Purinergic receptors and gastrointestinal secretomotor function

Abstract

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) (P2X_1-7, 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 A_2b, 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 A_2a 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.

Introduction

Gastrointestinal (GI) secretions in coordination with motility are important in digestion of food particles, lubrication, nutrient absorption, regulation of pH and solute concentration, and elimination of waste products. Diarrhea occurs in GI diseases to flush out noxious chemicals or unwanted microbes. Intestinal chloride secretion provides a driving force for fluid movement and obligate transport of water into the lumen. Under normal circumstances, mucosal chloride secretion is very low, but can reach extraordinary rates when challenged by enterotoxins or various secretogogues. Hallmarks of enteric infections or inflammatory bowel diseases (IBD) are malaise and diarrhea; the other extreme is constipation that occurs in patients with idiopathic constipation, constipation-predominant irritable bowel syndrome (IBS), or opioid-induced constipation. Structural or functional changes in the enteric nervous system (ENS), in neurotransmitters, signaling pathways, or other components of enteric neural reflexes may be the basis for the pathogenesis of disturbances in gut motor function.

The current review will focus primarily on the role of purines in secretomotor function in the GI system. The enterochromaffin–neural circuit–epithelial reflex pathway is our target for understanding secretomotor function in the intestinal tract. All cellular components of mucosal reflexes express purinergic receptors, and this review will focus on these receptors, their distribution, and function in mucosal reflexes and secretomotor function in the GI tract. The enteric neural circuits and cellular components involved in neurosecretory reflexes will first be briefly reviewed. A brief overview and basic knowledge of purinoceptor classification, pharmacology, receptor ligands and their selectivity is necessary in order to understand the concepts forwarded in this review. Emerging concepts will be reviewed in our understanding of purinergic regulation in mucosal reflexes, with particular emphasis on enterochromaffin cells (EC), mucosal reflexes, receptor distribution, and luminal P2 receptors involved in epithelial ion transport and fluid secretion. In addition, we review recent findings on purinergic regulation of gastric secretions, and in relation to gastric mucosal protection. Our review would not be complete without a very brief description of the emerging role of purinergic receptors as therapeutic targets in IBD [2] or implications of purine receptor abnormalities (up- or downregulation) in GI diarrhea diseases. The reader is also referred to more general reviews on purinergic signaling [12], GI secretomotor function and dysfunction [25, 26], epithelial secretion, and hepatobiliary function [92]. Other relevant reviews include adenosine neuromodulation [20], purines in mechanosensory signaling [26], ATP as a sensory transmitter [6], and P2Y₁ receptors in secretomotor function [113], or P2X receptors as potential targets in IBS [40].

Enterochromaffin–neural–epithelial reflex pathways

The secretory reflex is composed of sensory EC cells, intrinsic primary afferent neurons (IPANs), various interneurons, secretomotor neurons, interplexus neurons for coordinating secretion and motility or for secretomotor function [11], and the epithelial cells involved in ion transport and fluid secretion in the gut lumen (Fig. 1). Luminal stimulation by nutrients, changes in pH, mechanical forces, changes in solute concentration, luminal irritants, or invading enteropathogenic microorganisms will activate the EC cells to secrete serotonin (5-HT) among other mediators to trigger secretomotor reflexes leading to chloride, bicarbonate, potassium, mucin, and fluid secretion. EC cells communicate with both submucosal and myenteric IPANS to initiate secretomotor reflexes. Mucosal stimulation and activation of secretomotor reflexes via long myenteric pathways may play a role in the coordination of intestinal motility, secretion, and blood flow [88]. This review is restricted to submucous pathways and secretomotor reflexes. Under physiological circumstances, large fluctuations in secretions can occur depending on the stimulus. Crohn’s disease or Clostridium difficile toxin A (TxA) can cause diarrhea and alterations in secretion. However, the diarrhea observed in IBD is mainly malabsorptive not secretory; in the inflamed gut, secretion actually decreases. Hypersecretion due to infection is helpful in flushing to rid the lumen of a pathogen.

Fig. 1

Generic intestinal secretory reflex based on guinea pig intestine. Neural reflex pathway regulating secretion includes EC sensory cells that release 5-HT to activate IPANS projecting to the mucosa, to submucous ganglia, and to myenteric ganglia. IPANS synapse with several types of cholinergic (CHAT) and vasoactive intestinal peptide (VIP) secretomotor neurons and release acetylcholine (ACh) at muscarinic receptors (M3) and VIP at VIP receptors (VPACRs) on epithelial cells, or arterioles. VIP neurons are subdivided according to their ability to generate cAMP as VIP/cAMP (+) and VIP/cAMP (-) and may be further subdivided into VIP/cAMP/A2aRs and VIP/cAMP/A2aR (-). Cholinergic neurons are ChAT/calretinin (CALRET), CHAT/NPY, CHAT (-). Mechanical distortion such as brush stroking the mucosa is one stimulus for activation of the reflex. Myenteric secretomotor neurons project to the mucosa as well. CGRP calcitonin gene-related peptide, EC enterochromaffin cells, 5-HT 5-hydroxytryptamine, NK1 neurokinin-1, NPY neuropeptide Y, SP substance P, A2aRs adenosine A_2a receptors. (Modified from [25] by permission)

Release of sensory mediators from EC cells activates the afferent nerve endings of IPANs to trigger a secretory reflex. IPANs detect sensory information from the luminal environment in the form of action potentials carried in their primary afferent process. The sensory information is decoded and integrated in the cell soma and then relayed to interneurons or secretomotor neurons to elicit a secretory reflex. These IPANs in submucous ganglia have AH cell electrophysiological characteristics and comprise 15% of submucous neurons (and 25-30% of myenteric neurons). The axonal processes of submucous IPANs are known to project to other IPANs and to submucous or myenteric neurons. They have Dogiel type II morphology and project in all directions in the submucous plexus [25].

One type of IPAN in the submucous plexus has one sensory process projecting to the mucosa and responds to mucosal distortion/deformation at sites less than 1 mm² from the recording site. Another type of IPAN in the submucous plexus is sensitive to distention [26, 38, 112]. Notable differences exist in the chemical coding of these neurons in human, porcine, and rodent intestine [25]. After activation of IPANs, release of transmitters at synapses with interneurons or secretomotor neurons leads to epithelial secretion. 5-HT interplexus neurons send a process to synapse with secretomotor neurons expressing 5-HT3 receptors. Secretomotor neurons are S/type 1 neurons that release acetylcholine or VIP to activate the chloride-secreting crypt epithelial cells expressing muscarinic and VIP receptors. All cellular components of this reflex have functional adenosine (P1) or nucleotide (P2X and P2Y) receptor subtypes and are the subject of this review.

Purinergic receptors

Adenosine, adenosine 5′-triphosphate (ATP), ADP, AMP, uridine 5′-triphosphate (UTP), UDP, and UDP-glucose are endogenous purines that activate P1, P2X, or P2Y purinoceptor families that are widely and differentially distributed in the ENS and non-neuronal cells in the GI tract. The tissue distribution and/or biological experiments suggest that up to 14 of 18 purinoceptors may be involved in secretomotor reflexes in the GI tract. Still, our knowledge remains incomplete.

Adenosine interacts with four cell surface P1 receptors, designated as A₁, A_2a, A_2b, and A₃ receptors to influence a variety of physiological functions. The endogenous ligand is adenosine (or AMP, [41]). Receptor classification is based on receptor cloning, pharmacological studies, and mouse receptor knockout models. All four receptors are expressed in the GI tract, but their characterization is incomplete (see later). Adenosine receptors belong to the superfamily of seven transmembrane domain G protein-coupled receptors (GPCR) and they are emerging as potential therapeutic targets [53].

The nomenclature for the transmitter-gated ion channel P2X_1-7 receptors relies mostly on recombinant receptor pharmacology as reviewed by Khakh et al. [57]; the pharmacology of the P2X receptors in whole tissues is complicated by species differences, marked ectonucleotidase activity that interferes with agonist/antagonist potency profiles, and lack of very selective ligands for P2X receptor subtypes. Their review covers the functional properties of recombinant receptors, their pharmacology, ion effects (Zn²⁺, H⁺, Ca²⁺), and some native receptors in tissues.

A recent review summarizes the pharmacological profiles of cloned mammalian metabotropic P2Y receptors [110] that have been shown to mediate actions of nucleotides when expressed in functional systems. Eight P2Y receptors have been cloned and defined functionally. The P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptors are coupled to stimulation of phospholipase C (PLC). P2Y₁₂, P2Y₁₃, and P2Y₁₄ receptors are negatively coupled to adenylyl cyclase (AC); the P2Y₁₁ receptor is positively coupled to AC. Abbrachio et al. [1] provide an update on molecular mechanisms, pathophysiology, and therapeutics of P2Y receptors.

