This review is focused on purinergic neurotransmission, i.e., ATP released from nerves as a transmitter or cotransmitter to act as an extracellular signaling molecule on both pre- and postjunctional membranes at neuroeffector junctions and synapses, as well as acting as a trophic factor during development and regeneration. Emphasis is placed on the physiology and pathophysiology of ATP, but extracellular roles of its breakdown product, adenosine, are also considered because of their intimate interactions. The early history of the involvement of ATP in autonomic and skeletal neuromuscular transmission and in activities in the central nervous system and ganglia is reviewed. Brief background information is given about the identification of receptor subtypes for purines and pyrimidines and about ATP storage, release, and ectoenzymatic breakdown. Evidence that ATP is a cotransmitter in most, if not all, peripheral and central neurons is presented, as well as full accounts of neurotransmission and neuromodulation in autonomic and sensory ganglia and in the brain and spinal cord. There is coverage of neuron-glia interactions and of purinergic neuroeffector transmission to nonmuscular cells. To establish the primitive and widespread nature of purinergic neurotransmission, both the ontogeny and phylogeny of purinergic signaling are considered. Finally, the pathophysiology of purinergic neurotransmission in both peripheral and central nervous systems is reviewed, and speculations are made about future developments.
It is nearly 35 years ago that I published a paper entitled “Purinergic Nerves” in Pharmacological Reviews (241). It was a new hypothesis backed by some good evidence for ATP as a neurotransmitter in nonadrenergic, noncholinergic nerves supplying the gut and bladder and included every hint that I could find to support the possible involvement of purinergic signaling in different parts of the nervous system. However, it was regarded with skepticism by a large number of people over the next 20 years. So this current review of purinergic neurotransmission, with a huge and rapidly growing body of evidence for purinergic involvement in both physiological and pathophysiological neural mechanisms, is for me, a rather emotional vindication of my life's work. It is a comprehensive account, and therefore, I hope I will be forgiven for its length and for the long list of papers, although reference to review articles is made wherever possible to cover some of the earlier literature.
Extracellular actions of purine nucleotides and nucleosides were first described in a seminal paper by Drury and Szent-Györgyi in 1929 (481) in the cardiovascular system, and later in the uterus (451) and intestine (642). Studies of the effects of purines on the nervous system followed the early emphasis on their cardiovascular actions. There was early recognition for a physiological role for ATP at the skeletal neuromuscular junction. Buchthal and Folkow (226) injected ATP into the sciatic artery supplying the gastrocnemius muscle of the frog and reported tetanus-like contractions; they also observed that the sensitivity of the preparation to ACh was greatly increased by previous application of ATP (227, 228). Parts of the spinal cord were shown to be sensitive to ATP (225). Emmelin and Feldberg (518) found complex effects initiated by intravenous injection of ATP into cats affecting peripheral, reflex, and central mechanisms. Injection of ATP into the lateral ventricle of the cat produced muscular weakness, ataxia, and a tendency of the animal to sleep (538). The application of adenosine or ATP to various regions of the brain produced biochemical or electrophysiological changes (74, 610, 1563). ATP and related nucleotides were shown to have anti-anesthetic actions (981). The first hint that ATP might be a neurotransmitter in the peripheral nervous system (PNS) arose when it was proposed that ATP released from sensory nerves during antidromic nerve stimulation of the great auricular nerve caused vasodilatation in the rabbit ear artery (755, 756). The purinergic nerve hypothesis, with ATP as the transmitter responsible for nonadrenergic, noncholinergic (NANC) transmission to the smooth muscle of the gut and bladder, was proposed by Burnstock in 1972 (241). A brief historical review about the development of the concept of ATP as a neurotransmitter has been published recently (277).
A. Autonomic Neuromuscular Transmission
There was early recognition of atropine-resistant responses of the gastrointestinal tract to parasympathetic nerve stimulation (999, 1138, 1321). However, it was not until the early 1960s that autonomic transmission other than adrenergic and cholinergic was established. In 1963, 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, an inhibitory hyperpolarizing potential was observed (282, 283). The hyperpolarizing responses were blocked by tetrodotoxin (TTX), a neurotoxin that prevents the action potential in nerves without affecting the excitability of smooth muscle cells (233; Fig. 1A), indicating their neurogenic nature and establishing them as inhibitory junction potentials (IJPs) in response to NANC nerves. This work was extended by an analysis of the mechanical responses to NANC nerve stimulation of the taenia coli (284). NANC mechanical responses were also observed by Martinson and Muren in the cat stomach upon stimulation of the vagus nerve (1112), and NANC inhibitory innervation of the portal vein was also demonstrated (780).
The excitatory response of the mammalian urinary bladder to parasympathetic nerve stimulation was shown very early to be only partially antagonized by antimuscarinic agents (731, 1000). It was speculated that the subjunctional receptors, at which the endogenous ACh acts, were inaccessible to atropine (42, 420) or that atropine was displaced from these receptors by the high local concentrations of ACh released during parasympathetic nerve stimulation (781). However, it was later postulated that the atropine-resistant response may be due to the release of a noncholinergic excitatory transmitter, probably norepinephrine (NE) (43, 348).
By the end of the 1960s, evidence had accumulated for NANC nerves in the respiratory, cardiovascular, and urinogenital systems as well as in the gastrointestinal tract (239). The existence of NANC neurotransmission is now firmly established in a wide range of peripheral and central nerves, and fuller accounts of the development of this concept are available (see Refs. 248, 279, 288).
In the late 1960s, systematic studies were undertaken in an attempt to identify the transmitter utilized by the NANC nerves of the gut and urinary bladder. Several criteria, which must be satisfied before establishing a substance as a neurotransmitter (503), were considered (241, 285). First, a putative transmitter must be synthesized and stored within the nerve terminals from which it is released. Once released it must interact with specific postjunctional receptors, and the resultant nerve-mediated response must be mimicked by the exogenous application of the transmitter substance. Also, enzymes that inactivate the transmitter and/or uptake systems for the neurotransmitter or its derivatives must also be present, and finally, drugs that affect the nerve-mediated response must be shown to modify the response to exogenous transmitter in a similar manner.
Many substances were examined as putative transmitters in the NANC nerves of the gastrointestinal tract and bladder, but the substance that best satisfied the above criteria was the purine nucleotide ATP (285; Fig. 1, B and C). Nerves utilizing ATP as their principal transmitter were subsequently named “purinergic” (240), and a tentative model of storage, release, and inactivation of ATP for purinergic nerves was proposed (241; Fig. 2). Since then a great deal of evidence followed in support of the purinergic hypothesis (see Refs. 257, 260, 292, 488, 661, 1278, 1977), although there was considerable opposition to this idea in the first decade or two after it was put forward (see Refs. 242, 643). I believe that this was partly because biochemists felt that ATP was established as an intracellular energy source involved in various metabolic cycles and that such a ubiquitous molecule was unlikely to be involved in extracellular signaling. However, ATP was one of the biological molecules to first appear and, therefore, it is not surprising that it should have been used for extracellular, in addition to intracellular, purposes early in evolution (258). The fact that potent ectoATPases were described in most tissues in the early literature was also a strong indication for the extracellular actions of ATP. In more recent studies, transient clusters of receptors for ATP have been shown to accumulate on smooth muscle membranes opposite autonomic nerve varicosities in close contact with them (707, 1782).
B. Autonomic Ganglia
An effect of ATP on autonomic ganglia was first reported in 1948 when Feldberg and Hebb (537) demonstrated that intra-arterial injection of ATP excited neurons in the cat superior cervical ganglia (SCG). Later work from de Groat's laboratory showed that in the cat vesical parasympathetic ganglia and rat SCG, purines inhibited synaptic transmission through adenosine receptors, but high concentrations of ATP depolarized and excited the postganglionic neurons (1699, 1700). The earliest intracellular recordings of the action of ATP on neurons were obtained in frog sympathetic ganglia (24, 1566). ATP produced a depolarization through a reduction in K+ conductance. ATP was shown to excite mammalian dorsal root ganglia (DRG) neurons and some neurons from the dorsal horn of the spinal cord (819, 966). These responses were associated with an increase in membrane conductance (see sects. vA and viiH).
C. Central Nervous System
Following the early studies of Feldberg and Sherwood (538), there were reports that showed that adenosine acted via adenylate cyclase to produce cAMP in cerebral cortex slices and that this was antagonized by the methylxanthines, theophylline and caffeine (1504). Electrically evoked release of nucleotides and nucleosides from both brain slices and synaptosomes prepared from cerebral cortex raised the possibility that they may participate in intercellular transmission (983, 1384). These in vitro experiments were extended to the intact cerebral cortex (1665). It was shown that iontophoretic application of adenosine and several adenine nucleotides depressed the excitability of cerebral cortical neurons including identified Betz cells; cAMP, adenine, and inosine were less effective, whereas ATP caused an initial excitation followed by depression (1347). Adenosine and ATP also depressed firing in cerebellar Purkinje cells (959). About the same time microiontophoretic application of adenine nucleotides was shown to depress the spontaneous firing of corticospinal and other unidentified cerebral cortical neurons, although ATP had an additional excitant action on some neurons (1344, 1648). In other studies, adenosine and adenine nucleotides were shown to have an inhibitory action on the N-wave (a postsynaptic potential) amplitude in neurons of guinea pig olfactory cortex slices, but not on postsynaptic potentials in superior colliculus (1274, 1518). Schubert and Kreutzberg (1521) showed that after injections of tritiated adenosine into the visual cortex of rabbits, it was taken up and converted to radioactive nucleotides, which subsequently appeared in the thalamocortical relay cells of the lateral geniculate nucleus, consistent with synaptic transmission. This was supported by similar experiments in the somatosensory cortex (1875).
ATP was shown to activate units of the emetic chemoreceptor trigger zone of the area postrema of cat brain (174). Premature arousal of squirrels from periods of hibernation was evoked by adenosine nucleotides, but not by other purine nucleotides, and it was suggested that this effect was due to their action on neurons in the central nervous system (CNS) (1744). The infusion of cAMP into the hypothalamus of fowl induced behavioral and electrophysiological sleep, whereas dibutyryl cAMP produced arousal (1107). Local or systemic administration of adenosine in normal animals produced electroencephalogram (EEG) and behavioral alterations of the hypnogenic type (714). Cornforde and Oldendorf (385) demonstrated two independent transport systems across the rat blood-brain barrier, one for adenine and the other for adenosine, guanosine, inosine, and uridine. High levels of 5′-nucleotidase were demonstrated histochemically in the substantia gelatinosa of mouse spinal cord (1672). Early studies of the actions of purines on the CNS were reviewed by Burnstock (245), and important papers about the excitatory actions of ATP on subpopulations of spinal dorsal horn neurons were published in the early 1980s (608, 819, 1490) and excitation of single sensory neurons in the rat caudal trigeminal nucleus by iontophoretically applied ATP (1486). Although most of the early emphasis was about the neuromodulatory roles of adenosine, it was later recognized that fast synaptic transmission involving ATP was widespread in the CNS (see sects. vi and vii).
Early observations of mentally ill patients suggested that purines may play a role in the brain of man (see also sect. xiB5). Thus adenine nucleotides were implicated in depressive illness (7, 708, 1189). In the hypothesis proposed for the mechanism of depression by Abdulla and McFarlane (7), the effect of adenine nucleotides on prostaglandin biosynthesis was implicated. Blood levels of ATP and/or adenosine and urinary cAMP excretion were significantly elevated in patients diagnosed as schizophrenic or in psychotic and neurotic depression (6, 219, 709, but see also Ref. 830). Inherited disorders of purine metabolism in the brain were related to psychomotor retardation, athetosis, and self-mutilation (Lesch-Nyhan syndrome) (133, 1020, 1534). Antidepressant drugs such as imipramine and amitriptyline potentiated the suppression of neuronal firing in rat cerebral cortex by adenosine (1342, 1648). It was claimed that depressive symptoms in patients relate to hypoxanthine levels in the cerebrospinal fluid (1245). Competitive interactions between adenosine and benzodiazepines in cerebral cortical neurons were reported, and evidence was presented to suggest that morphine releases ATP, and that after breakdown to adenosine, depresses neurotransmission in the cortex (1350). It was suggested that adenosine was involved in the initial phase of seizure-induced functional hyperemia in the cortex (1519).
The majority of studies of the extracellular actions of ATP have been concerned with the short-term events that occur in neurotransmission and in secretion. However, there is increasing awareness that purines and pyrimidines can have potent long-term (trophic) roles in cell proliferation and growth and in disease and cytotoxicity (see Ref. 4; Table 1). An example of synergism between purines and trophic factors comes from studies of the transplantation of the myenteric plexus into the brain (1695, 1696). In these studies, which were originally designed to explore enteric nerves as a possible source for replacement of missing messengers such as dopamine for Parkinson's disease, the myenteric plexus was shown to cause a marked proliferation of nerve fibers in the corpus striatum. An analysis, using coculture of striatal neurons with various elements of the myenteric plexus and enteric neurotransmitters, showed that a growth factor released by enteric glial cells works synergistically with nitric oxide (NO) and ATP (via adenosine) released from NANC inhibitory nerves to promote nerve regeneration (760).
D. Purinergic Receptor Subtypes
Implicit in the concept of purinergic neurotransmission is the existence of postjunctional purinergic receptors, and the potent actions of extracellular ATP on many different cell types also implicates membrane receptors. Purinergic receptors were first defined in 1976 (244), and 2 years later a basis for distinguishing two types of purinoceptor, identified as P1 and P2 (for adenosine and ATP/ADP, respectively), was proposed (246). At about the same time, two subtypes of the P1 (adenosine) receptor were recognized (1069, 1766), but it was not until 1985 that a proposal suggesting a pharmacological basis for distinguishing two types of P2 receptor (P2X and P2Y) was made (291). A year later, two further P2 purinoceptor subtypes were identified, namely, a P2T receptor selective for ADP on platelets and a P2Z receptor on macrophages (661). Further subtypes followed, perhaps the most important being the P2U receptor, which could recognize pyrimidines such as UTP as well as ATP (1265, 1536). In 1994 Mike Williams made the point at a meeting that a classification of P2 purinoceptors based on a “random walk through the alphabet” was not satisfactory, and Abbracchio and Burnstock (3), on the basis of studies of transduction mechanisms (486) and the cloning of nucleotide receptors (194, 1080, 1763, 1843), proposed that purinoceptors should belong to two major families: a P2X family of ligand-gated ion channel receptors and a P2Y family of G protein-coupled receptors. This nomenclature has been widely adopted, and currently seven P2X subunits and eight P2Y receptor subtypes are recognized, including receptors that are sensitive to pyrimidines as well as purines (see Refs. 163, 275, 521, 1392). Receptors for diadenosine polyphosphates have been described on C6 glioma cells and presynaptic terminals in rat midbrain, although they have yet to be cloned (444).
1. P1 receptors
Four subtypes of P1 receptors have been cloned, namely, A1, A2A, A2B, and A3 (see Refs. 370, 423, 582, 1392). All P1 adenosine receptors couple to G proteins and, in common with other G protein-coupled receptors, they have seven putative transmembrane (TM) domains of hydrophobic amino acids, each believed to constitute an α-helix of ∼21–28 amino acids (see Fig. 3A). The NH2 terminus of the protein lies on the extracellular side, and the COOH terminus lies on the cytoplasmic side of the membrane. Typically, the extracellular loop between TM4 and TM5 and the cytoplasmic loop between TM5 and TM6 are extended. The intracellular segment of the receptor interacts with the appropriate G protein, with subsequent activation of the intracellular signal transduction mechanism. It is the residues within the transmembrane regions that are crucial for ligand binding and specificity and, with the exception of the distal (carboxyl) region of the second extracellular loop, the extracellular loops, the COOH terminus, and the NH2 terminus do not seem to be involved in ligand recognition (1275). Site-directed mutagenesis of the bovine A1 adenosine receptor suggests that conserved histidine residues in TM6 and TM7 are important in ligand binding. Specific agonists and antagonists are available for the P1 receptor subtypes (817; Table 2). For a specific review of P1 receptors in the nervous system, the reader is referred to Reference 1423.
2. P2X receptors
Members of the existing family of ligand-gated nonselective cation channel P2X1–7 receptor subunits show a subunit topology of intracellular NH2 and COOH termini possessing consensus binding motifs for protein kinases; two transmembrane-spanning regions (TM1 and TM2), the first involved with channel gating and the second lining the ion pore; a large extracellular loop, with 10 conserved cysteine residues forming a series of disulfide bridges; hydrophobic H5 regions close to the pore vestibule, for possible receptor/channel modulation by cations (magnesium, calcium, zinc, copper, and proton ions); and an ATP-binding site, which may involve regions of the extracellular loop adjacent to TM1 and TM2 (see Fig. 3B). The P2X1–7 receptors show 30–50% sequence identity at the peptide level. The stoichiometry of P2X1–7 receptor subunits is thought to involve three subunits that form a stretched trimer (see Refs. 109, 891, 1155, 1240).
It has become apparent that the pharmacology of the recombinant P2X receptor subtypes expressed in oocytes or other cell types is often different from the pharmacology of P2X receptor-mediated responses in naturally occurring sites. This is partly because heteromultimers as well as homomultimers are involved in forming the trimer ion pores (see below). Spliced variants of P2X receptor subtypes might play a part (334, 1578). For example, a splice variant of the P2X4 receptor, while it is nonfunctional on its own, can potentiate the actions of ATP through the full-length P2X4 receptors (1721). Third, the presence in tissues of powerful ectoenzymes that rapidly break down purines and pyrimidines is not a factor when examining recombinant receptors, but is in vivo (1979).
P2X7 receptors are predominantly localized on immune cells and glia, where they mediate proinflammatory cytokine release, cell proliferation, and apoptosis. P2X7 receptors, in addition to small cation channels, upon prolonged exposure to high concentrations of agonist, large channels, or pores are activated that allow the passage of larger molecular weight molecules. The possible mechanisms underlying the transition from small to large channels have been considered (508, 621).
