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

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 P2X(3)-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 P2X(3)-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 5ecto-ATPase inhibition by ARL67156. ARL67156 augmented and apyrase blocked Ca2+/5-HT mechanosensitive responses. 2-Methyl-thio-adenosine diphosphate 5-monophosphate-evoked (P2Y(1,12)) or mechanically-evoked responses were blocked or augmented by a P2Y(1,12) antagonist, MRS2179, in different cells or inhibited by U73122. A P2Y(12) antagonist, 2MeSAMP, augmented responses. A P2X(1,3) agonist, alpha,beta-MeATP, triggered Ca2+ responses, whereas a P2X(1,2/3,3) antagonist, 2,3-O-(2,4,6-trinitrophenyl)-ATP, blocked mechanical responses or cell-surface 5ATP-(TR) labeling. In hEC, alpha,beta-MeATP stimulated 5-HT release. In UC, P2X(3)-immunoreactivity decreased from 15% to 0.2% of 5-HT(+)hECs. Human mucosa and BON expressed P2X(1), P2X(3), P2X(4), P2X(5), P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), and P2Y(12)R-messenger RNA transcripts. Conclusions: ATP is a critical determinant of mechanosensation and 5-HT release via autocrine activation of slow P2Y(1)-phospholipase C/inositol-1,4,5-triphosphate-Ca2+ or inhibitory P2Y(12)-purinergic pathways, and fast ATP-gated P2X(3)-channels. UC downregulation of P2X(3)-channels (or A(2B)) is postulated to mediate abnormal 5-HT signaling.