P2Y₁ receptors are involved in platelet aggregation, vasodilation, neuromodulation, mechanosensitivity, and secretomotor reflexes. The P2Y₁ receptor is activated by ADP or its analog 2-methylthioADP (2MeSADP). A potent and selective antagonist is 2′-deoxy-N6-methyladenosine-3′5′-bisphosphate (MRS2179) or MRS2279. Adenosine 5′-O-(1-boranotriphosphate) (ATP-α-B) derivatives are novel P2Y₁ receptor agonists that may be of potential clinical relevance [78].

The P2Y₂ receptor plays an important role in Cl^- secretion from epithelial cells, and a P2Y₂ receptor agonist diquafosol is used for treatment of dry eye disease. It is activated by UTP and ATP and their actions are blocked by suramin, although suramin has affinity for non-purinergic receptors as well [54]. The P2Y₄ receptor is expressed in epithelia, and depending on the species, the receptor has a strong preference for UTP (human receptor) or equal preference for UTP and ATP (rat receptor). Unlike the P2Y₂ receptor it is not blocked by suramin. The P2Y₆ receptor is distributed in the cardiovascular system and brain. It has a preference for UDP and 1,2-di-(4-isothiocyanatophenyl)ethane (MRS2567) is a selective antagonist. UDP also acts on receptors for cysteinyl leukotrienes. The P2Y₁₁ receptor is blocked by suramin (and reactive blue 2) and it prefers ATP as an agonist. P2Y₁₂ and P2Y₁ receptors are similar in that they are both activated by ADP and more potently by 2MeSADP. P2Y₁₂ receptor antagonists are effective in clinical use to inhibit platelet aggregation; the receptor is also involved in inhibition of neuronal cells. Antagonists include N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-βγ-dichloromethylene-ATP (AR-C6931MX or cangrelor), AZD6140, and active metabolites of clopidogrel and prasugrel. The P2Y₁₃ receptor is found in nerve cells and immunocytes and is activated similar to the P2Y₁₂ receptor, but is selectively blocked by 6-(2′-chloro-5-nitro-azophenyl)-pyridoxal-α5-phosphate (MRS2211). The P2Y₁₄ receptor is activated by UDP-glucose.

Sensory enterochromaffin cells

5-HT release from EC cells activates secretory, peristaltic, and vasomotor reflexes and contributes to the coordination of these reflexes. As shown in Fig. 2, EC cells with long basally located fine projections containing serotonin and sometimes connecting to neuron-like structures could be observed; other cells had short blunt processes or were dividing EC cells. A provocative possibility is the recent speculation that EC cells may even function as primary neurons [44].

Fig. 2

Photomicrographs showing serotonin immunolabeled, formalin-fixed EC cells dispersed from the colonic mucosa. All cells have luminal projections. From the left, four serotonin-positive cells with one or two basally located, axon-like projections are seen. The fourth of these cells connects with a neuron-like structure. Right: a cell with short and blunt basal projections. The EC cells are in some cases attached to neighboring mucosa cells. (Modified from [44] by permission)

EC cell dysfunction is implicated in malignant carcinoid, dumping, and irritable bowel syndromes, as well as IBD. 5-HT secretion from carcinoid tumors is one of the putative mediators of carcinoid diarrhea that can be relieved by 5-HT antagonists. 5-HT3 antagonists or a partial 5-HT4 agonist (i.e., tegaserod) are beneficial in treating diarrhea and constipation predominant symptoms in patients, respectively [4, 67, 97]. Mucosal defects in 5-HT signaling occur in ulcerative colitis, diarrhea (or constipation) predominant IBS [24] or experimental colitis [67]. A better understanding of the signaling mechanisms, function, and dysfunction of EC cells is essential in understanding gut reflexes and symptoms of several diseases.

Despite the obvious importance of EC cells and 5-HT release in normal or disease states, our understanding of purinergic or other mechanisms involved in the regulation of 5-HT release is limited. Tissue studies are of limited use in helping us understand the complex regulation of 5-HT release because of potential interference from mediators released from neighboring cells. The human carcinoid BON cell line is a suitable EC cell model to study receptor and intracellular signaling pathways regulating 5-HT release [34].

A clone of BON/EC cells that is 99% enriched in 5-HT is a suitable EC model for population responses or for single cell functional studies. BON cells retain many of the mechanosensitive and chemosensitive properties of non-transformed cells, they respond to nutrients (glucose), mechanical activation (shaking, touch, stretch, volume changes, pressure), as well as receptor activation (purinergic, muscarinic, neurotensin, 5-HT, VIP, adrenergic, somatostatin, cholecystokinin, dopamine, bradykinin, etc.). A comparison of EC and BON cells indicates similarities in expression of receptors, ion transporters, ion channels, chemical mediators, or other mechanisms reviewed by Cooke and Christofi [25]. In addition to 5-HT, they secrete or contain adenosine, ATP neuron-specific enolase, synaptophysin, chromogranin A, neurotensin, vasoactive intestinal peptide (VIP), prostaglandins, and others. BON cell injection into nude mice can also serve as a model of carcinoid tumors [35].

Chemosensitivity of EC cells

EC cells have microvilli protruding into the lumen raising the possibility that they may “taste” the luminal contents. The nutrient monosaccharide D-glucose (10-100 mM) or the nonmetabolizable α-D-glucopyranoside (not fructose) caused a graded increase in 5-HT release in BON cells that was not caused by changes in osmolarity. Phloridzin blocked the effect of D-glucose by blocking D-glucose uptake [59, 87]. The role of purinergic modulation of nutrient activation of 5-HT release remains unknown.

Mechanosensitivity of EC cells

Mechanosensitivity in EC cells is poorly understood and the mechanosensitive elements remain unknown. Brush stroking, compression of the mucosa or villi, rotational stroking, or pressure/volume changes used to evoke mucosal secretomotor reflexes in the gut are not suitable to elicit 5-HT release in isolated EC/BON cells. Mechanical forces in the gut are complex and likely represent a composite of forces (pressure, shear force, centrifugal force, stretch, deformation, compression, touch, etc.). We succeeded in employing three different mechanical stimuli to elicit responses in EC cells to study purinergic regulation of mechanosensitive 5-HT release. Rotational shaking of cultured cells simulates forces that are generated in the intestinal lumen (i.e., shear forces, centrifugal forces, changes in hydrostatic pressure) and releases 5-HT. Pressures of 75 mmHg or greater (normally associated with painful stimulation) applied to cells in a pressure chamber or laminar shear stress of 1–2 dyne/cm² applied in a parallel plate flow chamber evoked 5-HT release [26].

Second messengers in mechanosensitivity

Balloon distension and puffs of nitrogen gas/pressure stimulate 5-HT release and trigger a reflex secretory response via submucous neurons in intact mucosa/submucosa preparations [84]. Rotational shaking of tissue preparations or the EC/BON cells in culture predominantly activates a Gαq/PLC/inositol triphosphate (IP₃)–Ca²⁺ signaling pathway leading to 5-HT release [59]. A separate Gs/Gi/AC/PKA (protein kinase A)/cAMP signaling pathway provides a minor contribution to mechanosensitive 5-HT release [18].

Dual modulation of mechanosensitivity by P2Y₁ and P2Y₁₂ receptors

Recent evidence from pharmacological and molecular studies support the hypothesis that dual modulation of 5-HT release occurs via excitatory P2Y₁ receptors coupled to the Gαq/PLC/IP₃-Ca²⁺ signaling pathway leading to 5-HT release [114] and inhibitory P2Y₁₂ receptors coupled to AC (Christofi, Wunderlich, Xue, Cooke, unpublished observations; [120]). Touch/stretch evokes a Ca²⁺ response in BON/EC cells that is used to study purinergic regulation of mechanosensitivity. In preliminary studies, touch Ca²⁺ responses in EC cells were inhibited by apyrase (ectonucleotidase) whereas a 5′-ectonucleotidase inhibitor ARL 67156 augmented responses indicating that a nucleotide is involved in mechanosensitivity. When single cells are examined and analyzed according to inhibition or augmentation of the Ca²⁺ response, dual modulation of the response is revealed in subsets of EC cells. Therefore, the P2Y₁ receptor antagonist MRS2179 could either abolish the touch Ca²⁺ response or augment the response in different subsets of EC cells. MRS2179 could block touch-evoked Ca²⁺ responses in cells responsive to the P2Y₁ receptor agonist 2MeSADP. The Ca²⁺ response evoked by 2MeSADP was either inhibited or augmented by MRS2179 in subsets of EC cells; an additional component of the touch Ca²⁺ response was blocked by the P2 receptor antagonist PPADS [114].

