Figure 6.
PKA activators increase GJ coupling and HC activities in Cx46 or Cx46 and Cx50 co-expressed cells, and this increase is alleviated by a PKA inhibitor
(A–D) CEF cells were infected with high-titer RCAS(A) retroviral vehicle (V) or recombinant RCAS(A) retroviruses containing Cx46 or co-infected with RCAS(A) containing Cx46 and Cx50, and cells were grown to confluence to maximize cell-cell contact. (A and B) Cells were treated with or without PKA activator: 8-Br-cAMP (1 mM) or forskolin (10 μM) for 2 h before scrape loading dye transfer assay using LY (green) as a tracer for GJs coupling and RD (red) as a tracer for originally dye-loaded cells. The extent of dye transfer was measured as the ratio of LY-labeled cells to that of RD-labeled cells. The data are presented as the mean ± SEM. (n = 3). ∗∗∗∗, p < 0.0001. (two-way ANOVA). (C and D) Cells were treated or not with 8-BrcAMP (1 mM) or forskolin (10 μM), with or without PKI (0.4 μM) for 2 h before scrape loading dye transfer assay. Scale bar: 50 μm. The extent of dye transfer was measured as the ratio of LY-stained cells to that of RD-labeled cells. The data are presented as the mean ± SEM. (n = 3). ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001 (two-way ANOVA).
(E and F) CEF cells infected with high-titer retrovirus RCAS(A) vehicle (V) or recombinant RCAS(A) containing Cx46 or co-infected with RCAS(A) containing Cx46 and Cx50 were cultured at low cell density with no physical contact. Cells were treated or not with 8-BrcAMP (1 mM) or forskolin (10 μM) with or without PKI (0.4 μM) for 2 h before FFSS at 1 dyne/cm2 for 30 min and followed by dye uptake assay with LY and RD. At least three microphotographs of fluorescence fields were captured. Scale bar: 50 μm. The LY uptake percentage was quantified by subtracting LY/RD double-positive cells from LY-positive cells. The data are presented as the mean ± SEM. (n = 3). ∗∗∗∗, p < 0.0001 (two-way ANOVA).