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. 2021 Sep 6;12:735876. doi: 10.3389/fphar.2021.735876

FIGURE 5.

FIGURE 5

EG selectively activates 5-HT1B signaling. (A) Stable cell lines harboring inducible VSV-G-mGluR5-5HT1B and corresponding vector constructs induced by 10 ng ml−1 doxycycline for 12 h, and another 12 h of serum starvation for quiescence, then stimulated for 5 min by graded concentrations of EG (10−12 to 10−7 M in 5-HT1B cells, 10−12 to 10−7 M in vector cells), and the 5-HT1B selective agonist CP-94253 at 10−7 M, using FBS as positive control and deionized water as negative control. Samples were then subjected to western blotting. The expression of 5-HT1B, phosphorylation of ERK1/2 MAP kinases and loading control of ERK1/2 were determined by indicated antibodies described above in legend of Figure 3. (B) Stable cell lines and the inducing and starvation treatments were similar, as described in A above. Cells were pre-incubated with the 5-HT1B selective antagonist SB 224289 at 10−7 M for 20 min, following stimulation and western blotting. (C−F) Stable cell lines harboring inducible VSV-G-mGluR5-5HT2A (C), VSV-G-mGluR5-D2 (D), VSV-G-mGluR5-MT2 (E), and VSV-G-mGluR5-OX2 (F) and the corresponding vector constructs treated similar to A and the effects of EG on the indicated GPCR mediated pathways determined by phosphorylation of ERK1/2 MAP kinases. The induced and quiescent stable cell lines were stimulated for 5 min by graded concentrations of EG (10−8 and 10−7 M), and the selective agonis at 10−7 M, that were DOI, dopamine, ramelteon and OxB, respectively corresponding to 5HT2A, D2, MT2 and OX2 cells, using FBS as positive control and deionized water as negative control. EG: emodin-8-O-β-d-glucopyranoside; FBS: fetal bovine serum; ERK: extracellular regulated protein kinases; CFP: cyan fluorescent protein; 5-HT1B: serotonin receptor 1B; 5-HT2A: serotonin receptor 2A; D2: dopamine receptor 2; MT2: melatonin receptor 2; OX2: orexin receptor 2; VSV: vesicular stomatitis virus; mGluR5: metabotropic glutamate receptor 5; FBS: fetal bovine serum; DOI (1-(4-iodo-2,5-dimethoxyphenyl) propan-2-amine; dopamine: 4-(2-Aminoethyl) benzene-1,2-diol; ramelteon (N-[2-[(8S)-2.6,7,8-tetrahydro-1H-cyclopenta [e] (Gadgaard and Jensen, 2020) benzoxol-8-yl]ethyl]propanamide).