In molecular signaling studies, when the human P2Y₁ receptor was overexpressed in BON cells with naitive P2Y receptors, it caused additional mobilization of Ca²⁺ in the cells and augmented 5-HT release. A P2Y₁ receptor silencing RNA reduced receptor expression leading to a reduction in 5-HT release. In heterologous 1321N1 cells, transfection of human P2Y₁ receptors enabled the cells to generate a Ca²⁺ response to mechanical stimulation. P2Y₁ receptor antagonists MRS2179 or A3P5P abolished the Ca²⁺ response in 1321N1 cells or 5-HT response in BON cells that overexpressed the human P2Y₁ receptor [120]. Overexpression of the P2Y₁₂ receptor in BON cells and/or HEK293 cells nearly abolished mechanically evoked 5-HT release (BON) and cAMP (both cell types). Blockade of P2Y₁₂ receptors by 2-MeSAMP augmented the cAMP responses. 5-HT release was augmented after knockdown of P2Y₁₂ receptors in BON cells; silencing of P2Y₁₃ receptors had no effect. Therefore, molecular studies are consistent with pharmacological studies and suggest that dual purinergic modulation of mechanosensory signaling via P2Y₁ and P2Y₁₂ receptors (that recognize both MRS2179 and 2MeSADP) leads to 5-HT release in EC cells. The P2Y₁ and P2Y₁₂ receptors may provide new targets for modulation of mucosal secretory reflexes and are potential therapeutic targets, as shown for platelets [1]. The role of other receptors expressed in EC/BON remains unknown. Messenger RNA transcripts for P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, and P2Y₁₃ exist in BON cells [25, 26].

Adenosinergic modulation of 5-HT release

Endogenous adenosine modulates the basal release of 5-HT from EC/BON cells. Adenosine deaminase caused a dose-dependent increase in 5-HT release (Fig. 3a). The nucleoside uptake inhibitor S-(p-nitrobenzyl)-6-thioinosine (NBTI) prevented mechanically evoked 5-HT release (Fig. 3b) by elevating endogenous adenosine (Fig. 3c). Overall, endogenous adenosine is sufficient to provide ongoing inhibition of basal and mechanically evoked 5-HT release. The adenosine A₃ receptor is activated by endogenous adenosine since baseline 5-HT release was augmented by an A₃ receptor antagonist MRS1191 and this was sensitive to an A₃ receptor agonist IB-MECA (Fig. 3d); the A₃ effect was restricted to the Ca²⁺ signaling pathway. The A₁ receptor also contributes, and an A₁ receptor antagonist (CPT), an A₃ receptor antagonist (MRS1191), or adenosine deaminase could also augment the touch/stretch Ca²⁺ response (Fig. 3e). Touch Ca²⁺ responses occur via a Gαq/PLC/IP₃ pathway that is the predominant mechanism in 5-HT release from EC cells. 5′-N-methylcarboxy aminoadenosine (MECA) elevated both 5-HT and cAMP levels in BON cells. A₂ receptor antagonists could suppress mechanically evoked 5-HT release indicating the endogenous adenosine can contribute to stimulation of 5-HT release via A₂ receptors. Together, these and other findings indicate that endogenous adenosine can activate inhibitory A₃/A₁ receptor–Gαq/PLC/IP₃–Ca²⁺ and excitatory A_2a/A_2b receptor–AC/cAMP signaling pathways on EC cells to regulate 5-HT release; A₁, A_2a, A_2b, and A₃ receptors or mRNA transcripts are present in both BON/EC cells, gastric and intestinal 5-HT-ir carcinoid tumors [18]. Recent isolation of ileal EC cells [58] may permit further study and comparison of purinergic signaling mechanisms in normal and diseased human ileum. Real-time electrochemical detection of local mucosal 5-HT release is also very promising [5].

Fig. 3

Regulation of 5-hydroxytryptamine (5-HT) release by endogenous adenosine release from BON cells via inhibitory A₁/A₃ receptors and excitatory A₂ receptors. a The enzyme ADA caused a dose-dependent increase in 5-HT release, indicating that release of endogenous adenosine provides an ongoing inhibition of 5-HT release. b In the same cells as a, NBTI blocked adenosine uptake and abolished mechanically evoked 5-HT release. c Adenosine release during resting/unstimulated conditions (Base) and mechanical stimulation (MS) is augmented in the presence of the adenosine uptake inhibitor S-(p-nitrobenzyl)-6-thio-inosine (1 μM NBTI). d Chemical stimulation with IB-MECA decreased basal 5-HT and the A₃R agonist cAMP levels. The A₃R antagonist MRS1191 (10 nM) augmented 5-HT release but not cAMP levels (p < 0.05). The MRS1191-evoked 5-HT response was sensitive to IB-MECA inhibition. e Adenosine A₁ and A₃R inhibitory components of touch-evoked Ca²⁺ responses in enterochromaffin/BON cells. The A₃R antagonist MRS1191 augmented the touch Ca²⁺ transient. Chronic exposure to the A₁ antagonist CPT for 24 h caused a significant augmentation in the touch Ca²⁺ response. The stimulatory effects of CPT and MRS1191 were additive on the touch Ca²⁺ response. Adenosine deaminase (5 units/ml)-induced augmentation is reduced by the A₃R agonist IB-MECA (50 nM). f A_2A/A_2B antagonists CGS and AL (alloxazine) inhibited mechanically evoked 5-HT release. Baseline values (pmol/well per 30 min) of 5-HT and/or cAMP are at the top of figures with the number of experiments indicated in parentheses. IB IB-MECA, MRS MRS1191, ME MECA, AL alloxazine, CGS CGS 21680, MS mechanical stimulation by rotational shaking at 80 rpm, NS not significant. (Modified from [59] by permission)

Intestinal secretory reflexes

Hubel’s [51] pioneering spirit and studies demonstrated that electrical field stimulation of tissue setup in Ussing chambers can stimulate transepithelial short-circuit current (Isc) changes indicative of ion transport. His studies provided proof that submucous neurons could regulate chloride secretion. This sparked the beginning of a new era of investigation on the influence of the ENS on epithelial secretion and secretomotor reflexes.

The secretory reflex can be activated by nutrients or mechanical stimulation or exposing the gut to food antigens regarded as foreign or to enteropathogenic microorganisms. The mucosal surveillance and immune defense system is exposed to an estimated 10¹² different antigens per day that mobilizes both intrinsic and extrinsic nervous systems, hormones, paracrine substances, and immune mediators to modulate chloride secretory rates.

The net flux of ions into the gut lumen is the balance of secretion and absorption. Basal secretion or “secretory tone” is also the net flux of ions and fluid into the intestinal lumen that is dependent on the spontaneous release of endogenous paracrine, autocrine, or neurocrine mediators. Secretory tone is assessed from changes in basal Isc, a measure of chloride secretion. The basal Isc is under ongoing neural stimulation. Data with P2 receptor antagonists (PPADS, suramin) or P1 receptor antagonists (CPT), nucleotidases, or nucleotidase inhibitors indicated that endogenous nucleotides stimulate, while endogenous adenosine inhibits basal Isc [29]. However, basal secretion is not affected by P2X or P2Y₁ receptors because neither of NF279 or MRS2179 influenced Isc. Overall, adenosine, nucleotides, somatostatin, and prostaglandins all contribute to “secretory tone” [26, 27].

P2Y₁ receptors and mucosal stroking-evoked neurosecretory reflexes in the colon

Mechanical or chemical activation of the mucosa can lead to an intestinal neural reflex and an increase in ion transport and fluid secretion. Very few studies have investigated purinergic regulation of secretomotor function [18, 27–29]. Mucosal distortion by brush stroking or distension can elicit the reflex (Fig. 4).

Fig. 4

Modified Ussing chambers used for mucosal stroking studies to evoke a reflex neurosecretory response. a Vertical configuration of the chamber permits a 2-mm brush to be placed on the mucosal surface. A single stroke represents the directional movement of the brush from one side to the other on the surface of the mucosa. b A complete 360° turn of the brush is another variation for stimulating reflexes; stroke (1 s) is reproducible every 5 min for a 40-min period. c Distention is accomplished by removing small volumes of fluid from the serosal side. Drugs are perfused separately on the serosal or mucosal surfaces of the mucosa-submucosa or whole thickness colonic or small bowel tissues. The Isc/short-circuit current nulled out the spontaneous potential difference (PD) and was used as a measure of chloride secretion in the distal colon. (Modified from [25] by permission)

Brush stroking of the mucosa causes an intestinal neural reflex response and an increase in Isc indicative of electrogenic chloride ion transport. The brush stroking reflex in the rat colon mucosa-submucosa is reduced by MRS2179 or apyrase and further inhibited by tetrodotoxin (TTX). 2MeSADP, a mixed P2Y₂/P2Y₄ agonist UTP, or ATP (P2 receptor agonist) evoked neural and non-neural secretory responses. Mucosal touch/distension evoked fluo-4/Ca²⁺ responses in submucous neurons were also inhibited by apyrase or blocked completely by MRS2179 (Fig. 5); MRS2179 only reduced Isc in stroking reflexes. The potency profile of nucleotides for reducing Isc is different from that for touch Ca²⁺ responses [18]. P2Y₁ immunoreactivity was identified in a majority of VIP, NOS, calretinin, NPY, or somatostatin neurons but not SP or calbindin submucous neurons. P2Y₄ immunoreactivity occurred mainly in the cell somas of NPY neurons (93%). P2Y₂ immunoreactivity occurred in a minority of SP, VIP, NPY CGRP varicose fibers (5-20%) and those surrounding calbindin neurons [18, 26]. Purinergic responses could be distinguished based on stroking reflexes, touch-evoked Ca²⁺ reflexes, actions of nucleotides, or sensitivity to antagonists. These and other data suggest that several nucleotides may contribute to mechanically evoked secretomotor reflexes. Nucleotides differentially activate P2Y₁, P2Y₂, and P2Y₄ receptors located at putative pre- or postsynaptic sites on submucous neurons. In rat hippocampus, ATP may activate presynaptic P2Y₁, P2Y₂, and P2Y₄ receptors to inhibit glutamate release [91]; presynaptic P2Y₁ and P2Y₂ receptors also occur in the rat submucous plexus. A working model of the purinergic neural circuitry in the rat colon is shown in Fig. 6.

