Liñán-Rico Andromeda, Ochoa-Cortes Fernando, Zuleta-Alarcon Alix, Alhaj Mazin, Tili Esmerina, Enneking Josh, Harzman Alan, Grants Iveta, Bergese Sergio, Christofi Fievos L
Department of Anesthesiology, The Wexner Medical Center at The Ohio State University, ColumbusOH, United States.
Molecular Virology, Immunology and Medical Genetics, The Wexner Medical Center at The Ohio State University, ColumbusOH, United States.
Front Pharmacol. 2017 Jul 13;8:429. doi: 10.3389/fphar.2017.00429. eCollection 2017.
Enterochromaffin cells (EC) synthesize and release 5-HT and ATP to trigger or modulate gut neural reflexes and transmit information about visceral/pain sensation. Alterations in 5-HT signaling mechanisms may contribute to the pathogenesis of IBD or IBS, but the pharmacologic or molecular mechanisms modulating Ca-dependent 5-HT release are not understood. Previous studies indicated that purinergic signaling via ATP and ADP is an important mechanism in modulation of 5-HT release. However, EC cells also respond to UTP and UDP suggesting uridine triphosphate receptor and signaling pathways are involved as well. We tested the hypothesis that UTP is a regulator of 5-HT release in human EC cells. UTP signaling mechanisms were studied in BON cells, a human EC model, using Fluo-4/Caimaging, patch-clamp, pharmacological analysis, immunohistochemistry, western blots and qPCR. 5-HT release was monitored in BON or EC isolated from human gut surgical specimens (hEC). UTP, UTPγS, UDP or ATP induced Caoscillations in BON. UTP evoked a biphasic concentration-dependent Caresponse. Cells responded in the order of UTP, ATP > UTPγS > UDP >> MRS2768, BzATP, α,β-MeATP > MRS2365, MRS2690, and NF546. Different proportions of cells activated by UTP and ATP also responded to UTPγS (P2Y, 50% cells), UDP (P2Y, 30%), UTPγS and UDP (14%) or MRS2768 (<3%). UTP Caresponses were blocked with inhibitors of PLC, IP3R, SERCA Capump, Lasensitive Cachannels or chelation of intracellular free Ca by BAPTA/AM. Inhibitors of L-type, TRPC, ryanodine-Capools, PI3-Kinase, PKC or SRC-Kinase had no effect. UTP stimulated voltage-sensitive Cacurrents (I), V-depolarization and inhibited I (not I) currents. An I7.2/7.3 K channel blocker XE-991 mimicked UTP-induced V-depolarization and blocked UTP-responses. XE-991 blocked I and UTP caused further reduction. La or PLC inhibitors blocked UTP depolarization; PKC inhibitors, thapsigargin or zero Cabuffer did not. UTP stimulated 5-HT release in hEC expressing TPH1, 5-HT, P2Y/P2YR. Zero-Cabuffer augmented Caresponses and 5-HT release. UTP activates a predominant P2YR pathway to trigger Caoscillations via internal Camobilization through a PLC/IP/IP3R/SERCA Casignaling pathway to stimulate 5-HT release; Cainflux is inhibitory. UTP-induced V-depolarization depends on PLC signaling and an unidentified K channel (which appears independent of Caoscillations or I/VOCC). UTP-gated signaling pathways triggered by activation of P2YR stimulate 5-HT release.
肠嗜铬细胞（EC）合成并释放5-羟色胺（5-HT）和三磷酸腺苷（ATP），以触发或调节肠道神经反射，并传递有关内脏/疼痛感觉的信息。5-HT信号传导机制的改变可能导致炎症性肠病（IBD）或肠易激综合征（IBS）的发病，但调节钙依赖性5-HT释放的药理或分子机制尚不清楚。先前的研究表明，通过ATP和二磷酸腺苷（ADP）的嘌呤能信号传导是调节5-HT释放的重要机制。然而，EC细胞也对三磷酸尿苷（UTP）和二磷酸尿苷（UDP）有反应，这表明尿苷三磷酸受体和信号通路也参与其中。我们验证了UTP是人类EC细胞中5-HT释放调节剂的假设。使用Fluo-4/钙成像、膜片钳、药理学分析、免疫组织化学、蛋白质免疫印迹和定量聚合酶链反应（qPCR），在人类EC模型BON细胞中研究了UTP信号传导机制。在从人体肠道手术标本（hEC）分离的BON或EC中监测5-HT释放。UTP、γ-硫代三磷酸尿苷（UTPγS）、UDP或ATP在BON中诱导钙振荡。UTP引起双相浓度依赖性钙反应。细胞对UTP、ATP的反应顺序为>UTPγS>UDP>>MRS2768、苯甲酰ATP（BzATP）、α,β-甲基ATP> MRS2365、MRS2690和NF546。UTP和ATP激活的不同比例的细胞也对UTPγS（P2Y，50%细胞）、UDP（P2Y，30%）、UTPγS和UDP（14%）或MRS2768（<3%）有反应。UTP钙反应被磷脂酶C（PLC）、三磷酸肌醇受体（IP3R）、肌浆网钙泵（SERCA钙泵）、拉敏感钙通道的抑制剂或BAPTA/AM螯合细胞内游离钙所阻断。L型、瞬时受体电位通道C（TRPC）、兰尼碱钙池（ryanodine钙池）、磷脂酰肌醇-3激酶（PI3-Kinase）、蛋白激酶C（PKC）或Src激酶的抑制剂无效。UTP刺激电压敏感性钙电流（I）、去极化，并抑制内向整流钾电流（I）（而非外向电流）。I7.2/7.3钾通道阻滞剂XE-991模拟UTP诱导的去极化并阻断UTP反应。XE-991阻断I，UTP导致进一步降低。镧或PLC抑制剂阻断UTP去极化；PKC抑制剂、毒胡萝卜素或零钙缓冲液则不能。UTP刺激表达色氨酸羟化酶1（TPH1）、5-HT、P2Y/P2Y受体（P2YR）的hEC中的5-HT释放。零钙缓冲液增强钙反应和5-HT释放。UTP通过PLC/肌醇三磷酸（IP）/IP3R/SERCA钙信号通路激活主要的P2YR途径，通过细胞内钙动员触发钙振荡，以刺激5-HT释放；钙内流具有抑制作用。UTP诱导的去极化取决于PLC信号传导和一种未确定的钾通道（其似乎独立于钙振荡或I/电压门控钙通道）。由P2YR激活触发的UTP门控信号通路刺激5-HT释放。
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