Fievos L. Christofi

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.

Sensory mechanisms: Transmitters, modulators and reflexes

The enteric nervous system in combination with inputs from parasympathetic and sympathetic nerves regulate the contractile, secretory and vasomotor activity of the gastrointestinal track via neural reflexes. Sensory elements which may be present in specialized neurones, enteroendocrine cells or mast cells detect changes in force, chemical composition or even foreign antigens. Sensory elements signal the enteric nervous system to correct these changes by altering contractile activity, secretion and blood flow. Advances have been made in understanding the sensory mechanisms that are involved in 5‐hydroxytryptamine (5‐HT) release from enterochromaffin cells (EC) or a model for EC cells. These advances relate to roles for ATP and its metabolites ADP and adenosine in mechanotransduction and a role for a sodium glucose cotransporter, a SGLT‐like protein, in chemotransduction.

Purinergic receptors and gastrointestinal secretomotor function

Secretomotor reflexes in the gastrointestinal (GI) tract are important in the lubrication and movement of digested products, absorption of nutrients, or the diarrhea that occurs in diseases to flush out unwanted microbes. Mechanical or chemical stimulation of mucosal sensory enterochromaffin (EC) cells triggers release of serotonin (5-HT) (among other mediators) and initiates local reflexes by activating intrinsic primary afferent neurons of the submucous plexus. Signals are conveyed to interneurons or secretomotor neurons to stimulate chloride and fluid secretion. Inputs from myenteric neurons modulate secretory rates and reflexes, and special neural circuits exist to coordinate secretion with motility. Cellular components of secretomotor reflexes variably express purinergic receptors for adenosine (A1, A2a, A2b, or A3 receptors) or the nucleotides adenosine 5′-triphosphate (ATP), adenosine diphosphate (ADP), uridine 5′-triphosphate (UTP), or uridine diphosphate (UDP) (P2X1-7, P2Y2, P2Y4, P2Y6, P2Y12 receptors). This review focuses on the emerging concepts in our understanding of purinergic regulation at these receptors, and in particular of mechanosensory reflexes. Purinergic inhibitory (A1, A3, P2Y12) or excitatory (A2, P2Y1) receptors modulate mechanosensitive 5-HT release. Excitatory (P2Y1, other P2Y, P2X) or inhibitory (A1, A3) receptors are involved in mechanically evoked secretory reflexes or “neurogenic diarrhea.” Distinct neural (pre- or postsynaptic) and non-neural distribution profiles of P2X2, P2X3, P2X5, P2Y1, P2Y2, P2Y4, P2Y6, or P2Y12 receptors, and for some their effects on neurotransmission, suggests their role in GI secretomotor function. Luminal A2b, P2Y2, P2Y4, and P2Y6 receptors are involved in fluid and Cl-, HCO3-, K+, or mucin secretion. Abnormal receptor expression in GI diseases may be of clinical relevance. Adenosine A2a or A3 receptors are emerging as therapeutic targets in inflammatory bowel diseases (IBD) and gastroprotection; they can also prevent purinergic receptor abnormalities and diarrhea. Purines are emerging as fundamental regulators of enteric secretomotor reflexes in health and 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.

Rabbit chronic ileitis leads to up-regulation of adenosine A1/A3 gene products, oxidative stress, and immune modulation.

