Purinergic signalling in the gastrointestinal tract and related organs in health and disease

Abstract

Purinergic signalling plays major roles in the physiology and pathophysiology of digestive organs. Adenosine 5′-triphosphate (ATP), together with nitric oxide and vasoactive intestinal peptide, is a cotransmitter in non-adrenergic, non-cholinergic inhibitory neuromuscular transmission. P2X and P2Y receptors are widely expressed in myenteric and submucous enteric plexuses and participate in sympathetic transmission and neuromodulation involved in enteric reflex activities, as well as influencing gastric and intestinal epithelial secretion and vascular activities. Involvement of purinergic signalling has been identified in a variety of diseases, including inflammatory bowel disease, ischaemia, diabetes and cancer. Purinergic mechanosensory transduction forms the basis of enteric nociception, where ATP released from mucosal epithelial cells by distension activates nociceptive subepithelial primary afferent sensory fibres expressing P2X3 receptors to send messages to the pain centres in the central nervous system via interneurons in the spinal cord. Purinergic signalling is also involved in salivary gland and bile duct secretion.

Synopsis

Introduction

Gastrointestinal tract

Smooth muscle

Non-adrenergic, non-cholinergic (NANC) neuromuscular transmission

Development of NANC transmission

Receptor identification

Sphincter control

Enteric plexuses

Enteric ganglia

Enteric glial cells

Interstitial cells of Cajal and fibroblast-like cells

Mucosal epithelium

Epithelium of oesophagus

Gastric acid secretion

Gastric mucous secretion

Intestinal secretion

Purinergic vascular control in gut

Pathophysiology

Inflammatory bowel disease

Chagas disease

Hirschsprung's disease

Motility disorders

Gastric ulcers

Diabetes

Nociception

Ischaemia

Injury

Cancer

Salivary glands

Parotid gland

Submandibular gland

Gall bladder and bile ducts

Gall bladder

Bile ducts

Summary and future directions

Introduction

Atropine-resistant responses of gastrointestinal smooth muscle to parasympathetic nerve stimulation were recognised early [419, 472, 544]. It was not until the early 1960s, however, that autonomic transmission other than adrenergic and cholinergic was identified. Electrical activity was recorded in the guinea pig taenia coli using the sucrose gap technique, and after stimulation of the intramural nerves in the presence of adrenergic and cholinergic blocking agents, inhibitory hyperpolarisations and relaxations were reported ([106, 107]; see [95]). These hyperpolarisations were blocked by tetrodotoxin (TTX), a neurotoxin that prevents the action potential in nerves without affecting the excitability of smooth muscle cells [77], establishing that they were inhibitory junction potentials (IJPs) in response to stimulation of non-adrenergic, non-cholinergic (NANC) nerves. Later NANC transmitters were shown to be present in intrinsic enteric neurons controlled by vagal or sacral parasympathetic nerves [108]. NANC mechanical responses were identified at about the same time in the stomach upon stimulation of the vagus nerve [459, 460].

Identification of the transmitter released during NANC inhibitory transmission in the gut was the next step. Several criteria were postulated by Eccles and also by Paton to be needed to be satisfied to establish a neurotransmitter: synthesis and storage in nerve terminals; release by a Ca²⁺-dependent mechanism; mimicry by the exogenously applied transmitter of the nerve-mediated responses; inactivation by neuronal uptake and/or ectoenzymes; and parallel block by drugs of responses to stimulation by nerves and exogenously applied transmitter [198]. Different substances were considered in the late 1960s, including amino acids, monoamines and neuropeptides, but none satisfied the criteria. However, hints in a paper by Drury and Szent-Györgyi [191] showing extracellular actions of purines on heart and blood vessels, papers showing extracellular actions of adenosine 5′-triphosphate (ATP) on autonomic ganglia [216] and a paper showing release of ATP during antidromic stimulation of sensory nerves supplying the rabbit ear artery [326] led Burnstock and his colleagues to look at ATP and this satisfied all the criteria needed to establish it as a transmitter involved in NANC inhibitory neurotransmission (Fig. 1; [109]). A review article was published formulating the purinergic neurotransmission hypothesis [82]. Few believed in this hypothesis over the next 20 years and it was often ridiculed at meetings and symposia. Resistance to this concept was understandable because ATP was well known as an intracellular energy source involved in the Krebs cycle, and it seemed unlikely that such a ubiquitous molecule would also act as an extracellular signaller. It is now recognised that ATP, an ancient biological molecule, appears to have evolved both as an intracellular energy source and an extracellular signalling molecule. Much evidence is now available in support of the purinergic hypothesis (see [1, 83, 84, 86, 88, 97, 103, 194, 289, 336, 511, 527, 646, 700, 760]).

Fig. 1.

Mimicry of inhibitory responses of various gastrointestinal smooth muscle preparations to transmural stimulation and ATP, often followed by rebound contractions. Hyoscine (1.3 μmol/l) and guanethidine (3.5 μmol/l) were present except where stated. a Guinea pig stomach preparations consisting of strips (4 × 40 mm) cut as a spiral around the mid portion of the stomach; transmural stimulation (S, 5 Hz for 30 s), ATP (5 μmol/l for 30 s). b Guinea pig taenia coli, transmural stimulation (S, 5 Hz for 15 s), ATP (1 μmol/l for 15 s). c Guinea pig ileum, acetylcholine (ACh, 0.006 μmol/l for 30 s, hyoscine omitted), noradrenaline (NA, 0.17 μmol/l for 30 s, hyoscine omitted), ATP (5 μmol/l for 30 s), transmural stimulation (S, 5 Hz for 30 s); d guinea pig colon, transmural stimulation (S, 5 Hz for 15 s), ATP (5 μmol/l for 15 s). e Biopsy specimen of human colon cut as 10 × 5 × 4 mm strips; transmural stimulation (S, 5 Hz for 2 min), ATP (400 μmol/l for 2 min). f Rat duodenum, transmural stimulation (S, 5 Hz for 20 s), ATP (10 μmol/l for 20 s). g Rat ileum, transmural stimulation (S, 5 Hz for 30 s), ATP (50 μmol/l for 30 s). h Rat rectum, transmural stimulation (S, 3 Hz for l min), ATP (200 μmol/l for l min). i Mouse colon, transmural stimulation (S, 5 Hz for 30 s), ATP (40 μmol/l for 30 s). j Mouse rectum, transmural stimulation (S, 5 Hz for 1 min), ATP (40 μmol/l for 1 min). Time markers, 1 min (Reproduced from [110], with permission from Wiley)

Purines can influence motility, secretion and absorption in a variety of direct and indirect ways. Purines can be released from intrinsic enteric nerves, sympathetic nerves or sensory motor nerves during axon reflexes, to act directly on smooth muscle purinoceptors mediating relaxation or contraction or on epithelial cell receptors. They act on prejunctional nerve terminals to modify transmitter release from motor and inhibitory neural control pathways. They participate in synaptic transmission in myenteric and submucosal ganglia that are involved in the control of gastrointestinal motility, mucosal secretion and absorption. They act on blood vessels or interstitial cells of Cajal (ICC) thereby indirectly modulating motility patterns. Purines also can act on sensory nerve endings in the gut wall after release from epithelial cells to initiate local and/or central reflex activity that alters gastrointestinal motility and secretory patterns and initiate nociception. Other signalling roles for ATP in the gut have emerged through the years, including synaptic transmission between neurons in myenteric and submucosal plexuses, control of epithelial cell secretion and absorption, as a sympathetic nerve cotransmitter in controlling intestinal vascular tone and initiating colic pain. The roles of purines in gut pain and inflammation and the possible roles of purinergic signalling in various gut diseases will be considered (see [99]).

There was early evidence for cotransmission in sympathetic nerves supplying the guinea pig taenia coli [647]. Stimulation of periarterial sympathetic nerves led to release of tritium from guinea pig taenia coli preincubated in [³H]adenosine (which is taken up and converted largely to [³H]ATP), and the release of both tritium and noradrenaline (NA) was blocked by guanethidine. It has been claimed that ATP is the sole transmitter in sympathetic nerves supplying arterioles in the submucosal plexus of the intestine, while NA release from these nerves acts as a modulator of ATP release [209]. ‘Axon reflex’ activity is widespread in autonomic effector systems and forms an important physiological component of autonomic control of blood vessels and visceral organs, including the gut [88, 328]. The early work of Holton [326] showing ATP release during antidromic stimulation of sensory collaterals, taken together with the evidence for glutamate in primary afferent sensory neurons, suggests that ATP and glutamate may be cotransmitters in these nerves. Most enteric neurons are derived from neural crest tissue that differs from that which forms the sympathetic and parasympathetic systems and form a local control system that is capable of acting independently [234]. Cotransmission occurs in enteric neurons and the concept of ‘chemical coding’ was proposed as a consequence of the patterns of co-localisation defining specific neuron types [235]. A subpopulation of intramural enteric nerves provides NANC inhibitory innervation of gastrointestinal smooth muscle. Three major cotransmitters are released from these nerves: (1) ATP producing fast IJPs; (2) nitric oxide (NO) also producing IJPs, but with a slower time course; and (3) vasoactive intestinal peptide (VIP) producing slow tonic relaxations [91]. The proportions of the effects mediated by these three transmitters vary considerably in different regions of the gut and in different species. For example, in some sphincters, the NANC inhibitory nerves primarily utilise VIP, in others they utilise NO, and in non-sphincteric regions of the intestine, ATP is more prominent. ATP and NO have been shown to co-mediate NANC relaxation of the circular muscle of the human sigmoid colon [49].

Gastrointestinal tract

A detailed account of purinergic neuromuscular transmission in different regions of the gut is available [103].

Smooth muscle

Non-adrenergic, non-cholinergic (NANC) neuromuscular transmission

NANC inhibitory nerves are prominent in many regions of the gut (see [87, 91, 337]), but NANC excitatory nerves have also been described, notably in the guinea pig ileum, and in the gastrointestinal tract of lower vertebrates (Fig. 2) [81, 89, 110, 628]. They are also found in neonatal development (see [89, 91]). While ATP, NO and VIP appear to be cotransmitters in many of the inhibitory NANC nerves, there is much variability in their proportional effects in different regions of the gut and between species. In general, it seems that in most species, NO is the dominant cotransmitter in anterior regions of the gut, while ATP is more prominent in the posterior regions. The P2Y₁ receptor is the main receptor subtype mediating NANC inhibitory responses in the mouse gut, partly by direct action on smooth muscle and partly by activating enteric neurons that release ATP and NO [271]. There is evidence that prostaglandin is responsible for the rebound contraction following stimulation of NANC inhibitory nerves [111].

Fig. 2.

Mimicry of excitatory responses of gut segments from lower vertebrates to transmural stimulation and ATP. Hyoscine (1.3 μmol/l) and guanethidine (3.5 μmol/l) were present throughout. a Lizard ileum; transmural stimulation (S, 10 Hz for 1 min), ATP (10 μmol/l for l min). b Toad duodenum, transmural stimulation (5 Hz for 15 s), ATP (10 μmol/l for 15 s). c Toad ileum, transmural stimulation (5 Hz for 15 s), ATP (25 μmol/l for 15 s). d Goldfish large intestine, transmural stimulation (10 Hz for 1 min), ATP (12 μmol/l for 1 min). Time markers: a–c, 5 min; d, 1 min (reproduced from [110], with permission from Wiley)

Two pathways of similar magnitude were involved in nerve-mediated relaxation of pig lower oesophageal sphincter: one via NO and one via an apamin-sensitive pathway, mediated by ATP and adenosine 5′-diphosphate (ADP), acting on P2Y₁ receptors [214]. A selective P2Y₁ receptor antagonist, MRS2179, reduced the non-nitrergic component in both pigs [214] and humans [206]. ATP and adenosine are reported to augment the contractions of the guinea pig oesophagus both to cholinergic nerve stimulation and applied acetylcholine (ACh) [375]. Immunoreactivity for P2X2 and P2X3 receptors was colocalised with vesicular glutamate transport 2, a specific marker for sensory intraganglionic laminar endings (IGLEs), in the mouse oesophagus [387, 693]. ATP may be a neuromodulator in IGLEs via a P2X2, P2X3 and/or P2X2/3 receptor-mediated pathway in the oesophagus.

Although many early papers did not favour purinergic involvement in NANC inhibitory transmission in the stomach (e.g. [11, 23, 229, 316, 344, 429, 430, 521]), there is good evidence that ATP is involved in most species in concert with NO and, to a lesser extent, VIP [45, 91, 161, 162, 211, 283, 480, 491, 523, 526, 532, 728, 751]. Vagally induced NANC gastric relaxation of cat stomach is inhibited by P2 receptor desensitisation with α,β-methylene ATP (α,β-meATP), but it is likely that this is due to interference with ganglionic transmission in the vagal pathway, rather than neuromuscular blockade [176, 177]. Responses to stimulation of enteric inhibitory neurons were reported to be substantially reduced by apamin, which blocks small conductance Ca²⁺-activated K⁺ channels [30], in the circular muscle coat of the antrum, but not fundus [154]. Studies of gastric volume from anaesthetised rabbits showed that the relaxations produced by vagal nerve stimulation were mimicked by ATP, but not VIP. Purinergic inhibitory neuromuscular transmission is lacking in the antrum of P2Y₁ receptor knockout mice [278].

There is evidence that ATP mediates the non-cholinergic component of the excitatory junction potential (EJP) and contraction of intestinal smooth muscle [747]. ATP also caused a fast contraction of rat ileum by stimulation of cholinergic interneurons in the myenteric plexus [595]. Evidence was presented for two types of P2 receptor in guinea pig ileum, one where α,β-meATP and 2-methylthio ATP (2-MeSATP) were equipotent in eliciting direct contraction of smooth muscle and another where α,β-meATP, but not 2-MeSATP, produced contractions by activating cholinergic nerves [384].

There is purinergic inhibitory neuromuscular transmission in the duodenum and jejunum of most species of laboratory animals [41, 91, 457, 522, 620, 705, 729, 730]. Purinergic NANC transmission has been reported in the ileum of pig [158, 217] and humans [729, 748]. ATP and NO are NANC cotransmitters in rat ileum [48, 627]. P1 (A₁) receptors mediate prejunctional inhibition of release of ACh, ATP and other transmitters including tachykinins from enteric nerve terminals [46, 91, 427, 605, 687], while presynaptic A_2A receptors mediate facilitation of cholinergic transmission [684].

ATP and NO are cotransmitters in NANC inhibitory nerves in the colon [65, 67, 200, 324, 382, 554, 586, 673, 746]. β-Nicotinamide adenine dinucleotide (β-NAD) may be the purinergic inhibitory neurotransmitter in the colon, but not in the caecum [291]. The results using P2Y₁ knockout mice support this view [245, 246, 278]. It has been claimed that β-NAD is the inhibitory neurotransmitter, rather than ATP, in human and non-human primate colons [196, 348]. However, evidence supporting this claim has been questioned [290]. Both purinergic and nitrergic components of NANC inhibitory transmission are inhibited by apamin, but it is more effective on the purinergic component [277]. Schisandrin, a Chinese herbal medicine, has been claimed to induce NANC relaxation of the rat colon, mediated by ATP and NO, but not VIP or adenosine [733].

