Jacqueline E. Wunderlich

ADOA3R as a Therapeutic Target in Experimental Colitis: Proof by Validated High-density Oligonucleotide Microarray Analysis

&NA; Adenosine A3 receptors (ADOA3Rs) are emerging as novel purinergic targets for treatment of inflammatory diseases. Our goal was to assess the protective effect of the ADOA3R agonist N(6)‐(3‐iodobenzyl)‐adenosine‐5‐N‐methyluronamide (IB‐MECA) on gene dysregulation and injury in a rat chronic model of 2,4,6‐trinitrobenzene sulfonic acid (TNBS)‐induced colitis. It was necessary to develop and validate a microarray technique for testing the protective effects of purine‐based drugs in experimental inflammatory bowel disease. High‐density oligonucleotide microarray analysis of gene dysregulation was assessed in colons from normal, TNBS‐treated (7 days), and oral IB‐MECA‐treated rats (1.5 mg/kg b.i.d.) using a rat RNU34 neural GeneChip of 724 genes and SYBR green polymerase chain reaction. Analysis included clinical evaluation, weight loss assessment, and electron paramagnetic resonance imaging/spin‐trap monitoring of free radicals. Remarkable colitis‐induced gene dysregulation occurs in the most exceptional cluster of 5.4% of the gene pool, revealing 2 modes of colitis‐related dysregulation. Downregulation occurs in membrane transporter, mitogen‐activated protein (MAP) kinase, and channel genes. Upregulation occurs in chemokine, cytokine/inflammatory, stress, growth factor, intracellular signaling, receptor, heat shock protein, retinoid metabolism, neural, remodeling, and redox‐sensitive genes. Oral IB‐MECA prevented dysregulation in 92% of these genes, histopathology, gut injury, and weight loss. IB‐MECA or adenosine suppressed elevated free radicals in ex vivo inflamed gut. Oral IB‐MECA blocked the colitis‐induced upregulation (≤20‐fold) of Bzrp, P2X1R, P2X4R, P2X7R, P2Y2R, P2Y6R, and A2aR/A2bR but not A1R or A3R genes or downregulated P2X2R, P2Y1R, and P2Y4R. Real‐time SYBR green polymerase chain reaction validated gene chip data for both induction of colitis and treatment with IB‐MECA for >90% of genes tested (33 of 37 genes). We conclude that our validated high‐density oligonucleotide microarray analysis is a powerful technique for molecular gene dysregulation studies to assess the beneficial effects of purine‐based or other drugs in experimental colitis. ADOA3R is new potential therapeutic target for inflammatory bowel disease.

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.

New bioinformatics approach to analyze gene expressions and signaling pathways reveals unique purine gene dysregulation profiles that distinguish between CD and UC

Background: Expression of purine genes is modulated by inflammation or experimental colitis and altered expression leads to disrupted gut function. We studied purine gene dysregulation profiles in inflammatory bowel disease (IBD) and determined whether they can distinguish between Crohns disease (CD) and ulcerative colitis (UC) using Pathway Analysis and a new Comparative Analysis of Gene Expression and Selection (CAGES) method. Methods: Raw datasets for 22 purine genes and 36 probe‐sets from National Center for Biotechnology Information (NCBI) GEO (Gene Expression Omnibus) (http://www.ncbi.nlm.nih.gov/projects/geo/) were analyzed by National Cancer Institute (NCI) Biological Resources Branch (BRB) array tools for random‐variance of multiple/36 t‐tests in colonic mucosal biopsies or peripheral blood mononuclear cells (PBMCs) of CD, UC or control subjects. Dysregulation occurs in 59% of purine genes in IBD including ADORA3, CD73, ADORA2A, ADORA2B, ADAR, AMPD2, AMPD3, DPP4, P2RY5, P2RY6, P2RY13, P2RY14, and P2RX5. Results: In CD biopsies, expression of ADORA3, AMPD3, P2RY13, and P2RY5 were negatively correlated with acute inflammatory score, Crohns Disease Activity Index (CDAI) or disease chronicity; P2RY14 was positively correlated in UC. In mucosal biopsies or PBMCs, CD and UC were distinguished by unique patterns of dysregulation (up‐ or downregulation) in purine genes. Purine gene dysregulation differs between PBMCs and biopsies and possibly between sexes for each disease. Ingenuity Pathway Analysis (IPA) revealed significant associations between alterations in the expression of CD73 (upregulation) or ADORA3 (downregulation) and inflammatory or purine genes (≤10% of 57 genes) as well as G‐protein coupled receptors, cAMP‐dependent, and inflammatory pathways; IPA distinguishes CD from UC. Conclusion: CAGES and Pathway Analysis provided novel evidence that UC and CD have distinct purine gene dysregulation signatures in association with inflammation, cAMP, or other signaling pathways. Disease‐specific purine gene signature profiles and pathway associations may be of therapeutic, diagnostic, and functional relevance.

