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

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

Background: 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(2+)-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. Methods: UTP signaling mechanisms were studied in BON cells, a human EC model, using Fluo-4/Ca(2+)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). Results: UTP, UTPγS, UDP or ATP induced Ca(2+)oscillations in BON. UTP evoked a biphasic concentration-dependent Ca(2+)response. 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 (P2Y4, 50% cells), UDP (P2Y6, 30%), UTPγS and UDP (14%) or MRS2768 (<3%). UTP Ca(2+)responses were blocked with inhibitors of PLC, IP3R, SERCA Ca(2+)pump, La(3+)sensitive Ca(2+)channels or chelation of intracellular free Ca(2+) by BAPTA/AM. Inhibitors of L-type, TRPC, ryanodine-Ca(2+)pools, PI3-Kinase, PKC or SRC-Kinase had no effect. UTP stimulated voltage-sensitive Ca(2+)currents (ICa), Vm-depolarization and inhibited IK (not IA) currents. An IKv7.2/7.3 K(+) channel blocker XE-991 mimicked UTP-induced Vm-depolarization and blocked UTP-responses. XE-991 blocked IK and UTP caused further reduction. La(3+) or PLC inhibitors blocked UTP depolarization; PKC inhibitors, thapsigargin or zero Ca(2+)buffer did not. UTP stimulated 5-HT release in hEC expressing TPH1, 5-HT, P2Y4/P2Y6R. Zero-Ca(2+)buffer augmented Ca(2+)responses and 5-HT release. Conclusion: UTP activates a predominant P2Y4R pathway to trigger Ca(2+)oscillations via internal Ca(2+)mobilization through a PLC/IP3/IP3R/SERCA Ca(2+)signaling pathway to stimulate 5-HT release; Ca(2+)influx is inhibitory. UTP-induced Vm-depolarization depends on PLC signaling and an unidentified K channel (which appears independent of Ca(2+)oscillations or Ica/VOCC). UTP-gated signaling pathways triggered by activation of P2Y4R stimulate 5-HT release.