The P2X receptor family shows many pharmacological and operational differences (634; see Table 2). The kinetics of activation, inactivation, and deactivation also vary considerably among P2X receptors. Calcium permeability is high for some P2X subunits and Cl− permeability for others, properties that are functionally important. For more specific reviews of the molecular physiology of the P2X receptor, the reader is referred to Khakh et al. (891), North (1257), Egan et al. (508), Stojilkovic et al. (1645), and Roberts et al. (1429).
3. P2Y receptors
Metabotropic P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) are characterized by a subunit topology of an extracellular NH2 terminus and intracellular COOH terminus, the latter possessing consensus binding motifs for protein kinases; seven transmembrane-spanning regions, which help to form the ligand-docking pocket; a high level of sequence homology between some transmembrane-spanning regions, particularly TM3, TM6, and TM7; and a structural diversity of intracellular loops and COOH terminus among P2Y subtypes, so influencing the degree of coupling with Gq/11, Gs, Gi, and Gi/o proteins (5; see Fig. 3C). Each P2Y receptor binds to a single heterotrimeric G protein (Gq/11 for P2Y1,2,4,6), although P2Y11 can couple to both Gq/11, and Gs, whereas P2Y12 and P2Y13 couple to Gi and P2Y14 to Gi/o. Many cells express multiple P2Y subtypes (see Refs. 5, 1800). P2Y receptors show a low level of sequence homology at the peptide level (19–55% identical) and, consequently, show significant differences in their pharmacological and operational profiles. Some P2Y receptors are activated principally by nucleoside diphosphates (P2Y1,6,12), while others are activated mainly by nucleoside triphosphates (P2Y2,4). Some P2Y receptors are activated by both purine and pyrimidine nucleotides (P2Y2,4,6), and others by purine nucleotides alone (P2Y1,11,12). In response to nucleotide activation, recombinant P2Y receptors either activate phospholipase C (PLC) and release intracellular calcium or affect adenylyl cyclase and alter cAMP levels. In recent years P2Y G protein-coupled receptors in neurons have been found to modulate the activity of voltage-gated ion channels in the cell membrane through certain actions of activated G proteins. For example, P2Y receptor subtypes that act via Gi/o proteins can involve N-type Ca2+ channels, while the M-current K+ channel can be inhibited through the activation of Gq/11-linked P2Y receptor subtypes (5). There is little evidence to indicate that P2Y5, P2Y9, and P2Y10 sequences are nucleotide receptors or affect intracellular signaling cascades and consequently have been dropped from International Union of Pharmacology (IUPHAR) P2Y receptor nomenclature and have been termed “orphans.”
2-Methylthio ADP (2-MeSADP) is a potent agonist of mammalian P2Y1 receptors and N6-methyl-2′-deoxyadenosine 3′,5′-bisphosphate (MRS2179), MRS2269 and MRS2286 have been identified as selective antagonists (221). At P2Y2 and P2Y4 receptors in the rat, ATP and UTP are equipotent, but the two receptors can be distinguished with antagonists, that is, suramin blocks P2Y2, while Reactive blue 2 blocks P2Y4 receptors (165, 1862). P2Y6 is UDP selective, while P2Y7 turned out to be a leukotriene receptor (1936). P2Y8 is a receptor cloned from frog embryos, where all the nucleotides are equipotent (164), but no mammalian homolog has been identified to date, apart from a report of P2Y8 mRNA in undifferentiated HL60 cells (13). The P2Y12 receptor found on platelets was not cloned until more recently (754), although it has only 19% homology with the other P2Y receptor subtypes. It seems likely to represent one of a subgroup of P2Y receptors, including P2Y13 and P2Y14, for which transduction is entirely through adenylate cyclase (2). It has been suggested that P2Y receptors can be subdivided into two subgroups, namely, one that includes P2Y1,2,4,6,11, the other includes P2Y12,13,14, largely on the basis of structural and phylogenetic criteria (see Ref. 5).
For a recent review of P2Y receptor molecular biology, pharmacology, cell distribution, and physiology, the reader should refer to Reference 5. The structure and properties of current P2Y receptor subtypes and the current status of P2 receptor subtype agonists and antagonists are summarized in Table 2.
4. Heteromultimeric receptors
The pharmacology of purinergic signaling is complicated because P2X receptor subunits can combine to form either homomultimers or heteromultimers (see Refs. 1257, 1800). Heteromultimers are clearly established for P2X2/3 receptors in nodose ganglia (1027, 1388), P2X4/6 receptors in CNS neurons (1006), P2X1/5 receptors in some blood vessels (699, 1718), and P2X2/6 receptors in the brain stem (916). P2X7 receptors do not form heteromultimers, and P2X6 receptors will not form a functional homomultimer without extensive glycosylation (1717).
P2Y receptor subtypes can also form heteromeric complexes (5, 1206), and most recently, adenosine A1 receptors have been shown to form a heteromeric complex with P2Y1 receptors (see Refs. 1381, 1941). Dopamine D1 and adenosine A1 receptors have also been shown to form functionally interactive heteromeric complexes (645).
E. ATP Storage, Release, and Breakdown
1. ATP storage and release
The cytoplasm of most neurons contains ∼2–5mM ATP, and higher concentrations of ATP (up to 100 mM) are stored in synaptic vesicles. Synaptic vesicles also contain other nucleotides such as ADP, AMP, Ap4A, Ap5A, and GTP, but at lower concentrations. Sperm, tumor cells, and the epithelial cells of the lens of the eye have exceptionally high intracellular levels of ATP and granules in adrenal chromaffin cells, Merkel cells, platelets, and pancreatic insulin-containing β-cells also contain significant amounts of ATP (see Refs. 1260, 1627). A recent paper has shown that ATP transport into brain synaptic vesicles can be distinguished from other neurotransmitter transport systems in terms of its mechanism and energy requirements (1947).
Release of ATP from exercising human forearm muscle was reported by Tom Forrester and colleagues (569) and from the perfused heart during coronary vasodilation in response to hypoxia was reported by Paddle and Burnstock (1288). However, until recently, it was usually assumed that the only source of extracellular ATP acting on purinoceptors was damaged or dying cells, but it is now recognized that ATP release from healthy cells is a physiological mechanism (see Refs. 160, 487, 1004, 1529). ATP is released from both peripheral and central neurons (285, 755, 881, 1302, 1652, 1855, 1857, 1889), but also from many nonneuronal cell types during mechanical deformation in response to shear stress, stretch, or osmotic swelling, as well as hypoxia and stimulation by various agents (160, 179).
ATP release by mechanical distortion of urothelial cells during distension of the bladder was first demonstrated by Ferguson and colleagues in 1997 (544) and later by Vlaskovska et al. (1797). ATP release by distension was demonstrated from urothelial cells in ureter (932) and from mucosal epithelial cells of the colorectum (1895). It is also released from osteoblasts (1441); astrocytes (373); epithelial cells in the tongue (1444), lung (56, 466), and kidney (127); keratinocytes in the skin; and glomus cells in the carotid body.
There is an active debate about the precise transport mechanism(s) involved in ATP release. There is compelling evidence for exocytotic vesicular release of ATP from nerves, but for ATP release from nonneuronal cells, various transport mechanisms have been proposed, including ATP-binding cassette (ABC) transporters, connexin, or pannexin hemichannels or possibly plasmalemmal voltage-dependent anion and P2X7 receptor channels, as well as vesicular release (see Refs. 160, 417, 439, 1004, 1475, 1529, 1631). Perhaps surprisingly, evidence was presented that the release of ATP from urothelial cells in the ureter is also largely vesicular, since monensin and brefeldin A, which interfere with vesicular formation and trafficking, inhibited distension-evoked ATP release, but not gadolinium, an inhibitor of stretch-activated channels, or glibenclamide, an inhibitor of members of the ABC protein family (932). Exocytotic vesicular release of ATP from endothelial cells (160, 261), osteoblasts (1441), fibroblasts (179), and astrocytes (373, 1169) has also been reported. There is increased release of ATP from endothelial cells during acute inflammation (159).
ATP released from nerves, or by autocrine and paracrine mechanisms from nonneuronal cells, is involved in a wide spectrum of physiological and pathophysiological activities, including synaptic transmission and modulation, pain and touch perception, vasomotor effects, platelet aggregation, endothelial cell release of vasorelaxants, immune defense, epithelial ion and water transport, as well as cell proliferation, migration, differentiation, and death.
2. ATP breakdown
ATP released from cells is regulated by a number of proteins that have their catalytic site on the outer side of the plasma membrane (121, 976, 1978). A recent review focuses on ectonucleotidases in the nervous system (1980). Extracellular nucleotides can be hydrolyzed by nonspecific enzymes, such as glycoslyphospatidylinositol-anchored ectoalkaline phosphatases and ecto-5′-nucleotidases or ectonucleotidases with more distinct characteristics that are now classified into two families (see Fig. 3D). E-NTPDases (CD39) family are ecto-nucleoside triphosphate diphosphohydrolases that hydrolyze nucleoside 5′-tri- and diphosphates. Another family of enzymes, E-NPP (with 3 subtypes), are ecto-nucleotide pyrophosphatase/phosphodiesterases with a broad substrate specificity. These can hydrolyze phosphodiester bonds of nucleotides and nucleic acids and pyrophosphatase bonds of nucleotides and nucleotide sugars, e.g., cleavage of ATP to AMP and PPi and conversion of cAMP to AMP. Some of these ectonucleotidases have distinct patterns of distribution in different cell types and are regulated during physiological and pathophysiological processes, probably in association with purine and pyrimidine signaling. The catalytic site of ectonucleotidases faces the extracellular medium, but some isoforms can be cleaved or released in a soluble form, in which case they can be regarded as ectonucleotidases. There are also other ectoenzymes that contribute to levels of extracellular nucleotides, such as interconversion of nucleotides by ecto-nucleoside diphosphokinase (ecto-NDPK) and ectoadenylate cyclase, possibly a production of ATP by F0-F1 ATP synthase, and use of ATP as a phosphate donor for ecto-protein kinase reactions. Ectoenzymes can act to regulate synaptic activity, controlling ATP and adenosine levels, depending on the synaptic plasticity developed in both physiological and pathophysiological conditions (171, 657). Excellent reviews are available summarizing the current status of extracellular ATP breakdown (see Refs. 1978, 1980; see also the recent Special Issue of “Purinergic Signaling” devoted to Ecto-nucleotidases, volume 2, number 2, 2006).
F. Plasticity of Purinergic Signaling
There was early recognition that the expression of purinergic cotransmitters and of receptors in the autonomic nervous system shows marked plasticity during development and ageing, in the nerves that remain following trauma or surgery, under the influence of hormones and in various disease situations (see Refs. 4, 254, 292). For plasticity of purinergic signaling in development and aging, see section xA1, and in disease, see section xi. Examples of neuronal plasticity occurring in healthy adults during pregnancy or following surgical interventions in visceral organs and the CNS follow.
Plasticity of purinergic signaling has been observed in the urinary bladder. Suppressed bladder contractility during pregnancy is associated with decreased muscarinic receptor density, while the affinity of purinergic receptors for ATP is increased (1716). The amplitude of NANC transmission in detrusor strips from mature female rats was diminished in ovariectomized animals (509). Pregnancy substantially increases the purinergic components of the response of the rabbit bladder to field stimulation (1022). In contrast, there was a decrease in excitatory junction potentials (EJPs), probably mediated by ATP, in guinea pig uterine artery (1686).
Capsaicin treatment of newborn rats leads to selective degeneration of some sensory nerve fibers. In a study of rat bladder in 3-mo-old rats treated at birth with capsaicin, contractions evoked by electrical field stimulation were significantly larger than those of control (vehicle-treated) animals, an effect which preferentially involves the cholinergic component of the response, although there was some increase, too, in the purinergic component (1975). However, since contractions in response to exogenous carbachol or ATP were not significantly different, this suggested that the changes involve prejunctional mechanisms. Capsaicin treatment, causing selective sensory denervation of the rat ureter, leads to increased sympathetic innervation (1496), perhaps involving an increase in release of both NE and ATP.
The urinary bladder of the rat, deprived of its motor innervation, increases severalfold in weight in response to distension; this increase in weight is due to both hyperplasia and hypertrophy of the smooth muscle (514). Since it is now known that distension of the bladder leads to substantial release of ATP from urothelial cells (see sect. iiE1) and ATP is known to have proliferative actions (4), purines seem likely to participate in the trophic changes that occur in the bladder. Incorporation of bowel tissue into the bladder wall has been used to increase bladder capacity and/or decrease bladder pressure; after 4–12 wk, the contractile response of the transplanted rabbit intestine underwent a partial change in the response to nerve stimulation and ATP from that of intestine towards that of detrusor (116).
Following chemical sympathectomy produced by long-term treatment with guanethidine and subsequent loss of the cotransmitter ATP in the vas deferens and spleen, there is an increase in density of P2X receptor sites, although there was a decrease in receptor affinity (1966).
Chronic food restriction alters P2Y1 receptor mRNA expression in the nucleus accumbens of the rat (969). Cerebellar lesion upregulates P2X1 and P2X2 receptors in the precerebellar nuclei of the rat, perhaps related to the survival of injured neurons (563). In vitro studies of organotypic cultures and in vivo experiments on hippocampus from gerbils subjected to bilateral common carotid occlusion showed that P2X2 and P2X4 receptors were upregulated by glucose/oxygen deprivation (321, 322). It was speculated that the changes in P2X receptor expression might be associated with ischemic cell death. There are data indicating a trophic role for ATP in the hippocampus (1584). It was shown that ATP and its slowly hydrolyzable analogs strongly inhibited neurite outgrowth and also inhibited aggregation of hippocampal neurons; it was suggested that the results indicate that extracellular ATP may be involved in synaptic plasticity through modulation of neural cell adhesion molecule (NCAM)-mediated adhesion and neurite outgrowth. Utilization of green fluorescent protein (GFP)-tagged P2X2 receptors on embryonic hippocampal neurons has led to the claim that ATP application can lead to changes in dendritic morphology and receptor distribution (891).
Propagation of intercellular Ca2+ waves between astrocytes depends on the diffusion of signaling molecules through gap junction channels (see sect. viiiA). Deletion of the main gap junction protein connexin-43 (Cx43) by homologous recombination results in a switch in mode of intercellular Ca2+ wave propagation to a purinoceptor-dependent mechanism. This compensatory mechanism in Cx43 knockout mice for intercellular Ca2+ wave propagation is related to a switch from P2Y1 to a UTP-sensitive P2Y4 receptor in spinal cord astrocytes (1654). Trophic effects of purines on neurons and glial cells have been reviewed (4, 1226, 1398).
Following chronic constriction injury to the sciatic nerve, the number of P2X3 receptor-positive small- and medium-diameter neurons increased in DRG, compared with sham-operated animals (1261, 1734). In addition, spinal cord immunoreactivity increased on the side ipsilateral to the ligated nerve, consistent with upregulation of purinergic receptors on presynaptic terminals of the primary sensory nerves. A decrease in P2X3 immunoreactivity and function in DRG of rats occurs after spinal nerve ligation (854). Changes in gene expression of multiple subtypes of P2X receptors on DRG neurons (L5) after spinal nerve ligation have been reported (904). After nerve injury, the mRNA for P2X5 receptors was increased, those for the P2X3 and P2X6 receptors were decreased, and those for P2X2 and P2X4 receptors were unchanged. However, immunostaining for receptor protein showed an increase from 23 to 73% P2X2 receptor-positive DRG neurons after nerve ligation. Two days following unilateral section of the cervical vagus nerve, there was a dramatic ipsilateral increase in P2X1, P2X2, and P2X4 receptor immunoreactivity in the cell soma of vagal efferent neurons in the dorsal vagal motor nucleus, but not in the nucleus ambiguous (72). Following surgical sympathectomy, 28% of the spontaneously active afferent fibers in sciatic nerve responded to ATP, compared with none in intact rats (343). After nerve injury, P2X4 receptor expression increased strikingly in hyperactive microglia, but not in neurons or astrocytes, in the ipsilateral spinal cord; this appears to be associated with tactile allodynia (1731 and see sect. xiB9).
The interactions of tyrosine kinase and P2Y2 receptor signaling pathways provide a paradigm for the regulation of neuronal differentiation and suggest a role for P2Y2 as a morphogen receptor that potentiates neurotrophin signaling in neuronal development and regeneration (64). P2Y2 receptors, which mediate the mitogenic effects of extracellular nucleotides on vascular smooth muscle, are upregulated in the synthetic phenotype in the neointima after balloon angioplasty (763).
III. ATP AS A COTRANSMITTER
The idea that neurons can synthesize, store, and release a single substance became known as “Dale's principle,” although Dale never explicitly suggested this; rather, he speculated that the same neurotransmitter would be stored and released from all the terminals of a single neuron. He was thinking in particular of primary afferent nerve fibers releasing the same transmitter in the spinal cord as from peripheral terminals in the skin during antidromic impulses in collaterals (419). It was only later that Eccles (502) introduced the term Dale's principle, and the notion that neurons utilize a single transmitter then dominated thinking until the late 1970s. However, there were a number of hints in the literature that this might not be universally true, and this together with the appeal of the general idea that neurons contain genes capable of producing more than one transmitter, but that during development and differentiation certain genes are triggered and others suppressed, led to a commentary by Burnstock introducing the cotransmitter hypothesis in 1976 (243). Judging from the increasing number of reviews that have appeared on this subject (252, 273, 329, 407, 604, 750, 982, 1077, 1282, 1371), it seems that this hypothesis is now widely accepted and that few neuroscientists today would venture to suggest that any neuron only utilizes one transmitter, albeit that a principal transmitter might dominate for much of its life-span. There is now a substantial body of evidence to show that ATP is a cotransmitter with classical transmitters in most nerves in the PNS and CNS, although the proportions vary between tissues and species as well as in different developmental and pathophysiological circumstances (see Refs. 112, 252, 273, 636). The spectrum of physiological signaling variations offered by cotransmission are discussed in these reviews.
