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Review Article |
Autonomic Neuroscience Centre, Royal Free and University College Medical School, London, United Kingdom
Abstract
Abstract I. Introduction II. Cardiovascular Diseases A. Thrombosis B. Heart Failure C. Hypertension D. Atherosclerosis and Restenosis III. Neurology A. Neuroprotection B. Migraine C. Pain D. Diseases of Special Senses 1. Eye. 2. Ear. 3. Nasal Organs. IV. Immune System and Inflammation V. Endocrinology, Diabetes, and Obesity A. Diabetes B. Obesity VI. Gastroenterology VII. Urogenital Diseases A. Kidney and Ureter B. Lower Urinary Tract C. Reproductive System VIII. Dermatology IX. Respiratory Diseases X. Musculoskeletal Diseases XI. Oncology XII. Future Developments
The concept of a purinergic signaling system, using purine nucleotides and nucleosides as extracellular messengers, was first proposed over 30 years ago. After a brief introduction and update of purinoceptor subtypes, this article focuses on the diverse pathophysiological roles of purines and pyrimidines as signaling molecules. These molecules mediate short-term (acute) signaling functions in neurotransmission, mechanosensory transduction, secretion and vasodilatation, and long-term (chronic) signaling functions in cell proliferation, differentiation, and death involved in development and regeneration. Plasticity of purinoceptor expression in pathological conditions is frequently observed, including an increase in the purinergic component of autonomic cotransmission. Recent advances in therapies using purinergic-related drugs in a wide range of pathological conditions will be addressed with speculation on future developments in the field.
A seminal article by Drury and Szent-Györgi in 1929
described the potent actions of purine nucleotides and nucleosides, ATP, and adenosine on the heart and blood vessels. Then, in 1970, evidence was presented for ATP as a neurotransmitter in nonadrenergic, noncholinergic (NANC1) nerves supplying the gut (Burnstock et al., 1970) and in 1972 the word "purinergic" was coined and the purinergic neurotransmission hypothesis was proposed by Burnstock (Burnstock, 1972). This concept met with considerable resistance for many years, because ATP had been established as an intracellular energy source involved in various metabolic cycles, and it was thought that such a ubiquitous molecule was unlikely to be involved in selective extracellular signaling. However, the concept is now widely accepted. Later, it was established that ATP was a cotransmitter with classic transmitters in both the peripheral and central nervous systems and that purines are also powerful extracellular messengers to non-neuronal cells, including exocrine and endocrine, secretory, endothelial, musculoskeletal, immune, and inflammatory cells (Burnstock and Knight, 2004).
Implicit in the purinergic hypothesis was the presence of purinoceptors (Ralevic and Burnstock, 1998). A basis for distinguishing P1 (adenosine) from P2 (ATP/ADP) receptors was proposed by Burnstock in 1978. This helped resolve some of the ambiguities in earlier reports, which were complicated by the breakdown of ATP to adenosine by ectoenzymes so that some of the actions of ATP were directly on P2 receptors, whereas others were due to indirect action via P1 receptors. Four subtypes of P1 receptors were cloned, namely, A1, A2A, A2B, and A3. In 1985, Burnstock and Kennedy proposed a basis for distinguishing two types of P2 purinoceptor, namely, P2X and P2Y, based largely on pharmacological criteria. In the early 1990s, studies of transduction mechanisms and cloning of both P2X and P2Y receptors were carried out, which led Abbracchio and Burnstock to put forward a new nomenclature system in 1994, which is now widely accepted (Fredholm et al., 1994; Schwiebert, 2003). They proposed that there were two families of P2 receptors, namely, P2X ionotropic ligand-gated ion channel receptors and P2Y metabotropic G protein-coupled receptors. This framework has allowed a logical expansion as new receptors were identified. Currently seven subtypes of P2X receptors and eight subtypes of P2Y receptors are clearly established (Table 1). It is recognized that the pyrimidine UTP is equipotent with ATP at P2Y2 and P2Y4 receptors and UDP is a potent agonist at P2Y6 receptors. The distribution and functional roles of P2 receptors in many cell types in the body have been described (Burnstock and Knight, 2004).
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Purinergic signaling is rapid in synaptic neurotransmission, in neuromuscular transmission leading to contraction or relaxation of smooth muscle, and in exocrine or endocrine secretion. However, there are now many examples of purinergic signaling regulating long-term events such as cell proliferation, differentiation, migration, and death in development, regeneration, and wound healing. Both P2X and P2Y receptors play prominent roles both directly and by modulation of other signaling systems in embryonic development, including the nervous system, cartilage in limb buds, the mesonephros, retina, myotubes, and neuromuscular junctions (Burnstock, 2001a).
There is increasing interest in the therapeutic potential of purinergic compounds (including receptor agonists and antagonists, ectoenzyme inhibitors and enhancers, and ATP transport inhibitors and enhancers) in a wide range of disease conditions in relation to both P1 and P2 receptors. A number of purine-related compounds have been patented. The autonomic nervous system shows marked plasticity. Dramatic changes occur in the expression of cotransmitters and receptors during development and aging, in nerves that remain after trauma or surgery and in disease conditions. There are several pathological conditions in which the purinergic component of cotransmission is increased (Burnstock, 2002b). The emphasis in this article is on the pathophysiology and therapeutic potential of P2 receptors (see also Burnstock and Williams, 2000; Boeynaems et al., 2001; Yerxa, 2001; Burnstock, 2002a; Jacobson et al., 2002; Ralevic and Burnstock, 2003) and readers are referred to recent reviews about the therapeutic potential of P1 receptors (Fredholm et al., 2002, 2005; Okusa, 2002; Pelleg et al., 2002; Dhalla et al., 2003; Ribeiro et al., 2003; Hutchinson and Scammells, 2004; McCallion et al., 2004).
ATP and adenosine are very much involved in the mechanisms underlying local control of vessel tone (Burnstock and Ralevic, 1994) (Fig. 1) as well as cell migration, proliferation, differentiation, and death during angiogenesis, atherosclerosis, and restenosis following angioplasty (Erlinge et al., 1998; Burnstock, 2002b). ATP, released as a cotransmitter from sympathetic nerves, constricts vascular smooth muscle via P2X receptors, whereas ATP released from sensory-motor nerves during "axon reflex" activity dilates or constricts some vessels. Furthermore, ATP released from endothelial cells during changes in flow (shear stress) or hypoxia acts on P2Y receptors in endothelial cells to release nitric oxide (NO), resulting in relaxation. Adenosine produced by the breakdown of extracellular ATP causes vasodilatation via smooth muscle P1 receptors. P2X receptors are also present on endothelial cells and appear to be associated with cell adhesion and permeability. ATP release from red blood cells is increased in pathological conditions such as subarachnoid hemorrhage, largely because there is widespread blood cell lysis (Sprague et al., 2003). This leads to transient constriction of arterioles via P2X receptors and sustained constriction of large cerebral vessels, largely through P2Y2 receptors. The differences in purinergic receptor distribution between macro- and microvessels in the cerebral circulation are likely to have important consequences in pathological conditions.
