Effects of MAPK pathway chemical inhibitors on nicotine-stimulated catecholamine secretion or chromogranin A transcription in pheochromocytoma cells
| Inhibitor | Chromogranin A transcription (luciferase/protein) | Catecholamine secretion | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Target | Compound | Basal | Nicotine (1 mm) | Ratio (nicotine/basal) | PMA (0.1 μm) | Ratio (PMA/basal) | Basal | Nicotine (60 μm) | Ratio (nicotine/basal) |
| % | |||||||||
| None | Mock | 233 ± 14.4 | 619 ± 28.41-b | 2.66 | 513 ± 22.71-b | 2.20 | 4.3 ± 0.25 | 13.1 ± 0.581-b | 3.0 |
| MAPK | Apigenin (12.5 μm) | 189 ± 10.81-a | 172 ± 7.541-a | 0.91 | 237 ± 12.2ab | 1.20 | 4.7 ± 0.31 | 13.8 ± 0.641-b | 2.9 |
| MEK | PD98059 (20 μm) | 170 ± 5.341-a | 198 ± 7.181-a | 1.16 | 195 ± 2.031-a | 1.10 | 4.0 ± 0.22 | 12.9 ± 0.411-b | 3.2 |
Chromogranin A transcription: After transfection of the 1133-bp mouse chromogranin A promoter/luciferase reporter, apigenin (MAP kinase inhibitor, 12.5 μm) or PD98059 (MAP kinase kinase inhibitor, 20 μm) was added and incubated for 48 hr. Nicotine (1 mm) was added at the same time as the inhibitors, whereas PMA (0.1 μm) was added 6 hr before cell harvest. Data shown are luciferase activity/mg of protein in mean ± standard error for four replicates/condition.
↵1-a Inhibitor treatment group significantly (p < 0.05) different from mock group for that stimulation (same column).
↵1-b Stimulated group significantly (p < 0.05) different from unstimulated group for that treatment (same row). Activation of this plasmid by nicotine or phorbol ester has been described previously (Tang et al., 1996, 1997).
Catecholamine secretion: Each group was preincubated with inhibitors for 1 hr. During the 30-min secretion period (with or without 60 μm nicotine), inhibitors also were maintained in each group. Percent norepinephrine secretion was calculated as [secreted dpm/(secreted dpm + cellular dpm)][100]. Ratio data are stimulated secretion/mock-stimulated secretion for each treatment. Results are mean ± standard error for three replicates/condition.ab, See above.