Impact of disrupting adenosine A3 receptors (A3−/−AR) on colonic motility or progression of colitis in the mouse

Background: Pharmacological studies suggest that adenosine A3AR influences motility and colitis. Functional A3−/−AR knockout mice were used to prove whether A3AR activation is involved in modulating either motility or colitis. Methods: A3AR was probed by polymerase chain reaction (PCR) genotyping, Western blot, and immunochemistry. Motility was assessed in vivo by artificial bead‐expulsion, stool‐frequency, and FITC‐dextran transit. Colitis was induced with dextran sodium sulfate (DSS) in A3−/−AR or wildtype (WT) age‐ and sex‐matched controls. Progression of colitis was evaluated by histopathology, changes in myeloperoxidase (MPO), colon length, CD4+‐cells, weight‐loss, diarrhea, and the guaiac test. Results: Goat anti‐hu‐A3 antiserum identified a 66 kDa immunogenic band in colon. A3AR‐immunoreactivity is expressed in SYN+‐nerve varicosities, s‐100+‐glia, and crypt cells, but not 5‐HT+ (EC), CD4+ (T), tryptase+ (MC), or muscle cells. A3AR immunoreactivity in myenteric ganglia of distal colon ⟩ proximal colon by a ratio of 2:1. Intestinal transit and bead expulsion were accelerated in A3−/−AR mice compared to WT; stool retention was lower by 40%–60% and stool frequency by 67%. DSS downregulated A3AR in epithelia. DSS histopathology scores indicated less mucosal damage in A3−/−AR mice than WT. A3−/−AR phenotype protected against DSS‐induced weight loss, neutrophil (MPO), or CD4+‐T cell infiltration, colon shortening, change in splenic weight, diarrhea, or occult‐fecal blood. Conclusions: Functional disruption of A3AR in A3−/−AR mice alters intestinal motility. We postulate that ongoing release of adenosine and activation of presynaptic‐inhibitory A3AR can slow down transit and inhibit the defecation reflex. A3AR may be involved in gliotransmission. In separate studies, A3−/−AR protects against DSS colitis, consistent with a novel hypothesis that A3AR activation contributes to development of colitis. (Inflamm Bowel Dis 2010)

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.

Neuropharmacology of Purinergic Receptors in Human Submucous Plexus: Involvement of P2X1, P2X2, P2X3 Channels, P2Y and A3 Metabotropic Receptors in Neurotransmission

RATIONALE The role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. EXPERIMENTAL APPROACH LSCM-Fluo-4/(Ca(2+))-imaging of postsynaptic Ca(2+) transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs). RESULTS Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPβS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,β-MeATP) agonist evokes fast, slow, biphasic Ca(2+) transients or Ca(2+) oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 >> P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7, P2Y1,2,12-14R. CONCLUSIONS Purines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are potential novel therapeutic targets for neurogastroenterology.