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There have been very promising developments concerning purinergic antithrombotic drugs. Platelets are known to express P2Y1, P2Y12, and P2X1 receptors (Hollopeter et al., 2001). "Mega" clinical trials CAPRIE (CAPRIE Steering Committee, 2001), CURE (Yusuf et al., 2001), and CREDO (Beinart et al., 2005) have provided clear evidence that the purinergic antithrombotic drugs clopidogrel and ticlopidine reduce the risks of recurrent strokes and heart attacks, especially when combined with aspirin (Kam and Nethery, 2003; Kunapuli et al., 2003; Boeynaems et al., 2005). These drugs are antagonists to the P2Y12 receptor that mediates platelet aggregation (Gachet, 2001). MRS2500, a highly potent and selective antagonist for the P2Y1 receptor, has also been shown recently to have antiaggregating activity on human platelets (Cattaneo et al., 2004). There are genetic variations in P2Y1 and P2Y12 receptor gene sequences in healthy subjects that explain variations in the platelet response to ADP; this may reflect individual variation in atherothrombotic risk and the efficacy of purinergic antithrombotic drugs (Fontana et al., 2003; Hetherington et al., 2004). A recent study has shown that there is a synergistic inhibition of ADP-induced platelet activation via P2Y1 and P2Y12 receptors and for thrombin and P2Y12 inhibition; it is suggested that there may be clinical benefit by combining these inhibitors, providing that bleeding problems do not outweigh this benefit (Nylander et al., 2004). Postoperative carotid thrombosis is a significant risk for stroke; it seems likely that clopidogrel or ticlopidine may provide an avenue for targeted antiplatelet therapy following vascular intervention (Hayes et al., 2003). Platelet aggregation in response to ADP is significantly inhibited in patients with peripheral vascular disease 2 to 4 h after a loading dose of clopidogrel, and ADP-induced platelet shape change is significantly inhibited following ingestion of clopidogrel (Matsagas et al., 2003). There is a synergistic interaction between ATP and noradrenaline in stimulating platelet aggregation, which suggests a prothrombotic role for ATP in stress (Birk et al., 2003). Platelet activation that occurs in human acute malaria infection is associated with elevated plasma ATP concentrations (Essien and White, 1998).
Much is now known about the ectonucleotidases that break down ATP released from non-neural cells as well as neurons (Zimmermann, 2001; Vorhoff et al., 2005). Several enzyme families are involved: ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), of which NTPDase1, 2, 3, and 8 are extracellular; ectonucleotide pyrophosphatase of three subtypes; alkaline phosphatases, ecto-5'-nucleotidase and ectonucleoside diphosphokinase. NTPDase1 hydrolyzes ATP directly to AMP and UTP to UDP, whereas NTPDase2 hydrolyzes ATP to ADP and 5'-nucleotidase AMP to adenosine. Ectonucleotidases are expressed by vascular endothelium, accessory vascular cells (e.g., monocytes, pericytes, and vascular smooth muscle cells), and dendritic cells; these are predominantly NTPDase1 and NTPDase2 (Sevigny et al., 2002). NTPDase1 hydrolyzes both tri- and diphosphonucleosides and blocks platelet aggregation responses to ADP. In contrast, NTPDase2, a preferential nucleoside triphosphatase, activates platelets by converting the competitive antagonist (ATP) of platelet ADP receptors to the specific agonist. Vascular NTPDase1 biochemical activity is rapidly lost from the endothelium of vascularized cardiac grafts subjected to oxidant stress. These changes are associated with thrombotic injury and platelet sequestration at sites of injury.
Up-regulation of P2X1 and P2Y2 receptor mRNA in the hearts of rats with congestive heart failure has been reported (Hou et al., 1999) and an increase in expression of P2X1 receptors in the atria of patients suffering from dilated cardiomyopathy (Berry et al., 1999). In congestive heat failure, the muscle mechanoreflex is accentuated and appears to be associated with enhanced P2X receptor-mediated responses of sensory nerves to 
,
-methylene ATP (Sinoway and Li, 2005). The positive inotropic response to ATP and ATP-induced increases in [Ca2+]i in cardiomyocytes are impaired in heart failure due to myocardial infarction; imidapril partially reverses this impairment (Saini et al., 2005). ATP and adenosine are widely used for the treatment of paroxysmal supraventricular tachycardia in both infants and adults (Belhassen and Pelleg, 1985; Vassort, 2001). The substantial enhancement of mechanical performance with 2-deoxy-ATP in cardiac muscle suggests that it may be a better substrate for contractility than ATP and suggests that ribonucleotide reductase may be a target for therapy in heart failure (Regnier et al., 2000).
Enhanced sympathetic nerve activity causes cardiac dysfunction, arrhythmias, and sudden cardiac death in myocardial ischemia. ATP is coreleased with noradrenaline (NA) and enhances NA release from sympathetic nerve terminals. A role for the ectonucleotidase, E-NTPDase1 at sympathetic nerve terminals may offer a novel therapeutic approach to hyperadrenergic states such as myocardial ischemia (Sesti et al., 2002). Because NTPDase1 also inhibits platelet aggregation, it is proposed for the treatment of thrombotic diatheses (Marcus et al., 2003). To examine the role of NTPDases after cardiac transplantation and grafts, several techniques to manipulate NTPDase1 expression have been used, for example, deletion of this enzyme in mice. These mutant mice exhibit major perturbations of P2 receptor-mediated signaling in the vasculature and immune systems and show hemostatic defects, thromboregulatory disturbances, and heightened acute inflammatory responses with a failure to generate cellular responses (Enjyoji et al., 1999); NTPDase1-null hearts have been shown to develop diffuse thrombotic infarcts and undergo rejection more rapidly than control grafts when transplanted into wild-type mice or into rats. In contrast, transgenic organs or cardiac grafts in which NTPDase1 has been up-regulated by gene therapy vectors enjoy a survival advantage post-transplantation (Imai et al., 2000).
ATP is routinely used to terminate spontaneous ventricular tachycardia, but it is not always successful, although no difference in efficacy was found between ischemic and nonischemic cardiomyopathy (Kouakam et al., 2003). By using transgenic overexpression of human P2X4 receptors, cardiac P2X4 receptors have been shown to have a beneficial life-prolonging role in heart failure; increased expression or activation of these ATP-stimulated receptor channels may represent a new therapeutic approach to the treatment of heart failure (Yang et al., 2004). Administration of ATP during sinus rhythm has been suggested as a useful bedside test for identifying patients with a concealed accessory pathway who are prone to develop atrioventricular reentrant tachycardia (Belhassen et al., 2000). The negative chronotropic effect of ATP on sinus node is much more pronounced in patients with sick sinus syndrome (Tan et al., 2004).
