FIGURE 4:

A₁AR-stimulated exocytosis is dependent on ADAM17. (A) Uroepithelial lysates obtained from rat tissues transduced in situ with scrambled shRNA or ADAM17 shRNA were resolved by SDS–PAGE and Western blots probed with rabbit-anti ADAM17 antibody (top) or mouse anti–β-actin (bottom). (B) Quantification of ADAM17 expression in rat tissue treated with scrambled shRNA or ADAM17 shRNA. Data are mean ± SEM (n ≥ 4). The means of the two treatment groups are significantly different (p < 0.05). (C) Immunolocalization of ADAM17 expression (red) in rat tissue treated with scrambled or ADAM17-specific shRNAs. Nuclei (blue) are labeled with TOPRO-3. Scale bar, 13 μm. The cell junctions are marked with arrows and the umbrella cells with an asterisk. (D) CCPA (500 nM) was added to rat tissue treated with scrambled or ADAM17 shRNA and total EGFR or receptor phosphorylated at Y¹¹⁷³ was detected by Western blot. (E) Quantification of effects of scrambled or ADAM17-specific shRNA treatment on EGFR activation. Data are mean ± SEM (n = 4). The two treatment groups were significantly different (p < 0.05). (F) WGA-FITC (50 μg/ml) was added to the mucosal hemichamber of untreated rat tissue (control) or that treated with 500 nM CCPA. After 120 min, the tissue was incubated at 4°C with N-acetylglucosamine to remove surface lectin, fixed, and then processed for immunofluorescence. In these whole-mounted tissues, internalized WGA-FITC is shown in green, phalloidin-labeled actin in red, and TOPRO-3-labeled nuclei in blue. Bar, 15 μm. Quantification of the fluorescence intensity of WGA-FITC below the apical membrane. Data are mean ± SEM (n ≥ 8). The two treatment groups were significantly different (p < 0.05). (G) Rat tissues were transduced with hGH alone (control) or transduced with hGH and either scrambled or ADAM17 shRNAs. The excised bladder tissue was mounted in an Ussing stretch chamber and left untreated (control), treated with CCPA (500 nM), or stretched by filling the mucosal hemichamber. The mucosal fluid was collected and concentrated, the tissues were lysed, and hGH was detected in the secreted fraction (1/20 of total) or tissue lysates (1/13 of total) using Western blot. (H) Quantification of effects of ADAM17-specific shRNAs on hGH secretion. Data are mean ± SEM (n ≥ 3). Statistically significant effects (p < 0.05) are indicated with an asterisk.