In keeping with the concept of purinergic cotransmission, there was early recognition that ATP and adenosine modulated prejunctional inhibition of ACh release from the skeletal neuromuscular junction (647, 1424) and of NE release from peripheral sympathetic nerves in a wide variety of tissues, including rabbit kidney, canine adipose tissue, guinea pig vas deferens (367, 724), and rabbit central ear artery, saphenous vein, portal vein, and pulmonary artery (1651, 1780). Prejunctional modulation of ACh release from peripheral cholinergic nerves by purines was observed in the isolated guinea pig ileum and the myenteric plexus longitudinal muscle preparation (1170, 1507, 1795). Clear evidence for this was presented by De Mey et al. (436) who showed that the prejunctional actions of purine nucleotides in canine saphenous vein were mediated largely by adenosine following the rapid breakdown of ATP, since slowly degradable analogs of ATP were ineffective. More recently, evidence has been presented for a prejunctional modulatory action by ATP itself, as well as adenosine, in the iris (589), rat vas deferens (1802), and tail artery (1560).
Purine nucleotides and nucleosides can also act on postjunctional receptors to modulate cholinergic and adrenergic neurotransmission. Purines were reported to increase ACh receptor activity in various preparations, including the rat diaphragm muscle (528), frog skeletal muscle (23), and rabbit iris sphincter (695). The interactions are Ca2+ dependent and may involve interaction with the allosteric site of the receptor-ion channel complex. Purine nucleotides and nucleosides were shown to interact with NE postjunctionally in vitro in the guinea pig seminal vesicles (1204), rabbit kidney (724), guinea pig and mouse vas deferens (751, 1589, 1877), rabbit mesenteric artery (965), and rat mesenteric bed (1391). These neuromodulatory actions of purines have been extensively reviewed (1319, 1420, 1635).
A. Sympathetic Nerves
It was recognized early that ATP was costored with catecholamines in adrenal medullary chromaffin cells (150, 741). Subsequently, ATP was shown to be coreleased with epinephrine from chromaffin cells (313, 468). The 1976 cotransmitter hypothesis included the suggestion that NE and ATP might be cotransmitters in sympathetic nerves, following the earlier demonstration that ATP was contained together with NE in sympathetic nerve terminals in a molar ratio estimated to be from 7:1 to 12:1, NE:ATP (627, 988, 1522, 1642). The first evidence for sympathetic cotransmission involving ATP together with NE came from studies of the taenia coli (1652). It was shown that 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 that the release of both tritium and NE was blocked by guanethidine. Soon after, the possibility that ATP might be coreleased with NE in chemical transmission from the hypogastric nerve to the seminal vesicle of the guinea pig was raised and that the substantial residual NANC responses of the cat nictitating membrane following depletion of NE by reserpine might be due to the release of ATP remaining in sympathetic nerves (998, 1204).
The most extensive evidence for sympathetic cotransmission, however, came from studies of the vas deferens, initially by Westfall and colleagues (536, 1853). Although it was not realized at the time, when EJPs were first recorded in smooth muscle cells of the vas deferens in response to stimulation of sympathetic nerves (289, 290; Fig. 1D), they were due to ATP rather than to NE. We were puzzled at the time that EJPs were not abolished by adrenoceptor antagonists; however, since they were abolished by the sympathetic neuron blocking agents bretylium and guanethidine (which are drugs that prevent nerve-mediated release of transmitter), we were correct in assuming they were produced by transmitter released from sympathetic nerves. Subsequent studies showed that EJPs are blocked by the ATP receptor antagonists arylazido aminopropionly-ATP (ANAPP3) and suramin and also following selective desensitization of the ATP receptor with the stable analog of ATP, α,β-methylene ATP (α,β-meATP) (872, 1593), but not by depletion of NE with reserpine (919, 1596). Furthermore, injection of ATP mimicked the EJP, whereas NE did not (1595). Direct evidence for concomitant release of ATP with NE and neuropeptide Y (NPY) from sympathetic nerves supplying the vas deferens was later presented (873). ATP has also been shown recently to be a cotransmitter with NE in sympathetic nerves supplying the human vas deferens (89). Sympathetic cotransmission to the seminal vesicles, epididymis, and prostate were also described (1777, 1778).
Evidence that soluble ectonucleotidases were released together with ATP and NE in the vas deferens was proposed (1711), although some later studies have contested this proposal. NTPDase1 was identified in cardiac synaptosomes (1543). The mechanisms underlying the synergistic postjunctional actions of NE and ATP on smooth muscle of the vas deferens have been explored (189, 1589). NPY is also colocalized in sympathetic nerve varicosities, but when released acts largely as a postjunctional neuromodulator, potentiating both the responses to NE and ATP in rat vas deferens (516), rat tail artery (189), and bovine chromaffin cells (1041), as well as acting as a prejunctional modulator of release of NE and ATP. Figure 4 A is a schematic illustrating sympathetic cotransmission.
Sympathetic purinergic cotransmission has also been clearly demonstrated in many different blood vessels (see Refs. 250, 253, 1594, 1651 for full accounts). The proportion of NE to ATP is extremely variable in the sympathetic nerves supplying the different blood vessels. The purinergic component is relatively minor in rabbit ear and rat tail arteries, is more pronounced in the rabbit saphenous artery, and has been claimed to be the sole transmitter in sympathetic nerves supplying arterioles in the mesentery and the submucosal plexus of the intestine, whereas NE release from these nerves acts as a modulator of ATP release (527, 1394). ATP is a prominent sympathetic cotransmitter in guinea pig vein, but not artery (1591). Sympathetic purinergic vasoconstriction of canine cutaneous veins is involved in thermoregulation (561, 944). Sympathetic cotransmission involves activation of vasoconstrictive P2X1 (and/or P2X3) and P2Y6-like receptors in mouse perfused kidney and short-term pretreatment with α,β-meATP (acting as a P2X1 and P2X3 agonist) potentiated P2Y6-like receptor-mediated vasoconstriction (1807). Decentralized rat tail arteries were hypersensitive to α,β-meATP (1932). β-Nicotinamide adenine dinucleotide (NAD) was shown recently to be released from sympathetic nerve terminals in canine mesenteric artery and proposed as a putative neurotransmitter or neuromodulator (1592).
The contributions of NE and ATP to postjunctional responses depend on the parameters of nerve discharge. For example, in the central ear artery, short pulse bursts (1 s) at low frequency (2–5 Hz) favor the purinergic component of the response, while long stimulation bursts at higher frequencies favor the noradrenergic component (889). Neurites from cultured sympathetic neurons can use local mechanisms for ATP synthesis that do not depend on a functional connection to the cell body (1712). ATP and NE are released from sympathetic nerves supplying the heart (440). In the pithed rat, there is selective blockade by nifedipine of the purinergic rather than adrenergic component of nerve-mediated vasopressor responses (236). Coreleased ATP can act both as a prejunctional modulator (mostly after breakdown to adenosine) and a postjunctional potentiator of sympathetic neurotransmission. The different prejunctional effects of agents such as PGE2, ANG II, and calcitonin gene-related peptide (CGRP) on the release of ATP and NE suggest that they are not stored in the same vesicles in the sympathetic nerve terminals (517). Full accounts of the progression of evidence in support of sympathetic cotransmission of ATP and NE are available (253, 1643, 1803). In addition, there is evidence that the preganglionic terminals on neurons in the SCG release ACh and ATP as cotransmitters (1796).
B. Parasympathetic Nerves
Parasympathetic nerves supplying the urinary bladder utilize ACh and ATP as cotransmitters, in variable proportions in different species (265, 286, 766, 786) and by analogy with sympathetic nerves, ATP again acts through P2X ionotropic receptors, whereas the slow component of the response is mediated by a metabotropic receptor, in this case muscarinic. There is also evidence to suggest that there is parasympathetic, purinergic cotransmission to resistance vessels in the heart and airways (802, 1476). Colocalization of P2X and nicotinic ACh receptors has been shown in rat vagal preganglionic nerves (1198).
C. Sensory-Motor Nerves
Since the seminal studies of Lewis (1028), it has been well established that transmitters released following the passage of antidromic impulses down sensory nerve collaterals during “axon reflex” activity produce vasodilatation of skin vessels. The early work of Holton (756) 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. We know now that “axon reflex” activity is widespread in autonomic effector systems and forms an important physiological component of autonomic control (1456). CGRP and substance P (SP) are well established as coexisting in sensory-motor nerves, and in some subpopulations, ATP is also likely to be a cotransmitter (255). Concurrent release of ATP and SP from guinea pig trigeminal ganglionic neurons in vivo has been described (1120).
D. Intrinsic Nerves in the Gut and Heart
Intrinsic neurons exist in most of the major organs of the body. Many of these are part of the parasympathetic nervous system, but certainly in the gut and perhaps also in the heart, some of these intrinsic neurons are derived from neural crest tissue that differs from those that form the sympathetic and parasympathetic systems and appear to represent an independent local control system.
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) NO also producing IJPs, but with a slower time course; and 3) vasoactive intestinal polypeptide (VIP) producing slow tonic relaxations (see Refs. 124, 266). The proportions of 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 utilize VIP, in others they utilize NO, and in nonsphincteric regions of the intestine, ATP is more prominent (see Ref. 266).
ACh and ATP are the major fast excitatory neurotransmitters to the distal colon myenteric ganglia of guinea pig (1263). In the intestinal myenteric plexus, all enteric neurons, except for the nitric oxide synthase (NOS)-immunoreactive inhibitory neurons supplying muscle, are choline acetyltransferase (ChAT) immunoreactive. Therefore, the authors suggest that purinergic inputs of myenteric origin come from neurons that utilize ACh and ATP as cotransmitters in presynaptic fibers. Fast excitatory postsynaptic potentials (EPSPs) with a purinergic component were found in myenteric neurons throughout the length of the guinea pig gut, but they were most prominent in the ileum and rare in the gastric corpus (1018). Fast synaptic transmitters, ACh and ATP, are released from the same nerve endings in the myenteric plexus of guinea pig ileum (1019).
In guinea pig submucosal and myenteric neurons, activation of 5-hydroxytryptamine (5-HT) and P2X receptors are interdependent (181), again raising the possibility that ATP and 5-HT are cotransmitters in presynaptic nerve terminals.
In the heart, subpopulations of intrinsic nerves in the atrial and intra-atrial septum have been shown to contain ATP as well as NO, NPY, ACh, and 5-HT. Many of these nerves project to the coronary microvasculature and produce potent vasomotor actions (252, 1476).
E. Peripheral Motor Nerves
In addition to the studies of Buchthol and Folkow (226, 227) mentioned earlier, there was later evidence that ATP was released together with ACh from cholinergic nerves in various tissues, including the electric organ of elasmobranch fish (470, 1976) and the phrenic nerve endings in rat diaphragm (1572), although there is also some release of ATP from muscle (1498).
Although it was accepted that ATP was stored in and released together with ACh from motor nerve terminals, it was not recognized at the time as a cotransmitter, but was considered rather as a molecule involved in the vesicular uptake and storage of the neurotransmitter ACh. 31P-NMR analysis of synaptic vesicles from Torpedo electric organ showed that they store ATP together with ACh associated in free solution at an acid pH (596).
Application of ATP or adenosine was shown to inhibit the release of ACh (647, 1424). The effect of ATP was dependent on hydrolysis to adenosine, which then acted on presynaptic receptors (1422, 1568). ATP was also shown to act postsynaptically to facilitate the action of ACh (1419). ATP facilitates both spontaneous and agonist-activated ACh channel opening. It was also shown that in early development of the neuromuscular junction, released ATP acted on P2X receptor ion channels as a genuine cotransmitter with ACh acting on nicotinic receptors, while in mature animals, ATP no longer acted as a cotransmitter, but rather as a modulator at both prejunctional and postjunctional sites (950, 1424). Later papers confirmed these findings, showing that ATP itself is involved in these postjunctional actions (see Refs. 727, 732, 1073, 1571). ACh and ATP release from Torpedo electric organ are both inhibited by the removal of extracellular Ca2+ or by the addition of the calmodulin antagonist trifluoperazine, suggesting that ACh and ATP are both released by exocytosis from synaptic vesicles (1526). However, it is interesting that ATP release (in contrast to ACh) is not blocked by botulinum toxin type A, and Ω-conotoxin also differentially blocks ACh and ATP release (1108). A high-affinity adenosine uptake system has been demonstrated in the synaptosomes for reconstitution of stored ATP. Isolated synaptic vesicles from Torpedo electric organ contain ∼200,000 molecules of ACh and ∼24,000 molecules of ATP; small amounts of ADP are also present (10% of ATP content) as well as traces of AMP. Direct postjunctional responsiveness to ATP reappears after denervation of chick skeletal muscle (1849). Corelease of ATP with ACh, prejunctional modulation of transmitter release by adenosine, and postjunctional potentiation of ACh release by ATP have also been demonstrated at the frog neuromuscular junction (see sect. ixB2c).
Recent papers have added some further details about the mechanisms underlying release of ATP from motor nerve terminals. For example, excitatory adenosine A2A receptors probably coexist with inhibitory (A1) receptors at the rat neuromuscular junction, modulating the evoked release of ACh, the balance of inhibition, or facilitation depending on the frequency of motor nerve stimulation (388). Depression of ACh release via presynaptic A1 receptors is by inhibition of N-type Ca2+ channels (1524), but is not the basis of tetanic fade at rat neuromuscular junctions (1095). Tetanic depression is overcome by tonic adenosine A2A receptor facilitation of Ca2+ influx through L-type channels at rat motor nerve terminals (1276). A presynaptic facilitating effect of P2 receptor activation on rat phrenic nerve endings was later also recognized (611, 1485), and P2X7-like receptors have been implicated at the mouse neuromuscular junction (1176). Evidence has been presented that ATP, via P2Y receptors, but not adenosine, inhibits nonquantal spontaneous ACh release at the neuromuscular junction of mouse (433, 678) and quantal release in frog (678).
Much of the evidence for purinergic involvement in skeletal neuromuscular transmission has come from studies of the fish electric organ and frog and chick neuromuscular junctions (see sect. xB2).
F. Nerves in the Brain and Spinal Cord
Evidence for purinergic cotransmission in the CNS has lagged behind that presented for purinergic cotransmission in the periphery. However, in the last few years a number of such studies have been reported.
Release of ATP from synaptosomal preparations and slices from discrete areas of the rat and guinea pig brain including cortex, hypothalamus, medulla, and habenula has been measured (97, 1626, 1855). In cortical synaptosomes, a proportion of the ATP appears to be coreleased with ACh, and a smaller proportion with NE (1372). In preparations of affinity-purified cholinergic nerve terminals from the rat caudate nucleus, ATP and ACh are coreleased (1426). There is also evidence for corelease of ATP with catecholamines from neurons in the locus coeruleus (1360) and hypothalamus (235, 1626). Purinergic and adrenergic agonist synergism for vasopressin and oxytocin release from hypothalamic supraoptic neurons is consistent with ATP cotransmission in the hypothalamus (867, 1605).
Corelease of ATP with GABA has been demonstrated in the rabbit retina (1334) and in dorsal horn and lateral hypothalamic neurons (835, 837). Possible mechanisms that underlie the balance between P2X receptor-mediated excitation and GABAA receptor-mediated inhibition were discussed in a Nature News and Views article (1488). P2X and GABA receptors are also colocalized in subpopulations of rat postnatal DRG neurons and their central terminals laminae I-III (986). The intracellular loop of GABAB subunits and the COOH-terminal domain of P2X2/P2X3 receptors are necessary for cross-talk between ATP and GABA-gated channels (182). There is evidence for corelease of ATP with glutamate in the hippocampus (1182) as well as widespread and pronounced modulatory effects of ATP on glutamatergic mechanisms (795). A recent study has shown that in central neuronal terminals, ATP is primarily stored and released from a distinct pool of vesicles and that the release of ATP is not synchronized either with the cotransmitters GABA or glutamate (1302). Cooperativity between extracellular ATP and N-methyl-d-aspartate (NMDA) receptors in long-term potentiation (LTP) induction in hippocampal CA1 neurons (592) is consistent with ATP/glutamate cotransmission. Colocalization of functional nicotinic and ionotropic nucleotide receptors have also been identified in isolated cholinergic synaptic terminals in midbrain (455). Interactions between P2X2 and both α3β4 and α3β2 nicotinic receptor channels has been shown in oocyte expression studies (892).
There is indirect evidence supporting the possibility that dopamine and ATP are cotransmitters in the CNS (968). After cerebellar lesions in rats producing axotomy of mossy and climbing fiber systems, nitrergic and purinergic systems were activated with similar time courses on precerebellar stations (1792). This raises the possibility that, as in a subpopulation of neurons in the gut, NO and ATP are cotransmitters.
It is speculated that postsynaptic selection of coreleased fast transmitters is used in the CNS to increase the diversity of individual neuronal outputs and achieve target-specific signaling in mixed inhibitory networks (489). Figure 4B summarizes current knowledge of purinergic cotransmission in the peripheral and central nervous systems.
IV. NEUROTRANSMISSION AND NEUROMODULATION IN AUTONOMIC GANGLIA
An earlier review, published in 2001, of purinergic signaling in autonomic ganglia is available (496).
A. Sympathetic Ganglia
Purinergic synaptic transmission was not demonstrated in sympathetic ganglia until the early 1990s (526, 1569). A number of subsequent studies have characterized the receptors present on sympathetic neurons, and it is now clear that there is a species difference between rat and guinea pig. In the guinea pig, α,β-meATP is an effective agonist on SCG (1404, 1968) and celiac ganglion neurons (896). In contrast, α,β-meATP evoked only a small slowly desensitizing response in a subpopulation of neurons from rat SCG (369, 896). In a study of rat and mouse celiac ganglion neurons, no responses to α,β-meATP were detected (1969). Most of the properties described for P2X receptors in rat sympathetic neurons (kinetics, agonist and antagonist profile, effect of Zn2+ and pH) are consistent with those of the recombinant P2X2 receptor. The presence of a small slowly desensitizing α,β-meATP response in rat SCG neurons can most easily be explained by the coexistence of some heteromeric P2X2/3 receptors (496).