Magnesium-ATP has been recommended for the treatment of ischemia, radiation injury, shock, and sepsis for many years (Harkema and Chaudry, 1992; Nalos et al., 2003). A recent study suggests that the protective effect of ATP on renal ischemic injury is, in part, related to inhibition of the inducible transcription factor, nuclear factor-
, via P2Y receptors in proximal tubule cells (Lee and Han, 2005). ATP was shown to inhibit atrioventricular conduction rather than the firing rate of sinoatrial nodes in patients with ischemic heart disease; injection of ATP is used only when a transient cardiac standstill is needed, such as for endovascular grafting surgery (Watanabe et al., 2002). Treatment of myocardial ischemia and reperfusion by ATP-MgCl2 is still recommended (Katircioglu et al., 2000), but many reports suggest that adenosine (a breakdown product of ATP) also mediates this effect (Liang and Jacobson, 1999; Broadley, 2000; Sommerschild and Kirkeboen, 2000; Thompson et al., 2002).
ATP plays a significant cotransmitter role in sympathetic nerves supplying hypertensive blood vessels. The purinergic component is increased in spontaneously hypertensive rats (Ralevic and Burnstock, 1998). The increase in sympathetic nerve activity in hypertension is well established, and there is an associated hyperplasia and hypertrophy of arterial walls (Julius and Nesbitt, 1996). ATP is a rapidly acting hypotensive agent that compares favorably with sodium nitroprusside (Kien et al., 1987). ATP-MgCl2 is a safe, effective, and preferential pulmonary vasodilator in children with pulmonary hypertension secondary to congenital heart defects; it has also been used for treating pulmonary hypertension after cardiac surgery (Brook et al., 1994). Red blood cells of humans with primary pulmonary hypertension fail to release ATP in response to mechanical deformation (Sprague et al., 2001). This is likely to result in the loss of local control of total pulmonary vascular resistance in the lung. Low-dose ATP exerts mitogenic actions on human pulmonary artery smooth muscle cells, which may be relevant to the pathophysiological basis of severe pulmonary hypertension (Zhang et al., 2004b). Pulmonary hypertension can be a problem in patients with chronic obstructive pulmonary disease, which also has other causes; it is a life-threatening condition, and intravenous ATP infusion produces a significant decrease in mean pulmonary arterial pressure and pulmonary vascular resistance without changing the mean systemic arterial pressure (Brook et al., 1994). ATP has been used to treat meconium aspiration-induced pulmonary hypertension in pigs (Kääpä et al., 1997). Eicosapentaenoic acid, one of the active components in fish oil that has antihypertensive effects, increases the release of ATP from vascular endothelial cells, leading to reduction of the blood pressure rise characteristic of aging (Hashimoto et al., 1998). It has been proposed that there is prognostic value of stress myocardial perfusion imaging using ATP at the beginning of hemodialysis treatment in patients with end-stage renal disease (Hase et al., 2004). P2X4 receptors are localized in the syncytiotrophoblast, stroma, and fetal capillary endothelial cells of human placenta. Placental P2X4 receptors are significantly up-regulated in mild preeclampsia (Roberts et al., 2005). The authors hypothesized that with preeclampsia, in which there is an increased potential for ATP release, P2X4 receptors may mediate a change in cellular homeostasis to limit cell damage.
D. Atherosclerosis and Restenosis
An early study reported that adenosine produces changes in cAMP and DNA synthesis in cultured arterial smooth muscle cells and suggested that this might result in the regulation of cell proliferation (Jonzon et al., 1985). The authors speculated that adenosine could be one of several regulatory factors in the development of atherosclerosis and might also regulate the release of a smooth muscle mitogen, platelet-derived growth factor. There is now good evidence that adenosine does regulate smooth muscle cell proliferation, but its properties differ from those for ATP and ADP (Burnstock, 2002a; Di Virgilio and Solini, 2002). Vascular injury represents a critical initiating event in the pathogenesis of various vascular diseases. Large amounts of ATP are released from injured cells and ATP and adenosine have potent actions in smooth muscle and endothelial cell growth, migration, proliferation, and death (Burnstock, 2002a). Apoptotic cell death is recognized to occur in a number of vascular diseases, including atherosclerosis and restenosis. Vascular endothelial cells are continuously exposed to variations in blood flow, which plays an important role in vessel growth or regression and in the local development of atherosclerosis. The shear stress that occurs during changes in blood flow leads to substantial release of ATP (and UTP) from endothelial cells (Burnstock, 1999), and these purines might mediate alterations in the balance between proliferation and apoptosis. Atherosclerotic damage results in the disappearance of endothelium-dependent responses to ATP. The release of ATP from endothelial cells has also been claimed to be impaired in atherosclerotic arteries and long-term supplementation with a high cholesterol diet decreases the release of ATP. In restenosis following balloon angioplasty, there is a peak in the proliferation and apoptosis of vascular smooth muscle cells at approximately 14 days. Clinical trials with clopidogrel and ticlopidine (P2Y12 receptor antagonists) in patients with atherosclerotic disease have shown significant benefit compared with aspirin.
Saphenous vein, internal mammary, and radial arteries have been used as grafts for coronary bypass surgery; the level of endothelial P2Y2 receptors is comparable in all three vessels, but endothelial P2X4 receptors vary from high in saphenous vein to significantly lower in the other two vessels. It has been suggested that P2X4 receptors play a more significant role in intense proliferation in arteriosclerosis and restenosis than P2Y2 receptors, as reflected by the susceptibility of saphenous vein grafts to atherosclerosis compared with internal mammary arteries (Ray et al., 2002). In another study, P2X1 and P2Y6 receptors mediated more prominent contractions in the saphenous vein compared with the internal mammary artery; it has been suggested that selective antagonists to these receptors may prevent vasospasm and restenosis in the saphenous vein during and after revascularization surgery (Borna et al., 2003). A novel role for P2Y2 receptors in the development of atherosclerosis has been suggested, whereby UTP induces vascular cell adhesion molecule-1 expression in coronary artery endothelial cells that mediate the recruitment of monocytes (Seye et al., 2003). The long-term (trophic) roles of purinergic signaling in vascular smooth muscle and endothelial cell proliferation and death have been implicated in atherosclerosis and restenosis and suggest the exploration of therapeutic strategies in relation to these events (Erlinge et al., 1998; Burnstock, 2002b; Hou et al., 2002; Wang et al., 2003b).
Newly developing vascular endothelia express very high levels of NTPDase1, also seen under hypoxic conditions (Eltzschig et al., 2003). Angiogenesis requires the dynamic interaction of endothelial cell proliferation and differentiation with orchestrated interactions between extracellular matrix and surrounding cells (such as vascular smooth muscle and/or pericytes). Such interactions could be coordinated by interplay between nucleotide release, P2 receptor modulation, and altered NTPDase expression (Goepfert et al., 2001). The role for adenosine in growth regulation in angiogenesis has been reviewed recently (Adair, 2005).