Development of NANC transmission

In developmental studies, NANC nerve-mediated effects were observed before birth in mouse and rabbit small intestine [263]. Quinacrine fluorescence, which indicates the presence of high levels of vesicle-bound ATP, appears before birth in enteric neurons of rabbit ileum and stomach, 3 days before catecholamine fluorescence [159]. At 17 days of gestation, NANC inhibitory and cholinergic excitatory innervation appeared simultaneously in rabbit and in mouse. The appearance of adrenergic innervation, however, lagged far behind the other two components. In an electrophysiological study of developmental changes in the innervation of the guinea pig taenia coli, the purinergic inhibitory system appeared before and matured faster than the cholinergic excitatory system [750]. P2X3 receptor-immunoreactive nerves in the embryonic (E) rat stomach are of both intrinsic and extrinsic origin [721]. Extrinsic sensory nerve fibres express P2X3 receptors as early as E12 and extend rapidly over the whole stomach by E14. The intrinsic enteric neuron cell bodies positive for P2X immunoreactivity did not appear until birth (P1). They peaked by P14 and then decreased in maturing animals. IGLEs and intramuscular arrays were first observed at P1 and P7, respectively. P2X3-positive neurons also expressed NO synthase (NOS) throughout perinatal development. Postnatal developmental changes in purinergic signalling in the small intestine have been described (see [92, 333]). In rat duodenal segments, ATP and ADP produced contractile responses at P1; the responses increased with age, but gradually decreased after P7 and were gone by day 21. In contrast, the relaxant responses to ATP and ADP appeared at day 21 and continued to increase thereafter. Responses to adenosine or adenosine monophosphate (AMP) were not elicited before day 14, which were small relaxations that increased with age. A_2B receptors were present at day 15 in the eat duodenum, but A₁ receptors did not appear until after day 20, both receptor subtypes mediating relaxation, while A_2B receptors mediated contraction of the muscularis mucosa from day 10. The longitudinal muscle of the colon relaxed via A_2B and P2Y receptors, while the muscularis mucosa contracted via A₁ and P2Y₂ or P2Y₄ receptors. From P3 to P8, P2Y₁ receptors mediated contraction of the mouse gastrointestinal tract, but there was relaxation of longitudinal muscle throughout the gastrointestinal tract from day 12 onwards [272]. The shift from contraction to relaxation occurred 1 week before weaning, associated with changes that take place in the gut when the food compositions change from maternal milk to solid food.

Receptor identification

ATP and ADP produce contractions of rabbit oesophageal muscularis mucosae [548] and purinoceptors were identified [704]. Adenosine, acting via A₁ receptors, contracted cat oesophageal smooth muscle [615].

The P2 receptor subtypes involved in gastric motility are still not entirely clear (see [532]). Most reports suggest that a P2Y receptor is involved in relaxation. It seems likely that a P2X receptor is involved in contraction [494]. There is immunostaining for P2X receptors in the muscularis externa and muscularis mucosa [91]. The muscularis mucosae of the rabbit stomach contracted in response to ATP and ADP, but not to AMP or adenosine [549]. Muscular P2Y receptors mediate relaxation in the mouse stomach [492]. A novel function of the P2Y₁₄ receptor, associated with the contractility of the rodent stomach, has been reported [43]. Cytidine-5′-diphosphocholine, an endogenous nucleotide, used for the treatment of neurodegenerative disorders, produced contractions of mouse gastric fundus through, at least in part, purinoceptors and Rho/Rho kinase signalling [303]. Uridine adenosine tetraphosphate produces contraction of gastric smooth muscle via P2Y receptors [744].

Analysis of the P2 receptor subtypes involved in motility in the small intestine revealed that:

1. P2Y₁ receptors mediate NANC inhibitory transmission to intestinal smooth muscle of laboratory animals and humans [243, 246, 690]. α,β-MeATP has a potent relaxant action in some preparations [366, 367, 535]. It seems likely that α,β-meATP is acting on P2X3 receptors [172, 644] on nerve varicosities to release ATP, which then acts on P2Y₁ receptors on smooth muscle eliciting relaxation (see [396]). Occupation of P2Y₁ receptors on the taenia coli activated phospholipase (PL) C, increased production of inositol 1,4,5-trisphosphate (InsP₃) and released intracellular Ca²⁺ ([Ca²⁺]_i) [406]. This led to enhanced production of spontaneous transient outward currents, which caused hyperpolarisation. It has been proposed that β-nicotinamide, an adenine dinucleotide, which acts on P2Y₁ receptors, is released together with ATP from NANC inhibitory nerves supplying the gut [496, 497]. The responses of rat ileal myocytes to ADP were not competitively blocked by pyridoxalphosphate-6-azophenyl-2′,5′-disulfonic acid (PPADS) [685]. High levels of P2Y₆ receptor mRNA were found in the human small intestinal muscle [147]. Uridine diphosphate (UDP) activation of P2Y₆ receptors produced contraction of mouse ileum [763].

2. P2Y₂ and/or P2Y₄ receptors mediate smooth muscle contractions in the small intestine of most lower vertebrates [81, 628], since they are activated by uridine 5′-triphosphate (UTP) as well as by ATP [366, 367, 384, 705].

3. Contraction of rat duodenal muscularis mucosae smooth muscle is mediated by P2X receptors [367].

4. Contraction is mediated by P2X receptors in guinea pig ileum [358, 376, 380, 488, 602, 702, 703]. α,β-meATP-induced ileal contractions were inhibited in P2X1 receptor knockout mice [683]. ATP and α,β-meATP produced contractions that were antagonised by atropine and it was concluded that P2 receptors mediated release of ACh from cholinergic enteric nerves [39, 40, 47, 384, 488, 512, 639, 640, 759].

Patch-clamped enzymatically dispersed smooth muscle cells from mouse ileum were activated by P2 purinergic agonists whose effects were attenuated by apamin [688]. Myocytes isolated from the longitudinal muscle of jejunum and ileum showed a slow transient increase in [Ca²⁺]_i in response to ATP and 2-MeSATP, suggesting P2Y receptor mediation [63]. The NANC relaxation of the human ileal longitudinal and circular muscle is inhibited by MRS2179, a selective P2Y₁ receptor antagonist [669]. Duodenal brush border intestinal alkaline phosphatase degrades ATP released from the epithelium and stimulates HCO₃⁻ secretion via P2Y receptor activation [483]. The ecto-purinergic system may regulate cell surface pH, maintaining a protective alkaline microclimate during acid stress.

Fast relaxations of the guinea pig taenia coli in response to ultraviolet light (UV) (340–380 nm) closely resembled the relaxations produced by NANC inhibitory nerve stimulation and ATP [105]. The responses to UV light were unaffected by TTX and were not due to ATP release, so it was proposed that UV light was probably acting on some components of the purinergic receptor complex. The first structure–activity studies of analogues of adenine nucleotides in taenia coli showed that di- or triphosphate groupings were of prime importance in binding adenine nucleotides to the putative smooth muscle receptor and that hydrolysis of the terminal phosphates was not a requirement for inhibitory activity. Later studies extended these findings [455, 599, 600] and the actions of enantiomers of 2-azido analogues on taenia were also examined [163]. Separate receptors for adenosine (P1) and ATP/ADP (P2) were proposed [85], and this was supported by later studies of the guinea pig taenia coli [75, 219, 600]. Theophylline blocked relaxations produced by adenosine, but not by ATP. The stereoselectivity of P2 and P1 receptors was studied in the taenia coli. It was shown that while P2 receptors mediating inhibitory responses in taenia coli showed marked stereoselectivity, those mediating excitatory responses in guinea pig bladder showed little stereoselectivity [112]. The A₂ receptor subtype was identified in the guinea pig taenia coli [112] and later adenosine analogues were shown to relax guinea pig taenia coli via P1 (A_2B) receptors [558]. β-NAD acts via Pl receptors, while β-nicotinamide adenine dinucleotide phosphate (NADP) acts as a P2 receptor agonist [100, 643]. The potent agonist N⁶-methylATP and the less potent agonist 2′-deoxyATP were shown to be selective for P2Y receptors in the taenia coli, but were inactive at P2X receptors [113]. Structure–activity relationships of pyridoxal-6-arylazo-5′-phosphate and phosphonate derivatives as P2 receptor antagonists showed that the phenylazo phosphate derivative and the ethyl phosphonate analogue of isoPPADS had antagonist actions on the guinea pig taenia coli P2Y receptor [391]. Diadenosine polyphosphates were claimed to act as P2Y agonists in the taenia coli with a potency order AP₃A = AP₄A > ATP > AP₄ = AP₅A, relaxations that were antagonised by suramin [335]. Comparison of the structure–activity relationships of ectonucleotidases with those of the P2 receptor was described on the guinea pig taenia coli [696]. Methylene isosteres of ATP and ADP resisted dephosphylation. Isopolar phosphonate analogues of ATP were inactive on P2Y receptors in taenia coli [164].

In murine colonic myocytes, there was a high potency of pyrimidines and it was suggested that ATP activated the low-threshold voltage-activated non-selective cation currents and depressed the relatively high-threshold voltage-activated (L-type) Ca²⁺ current via P2Y₄ receptors and stimulation of the PLC/protein kinase C (PKC) pathways [485]. Relaxation of the rat colon longitudinal muscle was elicited via P2Y and P1 (A₂) receptors [25]. ATP release of Ca²⁺ from intracellular stores was mediated by P2Y receptors, shown by employing single channel recording from cell patches of mouse colonic and ileal smooth muscle cells [44, 399, 688]. P2Y₁ receptors mediate inhibitory motor control of colonic excitability and transit in the mouse [349, 756], human [243, 244] and rat [294] colon. ATP and β-NAD and their metabolites, ADP and ADP-ribose, produced relaxation of murine colonic smooth muscle, and it was suggested that they might be involved in motility disorders [195]. It was concluded in a recent review that the P2Y₁ receptor-mediated inhibition may be a general phenomenon in the gut.

At least three subtypes of P2 receptors were claimed to be present in the circular muscle of the guinea pig colon [747], namely: P2 receptors, producing apamin-sensitive hyperpolarisation and relaxation, activated by ATP and sensitive to suramin and PPADS; P2 receptors, producing an apamin-sensitive hyperpolarisation and relaxation, which are activated by adenosine-5′-(β-thio)-diphosphate (ADPβS), but resistant to suramin and PPADS; and P2 receptors produce contractions, which are activated by ADPβS and are sensitive to suramin and PPADS. Canine colon circular myocytes expressed mRNAs for P2X2, P2X3 and P2X4 receptors, while longitudinal myocytes expressed mRNAs for P2X3 and P2X5 receptors, but no mRNA for P2X1, P2X6 or P2X7 receptors [425]. Activation of these receptors produced non-selective cation currents that depolarised and excited muscles in both layers. ATP also elicited contractions of the longitudinal muscle of the mouse distal colon acting directly on smooth muscle and indirectly via activation of cholinergic neurons [761]. Immunohistochemistry showed that P2Y₁ receptor proteins are dominant in smooth muscle cells of rat distal colon that mediate the potent effects of ADPβS, while neuronal P2X3 receptors might be involved in the relaxant response to α,β-meATP [674], probably via ATP release and activation of P2Y₁ receptors. It was also suggested that neuronal P2Y₂ receptors mediate relaxation, partially via NO release. RT-PCR and pharmacological characterisation of P1 receptors in the guinea pig distal colon led to the suggestion that adenosine mediates relaxation through two different receptor subtypes: A_l receptors on enteric neurons and A_2B receptors on smooth muscle [371].

Adenosine, ATP and related compounds produced contraction of the muscularis mucosae of the rat colon [24, 26, 334]. It was concluded that P1 (A₁) and P2Y receptors mediated these responses. However, immunohistochemical expression of P2X1 receptors in the smooth muscle of the muscularis mucosae, but not the muscularis externa, suggested that P2X1 rather than P2Y receptors were involved. The presence of P1 (A₁) receptors in rat colon muscularis mucosae, mediating contraction, was confirmed in a later study [566], although part of the response was claimed to be due to products of the cyclooxygenase pathway [567]. ATP inhibited swelling-activated Cl⁻ currents in canine colonic smooth muscle, and it was suggested that this may be related to the regulation of myogenic activation in response to distension [181]. Intestinal myofibroblasts form a monolayer network beneath the mucosal epithelium. mRNA for P2Y₂ receptors was expressed and ATP induced increases in [Ca²⁺]_i and contraction of these cells [502]. The human cathelicidin, LL-37, is involved in innate immune responses, angiogenesis and wound healing. It was suggested that LL-37 stimulated migration of the human colon cell line, Caco-2, via P2X7 receptors [499] affecting intestinal epithelial barrier integrity [533].

ATP released as a cotransmitter from nerves or by paracrine/autocrine release from non-neural cells and its breakdown product, adenosine, acted on guinea pig distal colon mucosal epithelial cells to increase short circuit currents corresponding to electrogenic Cl⁻ secretion and also activated electrogenic K⁺ secretion via P1 (A_2B) receptors on both apical and basolateral surfaces [753]. Intestinal epithelial cells form a permeable, but selective, barrier that functions as defence against pathogens as well as performing digestive functions. They secrete and respond to cytokines that recruit neutrophils and macrophages. ATP and UTP, via P2Y₂ receptors, serve as chemotactic agents by stimulating the migration of neutrophils and macrophages through the intestinal epithelial cell barrier [420]. It was suggested that this mechanism may contribute to the inflammatory mechanisms that contribute to inflammatory bowel disease. P2Y₄ receptors have also been identified immunohistochemically in the human bowel [145].

Postjunctional P1 (A₁ and A₂) receptors mediate relaxation of rat and mouse duodenum longitudinal muscle [308, 495, 762]. Adenosine-induced relaxation of possum duodenum is mediated by A₃, as well as A_2A receptors [711]. P1 (A_l and/or A_2B) receptors mediate contraction of rat ileal muscularis mucosae [508, 509]. P1 receptor-mediated contraction of the ileum of Suncus murinus, a primitive insectivore, has been reported [500].

Sphincter control

There is evidence for the involvement of ATP in the control of pyloric and internal anal sphincters [562, 629] and in the NANC inhibitory responses of the lower oesophageal sphincter [351, 743]. Studies of NANC inhibitory responses of the rat pyloric sphincter provided evidence for components mediated by both NO and ATP [354, 629] via P2Y₁ receptors, but P2X4 receptors were also expressed in this sphincter [596]. An ATP component was involved in relaxation of the rabbit sphincter of Oddi [352], and ATP and ADP were shown to have inhibitory actions on the cat sphincter of Oddi [551]. An excitatory response to ATP in the possum sphincter of Oddi involved P2X receptors, whereas the later inhibitory response was mediated by P2Y receptors [713]. ATP was considered early as a possible NANC inhibitory transmitter in the human internal anal sphincter [80]. Both ATP and adenosine were shown to produce concentration-dependent relaxations of the guinea pig [156], rabbit [59], rat [510, 529] and sheep [3] internal anal sphincter. Relaxation and hyperpolarisation during electrical field stimulation of the mouse internal anal sphincter was mediated by the cotransmitters ATP and NO [383]. ATP hyperpolarised and relaxed the internal anal sphincter of guinea pig [440, 562] and rat [171]. P2Y₁ receptors mediated the effects and both apamin-sensitive K⁺ channels and apamin-insensitive conductances were involved in hyperpolarisation and relaxation of the mouse internal anal sphincter [466].

Enteric plexuses

Enteric ganglia

Elegant electrophysiological studies, carried out during the past 20 years, demonstrated purinergic synaptic transmission between enteric neurons in both myenteric and submucous plexuses in both in situ and tissue culture preparations (see [68, 97, 98, 248, 250, 252, 341, 569, 570, 572, 671]). In a recent study of the development of the mouse enteric nervous system [313], almost all enteric neurons responded to ATP early at E11.5, E12.5, E15.5 and E18.5, and receptors for ATP were expressed early in E11.5 cultures, followed by the appearance of receptors to 5-hydroxytryptamine (5-HT). ATP is released together with ACh from the majority of presynaptic terminals [436].

Myenteric ganglia. P2X receptors

The effects of ATP in single myenteric neurons from guinea pig small intestine were first shown by Katayama and Morita [378], using intracellular electrodes. ATP produced hyperpolarisation in 80 % of AH neurons and depolarisation in 90 % of S neurons.