Fig. 5

Laser confocal imaging of touch-induced purinergic Ca²⁺ signals in neurons of the intact microdissected submucous plexus of the rat distal colon. a Perfusion chamber used to record touch-induced Ca²⁺ responses in the neurons. It includes a recording chamber made of a 35-mm microwell dish with a 1-cm, number 0.0 cover glass in the center of the bottom of the dish for visualizing the fluorescent neurons. A portion of the dish is embedded with a 2-mm thick layer of Sylgard shaped around a half moon at one end of the dish. Perfusion with oxygenated buffer or drugs is exactly opposite to the suction. The microdissected M-SMP preparation is stretched and fixed with many (15-20) micropins along the Sylgard layer. The remaining SMP without mucosa is stretched over the glass cover slip of the dish and secured with magnetic metal feet (not shown). A large ganglion of the intermediate layer of the submucous plexus is viewed with a 40× oil immersion objective (N.A. 1.3). b Inset shows Fluo-4/AM loaded neurons representing second order neurons that are visualized. c Side view of the tissue: The submucous plexus layer is located flat on the bottom of the dish and the mucosa/submucosa lifts over the Sylgard ends at each side of the half moon. The tissue is touched for 20 s on the mucosal surface of M-SMP portion that is stretched across the Sylgard with a fire-polished glass pipette to evoke a Ca²⁺ response in the neurons—the tissue is free to move/stretch downward during the touch, since it is located a distance of 2 mm above the glass bottom. d–g The P2Y₁ antagonist MRS2179 or TTX abolish the touch-induced Ca²⁺ fluorescence response. A representative example of the effects of MRS2179 and TTX on touch-induced Ca²⁺ transients in a single ganglion (seven neurons in one ganglion). A 20-min exposure to the selective P2Y₁ antagonist MRS2179 (10 μM) abolished the touch response in the neurons. In the presence of 0.2 μM TTX, the touch response decreased to below baseline levels. Arrows indicate touch-responsive cells. h Pooled data for the peak effects of MRS2179 and TTX; averaged transients from 30 neurons from 3 to 4 separate ganglia. i Breaking down endogenous nucleotides reduces the touch-induced Ca²⁺ response in the neurons of the intact M-SMP. The touch response was determined in the presence of 5 units/ml apyrase exposed for 10 min. Apyrase reduced by 50% the control touch response (n = 4 neurons, p < 0.01). Values are means ± SEM. (Modified from [18] by permission)

Fig. 6

Mechanical activation of purinergic neural circuitry in rat distal colon. Mucosal brush stroking or touch/stretch releases nucleotides that may include ATP, UTP, and ADP that activate neural P2Y₁, P2Y₂, and P2Y₄Rs at pre- or postsynaptic sites of submucous neurons leading to a net increase in Isc/chloride secretion. Intrinsic primary afferent neurons do not express P2YRs on their cell somas, but receive strong synaptic inputs from extrinsic fibers (i.e., pelvic origin) with P2Y₂Rs forming baskets of varicose fibers surrounding their cell somas. A very tiny subset of VIP secretomotor neurons expresses P2Y₂R. P2Y₂Rs, P2Y₁Rs, and P2Y₄Rs are coexpressed on > 90% of NPY/VIP putative secretomotor neurons that also express SP Rs. P2Y₁Rs are also expressed on putative somatostatin (SOM) neurons. Activation of P2Y₁Rs leads to a rise in intracellular free Ca²⁺ levels in the secretomotor neurons by ATP, UTP, or ADP. A nicotinic cholinergic synapse has not been ruled out. NOS neurons and CALR/CHAT cholinergic neurons also express P2Y₁Rs, but the functional role of these subsets of neurons is unknown. P2Y₂Rs are discretely localized on some extrinsic fibers of SP, VIP, NPY, CGRP, or cholinergic origin presumed to be involved in synaptic modulation of transmitter release. (Modified from [18] by permission)

ARL 67156 augmented, whereas apyrase, atropine, or TTX could inhibit stroking-evoked reflex Isc responses in guinea pig distal colon (Fig. 7a). The agonist potency profile of 2MeSADP > UTP ≥ UDP for stimulation of neural secretion supports a P2Y₁ receptor (Fig. 7b), but does not exclude others. MRS2179 reduced the stroking response by 54% and the effect of 2MeSADP by 70%. P2 receptor antagonists PPADS and suramin had additive inhibitory effects. The P2X_1/3 receptor agonist αβMeATP caused stimulation of Isc, and a desensitizing concentration reduced stroking-evoked secretion but did not affect the 2MeSADP response. Cell bodies of guinea pig colonic submucous neurons expressed P2Y immunoreactivity. About 20-50% of submucous neurons expressed P2Y₁ and P2Y₂ receptors. P2Y immunoreactivity (P2Y₁, P2Y₂, or P2Y₄) was virtually undetectable in any varicose fibers. P2Y₁ immunoreactivity was abundant in putative cholinergic secretomotor neurons and fewer than 2% of NPY/cholinergic secretomotor neurons [71]. In contrast, P2Y₂ immunoreactivity was found in all NPY secretomotor neurons and 30% of calbindin/intrinsic sensory neurons. P2Y₄ is of minor significance in submucous neurons. In guinea pig, mucosal stroking may activate putative P2X_1/3, P2Y₁, and P2Y₂ receptors located at postsynaptic membranes of submucous neurons leading to chloride secretion; P2Y₁ receptors on EC also contribute. Based on the available evidence, it was hypothesized that a separate P2Y₂ neural circuit pathway exists in guinea pig that is not activated by stroking the mucosa [25, 27]. In both rat and guinea pig, receptors or mRNA transcripts exist for P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₂ receptors in the submucosa, and therefore influence of other receptors in stroking reflexes deserves further attention. Species differences exist between mucosal stroking reflexes in rat [18] and guinea pig [27] distal colon in purinergic regulation, and no information exists for the human GI tract.

Fig. 7

Mucosal stroking releases endogenous ATP/nucleotides that activate excitatory neural P2Y₁Rs leading to an increase in Isc/chloride secretion in the guinea pig colon. a The ATPase/5′-nucleotidase inhibitor 6-N,N-diethyl-β,γ-dibromomethylene-D-adenosine-5′-triphosphate trisodium (10 μM, ARL 67156 or ARL) prevents hydrolysis of ATP and enhanced reflex-evoked Isc (n = 6, *p < 0.05), whereas 10 U/ml apyrase hydrolyzes ATP and inhibited reflex-evoked Isc responses (n = 4). This indicates that endogenous nucleotide release contributes to the stroking response. b Concentration-response curves for nucleotides in the presence of 1 μM CPT (n = 4-23), an A₁ receptor antagonist used to block eADO effects. The rank order of potencies of nucleotide agonists for stimulation of Isc was 2MeSADP = 2MeSATP > UTP = UDP that indicates a P2Y₁R interaction. c The selective P2Y₁R antagonist MRS2179 suppresses ~50% of the stroking response. d The Isc response to the P2Y₁R agonist 2MeSADP was nearly abolished by the P2Y₁R antagonist MRS2179 (10 μM, n = 4, p < 0.05). All experiments were done in the presence of 1 μM CPT to eliminate inhibitory influence of A₁ receptor activation by endogenous adenosine (n >/ 4, *p < 0.05) (Modified from [27] by permission)

Neurogenic mucosal secretion and P2Y₁ receptors in small intestine

In the guinea pig small intestine, it was shown that neurogenic mucosal secretion is mediated by the P2Y₁ receptor that may be expressed on VIP secretomotor neurons. MRS2179 blocked the neurally mediated ATP-evoked response with an IC₅₀ of 0.9 μM. TTX could suppress or abolish the ATP response. The P2Y₁ receptor was coupled to PLC/IP₃/Ca²⁺–calmodulin/protein kinase C signaling pathway [37] like that on EC/BON cells.