Varicose veins are characterized by hypoxia, inflammation, and cell death, all likely to lead to increases in extracellular ATP. P2X1 and P2X7 receptors are expressed by saphenous vein smooth muscle, and it has been suggested that the resultant P2X7 receptor activation contributes to the decrease in contractile myocytes and resulting disorganization of the vessel wall (Cario-Toumaniantz et al., 1998).
Functional implications for purinergic signaling in the nervous system include neuron-glia interactions, differentiation of stem cells, diseases of the nervous system and responses to injury, neuroimmune and neurovascular interactions, sympathetic transmission and plasticity, glial cell differentiation, intercellular communication between glia, and myelination (Fields and Stevens, 2000; Burnstock, 2003b; Newman, 2003; Pascual and Haydon, 2003; Bowser and Khakh, 2004; Mulligan and MacVicar, 2004; Davalos et al., 2005; Köles et al., 2005).
ATP is a cotransmitter in many nerve types, probably reflecting the early evolutionary presence of purinergic signaling (Burnstock, 1996a, 2004). There is evidence for the action of ATP as a cotransmitter with NA and neuropeptide Y in sympathetic nerves, with acetylcholine and vasoactive intestinal peptide in some parasympathetic nerves, with NO and vasoactive intestinal peptide in enteric NANC inhibitory nerves, and with calcitonin gene-related peptide (CGRP) and substance P in sensory-motor nerves. There is also evidence for the cotransmission of ATP with GABA in retinal nerves, and with glutamate, 5-hydroxytryptamine (serotonin), NA, or dopamine in nerves in the brain (Burnstock, 2003b). In sympathetically innervated tissues, such as the vas deferens or blood vessels, ATP produces fast responses mediated by P2X receptors, followed by a slower component mediated by G protein-coupled -adrenoceptors. Similarly, in the parasympathetic nerves supplying the urinary bladder, ATP provokes a fast transient response via P2X receptors, whereas the slower component is mediated by G protein-coupled muscarinic receptors. There are differences in the proportion of cotransmitters between species in development and aging and in different pathophysiological conditions. P2X3 and P2X2/3 receptors are selectively localized on sensory neurons in trigeminal, nodose, and dorsal root ganglia (DRG), and the terminals of these nociceptive neurons in the skin and visceral organs represent unique targets for novel analgesic agents that function as P2X3 and P2X2/3 receptor antagonists. Nonspecific P2 receptor antagonists, e.g., suramin and pyridoxal-5'-phosphate-6-azophenyl-2',4' disulfonic acid (PPADS), are antinociceptive, and P2X3 receptor-knockout mice reduce nociceptive inflammatory responses (Cockayne et al., 2000; Burnstock, 2001b).
The first clear evidence for nerve-nerve purinergic synaptic transmission was published in 1992 (Edwards et al., 1992; Evans et al., 1992; Silinsky et al., 1992). Synaptic potentials in the celiac ganglion and in the medial habenula in the brain were reversibly antagonized by suramin. Since then, many articles have described either the distribution of various P2 receptor subtypes in the brain and spinal cord or electrophysiological studies of the effects of purines in brain slices, isolated nerves, and glial cells (Burnstock, 2003b). Synaptic transmission has also been demonstrated in the myenteric plexus and in various sensory, sympathetic, and pelvic ganglia (Dunn et al., 2001). Adenosine, produced following ectoenzymatic breakdown of ATP, acts through presynaptic P1 receptors to inhibit the release of excitatory neurotransmitters in both the peripheral and the central nervous systems. P2Y receptors are expressed on both nonmyelinating and myelinating Schwann cells.
In the brain, P2 purinergic signaling is involved in the regulation of a variety of physiological and pathophysiological processes, including development and nervous tissue remodeling following trauma, stroke, ischemia, or neurodegenerative disorders (Burnstock, 2003a; Neary et al., 2004). Agonists and antagonists of adenosine and inhibitors of adenosine kinase are also being explored as therapeutic neuroprotective agents as well as for a number of other neurological conditions including epilepsy, sleep apnoea, pain, inflammation, aggression, anxiety, and memory loss (Kowaluk and Jarvis, 2000; Ribeiro et al., 2003; Fredholm et al., 2005; Köles et al., 2005). Microinjection of ATP analogs into the prepiriform cortex induces generalized motor seizures (Knutsen and Murray, 1997). P2X2, P2X4, and P2X6 receptors are expressed in the prepiriform cortex, suggesting that P2X receptor antagonists may have potential as neuroleptic agents. The hippocampus of chronic epileptic rats shows abnormal responses to ATP associated with increased expression of P2X7 receptors; it has been suggested that P2X7 receptors (perhaps on microglia) may participate in the pathophysiology of temporal lobe epilepsy (Vianna et al., 2002).
In nervous tissue, trophic factors ensure neuronal viability and regeneration. Neuronal injury releases fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor (Neary et al., 1999). In combination with these growth factors, ATP can stimulate astrocyte proliferation, contributing to the process of reactive astrogliosis, and to hypertrophic/hyperplastic responses. P2Y receptor antagonists have been proposed as potential neuroprotective agents in the cortex, hippocampus, and cerebellum by modulation of kainate and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced currents, excessive activation of glutamate receptor systems being implicated in neuronal cell death associated with stroke, epileptic seizures, and neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, and amyotrophic lateral scoliosis (Zona et al., 2000). Release of ATP from disrupted cells may cause cell death in neighboring cells expressing P2X7 receptors, leading to a necrotic volume increase, which has been proposed as a cellular mechanism in the pathogenesis of Parkinson's disease (Jun and Kim, 2004). Guanine nucleotides inhibit NMDA and kainate-induced neurotoxicity in cultured rat hippocampal and neocortical neurons and may be candidates for antagonizing glutamate receptor-mediated neurotoxicity (Morciano et al., 2004). P2 receptors have been claimed to mediate neuroprotective effects in the cerebellum and the possible therapeutic use of P2 receptor agonists as neuroprotective agents has been raised (Volonte et al., 1999, 2003). It has been suggested that adenine is involved in the control of Purkinje cell survival (Watanabe et al., 2003). Up-regulation of P2X1 and P2X2 receptors after cerebellar lesions has been reported (Florenzano et al., 2002). Purine derivatives are in clinical trials as memory-enhancing agents in Alzheimer's disease; two of these, propentofylline and AIT-082, appear to act as trophic effectors, increasing the production of neurotrophic factors in brain and spinal cord (Rathbone et al., 1999). It has been reported that aluminum can produce Alzheimer-like symptoms, and a mechanism has been proposed whereby the aluminum binds to ATP to act on P2 purinoceptors leading to formation of amyloid fibrils (Exley and Korchazhkina, 2001). The therapeutic potential of adenosine A2A receptor antagonists in Parkinson's disease is being explored (Simola et al., 2004; Xu et al., 2005).