A number of studies have demonstrated the presence of P2X receptors in sympathetic ganglia by immunohistochemistry. Immunoreactivity for P2X1, P2X2, P2X3, P2X4, and P2X6 receptors was detected in SCG and celiac ganglia of the rat (1896). In a study of cultured SCG neurons, P2X2 was the most highly expressed receptor; lower, although detectable, levels of all the other subunits, except P2X4, were present (1035). However, the extent to which the expression of P2X receptors may be influenced by tissue culture conditions is at present unclear. In a study of the guinea pig SCG, P2X2 and P2X3 immunoreactivity was detected (1968). P2X1-GFP has been used to study the time course of P2X1 receptor clustering (∼1 μm diameter) in plasma membranes of cultured sympathetic neurons from rat SCG and internalization of receptors following prolonged exposure to ATP (1035). In keeping with the histochemical evidence, mRNA for most P2X subunits has been detected in sympathetic neurons. P2X5 and P2X6 receptors were first isolated by PCR for celiac and SCG mRNAs, respectively (378). Fragments of P2X3 and P2X4 receptors have also been cloned from a rat SCG cDNA library (230, 1027). Three splice variants of the rat P2X2 receptor have been cloned, and all three were detected in SCG neurons by in situ hybridization (1578). Other in situ hybridization studies have detected P2X1, P2X4, and P2X6 mRNA in rat SCG neurons (230, 378).
Studies of the release and metabolism of endogenous ATP in rat SCG suggested that ATP and ACh were released simultaneously in response to stimulation of preganglionic nerve terminals, although release of ATP or ACh varies with stimulation frequency and temperature (1796), perhaps reflecting different vesicular storage. Endogenous adenosine can inhibit both posttetanic potentiation and LTP in rat SCG (748). Both excitatory P2X receptors (probably P2X2) (161) and inhibitory P2Y receptors (probably P2Y2) (162) have been described on presynaptic sympathetic nerve terminals. P2X7 receptors have also been claimed to be present on presynaptic terminals, but their function is unclear (34). From a study using P2X1 receptor knockout mice, at least three broad categories of SCG neurons were revealed: neurons with a P2X2 phenotype, α,β-meATP-sensitive neurons that suggested a P2X1 heteromeric receptor, and neurons that have no detectable P2X receptor expression (308).
P2Y receptors are also expressed on sympathetic neurons (162, 307). P2Y2 receptors on SCG neurons were shown to mediate inhibition of both N-type Ca2+ and M-type K+ currents (162, 557). Studies of sympathetic neurons cultured from thoracolumbal paravertebral ganglia show that while ATP release of NE is mediated by P2X receptors, UDP-induced release of NE is entirely due to generation of action potentials followed by calcium influx through voltage-gated channels (1801), perhaps mediated by P2Y6 receptors. A P2Y6 receptor was later shown to be expressed in SCG neurons and, like P2Y2 receptors, couples to both N-type Ca2+ and M-type K+ channels (1256). In a later study, P2Y4 receptors expressed in rat sympathetic neurons were shown to couple much more effectively to M-type K+ channels than to Ca2+ channels, in contrast to P2Y1, P2Y2, and P2Y6 receptors (556). Evidence for P2Y1, P2Y2, P2Y6 receptors and an atypical UTP-sensitive receptor in mouse cultured SCG neurons and glia has been presented (307).
B. Adrenal Chromaffin Cells
Chromaffin cells of the adrenal medulla can be regarded as a highly specialized form of sympathetic neuron. Although the P2X2 receptor was originally cloned from PC12 cells, which are a rat pheochromocytoma cell line, adrenomedullary chromaffin cells have received relatively little attention.
In one of the first immunohistochemical studies of P2X receptors, using antibodies raised against P2X1 and P2X2 receptors, Vulchanova et al. (1809) observed both P2X1 and P2X2 immunoreactivity in both differentiated PC12 cells and chromaffin cells of the adrenal medulla. This observation is not consistent with functional studies (see below) and was not substantiated in more recent immunohistochemical studies (14, 15) in which only limited expression of P2X5 and P2X7 was detected in rat chromaffin cells, while P2X6 immunoreactivity was detected in the guinea pig.
Brake et al. (194) cloned the P2X2 receptor from PC12 cells and detected weak expression of the mRNA in the adrenal gland by Northern blotting. P2X4 mRNA has also been detected (155). However, in both studies, it was not certain whether the mRNA was present in the medullary or cortical cells.
ATP can produce at least three different effects on adrenal chromaffin cells: inhibition of voltage-gated Ca2+ channels (411, 461, 1046), release of Ca2+ from internal stores (1407), and activation of a nonselective cation channel (1285). While the first two effects are most probably mediated by P2Y receptors, the third effect has the characteristics for the activation of P2X receptors.
Functional studies have demonstrated the presence of P2X receptors on bovine (1407) and guinea pig (1056, 1285) chromaffin cells. However, these receptors appear to be absent in the rat (753, 1056). The P2X receptor present on chromaffin cells can be activated by ATP and 2-methylthio ATP (2-MeSATP), but is much less sensitive or insensitive to α,β-meATP (1056, 1407). To date, the only detailed pharmacological study of P2X receptors on chromaffin cells has been carried out on the guinea pig. Here, the receptor is antagonized by pyridoxalphosphate-6-azonphenyl-2′,4′-disulfonic acid (PPADS), but suramin and Cibacron blue are quite weak antagonists. The response is potentiated by low pH, but inhibited by Zn2+. Thus while this receptor has some properties in common with the rat P2X2 receptor (agonist profile, effect of pH), the lack of potentiation by Zn2+ and the low sensitivity to the antagonists suramin and Cibacron blue are not. Although three spliced variants of the guinea pig P2X2 receptor have been cloned, and some pharmacological characterization has been carried out, there is at present insufficient information to identify the native P2X receptor present on guinea pig chromaffin cells. The pharmacological properties of the P2X receptor present on guinea pig chromaffin cells are very similar to that of the α,β-meATP-insensitive receptor found on pelvic ganglion neurons. It therefore seems likely that it is in fact the homomeric P2X2 receptor. ATP and catecholamines are released in parallel from adrenal chromaffin cells in response to stimulation by ACh, K+, or Ba2+ (871). Evidence has been presented that voltage-dependent Ca2+ channels are regulated in a paracrine fashion by ATP acting on P2X receptors in porcine adrenal chromaffin cells (1270).
Ecto-nucleotidases have been localized and characterized in pig adrenal glands (132, 1720). ARL 67156 is an effective inhibitor of ecto-nucleotidase activity in bovine chromaffin cells (475). Diadenosine polyphosphates are stored and released with ATP from chromaffin cells (825).
P2Y receptors mediate inhibition of exocytotic release of catecholamines from adrenal chromaffin cells by modulation of voltage-operated Ca2+ channels, rather than by a direct effect on the secretory machinery (1375, 1755). Exposure of bovine chromaffin cells to NPY results in a long-lasting increase in subsequent stimulation of inositol phosphate formation by ATP acting on P2Y receptors (476). P2Y2 receptors have been identified immunohistochemically on rat chromaffin cells (16), which is consistent with this effect. ATP stimulation also appears to act through adenylate cyclase to stimulate cAMP formation in bovine chromaffin cells (1956), so it is interesting that P2Y12 receptors that use this second messenger system have since been demonstrated in these cells (520).
C. Parasympathetic Ganglia
Prior to the cloning of P2X receptors, ATP was found to produce excitation in the vesical parasympathetic ganglion of the cat (1700). Responses to ATP have been recorded from dissociated neurons from the chick ciliary ganglia (10), rabbit vesical parasympathetic ganglion (1249), intramural ganglia from the guinea pig and cat urinary bladder (281, 1450), and guinea pig and rat cardiac neurons in culture (32, 511). In general, the results are very similar to those obtained in sympathetic neurons. Thus application of ATP evokes a rapid depolarization or inward current through the activation of P2X receptors. Although 2-MeSATP and ATP are approximately equipotent, α,β-meATP evoked only small responses when applied at high concentrations to rat neurons.
The neurons providing motor innervation to the bladder and other pelvic organs originate in the pelvic plexus. In the rat and mouse, this plexus consists of a pair of major pelvic ganglia and a number of small accessory ganglia. In the guinea pig and human, there are additional intramural ganglia within the wall of the bladder (404, 405). The pelvic ganglia receive sympathetic and parasympathetic inputs from preganglionic axons within the hypogastric and pelvic nerves, respectively.
Since they are smaller and have a more diffuse location, parasympathetic ganglia are much harder to study. Consequently, there is less immunohistochemical or molecular biological information about the presence of P2X receptors in these neurons. Although many neurons showed low levels of staining, a small percentage showed strong and specific staining. In keeping with these observations, P2X2 but not P2X1 receptor immunoreactivity was detected in axons and nerve terminals in the vas deferens (1809). High levels of P2X2 mRNA and protein have been identified in rat pelvic ganglion neurons (1971). Although some P2X4 message was also detectable, no staining was observed using probes directed against P2X1 and P2X3 receptor mRNA. P2X2 and heteromultimer P2X2/3 receptors are also dominant in mouse pelvic ganglion neurons (1969). In contrast, at least three P2X receptors are present in guinea pig pelvic neurons, P2X2, P2X3, and P2X2/3 receptors (1970).
In a study of ATP-evoked currents in parasympathetic neurons dissociated from rat submandibular ganglia, it was shown that the inorganic and organic cation permeability of the ATP-gated P2X receptor channel was similar to that of the cloned P2X2 receptor with a minimum pore diameter of 0.7 μm (1052). In the intact submandibular ganglia, ATP inhibits neurotransmitter release via presynaptic P2Y receptors but had no effect on postsynaptic neurons. However, upregulation of postsynaptic P2X receptors occurred in dissociated neurons (1586). In addition to P2X receptors, P1 and P2Y receptors are coexpressed postsynaptically in hamster submandibular ganglion neurons, and both receptors mediate inhibition of N- and P/Q-type voltage-dependent Ca2+ channels (9). In hamster submandibular ganglion neurons, ATP caused both depolarization and hyperpolarization: the depolarization was mediated via P2X receptors, the hyperpolarization via P2Y2 receptors (519).
In a comparative study of different parasympathetic ganglia, it was shown that neurons from intracardiac and paratracheal ganglia were insensitive to α,β-meATP, while all the neurons in otic and some neurons in sphenopalatine and submandibular ganglia responded (1085). Immunohistochemistry revealed strong staining for P2X2 receptors in all five ganglia and strong P2X3 staining in otic, sphenopalatine, and submandibular ganglia, suggesting that the receptor subtypes involved are homomeric P2X2 and heteromeric P2X2/3 receptors. Combined calcium imaging and immunohistochemistry indicated that both P2X3 and P2Y4 receptors were expressed in neurons from cat bladder intramural ganglia (1450; Fig. 5, A–C). It was shown that 100, 49, and 97% of P2X3 receptor immunopositive neurons coexpressed ChAT, NOS, and neurofilament 200 (NF200), respectively, while 100, 59 and 98% of P2Y4 immunopositive neurons coexpressed ChAT, NOS, and NF200, respectively. Application of α,β-meATP and UTP elevated intracellular Ca2+ in a subpopulation of dissociated cultured neurons. Immunohistochemistry revealed strong and specific staining for the P2X2 receptor subunit on rat parasympathetic neurons of the otic, sphenopalatine submandibular, intracardiac, and paratracheal ganglia (1085). Strong P2X3 receptor staining was seen on otic, sphenopalatine, and submandibular ganglia, but neurons in intracardiac and paratracheal ganglia were insensitive to α,β-meATP. The predominant P2 receptor subtypes are homomeric P2X2 and heteromeric P2X2/3 receptors. Thus P2X3 receptors are expressed in parasympathetic ganglia, in contrast to the widely held view that P2X3 and P2X2/3 receptor subtypes are restricted to sensory neurons. P2Y2 receptors have been identified on rat intracardiac neurons that mediate increases in intracellular Ca2+ and generation of inositol trisphosphate (IP3) (1053).
Adenosine was reported to mediate a slow hyperpolarizing synaptic potential in cat vesical parasympathetic ganglia by stimulating preganglionic nerves (25). In a later study of rat pelvic ganglion neurons, it was shown that adenosine inhibited N-type Ca2+ currents by activation of A1 receptors via a voltage-dependent and pertussis toxin (PTX)-sensitive pathway, which may explain how adenosine acts as an inhibitory modulator of ganglionic transmission in the pelvic plexus (1311).
D. Enteric Ganglia
Katayama and Morita (878) were the first to study the effects of ATP on single myenteric neurons from guinea pig small intestine using the intracellular electrophysiological recording technique. Myenteric neurons are classified into two groups electrophysiologically, and ATP elicited hyperpolarization in 80% of AH (type II) neurons and depolarization in 90% of S (type I) neurons in a dose-dependent manner. Quinidine reversibly depressed both the ATP-induced responses.
Several laboratories have extended these studies of purinergic signaling in the guinea pig myenteric and submucous neurons (see Refs. 266, 613, 771, 1168, 1411). Elegant whole cell and outside-out patch-clamp recordings were used to characterize the physiological and pharmacological properties of P2X receptors on myenteric neurons of the guinea pig ileum (95, 175). ATP and analogs evoked rapid inward currents in over 90% of the neurons studied.
P2X receptors and nicotinic cholinergic receptors are linked in a mutually inhibitory manner in guinea pig myenteric neurons (1972). Mulholland's group carried out studies of purinergic signaling in dispersed primary cultures of guinea pig myenteric plexus. Extracellular ATP was shown to mediate Ca2+ signaling via a PLC-dependent mechanism (911). Enteric neurons differed from one another in their ability to respond to combinations of ATP with ACh, ATP with SP, ATP with ACh, ATP with ACh and SP, ATP with bombesin, or ATP with ACh and bombesin. Evidence was presented from the laboratory of Christofi, Wood, and colleagues that two distinct types of P2 receptors are linked to a rise in [Ca2+]i in guinea pig intestinal myenteric neurons of both AH and S neuronal phenotypes and are not restricted to calbindin immunoreactive sensory neurons.
ATP regulates synaptic transmission by pre- as well as postsynaptic mechanisms in guinea pig myenteric neurons, i.e., ATP augments nicotinic fast depolarization produced by ACh, but inhibits muscarinic and SP-mediated depolarizations in both AH and S neurons (860). α,β-MeATP-sensitive P2X receptors (P2X2, P2X3, and P2X2/3 receptors) are prejunctional modulators of cholinergic neurotransmission between the myenteric plexus and longitudinal smooth muscle of the guinea pig intestine (130).
Exogenous and endogenous ATP, released during increase in intraluminal pressure, inhibits intestinal peristalsis in guinea pig via different apamin-sensitive purine receptor mechanisms. Exogenous ATP depresses peristalsis mostly via suramin- and PPADS-insensitive P2 receptors, whereas endogenous purines act via P2 receptors sensitive to both suramin and PPADS (729). Evidence has been presented that ATP plays a major role in excitatory neuroneuronal transmission in both ascending and descending reflex pathways to the longitudinal and circular muscles of the guinea pig ileum triggered by mucosal stimulation (1018, 1167, 1622). Distending inhibitory reflexes involve P2X receptor-mediated transmission from interneurons to motor neurons in guinea pig ileum (140). It has been proposed that ATP is a sensory mediator via P2X3 receptors on intrinsic sensory neurons in the enteric plexuses (138, 267). P2X3 receptor immunoreactivity has also been shown on sensory neurons in the human myenteric plexus (1935). Experiments with P2X2 and P2X3 receptor knockout mice showed that peristalsis is impaired in the small intestine, indicating that P2X3 and P2X2/3 receptors participate in the neural pathways underlying peristalsis, and it was suggested that these receptors may contribute to the detection of distension or intraluminal pressure increases and initiation of reflex contractions (139, 1411). Intraganglionic laminar nerve endings, which are prominently associated with myenteric ganglia, particularly in the stomach, express both P2X2 (317) and P2X3 receptors (1899). P2X3 receptors are dominant on neurons in the submucosal plexus of the rat ileum and distal colon, and ∼50% of the neurons express calbindin, a marker for enteric sensory neurons (1898).
Nicotinic ACh and P2X receptors play a central role in fast synaptic excitatory transmission in the myenteric plexus (615). Nicotinic receptors on S-type neurons on the guinea pig intestine are composed of at least the α3- and β4-subunits, while P2X receptors in S-type neurons are composed of P2X2 subunits (1411), and ATP acting on the receptors is the predominant fast excitatory neurotransmitter in the descending pathways. The P2X3 receptor subtype predominates in AH-type neurons (139) and probably participates in mechanosensory transduction (138, 1399). Fast EPSPs (fEPSPs) occur in bursts in the myenteric plexus during evoked motor reflexes in the guinea pig ileum. The amplitude of fEPSPs declines with repetitive stimulation. It is suggested that this synaptic “run-down” is not due to nicotinic or P2X receptor desensitization, but rather to depletion of a readily releasable pool of neurotransmitter (1412). Physical and functional interactions between P2X2 receptor channels and serotonin-gated 5-HT3 channels have been proposed as a basis for ionotropic cross-talk and a potential mechanism for regulating neuronal excitability and synaptic plasticity within the myenteric plexus (181).
Synaptosomal preparations from the guinea pig ileum myenteric plexus were first described by Dowe et al. (471) and Briggs and Cooper (207). ATP and adenosine were equipotent in their ability to inhibit the nicotinically induced release of [3H]ACh; the inhibition by both ATP and adenosine was reversed by theophylline, indicating that a P1 receptor was involved (1406). However, high concentrations of ATP caused a marked increase in the release of [3H]ACh, presumably mediated by a prejunctional P2 receptor.
