Inhibition of growth factor-stimulated PKA activity by H-89 in PC12 cells
| Growth factor treatment | PKA activity | ||
|---|---|---|---|
| Control | H-89-pretreated cultures | ||
| PKA activated | PKA activated | Inhibition versus control | |
| % | |||
| None | 20.0 ± 2.4 | 2.8 ± 1.0 | 86.2 ± 0.5 |
| NGF | 31.0 ± 2.4 | 2.2 ± 0.9 | 91.5 ± 2.5 |
| EGF | 29.0 ± 3.6 | 3.0 ± 0.7 | 89.6 ± 1.5 |
| PACAP38 | 31.0 ± 2.5 | 3.8 ± 1.1 | 85.2 ± 1.4 |
| PACAP38 + EGF | 51.1 ± 7.4 | 2.6 ± 0.2 | 94.9 ± 0.5 |
Cultures of PC12 cells were grown to high density (>70% confluency) in 10-cm-diameter dishes. Before the addition of test agents, cells were switched to a reduced-serum media (1% fetal bovine serum, 1% horse serum) and maintained overnight in this media. Cultures were incubated for 15 min in the absence or presence of 20 μmH-89 followed by an additional 5-min incubation with no addition (control), NGF (50 ng/ml), EGF (10 ng/ml), PACAP38 (5 nm), or PACAP38 + EGF. PKA activity was quantified using a protein kinase assay system (Life Technologies) as described in Materials and Methods.
Results are expressed as the mean ± standard error for data obtained from two independent experiments, with each sample was performed in triplicate.