Purinergic modulation of local inhibitory reflexes

Local inhibitory reflexes to the circular muscle evoked by mucosal application of nutrient amino acids in guinea pig jejunum are partially blocked by antagonists to P2X receptors suggesting involvement of ATP [46]. The receptor subtype(s) have not been characterized. Effects of nutrients on local secretomotor reflexes and involvement of purinergic signaling in the coordination of motility and secretion remain unclear, although some data are emerging on adenosine A₃ and A₁ inhibitory receptors (see later section on “Neurogenic diarrhea”; [8, 9]).

Fluo-4/Ca²⁺ imaging studies are providing new insights into the functional purine receptors involved in the excitability and synaptic transmission in intact neural circuits of the submucous plexus of the human gut. A recent study indicated that the submucous plexus from Roux-en-Y patients is a suitable model to study synaptic transmission in the human enteric nervous system [115]. It was shown that the P2Y₁ Gαq-PLC/IP₃ Ca²⁺ signaling pathway, N-Ca²⁺ channels, nicotinic receptors, and extrinsic nerves are involved in neurotransmission. Pharmacological findings suggest that inhibitory adenosine A₃ receptors modulate nucleotide and cholinergic transmission in the human enteric nervous system.

In the guinea pig small intestine, electrophysiological studies revealed that AMP activates presynaptic inhibitory A₁ receptors to suppress neurotransmission and postsynaptic A_2a receptors to elicit a slow excitatory postsynaptic potential (EPSP)-like response. Signaling pathways coupled to A_2a receptors include AC, PLC, PKC, and calmodulin-dependent kinases [41]; whether AMP can also activate A_2b or A₃ receptors remains unclear.

Endogenous adenosine and A₁ receptors in mucosal reflexes

The role of endogenous adenosine in the physiological regulation of mucosal secretory reflexes was investigated in pharmacological studies [29]. At nanomolar concentrations, the A₁ antagonist 8-chlorophenyltheophylline (CPT) caused a concentration-dependent enhancement of the stroking reflex. The effect still occurred in the presence of an A_2a receptor antagonist 8-(3-chlorostyrl) caffeine. Adenosine deaminase (5 U/ml) also enhanced the reflex secretory response, whereas the nucleoside uptake blocker NBTI inhibited it. The A₁ receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) inhibited reflex-evoked Isc (Fig. 8a; EC₅₀ = 6 nM) and its effect was also blocked by CPT. In experiments with either piroxicam to block the prostaglandin (PG)-mediated pathway or N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide to block the 5-HT mediated pathway, CCPA reduced or abolished the residual reflex Isc response (Fig. 8b). CCPA could abolish the reflex response to a pulse of 5-HT (Fig. 8c) without affecting TTX-insensitive epithelial responses to carbachol or forskolin. A₁ immunoreactivity was expressed in submucous neurons, in presynaptic varicose nerve endings (synaptophysin-positive neurons), and in glia. The chemical coding of those submucous neurons is not known. Overall, available data suggest that endogenous adenosine provides a physiological brake to suppress reflex-evoked Cl^- secretion elicited by stroking the mucosa by acting at A₁ receptors located on EC cells [17, 18] and in the ENS [29]. Adenosine acts at A₁ receptors to suppress both the PG and 5-HT sensory limbs of mucosal secretory reflexes [29]. Our understanding of the role of A₁ receptors in the modulation of mucosal reflexes is restricted to the guinea pig colon; however, it is likely this effect will occur in other regions, given that the A₁ receptor provides inhibition of synaptic transmission in myenteric and submucous neurons, and in other regions including stomach antrum, jejunum, and ileum [8, 20, 22].

Fig. 8

Effect of CCPA on stroking-evoked change in Isc. A Concentration-response curve (EC₅₀ = 6 nM, n = 3-6). B Effect of CCPA (0.1 μM) on 5-HT- and PG- mediated limb of reflex pathway. a Presence of 5-HT limb after blockade of prostaglandin synthesis with 10 μM piroxicam (PIR) (n = 5). b Presence of PG limb after blockade of 5-HT limb with 1–10 μM N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (HTP) (n = 6 or 7). Con control, p < 0.05 from control and PIR or HTP. C Effect of CCPA (0.1 μM) on change in Isc evoked by a mucosal pulse of 5-HT (1 μM) (n = 3); p < 0.05 (Modified from [29] by permission)

Distribution of adenosine, P2X, and P2Y receptors and gene products in ENS

Immunohistochemical, gene chip microarray, quantitative reverse transcriptase polymerase chain reaction (RT-PCR), or in situ hybridization data have identified various purinergic receptors in the gut of various species. These include A₁, A_2a, A_2b, A₃, P2X₂, P2X₃, P2X₅, P2X₇, P2Y₂, P2Y₄, P2Y₆, P2Y₁₂, and P2Y₁₄ receptors in the ENS of guinea pig, mouse, rat, or human intestine [14, 17–19, 25, 27, 29, 49, 93, 94, 108, 109, 117–119].

Differential gene and receptor expression of adenosine A₁, A_2a, A_2b, and A₃ receptors in the human enteric nervous system

Adenosine A₁, A_2a, A_2b, and A₃ receptor gene products were differentially expressed in neural and non-neural layers of the human jejunum, ileum, cecum, and colon as well as T-84, HT-29, T98G, and BON/EC cells or in PGP 9.5-ir enteric neurons [19]. Table 1 summarizes adenosine receptor mRNA expression levels in the different layers and cells using RT-PCR. With the exception of the A₁ receptor, mRNA transcripts for A_2b, A_2a, and A₃ receptors are expressed throughout the GI tract in epithelial cells, mucosa, and submucosa suggesting a significant role in secretomotor behavior of the human gut.

Table 1 RT-PCR to detect human adenosine receptor mRNA in human intestine

Cellular localization of adenosine receptor immunoreactivity is summarized in Table 2. Adenosine A₃ immunoreactivity is also expressed in a majority of substance P (~60%) and < 10% of VIP submucous neurons. In contrast to A₃R receptors, A_2b immunoreactivity is prominent in 50% of VIP neurons and not expressed in SP neurons in jejunum, but is expressed mainly in non-VIP neurons in colonic submucous neurons. Therefore, A_2b receptors and A₃ receptors may serve different roles in secretomotor function in the two regions by acting at putative secretomotor or sensory neurons, respectively.

Table 2 Cellular localization of adenosine A₁, A_2a, A_2b, and A₃ receptor immunoreactivities in human small and large intestine

A_2a immunoreactivity is found in a subset of VIP neurons. Varicose fibers in submucous ganglia label for A_2a or A_2b immunoreactivity. A_2a and A_2b immunoreactivity is prominent in submucous neurons, but only expressed in a minority of myenteric neurons. Immunoreactivities for A_2a and A_2b receptors are colocalized in submucous neurons of the jejunum and colon (and jejunal myenteric neurons) as well as in epithelial cells of the jejunum (only region studied). A_2b immunoreactivity was also intensely expressed in s-100 positive glia in both nerve plexuses.

A₁ immunoreactivity is abundantly expressed in submucous neurons of the colon, but was undetectable in submucous neurons of the jejunum. This is consistent with functional studies on synaptic Ca²⁺ responses in submucous neurons illustrating a lack of effect of A₁ receptor antagonists expected to block ongoing inhibitory effects of endogenous adenosine at neural receptors and enhance synaptic transmission, as revealed in other tissues with functional A₁ receptors (see later discussion; [9, 21]). In addition, strong A₁ immunoreactivity is found in epithelial cells of the human colonic mucosal glands, and mRNA and proteins expressed in two human epithelial cell lines. Therefore, A₁ receptors could potentially play a role in submucosal neural reflexes, leading to suppression of intestinal secretion in the human gut as reported for the guinea pig colon [29]. The diverse but receptor-specific localization and expression of the four adenosine receptors in submucous neurons, glial cells, or epithelial cells, and the colocalization of A_2a and A_2b receptors in submucous neurons or glandular epithelia, suggests a prominent and complex role for adenosine in the modulation of enteric neural reflexes that control secretomotor functions in the human digestive tract. Functional studies on adenosine receptors in human gut would be of physiological and clinical importance.

Distribution of P2X and P2Y receptors in the enteric nervous system

P2X₂ immunoreactivity has been identified in guinea pig ENS [14, 50, 111]. In submucous ganglia of the guinea pig ileum, P2X₂ immunoreactivity occurred in all VIP-positive neurons and calbindin-positive neurons making up about 50% of the neurons. In contrast, there was not any P2X₂ immunoreactivity in NPY or calretinin-positive neurons. Overall, P2X₂R are expressed in both nerve plexuses, in inhibitory motor neurons, IPANs of the myenteric plexus in stomach and intestine, on gastric endings of vagal afferent fibers, VIP noncholinergic secretomotor neurons, and IPANs of the submucous plexus [14]. In contrast to P2X₂, P2X₃ immunoreactivity in the submucous plexus occurs in most calretinin neurons that represent cholinergic neurons projecting to mucosa or arterioles, and make up ~12% of the population [10]; therefore, the activity of some secretomotor neurons is likely to be modulated by P2X₃ receptors in guinea pig.