Intracellular recordings from submucosal neurons in guinea pig small intestine showed that ATP induced fast transient depolarization of most AH-type neurons and fast transient depolarization followed by slower onset, longer lasting depolarization of S-type neurons (92, 1168). When whole cell patch recordings were employed, superfusion of ATP and analogs evoked rapidly desensitizing inward current, and ATP-induced single-channel currents were also recorded. In a whole cell patch-clamp study of ATP-induced membrane currents in guinea pig small intestinal submucous neurons by another group (655), the currents activated by ATP were not blocked by suramin and were often enhanced by Reactive blue 2. This could indicate the involvement of P2X4 or possibly P2X6 receptors. The functional interaction, between nicotinic and P2X receptors, has been investigated in freshly dissociated guinea pig submucosal neurons in primary culture: whole cell currents induced by ATP were blocked by PPADS and showed some interdependence on ACh-induced nicotinic currents blocked by hexamethonium (93, 1972). Inhibitory interactions between 5-HT3 and P2X channels in submucosal neurons have been described (94). Evidence has also been presented for two subtypes of P2X receptor in neurons of guinea pig ileal submucosal plexus (654). Slow EPSPs (sEPSPs) were mediated by P2Y1 receptors in neurons in the submucosal plexus of guinea pig small intestine (771, 1168). The purinergic excitatory input to these neurons came from neighboring neurons in the same plexus, from neurons in the myenteric plexus, and from sympathetic postganglionic neurons. ATP-mediated EPSPs occurred coincident with fast nicotinic synaptic potentials with noradrenergic IPSPs.
Immunohistochemical studies have demonstrated P2X2 receptors (318, 1898), P2X3 receptors (1364, 1769, 1898), and P2X7 receptors (770) in subpopulations of guinea pig and rat myenteric and submucous ganglion neurons (see Table 3). Enteric glial cells also express P2 receptors (910, 1773). P2X2 and P2X5 receptors have also been immunolocalized on interstitial cells of Cajal (ICCs) (293). P2X2 receptors were localized on smooth muscle in the mouse colon (637) and P2X5 receptors on nerve fibers that enveloped ganglion cell bodies and possibly glial cell processes in the mouse gastrointestinal tract (1452). ATP plays a major excitatory role, probably largely via P2X2 receptors, in rat myenteric neurons, whether sensory, motor, or interneurons (1271). Cross-inhibitory interactions between GABAA and P2X receptor channels in myenteric neurons of guinea pig small intestine have been described recently (869).
There is growing evidence for the expression of P2Y receptors on enteric neurons in addition to P2X receptors (1885). Fast and slow depolarizations and Ca2+ responses of cultured guinea pig ileal submucosal neurons to ATP were mediated by P2X and P2Y receptors, respectively (91). In the mouse gastrointestinal tract, P2Y1 receptors on NANC myenteric neurons appear to mediate relaxation through NO and ATP (637). Slow excitatory synaptic transmission is mediated by P2Y1 receptors in the guinea pig enteric nervous system (771). A P2Y1 receptor has been cloned and characterized recently from guinea pig submucosa (618). P2Y2 receptors are widely distributed on S-type (Dogiel type 1) neurons in the myenteric and submucosal plexuses throughout the guinea pig gut (1901). About 40–60% of P2X3 receptor immunoreactive neurons were immunoreactive for P2Y2 receptors in the myenteric plexus, and all P2X3 receptor immunoreactive neurons expressed P2Y2 receptors in the submucosal plexus. It seems likely that the S-type neurons with fast P2X receptor-mediated and slow P2Y receptor-mediated depolarizations are these neurons coexpressing P2X3 and P2Y2 receptor subunits. It has been shown recently that 30–36% of the ganglion cells in the myenteric, but not submucosal, plexus of the guinea pig gastrointestinal tract are labeled with P2Y6 receptor-immunoreactive neurons (see Fig. 5D), while ∼42–46% of the neurons in both myenteric and submucosal plexuses are immunoreactive for P2Y12 receptors (1903). About 28–35% of P2Y6 receptor-immunoreactive neurons coexist with NOS, but not with calbindin, while all P2Y12 receptor-immunoreactive neurons were immunopositive for calbindin and appear to be AH intrinsic primary afferent neurons. In a recent study of the rat distal colon, P2Y1 and P2Y6 immunoreactivity was found on smooth muscles, P2Y4 and P2Y6 receptor immunoreactivity on glial cells in both plexuses, P2Y4 receptors on ICCs, while P2Y2 and P2Y12 receptors were demonstrated on enteric neurons (1770, 1771). Some of the P2Y2 immunoreactive neurons, but none of the P2Y12 immunoreactive neurons exhibited neuronal (n)NOS.
Differential gene expression of A1, A2A, A2B, and A3 receptors in human enteric neurons have been reported (366), and fine-tuning modulation of myenteric motoneuron activity by endogenous adenosine has been claimed (387). Adenosine has dual effects on ACh release from myenteric motor neurons: prejunctional facilitating A2A and extrajunctional inhibitory A1 receptors (485). This group also showed that adenosine activation of A2A receptors downregulates nicotinic autoreceptor function at rat myenteric nerve terminals. Adenosine suppresses slow EPSPs in AH neurons via A1 receptors (see Ref. 1886). In the mouse distal colon, A2B receptors are located on enteric NANC inhibitory neurons (1983).
V. SENSORY NEURONS
Sensory neurons of the DRG share with neurons of the sympathetic, parasympathetic, and enteric ganglia, along with adrenomedullary chromaffin cells, a common embryological origin in the neural crest. In contrast, cranial sensory neurons are derived from the placodes. Although these sensory and autonomic neurons exhibit some common properties, they also show very diverse phenotypes commensurate with their diverse physiological roles.
There have been many reports characterizing the native P2X receptors in sensory neurons, including those from dorsal root, trigeminal, nodose, and petrosal ganglia. DRG and trigeminal ganglia contain primary somatosensory neurons, receiving nociceptive, mechanical, and proprioceptive inputs. Nodose and petrosal ganglia, on the other hand, contain cell bodies of afferents to visceral organs.
All P2X subtypes, except P2X7, are found in sensory neurons, although the P2X3 receptor has the highest level of expression (both in terms of mRNA and protein). P2X2/3 heteromultimers are particularly prominent in the nodose ganglion. P2X3 and P2X2/3 receptors are expressed on isolectin B4 (IB4) binding subpopulations of small nociceptive neurons (188). Species differences are recognized.
RT-PCR showed that P2Y1, P2Y2, P2Y4, and P2Y6 mRNA is expressed on neurons of DRG, nodose, and trigeminal ganglia, and receptor protein for P2Y1 is localized on over 80% of mostly small neurons (1451). Double immunolabeling showed that 73–84% of P2X3 receptor positive neurons also stained for the P2Y1 receptor (see Fig. 5, F–H), while 25–35% also stained for the P2Y4 receptor. Patch-clamp studies of cultured neurons from DRG were consistent with both P2X3 and P2Y1 receptors being present in a subpopulation of DRG neurons.
It has been shown that the sensory neurons have the machinery to form purinergic synapses on each other when placed in short-term tissue culture (1948). The resulting neurotransmitter release is calcium dependent and uses synaptotagmin-containing vesicles; the postsynaptic receptor involved is a P2X subtype. Experiments are needed to find out whether purinergic synapses form between sensory neurons in vivo, whether this is more common after nerve injury, and whether this has physiological or pathophysiological significance.
Comprehensive reviews of P2X receptor expression and function in sensory neurons in DRG, nodose, trigeminal and petrosal ganglia are available (262, 496). An account of the roles played by both adenosine and ATP in nociception can be found in section xi, A8 and B9).
A. Dorsal Root Ganglia
The P2X3 subunit that was first cloned using a cDNA library from neonatal rat DRG neurons shows a selectively high level of expression in a subset of sensory neurons, including those in DRG (335, 378, 1027). In DRG ganglia, the level of P2X3 transcript is the highest, although mRNA transcripts of P2X1–6 have been detected. Green fluorescence has been used to quantitate P2X receptor RNA in DRG (1751). The expression pattern of P2X3 receptors in sensory ganglia has also been studied by immunohistochemistry at both the light microscope (99, 188, 1261, 1811, 1812, 1896) and electron microscope (1062) levels. In DRG, intensive P2X3 immunoreactivity is found predominantly in a subset of small- and medium-diameter neurons, although it was absent from most large neurons. The P2X3 subunit is predominantly located in the nonpeptidergic subpopulation of nociceptors that binds the IB4 and is greatly reduced by neonatal capsaicin treatment (1812). The P2X3 subunit is present in an approximately equal number of neurons projecting to skin and viscera, but in very few of those innervating skeletal muscle (188). P2X3 receptors are strongly represented in sensory ganglia during rat embryonic neurogenesis (353; see sect. xA2).
P2X2 receptor immunoreactivity is observed in many small and large DRG neurons, although the level is lower than that of P2X3 (985, 1811). Some neurons seem to contain both P2X2 and P2X3 immunoreactivity. Although P2X3 immunoreactivity is the predominant type detected, variable levels of immunoreactivity for P2X1, P2X2, P2X4, P2X5, and P2X6 receptors have also been detected in DRG neurons (1338, 1896). These receptors are arranged in clusters 0.2–0.5 μm in diameter and rarely appear to colocalize (99). Both transient and sustained responses to P2 receptor agonists occur in DRG neurons (see Ref. 496). The transient response in DRG neurons is activated by ATP, α,β-meATP, and 2-MeSATP. The pharmacological evidence to date generally supports the hypothesis that this rapid desensitizing transient response is mediated by homomeric P2X3 receptors.
P2X receptors on the cell bodies of the sensory neurons have been studied extensively using voltage-clamp recordings from dissociated neurons of the DRG (238, 683, 1031, 1432). Rapid application of ATP evokes action potentials, and under voltage clamp, a fast-activating inward current, as well as depolarization and an increase in intracellular Ca2+ concentration.
mRNA for an orphan G protein-coupled receptor TGR7, which is specifically responsive to β-alanine, claimed to participate in synaptic transmission, is coexpressed in small-diameter neurons with P2X3 and vanilloid type 1 (VR1) receptors in both rat and monkey DRG (1559). Ca2+/calmodulin-dependent protein kinase II, upregulated by electrical stimulation, enhances P2X3 receptor activity in DRG neurons, and it is suggested that this may play a key role in the sensitization of P2X receptors under inflammatory conditions (1909). Rapid reduction of the excitatory action of ATP on DRG neurons by GABA, probably via GABAA anionic receptors, and slow inhibition of ATP currents via metabotropic GABAB receptors appear to be additional mechanisms of sensory information processing (986, 1600). Fibers project from DRG to the superficial lamina of the dorsal horn of the spinal cord where the receptors may function to modulate transmitter release near their central terminals. Oxytocin inhibits ATP-activated currents in DRG neurons (1921). In contrast, neurokinin B potentiates ATP-activated currents in DRG neurons (1827). 17β-Estradiol attenuates α,β-meATP-induced currents in rat DRG neurons (326, 1084). Ω-Conotoxin GVIA, known as a selective blocker of N-type calcium channels, potently inhibits the currents mediated by P2X receptors in rat DRG neurons (992), while neurokinin B potentiates ATP-activated currents (1827). Pentobarbital suppressed the fast-type current mediated by P2X3 receptors in rat DRG neurons and may contribute to its anesthetic and analgesic actions (922).
There are species differences in the responses of DRG neurons to ATP. Transient responses are the predominant type evoked by P2X agonists from DRG neurons of rat and mouse, with persistent and biphasic types seen less frequently (238, 683). In contrast, only sustained inward currents have been reported on DRG neurons from bullfrog (120, 1032). It is possible that distinct P2X receptors may be differentially distributed at cell soma and nerve terminals of the same neuron. The physiological significance of the heterogeneity in P2X receptor expression in sensory neurons is not yet clear.
Neurons and glial cells differentially express P2Y receptor subtype mRNA in rat DRG (939). P2Y1 and P2Y2 receptor mRNA was expressed in ∼20% of neurons; Schwann cells expressed P2Y2 mRNA, and nonneuronal satellite cells expressed P2Y12 and P2Y14 mRNA. ATP and UTP produce slow and sustained excitation of sensory neurons in DRG via P2Y2 receptors (1164). Colocalization P2Y1 and P2X3 immunoreactivity has been described in a subpopulation of DRG neurons (177, 1451). P2Y receptors contribute to ATP-induced increase in intracellular Ca2+ and subsequent release of CGRP from DRG neurons (1493). ATP and UTP were equipotent in increasing axonal transport in cultured DRG neurons, probably via P2Y2 receptors (1483). RT-PCR and immunohistochemistry studies have identified P2Y1 and P2Y4 mRNA and protein in DRG as well as nodose and trigeminal ganglia of the rat (1451). Other nucleoside triphosphates, including NTP, GTP, and CTP, and the diphosphates NDP, GDP, UDP, and CDP were also active in modulating sodium currents in DRG neurons (1313). Bradykinin and ATP, acting via P2Y receptors, accelerate Ca2+ efflux from rat sensory neurons via protein kinase C (PKC) and the plasma membrane Ca2+ pump isoform 4 and represent a novel mechanism to control excitability and augment their sensitivity to other stimuli (773, 1760). Inhibition of N-type voltage-activated calcium channels in DRG neurons by P2Y receptors has been proposed as a mechanism of ADP-induced analgesia (631). P2Y2 and P2Y4 receptors were strongly expressed in DRG of the cat, as well as P2X3 receptors (1449). Other P2X and P2Y receptor subtypes were also present in cat DRG, but there was low expression of P2Y1 receptors compared with >80% of P2Y1 receptor-positive neurons in rat DRG. Metabotropic P2Y1 receptors inhibit P2X3 receptor channels in rat DRG neurons via G protein activation (632). Extracellular ATP upregulated the TTX-resistant Na+ current recorded in cultured rat and mouse DRG neurons, consistent with P2Y receptor activation; the activation of PKC appears to be a necessary step in the GTP-dependent upregulation process (80). GFP studies have shown that there is ADP-induced endocytosis and internalization of P2Y receptors in DRG neurons (1825). An orphan G protein-coupled receptor, localized in rat DRG, has been proposed to be an adenine receptor (129).
Some nicotinic ACh receptor antagonists, such as α-bungarotoxin and (+)-tubocurarine, appear to be potent blockers of fast P2X receptor ATP-gated currents in DRG neurons (993). Adenosine-5′-O-(3-thiotriphosphate) (ATPγS) enhances nerve growth factor (NGF)-promoted neurite formation in DRG neurons, perhaps via its ability to increase NGF-promoted TrkA activation (63). NTPDase2 has been shown to be present in satellite glial cells in DRG (202), consistent with evidence for a functional role for ATP in satellite glial cells (705). Functional expression of P2X7 receptors on nonneuronal glial cells, but not on small-diameter neurons from rat DRG, has been reported (1960).
B. Nodose Ganglia
P2X2 and P2X3 receptors were shown to be expressed immunohistochemically in rat nodose ganglia (1811). ATP, α,β-meATP, and 2-MeSATP evoke sustained currents in rat nodose neurons. These responses are inhibited by suramin, PPADS, Cibacron blue, trinitrophenyl (TNP)-ATP, and Ca2+ (896, 1703, 1790), but not by diinosine pentaphosphate (Ip5I) (495). Therefore, the α,β-meATP-sensitive persistent responses in nodose neurons resemble the recombinant P2X2/3 receptors (1027). Neurons of the mouse nodose ganglion give persistent responses to both ATP and α,β-meATP similar to those seen in the rat and guinea pig (372, 1617, 1970). In P2X3 receptor-deficient mice, no nodose neurons respond to α,β-meATP at concentrations up to 100 μM, while the response to ATP is significantly reduced. The residual persistent responses to ATP have all the characteristics of recombinant P2X2 homomers. Thus the pharmacological evidence is consistent with the notion that both heteromeric P2X2/3 and homomeric P2X2 receptors are present in significant amounts in nodose neurons, although the proportions may vary from cell to cell (371). Most neurons in the rat nodose ganglia showed colocalization of P2X3 receptors and the IB4 from Griffonia simplicifolia type one (GS-IB4). Subpopulations of neurons expressed P2X1/3 and P2X2/3 heteromultimers (778). RT-PCR showed P2X1, P2X2, P2X4, and P2X7 receptors were expressed in rat nodose ganglia (71). Sensory neurons from nodose ganglia express, in addition to P2X3 receptor mRNA, significant levels of P2X1, P2X2, and P2X4 receptor mRNAs, and some of these mRNAs are present in the same cell. P2X2 and P2X3 receptor immunoreactivities are both present and are colocalized in the same neurons (1027, 1896).
P2Y1 receptors have been demonstrated immunohistochemically in rat and human nodose ganglia (565). Coexistence of functional P2Y receptors (acting via the IP3 pathway) and ryanodine receptors and their activation by ATP has been demonstrated in vagal sensory neurons from the rabbit nodose ganglion (747). RT-PCR has shown P2Y1, P2Y2, P2Y4, and P2Y6 receptor mRNA in rat nodose ganglia (1451). P2Y1 receptor immunoreactvity was found in >80% of the sensory neurons, particularly small diameter (neurofilament-negative neurons), while P2Y4 receptors were expressed in more medium- and large-diameter neurons. About 80% of the P2X3 receptor immunoreactive neurons also stained for P2Y1 receptors, while ∼30% of the neurons showed colocalization of P2Y4 with P2X3 receptors.
