Fig. 5.

EtdBr uptake by Cx43-EGFP cells in standard culture conditions is mediated by opening of hemichannels. (A) Phase contrast of a mixed culture of parental and Cx43-EGFP cells seeded 16 h before dye uptake assay. (B) Cx43-EGFP fluorescence of the field shown in A; six cells expressing different levels of EGFP are numbered 1–6. Cells 4–6 are in contact and show fluorescent gap junction plaques between them (arrows). The rounded-up cells on the left are dividing parental cells; flattened interphase parental cells are also present. (C) Cells were incubated in 10 μM EtdBr, and dye uptake was measured every 45 s as fluorescence emission of EtdBr binding to DNA [518 nm, arbitrary units (AU) of intensity]. Uptake was greatest in cell 3, which exhibited the most EGFP fluorescence, and less for the other cells. Uptake for parental cells (n = 15) was less than for any of the Cx43-EGFP cells. (D) Plot of EtdBr fluorescence (at 60 min) as a function of Cx43-EGFP fluorescence for 13 cells and for mean uptake by parental cells (n = 15). Uptake was linearly related to EGFP fluorescence (r = 0.79 for Cx43-EGFP cells). (E) Gap junction blockers La³⁺ (0.1 mM) and 18β-glycyrrhetinic acid (GA, 35 μM) reduced EtdBr uptake by Cx43-EGFP cells (P < 0.05 for parental vs. Cx43-EGFP, other pairings not significant by t test) but did not affect uptake by parental cells. (F) Dye uptake after a 75-min incubation with 10 μM EtdBr in mixed cultures of parental cells with EGFP-Cx43 cells (gray bars) or mixed cultures of parental with Cx43-EGFP cells (black bars). EtdBr uptake was low and not significantly different in EGFP-Cx43 and parental cells (P > 0.3, n = 20 for each cell type); EtdBr uptake was greater in Cx43-EGFP than in parental cells (P < 0.05, n = 20 for each cell type).