The studies of purinergic signalling in guinea pig myenteric neurons have been extended by several groups. Whole cell and outside-out patch-clamp recordings have been used to characterise the physiological and pharmacological features of P2X receptors on myenteric neurons of the guinea pig ileum [34]. Agonist rank order of potencies were as follows: adenosine-5′-(γ-thio)-triphosphate (ATPγS) = ATP = 2-MeSATP > > α,β-meATP = β,γ-methylene ATP, while adenosine and UTP were inactive. Fast excitatory postsynaptic currents (fEPSCs) were recorded in primary cultures of myenteric neurons from guinea pig intestine [435, 757]. Hexamethonium-resistant fEPSCs were abolished by PPADS. The slowly desensitising receptors that were α,β-meATP insensitive were likely to be P2X1 receptors [34], whereas the minority of rapidly desensitising receptors were probably P2X2 receptors. The fast excitatory postsynaptic potentials (EPSPs) mediated in part by P2X receptors were prominent in myenteric neurons along the small and large intestine, but were rare in the gastric corpus [435]. P2X and nicotinic receptors were shown to be linked in a mutually inhibitory manner in guinea pig myenteric neurons [758]. P2X2 receptors are expressed by subtypes of guinea pig enteric neurons, namely inhibitory motor neurons, vasomotor neurons, cholinergic secretomotor neurons, intrinsic sensory neurons and the endings of vagal afferent fibres in the stomach [120, 482]. Studies using P2X2 receptor knockout mice showed that P2X2 receptors contribute to fast synaptic excitation of myenteric neurons in small intestine [572]. P2X2 homomeric receptors appear to be the predominant receptors mediating fast synaptic excitation in the gut [248, 250, 524]. Intrinsic sensory neurons in the gut, identified as Dogiel type II neurons, express P2X2 receptors [237].

P2X3 receptors are expressed by excitatory and inhibitory motor neurons, ascending interneurons and cholinergic secretomotor neurons [557], but were claimed not to be expressed by intrinsic sensory neurons in guinea pig ileum [675]. Peristalsis was impaired in the small intestine of mice lacking P2X3 receptors [57]. The distribution of the mRNA and protein of P2X2 and P2X3 receptors has been described in the rat enteric nervous system [722]. It was shown that the P2X2 receptor was the dominant P2X receptor subtype in the myenteric plexus. Most myenteric S neurons in guinea pig small intestine expressed P2X3 receptors with about half of these being inhibitory motoneurons [571]. P2X5 receptors were present on nerve fibres that envelop ganglion cell bodies in the myenteric and submucous plexuses in mouse intestine, probably as heteromultimers with P2X2 receptors on enteric sensory neurons [587].

Studies of purinergic signalling in dispersed primary cultures of guinea pig myenteric plexus were carried out by the group of Mulholland. Extracellular ATP was shown to mediate Ca²⁺ signalling in primary cultures of neurons from guinea pig myenteric plexus via a PLC-dependent mechanism [393]. Different enteric neurons responded to combinations of ATP with ACh, ATP with substance P (SP), ATP with ACh, ATP with ACh and SP, ATP with bombesin or ATP with ACh and bombesin [392].

Two distinct types of P2 receptors are linked to a rise in [Ca²⁺]_i in guinea pig intestinal myenteric neurons. Both intestinal AH and S neuronal phenotypes responded to ATP by increases in [Ca²⁺]_i [139, 140]. ATP regulates synaptic transmission by both pre- and post-synaptic mechanisms in guinea pig myenteric neurons. Where ACh and ATP act as cotransmitters, there is an interaction between nicotinic and P2X receptors [173]. In the C-terminal tail of P2X2 receptors, there is cross-inhibition between α3β4 nicotinic and P2X2 receptors [173]. ATP augments nicotinic fast depolarisations, but inhibits muscarinic and SP-mediated depolarisations in both AH and S neurons [374].

Exogenous and endogenous ATP released during increases in intraluminal pressure inhibit intestinal peristalsis in guinea pig [318]. A major role is played by ATP in excitatory neuro-neuronal transmission in both ascending and descending reflex pathways to the longitudinal and circular muscles of the guinea pig ileum triggered by mucosal stimulation [143, 638]. Descending inhibitory reflexes involve P2X receptor-mediated transmission from interneurons to motor neurons in guinea pig ileum [58, 70]. Distension-evoked descending contractile responses of the circular and longitudinal muscle layers are regulated by separate sympathetic pathways, one mediated by P2 receptors, the other by 5-HT3 receptors [486]. Inhibitory interactions occur between P2X and γ-aminobutyric acid (GABA)-A receptors on myenteric neurons from the guinea pig small intestine [377].

IGLEs have been identified as specialised mechanosensitive endings of vagal afferent neurons arising from the nodose ganglion. P2X2 receptors are present on IGLEs in the mouse gastrointestinal tract, mainly in the stomach, some in the intestine [121]. IGLEs were first demonstrated at birth showing strong immunostaining for P2X3 receptors, and P2X3 receptors expressed on extrinsic nerves appeared as early as E12 in developing rat stomach in the trunk and branches of the vagus nerve [721].

Neuron cell bodies in the myenteric ganglia appear in the first trimester. Neurons expressing P2X3 receptors peaked at 45 % during development, but at P60 only 11 % were P2X3 receptor immunoreactive. Several enteric neurotransmitters have been claimed to modulate ATP release by acting on NANC neuronal cell bodies in the myenteric plexus. For example, morphine or enkephalin inhibition of NANC-evoked relaxations was reversed by nalaxone [356, 616]. Enkephalin is very effective in inhibiting NANC IJPs evoked in human colon [339]. ATP transiently facilitates ACh release from myenteric motoneurons via prejunctional P2X2 receptors, and following breakdown to ADP and adenosine, there is inhibition of ACh release via P2Y₁ and P1 receptors [192]. Evidence has been presented that 5-HT released ATP from nerve varicosities isolated from the myenteric plexus of the guinea pig ileum [8, 247]. GABA receptors mediate relaxation of rat duodenum by activating intramural NANC neurons in guinea pig intestine [410], rat duodenum [454], dog ileocolonic junction [64] and guinea pig distal colon [481].

Myenteric ganglia. P2Y receptors

Evidence for the expression of P2Y receptors on enteric neurons in addition to P2X receptors has been presented [255, 676, 710, 723, 724]. In the mouse gastrointestinal tract, P2Y₁ receptors on NANC myenteric neurons mediate relaxation [271]. In the guinea pig enteric nervous system, slow excitatory synaptic transmission on S-type neurons is mediated by P2Y₁ receptors [341]. P2Y₁ receptors mediate slow excitatory synaptic potentials on interneurons during descending inhibition in guinea pig ileum [661]. P2Y₂ receptors are expressed by S-type (Dogiel type I) neurons in both the myenteric and submucosal plexuses throughout the guinea pig gut. In the myenteric plexus, 40–60 % of P2X3 receptor-immunoreactive neurons were immunoreactive for P2Y₂ receptors and all P2X3 receptor-immunoreactive neurons expressed P2Y₂ receptors in the submucosal plexus [723]. Thirty to 36 % of neurons in ganglia in the myenteric, but not submucosal plexus of the guinea pig gut, expressed P2Y₆ receptors [724]. Forty to 46 % of the neurons in both myenteric and submucosal plexuses were immunoreactive for P2Y₁₂ receptors. Twenty-eight to 35 % of P2Y₆ receptor-immunoreactive neurons coexist with NOS, while all P2Y₁₂ receptor-immunoreactive neurons were immunopositive for calbindin, probably AH intrinsic sensory neurons. In the rat distal colon, P2Y₁ and P2Y₆ immunoreactivity was located on smooth muscles, P2Y₄ and P2Y₆ receptor immunoreactivity on glial cells in both plexuses, P2Y₄ receptors on ICCs, while P2Y₂ and P2Y₁₂ receptors were identified on enteric neurons [676]. There is a shift from contraction to relaxation via P2Y₁ receptors during postnatal development of mouse intestinal smooth muscle 1 week before weaning, perhaps associated with the change from maternal milk to solid food [272]. The role of both P2X and P2Y receptors in sympathetic transmission at functionally identified synapses in the enteric nervous system has been reviewed [310].

Myenteric ganglia. Adenosine (P1) receptors

P1 (adenosine) receptors on myenteric neurons were claimed following the demonstration that in the guinea pig ileum, methylxanthines (P1 receptor blockers) antagonised the dipyridamole (adenosine uptake inhibitor)-induced inhibition of peristaltic activity [618, 678]. Adenosine inhibited forskolin-induced excitation of myenteric nerves suggesting that adenosine acts to prevent activation of adenylate cyclase by substances mediating slow EPSPs [745]. Adenosine applied to AH (type II) neurons, but not to S (type I) neurons, resulted in membrane hyperpolarisation and decreases in input resistance following opening of K⁺ channels [537]. Adenosine suppressed nicotinic synaptic transmission in myenteric ganglia of the guinea pig gastric antrum and small intestine, by interacting with presynaptic P1 receptors on AH type II neurons [134–137]. A minority subset of AH neurons also express A₂ subtype receptors coupled to adenylate cyclase mediating excitation of these neurons [138]. Adenosine acting at A₁ presynaptic receptors suppressed slow EPSPs and amplified slow inhibitory postsynaptic potentials in myenteric neurons [134, 374].

Adenosine suppressed cyclic AMP (cAMP) formation in myenteric ganglia in vitro [720]. Reduction of cholinergic synaptic transmission via prejunctional A₁ receptors involves the activation of pertussis toxin-insensitive G proteins [35]. Differential gene expression of A₁, A_2A, A_2B and A₃ receptors in human enteric neurons has been reported [141]. Fine-tuning modulation of myenteric and submucosal motoneuron activity by adenosine has been claimed acting via presynaptic A₁ receptors [153, 256]. Synaptosomal preparations from the guinea pig ileum myenteric plexus have been described [74, 189]. Adenosine inhibited the nicotinically induced release of [³H]ACh from synaptosomes [565, 617]. Both A₁ and A₂ subtypes appear to be involved [131, 132].

A neuroprotective role for adenosine in ischaemia has been postulated [178]. The P1 agonist, 5′-N-ethylcarboxamidoadenosine (NECA), is a potent inhibitor of morphine withdrawal-induced diarrhoea in rats [664]. A_2B receptors mediate inhibition of secretion and it was suggested that A_2B adenosine agonists may be of clinical value in the management of some types of diarrhoea [312].

Submucosal ganglia

Adenosine depolarised submucosal neurons by acting at P1 (A₂-like) receptors and to act presynaptically via P1 (A₁) receptors to inhibit the release of ACh from intramural nerves and of NA from sympathetic nerves in the submucosal plexus [31, 32].

Slow postsynaptic inhibitory and excitatory potentials in S neurons of the submucous plexus of the guinea pig caecum were mimicked by various transmitters, the non-reversing type of slow excitatory postsynaptic potential was mimicked only by ATP [478]. ATP-induced fast transient depolarisation of most AH-type neurons and fast transient depolarisation followed by slower onset, longer lasting depolarisation of S-type neurons was reported [33], mediated by P2X and P2Y receptors, respectively [37]. Many neurons in the submucous plexus were immunopositive for P2X3 receptors and were colocalised with calretinin and calbindin, indicating labelling of intrinsic sensory neurons [722]. Using whole-cell patch recording, superfusion of ATP and analogues was shown to evoke rapidly desensitising inward current, and ATP-induced single channel currents were also recorded [33, 285], perhaps involving P2X4 or P2X6 receptors (see [90]). Functional interactions between nicotinic and P2X receptors have been demonstrated in freshly dissociated guinea pig submucosal neurons in primary culture [36, 285, 758]. Later, two subtypes of P2X receptors were identified in neurons of guinea pig ileal submucosal plexuses [286]. Fast inhibitory interactions between P2X and 5-HT₃ receptors in guinea pig submucosal neurons were described [38]. Fast, slow and intermediate EPSPs were recorded in neurons of the submucous plexus of the guinea pig ileum [487]. P2X receptors mediated a subpopulation of fast EPSPs. The slow EPSPs and intermediate EPSPs were blocked by MRS2179, a P2Y₁ selective antagonist. A P2Y₁ receptor has been cloned and characterised from guinea pig submucosa. P2Y₁ receptor signalling involved in synaptic transmission in the human submucous nerve plexus is a predominant pathway, and the A₃ receptor inhibits purinergic and cholinergic transmission in the human enteric nervous system [717].

Intrinsic sensory neurons

Both intrinsic and extrinsic sensory nerves are present in the enteric nervous system (see [62, 412]). Intrinsic sensory neurons are located in both the submucosal and myenteric ganglia [236]. Their terminals are largely in a subepithelial plexus. They mediate enteric reflex activities, including peristalsis. Extrinsic sensory nerves also have terminals in the subepithelial plexus. Their cell bodies are in dorsal root and nodose ganglia. They, too, can evoke enteric reflex activities via the spinal cord and brainstem and they mediate visceral pain. Most of the data about intrinsic enteric sensory nerve activities has been reported for the guinea pig ileum. However, this may or may not represent comparable activities in other species and regions of the gastrointestinal tract.

The intrinsic sensory neurons have been identified electrophysiologically as AH-type and morphologically as Dogiel type II cells, while S-type (that include Dogiel type I neurons) are motoneurons or interneurons. Depending on the species, most AH cells express calbindin and/or calretinin. Adenosine acts presynaptically via A₁ receptors and postsynaptically via A₁, A_2A and A₃ receptors on intrinsic sensory neurons [129]. Synaptic transmission to intrinsic sensory neurons is mediated by P2X receptors [54], perhaps of the P2X2 subtype in guinea pig intestine [120]. Postsynaptic inhibition via P2Y receptors has also been claimed to be present on intrinsic sensory nerves [52, 53]. P2X3 receptors are expressed by intrinsic sensory nerves in rat ileum and distal colon [722] and on sensory neurons in human myenteric plexus. P2Y₁₂ receptors are expressed by sensory neurons in guinea pig myenteric plexus [724].

Mucosal terminals of intrinsic sensory neurons in the guinea pig intestine are activated by ATP and α,β-meATP [54], which supports the hypothesis of Burnstock [91, 93] that ATP released from mucosal epithelial cells has a dual action on P2X3 and/or P2X2/3 receptors on subepithelial sensory nerve terminals. It was proposed that ATP acts on the terminals of low-threshold intrinsic enteric sensory neurons to initiate or modulate intestinal reflexes, while it acts on the terminals of high-threshold extrinsic sensory fibres to initiate pain. Support for this hypothesis was gained from a rat pelvic sensory nerve–colorectal preparation [718]. Distension of the colorectum led to an increase in the release of ATP from mucosal epithelial cells and also evoked pelvic nerve excitation, which was mimicked by application of ATP and α,β-meATP and attenuated by the selective P2X3 and P2X2/3 antagonist, 2′(3′)-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), and by PPADS. Purinergic mechanosensory transduction has also been implicated in reflex control of intestinal secretion [149, 726]. Extrinsic and possibly intrinsic sensory nerves associated with mucosal epithelial cells appear to be sensitive to pH, involving P2X2 and P2X2/3 receptors [329].

