Figure 2. 2-Me-5-HT enhances 5-HT₃R-calmodulin (CaM) colocalization in a palonosetron-sensitive manner in least shrew brainstem and intestine.

Graphs A and B) Effects of the 5-HT₃R agonist 2-Me-5-HT and the 5-HT₃R antagonist palonosetron on 5-HT₃R-CaM interaction in the least shrew brainstem as revealed by co-immunoprecipitation (IP). Palonosetron (Palo, 5 mg/kg, s.c) or its vehicle (Veh) was administered 30 min prior to 2-Me-5-HT (or its vehicle) in different groups of shrews. Twenty minutes following 2-Me-5-HT administration (5 mg/kg, i.p.), brainstems were collected from the Control (Ctl) group (Veh + Veh), 2-Me-5-HT group (Veh + 2-Me-5-HT), Palonosetron group (Palo + Veh) and Palonosetron + 2-Me-5-HT group (Palo + 2-Me-5-HT). Proteins were immunoprecipitated by rabbit anti-5-HT₃R antibody and Western blots were developed on 5-HT₃R immunoprecipitates using goat anti-5-HT₃R antibody and mouse anti-CaM antibody. The ratio of optical density for CaM (17 kD) to 5-HT₃R (55 kD) was acquired and expressed as fold change of control. A) The representative Western blot, and B) Summarized data. *P<0.05 vs. the Control. Graphs C and D show the immunohistochemical analysis of 5-HT₃R-CaM colocalization in brainstem (C) and intestinal slices (D) from shrews treated as described for A and B. 10 µm thick cryo-sections of brainstem and intestine were co-labeled with rabbit anti-5-HT₃R and mouse anti-CaM antibodies. Representative high magnification fluorescence images (200×) show colocalization of 5-HT₃R and CaM in the area postrema (AP) region of brainstem (C) and jejual segment of intestine (D) which were increased following 5-HT₃R stimulation by 2-Me-5HT (5 mg/kg, i.p.). A 30 min prior exposure to the 5-HT₃R antagonist palonosetron (5 mg/kg, s.c.) abolished the 2-Me-5-HT-induced enhancement of the 5-HT₃R-CaM colocalization. Scale bar, 10 µm.