Additions | Melatonin production (%) | NAT activity (%) |
---|---|---|
Experiment 1 | ||
Control | 100 | 100 |
Acetylcholine | ||
50 μm | 60.4 ± 2.9 | 65.3 ± 4.4 |
100 μm | 43.3 ± 3.8 | 51.3 ± 3.2 |
200 μm | 25.4 ± 1.8 | 32.6 ± 4.9 |
500 μm | 27.4 ± 0.9 | 32.7 ± 2.5 |
Acetylcholine + tubocurarine | 86.3 ± 6.3 | 84.0 ± 7.9 |
Acetylcholine + MCCG | 58.9 ± 8.3 | 79.0 ± 6.9 |
Experiment 2 | ||
Control | 100 | 100 |
Nicotine | ||
50 μm | 53.2 ± 7.6 | 61.5 ± 5.8 |
100 μm | 40.1 ± 4.9 | 43.1 ± 6.2 |
200 μm | 26.3 ± 5.9 | 24.1 ± 2.9 |
500 μm | 28.4 ± 1.3 | 31.0 ± 2.7 |
Muscarine | 76.3 ± 3.2 | 82.7 ± 2.4 |
Carbachol | 53.4 ± 2.9 | 44.9 ± 4.2 |
Oxotremorine | 73.0 ± 5.2 | 70.6 ± 3.8 |
Nicotine + tubocurarine | 79.4 ± 8.3 | 65.8 ± 4.8 |
Nicotine + MCCG | 72.8 ± 6.4 | 60.7 ± 6.4 |
Pineal glands (3 glands per experiment) were incubated in 1 ml of DMEM for 1 hr. After glands were washed with DMEM, 10 μmNE (experiments 1 and 2) plus the indicated compound (200 μm each, unless stated otherwise) were added. In some cases, organs were preincubated with 2 mm MCCG or 400 μm tubocurarine for 30 min, and then the indicated compounds were added. After further incubation for 6 hr, the medium was collected and melatonin content was determined. Simultaneously, glands were homogenized and NAT activity was assayed. The results are presented as relative activity ± SEM (4 independent experiments) with 100% activity of 0.60 and 0.65 nmol/ml of melatonin synthesis, and 2.4 and 2.1 nmol · min−1 · mg−1 protein of NAT activity in experiments 1 and 2, respectively. Neither acetylcholine nor AchR agonists affected the level of these activities in the absence of NE. In experiment 1, 50 μm eserine was also included in the assay medium.