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

Abstract

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 (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. 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 Vm-depolarization depends on PLC signaling and an unidentified K channel (which appears independent of Ca2+oscillations or Ica/VOCC). UTP-gated signaling pathways triggered by activation of P2Y4R stimulate 5-HT release.

A population of gut epithelial enterochromaffin cells is mechanosensitive and requires Piezo2 to convert force into serotonin release

“…Because EE and EC cells are electrically excitable ( 21 , 28 , 47 ), it is possible that Piezo2 generates a receptor potential that activates voltage-gated sodium channels, which mediate seconds-long EC cell-bursting activity ( 21 , 28 ), and activate voltage-gated calcium channels, which have been implicated in EC cell chemosensitivity ( 28 , 48 , 49 ). Another possibility is that Ca 2+ flux through Piezo2 directly activates EC cell Ca 2+ -induced Ca 2+ release ( 50 ) or ATP release and subsequent autocrine P2X activation ( 51 ). These possibilities are not mutually exclusive, and clarifying the involvement of these mechanisms in EC mechanotransduction may help delineate EC cell roles within the neuroepithelial ( 28 , 52 ) and endocrine ( 7 , 39 ) systems.…”

The Mechanism of Secretion and Metabolism of Gut-Derived 5-Hydroxytryptamine

“…EC cells represent around 50% of all EE cells that sense diverse dietary nutrients and metabolites to produce ~95% of total body 5-HT [ 14 ]. Studies conducted on human primary colonic EC cells and BON cells (immortalized cell line models of EC cells) found that 5-HT is released from EC cells in response to stimulation of luminal nutrients [ 11 , 14 , 15 , 85 ]. Unlike human primary colonic EC cells, BON cells release 5-HT following the stimulation of luminal D-glucose through sodium-glucose-linked transporter 1 (SGLT1) [ 86 ].…”

Enterochromaffin Cells: Sentinels to Gut Microbiota in Hyperalgesia?

“…ATP and its breakdown products are a purinergic transmitter in the ENS, which can initiate enteric nerve reflex or activate afferent nerve endings to transmit pain to the brain. Through BON cells or EC cells isolated from human intestinal surgery specimens, it is verified that ECs can respond to ATP, uridine triphosphate (UTP) and uridine diphosphate (UDP), mainly by activating P2X3, P2Y4R and PLC/IP3/IP3R/SERCA Ca 2+ signaling pathways, to involve in visceral sensitization and pain production ( Xu et al., 2008 ; Liñán-Rico et al., 2017 ). In addition, metabolites of norepinephrine and isovalerate activate ECs to release 5-HT ( Bellono et al., 2017 ).…”