Background: Alterations in 5-hydroxytryptamine (HT) signaling in inflamed gut may contribute to pathogenesis of inflammatory bowel diseases. Adenosine 5′-triphosphate (ATP) regulates mucosal-mechanosensory reflexes and ATP receptors are sensitive to mucosal inflammation. Yet, it remains unknown whether ATP can modulate 5-HT signaling in enterochromaffin cells (EC). We tested the novel purinergic hypothesis that ATP is a critical autocrine regulator of EC mechanosensitivity and whether EC expression of ATP-gated P2X3-ion channels is altered in inflammatory bowel diseases.
Methods: Laser confocal (fluo-4) Ca2+ imaging was performed in 1947 BON cells. Chemical stimulation or mechanical stimulation (MS) was used to study 5-HT or ATP release in human BON or surgical mucosal specimens, and purine receptors by reverse transcription-polymerase chain reaction, Western Blot, or P2X3-immunoreactivity in BON or 5-HT+ human EC (hEC) in 11 control and 10 severely inflamed ulcerative colitis (UC) cases.
Results: ATP or MS triggered Ca2+-transients or 5-HT release in BON. ATP or adenosine diphosphate increased 5-HT release 5-fold. MS caused ATP release, detected after 5′ecto-ATPase inhibition by ARL67156. ARL67156 augmented and apyrase blocked Ca2+/5-HT mechanosensitive responses. 2-Methyl-thio-adenosine diphosphate 5′-monophosphate-evoked (P2Y1,12) or mechanically-evoked responses were blocked or augmented by a P2Y1,12 antagonist, MRS2179, in different cells or inhibited by U73122. A P2Y12 antagonist, 2MeSAMP, augmented responses. A P2X1,3 agonist, α,β-MeATP, triggered Ca2+ responses, whereas a P2X1,2/3,3 antagonist, 2′,3′-O-(2,4,6-trinitrophenyl)-ATP, blocked mechanical responses or cell-surface 5′ATP-TR labeling. In hEC, α,β-MeATP stimulated 5-HT release. In UC, P2X3-immunoreactivity decreased from 15% to 0.2% of 5-HT+hECs. Human mucosa and BON expressed P2X1, P2X3, P2X4, P2X5, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12R-messenger RNA transcripts.
Conclusions: ATP is a critical determinant of mechanosensation and 5-HT release via autocrine activation of slow P2Y1-phospholipase C/inositol-1,4,5-triphosphate-Ca2+ or inhibitory P2Y12-purinergic pathways, and fast ATP-gated P2X3-channels. UC downregulation of P2X3-channels (or A2B) is postulated to mediate abnormal 5-HT signaling.
Article first published online 2 August 2013
Departments of *Anesthesiology and
†Pathology, The Wexner Medical Center at The Ohio State University, Columbus, Ohio;
‡Center for Perinatal Research,
§Division of Pediatric Gastroenterology, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio; and
‖Departments of Surgery and
**Neuroscience, The Wexner Medical Center at The Ohio State University, Columbus, Ohio.
Reprints: Fievos L. Christofi, PhD, AGAF, Department of Anesthesiology, The Wexner Medical Center at The Ohio State University, 226 Tzagournis Medical Research Facility, 420 West 12th Avenue, Columbus, OH 43210 (e-mail: Fedias.Christofi@osumc.edu).
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Supported by the National Institutes of Health on NIH DK093499-01, DK044179-15, and NCRR S10RR11434 to F.L.C. and 5K08 DK078035-05 to K.W.
The authors have no conflicts of interest to disclose.
A. Liñán-Rico and J.E. Wunderlich have contributed equally to this work.
Received May 03, 2013
Accepted June 03, 2013