AUTHOR=Liñán-Rico Andromeda , Ochoa-Cortes Fernando , Zuleta-Alarcon Alix , Alhaj Mazin , Tili Esmerina , Enneking Josh , Harzman Alan , Grants Iveta , Bergese Sergio , Christofi Fievos L. 

TITLE=UTP – Gated Signaling Pathways of 5-HT Release from BON Cells as a Model of Human Enterochromaffin Cells

JOURNAL=Frontiers in Pharmacology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2017.00429

DOI=10.3389/fphar.2017.00429

ISSN=1663-9812

ABSTRACT=<p><bold>Background:</bold> 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<sup>2+</sup>-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.</p><p><bold>Methods:</bold> UTP signaling mechanisms were studied in BON cells, a human EC model, using Fluo-4/Ca<sup>2+</sup>imaging, 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).</p><p><bold>Results:</bold> UTP, UTPγS, UDP or ATP induced Ca<sup>2+</sup>oscillations in BON. UTP evoked a biphasic concentration-dependent Ca<sup>2+</sup>response. Cells responded in the order of UTP, ATP &gt; UTPγS &gt; UDP &gt;&gt; MRS2768, BzATP, α,β-MeATP &gt; MRS2365, MRS2690, and NF546. Different proportions of cells activated by UTP and ATP also responded to UTPγS (P2Y<sub>4</sub>, 50% cells), UDP (P2Y<sub>6</sub>, 30%), UTPγS and UDP (14%) or MRS2768 (&lt;3%). UTP Ca<sup>2+</sup>responses were blocked with inhibitors of PLC, IP3R, SERCA Ca<sup>2+</sup>pump, La<sup>3+</sup>sensitive Ca<sup>2+</sup>channels or chelation of intracellular free Ca<sup>2+</sup> by BAPTA/AM. Inhibitors of L-type, TRPC, ryanodine-Ca<sup>2+</sup>pools, PI3-Kinase, PKC or SRC-Kinase had no effect. UTP stimulated voltage-sensitive Ca<sup>2+</sup>currents (I<sub>Ca</sub>), V<sub>m</sub>-depolarization and inhibited I<sub>K</sub> (not I<sub>A</sub>) currents. An I<sub>Kv</sub>7.2/7.3 K<sup>+</sup> channel blocker XE-991 mimicked UTP-induced V<sub>m</sub>-depolarization and blocked UTP-responses. XE-991 blocked I<sub>K</sub> and UTP caused further reduction. La<sup>3+</sup> or PLC inhibitors blocked UTP depolarization; PKC inhibitors, thapsigargin or zero Ca<sup>2+</sup>buffer did not. UTP stimulated 5-HT release in hEC expressing TPH1, 5-HT, P2Y<sub>4</sub>/P2Y<sub>6</sub>R. Zero-Ca<sup>2+</sup>buffer augmented Ca<sup>2+</sup>responses and 5-HT release.</p><p><bold>Conclusion:</bold> UTP activates a predominant P2Y<sub>4</sub>R pathway to trigger Ca<sup>2+</sup>oscillations via internal Ca<sup>2+</sup>mobilization through a PLC/IP<sub>3</sub>/IP3R/SERCA Ca<sup>2+</sup>signaling pathway to stimulate 5-HT release; Ca<sup>2+</sup>influx is inhibitory. UTP-induced V<sub>m</sub>-depolarization depends on PLC signaling and an unidentified K channel (which appears independent of Ca<sup>2+</sup>oscillations or I<sub>ca</sub>/VOCC). UTP-gated signaling pathways triggered by activation of P2Y<sub>4</sub>R stimulate 5-HT release.</p>