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.
Highlights
Enterochromaffin cells (EC) synthesize and release 5-HT, ATP and other mediators involved in gut neural reflexes and transmission of information about visceral/pain sensation (Kellum et al, 1999; Kim et al, 2001a; Raybould et al, 2004; Cooke and Christofi, 2006; Christofi, 2008)
Emerging evidence suggests that alterations in 5-HT release or handling mechanisms may contribute to inflammatory bowel diseases (IBD), Irritable Bowel Syndrome (IBS) and the diarrhea associated with bacterial toxin enterocolitis
Patient consent was obtained for each human subject and surgical tissue that is otherwise discarded by pathology was used to isolate human EC cells (hEC) for studies on 5-HT release, or western blot analysis in human mucosa for P2Y receptors
Summary
Enterochromaffin cells (EC) synthesize and release 5-HT, ATP and other mediators involved in gut neural reflexes and transmission of information about visceral/pain sensation (Kellum et al, 1999; Kim et al, 2001a; Raybould et al, 2004; Cooke and Christofi, 2006; Christofi, 2008). Abnormal regulation of 5-HT occurs in gastrointestinal disorders and inflammatory bowel diseases (IBD), where 5-HT signaling may represent a key mechanism in the pathogenesis of intestinal inflammation (Mawe and Hoffman, 2013; Liñán-Rico et al, 2016). 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. We tested the hypothesis that UTP is a regulator of 5-HT release in human EC cells
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