P2X₂ and P2X₃ immunoreactivity was also identified in nerve fibers in enteric ganglia, interganglionic fiber tracts, in the muscularis as well as in the perivascular plexus of the guinea pig gallbladder. P2X₂ or P2X₃ neurons displayed immunoreactivity for VIP (> 90%) and NOS (~50-60%) [93]. The function of these receptors remains unknown.

P2X₂ and P2X₃ immunoreactivity was also localized in rat ENS, by in situ hybridization or mRNA levels [119]. Receptors were identified throughout the GI tract in both myenteric and submucous neurons. P2X₂ immunoreactivity (56% ileum, 45% distal colon) and P2X₃ immunoreactivity (62%, 40%) was abundant in submucous neurons. P2X₂ immunoreactivity in submucous plexus colabeled for calbindin or calretinin in 30-50% of neurons. P2X₃ immunoreactivity neurons expressed calretinin (40%) and calbindin (30-75%). Coexpression of P2X₃ immunoreactivity in calbindin neurons also occurred in colorectum [116] but not in those in rat ileum [85, 108]. At least in the mouse, AH sensory (not S/type 1) neurons did express P2X₃ immunoreactivity [7]. Species and region differences may exist in distribution of the P2X₂ and P2X₃ receptors in submucous neurons (see [7, 14, 49, 93, 111, 118]). The P2X₁, P2X₄, or P2X₆ receptor was undetectable in mouse ENS. The P2X₅ receptor is widely distributed in mouse GI tract in enteric ganglia in both nerve plexuses. In the submucous plexus, P2X₅ immunoreactivity discretely colocalize with 22% of calretinin, 9% of calbindin, 6% of nitric oxide synthase, and 68% of VIP-positive neurons. Therefore, the P2X₅ receptor may be distributed in secretomotor and intrinsic sensory neurons. The mouse P2X₅ receptor could form heteromultimers with a unique pharmacology [94].

P2X₃ immunoreactivity has also been identified in submucous (and myenteric) neurons as well as in some axons/dendrites of the human colon [36, 122], but the identity of the subtypes of human neurons expressing P2X₃ immunoreactivity remains unclear. Future studies should include detailed analysis of the distribution of purinergic receptors in the human ENS.

P2Y₂ immunoreactivity occurs in neurons and fibers in both myenteric and submucous plexuses in the corpus of the stomach, jejunum, ileum, and colon of the guinea pig [118]. P2Y₂ immunoreactivity is prominent in neurons with Dogiel type I morphology. All P2X₃ neurons and none of the P2X₂ neurons in the submucous plexus (or ~50% in myenteric neurons) coexpressed P2Y₂ immunoreactivity throughout the GI tract. A majority of calretinin and NPY submucous neurons also expressed P2Y₂ immunoreactivity. Submucosal neurons with P2X₃ and P2Y₂ receptor immunoreactivity with Dogiel type I morphology may represent a subset of S/type 1 neurons with Dogiel type I morphology that display both a fast P2X and slow P2Y membrane depolarization [3]. It has been suggested that a P2X₂ or P2X₇ receptors may mediate the fast P2X response in neurons that only display a fast response [3, 14, 49, 119]. Lack of P2Y₂ immunoreactivity in calbindin submucosal neurons of the guinea pig gut argues against their expression on IPANs [39]. Regional differences occur in the colocalization of P2Y₂ immunoreactivity with P2X₃ immunoreactivity or in other types of neuronal markers. These receptors may play a role in regulation of mucosal secretory glands and the local vasculature.

The P2Y₆ receptor is widely distributed in myenteric but not submucous ganglia of the stomach, jejunum, ileum, and colon (in guinea pig), whereas the P2Y₁₂ receptor is also widely distributed in submucous ganglia. P2Y₁₂ and P2Y₆ receptors and neurons likely play different roles in the gut. A distinction between their localization is that P2Y₁₂ immunoreactivity (not P2Y₆) is also expressed in calbindin intrinsic sensory neurons and not calretinin or NOS (in both plexuses). P2Y₆ immunoreactivity is instead found in NOS and calretinin-positive neurons. Therefore, the P2Y₁₂ receptor may play a role in sensory signaling in mucosal secretomotor reflexes.

Mucosal epithelial cell lines and ion transport

In human epithelial carcinoma cell lines (HCT8 and Caco-2), cells express mRNA for P2X₁, P2X₃, P2X₄, P2X₅, P2X₆, and P2X₇ and P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, and P2Y₁₂ receptors. In addition, P2Y₁, P2Y₂, and P2X_1-7 receptor proteins are expressed in these cells. Functional studies on cell proliferation and apoptosis using selective agonists and antagonists suggested involvement of P2X₇, P2Y₁, P2Y₂, and P2Y₄ receptors but their role in secretion awaits further study [31].

ENaC and CFTR channels

ENaC mediate electrogenic absorption of Na⁺ in intestine (and other epithelial tissues). ENaC in colonic epithelia is expressed together with the CFTR Cl^- channels. Stimulation of purinergic receptors by ATP or activation of CFTR stimulate chloride secretion and inhibit amiloride-sensitive Na⁺ transport (ENaC). Inhibition of ENaC by nucleotides is not via CFTR-mediated ATP release [62]. ATP or UTP acting at P2YRs also activate Ca²⁺-dependent Cl^- channels. Na⁺ absorption in mouse colon (and airway epithelia) is inhibited by cell shrinkage (change in volume) by a mechanism that does not interfere with purinergic (or CFTR) mediated inhibition of ENaC [101]. A recent study in P2Y₂ and P2Y₄ knockout mice suggests that only the stimulation of the luminal P2Y₂ receptor mediates inhibition of electrogenic Na⁺ absorption via epithelial Na⁺ channels (ENaC) in mouse colon. In contrast, luminal P2Y₂ and P2Y₄ receptors stimulate K⁺ secretion [74].

Luminal P2 and ion transport

P2 receptors are found in both the basolateral and luminal membranes. A recent review by Leipziger (2003) summarizes the different P2X and P2Y receptors, their functions, and intracellular signaling pathways in all epithelial tissues.

Relevant information on gut luminal P2 receptors is summarized in Table 3. Several generalizations can be made from Table 3. Epithelial cells respond well to ATP and UTP. P2Y₂ and P2Y₄ receptors are very prevalent luminal receptors involved in Cl^-, HCO₃ ^-, K⁺ secretion, or mucin secretion. Nucleotide stimulation of K⁺ secretion occurs in the distal colon, K⁺ secretion and HCO₃ ^- secretion in the gallbladder (Table 3; [17, 18, 75, 90]); HCO₃ ^- secretion also occurs in ducts and is involved in the formation of pancreatic juice. Stimulation of Cl^- secretion occurs in the gallbladder, small intestine, and duct cells. Ca²⁺ is the main second messenger coupled to P2Y receptors, although cAMP can be involved as well. Region and species differences occur especially in P2X₇ and P2Y₆ receptors. Functional discrimination between P2Y₂ and P2Y₄ receptors is difficult because of the similar agonist potency profiles [102]. The persistence of a Cl^- secretory response to UTP or ATP in P2Y₂ ^-/- mice suggested that the response in mice could be mediated by P2Y₄ receptors [32]. Indeed, loss of nucleotide regulation of epithelial chloride transport occurs in the jejunum of P2Y₄-null mice [90]. P2Y₄ mRNA are expressed in rat and guinea pig submucosa [17, 18, 27], murine colonic crypts [75], stomach and intestine [90, 105], and more recently in enteric glial cells [108, 109]. In the guinea pig small intestine, P2Y₄ immunoreactivity is expressed in enteric glial cells (EGCs) in various regions of the GI tract except esophagus. EGCs are important regulators of barrier function, mucosal permeability, neuronal activity, and epithelial cell growth and are active participants in intestinal inflammation [13, 95]. Some discrepancy exists between studies on whether P2Y₄ immunoreactivity occurs in epithelial cells, but EGCs may play a role in the modulation of ion transport in gut epithelial cells (for review see [95]).

Table 3 Functional distribution of luminal P2 receptors in GI epithelial cells

Little evidence exists that luminal P2 receptors activate absorption, other than the preposition that a P2X₇ receptor is involved in stimulation of Na⁺/H⁺ exchanger type 3-mediated Na⁺ absorption in rat submandibular gland ducts [66]. In contrast, data suggest that luminal fluid secretion and a diarrhea response would occur with activation of luminal P2Y receptors in small intestine (Cl^- secretion) or colon (inhibition of Na⁺ absorption via ENaCs and K⁺ secretion); a diarrhea response may play a role in host defense reactions against potential pathogenic invading organisms. Another possibility is that the P2X₇ receptor in villus tip cells that undergo programmed cell death and exfoliation may act as a “death signal” to insure villus cell regeneration [43]. The P2Y₆ receptor is a potential therapeutic target in the treatment of cystic fibrosis gallbladder disease because UDP retained its ability to promote/stimulate Isc/ion transport changes in cystic fibrosis gallbladder epithelial cells from CFTR-deficient mice [65].