ATP inhibits the release of the excitatory transmitter, glutamate, and stimulates release of the inhibitory transmitter, GABA, from hippocampal nerves, thus serving a protective role (Inoue, 1998). In addition, ATP coreleased with glutamate induces long-term potentiation (LTP) in CA1 neurons associated with learning and memory (Fujii et al., 1999; Mori et al., 2001). Nanomolar concentrations of ATP induce long-lasting enhancement of LTP in hippocampal neurons; the P2 antagonist, suramin, inhibited activity of the ectoenzyme, apyrase, which has been shown to participate in the mechanisms of memory acquisition (Bonan et al., 1999). It has been suggested that ATP coreleased with glutamate activates CA1 pyramidal hippocampal neurons, allowing calcium to enter postsynaptic cells and thereby inhibiting the effectiveness of NMDA receptors in inducing LTP (Pankratov et al., 2002). Because P2X receptors contribute to synaptic transmission, mainly at low frequencies of stimulation, they may act as a dynamic low-frequency filter, preventing weak stimuli from inducing long-lasting changes in synaptic efficacy. It has also been claimed that ATP analogs can facilitate LTP through P2 receptor activation that triggers adenosine release, leading to activation of P1 (A2A) receptors (Almeida et al., 2003). Large rises in [Ca2+]i in CA1 neurons induce LTP, but small rises induce long-term depression (Yamazaki et al., 2002). ATP and activation of glutamate NMDA receptors leads to potentiation of LTP in CA1 neurons (Fujii et al., 2002) in keeping with the synergism that often occurs between cotransmitters (Burnstock, 2004). There is expression of functional P2X receptor channels in the axons of CA3 neurons branching to their postsynaptic targets and predominantly in nerve terminals forming synapses with interneurons (Khakh et al., 2003).
ATP, released from astrocytes, acts as an activity-dependent signaling molecule in neuron-glia communication, resulting in astrocyte Ca2+ waves and synaptic modulation; neuron-glia cross talk may represent an integral part of activity-dependent plasticity of neural networks (Zhang et al., 2003). Clearly there are multiple roles for P2 and P1 receptors in relation to learning and memory, but the way that therapeutic manipulation of purinergic mechanisms can be used to improve these functions is still unresolved. Higher order cognitive functions, including learning and memory in the prefrontal cortex appear to involve P2Y receptor signaling (Wirkner et al., 2003).
ATP injected into the supraoptic nucleus of the hypothalamus has antidiuretic effects (Mori et al., 1994). Purinergic and adrenergic synergism for vasopressin and oxytocin release is consistent with ATP cotransmission in the hypothalamus (Kapoor and Sladek, 2000). Purinergic signaling appears to play a significant role in the regulation of body temperature during fever by central hypothalamic and brainstem nuclei (Gourine et al., 2002, 2004). Functional interactions seem likely to occur between purinergic and nitrergic neurotransmitter systems; they may be important for regulation of hormone secretion and body temperature at the hypothalamic level and for cardiovascular and respiratory control at the level of the brainstem (Yao et al., 2003). ATP is coreleased with GABA or NA to act on P2 receptors, which are strongly expressed in most nuclei in the hypothalamus, including arcuate, paraventricular, retrochiasmatic, supraoptic, ventromedial, and dorsomedial (Matsumoto et al., 2004).
P2X and GABAA receptors play an important role in CO2 chemoreception and are involved in mediation of the ventilatory response to hypercapnia (Gourine and Spyer, 2003). P2X receptors expressed in neurons in the trigeminal mesencephalic nucleus may be involved in the processing of proprioceptive information (Khakh et al., 1997). The nucleus tractus solitarius (NTS) is a major integrative center in the brainstem that is involved in reflex control of the cardiovascular system; stimulation of P2X receptors in the NTS evokes hypotension with decreases in both cardiac output and total peripheral resistance (Kitchen et al., 2001). Different NTS purinoceptor subtypes may contribute to patterned autonomic responses observed in specific physiological or pathological situations (Scislo et al., 2001). Evaluation of the roles of purinergic signaling in processing of the sympathoexcitatory component of the chemoreflex at the NTS level may illuminate the mechanisms underlying the sympathetic overactivity observed in pathophysiological conditions such as hypertension, obstructive sleep apnea, and heart failure (De Paula et al., 2004).
In the striatum, extracellular ATP and adenosine are involved in the regulation of the feeding-associated mesolimbic neuronal activity in an antagonistic manner (Kittner et al., 2003). PPADS suppresses the feeding-evoked dopamine release in the nucleus accumbens, a brain region regarded as important for the regulation of appetite behavior and reinforcement (Kittner et al., 2000). Adenosine-dopamine interactions in the ventral striatum have been implicated in schizophrenia (Tsai, 2005). A hypothesis in which dysfunction of purinergic signaling (for example, decreased ATPase activity in erythrocytes, leading to increased levels of ATP and decreased adenosine) may lead to schizophrenia has been put forward (Lara and Souza, 2000).
It has been claimed that purinergic signaling dysfunction (perhaps largely reduced adenosinergic activity) is involved in mania and aggressive behavior (Machado-Vieira et al., 2002). Endogenous ATP has been claimed to be involved in the regulation of anxiety via stimulation of P2Y1 receptors in the dorsomedial hypothalamus in rats (Kittner et al., 2003). Chronically administered guanosine has anticonvulsant, amnesic, and anxiolytic effects in mice, perhaps associated with modulation of glutamatergic excitation (Vinade et al., 2003).
Multiple P2X and P2Y receptors have been identified on single cerebellar granule cells (Hervas et al., 2003). It has been reported that ATP continuously modulates the cerebellar circuit by increasing the inhibitory input to Purkinje neurons, probably via P2X5 and P2Y2 and/or P2Y4 receptor subtypes, thus decreasing the main cerebellar output activity, which contributes to locomotor coordination (Brockhaus et al., 2004).
Spinal cord traumas are a major health problem. ATP-MgCl2 has been shown to decrease lipid peroxidation in spinal cord injury and protect the spinal cord from secondary injury after trauma; it was concluded that ATP-MgCl2 should be explored for the treatment of spinal cord injuries in conjunction with other treatment modulators (Cakir et al., 2003). In a recent study, topical application of ATP after spinal cord injury significantly improved locomotor function (Shields et al., 2004).
Phenylketonuria is an innervated deficiency of phenylalanine hydroxylase activity in the liver, which causes increased brain levels of phenylalanine and its metabolites, leading to permanent brain damage in the early period of postnatal brain development. Phenylalanine has been shown to inhibit ATP diphosphohydrolase, resulting in increases in ATP levels, perhaps the neurotoxic mechanism underlying brain damage in this disease (Berti et al., 2001).
Acanthamoeba is a protozoan parasite that can cause fatal granulomatous amebic encephalitis. It has been shown recently to hydrolyze ATP, and it was suggested that this ecto-ATPase activity may play a role in the pathogenesis of this disease (Sissons et al., 2004).
Diadenosine tetraphosphate (Ap4A) protects against injuries induced by ischemia and 6-hydroxytryptamine in rat brain and has been suggested as a potentially useful target molecule in the therapy of stroke (Wang et al., 2003b). Up-regulation of P2X2 and P2X4 receptors in organotypic cultures of hippocampus, cortex, and striatum is associated with ischemic cell death and was prevented by P2 receptor antagonists (Cavaliere et al., 2003).