Enteric glial cells

Enteric glia, in about a 2:1 ratio with enteric neurons (depending on species) [240], display morphological and molecular similarities to astrocytes in the central nervous system (CNS) and they stain for glial fibrillary acidic protein [362, 505, 588]. They respond to ATP and UTP via P2 receptors by increasing intracellular calcium, probably via P2Y₂ or P2Y₄ receptors [393]. Later evidence showed release of Ca²⁺ from intracellular stores supporting the involvement of P2Y receptors [598]. Cultured enteric glia responded to ATP [288, 755]. Immunohistochemical studies showed expression of P2X7 receptors on enteric glial cells [679] as well as P2Y₄ receptors [677]. Ectonucleotide NTPDase2 is exclusively localised on the surface of enteric glial cells, suggesting that enteric glia regulate the responses to ATP and UTP [72]. Evidence was presented to suggest that enteric glia release ATP, to participate in intercellular propagation of Ca²⁺ waves between enteric glial cells and Ca²⁺ wave-induced ATP release was shown to elicit neuronal responses [755]. ATP release from enteric glia was also proposed to produce a feedback system for ICCs to modulate slow wave activity [101]. It has been suggested that ATP released from sympathetic nerves activates enteric glia [301]. Purinergic neuron–glia interactions in the enteric nervous system have been described, reflecting similar mechanisms in the CNS [300]. It was shown that stimulation of enteric neurons elicited increased [Ca²⁺]_i in enteric glial cells, mimicked by exogenously applied ATP, probably by P2Y₄ receptors. Parasympathetic and sympathetic varicosities in the myenteric plexus co-release ATP with ACh and NA, respectively ([8, 515]; see [98]). It was concluded from an electrophysiological study of a mouse enteric neuron–glial culture preparation that neuronal cells primarily express P2X receptors, while glial cells primarily express P2Y receptors [241].

Interstitial cells of Cajal and fibroblast-like cells

ICCs are a specialised cell type that act as pacemakers to regulate the activities of smooth muscle cells in the gut. P2X2 and P2X5 receptors were shown to be expressed on ICC’s in guinea pig intestine [101] and more recently P2Y₄ receptors were also identified on ICCs in guinea pig gastrointestinal tract mediating modulation of intracellular Ca²⁺ oscillations [677]. This is consistent with ATP being released as a cotransmitter from enteric nerves and glial cells to regulate the activities of these cells [101]. Purinergic modulation of pacemaker [Ca²⁺]_i activity in ICC’s was mediated by P2X receptors [239]. ICCs in human and murine small intestine express P2Y₁ and P2Y₄ receptors [126].

P2Y₁ receptors have been identified on ‘fibroblast-like cells’ that form a network of cells distinct from ICCs located between intestinal circular and longitudinal smooth muscle, near terminals of enteric motor neurons and with gap junction connectivity with muscle cells [414]. Apamin and MRS2500, a selective P2Y₁ antagonist, blocked the activation of currents and increase in [Ca²⁺]_i by purine nucleotides (see Fig. 3). The majority of subserosal interstitial cells, probably fibroblast-like cells, in the guinea pig proximal colon respond to ATP via P2Y₁ receptors and may thereby contribute to smooth muscle relaxation [652]. Three cell types form a syncytium in mouse colon, namely smooth muscle cells, ICCs and platelet-derived growth factor receptor α-positive cells, and these cells are claimed to show differential expression of genes related to purinergic signalling [553].

Fig. 3.

a Effects of ATP on fibroblast-like cells were concentration-dependent and repeatable. a Brief exposures (20 s) to ATP (0.1, 1 and 10 μm and 1 mm) at a holding potential of −50 mV (approximate resting potential of murine colonic muscles) caused large outward currents, resolved at 0.1 μm and nearly maximal at 10 μm. b ATP concentration vs. current response in six cells. The X-axis is the log of ATP concentration (metre), and the Y-axis is the integral of ATP response current (area under the curve; AUC) normalised to the maximum response integral. Data were fitted with a Boltzmann function and EC₅₀ was calculated to be 1.96 μm (Hill slope = 1.19). Averaged AUC at maximal ATP concentration (1 mm) was 55.19 ± 33.1 pA min (n = 6). c, d Outward currents elicited in a platelet-derived growth factor receptor α-positive (PDGFRα⁺) cell by alternating exposures to ATP (10 μm) and ADP (10 μm). ATP and ADP had similar effects and repetitive application yielded reproducible responses. Note responses were often spontaneous transient outward current-like and often extended past the period of exposure. d Averaged current responses to ATP and ADP in seven cells. There were no significant difference in maximum current elicited by either ATP or ADP (ATP = 47.8 ± 20.0 pA pF⁻¹ and ADP = 47.8 ± 20.3 pA pF⁻¹; P = 0.4337) or in integrated current responses (ATP = 165.9 ± 82.7 pA min and ADP = 133.5 ± 60.1 pA min; P = 0.6416; n = 7). b Blockade of ATP responses by small conductance Ca²⁺-activated K⁺ channel blocker and P2Y₁ antagonist. a Brief exposures (20 s) to ATP (10 μm) elicited reproducible large outward currents in PDGFRα⁺ cells (average 26.0 ± 5.8 pA pF⁻¹, n = 6) that were reduced by apamin (300 nm) (7.8 ± 6.1 pA pF⁻¹, n = 6; P = 0.0008). b MRS2500 (1 μm) blocked outward currents elicited by ATP (control ATP response 37.5 ± 19.2 pA pF⁻¹; after MRS2500 1.0 ± 1.0 pA pF⁻¹, n = 8; P < 0.0001) (reproduced from [414], with permission from The Physiological Society)

Mucosal epithelium

There are a wide variety of signalling roles for purines and pyrimidines in mucosal and glandular epithelial cells in most regions of the gastrointestinal tract [69, 130, 133, 583]. ATP modulates gastric acid and intestinal secretion and both P2Y and P2X receptors are expressed by mucosal epithelial cells and gastric glands ([280, 298, 672]; see [91]). ATP and adenosine are potent stimulants of fluid and electrolyte secretion in the colon following release from both local cells and nerves (see [91, 233, 583]).

Epithelium of oesophagus

Extracellular ATP has long been recognised as a stimulant of ciliary activity in frog oesophagus and in water and mucous-transporting epithelia (see [437]). Other studies have been carried out on monolayer tissue cultures of epithelial cells grown from frog oesophagus [534, 695]. ATP enhances ciliary beat frequency two- to threefold and induces pronounced changes in the metachronal wave parameters [269]. In addition, membrane fluidisation was induced, and increases in cytosolic Ca²⁺, principally from internal stores, coupled to membrane hyperpolarisation were necessary to activate all these cellular effects [9, 655]. Studies from this group have also established the existence of two P2 receptors, one of which is probably a P2Y receptor [270] and that the actions of ATP depends on PKC producing sustained enhancement of ciliary beat frequency via activation of calcium influx through non-voltage-operated Ca²⁺ channels [437]. HCl-induced activation of transient receptor potential vanilloid (TRPV) 1 causes ATP release from oesophageal epithelial cells [450].

Gastric acid secretion

Purinergic modulation of gastric acid secretion was first reported by Kidder [388], who showed that ATP or the ATP analogue 5′-adenylyl methylene diphosphonate added to the serosal bathing solution of the bullfrog gastric mucosa inhibited gastric acid secretion, although they were unaware at that time of purinergic receptors and did not explain their findings in these terms. Another study at this time of the effect of vagal nerve stimulation on gastric acid secretion in anaesthetised dogs led to the conclusion that, in addition to cholinergic nerves, an unsuspected second neural pathway existed which was capable of influencing gastric acid secretion [654]. Gastric hypersecretion of pylorus-ligated rats was inhibited dose-dependently by ADP and AMP [489]. ATPases are involved in the regulation of the gastric acid secretory process [227, 490, 504, 591]. NTPDase has been localised in the gastric mucosa and probably plays a role in the control of acid and pepsin secretion and mucous production, as well as contractility of the stomach [603].

There were also early suggestions that extracellular receptors to adenosine were responsible for modulation of acid secretions to the secretagogues, histamine and methacholine [261, 262, 624] using a dog gastric fundus preparation; theophylline was shown to block the adenosine actions. In a study of basal acid secretion in whole rat stomach, it was shown that, while adenosine caused a significant reduction in basal acid secretion, ATP and ADP significantly increased basal acid secretion [253]. Vagally mediated stimulation of gastric acid secretion by intravenously administered adenosine derivations was demonstrated in anaesthetised rats [561]. The authors took this to indicate that adenosine can stimulate gastric acid secretion by activating the vagus nerves via adenosine receptors in afferent pathways. The potent effects of the adenosine analogue N⁶-phenylisopropyladenosine (R-PIA) on inhibitory gastric acid secretion in the rat was taken to indicate that the P1 receptor involved was of the A_l subtype [606]. Gastric acid secretion was measured in conscious rats with an indwelling gastric cannula [284]. The potent P1 receptor antagonist 8-phenyltheophylline augmented gastric acid output, supporting a role for adenosine as a regulator of gastric acid secretion. Data was presented to suggest that endogenous adenosine generated by gastric cells interacts with parietal cell adenosine receptors to mediate acid secretion to histamine [260, 262].

Following up their earlier study of the effect of intravenous adenosine in anaesthetised rats, Puurunen and Huttunen [560] presented evidence to indicate that adenosine inhibits gastric acid secretion by a decrease in stimulation of vagal impulses to the stomach and that it acts in the brain via P1 receptors insensitive to xanthine. Another study, using unanaesthetised rats with indwelling gastric cannulas, showed a rank order for P1 receptor agonists in decreasing gastric acid output of NECA = R-PIA > 2-chloroadenosine > S-PIA [699]. NECA decreased the volume of gastric secretion, whereas R-PIA had no effects on volume, but significantly increased the pH of the secretions. In an attempt to characterise the effects of adenosine, ATP and ADP on acid secretion in isolated rabbit gastric cells, it was claimed that there were stimulating receptors to adenosine that were inhibited by methylxanthines, perhaps mediated via P1 receptors, and inhibitory receptors to ATP, α,β-meATP and ADP, which were reduced by indomethacin, perhaps mediated via P2 receptors [6, 7, 279]. It was claimed that there were P1 (A₂ subtype) receptors on rabbit parietal cells which mediate the stimulatory effects of adenosine and analogues on gastric acid production [7, 530]. Data was also presented to suggest that ATP selectively inhibits histamine-stimulated gastric acid secretion by acting directly on parietal cells, perhaps mediated by P2Y receptors with some part due to prostaglandin production [281].

Adenosine has been shown to decrease or increase production of gastrin, a known stimulant of gastric secretions, perhaps via A₁ or A₂ subtypes, thereby indirectly modulating gastric acid secretion [608, 698]. It was later shown that adenosine may suppress immunoreactive gastrin release by activating A₁ receptors on G cells, leading to inhibition of gastric acid secretion [739]. Adenosine may also act via A_2A receptors to augment somatostatin release and consequently influence gastric acid secretion [732, 738].

It has been suggested that muscularis mucosae may augment gastric acid secretion, and in a study designed to test this hypothesis, it was shown that there are contractions of the muscularis mucosae to ATP and ADP, but it was concluded that muscularis mucosae relaxation, rather than contraction, might be allied to acid secretion [549].

Gastric mucous secretion

ATP stimulated mucous glycoprotein secretion by rabbit gastric mucous cells in primary culture (Fig. 4a) [531]. The order of potency of ATP analogues was α,β-meATP > ATP > 2-MeSATP; the efficacy of ATP analogues to increase [Ca²⁺]_i was similar. A study of mucin secretion in the goblet cell line, HT29-C1.16E, suggested that both ATP and carbachol produce exocytotic release of mucin by acting on the same granular pool [51]. P1 receptor agonists had no effect. P2 receptor-mediated stimulation of mucous secretion appeared to be mediated by intracellular calcium, not by endogenous prostaglandin E₂. An autoradiographic study of sections of rabbit fundus with [³⁵S]2′-deoxy adenosine-5′-O-(1-thiotriphosphate), regarded as a radioligand for P2Y receptors, shows a selective distribution over the mucosa, but not muscle layer, and was paralleled by high-density binding on gastric gland plasma membranes [672].

Fig. 4.

a Dose-dependent effect of ATP on mucous secretion from rabbit gastric mucous cells in primary culture. Cells pre-labelled with [³H]glucosamine were incubated with ATP for 30 min. Secreted proteins (solubilised in with NaOH and neutralised with 1 N HCl) were measured using a scintillation counter and expressed as counts per minute per well. Values are means ± SEM for four determinations (reproduced from [531], with permission from Elsevier). b Time course of the change in potential difference across the wall of the rat jejunum following the addition of l mmol/l ATP to the mucosal bathing medium. Positive values for potential indicate that the serosal side is positive with respect to the mucosal side (reproduced from [400], with permission from Wiley)

Intestinal secretion

The first hint that extracellular ATP might be involved in electrolyte secretion in the intestine was the observation that ATP, either in the mucosal or the serosal fluid, caused a transient increase in the potential difference and short circuit current across the wall of rat small intestine or colon (Fig. 4b) [400]. Later, ATP, ADP and AMP, but not adenosine, were shown to increase cAMP-mediated stimulation of active ion transport in dispersed enterocytes prepared from the guinea pig small intestine [407], and later, ATP was shown to stimulate Ca²⁺ uptake in isolated rat intestinal epithelial cells [577].

Rabbit ileal mucosa, when mounted in a flux chamber and subjected to electrical field stimulation, secreted Cl⁻, a change reflected in an increase in short circuit current, and it was suggested that the mediator was likely to be a combination of ACh and NANC neurotransmitters released from nerves lying close to the secretory epithelium [342], the major NANC transmitter involved being VIP [233]. Differential effects of apical and basolateral UTP on intestinal epithelial Cl⁻ secretion have been described [626]. There is a loss of regulation of Cl⁻ transport by ATP and UTP in the jejunum of P2Y₄-null mice [580]. Mechanical stimulation releases nucleotides to activate neural P2Y₁ and P2X1 or P2X3 receptors to trigger neural reflex Cl⁻ secretion in guinea pig distal colon [150]. In another study from this group, mechanically evoked reflex Cl⁻ secretion in rat distal colon was claimed to be triggered by endogenous nucleotides acting via P2Y₁, P2Y₂ and P2Y₄ receptors [142]. In a later study, using P2Y₄ knock-out mice, it was shown that the P2Y₄ receptor fully mediates the chloride-secreting response to UTP in both small and large intestines, except on the basolateral side of the jejunum, where both P2Y₂ and P2Y₄ receptors are involved [268]. Apical targeting of the P2Y₄ receptor is controlled by hydrophobic and basic residues in the cytoplasmic tail [193]. Further, K⁺ secretion was activated via luminal P2Y₂ and P2Y₄ receptors in mouse colon [462]. Activation of P2Y₂ receptors on mouse duodenocytes enhances bicarbonate secretion via elevation of [Ca²⁺]_i [185].

Experiments carried out by Cuthbert and Hickman [165] confirmed the earlier reports about the effects of ATP on transepithelial ion transport but, since they found that TTX virtually abolished the effects of ATP on electrogenic chloride secretion, they suggested that the effects of ATP were indirect, via neural elements in the intramural plexus.

Under resting conditions, the mammalian distal colon has a NaCl⁻ absorptive epithelium, the absorption occurring at surface cells in colonic crypts, and intracellular Ca²⁺ or cAMP are important second messengers that activate NaCl⁻ secretion [60]. ATP released from the luminal side of epithelial cells in guinea pig colon by hypotonic stimulation appears to exert an inhibitory effect on electrogenic Na⁺ absorption, probably via P2Y₂ receptors on the apical membranes [731]. P2Y₆ receptors mediate colonic NaCl secretion in rat colon, as evidenced by RT-PCR localisation of P2Y₆ receptor mRNA and activation by UDP [409]. Activation of P2Y receptors may improve the absorption of water-soluble and high molecular weight compounds from the rat ileum [397]. Distal colonic Na⁺ absorption is inhibited by P2Y₂, but not P2Y₄, receptors [463]. A study of ATP actions in isolated crypts of rat distal colon, using the fura-2 technique to measure [Ca²⁺]_i (Fig. 5) [431] led to the following conclusions that basolateral ATP induces [Ca²⁺]_i in isolated crypts and acts as a secretagogue in the distal rat colon; basolateral P2Y receptors are responsible for this ATP-induced NaCl secretion; ATP action is not mediated by adenosine; and ATP-induced [Ca²⁺]_i signals are mostly located at the crypt base, which is the secretory part of the colonic crypt. The rank order of potencies for these actions was 2-MeSATP > ADP > ATP > > UTP, suggesting that a P2Y₁ receptor might be involved. In a later abstract, this group reported that luminal ATP induces K⁺ secretion via a P2Y₂ receptor in rat distal colonic mucosa [385]. It is interesting that TNP-ATP, which has since been identified as a potent antagonist at P2X1 and P2X3 receptors [686], was shown to block colonic Cl⁻ channels [682], although there do not appear to be reports of the effects of TNP-ATP on P2Y receptors. In situ hybridisation studies have shown that the mRNA for P2X4 receptors is localised in rat intestinal crypts [653].