A rabbit model of chronic ileitis has helped decipher the mechanism of alteration of multiple electrolyte and nutrient malabsorptions in inflammatory bowel disease (IBD). This study examined alterations in the adenosine A1/A3 receptor, oxidant, antioxidant, and immune-inflammatory pathways in chronic ileitis. Chronic ileal inflammation was induced 13-15 days after infection with 10,000 Eimeria magna oocytes. Quantitative analysis in 16 rabbits was done for oxidants, antioxidants, A1 and A3 transcripts, transport, injury, and inflammatory mediators. Inflamed gut had villus blunting, crypt hyperplasia and fusion, and immune cell infiltration. Alkaline phosphatase and Na-glucose co-transport were reduced by 78% (P=0.001) and 89% (P=0.001), respectively. Real-time fluorescence monitoring (TaqMan)-polymerase chain reaction revealed a transcriptional up-regulation of 1.34-fold for A1 and 5.40-fold for A3 receptors in inflamed gut. Lipid peroxidation increased in the mucosa (78%, P=0.012), longitudinal muscle-myenteric plexus (118%, P=0.042), and plasma (104%, P=0.001). Mucosal antioxidants were altered by inflammation: reductions occurred in superoxide dismutase (32%, P=0.001) and catalase (43%, P=0.001), whereas increases occurred in glutathione (75%, P=0.0271) and glutathione reductase (86%, P=0.0007). Oxidant enzyme activities were elevated by 21% for xanthine oxidase (P=0.004), 172% for chloramine (P=0.022), 47% for gelatinase (P=0.041), and 190% for myeloperoxidase (P=0.002). Mast cell tryptase increased by 79% (P=0.006). Increases occurred in the plasma concentration of leukotriene B(4) (13-fold, P=0.003), thromboxane B(2) (61-fold, P=0.018), and tumor necrosis factor-alpha (9-fold, P=0.002). In conclusion, chronic ileitis and tissue injury are associated with discrete alterations in complex multi-level oxidant, antioxidant, and immune inflammatory components. The rabbit ileitis model is a suitable model to gain further insight into chronic inflammation and IBD. We hypothesize that adenosine A3 and A1 receptors may provide a novel target for therapy in chronic ileitis and perhaps IBD.

Enteric Glial Cells: A New Frontier in Neurogastroenterology and Clinical Target for Inflammatory Bowel Diseases

Abstract:The word “glia” is derived from the Greek word “&ggr;&lgr;o&igr;&agr;,” glue of the enteric nervous system, and for many years, enteric glial cells (EGCs) were believed to provide mainly structural support. However, EGCs as astrocytes in the central nervous system may serve a much more vital and active role in the enteric nervous system, and in homeostatic regulation of gastrointestinal functions. The emphasis of this review will be on emerging concepts supported by basic, translational, and/or clinical studies, implicating EGCs in neuron-to-glial (neuroglial) communication, motility, interactions with other cells in the gut microenvironment, infection, and inflammatory bowel diseases. The concept of the “reactive glial phenotype” is explored as it relates to inflammatory bowel diseases, bacterial and viral infections, postoperative ileus, functional gastrointestinal disorders, and motility disorders. The main theme of this review is that EGCs are emerging as a new frontier in neurogastroenterology and a potential therapeutic target. New technological innovations in neuroimaging techniques are facilitating progress in the field, and an update is provided on exciting new translational studies. Gaps in our knowledge are discussed for further research. Restoring normal EGC function may prove to be an efficient strategy to dampen inflammation. Probiotics, palmitoylethanolamide (peroxisome proliferator-activated receptor–&agr;), interleukin-1 antagonists (anakinra), and interventions acting on nitric oxide, receptor for advanced glycation end products, S100B, or purinergic signaling pathways are relevant clinical targets on EGCs with therapeutic potential.

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.

Adenosine A2 receptor-mediated excitation of a subset of AH/Type 2 neurons and elevation of cAMP levels in myenteric ganglia of guinea-pig ileum