ATP is an extracellular signaling molecule between neurons and glial cells. ATP released by cellular damage and from astrocytes may be important in triggering cellular responses to trauma and ischemia by initiating and maintaining reactive astrogliosis, which involves striking changes in proliferation and morphology of astrocytes and microglia. Some of the responses to ATP released during brain injury are neuroprotective, but in some cases ATP contributes to the pathophysiology initiated after trauma (Fields and Stevens-Graham, 2002; Murakami et al., 2003; Neary et al., 2003; Meme et al., 2004). It has been claimed that P2Y2 receptors activate neuroprotective mechanisms in astrocytic cells (Chorna et al., 2004). In contrast, experimental infusion of ATP into nucleus accumbens or cerebral hemisphere of rats suggests that purines might be a signal for induction of malignant brain tumors. It has been claimed recently that ischemic brain injury is regulated by extracellular ATP-mediated interleukin (IL)-10 expression in microglia (Han et al., 2004). Multiple P2X and P2Y receptor subtypes are expressed by astrocytes, oligodendrocytes, and microglia (James and Butt, 2002). P2Y receptors mediate reactive astrogliosis via induction of COX-2, and P2Y receptor antagonists might counteract excessive COX-2 activation in both acute and chronic neurological disease (Brambilla et al., 1999). P2 receptors also mediate regulation of COX-2 in microglia (Choi et al., 2003). IL-1-induced astrocyte activation is regulated by purinergic signaling; this is compatible with the hypothesis that nucleotides released under inflammatory conditions activate autocrine or paracrine signaling pathways that modulate inflammation (John et al., 2001).
Recent experiments suggest that astrocytes can sense the severity of damage in the central nervous system (CNS) via ATP release from damaged cells and can modulate the tumor necrosis factor--mediated inflammatory response, depending on the extracellular ATP concentration and corresponding type of astrocyte P2 receptor activated (Kucher and Neary, 2005). Thus, micromolar ATP/P2Y receptor activation may act to boost a moderate inflammatory response, whereas millimolar ATP/P2X receptor activation may prevent the perpetuation of a comparatively large inflammatory response perhaps by induction of apoptosis. Protein kinase B/Akt is a key signaling molecule that regulates cell survival, growth and metabolism and inhibits apoptosis. Traumatic brain injury activates Akt. When cortical astrocytes were subjected to mechanical strain, ATP was released, leading to Akt activation; PPADS attenuated the Akt activation (Neary et al., 2005). ATP can activate P2X7 receptors in astrocytes to release glutamate, GABA, and ATP, which regulate the excitability of neurons (Wang et al., 2002). ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines and prostaglandin E2 in astrocytes and may contribute to the chronic inflammation seen in Alzheimer's disease (Xu et al., 2003). Astrocytic gap junctions are involved in the neuroprotective process, in particular, to protect neurons from oxidative stress and glutamate toxicity (Naus et al., 2001). ATP released from astrocytes is essential in mediating the injury-induced responses of microglial processes (Davalos et al., 2005).
Microglial cells are the major cellular elements with immune functions inside the CNS and play important roles in orchestrating inflammatory brain responses to trauma and hypoxia. They express multiple P2 receptors (Bianco et al., 2005) and are activated by purines and pyrimidines to release inflammatory cytokines such as IL-1 and IL-6 and tumor necrosis factor-. Activated microglia can also act as scavenger cells that induce apoptosis in damaged neurons by releasing toxic factors, including NO (Sanz and Di Virgilio, 2000; Inoue, 2002). Thus, whereas microglia may play an important role against infection in the CNS, overstimulation of this immune reaction may accelerate the neuronal damage caused by ischemia, trauma, or neurodegenerative diseases such as Alzheimer's and Parkinson's disease, human immunodeficiency virus encephalopathy, multiple sclerosis, and amyotrophic lateral scoliosis, which exhibit microglial proliferation and activation (Ogata et al., 2003). These authors showed that ATP inhibits cytokine release from lipopolysaccharide-activated microglia via P2Y receptors and suggested that P2Y agonists may be a potential treatment for toxic immunoreactions. P2X4 receptors, induced in spinal microglia, gate tactile allodynia after nerve injury (Tsuda et al., 2003) (see section II.B.). P2X7 receptors mediate superoxide production in primary microglia and are up-regulated in a transgenic model of Alzheimer's' disease, particularly around -amyloid plaques (Parvathenani et al., 2003). Stimulation of microglial P2X7 receptors also leads to enhancement of interferon-
-induced type II nitric oxide synthase activity (Gendron et al., 2003). P2X7 receptors may therefore provide a therapeutic target for inflammatory responses seen in neurodegenerative disorders.
P2 receptors are expressed by oligodendrocytes. However, P2 receptors on oligodendrocytic progenitor cells mediate an increase in [Ca2+]i and may mediate the formation of myelin, raising the possibility that activation of P2 receptors may offer new approaches to the treatment of demyelinating diseases in the CNS, such as multiple sclerosis (Stevens et al., 2002; Agresti et al., 2005). P2X receptors expressed by Schwann cells may regulate the synthesis and release of cytokines during pathophysiological events (Colomar and Amedee, 2001; Irnich et al., 2001).
Classical migraine is associated with two distinct cerebrovascular phases: an initial vasoconstriction (not associated with pain) followed by vasodilatation (reactive hyperemia) associated with pain. The "purinergic" hypothesis for migraine was originally put forward in 1981 as a basis for the reactive hyperaemia and pain during the headache phase (Burnstock, 1981). It was suggested that ATP and its breakdown products adenosine 5'-monophosphate and adenosine were strong contenders for mediating the vasodilatation following the initial vasospasm and subsequent hypoxia. ATP was also implicated in the pathogenesis of pain during migraine via stimulation of primary afferent nerve terminals located in the cerebral microvasculature. Later studies have shown that the ATP-induced cerebral vasodilatation is endothelium-dependent via activation of P2Y receptors on the endothelial cell surface and subsequent release of endothelium-derived relaxing factor and that the endothelial cells are the main local source of ATP involved, although adenosine 5'-diphosphate and ATP released from aggregating platelets may also contribute to this vasodilatation. These findings have extended the purinergic hypothesis for migraine in two ways. First, they have clarified the mechanism of purinergic vasodilatation during the headache phase of migraine. Second, they suggest that a purinergic mechanism may also be involved in the initial local vasospasm, via P2X receptors on smooth muscle cells occupied by ATP released either as a cotransmitter with NA from perivascular sympathetic nerves or from damaged endothelial cells (Burnstock, 1989). The hypothesis has gained further support by the identification of P2X3 receptors on primary afferent nerve terminals arising from trigeminal, nodose, and spinal ganglia (Chen et al., 1995; Burnstock, 2001b). Thus, P2X3 receptor antagonists may be candidates for antimigraine drug development (Waeber and Moskowitz, 2003). There is also recent evidence that migraine is a chronic sympathetic nervous system disorder, with which there is an increase in release of sympathetic cotransmitters, including ATP (Peroutka, 2004), which may contribute to the initial vasospasm (Macdonald, 2000).