182 Neural Circuit Behavior in Enteric Neural Plexus Developed From Neural Crest-Derived Stem Cells (ENPCs)

the circumferentially projecting axons of DII neurons achieved full length by P10-11, but the orally and anally projecting axons of uniaxonal neurons grew significantly between P1011 and adult. Neither increase closely matched the increase in size of the small intestine. Uniaxonal neurons underwent major changes in dendritic morphology. While most adult uniaxonal neurons (>70%) had only lamellar dendrites, P0 uniaxonal neurons rarely had lamellar dendrites but have filamentous dendrites instead. Electrophysiological properties of postnatal DII neurons differed markedly from their adult counterparts. At P0, DII neurons rarely had AHPs; and at P10-11, AHPs were only evoked by trains of action potentials. Postnatal DII neurons had prominent Ca2+-mediated afterdepolarizing potentials (ADPs, up to 70 mV in amplitude from a starting membrane potential of -100 mV) and rarely exhibited slow EPSPs. Adult AH/DII neurons displayed AHPs and slow EPSPs, and only a small ADP. Like adult S neurons, at P10-11, S neurons exhibited fast EPSPs which were mainly mediated by nicotinic receptors. Conclusions: This is the first study to examine the postnatal development of electrophysiological andmorphological properties in the intact ENS. At P0, functional synapses are present and neurons can be differentiated electrophysiologically and morphologically into two classes resembling adult classifications, but major modifications occur later in development. When compared their adult counterparts, P10-11 DII neurons are mature morphologically, but not electrophysiologically, while the morphology of S/uniaxonal neurons remains immature.

183 Glial Transmission in Human Enteric Nervous System

the circumferentially projecting axons of DII neurons achieved full length by P10-11, but the orally and anally projecting axons of uniaxonal neurons grew significantly between P1011 and adult. Neither increase closely matched the increase in size of the small intestine. Uniaxonal neurons underwent major changes in dendritic morphology. While most adult uniaxonal neurons (>70%) had only lamellar dendrites, P0 uniaxonal neurons rarely had lamellar dendrites but have filamentous dendrites instead. Electrophysiological properties of postnatal DII neurons differed markedly from their adult counterparts. At P0, DII neurons rarely had AHPs; and at P10-11, AHPs were only evoked by trains of action potentials. Postnatal DII neurons had prominent Ca2+-mediated afterdepolarizing potentials (ADPs, up to 70 mV in amplitude from a starting membrane potential of -100 mV) and rarely exhibited slow EPSPs. Adult AH/DII neurons displayed AHPs and slow EPSPs, and only a small ADP. Like adult S neurons, at P10-11, S neurons exhibited fast EPSPs which were mainly mediated by nicotinic receptors. Conclusions: This is the first study to examine the postnatal development of electrophysiological andmorphological properties in the intact ENS. At P0, functional synapses are present and neurons can be differentiated electrophysiologically and morphologically into two classes resembling adult classifications, but major modifications occur later in development. When compared their adult counterparts, P10-11 DII neurons are mature morphologically, but not electrophysiologically, while the morphology of S/uniaxonal neurons remains immature.

A Novel eADO Biosensor Microelectrode Array (MEA) for Real-Time Electrochemical Detection of eADO Release From Mouse or Human Gut

Salmonella typhimurium (S. typhi) at 107 cfu for 3 days. RESULTS: Although ubiquitously expressed, immunofluorescence confirmed high expression of ZBP-89 protein with serotonin in both mouse and human gut EC cells. ZBP-89Δcolon mice exhibited a 50% reduction in the numbers of serotonin-positive EC cells in the colon, but not the small intestine. Serotonin tissue content in the colon determined by ELISA was also reduced. A microarray analysis of WT versus ZBP-89Δcolon mice revealed an ~4-fold decrease in TPH1 and chromograninA mRNA. Quantitative PCR was used to verify the decrease in ZBP-89 and TPH1 mRNA in 4 mice. Two ZBP-89 consensus elements were identified in the TPH1 promoter. EMSAs and DAPA confirmed ZBP-89 binding to both elements along with Sp1 and Sp3 transcription factors. Mutation of both GC-rich DNA elements in the TPH1-Luc reporter abolished promoter activity. Infecting the ZBP-89Δcolon mice with S. typhi resulted in less submucosal edema than in the ceca of infected WT mice. CONCLUSIONS: ZBP-89 is required for basal TPH1 expression and serotonin production. Reduced TPH1 expression in the ZBP-89Δcolon mice appeared to mitigate the acute mucosal effects of S. typhi infection, presumably due to less serotonin production and fluid secretion.