Fig. 4.

NO induces astrocyte dye uptake. (a) Confluent astrocyte cultures were photographed after incubation with 100 μM GSNO, an NO donor, for 50 min, and then exposed to 100 μM EtdBr for 5 min (Left), incubated with La³⁺ for the last 5 min of 50 min of GSNO and then exposed to EtdBr for 5 min (Middle), or incubated with DTT for the last 5 min of 50 min of GSNO and then EtdBr for 5 min (Right). La³⁺ and DTT largely prevented dye uptake, indicating rapid reduction of hemichannel permeability. n = 3. (b) Time-lapse measurement of dye uptake in 10 μM EtdBr. Dye uptake at a low basal rate was increased a few minutes after addition of GSNO. The rate of uptake was markedly reduced by DTT (10 mM) replacing GSNO at ≈48 min. Each point corresponds to mean fluorescence intensity of 21 cells in each of three independent experiments ± SE. (c) Western blot analysis of cell surface Cx43 pulled down with biotin from astrocytes under control conditions (Control), treated for 50 min with 100 μM GSNO, treated for 50 min with 100 μM GSNO, and with 10 mM DTT during the last 10 min or with 10 mM DTT for 10 min. Representative results of three experiments are shown.