The involvement of ATP in the initiation of pain was recognized early (Collier et al., 1966; Bleehen and Keele, 1977; Burnstock, 1981; Jahr and Jessell, 1983). A major advance was made when the P2X3 ionotropic receptor was cloned in 1995 (Chen et al., 1995; Lewis et al., 1995) and shown later to be predominantly localized in the subpopulation of small nociceptive sensory nerves that label with isolectin B4 (IB4) in DRG (Bradbury et al., 1998). Burnstock (1996b) put forward a unifying purinergic hypothesis for the initiation of pain by ATP acting via P2X3 and P2X2/3 receptors associated with causalgia, reflex sympathetic dystrophy, angina, migraine, and pelvic and cancer pain. This has been followed by an increasing number of papers expanding on this concept for acute, inflammatory, neuropathic, and visceral pain (see reviews by Burnstock and Wood, 1996; Chizh and Illes, 2000; Burnstock, 2001c; Dunn et al., 2001; Cooke et al., 2003; Gu, 2003; Inoue et al., 2003; Jarvis, 2003; Sawynok and Liu, 2003; Ueda and Rashid, 2003; North, 2004). Sensory terminals are sensitive to ATP released from local cells during mechanical stress and to ,-meATP in the tongue (Rong et al., 2000), tooth pulp (Alavi et al., 2001; Renton et al., 2003), bladder (Cockayne et al., 2000; Vlaskovska et al., 2001; O'Reilly et al., 2002; Rong et al., 2002), ureter (Knight et al., 2002; Rong and Burnstock, 2004), gut (Wynn et al., 2003, 2004), and uterine cervix (Papka et al., 2005). Both P2X3 (homomultimer) and P2X2/3 (heteromultimer) receptors mediate nociceptive afferent responses, but the proportions vary in different organs. P2Y1 receptors have also been demonstrated in a subpopulation of sensory neurons that colocalise with P2X3 receptors (Ruan and Burnstock, 2003; Gerevich et al., 2004).
The search is on for selective P2X3 and P2X2/3 receptor antagonists that do not degrade in vivo. PPADS is a nonselective P2 antagonist, but has the advantage that it associates and dissociates approximately 100 to 10,000 times more slowly than other known antagonists (Spelta et al., 2002). The trinitrophenyl-substituted nucleotide TNP-ATP is a very potent antagonist at both P2X3 and P2X2/3 receptors (Mockett et al., 1994; King et al., 1997; Virginio et al., 1998; Burgard et al., 2000; Honore et al., 2002). A-317491 is a potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, and it reduces chronic inflammatory and neuropathic pain in the rat (Jarvis et al., 2002, 2004; McGaraughty et al., 2003). Antisense oligonucleotides have been used to down-regulate the P2X3 receptor, and in models of neuropathic (partial sciatic nerve ligation) and inflammatory (complete Freund's adjuvant) pain, inhibition of the development of mechanical hyperalgesia as well as significant reversal of established hyperalgesia, were observed within 2 days of treatment (Barclay et al., 2002; Honore et al., 2002; Stone and Vulchanova, 2003). P2X3 antisense oligonucleotides or antagonists appear to be less effective for treating discogenic (lumbar intervertebral disc) than cutaneous tissue pain (Aoki et al., 2003). Combined antisense and RNA interference-mediated treatment for specific inhibition of the recombinant rat P2X3 receptor appears to be promising for pain therapy (Hemmings-Mieszczak et al., 2003). P2X3 double-stranded short interfering RNA relieves chronic neuropathic pain and opens up new avenues for therapeutic pain strategies in man (Dorn et al., 2004). Cytotoxic targeting using the IB4-saporin conjugate of isolectin IB4-binding nociceptive sensory neurons decreases the severity to noxious stimuli (Vulchanova et al., 2001; Nishiguchi et al., 2004). Antagonism of P2X1 and P2X3 receptors by phenol red has recently been reported (King et al., 2005) and shown to cause significant increases in the pressure and volume threshold required to initiate the micturition reflex in female urethra-anesthetized rats (King et al., 2004). Modulation of neurotransmission through P2X3 receptors in central and peripheral nervous systems may contribute to the anesthesia and analgesia produced by barbiturates (Kitahara et al., 2003). Tetramethylpyrazine, a traditional Chinese medicine used as an analgesic for dysmenorrhea, was shown to block P2X3 receptor signaling, inhibit significantly the first phase of nociceptive behavior induced by 5% formalin, and attenuate slightly the second phase in the rat hindpaw pain model (Liang et al., 2004, 2005). Interactions between vanilloid and metabotropic P2Y receptors are also being explored in terms of treatments for chronic pain (Premkumar, 2001) and thermal hypersensitivity (Moriyama et al., 2003). It has been reported recently that antagonism of spinal P2X3/P2X2/3 receptors results in an indirect activation of the opioid system to alleviate inflammatory hyperalgesia and chemogenic nociception (McGaraughty et al., 2005).
For neuropathic pain, the tactile allodynia that follows peripheral nerve injury is reduced by A-134974, a novel adenosine kinase inhibitor acting at spinal sites (Zhu et al., 2001). Endogenous ATP acting on P2X receptors appears to be necessary for the induction of the postoperative pain characterized by mechanical allodynia (Tsuda et al., 2000, 2001). Up-regulation of P2Y1 receptor expression in the DRG occurs after transection of sciatic nerves (Xiao et al., 2002). P2X4 receptors are induced in spinal microglia that appear to gate tactile allodynia after nerve injury (Tsuda et al., 2003, 2005; Inoue et al., 2004, 2005). Intraspinal administration of p38 inhibitor suppressed allodynia, which suggests that neuropathic pain hypersensitivity depends on the activation of the p38 signaling pattern in microglia in the dorsal horn following peripheral nerve injury (Inoue et al., 2003). Suramin inhibits spinal cord microglia activation and long-term hyperalgesia induced by inflammation produced by formalin injection (Wu et al., 2004; Guo et al., 2005). Analgesic effects with intrathecal administration of P2Y receptor agonists UTP and UDP in normal and the neuropathic pain rat model have been reported, suggesting that P2Y2 (and/or P2Y4) and P2Y6 receptors produce inhibitory effects in spinal pain transmission (Okada et al., 2002). Disruption of the P2X7 receptor gene abolishes chronic inflammatory and neuropathic pain (Chessell et al., 2005). Purinergic mechanisms are also beginning to be explored in relation to cancer pain (Burnstock, 1996; Cain et al., 2001; Mantyh et al., 2002; Gilchrist et al., 2005).