C. Trigeminal Ganglia
Most of the facial sensory innervation is provided by nerve fibers originating in the trigeminal ganglion, comprised of neurons that transduce mechanical, thermal, and chemical stimuli, probably including odorant molecules. In trigeminal ganglia, P2X3 receptor immunoreactivity is found in the cell bodies of both small and large neurons (383, 833, 1062, 1896). Lower levels of immunoreactivity to P2X1, P2X2, P2X4, and P2X6 receptors appear to be present in these neurons. Whole cell patch-clamp studies of trigeminal neurons showed ATP-activated (both fast and slow) desensitizing currents in the majority of cells examined, but outward or biphasic currents also occurred in a small number of cells (688). P2X3 receptor mRNA increased in trigeminal ganglia (and DRG) after nerve transection, suggesting that they play a role in the pathomechanism of postnerve injury hypersensitivity (1734). A subpopulation of neurons cultured from rat trigeminal ganglia have been identified which lack the typical nociceptive characteristics and express homomeric P2X2 receptors (1619). The authors speculate that trigeminal neurons are equipped with a repertoire of receptors that fulfill multiple tasks affecting different sensory modulators. Expression of P2X3 receptors in the rat trigeminal ganglia after inferior alveolar nerve injury decreased by ∼35% (522). One day after ischemic insult, the number of P2X3 receptor immunoreactive neurons in trigeminal ganglia of the Mongolian gerbil decreased by ∼67%, and by day 5, only a few neurons showed weak immunoreactivity (782).
P2Y1 and P2Y4 receptor mRNA and protein are also expressed in rat trigeminal ganglia with many neurons showing colocalization with P2X3 receptors (1451). Striking differences between P2X3 receptors in trigeminal and DRG neurons were highlighted, questioning the validity of extrapolating spinal cord models of P2X3 function to the craniofacial region (44). In particular, only a small percentage of IB4-binding neurons express P2X3 receptors, whereas many peptidergic neurons express P2X3 receptors.
Evidence has been presented that satellite glial cells in mouse trigeminal ganglia express P2Y receptors (possibly the P2Y1 subtype), although their precise role is not yet clear (705, 1845; see sect. viii for further discussion). Recently, single-cell calcium imaging demonstrated that both P2Y1 and, to a lesser extent, P2Y2,4,6,12,13 receptors on satellite glial cells contribute to ATP-induced calcium-dependent signaling in mixed neuron-glia primary cultures from mouse trigeminal ganglia (324).
D. Petrosal Ganglia
The petrosal ganglion provides sensory innervation of the cortical sinus and carotid body through the carotid sinus nerve. P2X receptors have been identified on neurons in the rat petrosal ganglia (1955). ATP activates cat and rabbit petrosal ganglia neurons in vitro (29, 31) and evokes ventilatory reflexes in situ, which are abolished after bilateral chemosensory denervation (1613). Dopamine inhibits ATP-induced responses of neurons of the cat petrosal ganglia (30).
E. Retinal Ganglia
Retinal ganglion cells on the eye receive information from both rods and cones, and early papers about purinergic transmission in the retina have been reviewed (1354). P2X2 receptors have been identified in retinal ganglion cells (198, 675), particularly within cone pathways (1386). Functional studies have also identified P2X2/3 heteromultimeric receptors in cultured rat retinal ganglion cells (1687). P2X2 receptors are expressed on cholinergic amacrine cells of mouse retina (862) and also GABAergic amacrine cells (1386).
It was proposed that ATP, coreleased with ACh from retinal neurons, modulates light-evoked release of ACh by stimulating a glycinergic inhibitory feedback loop (1219). RT-PCR at the single-cell level revealed expression of P2X2, P2X3, P2X4, and P2X5 receptor mRNA in approximately one-third of the bipolar cells (1854); P2X7 receptors were identified on both inner and outer retinal ganglion cell layers of the primate (812) and rat (199), and electron microscope analysis suggested that these receptors were localized in synapses, suggesting that purines play a significant role in neurotransmission within the retina and may modulate both photoreceptor and rod bipolar cell responses (1387). Stimulation of P2X7 receptors elevated Ca2+ and killed retinal ganglion cells (1958) and may be involved in retinal cholinergic neuron density regulation (1414).
P2X3 receptors are present on Müller cells (815). Müller cells release ATP during Ca2+ wave propagation (1235). While the potent P2X7 agonist 3′-O-(4-benzoyl)benzoyl ATP (BzATP) killed retinal ganglion cells, this was prevented by the breakdown product adenosine via A3 receptors (1959).
Evidence has been presented recently for the involvement of P2X7 receptors in outer retinal processing: P2X7 receptors are expressed postsynaptically on horizontal cell processes as well as presynaptically on photoreceptor synaptic terminals in both rat and marmoset retinas (1387).
F. Sensory Nerve Fibers and Terminals
Sensory nerve terminals express purinoceptors and respond to ATP in many situations (see Ref. 262). However, it has been shown that ATP sensitivity is not necessarily restricted to the terminals; increased axonal excitability to ATP and/or adenosine of unmyelinated fibers in rat vagus, sural and dorsal root nerves, as well as human sural nerve has been described (810, 1725).
During purinergic mechanosensory transduction, the ATP that acts on P2X3 and P2X2/3 receptors on sensory nerve endings is released by mechanical distortion from urothelial cells during distension of bladder and ureter and from mucosal epithelial cells during distension of the colorectum (see sect. xiA8). It is probably released from odontoblasts in tooth pulp, from epithelial cells in the tongue, epithelial cells in the lung, keratinocytes in the skin, and glomus cells in the carotid body. Released ATP is rapidly broken down by ectoenzymes to ADP (to act on P2Y1, P2Y12, and P2Y13 receptors) or adenosine (to act on P1 receptors) (1980).
Pulmonary neuroepithelial bodies (NEBs) (215, 218) and more recently subepithelial receptor-like endings associated with smooth muscle (SMARs) (216) have been shown to serve as sensory organs in the lung, and P2X3 and P2X2/3 receptors are expressed on a subpopulation of vagal sensory fibers that supply NEBs and SMARs with their origin in the nodose ganglia. Quinacrine staining of NEBs indicates the presence of high concentrations of ATP in their secretory vesicles, and it has been suggested that ATP is released in response to both mechanical stimulation during high-pressure ventilation and during hypoxia (1425). NEBs are oxygen sensors especially in early development, before the carotid system has matured (217, 588). In a study of bronchopulmonary afferent nerve activity of a mouse isolated perfused nerve-lung preparation, it was found that C fibers could be subdivided into two groups: fibers that conduct action potentials at <0.7 ms−1 and are responsive to capsaicin, bradykinin, and ATP; and fibers that conduct action potentials on an average of 0.9 ms−1 and respond vigorously to ATP, but not to capsaicin or bradykinin (952). Both the transient receptor potential vanilloid 1 (TRPV1) receptor and P2X receptors mediate the sensory transduction of pulmonary reactive oxygen species, especially H2O2 and OH, by capsaicin-sensitive vagal lung afferent fibers (1454).
Vagal C-fibers innervating the pulmonary system are derived from cell bodies situated in two distinct vagal sensory ganglia: the jugular (superior) ganglion neurons project fibers to the extrapulmonary airways (larynx, trachea, bronchus) and the lung parenchymal tissue, while the nodose (inferior) neurons innervate primarily structures within the lungs (1757). Nerve terminals in the lungs from both jugular and nodose ganglia responded to capsaicin and bradykinin, but only the nodose C-fibers responded to α,β-meATP.
Sensory afferent fibers within the respiratory tract, which are sensitive to ATP, probably largely via P2X2/3 receptors, have been implicated in vagal reflex activity (1327), in the cough reflex (858, 859), and in the bradypneic reflex (1455).
ATP and α,β-meATP activated submucosal terminals of intrinsic sensory neurons in the guinea pig intestine (138), supporting the hypothesis of Burnstock (266) that ATP released from mucosal epithelial cells has a dual action on P2X3 and/or P2X2/3 receptors in the subepithelial sensory nerve plexus. ATP acts on the terminals of low-threshold intrinsic enteric sensory neurons to initiate or modulate intestinal reflexes and acts on the terminals of high-threshold extrinsic sensory fibers to initiate pain (see sect. xiA8c). Further support comes from the demonstration that peristalsis is impaired in the small intestine of mice lacking the P2X3 subunit (139) and that up to 75% of the neurons with P2X3 receptor immunoreactivity in the rat submucosal plexus expressed calbindin (1898); calbindin is regarded as a marker for intrinsic sensory neurons, at least in the guinea pig (see Ref. 603). Thirty-two percent of retrogradely labeled cells in the mouse DRG at levels T8-L1 and L6-S1, supplying sensory nerve fibers to the mouse distal colon, were immunoreactive for P2X3 receptors (1433). Intraganglionic laminar nerve endings are specialized mechanosensory endings of vagal afferent nerves in the rat stomach, arising from the nodose ganglion; they express P2X3 receptors and are probably involved in physiological reflex activity, especially in early postnatal development (1899).
Purinergic mechanosensory transduction has also been implicated in reflex control of intestinal secretion, whereby ATP released from mucosal epithelial cells acts on P2Y1 receptors on enterochromaffin cells to release 5-HT, which leads to regulation of secretion either directly or via intrinsic reflex activity (384).
3. Carotid body
The ventilatory response to decreased oxygen tension in the arterial blood is initiated by excitation of specialized oxygen-sensitive chemoreceptor cells in the carotid body that release neurotransmitter to activate endings of the sinus nerve afferent fibers. ATP and adenosine were shown early on to excite nerve endings in the carotid bifurcation (828, 1137) and subsequently α,β-meATP (1623).
Large amounts of adenine nucleotides were localized in glomus cells, stored within specific granules together with catecholamines and proteins (158). Evidence of ATP release from carotid chemoreceptor cells has been reported (300), and corelease of ATP and ACh is the likely mechanism for chemosensory signaling in the carotid body in vivo (see Ref. 1264). ATP coreleased with ACh from type I glomus chemoreceptor cells during hypoxic and mechanical stimulation was shown to act on P2X2/3 receptors on nerve fibers arising from the petrosal ganglion mediating hypoxic signaling at rat and cat carotid body chemoreceptors (1377, 1775, 1955). Immunoreactivity for P2X2 and P2X3 receptor subunits has been localized on rat carotid body afferents (1377). These findings were confirmed and extended in a recent study where P2X2 receptor deficiency resulted in a dramatic reduction in the responses of the carotid sinus nerve to hypoxia in an in vitro mouse carotid body-sinus nerve preparation (1444). ATP mimicked the afferent discharge, and PPADS blocked the hypoxia-induced discharge. Immunoreactivity for P2X2 and P2X3 receptor subunits was detected on afferent terminals surrounding clusters of glomus cells in wild-type but not in P2X2 and/or P2X3 receptor-deficient mice. ATP induces [Ca2+]i rise in rat carotid body cultured glomus cells (1163). Support for this mechanism in hypercapnia, as well as in hypoxia, came from CO2/pH chemosensory signaling in cocultures of rat carotid body and petrosal neurons (1954).
ATP, acting on P2X2 receptors, contributed to modified chemoreceptor activity after chronic hypoxia, indicating a role for purinergic mechanisms in the adaptation of the carotid body in a chronic low-O2 environment (721). It was concluded in recent reviews that ATP is emerging as an important excitatory transmitter for afferent nerve activation by hypoxia, but that further studies are needed to analyze the interactions between putative O2 sensors and excitatory and inhibitory transmitters in the carotid body (1376). Adenosine also stimulates carotid chemoreceptors, probably via postsynaptic A2A receptors (938, 1463) and presynaptic A2B receptors (381).
An ATP-triggered vagal reflex has been described leading to suppression of sinus mode automaticity and atrioventricular nodal conduction (1326). This is probably mediated by P2X2/3 receptors located on vagal sensory nerve terminals in the left ventricle, supporting the hypothesis that ATP released from ischemic myocytes is a mediator of atropine-sensitive bradyarrhythmias associated with left ventricular myocardial infarction (1911).
5. Skin, muscle, and joints
Ca2+ waves in human epidermal keratinocytes mediated by extracellular ATP produce [Ca2+]i elevation in DRG neurons, suggesting a dynamic cross-talk between skin and sensory neurons mediated by extracellular ATP (947).
P2 receptors on the endings of thin fiber muscle afferents play a role in evoking both the metabolic and mechanoreceptor components of the exercise pressor reflex (706). PPADS attenuated the pressor response to contraction of the triceps muscle. ATP has been shown to be an effective stimulant of group IV receptors in mechanically sensitive muscle afferents (1038, 1408). Arterial injection of α,β-meATP in the blood supply of the triceps surae muscle evoked a pressor response that was a reflex localized to the cat hindlimb and was reduced by P2X receptor blockade (1038). In this study, ATP was also shown to enhance the muscle pressor response evoked by mechanically sensitive muscle stretch, which was attenuated by PPADS.
Sensory nerve fibers arising from the trigeminal ganglion supplying the temporomandibular joint have abundant receptors that respond to capsaicin, protons, heat, and ATP; retrograde tracing revealed 25, 41, and 52% of neurons supplying this joint exhibited VR1 and P2X3 receptors, respectively (785).
6. Inner ear
ATP has been shown to be an auditory afferent neurotransmitter, alongside glutamate (see Ref. 765). There are ∼50,000 primary afferent neurons in the human cochlear and about one-half express P2X2 (or P2X2 variants) and, debatably, P2X3 receptors. ATP is released from K+-depolarized organ of Corti in a Ca2+-dependent manner, and an increase in ATP levels in the endolymph has been demonstrated during noise exposure, perhaps released by exocytosis from the marginal cells of the stria vascularis (1194). The P2 receptor antagonist PPADS attenuated the effects of a moderately intense sound on cochlea mechanics (157). NO enhances the ATP-induced [Ca2+]i increase in outer hair cells (1549). Spiral ganglion neurons, located in the cochlear, convey to the brain stem the acoustic information arising from the mechanoelectrical transduction of the inner hair cells express P2X receptors (1897) and are responsive to ATP (491, 814).
7. Nasal organ
Odorant recognition is mediated by olfactory receptors predominantly situtated on the microvilli of olfactory receptor neurons in the nasal organ. Nucleotides act via purinoceptors on olfactory neurons as well as sustentacular supporting cells (412, 624, 725). Purinergic receptors appear to play an integral role in signaling acute damage in the olfactory epithelium by airborne pollutants. Damaged cells release ATP, thereby activating purinergic receptors on neighboring sustentacular cells, olfactory receptor neurons, and basal cells, initiating a stress-signaling cascade involving heat shock proteins for neuroprotection (726). The majority of nasal trigeminal neurons lacked P2X3 receptor-mediated currents but showed P2X2-mediated responses when stimulated by ATP (424).
8. Taste buds
Taste bud cells and associated sensory nerve fibers express P2 receptors, including P2X2 and P2X3 receptor subunits (153) and P2Y1 receptors (877). ATP is the key transmitter acting via P2X2 and P2X3 receptors on taste receptor cells detecting chemicals in the oral cavity (558). These authors showed that genetic elimination of P2X2 and P2X3 receptors abolished responses of the taste nerves, although the nerves remained responsive to touching, temperature, and menthol and reduced responses to sweeteners, glutamate, and bitter substances. They also showed that a bitter mixture containing denatonium and quinine stimulated release of ATP from the taste epithelium. Ectonucleotidases are known to be abundantly present in taste buds (110). Dystonin disruption, produced in mutant mice, resulted in a decrease in the number of vagal and glossopharyngeal sensory neurons and in the number of taste buds as well as in the number of P2X3 receptor-labeled neurons and their peripheral endings in taste bud epithelium (784). Other papers present data that suggest that P2Y2 and P2Y4 receptors also play a dominant role in mediating taste cell responses to ATP and UTP (113, 302, 909). NTPDase2 has been shown to have a dominant presence on type 1 cells in mouse taste papillae (111).
VI. NEUROTRANSMISSION AND NEUROMODULATION IN THE CENTRAL NERVOUS SYSTEM
The actions of adenosine in the CNS have been recognized for many years (see review Refs. 497, 1102, 1343, 1350, 1533, 1597, 1866). However, consideration of the role(s) of ATP in the CNS received less attention until more recently (see Refs. 1, 154, 257, 272, 378, 505, 639, 792, 796, 804, 1117, 1258, 1431). In particular, fast purinergic synaptic transmission has been clearly identified in the brain (890). It was first observed in the medial habenula (506) and has now been described in a number of other areas of the CNS, including spinal cord (102), locus coeruleus (1243), hippocampus (1182, 1299), and somatic-sensory cortex (1301). Electron microscopic immunocytochemical studies support these functional experiments (see Fig. 6). Although adenosine, following ectoenzymatic breakdown of ATP, is the predominant, presynaptic modulator of transmitter release in the CNS (see Refs. 497, 1422), ATP itself can also act presynaptically (409). A strong case is made for coordinated purinergic regulatory systems in the CNS controlling local network behaviors by regulating the balance between the effects of ATP, adenosine, and ectonucleotides on synaptic transmission (879, 1121). Two examples are given: one showing synergistic modulation of excitatory and inhibitory synaptic inputs in the hippocampus, and the other showing diametrically opposite effects on excitatory transmission in the caudal nucleus of the solitary tract.
ATP is present in high concentrations within the brain, varying from ∼2 mM/kg in the cortex to 4 mM/kg in the putamen and hippocampus (945). Much is now known about the breakdown of ATP released in the CNS (977). Cortex and hippocampus synaptic membranes exhibit higher activities of NTPDase1 and NTPDase2 than cerebellum and medulla oblongata, with ecto-5′-nucleotidases and adenosine deaminase were found in most brain regions.
In situ hybridization of P2 receptor subtype mRNA and immunohistochemistry of receptor subtype proteins have been carried out in recent years to show wide, but heterogeneous, distribution in the CNS of both P2X receptors (292, 864, 1006, 1062, 1064, 1257, 1457, 1896) and P2Y receptors (272, 989, 1172, 1179). P2X2, P2X4, and P2X6 receptors are widespread in the brain and often form heteromultimers. P2X1 receptors are found in some regions such as cerebellum and P2X3 receptors in the brain stem. P2X7 receptors are probably largely prejunctional. P2Y1 receptors are also abundant and widespread in the brain. The hippocampus expresses all P2X receptor subtypes and P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12 receptors.
