Jianjing Xue

Differential gene expression of adenosine A1, A2a, A2b, and A3 receptors in the human enteric nervous system.

Adenosine receptors (ADORs) in the enteric nervous system may be of importance in the control of motor and secretomotor functions. Gene expression and distribution of neural adenosine A1, A2a, A2b, or A3 receptors (Rs) in the human intestine was investigated using immunochemical, Western blotting, RT‐PCR, and short‐circuit current (Isc) studies. Adenosine A1R, A2aR, A2bR, or A3R mRNAs were differentially expressed in neural and nonneural layers of the jejunum, ileum, colon, and cecum and in HT‐29, T‐84, T98G, and Bon cell lines. A1R, A2aR, A2bR, and A3R immunoreactivities (IRs) were differentially expressed in PGP 9.5‐immunoreactive neurons. A2bR IR occurs exclusively in 50% of submucosal vasoactive intestinal peptide (VIP) neurons (interneurons, secretomotor or motor neurons) in jejunum, but not colon; A2aR is also found in other neurons. A3R IR occurs in 57% of substance P‐positive jejunal submucosal neurons (putative intrinsic primary afferent neurons) and less than 10% of VIP neurons. Western blots revealed bands for A3R at 44 kDa, 52 kDa, and 66 kDa. A2aR and A2bR are coexpressed in enteric neurons and epithelial cells. 5′‐N‐methylcarboxamidoadenosine or carbachol evoked an increase in Isc. A2bR IR is more prominent than A2aR IR in myenteric neurons, nerve fibers, or glia. A1R is expressed in jejunal myenteric neurons and colonic submucosal neurons. Regional differences also exist in smooth muscle expression of ADOR IR(s). It is concluded that neural and nonneural A1, A2a, A2b, and A3Rs may participate in the regulation of neural reflexes in the human gut. Clear cell and regional differences exist in ADOR gene expression, distribution, localization, and coexpression. J. Comp. Neurol. 439:46–64, 2001.

Mechanically evoked reflex electrogenic chloride secretion in rat distal colon is triggered by endogenous nucleotides acting at P2Y1, P2Y2, and P2Y4 receptors

Mechanical activation of the mucosal lining of the colon by brush stroking elicits an intestinal neural reflex and an increase in short circuit current (Isc) indicative of electrogenic chloride ion transport. We tested whether endogenous nucleotides are physiologic regulators of mucosal reflexes that control ion transport. The brush stroking‐evoked Isc response in mucosa and submucosa preparations (M‐SMP) of rat colon was reduced by the P2Y1 receptor (R) antagonist 2′deoxy‐N6‐methyl adenosine 3′,5′‐diphosphate diammonium salt (MRS 2179) and further blocked by tetrodotoxin (TTX). M‐SMP Isc responses to serosal application of the P2Y1 R agonist 2‐methylthioadenosine‐diphosphate (2MeSADP) or the P2Y2/P2Y4 R agonist 5′uridine‐triphosphate (UTP) were reduced but not abolished by TTX. The potency profile of nucleotides for increasing Isc was 5′adenosine‐triphosphate (ATP; effective concentration at half maximal response [EC50] 0.65 × 104 M) ≅ UTP (EC50 1.0 × 10−4 M) ≅ 2MeSADP (EC50 = 1.60 × 10−4 M). Mucosal touch and distention‐induced Ca2+ transients in submucous neurons were reduced by apyrase and prevented by blocking the P2Y1 R with MRS 2179 and TTX; denervation of the mucosa. It did not occur by touching a ganglion directly. 2MeSADP Ca2+ responses occurred in subsets of neurons with or without substance P (SP) responses. The potency profile of nucleotides on the neural Ca2+ response was 2MeSADP (5 × 10−7 M) > UTP (6 × 10−6 M) > ATP (9 × 10−5 M). The expression of P2Y R immunoreactivity (ir) in nerve cell bodies was in the order of P2Y1 R > P2Y4 R ≫ P2Y2 R. P2Y1R ir occurred in the cell somas of more than 90% of neuronal nitric oxide synthase, vasoactive intestinal peptide (VIP), calretinin, or neuropeptide Y (NPY)–ir neurons, 78% of somatostatin neurons, but not in calbindin or SP neurons. P2Y2 R ir was expressed in a minority of SP, VIP, NPY, vesicular acetylcholine transporter, and calcitonin gene‐related peptide–ir varicose fibers (5–20%) and those surrounding calbindin (5–20%) neurons. P2Y4 ir occurred mainly in the cell somas of 93% of NPY neurons. Reverse transcriptase polymerase chain reaction of the submucosa demonstrated mRNA for P2Y1R, P2Y2, P2Y4, P2Y6, and P2Y12 Rs. Expression of P2Y1, P2Y2, and P2Y4 protein was confirmed by western blots. In conclusion, endogenous nucleotides acting at P2YRs transduce mechanically evoked reflex chloride ion transport in rat distal colon. Nucleotides evoke reflexes by acting primarily at postsynaptic P2Y1 Rs and P2Y4 R on VIP+/NPY+ secretomotor neurons, at P2Y2 Rs on no more than 2% of VIP+ secretomotor neurons, and 2Y2 Rs mainly of extrinsic varicose fibers surrounding putative intrinsic primary afferent and secretomotor neurons. During mucosal mechanical reflexes, it is postulated that P2Y1 R, P2Y2 R, and P2Y4 R are activated by endogenous ATP, UTP, and 5′uridine‐diphosphate. J. Comp. Neurol. 469:16–36, 2004.

