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

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 Ca²⁺-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/Ca²⁺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 Ca²⁺oscillations in BON. UTP evoked a biphasic concentration-dependent Ca²⁺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 Ca²⁺responses were blocked with inhibitors of PLC, IP3R, SERCA Ca²⁺pump, La³⁺sensitive Ca²⁺channels or chelation of intracellular free Ca²⁺ by BAPTA/AM. Inhibitors of L-type, TRPC, ryanodine-Ca²⁺pools, PI3-Kinase, PKC or SRC-Kinase had no effect. UTP stimulated voltage-sensitive Ca²⁺currents (I_Ca), V_m-depolarization and inhibited I_K (not I_A) currents. An I_Kv7.2/7.3 K⁺ channel blocker XE-991 mimicked UTP-induced V_m-depolarization and blocked UTP-responses. XE-991 blocked I_K and UTP caused further reduction. La³⁺ or PLC inhibitors blocked UTP depolarization; PKC inhibitors, thapsigargin or zero Ca²⁺buffer did not. UTP stimulated 5-HT release in hEC expressing TPH1, 5-HT, P2Y₄/P2Y₆R. Zero-Ca²⁺buffer augmented Ca²⁺responses and 5-HT release. Conclusion: UTP activates a predominant P2Y₄R pathway to trigger Ca²⁺oscillations via internal Ca²⁺mobilization through a PLC/IP₃/IP3R/SERCA Ca²⁺signaling pathway to stimulate 5-HT release; Ca²⁺influx is inhibitory. UTP-induced V_m-depolarization depends on PLC signaling and an unidentified K channel (which appears independent of Ca²⁺oscillations or I_ca/VOCC). UTP-gated signaling pathways triggered by activation of P2Y₄R stimulate 5-HT release.