Figure 6.

Interaction between parasympathetic stimulation and 5-HT. (A) Stimulated tear secretion by 5-HT, CCH, or simultaneous 5-HT with CCH under a condition whereby both parasympathetic and 5-HT stimuli were decreased. Each drug was injected after 1 day of combined PGD and 3 days of Trp-free feeding (n = 5 mice). Sum of tear secretion for 20 minutes after each injection. (B) Change in LG weight (n = 6–8 LG) and (C) morphology of the LG (scale bar is 10 µm). (D) Change in the phosphorylation levels of mTOR and S6 (n = 6 LG). (D) Change in expression levels of p62 and LC3-II (n = 6 LGs). Change in expression level of each protein (D,E) was analyzed by western blot at day 1 of combined PGD and 4 days of Trp-free feeding. Representative western blot image and quantitative analysis of western blot (bar chart). The LG was analyzed at day 1 after PGD (PGD), at day 4 after feeding a Trp-free diet (Trp-free), at day 1 after combination of PGD and 4 days of Trp-free feeding (Trp-free + PGD) (B–E). (F) Interaction between ACh and 5-HT on [Ca²⁺]i elevation in LG acinar cells (n = 33–35 acini). Acinar cells isolated from normal rat LG were stimulated by ACh at dosages between 0.1 µM to 1 mM with or without 5-HT at a constant dosage (1 µM); each stimulation was performed for 1 minute. The upper panel shows pseudocolored images of [Ca²⁺]i (scale bar, 50 µm). The lower trace shows the typical response to each stimulus. The arrow indicates the time at which ACh with or without 5-HT was applied to the cells. The line chart show the summarized amplitudes of [Ca²⁺]i responses. All data represent the mean ± SD, ***P < 0.001 versus vehicle, ^††† P < 0.001 versus 5-HT, ^§§§ P < 0.001 versus CCH (A), *P < 0.05, ***P < 0.001 versus control, ^§§§ P < 0.01 versus PGD, ^§§§ P < 0.001 versus Trp-free (B,D,E), ***P < 0.001 versus without 5-HT (F).