Basolateral P2Y₆ stimulates NaCl secretion

Recent evidence suggests that basolateral P2Y₆ receptors that are sensitive to UDP in colonic epithelia cells stimulate sustained NaCl secretion by evoking a synergistic increase in intracellular free Ca²⁺ and cAMP. In Xenopus oocytes coexpressing P2Y₆ with the cystic fibrosis transmembrane conductance regulator (CFTR), UDP transiently activated the Ca²⁺-activated Cl^- current and subsequently CFTR.

A_2b receptors in apical secretion

Adenosine is generated at sites of tissue injury or stress including ischemia, inflammation, and tissue remodeling by ATP catabolism. Adenosine at inflammatory sites can have either proinflammatory or anti-inflammatory effects depending on the receptor or tissue. In crypt abscesses during periods of active inflammation, ectonucleotidases convert ATP derived from neutrophils to adenosine. Adenosine causes vectorial chloride and interleukin-6 secretion [33, 73, 103]. In human jejunum, an A₂ receptor agonist 5′-N-methylcarboxamidoadenosine stimulates chloride secretion. A_2b immunoreactivity is prominent in human mucosa or epithelial cells of the cecum, colon, and jejunum (Christofi et al. 1999; [89, 104]).

At rest, in epithelial cells, the A_2b receptor is intracellular and inactive, and agonist stimulation on the apical or basolateral side of human epithelial cells induces polarized trafficking and surface expression of the A_2b receptor in intestinal epithelial cells [33]. N-ethylmaleimide attachment receptor (SNARE) proteins participate in recruitment of A_2b receptors that may be necessary for its signaling. The A_2b receptor signals through the cAMP/protein kinase A/cAMP response-element binding protein to cause apical chloride and interleukin-6 secretion. It has been suggested that interleukin-6 would promote neutrophil degranulation and enhance microbicidal activity of neutrophil trafficking to the intestinal mucosa [103].

Purinergic regulation of secretion from neuroendocrine cells in gastric mucosa

A1/gastrin release

In the stomach, adenosine protects against stress-induced gastric ulcer formation by inhibiting gastric acid secretion by direct action on parietal cells in some species (guinea pigs and dogs, [42, 48]) but not others (rats, [86]). A recent study using selective A₁ (N6-cyclopentyladenosine), A_2a (CGS 2168), and A₃ (IB-MECA) receptor agonists provided pharmacological proof that an A₁ receptor is negatively coupled to gastrin release in rat stomach. An A₁ receptor selective antagonist 8-cyclopentyl-1,3-dipropyl-xanthine abolished adenosine effects on gastrin release, and A₁ immunoreactivity was found on mucosal G cells (gastrin) and D cells (somatostatin), but not parietal cells (H⁺ ions, acid). Therefore, adenosine may suppress gastrin release by activating A₁ receptors on G cells, leading to inhibition of gastric acid secretion [123, 125].

Omeprazole-induced achlorhydria suppresses gene expression of both A₁ and A_2a receptors in the antrum and corpus mucosa. In the vascularly perfused rat stomach, omeprazole also significantly attenuated the A_2a receptor-mediated stimulation of somatostatin release along with a corresponding decrease in A_2a receptor mRNA expression. This acid-dependent change in A_2a receptor expression is a regulatory feedback mechanism to control gastric acid secretion [124]. The A_2a receptor agonist ATL-146e could almost prevent aspirin-induced gastric mucosal lesions [83].

Clinical implications of adenosine deaminase (ADA) activity

Clinical studies suggest that adenosine inhibits gastric acid secretion and may act as a gastroprotective agent. For instance, patients suffering from acid hypersecretion have higher ADA activity. There is a direct correlation between ADA activity and gastric acid output in fundic mucosa of patients with ulcer, gastritis, and achlorhydria [79]. In patients with gastric ulcers, corpus mucosa ADA activity is reduced after H2 blocker ranitidine treatment [80].

A_2a receptors and somatostatin release

Adenosine administration to the isolated vascularly perfused rat stomach inhibits gastrin release and stimulates somatostatin release [64], a potent inhibitor of gastric acid secretion. Adenosine analogs augment somatostatin-like immunoreactivity release with a potency profile of CGS 21680 = 5′-N-ethylcarboxamidoadenosine > 2-chlroadenosine > R-(-)-N6-(2-phenylisopropyl)adenosine >1-deoxy-1-[6-[[3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide > N6-cyclopentyladenosine = N6-cyclohexyladenosine > S-(+)-N6-(2-phenylisopropyl) adenosine, suggesting an A_2a receptor involvement. The A_2a receptor antagonist ZM 241385 abolished the CGS 21680 or adenosine stimulation of somatostatin release. Immunochemical distribution of A_2a receptors suggests that adenosine can act on D cells or indirectly on myenteric neurons to stimulate somatostatin release and consequently suppress gastric acid secretion [125].

Gastric preconditioning induced by short ischemia (brief occlusion of celiac artery for 5 min) protects against mucosal damage induced by severe ischemia/reperfusion or topical mucosal irritants in the stomach. Adenosine release among other mediators [63] contributes to protection. Adenosine pretreatment (10 mg/kg i.g.) reduced lesions and improved gastric blood flow similar to ischemic preconditioning. The adenosine receptor antagonist 8-phenyltheophiline (10 mg/kg i.g.) attenuated the gastroprotection due to preconditioning. Furthermore, the selective A_2a receptor agonist ATL-146e (5 μg/kg) attenuated stress-induced gastric lesions and damage and reduced production of proinflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-1β] and neutrophil infiltration, suggesting a protective effect via A₂ receptors against ulcer formation [82].

Reflex-driven intestinal diarrhea and clinical relevance of purines

The therapeutic potential of purinergic receptors in intestinal secretomotor disorders remains to be established, but evidence is building in support of endogenous adenosine, A_2a, and A₃ receptors as therapeutic targets. Toxin A (TxA) release from Clostridium difficile (a gram-positive anaerobic bacillus) can contribute to antibiotic-induced diarrhea and pseudomembranous colitis; the latter condition is associated with significant mucosal secretion and inflammation. The neural circuit mechanism deduced from the available evidence is shown in Fig. 9. Briefly, activation of nuclear factor NF-κB initiates a cascade of events involving epithelial cells, macrophages, neutrophils, mast cells, extrinsic primary afferent neurons (EPANs), and the ENS including cholinergic secretomotor neurons. Eventual hyperexcitability of neural circuits leads to amplification of the secretory response and profuse secretion to flush out the pathogenic organism [25]. The selective A_2a receptor agonist ATL 313 (0.5-5 nM) also reduced Clostridium difficile TxA-induced secretion and edema, prevented the mucosal damage and neutrophil infiltration, TxA-induced cell death, TNF-α production, as well as increased ileal ADA activity, in murine ileal loops exposed to TxA. Effects were reversed by the selective A_2a receptor antagonist ZM241385 (5 nM) [15].

Fig. 9

1Clostridium difficile toxin A (TxA) stimulates reflex-evoked intestinal secretion and causes diarrhea. 2 After TxA is internalized in enterocytes, TxA activates NF-kB and releases LTB4, PGE2, TNF. 3 After NF-kB activation, the proinflammatory mediators interleukins (IL) are released from monocytes/macrophages (MO). 4 Stimulation of processes of EPANS occurs, followed by release of substance P (SP)/CGRP that degranulates mast cells (MC) by activating NK1 receptors. 5 SP-containing EPANS release SP on cholinergic secretomotor neurons. The mediators released from mast cells include histamine, 5-HT, PGE2, and tryptase. 6 Tryptase acts on PAR2 receptors on EPANS and on IPANS to stimulate further release of PGE2. 7 The sympathetic innervation with norepinephrine release and its binding to α2-adrenergic receptors normally reduces secretion via VIP secretomotor neurons. ILs suppress norepinephrine release. These cytokines act on sympathetics to lift the sympathetic brake exerted by norepinephrine. TxA-induced release of proinflammatory mediators degranulates mast cells that amplify neural and secretory responses in order to maximize secretion in defense of the host. Treatment with an A_2a receptor agonist attenuates the TxA-induced diarrhea presumably by acting in this reflex pathway. (Modified from [25] by permission)

Recent studies provided convincing evidence that activation of an A_2a receptor attenuates intestinal inflammation in animal models of IBD and may serve as a novel therapy for IBD [15, 77, 82]. In general, adenosine has diverse functions in the GI tract in the regulation of secretion, motility, innate mucosal immunity, is protective against experimental IBD, and is important in mediating inflammatory responses at sites of injury, and has anti-inflammatory effects in epithelial cells.

