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Purinergic Autocrine Regulation of Mechanosensitivity and Serotonin Release in a Human EC Model: ATP-gated P2X3 Channels in EC are Downregulated in Ulcerative Colitis

Liñán-Rico, Andrómeda PhD*; Wunderlich, Jacqueline E. MD, PhD*; Grants, Iveta S. BSc*; Frankel, Wendy L. MD; Xue, Jianjing MS; Williams, Kent C. MD*,§; Harzman, Alan E. MD; Enneking, Joshua T.*; Cooke, Helen J. PhD**; Christofi, Fievos L. PhD*

doi: 10.1097/MIB.0b013e31829ecf4d
Original Basic Science Articles

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).

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.ibdjournal.org).

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

© Crohn's & Colitis Foundation of America, Inc.
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