Helen J. Cooke

Neurobiology of the Intestinal Mucosa

The importance of the enteric nervous system for coordinating and programming the digestive modes of the gastrointestinal effecters is well recognized. Attention has been focused in the past on the involvement of intrinsic nerves in generating specific motility patterns of the smooth muscle effecters, with little regard for the influence of intrinsic nerves on mucosal transport processes. The advent of immunohistochemical techniques for identifying neuronal types and their projections has documented the intrinsic innervation of the mucosa and has renewed interest in the epithelium as a neuroeffector system. The first definitive evidence of the direct involvement of the enteric nervous system in the regulation of intestinal mucosal ion transport was provided by Hubel (1) who adapted conventional electrical field stimulation techniques that were used routinely to examine the neural regulation of smooth muscle contractile activity to whole-thickness sheets of intestine in Ussing chambers. This technique, with its subsequent modifications by Cooke et al. (2), has been the basis for current understanding of the involvement of the enteric nervous system in the regulation of mucosal transport function. Significant progress has been made over the last decade to develop the foundation on which to build a working model of neural mechanisms that influence the mucosa; however, physiologic experiments have not kept pace with the morphologic studies that have identified a multiplicity of putative neurotransmitters within submucosa neurons, and, therefore, the

Neurotransmitters in Neuronal Reflexes Regulating Intestinal Secretion

Abstract: The intestinal crypt cell secretes chloride into the lumen, resulting in accumulation of fluid that normally thins out mucus or, at higher secretory rates, flushes out the contents. The regulation of chloride secretion occurs by neural reflex pathways within the enteric nervous system. Mechanical stimulation releases 5‐hydroxytryptamine (5‐HT) from enterochromaffin cells with subsequent activation of intrinsic primary afferents that carry electrical signals to submucosal ganglia. After processing, interneurons activate cholinergic and vasoactive intestinal peptide (VIP) secretomotor neurons. Acetylcholine and VIP bind to epithelial receptors and stimulate sodium chloride and fluid secretion. Reflex‐evoked secretory rates can be modulated by a variety of mediators at the level of the enterochromaffin cells, neurons within the reflex pathway, or epithelial cells. Understanding the complex regulatory mechanisms for chloride secretion is likely to provide mechanistic insights into constipation and diarrhea.

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.

5‐HT activates neural reflexes regulating secretion in the guinea‐pig colon

The role of 5‐hydroxytryptamine (5‐HT) in neural reflexes regulating secretion was examined in muscle‐stripped segments of guinea‐pig colon set up in modified flux chambers. A 15‐μL pulse of 5‐HT (100 μM) to the mucosal bath (1.5 mL), which was continuously perfused, evoked an increase in short‐circuit current (Isc). The 5‐HT‐induced increase in Isc was inhibited by tetrodotoxin, N‐acetyl‐5‐hydroxytryptophyl‐5‐hydroxytryptophan amide (5‐HTP‐DP), GR82334 and atropine, but not by tropisetron. 5‐HTP‐DP reduced the response to a 5‐HT pulse over the concentration range of 1 nM to 1 μM. The Isc response to a 5‐HT pulse was unaffected by the cyclooxygenase inhibitor, piroxicam. This contrasted with a reduction in the Isc response to mucosal stroking with a brush by piroxicam. The results suggest that a 5‐HT pulse, like mucosal stroking, activates a secretory reflex that includes tachykinin and cholinergic neurons but, unlike mucosal stroking, does not release prostaglandins.

Neuroimmune communication in the submucous plexus of guinea pig colon after infection with Trichinella spiralis

BACKGROUND/AIMS Enteric neuroimmune communication in gastrointestinal hypersensitivity responses includes antigen detection by mast cells and release of chemical messages to the enteric nervous system. The aim of this study was to analyze the electrical and synaptic behavior of neurons in the colonic submucous plexus during exposure to Trichinella spiralis antigen in animals infected earlier with the parasite. METHODS Microelectrodes were used to record in submucous neurons of guinea pig distal colon during application of Trichinella antigen. RESULTS Neurons in sensitized animals were more excitable than in controls. Hyperexcitability was seen as a greater probability of spontaneous action potential discharge and repetitive firing to depolarizing current or exposure to acetylcholine. Application of histaminergic antagonists reversed the augmented excitability, suggesting endogenously released histamine as a responsible factor. Antigenic exposure increased neuronal excitability and suppressed nicotinic transmission at fast cholinergic synapses only in sensitized animals. Effects on excitability, but not presynaptic inhibitory effects, were blocked by cimetidine. CONCLUSIONS Signaling between mucosal mast cells and the enteric nervous system is involved in colonic anaphylactic responses to sensitizing antigens. Histamine is a paracrine signal in the communication pathway.

Role of the "little brain" in the gut in water and electrolyte homeostasis.

The enteric nervous system plays a key role in maintenance of body fluid homeostasis by regulating the transport of ions by the intestinal epithelium. The epithelial cells normally absorb large volumes of fluid and ions daily, but tonically active submucosal neurons continuously suppress ion transport and limit the absorptive capacity of the intestine. Specialized nerve endings detect chemical, osmotic, or thermal alterations of the luminal contents or mechanical activity of the gut wall and encode this information as action potentials that propagate along nerve processes to the ganglia. Information transfer within the ganglia occurs at nicotinic cholinergic or other synapses. Ion transport is altered when neurotransmitters released from motor neurons interact with receptors on epithelial cells to initiate stimulus‐response coupling. The signals that transduce changes in epithelial ion transport are largely unknown, except for acetylcholine, but may include vasoactive intestinal peptide or other peptides. These trigger changes in intracellular messengers that influence the state of ionic channels in the epithelial cells and thereby inhibit absorptive processes or stimulate secretory mechanisms. When conservation of salt and water is necessary, command signals from the central nervous system, and perhaps from the myenteric ganglia, will shut down the synaptic circuits in the submucosal ganglia and enhance the absorptive capacity of the bowel.—Cooke, H. J. Role of the “little brain” in the gut in water and electrolyte homeostasis. FASEB J. 3: 127‐138; 1989.

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.

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.