Adenosine is also reported to be a conditionally essential nutrient for the gut and has been shown to be beneficial in alleviating severe diarrhea in children with cholera [20, 25, 98].

Recent findings indicate that lumenal adenosine or AMP can rapidly increase glucose transport by small intestine and increase systemic availability of 3-O-methyl glucose after an oral administration to mice. The putative A₂ receptor involved is linked to a signaling pathway involving cAMP production. Adenosine causes a rapid increase in carrier-mediated glucose uptake that is suggested to be of clinical relevance [60]. In contrast to other modulators, adenosine is a natural component of the diet, and this alone or as supplement in the diet may suggest potential applications in treatment of malabsorption in chronic intestinal inflammatory diseases. Daily intake of purines is 600-1,000 mg/day by adult individuals in the USA [99]. It has been estimated that diet and endogenous adenosine production can contribute 5-6 mM of adenosine in the lumen in individuals on a Western diet. A concentration of 1 mM luminal adenosine caused a maximum effect on glucose uptake [60].

In human intestinal epithelial cells, adenosine affects immune function and exerts anti-inflammatory effects by acting as a negative regulator of NF-κB and MAPK signaling. Adenosine pretreatment in HT-29 cells caused a reduction in stimulated IL-8 expression and secretion [55].

Neurogenic diarrhea

The adenosine A₃ receptor is one of the least understood receptors in secretomotor reflexes. The physiological relevance of A₃ receptor is unclear, and current opinion is that it may function primarily in disease or abnormal states such as ischemia/reperfusion injury, tissue damage, gut inflammation, and extremes in motor activity of the gut when endogenous adenosine release is sufficiently high to activate the low affinity receptor [53]. It has been reported by Cooke et al. [30] that dimaprit–H₂R activation of submucous neurons leads to a stereotypical cyclical pattern of secretion in coordination with motility that can last for several hours. The underlying electrophysiological correlate is cyclical depolarization and excitation of submucous neurons. This is a model of neurogenic diarrhea involving an immune-neural circuit. It proved useful in elucidating a functional role of A₃ receptors in addition to A₁ receptors in the secretomotor function and coordination of motility and secretion [8, 9]. Dimaprit was used to elicit a stereotypical cyclical increase in short-circuit current (Isc = chloride secretion) in coordination with motility. Isc was recorded in guinea pig distal colon simultaneously with muscle length in the circular orientation by sonomicrometry. A₁ receptor-selective antagonists 8-cyclopentyltheophylline (CPT) or 1,3-dipropyl-8-(2-amino-4-chlorophenyl) xanthine (PACPX) or the irreversible antagonist FSCPX caused a concentration-dependent augmentation of coordinated responses; 100 nM antagonist caused maximum blockade of A₁ receptors and enhanced Isc 133% and ICD 64% with ED₅₀ values in the nanomolar concentration range. Dimaprit responses were abolished by 10 nM A₁ receptor agonist CCPA and reversed by A₁ receptor antagonists. After knockdown of A₁ receptors with FSCPX (1 μM), the selective A₃ receptor agonist IB-MECA could still abolish coordinated responses (IC₅₀-Isc = 2.1 μM; IC₅₀-ICD = 7.1 μM) that could be reversed by the A₃ receptor antagonist MRS1191. The A₃ receptor antagonist alone could also enhance Isc and coordinated muscle responses. The coordinated response to dimaprit is abolished by nerve blockade with TTX, and remaining myogenic activity was reduced by ~50% by IB-MECA after A₁ receptor knockdown with FSCPX. MRS1191 (1 μM) could enhance TTX-sensitive distension-evoked Isc responses. A₃ receptor immunoreactivity occurs in myenteric and submucous neurons of guinea pig, mouse, rat, and human colon (Christofi, unpublished observations). Availability of knockout models for adenosine receptor subtypes should provide unequivocal proof of function.

In summary, preliminary data suggest that in a model of neurogenic diarrhea endogenous adenosine release is sufficient to act at inhibitory A₁ and A₃ receptors to modulate the stereotype neural-motor behavior triggered by the mast cell mediator histamine. Adenosine A₃ receptors may be targets for modulation of distension-evoked neurosecretory reflexes as well. The role of A₃ receptors in bowel disorders associated with diarrhea or diarrhea predominant IBS warrants further investigation.

Differential dysregulation of purinoceptors in GI disease states

As already described, purinergic signaling regulates all important physiological functions of the GI tract including epithelial transport, 5-HT release from EC cells, mucosal reflexes, sensory signaling in the ENS, synaptic transmission, as well as coordination of motility and secretion. It is reasonable then to suggest that these abnormalities in purinergic receptors or signaling pathways would have a significant impact on the overall behavior of the gut, enteric reflexes, and secretomotor behavior of the gut in bowel diseases. Purinergic receptors in gastrointestinal inflammation were the subject of a recent review by Kolachala et al. [61]. A recent study by Guzman et al. [45] using high-density oligonucleotide microarray analysis and SYBR-Green RT-PCR validation provided direct proof for differential dysregulation (upregulation or downregulation depending on the purine gene) of P2X₁, P2X₂, P2X₄, P2X₇, P2Y₁, P2Y₂, P2Y₄, P2Y₆, A_2a, A_2b, A₁, or A₃ receptors in whole thickness gut specimens of experimental trinitrobenzene sulfonic acid (TNBS) colitis in rats . Expression of A₁ and A₃ receptor gene products is also altered in a chronic ileitis model of Crohn’s disease [106]. The ATP-gated ion channel P2X₃ is increased in human IBD [122] as is the case for TNBS colitis [45]. In submucosal neurons of TNBS colitis, there is emergence of a purinergic component of the fast EPSP that is cholinergic in nature in normal animals [69, 70]. Therefore, there is plasticity of purinergic neurotransmission. Abnormalities in the neural regulation of the gastrointestinal vasculature could contribute to the pathogenesis of IBD. Recent data suggest that reduced purinergic neurotransmission via putative P2X₁ receptors may underlie a defect in sympathetic regulation of gut vasomotor function during TNBS colitis [68]. Changes in vasomotor function could also impact on secretomotor responses. Further studies are needed to identify the location of these receptors in the gut wall and better understand the functional relevance of these and other receptor abnormalities on secretomotor and vasomotor function in various species and in human gut.

It was recently shown that purinergic gene dysregulation in experimental colitis is sensitive to oral administration of the A₃ receptor agonist IB-MECA, a potential therapeutic target for IBD [45]. IB-MECA protects against tissue injury, development of colitis, and prevents dysregulation of 92% of genes in TNBS colitis, including purine genes. This illustrates a new important benefit of A₃ receptor agonists in preventing abnormalities in purine gene expression in colitis. A review by Antonioli et al. [2] discusses the pharmacological modulation of adenosinergic pathways in the therapeutic management of IBD. Adenosine A₁, A_2a, A_2b, and A₃ receptors are potential therapeutic targets as regulators of gut enteric immune responses and all components of gut motor reflexes [2, 27].

Summary and conclusions

A secretomotor reflex is activated by mucosal stroking, distension, or chemical stimulation of the mucosa leading to release of 5-HT or other mediators, subsequent activation of IPANs in synaptic communication with 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 and vasomotor functions. All cellular components of secretomotor reflexes express multiple purinergic receptors for adenosine or the nucleotides ATP, ADP, UTP, or UDP. This review focused on the emerging concepts in our understanding of purinergic regulation at these receptors, and in particular of mechanosensory reflexes. Endogenous purines can activate inhibitory A₁, A₃, and P2Y₁₂ receptors or stimulatory A₂ and P2Y₁ receptors to modulate 5-HT release. The resting “secretory tone” is influenced by endogenous purines. Stimulatory P2Y₁ receptor or inhibitory A₁/A₃ receptors are involved in mechanically evoked secretomotor reflexes and a model of neurogenic diarrhea. Recent studies identified the neural and/or non-neural distribution profiles of P2X₂, P2X₃, P2X₅, P2Y₁, P2Y₂, P2Y₄, P2Y₆, or P2Y₁₂ receptor immunoreactivity in the GI tract suggesting their role in secretomotor function. Significant species and regional differences exist in distribution and function of the receptors. Abnormal expression of purinergic receptors may contribute to functional abnormalities and diarrhea in IBD; functional studies are lacking. Adenosine A_2a or A₃ receptors are emerging as therapeutic targets in IBD and protect against purinergic receptor abnormalities or profuse diarrhea. Purines are emerging as fundamental regulators of enteric secretomotor reflexes in health and disease.

Future studies should be aimed at better understanding the function of other purinergic receptors expressed in the enteric nervous system or non-neuronal components of the reflex, in both rodents and human gut. More integrative studies on purinergic regulation of chemosensory or mechanosensory secretomotor reflexes are desperately needed. This should pave the way to better understand purine receptor biology, abnormalities in disease states, or as therapeutic targets.