Figure 4

Increase in cAMP enhances connexin gene and protein expression. Cultures of the mouse TEC line were treated with either 8-Br-cAMP (1 mM) or forskolin (10 μM) for 6 hrs at 37°C. Cells were then fixed, permeabilized and labeled with the anti-Cx43 polyclonal antibody, ultimately revealed with the Alexa 488-conjugated secondary antibody. The fluorescence microscopy images (A, C, E) and the corresponding phase contrast images are depicted (B, D, F). The mouse TEC treated with 8-Br-cAMP (C, D) and forskolin (E, F) presented an increased punctate labeling of Cx43, mainly at cell-to-cell contact regions, when compared with the untreated controls (A, B). Inserts in A and B show the fluorescence microscopy image and respective phase contrast image of TEC subjected to isotype control primary antibody and Alexa-488-coupled secondary antibody (Magnification, ×400). Panel G show by northern blot analysis that 8-Br-cAMP also enhances Cx43 gene transcription. Mouse TEC were treated or not (Ct) with 8-Br-cAMP (1 mM) and cultured for 1, 6 or 24 hrs at 37°C. The total RNA was then extracted, and 10 - 20 μg of total RNA was loaded in 1.2% agarose gel and plotted onto nylon membrane. For detection of Cx43 mRNA, the membrane was hybridized with a ³²P-labeled cDNA probe for Cx43. Alternatively, the membrane was also hybridized with a ³²P-labeled cDNA for GAPDH. The amount of Cx43 mRNA in IT-76M1 cells was increased after 1, 6, and 24 hours of treatment with 8-Br-cAMP.