Abstract The aim of the study was to test the hypothesis that excitatory A2 and inhibitory A1 receptors coexist on myenteric AHIType 2 neurons, and are positively coupled to adenylate cyclase to stimulate cAMP formation. The A2 agonists NECA and CGS 21680 increased excitability and depolarized the membrane in 40% of 71 AH/Type 2 neurons. In the remainder, the agonists depressed excitability and hyperpolarized the neurons. In 13% of neurons, A2 agonists caused a concentration‐dependent depolarization at nanomolar concentrations, followed by hyperpolarization at higher concentrations. CGS 21680 (EC50=0.15 nM) was 133‐fold more potent than NECA (EC50= 20 nM) in depolarizing AH/Type 2 neurons. The A1 agonist, CCPA, caused hyperpolarization and depressed excitability in more than 90% of neurons. The potency profile of agonists for depolarization was CGS 21680 ≫ NECA ≫> CCPA. NECA augmented at nanomolar and inhibited at micromolar concentrations, excitatory depolarizing responses to forskolin in AH/Type 2 neurons; whereas, CCPA only inhibited the action of forskolin. In parallel studies on enzymatically dissociated myenteric ganglia, when the ganglia were exposed to priming concentrations of forskolin (5 μM) in the presence of Ro‐20 1724, NECA enhanced the stimulatory action of forskolin on CAMP formation. This effect was abolished by the adenosine receptor antagonist DPSPX. The potency of NECA for stimulation of adenylate cyclase equalled that for depolarization of the AH/Type 2 neurons. The results suggest that high affinity excitutory A2 receptors are coupled to adenylate cyclase in a minority subset of AH/Type 2 myenteric neurons, and that inhibitory A1 and excitatory A2 receptors are co‐localized on some AH/Type 2 neurons.

Activation of neuronal adenosine A1 receptors suppresses secretory reflexes in the guinea pig colon

The role of adenosine A1 receptors (A1R) in reflex-evoked short-circuit current (Isc) indicative of chloride secretion was studied in the guinea pig colon. The A1R antagonist 8-cyclopentyltheophylline (CPT) enhanced reflex-evoked Isc. Adenosine deaminase and the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine enhanced and reduced reflex-induced Isc, respectively. The A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA) inhibited reflex-evoked Isc at nanomolar concentrations, and its action was antagonized by CPT. In the presence of either N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide to block the 5-hydroxytryptamine (5-HT)-mediated pathway or piroxicam to block the prostaglandin-mediated pathway, CCPA reduced the residual reflex-evoked Isc. CCPA reduced the response to a 5-HT pulse without affecting the tetrodotoxin-insensitive Isc responses to carbachol or forskolin. Immunoreactivity for A1R was detected in the membrane (10% of neurons) and cytoplasm (90% of neurons) of neural protein gene product 9.5-immunoreactive (or S-100-negative) submucosal neurons, in glia, and in the muscularis mucosa. A1R immunoreactivity in a majority of neurons remained elevated in the cytoplasm despite preincubation with adenosine deaminase or CPT. A1R immunoreactivity colocalized in synaptophysin-immunoreactive presynaptic varicose nerve terminals. The results indicate that endogenous adenosine binding to high-affinity A1R on submucosal neurons acts as a physiological brake to suppress reflex-evoked Isc indicative of chloride secretion.The role of adenosine A1 receptors (A1R) in reflex-evoked short-circuit current ( I sc) indicative of chloride secretion was studied in the guinea pig colon. The A1R antagonist 8-cyclopentyltheophylline (CPT) enhanced reflex-evoked I sc. Adenosine deaminase and the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine enhanced and reduced reflex-induced I sc, respectively. The A1R agonist 2-chloro- N 6-cyclopentyladenosine (CCPA) inhibited reflex-evoked I sc at nanomolar concentrations, and its action was antagonized by CPT. In the presence of either N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide to block the 5-hydroxytryptamine (5-HT)-mediated pathway or piroxicam to block the prostaglandin-mediated pathway, CCPA reduced the residual reflex-evoked I sc. CCPA reduced the response to a 5-HT pulse without affecting the tetrodotoxin-insensitive I sc responses to carbachol or forskolin. Immunoreactivity for A1R was detected in the membrane (10% of neurons) and cytoplasm (90% of neurons) of neural protein gene product 9.5-immunoreactive (or S-100-negative) submucosal neurons, in glia, and in the muscularis mucosa. A1R immunoreactivity in a majority of neurons remained elevated in the cytoplasm despite preincubation with adenosine deaminase or CPT. A1R immunoreactivity colocalized in synaptophysin-immunoreactive presynaptic varicose nerve terminals. The results indicate that endogenous adenosine binding to high-affinity A1R on submucosal neurons acts as a physiological brake to suppress reflex-evoked I scindicative of chloride secretion.