Mechanical stimulation releases nucleotides that activate P2Y1 receptors to trigger neural reflex chloride secretion in guinea pig distal colon.

Stroking the mucosal lining of the guinea pig colon with a brush elicits an intestinal neural reflex, and an increase in short‐circuit current (Isc) indicative of chloride secretion. We tested whether endogenous and exogenous nucleotides are physiologic regulators of mucosal reflexes that modulate chloride secretion. The basal Isc was augmented by 6‐N,N‐diethyl‐β,γ‐dibromomethylene‐D‐adenosine‐5′‐triphosphate (ARL67156) inhibition of nucleotide breakdown or adenosine A1 receptor blockade and reduced by apyrase inactivation of nucleotidases, P2 receptor antagonists, tetrodotoxin (TTX), or piroxicam. ARL67156 augmented, and apyrase inhibited, stroking‐evoked Isc responses. TTX and atropine inhibited nucleotide‐evoked Isc responses. The agonist potency profile for Isc, 2‐methylthioadenosine‐diphosphate (2MeSADP) = 2‐methioadenosine‐triphosphate ≫ 5′adenosine‐triphosphate (ATP) ≥ 5′adenosine‐diphosphate > 5′uridine‐triphosphate ≥ 5′uridine‐diphosphate, supports a P2Y1 receptor (R). The P2 receptor antagonists suramin and pyridoxalphosphate‐6‐azophenyl‐2′4′‐disulfonic acid, reduced stroking responses (36%) and their effects were additive. The selective P2Y1 R antagonist, 2′deoxy‐N6‐methyl adenosine 3′,5′‐diphosphate diammonium salt, reduced stroking (54%) and 2MeSADP (70%) responses at P2Y1 Rs. The P2X1/3 R agonist, α,βMeATP, increased Isc. A desensitizing dose of α,βMeATP reduced stroking Isc responses but did not prevent the 2MeSADP‐evoked Isc response. Reverse transcriptase polymerase chain reaction analysis revealed mRNAs for P2Y1 R, P2Y2 R, P2Y4 R, P2Y6 R, and P2Y12 R in submucosa. The expression of P2Y R immunoreactivity (ir) in cell bodies of submucous neurons followed the order of P2Y1 = P2Y2 ≫ P2Y4 R ir; P2Y1 Rs and P2Y2 R ir were abundant (21–50% of neurons). P2Y1 R ir was abundant in cholinergic secretomotor neurons and fewer than 2% of neuropeptide Y (NPY)/choline acetyltransferase secretomotor neurons, and P2Y2 R ir was expressed in virtually all NPY secretomotor neurons and approximately 30% of calbindin/intrinsic primary afferent neurons. P2Y4 R ir was present in NPY‐positive neurons. P2Y ir was rare or absent in varicose nerve fibers. The functional data support the hypothesis that mechanical stimulation with a brush releases nucleotides that act predominantly at P2Y1 Rs and to a lesser extent on P2X1/3 Rs to mediate reflex chloride secretion. A separate P2Y2 R neural circuit pathway exists that is not activated by mechanical forces. Other receptors including P2Y4, P2Y6, P2Y12, or P4 Rs cannot be excluded. J. Comp. Neurol. 469:1–15, 2004.

Autonomic Nervous System and Secretion across the Intestinal Mucosal Surface

Chloride secretion is important because it is the driving force for fluid movement into the intestinal lumen. The flow of accumulated fluid flushes out invading micro-organisms in defense of the host. Chloride secretion is regulated by neurons in the submucosal plexus of the enteric nervous system. Mechanosensitive enterochromaffin cells that release 5-hydroxytryptamine (5-HT) and activate intrinsic afferent neurons in the submucosal plexus and initiate chloride secretion. Mechanical stimulation by distention may also trigger reflexes by a direct action on intrinsic afferent neurons. Dysregulation of 5-HT release or altered activity of intrinsic afferents is likely to occur in states of inflammation and other disorders.

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 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, &agr;,&bgr;-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, &agr;,&bgr;-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.

Mechanically evoked 5‐hydroxytryptamine release is mediated by caveolin‐associated cholesterol rich membrane domains

Abstract  5‐Hydroxytryptamine (5‐HT) from enterochromaffin cells activates neural reflexes that govern intestinal motility and secretion. Mechanical stimulation of human enterochromaffin cell‐derived BON cells activates a Gαq‐signalling pathway coupled to 5‐HT release. Molecular mechanisms identifying elements of mechanosensory transduction are unknown. The aim of this study was to determine the role of caveolin and caveolin‐associated cholesterol rich microdomains in mechanically stimulated 5‐HT release from BON cells. Caveolin‐1 transcripts and immunofluorescence were found in BON cells. In the static state, caveolins‐1 and ‐2 co‐precipitated with Gαq in cholesterol rich cell fractions, but not with Gαs, Gαi/o and Gβ. Mechanical stimulation transiently uncoupled Gαq from caveolin‐1 and increased 5‐HT release. Disassembly of caveolin‐associated membrane microdomains by filipin or by cholesterol depletion with methyl‐β‐cyclodextrin decreased mechanically evoked 5‐HT release. These results suggest that caveolin and caveolin‐associated cholesterol rich membrane microdomains are key regulators in mechanically evoked 5‐HT release from enterochromaffin cells.