Fig. 5.

ATP-induced (10 μmol/l) [Ca²⁺]_i increase in the rat isolated, intact colonic crypt was measured with the Ca²⁺-sensitive dye fura-2 and a video imaging set-up along the axis of the crypt. The cells were classified as basal, middle and surface cells according to their location. Base refers to the very base, middle refers to 50 % of total length in the middle of the crypt and surface refers to only cells at the very surface. Both the ATP-induced [Ca²⁺]_i peak and plateau decrease along the crypt axis. Asterisks indicate significant differences of peak and plateau [Ca²⁺]_i increases compared between basal and middle and middle and surface cells (reproduced from [431], with permission from Springer)

ATP regulation of Cl⁻ secretion has also been demonstrated in a human intestinal epithelial cell line, Caco-2, grown on permeable membrane supports and assayed for Cl⁻ secretion by measuring short circuit current [353]. The potency order on the apical side was UTP > ATP > UDP > 2-MeSATP = ADP and on the basolateral side UTP = 2-MeSATP = ATP > ADP > > UDP, suggesting that two different P2Y receptor subtypes are involved. UDP increases [Ca²⁺]_i, leading to increase in Cl⁻ secretion from mouse intestinal epithelium [76], suggesting that P2Y₆ receptors might be involved.

The strong presence of ecto-diphosphohydrolase (apyrase) in rat small intestinal brush-border membranes has been demonstrated [611], consistent with the view that nucleotides have potent actions in mucosal epithelial cells. A study of goblet cell-like clone derived from colonic HT-29 cells led to the conclusion that ATP-stimulated increase in Cl⁻ current does not require an increase in [Ca²⁺]_i suggesting the involvement of either another signalling pathway or direct activation of Cl⁻ channels via purinergic receptors [305]. ATP-stimulated electrolyte and mucin secretion by this human intestinal goblet cell line has been reported [474]. Exogenous ATP added to the medium bathing the mucosal surface of the intestine inhibits calcium transport to reduce the unidirectional flux of Ca²⁺ from the mucosal to serosal side [715]. Inhibition of uptake of amino acids (including leucine, lysine, alanine, valine and isoleucine) from isolated intestinal epithelial cells by extracellular ATP has been demonstrated [568] as well as regulation of Na⁺-dependent sugar transport [394]. ATP synthase generates extracellular ATP to regulate bicarbonate secretion in rat duodenum [694], via P2Y₁ receptors in guinea pig duodenum [215]. ATP-induced muscularis mucosae contraction evokes epithelial secretion in rabbit distal colon via NANC secretomotor nerve stimulation and prostaglandin synthesis [550]. ATP is released as a neurotransmitter to stimulate mucosal secretion of electrolytes and H₂O via P2Y₁ receptors expressed by VIPergic secretomotor nerves [213, 712]. ATP is released by mechanical deformation from enterochromaffin cells to act as an autocrine or paracrine messenger to stimulate release of 5-HT from enterochromaffin cells via P2Y₁ receptors or on P2X3 receptors on sensory nerve subepithelial nerve terminals [726].

Adenosine was claimed to stimulate electrolytic secretion in isolated epithelia of rabbit colon [295] and the P1 receptor antagonist, theophylline, caused an increase in short circuit current and reversed the direction of net Cl⁻ movement in rabbit ileum [5]. An examination of the effects of various analogues of adenosine led to the conclusion that the A₁ receptor subtype is present in rat jejunal mucosal epithelial cells [575]. Earlier studies showed that increases in short circuit current, in both small and large intestine, were preferential to ATP, with adenosine having significantly less effect [165, 400]. Adenosine has been shown to inhibit intestinal fluid secretion and a study of the relative actions of various adenosine agonists and antagonists led to the conclusion that the P1 (A_2B) receptor subtype is involved [312]. Neutrophil–epithelial cross-talk at the intestinal lumen surface is mediated by secretion of adenosine and interleukin (IL)-6 from inflamed epithelial cells [622]. A_2B receptors mediate signalling through the adenylate cyclase 6 isoform in intestinal epithelial cells and the authors suggest that this may have therapeutic implications for intestinal inflammation and diarrhoea, where the A_2B receptor is upregulated [402]. Luminal adenosine stimulates chloride secretion through A₁ receptors in mouse jejunum [267] and rapidly increases glucose transport [395]. Adenosine is a negative regulator of mitogen-activated protein kinase (MAPK) and pro-inflammatory signalling in human epithelial cells [364]. Clostridium difficile causes widespread infection by releasing toxins that break down epithelial tight junctions and compromising the intestinal epithelial barrier. CD73-mediated liberation of adenosine has been shown to protect intestinal epithelial cells from C. difficile toxin-induced damage [607]. Neurotensin stimulates Cl⁻ secretion in human colonic mucosae, involving mucosal nerves, adenosine and prostaglandins [579]. Mechanical stimulation of human enterochromaffin cells releases ATP which breaks down to adenosine to act via A₃ receptors to modulate 5-HT release [716]. In addition to roles in secretion, UDP has been shown to promote intestinal epithelial cell migration via the P2Y₆ receptor [503].

Purinergic vascular control in gut

The possibility that nucleotides were responsible for the initial rapid response, particularly at low frequency stimulation of sympathetic nerves, of the perfused intestinal microcirculation of anaesthetised cats was first raised by Taylor and Parsons [656], a secondary, slower phase being mediated by adrenoceptors. The initial rapid phase was completely abolished by selective desensitisation of the ATP receptor with α,β-meATP. In a follow-up study, these authors demonstrated that functional P2X receptors were present in both arterial and venous blood vessels of the cat intestinal circulation [657].

An important paper was published in 1992 in which it was shown that the postjunctional responses (EJPs as well as constrictions) of guinea pig submucosal arterioles to sympathetic nerve stimulation were mediated solely through the activation of P2X receptors by ATP or a related purine nucleotide (Fig. 6); the function of neurally released NA was to act through prejunctional α₂-adrenoceptors to depress transmitter release [209]. The finding was confirmed and extended by another laboratory, which examined the relative potencies of a number of purinergic agonists on guinea pig submucosal arterioles and showed that the constrictions were antagonised by suramin and PPADS [251]. Surprenant and her colleagues speculated in review articles [648, 681] that sympathetic purinergic control of arterioles is involved in extrinsically mediated mucosal reflex activity, particularly during inflammatory conditions. In another paper, it was considered that ACh inhibits neurogenic constriction of guinea pig interstitial submucosal arterioles by prejunctional modulation of ATP release from the perivascular sympathetic nerves with no major role for endothelial paracrine factors [408].

Fig. 6.

Constrictions of submucosal arteries of guinea pig in response to nerve stimulation are not mediated by noradrenaline but through the activation of P2 receptors. a Nerve-evoked constrictions (NS, 100 pulses at 10 Hz) were unaffected by the α₁-adrenoceptor antagonist, prazosin (0.1 μmol/l). b Constrictions to exogenously applied ATP (3 μmol/l) were abolished by the P2 receptor antagonist, suramin (100 μmol/l). c Suramin (100 μmol/l) had no effect on the contraction evoked by the exogenous application of noradrenaline (NA, 3 μmol/l). Vessel diameter of isolated superfused submucosal arteries was measured using an on-line computer analysis of TV images with an Imaging Technology system, sampling data at 10–2 0Hz with a resolution of less than 1 μm (reproduced from [209], with permission from John Wiley and Sons)

It has been known for some time that ATP, when injected inter-arterially, elicited pronounced vasodilatation in the denervated cat small intestine [225]. In terms of current knowledge, this could be due to several possible mechanisms, such as action of ATP on endothelial P2Y receptors leading to release of NO, a direct action on P2Y receptors located on vascular smooth muscle or breakdown of ATP by ectoenzymes to adenosine to act on P1 receptors (see [102]). ATP applied to equine colonic arterial and venous rings induced a biphasic response, contraction followed by sustained relaxation [660]. The relaxant response was reduced, but not eliminated in endothelium-free preparations, suggesting that a mechanism other than NO was involved.

Adenosine acting through P1 receptors can increase blood flow in interstitial vessels in two ways: by direct action on A₂-like receptors on vascular smooth muscle to produce vasodilatation; and by indirect action on A₁ prejunctional receptors on sympathetic vasoconstrictor nerves to inhibit release of the cotransmitters NA and ATP [293, 547, 559, 604]. A₁ and to a lesser extent A_2A and A_2B receptors contribute to adenosine-mediated vasodilatation of vessels in the rat jejunum [438].

Sensory nerves mediate protective vasodilatation in rat gastric mucosa [330], and there was an earlier report that ATP causes an increase in gastric blood flow [734]. This is of interest since ATP is a cotransmitter in sensory motor nerves and, upon release, acts on P2Y receptors present in the vascular smooth muscle [104].

Mesenteric arteries supplying the gastrointestinal tract of rat, guinea pig, rabbit and dog have been shown to be innervated by sympathetic nerves in which ATP is a major cotransmitter with NA [78, 79, 187, 357, 411, 453, 493, 528, 623, 689]. For the sympathetic nerves in the jejunal branches of the rabbit mesenteric artery, like those supplying submucosal arterioles, ATP appears to be the sole transmitter, while NA acts prejunctionally [564]. In a later paper [208], it was concluded that, while contraction of the rabbit jejunal artery to short trains of stimuli is predominantly purinergic, a noradrenergic component can be revealed at higher frequencies of stimulation or during longer trains of stimuli.

Pathophysiology

A limited number of studies have been conducted to date on changes in purinergic signalling in the diseased gut. ATP and adenosine have been implicated in the development of gastric ulcers, Hirschsprung's and Chagas diseases, ischaemia and colonic tumours [91]. Extracellular nucleotides and their receptors have been implicated in the pathogenesis of inflammatory bowel disease (IBD) [634]. P2Y receptors on smooth muscle and ATP production in myenteric neurons increase in postoperative ileus, probably contributing to delayed colonic transit [691]. Several reviews have highlighted the potential of purinergic drugs for the treatment of functional bowel disorders [99, 249, 327, 398]. Malnutrition affects millions of people. In an undernourished rat model, the density of P2X2 and P2X7-immunoreactive neurons in the enteric plexuses was increased, and these changes were reversible in re-fed rats [282]. The P2X2 and P2X7 receptors were expressed on NOS-positive inhibitory neurons, intrinsic sensory neurons and cholinergic secretomotor neurons. There are reviews of the purinergic literature about gut disorders [13, 17, 91, 96, 99, 103, 299, 327].

Inflammatory bowel disease

Acid sensing is of critical importance for the survival of the epithelial cells throughout the gastrointestinal tract, and its importance for mucosal defence, lipid uptake and cystic fibrosis has been discussed [379]. Nearly all the acid sensors occur on intrinsic sensory neurons and P2X3 receptors are upregulated in inflammation and hypersensitivity [719]. This has been taken to suggest that the aberrant function of molecular acid sensors may contribute to abnormal hyperalgesia and pain [329]. Intestinal inflammation increases the expression of P2Y₆ receptors on epithelial cells and the release by UDP of CXCL8 (a chemokine known for its chemoattraction ability to recruit neutrophils during the acute phase of colitis) [296, 297]. Intraduodenal administration of ATP concomitantly with ingestion of non-steroidal anti-inflammatory drugs (NSAIDs) attenuated the NSAID-induced increase in intestinal permeability in healthy humans, and it was suggested that ATP may also be beneficial in the treatment of intestinal disorders where intestinal permeability changes were involved [71]. P2Y₂ receptor expression is upregulated in intestinal epithelial cells by the transcription factor C/EBPβ during inflammation [174].

Nucleotides and their receptors have been explored in the pathogenesis of IBD. P2X3 receptor expression was increased in enteric plexuses of human IBD, suggesting a role in dysmotility and pain [737]. The possibility that P2X receptor antagonists could be used for the treatment of IBS was raised [249]. P2X receptors on intrinsic enteric neurons may elicit enhanced gastrointestinal propulsion and secretion, and it has been suggested that they might be used for treating constipation-predominant IBS, while P2X antagonists might be useful for treating diarrhoea-predominant IBS. Peripheral sensitisation of P2X3 receptors on vagal and spinal afferents in the stomach may contribute to the development of visceral hyperalgesia [168]. During chronic interstitial inflammation induced by infection of mice with the parasite Schistosoma mansoni, purinergic modulation of cholinergic nerve-mediated effects was impaired [172]. In inflamed gastrointestinal tract, glial cells proliferate and produce cytokines, suggesting that P2X7 receptors may play a role in the response of enteric glia to inflammation [679].

In trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice, the purinergic component of sympathetic cotransmission to colonic submucosal arterioles was reduced, perhaps due to increased degradation of extracellular ATP and P2X1 receptor expression was increased [447]. Propulsive motility is attenuated in the ulcerated region of the TNBS-inflamed colon, and this is associated with a decrease in the purinergic component of the descending inhibitory limb of the peristaltic reflex circuit [645]. P2X3 receptor mRNA expression in dorsal root ganglia (DRG) was significantly decreased in the ovariectomised rat model of colitis, an effect that was reversed by oestrogen [212]. It has been suggested that ATP is a critical autocrine regulator of mechanosensitive 5-HT release, which is involved in the pathogenesis of IBD and that P2X3 receptors on enterochromaffin cells are downregulated in ulcerative colitis [442]. CD39 (NPTDase1) was upregulated in the submucosa during colitis that contributed to impaired sympathetic regulation of gastrointestinal blood flow, compromising epithelial barrier function [506]. Increase in sympathetic innervation of the mesenteric arteries supplying the colon was reported in inflamed human bowel [61]. Dysregulation occurs in 59 % of purinoceptor genes in IBD, including P2Y₆, P2Y₁₃, P2Y₁₄, P2X5, A_2A and A_2B receptors [589].

P2X7 receptors play a pivotal role in intestinal inflammation and are involved in the development of visceral hypersensitivity [381]. P2X7 receptors on epithelial and immune cells are implicated in the pathogenesis of diseases based on the dysregulation of immune responses in inflammatory bowel disease [170]. Activation of neuronal P2X7 receptor/pannexin 1 mediates death of enteric neurons during colitis [302]. This supported an earlier study of TNBS-induced colitis, using high-density oligonucleotide microassay analysis, and oral N⁶-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine blocked the colitis-induced upregulation of P2X1, P2X4, P2X7, P2Y₂ and P2Y₆ receptors [309]. Extracellular ATP evokes cell death in human intestinal epithelial cells, largely via P2X7 receptors, and the implications of this in inflammatory conditions and immune responses were considered [635]. It has also been reported that ATP mediates mast cell-dependent intestinal inflammation via P2X7 receptors [415]. An adenosine A₃ agonist has been claimed to be protective in two murine models of colitis [452].