Evidence has been presented that nucleotides can act synergistically with growth factors to regulate trophic events (1228, 1398). However, a recent paper has shown that ATP can also stimulate neurite outgrowth from neuroblastoma cells independent of NGF (991). In addition to P2 receptors and the release of ATP from neurons in the brain, there is abundant evidence for P2 receptors on, and release of ATP from, glial cells, suggesting that both short- and long-term (trophic) neuronal-glial cell interactions are taking place (see sect. vii).
There are many reports of ATP acting as a cotransmitter with classical transmitters in neurons in the CNS (see sect. iiiF). Description of neurotransmission and neuromodulation in the brain stem, hypothalamus, and spinal cord can be found in section vii concerned with CNS control of autonomic function.
Early studies carried out about purinergic signaling in the cortex have been reviewed in section iiC. Although two earlier papers mentioned some direct excitatory effect of ATP in cortex (1350, 1648), serious attention to these actions rather than those of adenosine did not appear until the 1990s. ATP diphosphohydrolase was shown to be one of the ectoenzymes involved in breakdown of ATP released from rat cortical synaptosomes (117). A novel group of pyrimidine compounds has been shown to act as inhibitors of ATP and ADP hydrolysis by NTPDases in synaptosomes from rat cerebral cortex (323). NTPDases1 and -2 have been purified and characterized from porcine cortex synaptosomes (977).
The expression of P2X7 receptors in the CNS is hotly debated (see Refs. 45, 1495, 1628). The polymorphic variations in P2X7 receptor expression might provide an explanation, at least in part, for some of the ambiguous findings (1863). P2X7 receptors have been identified in rat cortical synaptosomes (1078) and have been claimed to mediate caspase-8/9/3-dependent apoptosis in rat primary cortical neurons (956). A recent study claims that P2X7 receptors exert a permissive role on the activation of release-enhancing presynaptic α7 nicotinic receptors coexisting in rat neocortex glutaminergic terminals (1322). Evidence has been presented that diadenosine pentaphosphate (Ap5A) and ATP activate P2X and possibly specific dinucleotide receptors on human cerebrocortical synaptic terminals (1356).
Evidence has also been presented for P2Y receptor-mediated inhibition of NE release in the rat cortex (1802). Release of glutamate from depolarized nerve terminals has been claimed to be inhibited by agonists acting on both P1 and P2Y receptors (131). Release of ATP from cortical synaptosomes is decreased by vesamicol, which also inhibits ACh, but not glutamate release (1484). ATP receptor-mediated Ca2+ concentration changes in pyramidal neocortical neurons in rat brain slices from 2-wk-old rats were mediated by both P2X and P2Y receptors (994). Responses of cultured rat cerebral cortical neurons to UTP (1251) indicated the presence of P2Y2 and/or P2Y4 receptors. P2Y receptors were identified on synaptosomal membranes from rat cortex (1511). A P2Y receptor from the adult bovine corpus callosum was cloned and characterized and the mRNA identified in the frontal cortex (448). P2Y and muscarinic receptor activation evoke a sustained increase in intracellular calcium in rat neocortical neurons and glial cells, and it was proposed that a common calcium entry pathway was involved (1382). Interaction between P2Y and NMDA receptors in layer V pyramidal neurons of rat prefrontal cortex have been demonstrated (1081). ATP induces postsynaptic gene expression in neuron-neuron synapses via P2Y1 receptors, which could regulate the acetylcholinesterase (AChE) promotor activity in cultured cortical neurons (1581). P2Y1 receptors mediate inhibition of both strength and plasticity of glutamatergic synaptic neurotransmission in the rat prefrontal cortex (697). Extracellular ATP upregulates the expression of egr-1, egr-2, and egr-3, members of the early growth response family, in cultured rat primary cortical neurons (1133).
Adenosine has been shown to inhibit release of transmitters from slices of rat and rabbit cerebral cortex, including NE, ACh, GABA, and amino acids, probably involving A1 and A3 receptors (197) and for glutamate release A1 and A2A receptors (1105). Thalamocortical excitation is regulated by presynaptic adenosine (A1) receptors and provides a mechanism by which increased adenosine levels can directly reduce cortical excitability (566). A high level of endogenous adenosine, which occurs during hypoxia, activates A3 receptors, which inhibit synaptic potentials in pyramidal cells from the cingulate cortex (733). In vivo imaging of A1 receptors in the human cortex has been achieved with the use of the selective A1 antagonist 8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propylxanthine (118). Adenosine suppresses GABAA receptor-mediated responses in rat sacral dorsal commissural neurons (1036). In contrast, P2X receptors can act presynaptically in olfactory bulbs to enhance the release of glutamate (156). Evidence has been presented for uridine activation of fast transmembrane Ca2+ fluxes in rat cortical homogenates (870) and later identification of a uridine-specific binding cite in rat cerebrocortical homogenates (962).
There are many papers describing the actions of adenosine via P1 receptors in the hippocampus, including presynaptic modulation with both inhibition and enhancement of transmitter release by acting on A1 and A2A receptors, respectively (1070, 1421), influencing both LTP and long-term depression (LTD) in CA1 neurons and synaptic plasticity (see Refs. 392, 435, 1421). Presynaptic A3 receptors have also been implicated in modulation of LTP and LTD (391). Most workers recognize that adenosine is produced following rapid ectoenzymatic breakdown of released ATP in the hippocampus (see Refs. 410, 499). Adenosine inhibits excitatory, but not inhibitory, synaptic transmission by decreasing neurotransmitter release in the rat hippocampus (1937).
A1 and A2A receptors are coexpressed in pyramidal neurons and colocalized on glutamatergic nerve terminals of the rat hippocampus (1401). Modulation of hippocampal cholinergic, glutamatergic, and GABAergic transmission by ATP is dependent largely on prejunctional A1 receptors (579, 974, 1116). A1 receptor-mediated inhibition of glutamate release at rat hippocampal CA3-CA1 synapses is primarily due to inhibition of N-type Ca2+ channels (1100). Interactions between adenosine and metabotropic glutamate receptors in rat hippocampal slices have been reported (1545). A2A and mGlu5 receptors are colocalized and mediate synergistic actions and suggest that A2A receptors play a permissive role on mGlu5R receptor-mediated potentiation of NMDA effects in the hippocampus (1690). The physiological features of mossy fiber synapses are due largely to the tonic action of adenosine acting via presynaptic A1 receptors, which maintain a low basal probability of transmitter release (1174). It has been claimed that A1 receptors are strategically located on the presynaptic component of the active zone for inhibition of transmitter release as well as in the postsynaptic density to influence NMDA receptor firing and dendritic integration (1401). Activation of A2A receptors facilitates brain-derived neurotrophic factor (BDNF) modulation of synaptic transmission in hippocampal slices (460). Deletion of presynaptic A1 receptors impairs the recovery for synaptic transmission in the hippocampus after hypoxia (61). Hypoxia leads to a rapid (<90 min) homologous desensitization of A1 receptor-mediated inhibition of synaptic transmission that is likely to be due to an internalization of A1 receptors in nerve terminals (374). Lee and Chao (1008) have shown that purinergic signaling interacts with neurotrophin signaling, by transactivating Trk A and Trk B receptors via A2A receptor stimulation.
In an analysis of the role of ATP as a transmitter in the hippocampus and its role in synaptic plasticity, Wieraszko (1858) concluded that the purinergic system is particularly involved in the long-term maintenance, rather than the initial induction, of LTP. ATP receptor activation can stimulate or inhibit glutamate release from rat hippocampal neurons, and ATP release has been implicated in hippocampal LTP (1859). P2X1, P2X2/3, and P2X3 receptors can act presynaptically to facilitate glutamate release, and P2Y1, P2Y2, and P2Y4 receptor activation can inhibit release from hippocampal neurons (1439). Stimulation of Schaffer collaterals of rat and mouse hippocampal slices resulted in release of ATP and an increase in the size of LTP (1859, 1860). There is preferential release of ATP upon high- but not low-frequency stimulation of rat hippocampal slices (410). Acute ATP hydrolysis is required for the regulation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propioic acid (AMPA) receptors at hippocampal synapses; this requirement is selective for AMPA over NMDA receptors and is necessary both for LTP and LTD (1045).
ATP released from presynaptic terminals during burst stimulation (or applied extracellularly) is involved in the induction of LTP and CA1 neurons of guinea pig hippocampus through phosphorylation of extracellular domains of synaptic membrane proteins as the substrate for ectoprotein kinase (591, 1920). Extracellular ATP inhibits release of excitatory glutamate from hippocampal neurons, but stimulates release of inhibitory GABA (799, 805).
Evidence for the presence of a P2Y receptor in hippocampus neurons was presented (790, 1157). ATP inhibited K+ channels in cultured rat hippocampal neurons through P2Y receptors that showed equipotency to UTP and ATP (1213), suggesting that a P2Y2 or P2Y4 subtype was involved. It was proposed that glutamate releases ATP from hippocampal neurons to act as a neurotransmitter (803). Evidence using P2 receptor antagonists was presented to suggest that both presynaptic and postsynaptic P2 receptors in the hippocampus modulate the release and action of endogenous glutamate (1188). ATP inhibits synaptic release of glutamate by acting on P2Y receptors on pyramidal neurons in hippocampal slices (1143). Presynaptic inhibitory P2 receptors in the hippocampus inhibit calcium oscillations produced by release of glutamate (949). P2 receptor-mediated inhibition of NE release in rat hippocampus has been reported (940). While presynaptic P2Y receptors mediate inhibition of transmitter release, P2X receptors mediate facilitation of transmitter release (409). Activation of P2Y1 receptors induces inhibition of the M-type K+ current in rat hippocampal pyramidal neurons (555). Examination of the effect of ATP on the voltage-clamped, dissociated rat hippocampal neurons showed that over 30% possessed P2X receptors (82). ATP can produce facilitation of transmission in rat hippocampal slice neurons, which may require the simultaneous activation of P1 and P2 receptors (1244). Ap5A enhanced the activity of N-type Ca2+ channels in rat CA3 hippocampal neurons (1297).
A purinergic component of the excitatory postsynaptic current (EPSC) was identified mediated by P2X receptors in CA1 neurons of the rat hippocampus; EPSCs, which were blocked by PPADS, were elicited by stimulating the Schaffer collateral at a frequency below 0.2 Hz (1299). Evidence for fast synaptic transmission mediated by P2X receptors in CA3 pyramidal cells of rat hippocampal slice cultures has been reported (1182).
Binding studies have shown mRNA for several P2 receptor subtypes in the hippocampus, including binding to α,β-[3H]meATP (83, 154). Immunolocalization of the P2X4 receptor was widespread in the hippocampus; immunopositive cells were prominent in the pyramidal cell layer (in interneurons as well as pyramidal cells), scattered through CA1, CA2, and CA3 subfields as well as within the granule cell layer and hilus of the gentate gyrus (1006). P2X4 receptors are located at the subsynaptic membrane somewhat peripherally to AMPA receptors in the CA1 area of the hippocampus. Experiments with P2X4 receptor knock-out mice show that LTP at Schaffer collateral synapses is reduced, and ivermectin, which potentiates currents at P2X4 receptors, had no effect on P2X4 knock-out mice, but increases LTP in wild-type mice (1575). The authors suggest that calcium entry through subsynaptic P2X4 receptors during high-frequency stimulation contributes to synaptic strengthening.
Arguments have been presented that ATP may have a role in the protection of the function of the hippocampus from overstimulation by glutamate (799). ATP produces an initial rise and later reduction in serotonin release from perfused rat hippocampus mediated by P2X and P1(A1) receptors, respectively (1272). Noradrenergic terminals of the rat hippocampus are equipped with presynaptic P2X receptors that facilitate NE release (1307).
There are some exciting data indicating a trophic role for ATP in the hippocampus (1584). It was shown that ATP and its slowly hydrolyzable analogs strongly inhibited neurite outgrowth and also inhibited aggregation of hippocampal neurons; it was suggested that the results indicate that extracellular ATP may be involved in synaptic plasticity through modulation of NCAM-mediated adhesion and neurite outgrowth. Changes in [Ca2+]i during ATP-induced synaptic plasticity in guinea pig hippocampal CA1 neurons have been claimed (1919).
A study of the single-channel properties of P2X receptors in outside-out patches from rat hippocampal granule cells has been carried out that suggests the presence of P2X2/4 and/or P2X2/6 and/or P2X4/6 heteromultimers (1884). This has been supported by P2X receptor expression studies (1616). P2X7 receptors have been implicated in the regulation of neurotransmitter release in the rat hippocampus (1628). Activation of presynaptic P2X7-like receptors depresses mossy fiber-CA3 synaptic transmission through P38 mitogen-activated protein kinase (58). The relatively stable P2X7 agonist BzATP is enzymatically converted to adenosine in hippocampal slices so that its effects on mossy fiber terminals may be via A1 rather than P2X7 receptors (975). A stabilizing effect of extracellular ATP on synaptic efficacy and plasticity has been described in hippocampal pyramidal neurons under hypoxic conditions where there is depletion of intracellular ATP (1109).
ATP-gated presynaptic P2X2 channels facilitate excitatory transmission onto stratum radiatum interneurons, but not onto CA1 pyramidal neurons (893). This demonstration of preferential expression of functional P2X channels is a novel finding that might have wider physiological implications for purinergic signaling in the brain.
NTPDase2 and functional P2X receptors have been identified on proliferating hippocampal progenitor cells in the dentate gyrus, which may play a role in the control of hippocampal neurogenesis (1565).
ATP-induced [3H]GABA and [3H]glutamate release is absent in P2X7 receptor knockout mice, suggesting that ATP facilitates GABA and glutamate release by a presynaptic mechanism involving P2X7 receptors (1308).
A high density of P2X receptor binding was found in the cerebellar cortex (83). Electron-immunocytochemistry showed localization of P2X1 receptors in subpopulations of synapses on both presynaptic and postsynaptic sites as well as on some astrocyte processes (1064). P2X4 mRNA was found in the cerebellar cortex in Purkinje, granular, and stellate basket cells and P2X6 receptor immunoreactivity, which was confined mainly to Purkinje cells (620, 1616). Functional P2X3 and P2X7 receptors in rat cerebellar synaptic terminals have also been reported (737). A lack of correlation between glutamate-induced release of ATP and neuronal death in cultured cerebellar neurons was reported (1103).
Cerebellar Purkinje neurons were shown experimentally to have P2Y receptors (918) as well as P2X (probably mainly P2X2) receptors (620). Patch-clamp studies of dissociated cerebellar neurons revealed P2Y receptor-operated potassium channels (789). There appears to be a molecular interplay between the P2Y4 receptor and the R1 subunit of the NMDA receptor during glucose deprivation with P2Y4 receptor involvement in cell death under conditions of metabolism impairment (319).
ATP increased the release of aspartate from cultured cerebellar granule neurons and also potentiated its release by glutamate; it was concluded that this was consistent with a cotransmitter role of ATP in the cerebellum (1145). This group also showed that a P2 receptor antagonist prevented glutamate-evoked cytotoxicity in these cultured neurons (39).
cDNAs encoding three splice variants of the P2X2 receptor were isolated from rat cerebellum (1578). Ecto-5′-nucleotidase has been localized on cell membranes in cultures of cerebellar granule cells and also ectophosphorylated protein and ecto-ATPase (1982), consistent with purinergic signaling. Single-channel properties of P2X receptors in rat cerebellar slices suggested that they may be P2X4/6 heteromultimer receptors (700). Both ADP and adenosine prevent apoptosis of cultured rat cerebellar granule cells via P2X and A1 receptors, respectively (1794). In a study by Florenzano et al. (563), it was claimed that cerebellar lesion upregulates P2X1 and P2X2 receptors in precerebellar nuclei.
P2Y receptors mediate both short-term presynaptic and long-term postsynaptic enhancement of GABAergic transmission between cerebellar interneurons and Purkinje cells (1481). Coexpression of functional P2X and P2Y receptors has been identified in single cerebellar granule cells (736). Modulation of synaptic activity in Purkinje neurons by ATP has been reviewed recently (442).
ATPγS recapitulates in cultured cerebellar granule neurons many warning signs of cellular neurodegeneration occurring in vivo, including morphological abnormalities, mitochondrial impairment, free radical generation, and oxidative stress; PPADS can efficiently postpone or prevent the progression of neuronal death in a cell culture model (40).
Both P2Y1 and P2X7 receptors induce calcium (calmodulin-dependent protein kinase II) activation in cerebellar granule neurons, although there are differences in subcellular distribution and duration of effects between the two receptor subtypes (1017). The P2X7 activation was not associated with pore formation, but its abundant presence at synaptic structure suggests a role in synaptic plasticity.
P1(A1) receptors were identified on cerebellar granule cells (1881), and adenosine was shown to selectively block parallel fiber-mediated synaptic potentials in the rat cerebellar cortex (942) and inhibit Purkinje cell firing and glutamate release from cultured cerebellar neurons (464).
D. Basal Ganglia
In vivo release of adenosine from cat basal ganglia was taken as early support for the existence of purinergic nerves in the brain (96). Autoradiographic labeling of P1(A2) receptors showed them to be exclusively restricted to the human caudate nucleus, putamen, nucleus accumbens, and globus pallidus as well as the olfactory tubercle (1111). Adenosine A2A receptor modulation of electrically evoked GABA release from slices of rat globus pallidus was described (1127). A2A receptors also mediate inhibition of the NMDA component of excitatory synaptic currents in rat striatal neurons (1872). However, A2A receptors are located largely outside the active zone at synapses in contrast to the location of hippocampal A2A receptors, which are mostly located in the presynaptic active zone (1402). A2A receptors were also shown to be prominent in dopamine-innervated areas of the basal ganglia (1675), and adenosine-dopamine receptor-receptor interactions have been proposed to be an integrative mechanism for motor stimulation actions in basal ganglia (548). Dopamine D1, D2, and D3 knockout mice showed an increase in A2A receptor binding in the caudate putamen, nucleus accumbens, and olfactory tubercle, while in A2A receptor knockout mice there was an increase in D2 receptor mRNA in these regions of the basal ganglia (1564). Dopaminergic principal neurons in the ventral tegmental area do not possess somatic P2 receptors, in contrast to peripheral and central noradrenergic neurons (1359).
