Figure 2.

Progressive EtdBr uptake is induced by metabolic inhibition, but not by membrane depolarization or low extracellular calcium. Uptake is sensitive to a gap junction blocker but not to a blocker of P2X ATP receptors. Astrocyte monolayers in 96-well plates were treated with metabolic inhibitors for different periods of time with EtdBr applied for the last 15 min before fluorescence was measured. (A–D) A progressive increase in fluorescence intensity and number of labeled cells was observed from time 0 to 15, 60 and 90 min of metabolic inhibition. (E) Time course of EtdBr uptake, measured as fluorescence, by astrocytes during metabolic inhibition. Each point represents the average value for five wells in each of three independent experiments corrected for initial control values and normalized to the maximal value measured at 120 min and plotted ±SD. (F) Uptake of EtdBr measured after 75 min metabolic inhibition was markedly reduced by 5 min prior treatment with 35 μM 18α-glycyrrhetinic acid (AGA), but not with 300 μM oxidized ATP (o-ATP). Uptake of EtdBr in astrocytes bathed for 2 h with a solution containing 55 mM K⁺, zero extracellular Ca²⁺ plus 5 mM EGTA (0 mM Ca²⁺), or both high K⁺ and zero Ca²⁺ was similar to that observed in astrocytes maintained under control conditions. Each point represents the normalized value obtained in three experiments ±SD.