Tumour necrosis factor-α upregulates A_2B receptor expression and signalling in intestinal epithelium in colitis [401]. Blockade of A_2B receptors ameliorates mouse colitis [403] as does A_2B gene deletion [404]. The inhibitory effects of adenosine on enteric neuromuscular activities are reduced in inflamed colon [12]. It has been reported that oxidative stress disrupts purinergic neuromuscular transmission in the inflamed colon [581]. It was claimed that this can be prevented by treatment with a free radical scavenger, resulting in improved motility. It has been suggested that A_2B receptors play a role in the control of T cell-mediated colitis by suppressing the expression of pro-inflammatory cytokines, while sparing anti-inflammatory activity mediated by IL-10 and transforming growth factor-β [498]. A_2A receptors also mediate the inhibitory effects of adenosine on colonic motility in the TNBS model of experimental colitis [13, 563]. Inhibition of adenosine deaminase attenuates inflammation in experimental colitis [14]. Adenosine, acting via A₃ receptors, has been implicated in intestinal anti-inflammation activities [265, 309]. A_2A receptors have also been implicated in the anti-inflammatory actions of adenosine [519], and A_2A receptor agonists have been developed for the treatment of inflammatory bowel disease [201]. A_2B receptors mediate regulation of 5-HT synthesis and release from hypoxic enterochromaffin cells in IBD [167]. A_2B receptor antagonists appear to be effective against murine colitis [405]. The involvement of adenosine A₁ and A_2A receptors [16] and A₃ receptors [573] in colitis has been described. Reviews of the roles of adenosine signalling in gastrointestinal inflammation are available [146, 207]. Blockade of adenosine deaminase reduces chronic experimental colitis through the recruitment of A_2A and A₃ receptors [15]. There has been an investigation of adenosine deaminase in patients with Crohn's disease [458]. The inhibition of adenosine kinase by GP515 has been explored as a potential target for the treatment of colitis [621]. In a review about purinergic receptors in gastrointestinal inflammation [403], it was concluded that P1 (A_2A and A_2B) and P2Y receptor-based therapy is highly promising for the treatment of inflammatory conditions of the gut, as well as for fibrotic liver diseases (see [477]). Serum adenosine deaminase activity has been claimed to be a predictor of disease severity in ulcerative colitis [55]. It has been reported that ENTPase 7 is preferentially expressed in epithelial cells of mouse small intestine [417]. ATP released from colonic mucosal epithelial cells of IBS patients excites enteric cholinergic motor neurons via P2X receptors [29]. The role of adenosine as an immune modulator of IBD has been reviewed [736]. Genetic polymorphisms of CD39 have been linked to Crohn's disease [413]. A large migration of neutrophils into the intestinal mucosa is a feature of IBD. It has been shown that release of ATP by activated neutrophils and necrotic intestinal epithelial cells stimulates epithelial cell P2X7 receptors leading to activation of caspase 1 and secretion of pro-inflammatory cytokines, such as IL-1β [123]. P2X7 receptor expression was shown to be weak in intestinal biopsies obtained during the active phase of IBD.

Chagas disease

Chagas disease is caused by the protozoan parasite, Trypanosoma cruzi. Transmission to humans occurs through blood-sucking reduviid bugs, but it may also occur through blood infusion or organ transplant. Little is known about the neurotransmitters most affected in Chagas disease, but there are hints that purinergic signalling might be impaired and there is evidence for a preferential destruction of intrinsic inhibitory neurons [166]. Both low affinity Mg²⁺-activated ATPase and high affinity (Ca²⁺-Mg²⁺) ATPase [122, 228] as well as adenosine kinase [389] are present in T. cruzi, which rapidly break down extracellular nucleotides. E-NTPDase (CD39) and ecto-adenosine deaminase activity are decreased in lymphocytes of patients with the indeterminate form of Chagas disease [637]. Regulation of these extracellular nucleotides through ectonucleotidase activities on the platelets of patients with the indeterminate form of Chagas disease represents control of purine-mediated thrombogenic function in the cardiovascular system [636]. Enhancement of P2X7 receptor-associated cell permeabilisation occurs during the acute phase of Chagas disease [155]. Purinergic signalling through other P2X receptor subtypes and P2Y receptors may also be impaired, perhaps because the parasite protozoan that causes the disease contains high levels of ATPases. Thymus atrophy induced by T. cruzi infection may involve ATP-induced cell death via P2X7 receptors [456]. However, experiments using P2X7 knockout mice suggested that P2X4 and P2Y receptors may also be involved [119].

Hirschsprung's disease

Hirschsprung's disease is a congenital abnormality of the enteric nervous system and is characterised by the absence of ganglion cells in the submucosal and myenteric plexuses of the hind gut and by chronic constriction of the aganglionic region. There is hyperinnervation of the Hirschsprung's human gut by extrinsic sympathetic nerves and preganglionic parasympathetic nerves (see [254, 350]). Enteric nerves arising from intrinsic neurons containing various neurotransmitters including ATP show substantial reductions in density in aganglionic segments [421, 422, 582]. IJPs were not recorded in aganglionic segments of human colon [230, 525], and ATP caused contraction of the muscle [749]. IJPs were also not evoked in aganglionic segments of piebald-lethal mouse colon [116, 525, 578]. In the aganglionic intestine, there was only weak P2X3 receptor immunostaining in the myenteric and submucous plexuses compared to normal intestine [210]. The absence of expression of P2Y₁ and P2Y₂ receptors in the aganglionic intestine in Hirschsprung's disease has been described [518], which suggests that purinergic inhibitory neurotransmission is absent and may account for the contracted state of the aganglionic gut in Hirschsprung's disease.

Motility disorders

Bile evokes ATP depletion and contributes to the early mucosal permeability alteration and barrier lesions that occur during experimental oesophageal reflux [649]. It has been suggested that purinergic signalling might be involved in achalasia or symptomatic diffuse oesophageal spasm [182, 222]. ATP production in myenteric neurons and P2Y receptor expression on smooth muscle in postoperative ileus increase, contributing to delayed colonic transit [691]. Agonists acting on P2X receptors on intrinsic enteric neurons may enhance gastrointestinal propulsion and might be useful for treating constipation, while P2X antagonists might be useful for treating diarrhoea. P2Y receptor stimulation has been proposed to be beneficial for the treatment of constipation [249]. Increased apoptotic cell death in enteric neurons and ICCs from the colon of patients with slow transit constipation has been reported [275], probably as the result of activation of P2X7 receptors. Prejunctional P2Y₁ receptors modulate the activity of excitatory enteric motoneurons and might be therapeutic targets for patients with functional disorders affecting colonic motility [21]. Disturbed motility occurs with intestinal anaphylaxis. Allergic diarrhoea, in a model of food allergy, was accompanied by chronic inflammation and mast cell hyperplasia in the colon [434], and it was suggested that sustained alteration in purinergic neurotransmission contributed to the disturbed motility characterised by this condition. Herpes simplex virus type-1 infects the enteric nervous system and affects gut motor function; contractions mediated by adenosine acting via A₁ or A_2A receptors on smooth muscle and A_2A and A₃ receptors in the myenteric plexus were impaired in virus-infected rats [764].

Gastric ulcers

Helicobacter pylori infection and gastric hyperacidity results in the development of gastric ulceration [287, 465]. ATP was shown to be involved in the development of gastric hypersecretion and ulceration in pylorus-ligated rats [489]. It was shown that in pylorus-ligated rats, gastric acid secretion was an ATP-dependent process and that adenosine acting via P1 receptors inhibited the development of ulceration. Methylxanthines, which blocked the action of adenosine, stimulated the acid content of gastric secretions [365] and promoted gastric ulceration [204, 319]. Dipyridamole, which leads to an increase in extracellular adenosine, significantly reduced the extent of gastric bleeding and ulcer formation [538]. Intracerebral or subcutaneous administration of adenosine increased stress-induced gastric lesions [670]. However, in contrast, it was reported that adenosine receptor activation in the brain reduced stress-induced ulcer formation [259, 697].

Diabetes

Relaxations in response to NANC nerve stimulation were reduced in longitudinal strips of gastric fundus from 8-week streptozotocin-induced diabetic rats [360], but NANC contractions were enhanced [361]. IJPs of reduced amplitude were observed in gastric smooth muscle from streptozotocin-induced diabetic rats [727]. The rate of hyperpolarisation of single IJPs was slower in the circular muscle of the caecum of streptozotocin diabetic (8-week) rats [338]. Maximum relaxant responses and sensitivity of the colon to ATP were unchanged in 8-week streptozotocin diabetic rats, but the responses to adenosine were reduced [307]. Diabetic enteric neuropathy was associated with apoptosis in the myenteric plexus of the rat colon [304].

Nociception

Submucosal intrinsic sensory neurons and extrinsic sensory nerves both show positive immunoreactivity for P2X3 receptors [722]. It has been proposed that during excessive intestinal distension, high-threshold extrinsic enteric sensory fibres are activated via P2X3 and P2X2/3 receptors by ATP released from mucosal epithelial cells, leading to initiation of nociceptive impulses that pass messages through the DRG to pain centres in the CNS [93]. This hypothesis was supported by experiments on a rat pelvic sensory nerve–colorectal preparation ([718]; Fig. 7). Colorectum distension led to pressure-dependent increase in the release of ATP from mucosal epithelial cells and evoked pelvic sensory nerve excitation. This excitation was mimicked by application of ATP and attenuated by the selective P2X3 and P2X2/3 antagonist, TNP-ATP, and by PPADS. The sensory activity in the nerves was potentiated by ARL-67156, an ATPase inhibitor. It has been claimed recently that subepithelial fibroblasts in rat ductal villi also release ATP by mechanical stimuli, which has actions on P2Y₁ receptors expressed by the fibroblasts, as well as activating P2X3 receptors on subepithelial sensory neurons [238]. ATP release and P2X3 and P2X2/3 receptor-mediated nociceptive sensory nerve responses were enhanced in the rat TNBS model of colitis [719]. Different mechanosensory information from the colon to the spinal cord is conveyed by lumbar splanchnic (LSN) and sacral pelvic (PN) nerves. Forty percent of LSN afferents responded to α,β-meATP compared to 7 % of PN afferents [73]. Enhancement of P2X3 receptor-mediated signalling in an animal model of colonic inflammation has been reported. This was due, at least in part, by the appearance of P2X3 receptor expression in a greater number of calcitonin gene-related peptide-labelled small nociceptive neurons in the DRG [719]. Purinergic mechanosensory transduction has also been shown to contribute to post-infectious mechano-hypersensitivity [585]. P2X3 receptor expression was increased in human IBD enteric plexuses suggesting a potential role in dysmotility and pain [737]. Substances are released from various sources under these conditions that often act synergistically to cause sensitisation of afferent nerves to mechanical or chemical stimuli. Receptors to a variety of substances (including ATP released during gut distension) represent potential targets for drug treatment for abnormal bowel function and visceral pain (see [327, 398]). The sensitising effects of P2X3 receptor agonists on mechanosensory function were demonstrated in oesophagitis [536]. Visceral hyperalgesia is associated with an increase in ATP activity and enhanced expression of P2X3 receptors in colonic sensory neurons [725]. Selective P2X3 and P2X2/3 receptor antagonists that are orally bioavailable and do not degrade in vivo are in clinical trials for the treatment of pain (see [186, 266]).

Fig. 7.

Schematic of a novel hypothesis about purinergic mechanosensory transduction in the gut. It is proposed that ATP released from mucosal epithelial cells during moderate distension acts preferentially on P2X3 and/or P2X2/3 receptors on low-threshold subepithelial intrinsic sensory nerve fibres (labelled with calbindin) to modulate enteric reflexes. ATP released during extreme (colic) distension also acts on P2X3 and/or P2X2/3 receptors on high-threshold extrinsic sensory nerve fibres (labelled with isolectin B4 (IB₄)) that send messages via the dorsal root ganglia (DRG) to pain centres in the central nervous system (reproduced from [94], with permission from Wiley-Liss, Inc.)

Ischaemia

Purinergic signalling has been identified in the development of intestinal ischaemia–reperfusion injury. For example, adenosine acts via both A_2A and A_2B receptors, and A_2A receptors provide potential protection and is a novel therapeutic target for intestinal ischaemia–reperfusion injury [180, 203, 314]. ATP attenuated intestinal dysfunction produced by ischaemia, but not that caused by reperfusion in rabbits [650]. In ischaemia–reperfusion of the intestine, there was a decrease in P2X2 receptor expression in the myenteric and submucosal plexus [545].

Injury

P2Y₁ receptor signalling has been shown to mediate wound-induced cyclooxygenase (COX)-2 expression through both p38 MAPK and PKC pathways in intestinal subepithelial myofibroblasts [359]. It was suggested that this might indicate a novel treatment for intestinal barrier dysfunction during inflammation.

Cancer

Colorectal cancer is a major disease. [Ca²⁺]_i was increased in the HT-29 human colonic adenoma cell line by ATP and ADP [325]. HT-29 cells were depolarised by UTP > ATP > ADP > adenosine [446]. Cultured human colonic tumour cells (LoVo) were resistant to ATP cytotoxicity, but verapamil increased sensitivity to ATP [152]. P2U (i.e. P2Y₂ and/or P2Y₄) receptors are expressed by HT-29 cells [160, 516, 542].

ATP transiently increased Cl⁻ conductance in the highly differentiated sub-clone of the HT-29 colonic cancer cell line, HT-29-C116E [305, 306]. ATP activation of Cl⁻ conductance was also reported in the T84 human colonic adenocarcinoma cell line [179]. A decrease of intracellular Cl⁻ and Na⁺ and an increase in Ca²⁺ in HT-29 cells response to both ATP and UTP via P2U (P2Y₂ and/or P2Y₄) receptors was shown [754]. P2U receptor mRNA in both primary cultures of human colorectal carcinoma cells and HT-29 cells was reported, where they play a role in the regulation of cell proliferation and apoptosis [331]. Resistance to ursolic acid-induced apoptosis in HT-29 cells was mediated by P2Y₂ receptors [441]. ATP induced apoptosis and inhibited growth of primary cultures of colorectal carcinomas [331], probably via P2Y₂ receptors [332]. P2Y₂, P2Y₄ and P2Y₆ receptor mRNAs were located on the apical membranes of human colonic Caco-2 adenocarcinoma cells [114, 464]. The hypotonicity-induced release of ATP from basolateral, but not apical, membranes of Caco-2 cells was facilitated by caveolin-1 [668]. P2Y₂ and P2Y₄ receptors were upregulated in human colon cancer [517]. Gβγ-Subunits mediate regulation of increase in [Ca²⁺]_i during P2Y₂ receptor activation [340]. P2Y₂ receptors have oncogenic potential mediating transformation of colorectal RKO cancer cells [315]. Proliferation of Caco-2 cells is evoked by ATP acting via P2Y receptors [115]. Tissue from patients with colorectal cancers showed increased expression of an ATP-binding cassette super-family transporter, multidrug resistance protein-2 [323]. CD39 (NTPDase1) modulated colorectal tumour growth and liver metastasis and the expression of both P2Y₂ and P2X7 receptors [413]. The activities of CD73 and adenosine deaminase were higher in primary human colorectal tumours [205] and in human colorectal adenocarcinomas [658]. Gene expression of adenosine kinase is significantly increased in human colorectal cancer [276]. There is heterogeneity of chemosensitivity of colorectal adenocarcinoma, and this may be used to identify patients who would benefit from specific chemotherapeutic agents alone or in combination [128, 343, 701]. Surgeons often wash the abdominal cavity with distilled water to lyse colorectal cancer cells remaining after surgery, and it has been shown that water induces release of ATP from epithelial cells, which then causes cell death of tumour cells via P2X7 receptors [613].

