025 mM hydrogen peroxide and the enhanced rate of Ca2+ efflux rapidly decreased to pre-exposure values. Sustained and enhanced Ca2+ effluxes from those cells exposed to 0.005 mM or 0.01 mM hydrogen peroxide were also consistent with regulatory pumping of Ca2+ out of the cell although contraction and blebbing of neurites and swelling of the soma may indicate that a proportion of the efflux arose from release of Ca2+ from disrupted intracellular stores. The vibrating electrode is a useful additional technique for the study of the pathogenesis of neurological conditions, as ionic fluxes across single nerve cells exposed to physiologically-relevant concentrations of free radicals can be monitored non-invasively for prolonged periods.