Fig. 5.

Treatment with a PKA inhibitor suggests dependence on PKA for the increase in S-phase induced by brief treatment with forskolin. A, B, Timelines are shown. A, Pieces of epithelium were incubated for 15 min with 1 μmforskolin (Forsk) and then maintained in a standard medium for the remainder of the 72 hr culture period. Other pieces of utricle were preincubated for 60 min with 0.5, 1, or 5 μmKT5720 in a medium containing 2.5% FBS but no BrdU. The medium was then changed for a standard medium containing the inhibitor and 1 μm forskolin for 15 min. The forskolin was removed, and the pieces of epithelium were maintained in a standard medium containing KT5720 for 72 hr. B, Pieces of epithelium were treated for 1 hr with 0.5 mm Br-cAMP in a standard medium. The Br-cAMP-containing medium was then replaced with a standard medium for the remainder of the 72 hr culture period. Other pieces of epithelium were preincubated for 60 min with 5 μm KT5720 in a medium containing 2.5% FBS but no BrdU. The medium was then changed for a standard medium containing the inhibitor and 0.5 mm Br-cAMP for 1 hr. The Br-cAMP was removed, and the pieces of epithelium were maintained in a standard medium containing 5 μm KT5720 for 72 hr. C, Exposure to KT5720 induced a dose-dependent inhibition, reducing the forskolin-induced effect by 52% at 1 μm KT5720 and by 92.4% at 5 μm KT5720. Treatment with 5 μm KT5720 reduced the short-term Br-cAMP effect by 85.2% *, Significant difference when compared with Forsk 15 min(p < 0.05). **, Significant difference when compared with Br-cAMP 1 hr (p< 0.001). F, Fixed; S, start.