Adenosine facilitates tumour survival [443, 641]. Proliferation in poorly differentiated HT-29 cells is promoted by adenosine via A₁ receptors and there is inhibition of tumour growth by adenosine deaminase or A₁ receptor antagonists [432]. However, adenosine had less effect on more differentiated cells [433]. It has been claimed that adenosine suppresses growth of CW2 human colonic cancer cells by inducing apoptosis via A₁ receptors [594]. A_2B receptor expression is enhanced in proliferating colorectal cancer cells and A_2B receptor antagonists are being explored for the treatment of colorectal cancer therapy [449]. There is significant inhibition of the growth of xenografted subcutaneous human colon adenocarcinoma cell line, HCT116, in nude mice by a single low-level intravenous dose of [³²P]ATP [127]. Growth of colorectal cancer cell lines HCT116 and 80514 were inhibited in vitro and in vivo by 8-chloro-adenosine [117]. Primary colon carcinoma growth was inhibited by A₃ receptor agonists [223, 520], although a later paper claimed that A₃ receptors mediated proliferation of Caco-2, DLD1 and HT-29 colorectal tumour cell lines [264]. The A₃ receptor agonist CF101 stabilised the tumour in 35 % of the patients with refractory metastatic colorectal cancer in a phase II, multi-centre study [642]. Adenosine has been claimed to induce apoptosis in Caco-2 colonic cancer cell [735]. Adenosine upregulates CXCR4, which is a chemokine receptor that plays a crucial role in determining the ability of cancer cells to metastasize from the primary tumour. CXCR4 enhances the proliferative and migratory responses of HT-29 cells [576]. Adenosine can stimulate migration of colon cancer cells and caffeine significantly inhibits this action [473].

ATP produces hyperpolarisation of the human gastric signet ring cell carcinoma cell line (JR-1), probably mediated by P2Y receptors [311]. Proliferation was reduced and apoptosis induced in the human gastric carcinoma cell line (HGC-27) by ATP and adenosine [593, 692]. The benefits of chemotherapeutic drugs in patients with gastric cancer have been examined using an ATP-based chemotherapy response assay [426, 540]. Infection of the gastric body by Heliobacter pylori contributes to the progression of gastric carcinoma [592]. Gastric cancer cells show a loss of A₃ receptors [151]. Kyse-140, a human oesophageal squamous carcinoma cell line, and cancer cell primary cultures both expressed P2Y₂ receptors, which mediated inhibition of growth [451]. Using the ATP-tumour chemosensitivity assay, heterogeneity of chemosensitivity in oesophageal cancer has been reported [444]. Neuroendocrine tumours are a heterogeneous group of neoplasms originating from enteric chromaffin cells and these tumours express A_2A and A_2B receptors, the activation of which leads to increased proliferation [372], a potential target for therapy [373]. Gastrointestinal stromal tumours that originate from pacemaker cells of the gastrointestinal tract release ATP, which may be important for tumour homeostasis and immune surveillance escape [50].

Salivary glands

Salivary acinar and ductal epithelial cells are responsible for the controlled secretion of fluid and electrolytes and of specific proteins and growth factors [148]. There are several Ca²⁺-mobilising receptors involved in these activities, including muscarinic receptors, α-adrenoceptors and SP receptors, but it is now well recognised that there are also Ca²⁺-mobilising receptors for extracellular ATP in rat and mouse parotid acini [232, 242, 470, 631] and in rat, mouse and human submandibular acinar and duct cells [273, 347, 416, 665, 666, 740]. Stimulation of the NANC component of parasympathetic nerves produced increased production of saliva from parotid, submandibular glands [199]. Potassium-evoked release of purines from rat submaxillary gland has been demonstrated, although it was not possible in the experiments described to discriminate between neuronal and non-neuronal elements as the source of purines released by depolarisation [220]. Zinc ions are present in high concentrations in acinar secretory vesicles. They are co-released during salivation and play a physiological role in salivary secretions. The zinc sensing receptor enhances secretion of ATP from ductal cells [614]. Intra-arterial administration of various nucleotides, including ATP and ADP, to the cat submandibular salivary gland led to an increase in blood flow, possibly mimicking neurally released ATP as a cotransmitter in parasympathetic and/or sympathetic nerves [368]. NTPDase3 was co-expressed with NTPDase2 and 5′-nucleotidase in subpopulations of epithelial cells in the salivary glands of mice and guinea pigs [423]. Reviews of the distribution and function of P2 nucleotide receptors in salivary glands are available [513, 667].

Parotid gland

In a seminal paper, Gallacher [242] showed that in acinar cells of the parotid gland, ATP evoked a marked increase in membrane conductance, K⁺ efflux and amylase secretion; a P2 receptor was implicated, since adenosine had no effect and the responses could be blocked by quinidine, but not by theophylline. Extracellular ATP was later shown to elevate intracellular free calcium in rat parotid acinar cells and the possibility that ATP plays a neurotransmitter role in the parotid gland raised [470]. In fact, ATP was found to be more effective than muscarinic and α-adrenergic agonists and SP as a stimulus for elevating [Ca²⁺]_i levels [471]. ATP induces oscillatory changes in [Ca²⁺]_i in HSY cells, a salivary ductal cell line from human parotid [663]. Purinoceptors mediate spontaneous Ca²⁺ oscillations and associated cell swelling in rat parotid ductal cells and regulation of electrolyte reabsorption from the primary saliva in the resting state [619]. Coomassie brilliant blue G was a more potent antagonist of P2 receptor-mediated responses of rat parotid acinar cells than Reactive blue 2 (Cibacron blue 3GA) [630]. Further studies by this group led them to suggest that ATP may function as a neurotransmitter to modulate salivary fluid secretion by stimulating Ca²⁺-sensitive Cl⁻ and K⁺ channels and multiple Na⁺ uptake pathways in the rat parotid acinar cell [631]. They showed that some of these pathways were similar to those activated by carbachol while others were unique to ATP. Extracellular ATP increases the conductance to both Na⁺ and Cl⁻ in parotid acinar cells through independent mechanisms [19]. A later paper showed that P2X7 receptors were essential from anion activation and that Na⁺ regulates anion conductivity and permeation through this receptor [574]. It was suggested that the source of the ATP could be as a cotransmitter from nerves or directly from acinar vesicles or secretory granules into the lumen following muscarinic stimulation [513].

Two distinct [Ca²⁺]_i responses to ATP were distinguished in rat parotid acinar cells raising the possibility that both P2X7 and P2Y receptors were implicated [469, 632]. To learn more about the ATP-binding site of the P2X7 receptor in these acinar cells, the isothiocyanate compound, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid, was examined and found to be an effective antagonist at the parotid P2X7 receptor [633]. A study by another group showed an inhibitory effect of ATP⁴⁻ on the ACh-mediated response of rat parotid acini and presented evidence to suggest that this was due to interactions of the activated P2X7 receptor with the PLC-coupled processes underlying the muscarinic cholinergic response [369]. 2′(3′)-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (BzATP), acting via P2X7 receptors on rat parotid acinar cells, leads to the formation of large pores [274]. ATP, acting through P2X7 receptors, causes Na⁺ entry by opening cation-permeable channels, and thereafter, the increase in [Na⁺]_i triggers Ca²⁺ release from intracellular ryanodine-sensitive stores, while UTP acting through P2U (= P2Y₂ and/or P2Y₄)-type receptors caused Ca²⁺ release independent of external Na⁺ [232]. In a somewhat conflicting paper, activation of P2X7 receptors on rat parotid acinar cells was claimed to cause a large entry of Ca²⁺ into the cells [662]. In an abstract, a P2X7 receptor was identified in rat parotid salivary glands which was modulated by Mn²⁺ and Ni²⁺, but not by Cd²⁺ [18]. Activation of P2X7 receptors by ATP in mouse parotid acinar cells occurs in two steps: slow assembly (which requires an intact cytoskeleton) and rapid gating (which does not) [439]. Duct cell P2X7 receptors are pre-assembled and therefore continuously subject to rapid gating by ATP. A study of P2X7 receptor knockout mice led to the conclusion that cholinergic stimulation leads to the release of ATP that can, via P2X7 receptors, upregulate parotid salivary secretion [514].

The results of a study using RT-PCR showed strong expression of P2X4 and P2X7 receptor mRNA in parotid glands, which correlated well with the responses of the parotid acinar cells to extracellular ATP [659]. It was further shown that parasympathetic denervation of the parotid gland increased the number of cells with P2X4 responses and the levels of P2X4 mRNA, opening up important general issues about trans-synaptic regulation of P2X receptor expression. Another study describes how ATP, acting through P2X7 receptor-mediated PLD, may produce a Ca²⁺-independent PKC to account for the finding that ATP shortened the duration and decreased the magnitude of ACh-induced Ca²⁺ release from rat parotid acinar cells [231]. Functional interactions between P2X4 and P2X7 receptors in mouse parotid acinar cells have been described [118, 556]. Ectonucleotidase and 5′-nucleotidase levels in parotid acini have been reported [188]. A recent study claims that P2X4 receptors are largely localised on the basal and basolateral surfaces of mouse parotid acinar cells, activated by ATP released as a cotransmitter from autonomic unmyelinated nerve fibre varicosities that surround acini, while P2X7 receptors are located largely on the apical surface of acini cells, activated by autocrine/paracrine ATP release from acinar cells ([56]; Fig. 8).

Fig. 8.

a–d P2X receptor [Ca²⁺]_i-evoked changes exhibit similar kinetics in mouse parotid acinar cells. a Acinar cells were loaded with fura-2 AM and ratio images were obtained at 2 Hz. The ratio images depict [Ca²⁺]_i rise following a sequential application of ATP (1 mm) stimulation in the same acinar cell cluster. Scale bar for the acinar unit, 17 μm. b Representative line trace depicting [Ca²⁺]_i responses in parotid acinar clumps evoked by ATP (1 mm). Scale bar indicates fluorescence ratio units (r.u.) and time in seconds. c Peak [Ca²⁺]_i amplitudes evoked by 1 mm ATP compared with that evoked by treatment with 300 μm UTP (n = 3), 100 μm α-methylthio-ATP (2-MeSATP; n = 7). The results are represented as a fold change in peak [Ca²⁺]_i amplitudes. Asterisks denote significant difference from control values. d Peak [Ca²⁺]_i amplitudes evoked following selective P2X7 receptor activation using 500 μm 2′(3′)-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (BzATP) with (n = 5) or without (n = 5) prior Ca²⁺ store depletion using 30 μm cyclopiazonic acid. e Confocal images from mouse parotid gland slice preparations (10 μm thickness) labelled with P2X4 (red) and P2X7 (green) receptor antisera and F-actin (cyan) using Alexa Fluor 647-conjugated phalloidin. Asterisks mark ducts in the parotid lobules. Scale bar is 25 μm (reproduced from [56], with permission from The Physiological Society)

Using a rat parotid secretory granule preparation, ATP was shown to activate Ca²⁺-independent membrane-associated PLA₂ [484]. ATPγS was active to a lesser extent, while UTP, cytosine triphosphate and cytosine 5′-O-(thiotriphosphate) showed little activation. It was suggested that the PLA₂ located in the granular membranes may participate in the liberation of arachidonic acid in parietal cells that is regulated through a mechanism mediated by ATP. Nucleotides are important modulators of Ca²⁺ release from parotid salivary glands under physiological conditions and a decrease in ATP levels may impact Ca²⁺ signalling in pathological situations [539].

Submandibular gland

The effect of ATP on various types of preparations from submandibular salivary glands has been reported, including the human submandibular duct cell line, HSG-PA [390, 416, 740], rat submandibular gland acini [345–347], crude cell suspensions of whole rat submandibular glands [175, 418, 475, 665, 752] or ductal cells [10], a mouse submandibular epithelial salivary cell line, ST₈₈₅ [273], and an immortalized cell line SMG C10 cells, originally obtained from the rat submandibular salivary gland [22].

The presence of a P_2U-like receptor, where the agonist profile was UTP = ATP > ATPγS > ADP > ADPβS with both α,β-meATP and 2-MeSATP having little or no effect, was identified in a human submandibular duct cell line HSG-PA [740]. The cells challenged by UTP hyperpolarised which provided the driving force for net Cl⁻ efflux [390]. In the most recent paper on HSG cells, in addition to a P2U receptor mediating InsP₃ formation to nucleotides, the authors suggested that Ca²⁺ influx might be mediated by a second, perhaps P2X receptor [416].

Studies of rat submandibular gland acini identified, as for parotid acini, a P2X7 receptor activated by ATP⁴⁻ which promoted Ca²⁺ and Na⁺ influx, but not release, from intracellular stores [345–347]. A later paper showed activation of PLD by P2X7 agonists in rat submandibular gland acini [552] and ductal cells [555]. Activation of P2X7 receptors in mouse submandibular glands triggers an intracellular signalling cascade involving PKC and MAPK leading to stimulation of NADPH oxidase and the subsequent generation of reactive oxygen species [612]. ATP also acts via P2X7 receptors to inhibit muscarinic-induced fluid secretion in murine submandibular glands [501]. The presence of two populations of P2X7 receptors in the plasma membrane of rat submandibular gland has been claimed, in raft and non-raft compartments [258]. There is also an unusual report that P2X7 receptors mediate depolarisation of mitochondrial as well as plasma membranes [257], which is interesting in view of earlier reports of intracellular immunolocalisation of P2X7 receptors [20]. ATP via P2X7 receptors increased the production of reactive oxygen species in rat submandibular glands, and the authors speculate that purinergic receptors could be regulators of the bactericidal properties of saliva by promoting the secretion of peroxidase from acinar cells and by activating Duox2 [226]. It was reported that P2X7 receptor activation induces inflammatory responses in mouse submandibular gland cells [714].

For mixed duct and acinar cell suspensions, again a P2X7 receptor coupled to a non-selective cation channel was described, occupation of which by ATP potentiates the responses to both carbachol and SP [418, 475]. In a subsequent paper from this group, using suspensions of submandibular ductal cells only, two purinergic receptors were identified, a metabotropic, probably P2Y₁ receptors and a P2X ionotropic receptor coupled to a manganese-permeant calcium channel and to kallikrein secretion [10]. In the most recent study by another group, coordinated actions of P2X7 (luminal) and P_2U-like (basolateral) receptors were proposed that mediate part of the transcellular cystic fibrosis transmembrane regulator (CFTR)-like Cl⁻ transport by acinar and duct cells to determine the final electrolyte composition of salivary fluid [752]. P2Y₂ receptors (= old P2U receptor, including P2Y₄ receptors) identified in both acinar and ductal cells of rat submandibular gland increased with time in culture, and it was speculated that changes in expression of the P2Y₂ receptor on salivary gland cells may be related to pathological challenges to the gland in vivo [665]. This group included a review of the field in an experimental paper, their main observations being:

1. P2Y₁ receptor activity is present in submandibular glands, although it tends to decline with age.

2. P2Y₂ receptors are present in cell lines and are upregulated during short-term culture of normal glands and following ligation of the main secretory duct of submandibular gland.

3. The P2X subtypes, P2X4 and P2X7, and the P2Y subtypes, P2Y₁ and P2Y₂, are co-expressed in salivary glands and salivary cell lines, and exhibit distinct basolateral, as opposed to apical, localisation in polarised cell monolayers as well as having discrete patterns of intracellular signalling [666].

In mouse submandibular ductal cells, P2X7 receptors are present, but P2X4 receptors are also involved in some ATP effects [555].

The conclusion from a study of nucleotide actions of the mouse submandibular salivary cell line, ST₅₈₈, was that two P2 receptor subtypes were probably present, one where ATP and UTP were equipotent (probably P2Y₂) and another where 2-MeSATP was active (possibly a P2Y₁ receptor) [273]. P2Y₂ receptors are upregulated in duct-ligated rat submandibular gland, and it was suggested that this may be an important component of the response to injury and that during recovery there is a return to P2Y₂ receptor levels [4]. P2Y₂ receptor activation upregulates vascular cell adhesion molecule-1 expression and enhances lymphocyte adherence from a human submandibular gland cell line [27]. Mechanical stimulation in submandibular gland cells results in the release of ATP, which then acts via P2Y₂ receptors to produce Ca²⁺ waves resulting in synchronised salivary gland cell function [590]. It was suggested that P2Y₂ receptors may be a novel target for dry mouth symptoms.