1. Eye.
Purinergic signaling is widespread in the eye (Pintor, 2000) and novel therapeutic strategies are being developed for glaucoma, dry eye, and retinal detachment (Pintor et al., 2003a). P2Y receptors on human corneal epithelial cells appear to play a critical role in the injury-repair process (Klepeis et al., 2004).
ATP, acting via both P2X and P2Y receptors, modulates retinal neurotransmission, affecting retinal blood flow and intraocular pressure. The ATP analog ,-methylene ATP is more effective in reducing intraocular pressure (40%) than muscarinic agonists such as pilocarpine (25%) and -adrenoceptor blockers (30%), raising the potential for the use of purinergic agents in glaucoma (Pintor and Peral, 2001). Dinucleoside polyphosphates acting via P2Y1 receptors on trabecular network cells increase aqueous humor outflow and may be another target for antiglaucomatous drugs (Soto et al., 2005). Suramin, a P2 receptor antagonist, has been shown to inhibit the fibrotic wound healing reactions that sometimes follow trabeculectomies for surgically treating eyes with glaucoma (Mietz et al., 1998). Topical application of Ap4A has been proposed for the lowering of intraocular pressure in glaucoma (Pintor et al., 2003b). The formation of P2X7 receptor pores and apoptosis is enhanced in retinal microvessels early in the course of experimental diabetes, suggesting that purinergic vasotoxicity may play a role in microvascular cell death, a feature of diabetic retinopathy (Sugiyama et al., 2004).
P2Y2 receptor activation increases salt, water, and mucus excretion and thus represents a potential treatment for dry eye conditions (Murakami et al., 2000; Yerxa, 2001). In the pigmented layer of the retina, P2Y2 receptor activation promotes fluid absorption and may be involved in retinal detachment. INS37217, a long-lasting synthetic P2Y2 receptor agonist, stimulates the retinal pigment epithelium by activating P2Y2 receptors at the apical membrane, and in vivo treatment enhances the rate of subretinal fluid reabsorption in experimentally induced retinal detachments and may be useful for treating a variety of retinal diseases that result in fluid accumulation in the subretinal space (Maminishkis et al., 2002). Reactive responses of Müller cells occur within 24 h of retinal detachment. Suramin inhibits some of these responses and may provide a therapeutic candidate to limit the detrimental effects of immune cell activation and Müller cell gliosis during retinal detachment (Uhlmann et al., 2003).
ATP and UTP restore the rates of both net Cl- and fluid secretion in adenovirus type 5-infected conjunctival tissues and are considered as potential therapeutic modulators for the treatment of various transport defects encountered in ocular tissues in diseased and/or inflamed states (Kulkarni et al., 2003). UTP and Ap4A accelerate wound healing in the rabbit cornea, by regulating the rate of epithelial cell migration (Pintor et al., 2004).
The UPL rat is a dominant hereditary cataract model derived from Sprague-Dawley rats and has been used to show that Ca2+-ATPase expression increases, whereas ATP control decreases in lenses during the development of the cataract and opacification; disulfiram and aminoguanidine, which inhibit inducible NO and scavenge reactive oxygen species, attenuate the decrease in ATP, resulting in a delay in cataract development (Nabekura et al., 2004).
A recent article has raised the possibility that alterations in sympathetic nerves may underlie some of the complications observed in diabetic retinopathy (Wiley et al., 2005); ATP is well established as a cotransmitter in sympathetic nerves.
2. Ear.
In the auditory system ATP, acting via P2Y receptors, depresses sound-evoked gross compound action potentials in the auditory nerve and the distortion produces otoacoustic emission, the latter being a measure of the active process of the outer hair cells (Housley, 2000; Sueta et al., 2003). P2X splice variants are found on the endolymphatic surface of the cochlear endothelium, an area associated with sound transduction. Both P2X and P2Y receptors have been identified in the vestibular system (Xiang et al., 1999). ATP may regulate fluid homeostasis, cochlear blood flow, hearing sensitivity and development, and thus may be useful in the treatment of Ménière's disease, tinnitus, and sensorineural deafness.
Sustained loud noise produces an up-regulation of P2X2 receptors in the cochlea, particularly at the site of outer hair cell sound transduction. P2X2 expression is also increased in spiral ganglion neurons, indicating that extracellular ATP acts as a modulator of auditory neurotransmission that is adaptive and dependent on the noise level (Wang et al., 2003a). Excessive noise can irreversibly damage hair cell stereocilia leading to deafness. Data have been presented showing that release of ATP from damaged hair cells is required for Ca2+ wave propagation through the support cells of the organ of Corti, involving P2Y receptors, and this may constitute the fundamental mechanism to signal the occurrence of hair cell damage (Munoz et al., 2001; Gale et al., 2004). ATP is claimed to mitigate the effects of noise trauma (Jakobi et al., 1977; Sugahara et al., 2004), although the mechanisms involved are not clear.
3. Nasal Organs.
Purinergic receptors have been described in the nasal mucosa, including the expression of P2X3 receptors on olfactory neurones (Gayle and Burnstock, 2005). Enhanced sensitivity to odors in the presence of P2 purinergic antagonists suggests that low-level endogenous ATP normally reduces odor responsiveness (Hegg et al., 2003a). It appears that the induction of heat-shock proteins by noxious odor damage can be prevented by the in vivo administration of P2 receptor antagonists (Hegg et al., 2003b). The predominantly suppressive effect of ATP in odor responses could play a role in the reduced odor sensitivity that occurs during acute exposure to noxious fumes and may be a novel neuroprotective mechanism.
IV. Immune System and Inflammation
ATP and adenosine are released at sites of inflammation. ATP is involved in the development of inflammation through a combination of actions: release of histamine from mast cells, provoking production of prostaglandins; and the production and release of cyokines from immune cells (Di Virgilio et al., 1998). In contrast, adenosine exerts anti-inflammatory actions. P2X7 and P2Y1 and P2Y2 receptors located on inflammatory and immune cells play a pivotal role in inflammation and immunomodulation (Di Virgilio et al., 1998; Luttikhuizen et al., 2004). In addition, NTPDases have effects on immune reactions (Robson et al., 2001). NTPDase1 deficiency appears to have two opposing impacts on nucleotide-mediated signaling, that is, inhibition via desensitization of some, but not all, P2 receptors and augmentation of certain other responses via impaired hydrolysis of their ligands. Consequently, there are defects in dendritic cell function, antigen presentation, T-cell responses to haptens (type IV hypersensitivity reactions), and delayed cellular rejection responses under partial costimulation blockade in NTPDase1-null mice.
In addition to the roles of purines in inflammation, they have a broad range of functions carried out through purinergic receptors on immune cells, including killing intracellular pathogens by inducing apoptosis of host macrophages, chemoattraction, and cell adhesion (Burnstock, 2001d; Di Virgilio et al., 2001). Purinergic compounds may turn out to be useful for the treatment of neurogenic inflammation, rheumatoid arthritis, and periodontitis (Dubyak and El Moatassim, 1993).
ATP-induced apoptosis in macrophages via P2X7 receptors, also results in killing of the mycobacteria contained within them,
