Figure 1: Regulation of 5-HT uptake by H₃R/H₄R ligands.

(A, B) Dose-dependent effect of H₃R/H₄R-agonists on SERT activity. Synaptosomes prepared from rat hippocampus were preincubated with vehicle or various concentrations of R-mHA or immepip (nM: 10 to 2000) for 15 min at 37°C before performing the 5-HT uptake assays. 5-HT uptake was initiated by adding [³H]labeled 5-HT (50 nM) and continued for 5 min at 37°C, and then SERT specific 5-HT uptake was determined as described in the Methods. The results are presented as a percentage change from corresponding vehicle-treated controls. Values are expressed as mean ± S.E.M. *P< 0.05, **P< 0.01, ***P< 0.0001 compared with vehicle (One-Way ANOVA with Bonferroni multiple comparison tests). (C, D) Time course of the effect of H₃R/H₄R-agonists on SERT activity. Synaptosomes were preincubated with R-mHA (1000 nM) or immepip (500 nM) for the time indicated followed by [³H]5-HT (50 nM) uptake for 5 min at 37°C. The results are shown as a percentage of specific SERT-mediated uptake value relative to that of corresponding vehicle-treated control at each time point. ***P< 0.0001 compared with vehicle (One-Way ANOVA with Bonferroni multiple comparison tests). (E) Effect of H₃R/H₄R inverse agonist thioperamide on Immepip or R-mHA-mediated downregulation of SERT function. The synaptosomes were preincubated for 10 min with thioperamide (1000 nM) prior to the addition of vehicle or immepip (100 nM) or R-mHA (100 nM). Following additional 5 min incubation with [³H]5-HT (50 nM), SERT activity was measured and compared with vehicle control treatment. **P< 0.009 compared with control vehicle; ^P< 0.03 versus control vehicle; §P< 0.02 compared with thioperamide alone; ¶P< 0.01 compared with immepip or R-mHA alone (One-Way ANOVA with Bonferroni multiple comparison tests). (F) Effect of H₃R/H₄R agonists R-mHA and immepip on SERT activity in HEK-293 cells. To express SERT, HEK-293 cells were transiently transfected with hSERT as previously described³⁴. After 24 hrs, cells were treated with vehicle and R-mHA or immepip (10, 20 μM) for 15 min at 37°C followed by 10 min 5-HT uptake assays. R-mHA: (R)-(−)-α-methyl-histamine, Thio: thioperamide, ns: nonsignificant.