There is convincing evidence for the expression of Na⁺–H⁺ exchanger (NHE) isoforms in the basolateral membrane of rat submandibular gland duct and acinar cells (NHE1) and NHE2 and NHE3 in the luminal membrane of these cells which shows that the activities of the basolateral and luminal NHEs are regulated by P2 receptors (P2U receptors in the isolated membrane and P2X7 receptors in the luminal membrane) in a membrane-specific manner, which may play an important role in co-ordinating the overall process of Na⁺ absorption [428]. Another study [370] has shown that extracellular ATP and BzATP, a potent agonist for P2X7 receptors, substantially increased the release of arachidonic acid from rat submandibular gland ductal cells; these effects involved activation of PLA₂ by the purinergic agonists. It has been suggested that propofol, a widely used intravenous anaesthetic agent, potentiates the response, probably mediated by P2X4 receptors, of submandibular acinar cells [218].

An RT-PCR and pharmacological study of postnatal development of purinergic signalling in salivary glands, using dispersed cell aggregate preparations from the submandibular–sublingual gland complex of 1-day-old and 1-, 2-, 3- and 4-week old rats, showed that functional P2Y₁ receptors were expressed in immature (1 day postnatal) salivary glands and that receptor activity decreased as the glands matured, suggesting that P2Y₁ receptors may have an important role during salivary gland development [541]. P2Y₁ receptors have been shown to play important roles in embryonic chick development [476].

Ecto-ATP diphosphohydrolase and ecto-5′-nucleotidase have been identified in cultured rat submandibular glands, which hydrolyse ATP released as a cotransmitter from nerve terminals at the basal border of cells [320]. Ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) is colocalised with NTPDase and ecto-5′-nucleotidase in cells cultured from submandibular salivary glands [321]. Dry mouth is a common side effect caused by antidepressant therapy, and antidepressant drugs have been shown to modulate E-NPPs from submandibular gland cells [322].

Neurons in the parasympathetic submandibular ganglion innervate the submandibular gland to control secretion of saliva. ATP, probably released as a cotransmitter with ACh in preganglionic nerves, acts on both postsynaptic P2X [445, 625] and P1 and P2Y (probably P2Y₂) [2] receptors. Occupation of P1 and P2Y receptors led to inhibition of N- and P/Q-type voltage-dependent Ca²⁺ channel currents via G_i/o proteins [2].

Both P2X and P2Y subtypes are expressed by cells of the salivary glands, and opportunities for utilisation of these receptors as pharmaceutical targets for diseases involving salivary gland dysfunction appear promising (see [19, 667]). Primary Sjögren's syndrome is a common inflammatory autoimmune disorder characterised by decreased secretion of saliva leading to symptoms of dry mouth. ATP, probably released as a cotransmitter in sympathetic and/or parasympathetic nerves, increased [Ca²⁺]_i in the acini of labial salivary glands via P2Y receptors to activate saliva production in both saliva glands from healthy and Sjögren's patients [546]. It was speculated that there may be some abnormality in the innervation of salivary glands in Sjögren's syndrome. Another study suggested that the P2Y₂ receptor is upregulated in the submandibular gland of the NOD B10 mouse model of Sjögren's syndrome [610].

Gall bladder and bile duct

Gall bladder

Stimulation in vivo of the cervical vagus nerve produces contraction of the guinea pig gall bladder. Following block of this response by atropine, a relaxation is revealed which is not affected by the adrenergic neuron blocker, guanethidine. This relaxation is, however, blocked by hexamethonium, indicating that the NANC inhibitory neurons are located in the wall of the gall bladder. There was early evidence to suggest that the intrinsic NANC inhibitory neurons, like those in the gut, were purinergic [169]. The NANC relaxation was mimicked by ATP and intrinsic nerve cell bodies exhibit fluorescence for quinacrine which binds to high levels of granule-bound ATP. Release of ATP from strips of guinea pig gall bladder during transmural stimulation of intrinsic nerves was demonstrated [651]. ATP release was stimulation frequency-dependent and both ATP release and contractions were completely abolished in Ca²⁺-free medium; this suggests mediation by P2X receptors. It is likely that prostaglandins participate in the contractile response, since responses of the guinea pig gall bladder to ATP were antagonised by indomethacin [184]. Activation of an apical Cl⁻ conductance by extracellular ATP in Necturus gall bladder is mediated by cAMP, not by [Ca²⁺]_i [680]. A recent paper has shown that ATP stimulates P2Y₄ receptors within the gall bladder musculature and, in turn, stimulates prostanoid production via COX-1 leading to increased excitability of gall bladder smooth muscle [42].

P2Y₆ receptor mRNA was identified on mature gall bladder epithelial cells and UDP shown to promote current changes in cystic fibrosis gall bladder epithelia, suggesting that the P2Y₆ receptors may be a target for the treatment of cystic fibrosis gall bladder disease [424].

Biliary duct

The extra-hepatic biliary tract is innervated by dense networks of extrinsic and intrinsic nerves that regulate both smooth muscle tone and epithelial cell function [28]. ATP activates ion permeabilities in rat biliary epithelial cells (cholangiocytes that form the intrahepatic bile ducts) via two pathways, extracellular Ca²⁺-dependent and Ca²⁺-independent [467], implicating P2X and P2Y receptors. Cl⁻ secretion, measured by both electrophysiological and radio-nucleotide methods, is stimulated though the activation of P2Y₂ receptors in rat bile duct epithelial cells [224]. Ca²⁺-dependent activation of chloride currents in rat biliary epithelial cells is regulated by calmodulin-dependent protein kinase II [468]. Extracellular nucleotides modulate secretory and absorptive functions of cholangiocytes by activating Na⁺/H⁺ exchange mechanisms [202]. Apical P2Y₂ and basolateral P1(A₁) receptors regulate Na⁺/H⁺ exchange activity (acid/base transport) in rat cholangiocytes [765]. Basolateral ADP was more potent in stimulating transepithelial currents [597], consistent with mediation by P2Y₁, P2Y₁₂ or P2Y₁₃ receptors. ATP, ADP and AMP are present in rat, pig and human bile, perhaps released by paracrine and/or autocrine activities, in sufficient concentrations to regulate biliary secretion [125]. Evidence has been presented to suggest that tachykinins and ATP may be excitatory cotransmitters in NANC nerves supplying the guinea pig common bile duct [543]. ATP is released into the bile by both hepatocytes and cholangiocytes, where it functions as a potent paracrine/autocrine stimulator for cholangiocyte secretion (Fig. 9) [583, 584]. Both small and large cholangiocytes show mechanosensitive vesicular ATP release, but this is greater in small cholangiocytes [709].

Fig. 9.

ATP stimulates transepithelial secretion across cholangiocyte monolayers. In this example, polarised cell monolayers are mounted in an Ussing chamber, and the transepithelial movement of Cl⁻ ions from the basolateral to apical space is measured as short circuit current (Isc, A). Under basal conditions, Isc is low (b). However, addition of ATP (bar) activates P2Y₂ receptors (P2R) in the apical membrane and elicits a brisk Cl⁻ secretory response as demonstrated by a marked increase in Isc. Apical Cl⁻ secretion occurs via opening of apical membrane Cl⁻ channels and is inhibited by application of the selective anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, bar) (reproduced from [583], with permission from Elsevier)

A key role in modifying the volume and composition of bile is played by fluid absorption and secretion across intrahepatic bile duct units (IBDUs). P2Y₁, P2Y₂, P2Y₄, P2Y₆ and P2X4 receptor mRNAs were expressed in isolated, microperfused IBDUs using RT-PCR [190]. In human intrahepatic biliary epithelial cell lines [706], ATP and UTP increase [Ca²⁺]_i, probably via P2Y₂ or P2Y₄ receptors. Purinoceptors mediate activation of cholangiocytes to secrete Cl⁻ and HCO₃⁻ in the intrahepatic bile ducts [597]. Vesicles containing ATP within the biliary epithelial cells are in part responsible for the initiation of purinergic signalling in the biliary system [601].

RT-PCR from cultured rat cholangiocytes detected transcripts for P2X2, P2X3, P2X4 and P2X6 receptors, and immunohistochemistry showed that the P2X4 receptor protein was dominant, particularly in intrahepatic bile ducts, and functional studies implicated the P2X4 receptor in modulation of biliary secretion [183]. Fluid flow (shear stress) induces cholangiocyte mechanosensitive ATP release through calcium signalling and chloride transport via PKC-dependent pathways [707]. Portal fibroblasts inhibit the proliferation of bile duct epithelia via blockade of P2Y activation and expression of NTPDase2 [363]. TRPV4 is expressed on cholangiocyte cilia in intrahepatic bile duct units and its activation induces increases in bile flow, ATP release and bicarbonate secretion [292]. It has been suggested that cholangiocyte primary cilia are chemosensory organelles that detect biliary nucleotides via P2Y₁₂ receptors [461]. The existence of a P2 receptor signalling axis was proposed, present along the intrahepatic biliary tree, with upstream small cholangiocytes releasing ATP, which then serves as a signalling molecule for downstream large cholangiocytes [708].

It was suggested that ATP release may be a key regulator of biliary secretion and a target to modulate bile flow in the treatment of cholestatic liver disease. It has been claimed that extracellular ATP induces IL-6 transcription in bile duct epithelial cells via the P2Y₁₁ receptor [742]. P2Y₁₃-deficient mice exhibit a decrease in hepatic high-density lipoprotein cholesterol uptake, hepatic cholesterol content and biliary cholesterol output [66]. Pharmacological activation of P2Y₁₃ receptors with ADP increases reverse cholesterol transport and it was suggested that P2Y₁₃ agonists may have a potential role as a novel target for the treatment of dyslipidemia. Cholangiopathies are characterised by impaired cholangiocyte secretion. Ursodeoxycholic acid (UDCA) is widely used for cholangiopathy treatment. Data has been presented that indicates that UDCA stimulates a CFTR-dependent apical ATP release from cholangiocytes to act on P2Y receptors, which, through [Ca²⁺]_i increase and PKC activation, stimulates Cl⁻ efflux and fluid secretion [221]. Bile ductular proliferation is markedly upregulated in biliary fibrosis and cirrhosis. The ectonucleotidase NTPDase2 has been shown to be a critical regulator of bile ductular proliferation and IL-6 to downregulate NTPDase2 mRNA expression [741]. The apical P2Y–InsP₃ receptor signalling pathway mediating Cl⁻ transport may be a potential target for increasing secretion for the treatment of cholestatic liver disease [197]. Bicarbonate secretion is a function of cholangiocytes and it has been reported that cAMP regulates bicarbonate secretion from cholangiocytes via release of ATP into the bile [479].

Summary and future directions

Purinoceptors are widely expressed by non-neuronal cells as well as neurons in the gut and associated organs (see Tables 1, 2 and 3). Exploration of the therapeutic potential of purinergic-related drugs for inflammatory and motility disorders is in its infancy and should be encouraged in future investigations.

Table 1.

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

	A 1	A 2a	A 2b	A 3
Jejunum
Whole thickness	− (−)	− (+)?	++	++
Mucosa/submucosa	− (−)	++	++	+
Mucosa	− (−)	+	++	+
Mucosa	− (+)?	++	++	++
Submucous plexus	− (+)	− (+)	+	+
Longitudinal/circular	+ (++)	++	++	+
Ileum
Whole thickness	+ (+)	++	+	+
Mucosa/submucosa	ND	ND	ND	ND
Mucosa	− (+)	++	+	+
Submucous plexus	ND	ND	ND	ND
Caecum
Whole thickness	− (−)	+	++	+
Mucosa/submucosa	− (+)	++	+	+
Mucosa	− (−)	+	++	+
Submucous plexus	− (+)	− (−)	+	− (+)
Colon
Whole thickness	− (−)	− (−)	+	− (+)?
Mucosa/submucosa	− (−)	− (−)	++	+
Mucosa	ND	ND	ND	ND
Submucous plexus	− (+)	− (+)?	++	+
Human cell lines
HT-29 (colonic epithelium)	+ (+)	+++	+++	− (−)?
T-84 (colonic epithelium)	− (+)	+	+++	− (−)?
T98G (glioblastoma)	++	+++	+++	− (+)

ND not done, + or − indicates receptor mRNA detected or not detected, respectively. Failure to detect adenosine receptor mRNA after the first round of PCR reflects either absence or low expression. If the results of RT-PCR were negative, an aliquot of the first PCR reaction was amplified a second time, and the result is indicated in parentheses. Failure to detect adenosine receptor mRNA after the second round of PCR is likely to be due to its absence. A question mark indicates a possible faint expression of receptor mRNA (reproduced from [130], with permission from Springer)

Table 2.

Cellular localisation of adenosine A₁, A_2A, A_2B and A₃ receptor immunoreactivities in human small and large intestine

Human intestinal region/cell type	A 1	A 2a	A 2b	A 3
Jejunum				+
Longitudinal muscle	±	±	−
Myenteric plexus neurons	+	+	+
Glia	−	−	+++
Circular muscle	+	−	−
Submucous plexus neurons	−	+++	+++
Nerve fibres/neurites	−	+	+	+
Epithelia	−	+	+	+
Colon				+
Longitudinal muscle	±	−	−
Myenteric plexus neurons	−	+	+
Glia	−	−	+
Circular muscle	+	+	+
Submucous plexus neurons	+++	+	+
Epithelia	+
T98G				+
U373				+
BON cells				+

− absent, + present (or present ≤2 neurons), ± marginally detectable, ++ three to six neurons, +++ >6 neurons (reproduced from [130], with permission from Springer)

Table 3.

Functional distribution of luminal P2 receptors in gastrointestinal epithelial cells

Tissue	Species	P2 receptor	Endogenous agonist	Function	Signalling	Reference
Jejunum	Mouse	P2Y4	ATP = UTP	Cl− secretion ↑		[157]
Duodenal villus	Rat	P2X7		Apoptosis ??		[298]
Pancreatic duct	Guinea pig	P2Y2	ATP = UTP	HCO3 − secretion ↑	Ca2+ ↑	[355]
	Rat	P2X7	Bz-ATP		Ca2+ ↑	[317]
	Rat	P2Y2, P2Y4, P2X7	UTP/ATP	Cl− secretion ↑	Ca2+ ↑	[448]
	Dog		ATP	Mucin secretion ↑	Ca2+ ↑	[507]
PDEC	Human	P2Y2/P2Y4	UTP/ATP	Cl− secretion ↑	Ca2+ ↑	[124]
CFPAC-1	Mouse	P2Y2/P2Y6	ATP = UTP/UDP	Cl− secretion ↑		[157]
Gall bladder	Mouse	P2Y2	ATP/UTP	HCO3 − secretion ↑	Ca2+ ↑	[144]
	Necturus	P2	ATP	Cl− secretion ↑	cAMP ↑	[680]
Bile duct	Rat	P2Y1, P2Y2	ATP, UTP	HCO3 − secretion ↑
		P2Y4, P2Y6	ADP, UDP
			2MeSATP
	Rat	P2Y2/P2Y4	UTP/ATP	Cl− secretion ↑		[609]
Colon	Rat, mouse	P2Y2/P2Y4	UTP/ATP	K+ secretion ↑	Ca2+ ↑	[386]
				Na+ absorption ↓	?
Caco-2	Human	P2Y2, P2Y4, P2Y6	UTP/ATP	Cl− secretion ↑	Ca2+ ↑	[464]

Reproduced from [130], with permission from Springer