Purinergic Autocrine Regulation of Mechanosensitivity and Serotonin Release in a Human EC Model

Abstract

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.

Potential for Developing Purinergic Drugs for Gastrointestinal Diseases

“…However, in patients with ulcerative colitis (UC), P2X 3 could no longer be detected by a selective P2X 3 antiserum indicating that the fast-purinergic autocrine regulation of 5-HT release is impaired. 130 This needs confirmation in functional studies, but if so, it may be an important mechanism in the pathophysiology of UC. Alternatively, impairment in the P2X 3 mechanism may actually be a compensatory mechanism in a futile attempt to try and restore normal 5-HT signaling that is known to be altered in IBD (and IBS).…”

“…Therefore, these receptors are likely to play a critical role in the physiological regulation of peristaltic and secretory reflexes. 130 Any change in the expression of these receptors or signaling pathways in disease states such as IBD (or IBS) would be expected to have significant consequences. So for instance, P2X 3 – immunoreactivity is normally expressed in 15% of human EC cells lining the colonic mucosa.…”

Purinergic signalling in the gastrointestinal tract and related organs in health and disease

“…P2X3 receptor mRNA expression in dorsal root ganglia (DRG) was significantly decreased in the ovariectomised rat model of colitis, an effect that was reversed by oestrogen [212]. It has been suggested that ATP is a critical autocrine regulator of mechanosensitive 5-HT release, which is involved in the pathogenesis of IBD and that P2X3 receptors on enterochromaffin cells are downregulated in ulcerative colitis [442]. CD39 (NPTDase1) was upregulated in the submucosa during colitis that contributed to impaired sympathetic regulation of gastrointestinal blood flow, compromising epithelial barrier function [506].…”

What goes around comes around: novel pharmacological targets in the gut–brain axis

“…activating synaptotagmins capable of promoting cell and 5-HT vesicle membrane fusion, required for 5-HT release [Linan-Rico et al 2013]. Interestingly, using intestine explants Patel demonstrated that ileum 5-HT release depends on ATP whereas colon does not release 5-HT upon ATP stimulation [Patel, 2014].…”