Manzella Christopher R, Ackerman Max, Singhal Megha, Ticho Alexander L, Ceh Justin, Alrefai Waddah A, Saksena Seema, Dudeja Pradeep K, Gill Ravinder K
Department of Physiology & Biophysics, University of Illinois at Chicago, Chicago, IL, USA.
Division of Gastroenterology & Hepatology, University of Illinois at Chicago, Chicago, IL, USA.
Cell Physiol Biochem. 2020 Feb 5;54(1):126-141. doi: 10.33594/000000209.
BACKGROUND/AIMS: Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter and hormone with important physiological functions in many organs, including the intestine. We have previously shown that 5-HT activates the aryl hydrocarbon receptor (AhR) in intestinal epithelial cells (IECs) via a serotonin transporter (SERT)-dependent mechanism. AhR is a nuclear receptor that binds a variety of molecules including tryptophan (TRP) metabolites to regulate physiological processes in the intestine including xenobiotic detoxification and immune modulation. We hypothesized that 5-HT activates AhR indirectly by interfering with metabolic clearance of AhR ligands by cytochrome P450 1A1 (CYP1A1).
Inhibition of CYP1A1 activity by 5-HT was assessed in the human intestinal epithelial cell line Caco-2 and recombinant CYP1A1 microsomes using both luciferase and LC-MS/MS. Degradation of 5-HT by recombinant CYP1A1 was measured by LC-MS/MS. For in vitro studies, CYP1A1 and CYP1B1 mRNA expression levels were measured by RT-PCR and CYP1A1 activity was measured by ethoxyresorufin-O-deethylase (EROD) assays. For in vivo studies, AhR ligands were administered to SERT KO mice and WT littermates and intestinal mucosa CYP1A1 mRNA was measured.
We show that 5-HT inhibits metabolism of both the pro-luciferin CYP1A1 substrate Luc-CEE as well as the high affinity AhR ligand 6-formylindolo[3,2-b] carbazole (FICZ). Recombinant CYP1A1 assays revealed that 5-HT is metabolized by CYP1A1 in an NADPH dependent manner. Treatment with 5-HT in TRP-free medium, which is devoid of trace AhR ligands, showed that 5-HT requires the presence of AhR ligands to activate AhR. Cotreatment with 5-HT and FICZ confirmed that 5-HT potentiates induction of AhR target genes by AhR ligands. However, this was only true for ligands which are CYP1A1 substrates such as FICZ. Administration of β-napthoflavone by gavage or indole-3-carbinol via diet to SERT KO mice revealed that lack of SERT impairs intestinal AhR activation.
Our studies provide novel evidence of crosstalk between serotonergic and AhR signaling where 5-HT can influence the ability of AhR ligands to activate the receptor in the intestine.
背景/目的：血清素（5-羟色胺，5-HT）是一种神经递质和激素，在包括肠道在内的许多器官中具有重要的生理功能。我们之前已经表明，5-HT通过血清素转运体（SERT）依赖性机制激活肠道上皮细胞（IECs）中的芳烃受体（AhR）。AhR是一种核受体，可结合包括色氨酸（TRP）代谢物在内的多种分子，以调节肠道中的生理过程，包括外源性物质解毒和免疫调节。我们假设5-HT通过干扰细胞色素P450 1A1（CYP1A1）对AhR配体的代谢清除来间接激活AhR。
使用荧光素酶和液相色谱-串联质谱法（LC-MS/MS）在人肠道上皮细胞系Caco-2和重组CYP1A1微粒体中评估5-HT对CYP1A1活性的抑制作用。通过LC-MS/MS测量重组CYP1A1对5-HT的降解。对于体外研究，通过逆转录聚合酶链反应（RT-PCR）测量CYP1A1和CYP1B1 mRNA表达水平，并通过乙氧基异吩恶唑酮-O-脱乙基酶（EROD）测定法测量CYP1A1活性。对于体内研究，将AhR配体给予SERT基因敲除小鼠和野生型同窝小鼠，并测量肠道黏膜CYP1A1 mRNA。
我们表明，5-HT抑制荧光素酶原CYP1A1底物Luc-CEE以及高亲和力AhR配体6-甲酰基吲哚并[3,2-b]咔唑（FICZ）的代谢。重组CYP1A1测定表明，5-HT以NADPH依赖性方式被CYP1A1代谢。在不含微量AhR配体的无TRP培养基中用5-HT处理表明，5-HT需要AhR配体的存在才能激活AhR。5-HT与FICZ共同处理证实，5-HT增强了AhR配体对AhR靶基因的诱导。然而，这仅适用于CYP1A1底物的配体，如FICZ。通过灌胃给予β-萘黄酮或通过饮食给予吲哚-3-甲醇给SERT基因敲除小鼠表明，缺乏SERT会损害肠道AhR的激活。
我们的研究提供了血清素能信号与AhR信号之间相互作用的新证据，其中5-HT可以影响AhR配体在肠道中激活受体的能力。