The extracellular actions of adenosine via P1 receptors on the striatum were the first instance in which active purines were recognized; they were largely involved in presynaptic modulation of release of dopamine, ACh, GABA, and glutamate (607). The P1 receptor subtype involved is predominantly A1, but A2 receptors were shown to mediate stimulation of dopamine synthesis (364, 1675). A1 receptors play a major modulatory role in dopamine and adenosine receptor signaling in the neostriatum (1915). Inactivation of adenosine A2A receptor impairs LTP in the nucleus accumbens without altering basal synaptic transmission (414).
ATP release was demonstrated from affinity-purified rat cholinergic nerve terminals from rat caudate nucleus and adenosine resulting from ectoenzymatic breakdown of ATP, acted on prejunctional A1 receptors to inhibit ACh release (1426). It was shown that ATP was released from cultured mouse embryonic neostriatal neurons (1952) and that adenosine is produced from extracellular ATP at the striatal cholinergic synapse (822). ATP-evoked potassium current in rat striatal neurons was shown to be mediated by a P2 purinergic receptor (788). ATP increases the extracellular dopamine level in rat striatum through stimulation of P2Y receptors (1963), although it has been claimed to inhibit dopamine release in the neostriatum (1724). Dopamine facilitates activation of P2X receptors by ATP (807). Intra-accumbens injection of 2-MeSATP leads to release of dopamine (928). ATP induces neurotoxicity in vivo in the rat striatum via P2 receptors (1473).
Neostriatal medium-spiny neurons and cholinergic interneurons express P2X2 and P2Y1 receptors, but it appears that they only become functional under certain as yet unknown conditions (1512). Accumbal neuronal output, reflected by both dopamine release and neuronal electrical activity, is modulated in a functionally antagonistic manner by P2 and P1 receptor stimulation (968).
GABAergic synaptic terminals from rat midbrain exhibit functional P2X and dinucleotide receptors, able to induce GABA secretion (659). Coexistence of ionotropic nucleotidic and nicotinic receptors in isolated synaptic terminals from midbrain has been demonstrated (455). GABAB receptor-mediated presynaptic potentiation of P2X receptor-mediated responses in rat midbrain synaptosomes has been reported (659). With the use of these synaptosomal preparations, it was also shown that aminergic nerve terminals possess modulatory presynaptic nucleotide and dinucleotide receptors (649). P2X3 receptors have been identified on rat midbrain presynaptic terminals (455). P2X7 receptors have been identified immunohistochemically and by Ca2+ imaging on midbrain synaptosomes and on axodendritic prolongations of cerebellar granule cells (1156). ATP and the diadenosine polyphosphate Ap5A induce concentration-dependent glutamate release from synaptosomal populations, which was inhibited by PPADS and Ip5I, respectively (689). Ap4A was shown to be active on rat midbrain synaptosomal preparations, probably acting via P2X1 or P2X3 receptor subtypes or possibly by another unidentified P2X subtype (1356). Subtypes P2X1–6 were detected in the periaqueductal gray area of the midbrain (1887).
Extracellular ATP increased cytosolic Ca2+ concentration on ventral tegmental neurons of rat brain (1611). Stimulation of P2Y1 receptors in the ventral tegmental area enhances dopaminergic mechanisms in vivo (967).
Adenosine mediated presynaptic modulation of glutamatergic transmission in the laterodorsal tegmentum (62). An inhibitory GABAergic feedback projection to the ventral tegmental area is stimulated by adenosine either directly or indirectly via glutamate release (968).
Adenosine promotes burst activity in guinea pig geniculocortical neurons (1305). Adenosine can downregulate inhibitory postsynaptic responses in thalamus and exert antioscillatory effects (1756). Adenosine inhibits synaptic release of GABA and glutamate by stimulation of presynaptic A1 receptors in the subthalamic nucleus (1550).
P2X receptors have been localized in thalamus using α,β-[3H]meATP binding (154), and P2Y receptors have also been described in thalamic neurons (1157). Nociceptive activity was elicited by electrical stimulation of afferent C-fibers in the sural nerve and recorded from single neurons in the rat ventrobasal complex of the thalamus; the P2 receptor antagonist Reactive red, administered intrathecally, produced significant reduction of the evoked activity in thalamic neurons (479).
The first clear demonstration of ATP receptor-mediated fast synaptic currents in the CNS was described in the rat medial habenula (506). These synaptic currents were mimicked by ATP and reversibly blocked by suramin and by α,β-meATP desensitization. The evidence was extended by demonstration of ATP release from an isolated rat habenula preparation during electrical field stimulation (1624, 1796). This group later showed that the projections from the triangular septal and septofimbrial nucleus to the habenula are the major source of ATP in the rat habenula and utilize ATP as a fast transmitter, probably with glutamate as a cotransmitter (1626). It was concluded by another group that the P2X receptor-mediated synaptic currents were the only calcium-permeable fast transmitter-gated currents in these neurons (1430). LTP of glutamatergic synaptic transmission induced by activation of presynaptic P2Y receptors in the rat medial habenula nucleus has also been claimed (1379).
H. Behavioral Studies
While the involvement of purinergic signaling in neurotransmission and neuromodulation in the CNS is now well established, there are relatively few studies of the involvement of purinergic signaling in behavioral pathways, apart from brain stem control of autonomic functions (see sect. vii), although behavioral changes have been reported in pathological situations (see sect. xiB5).
1. Learning and memory
ATP and adenosine are involved in mechanisms of synaptic plasticity and memory formation (410, 1859). ATP coreleased with glutamate induces LTP in CA1 neurons associated with learning and memory (592, 1182, 1929; see also sect. viB). 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 (171). 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 (1300). 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 LTP and long-lasting changes in synaptic efficacy. Mice lacking the P2X3 receptor exhibit abnormalities in hippocampal synaptic plasticity, but not in special learning (1832).
Large rises in [Ca2+]i in CA1 neurons induce LTP, but small rises induce LTD (1919). 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 (893).
It has been shown that ATP analogs can facilitate LTP through P2 receptor activation that triggers adenosine release, leading to activation of P1(A2A) receptors (37), which are claimed to be involved in modulating spatial recognition memory in mice (1823). Activation of adenosine receptors in the posterior cingulate cortex impairs memory retrieval in the rat (1333).
Tetanus-induced heterosynaptic depression in the hippocampus is a key cellular mechanism in neural networks implicated in learning and memory. ATP release from glial cells, degradation to adenosine, and activation of A1 receptors on Schaffer collaterals appear to underlie heterosynaptic depression (1542). Mice lacking the A1 receptor have normal spatial learning and plasticity, but they habituate more slowly (644). LTP is impaired in middle-aged rats and provides a possible explanation for memory losses during normal aging and indicate that, with regard to plasticity, different segments of pyramidal neurons age at different rates (1416).
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 (1874).
2. Sleep and arousal
The hypnotic/sedative (somnogenic) actions of adenosine are well known as are the central stimulant actions of methylxanthine antagonists (see Refs. 114, 500, 538). Adenosine, acting through A1 receptors, is an endogenous, homeostatic sleep factor, mediating the sleepiness that follows prolonged wakefulness. Perfusion of antisense oligonucleotide to the A1 receptor in the basal forebrain of the rat confirmed the role of A1 receptors in promoting sleep (1697), and cyclopentyl-l,3-dimethylxanthine, an A1 specific antagonist, in wild-type mice inhibited rebound sleep (1638). Sleep deprivation induces an increase in A1 receptor mRNA in basal forebrain (114). The effect of sleep deprivation on the “righting reflex” in the rat is partially reversed by administration of A1 and A2 receptor antagonists (1736). Old rats have higher extracellular levels of adenosine compared with young rats across the 24-h diurnal sleep cycle, but a reduction and sensitivity of the adenosine receptors may be a contributing factor to the decline in sleep drive in the elderly (1196).
The basal forebrain as well as neurons in the cholinergic laterodorsal tegmental nuclei are essential areas for mediating the sleep-inducing effects of adenosine by inhibition of wake-promoting neurons (60). It has been suggested that adenosine may promote sleep by blocking inhibitory inputs on ventrolateral preoptic area sleep-active neurons (1177). A2A receptors in the subarachnoid space below the rostral forebrain, activating cells in the nucleus accumbens that increase activity of ventrolateral preoptic area neurons, may also play a role in the somnogenic effect of adenosine (1510). The sleep-promoting process induced by the A2A receptor agonist CGS21680 was associated with a decline in the activity of orexin neurons (1503). Direct administration of adenosine into the brain elicits an EEG profile indicative of deep sleep, i.e., an increase in rapid-eye-movement (REM) sleep with a reduction in REM sleep latency, resulting in an increase in total sleep (312). In vivo microdialysis measurements in freely behaving cats showed that adenosine extracellular concentrations in the basal forebrain cholinergic region increased during spontaneous wakefulness and during prolonged wakefulness and declined slowly during recovery sleep (1369). It has been suggested that diurnal and age-related variation of the activity of ecto-5′-nucleotidase in the basal forebrain may underlie the role that adenosine plays in promoting sleep and allowing wakefulness (1089). A functional genetic variation of adenosine deaminase affects the duration and intensity of deep sleep in humans (1415). Adenosine and caffeine modulate circadian rhythms in the Syrian hamster (49), and A1 receptors regulate the response of the hamster and mouse circadian clock to light (1567).
P2X2 receptor mRNA and protein are expressed by all hypothalamic hypocretin/orexin neurons and might therefore be involved in the regulation of the functions of orexin associated with arousal and wakefulness (564, 1882). A recent study has identified P2Y1 and P2Y4 receptors on histaminergic neurons located on the tuberomamillary nucleus of the posterior hypothalamus that mediate increase firing (1541). These neurons are tonically active during wakefulness, but cease firing during sleep; the authors suggest that excitation of the wake-active tuberomamillary neurons by nucleotides and the lack of adenosine action may be an important factor in sleep-wake regulation.
The central inhibitory effects of adenosine on spontaneous locomotor activity of rodents and antagonism by caffeine have been known for some time (e.g., Refs. 105, 1598). Later A2A receptors on the nucleus accumbens were shown to mediate locomotor depression (107). Modulation of striatal A1 and A2 receptor-mediated activity induces rotational behavior in response to dopaminergic stimulation in intact rats (1367). Interactions between adenosine and L-type Ca2+ channels in the locomotor activity of rat was demonstrated (525). A predominant role for A1 receptors in the motor-activity effects of acutely administered caffeine in rats has been reported (50). A combination of A1 and A2A receptor blocking agents induces caffeine-like spontaneous locomotor activity in mice (984).
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 (210). P2X2 receptor immunoreactivity in the cerebellum was demonstrated and claimed to be consistent with a role for extracellular ATP acting as a fast transmitter in motor learning and coordination of movement (865). Administration of the P2 receptor agonist 2-MeSATP into the nucleus accumbens of rats raises the extracellular level of dopamine and enhances locomotion (928, 1610). Enhanced motor activity is also produced by the psychostimulant amphetamine, but the P2 receptor antagonist PPADS blocks these motor effects (930). Adult rats trained in a step-down inhibitory avoidance task or submitted to isolated foot-shock showed increased ATP hydrolysis in synaptosomes prepared from the cingulate cortex, suggesting that the ectonucleotidase pathway may be involved in memory consolidation of step-down inhibitory avoidance in the cortex (1332). Inhibitory avoidance training led to decreased ATP diphosphohydrolase activity in hippocampal synaptosomes, suggesting involvement of this enzyme in the formation of inhibitory avoidance memory (169). Intrahippocampal infusion of suramin, acting either by blocking purinergic neurotransmission or as an inhibitor of ATP degradation, modulated inhibitory avoidance learning in rats (170). The inhibitory avoidance task is associated with a decrease in hippocampal nucleotidase activities in adult male, but not female, rats (1458).
ATP is released during swimming of frog embryos, to activate P2Y receptors and produce an increase in excitability of the spinal motor circuits; while adenosine, produced following the breakdown of ATP, lowers the excitability of the motor circuits (422). It was suggested that a gradually changing balance between the actions of ATP and adenosine underlies the rundown of the motor pattern for swimming in Xenopus.
Adenosine given centrally can result in a decrease in food intake (1024). In a later paper this group showed that the adenosine agonist N6-R-phenylisopropyladenosine (R-PIA) stimulated feeding in rats; this effect was not blocked by caffeine, but the opioid antagonist naloxone did block R-PIA-induced eating (1023).
In the striatum, extracellular ATP and adenosine are involved in the regulation of the feeding-associated mesolimbic neuronal activity in an antagonistic manner (929). The ATP-induced increase in cytosolic Ca2+ concentration (1610) and feeding-evoked dopamine release (967) have been demonstrated in the rat nucleus accumbens. 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 (928). It has been reported that feeding behavior relies on tonic activation of A2A receptors in the nucleus accumbens in rats (1200). NTPDase3 and 5′-ectonucleotidase regulate the levels of adenosine involved in feeding behavior in rat brain (126). Enhanced food intake after stimulation of hypothalamic P2Y1 receptors in rats has been described recently (927). Expression of P2Y1 receptors in the hypothalamus of the rat is enhanced by reduced food availability (1535).
Neonatal rat handling, a brief separation from the mother in the neonatal period, can lead to increased sweet food consumption in adulthood; this appears to be associated with decreased hydrolysis of AMP in the nucleas accumbens and P1 receptor-mediated modulation of dopamine neurotransmission (1574).
5. Mood and motivation
Adenosine has been reported to interact with the psychotomimetic phencyclidine and with alcohol, both agents being potent mood regulators (see Ref. 1867; see also sect. xE2). Striatal A2A receptors appear to be an important mediator of the molecular and behavioral sequelae following administration of the antipsychotic drug haloperidol (1834). There is selective attenuation of psychostimulant-induced behavioral responses in mice lacking A2A receptors (339). Caffeine, a P1 receptor antagonist, has been considered as the most widely used psychologically active drug of benefit for many psychomotor variables, including choice reaction time, mood state, and sensory vigilance (see Refs. 579, 847). Evidence was presented that purinergic stimulation via inosine and hypoxanthine can produce an anxiety response that is related to the benzodiazepine receptor (1814). Mice lacking the A1 receptor showed signs of increased anxiety (839). Stimulation of P2Y1 receptors causes anxiolytic-like effects, which appear to involve P2Y1 receptor-mediated NO production (926). An antidepressant effect of adenosine has been reported in mice, apparently involving A1 and A2A receptors (875; see sect. xiB5). The inhibitory action of dilazep on clonidine-induced aggressive behavior was claimed to be substantially attributed to central purinoceptor stimulation (1761). Suramin blocked the conditioned fear response in a rat model, suggesting that P2 receptors might be involved in fear behavior (1986). A1 receptor activation selectively impairs the acquisition of fear conditioning in rats (386). Reduced adenosinergic activity, mostly at A1 receptors, is associated with the complex network of changes in neurotransmitter pathways related to manic behavior (1088). An A2A receptor genetic polymorphism has been implicated in “panic disorder.”
P2 receptors of the mesolimbic-mesocortical system, probably of the P2Y1 subtype, are involved in the release of transmitters such as dopamine and glutamate, which are responsible for the generation and pattern of the behavioral outcome after motivation-related stimuli (970). Antagonism of A2A receptors by KW-6002 given systemically enhances motor and motivational responses in the rat (1266). Evidence from A2A receptor knockout mice suggests that A2A receptors are involved in goal-directed behavior (1564).
VII. CENTRAL CONTROL OF AUTONOMIC FUNCTION
Research into the central control of autonomic function by purinergic signaling has attracted particular attention in recent years. Furthermore, since adenosine, rather than ATP, was considered for many years to be the principal receptor present in the brain, the early papers concerned with central autonomic control were largely concerned with the effects of adenosine on P1 receptors, but there are more recent studies of the involvement of P2 receptors (664, 1605, 1926).
While the main areas of the brain concerned with control of autonomic function are the brain stem and hypothalamus, the prefrontal cortex is implicated in the integration of sensory, limbic, and autonomic information (see Ref. 679). However, no studies into the possible involvement of purinergic signaling by the prefrontal cortex appear to have been undertaken yet.
A. Ventrolateral Medulla
The ventrolateral medulla (VLM) contains a network of respiratory neurons that are responsible for the generation and shaping of respiratory rhythm; it also functions as a chemoreceptive area mediating the ventilating response to hypercapnia. Evidence has been presented that ATP acting on P2X2 receptors expressed in VLM neurons influences these functions (665).
Iontophoretic application of ATP excited the spinal cord-projection neurons in the rostral VLM (RVLM) of the medulla oblongata causing powerful vasopressor actions, a response that was mimicked and then blocked by α,β-meATP as well as by suramin (1668). A further study suggested that two P2X receptor subtypes might be present in RVLM neurons, one sensitive to both ATP and α,β-meATP and the other sensitive to ATP, but not to α,β-meATP (1393). Activation of P2X receptors in the VLM was shown to be capable of producing marked excitation of both sympathoexcitatory and sympathoinhibitory neurons (761). However, P2Y as well as P2X receptors appear to be involved in neural activity in the RVLM (1393). Evidence was presented to suggest that CO2-evoked changes in respiration are mediated, at least in part, by P2X receptors in the retrofacial area of the VLM (1705). CO2-P2X-mediated actions were observed only in inspiratory neurons that have purinoceptors with pH sensitivity (characteristic of the P2X2 receptor subtype) that could account for the actions of CO2 in modifying ventilatory activity. Not surprisingly, adenosine was shown to be a neuromodulator in the RLVM (1704). It has been shown that a high percentage of NOS-immunoreactive neurons in the RVLM and supraoptic nucleus (SON) of the hypothalamus are also P2X2 receptor immunoreactive (1927). During hypoxia, release of ATP in the VLM plays an important role in the hypoxic ventilatory response in rats (664, 667, 668). It was suggested that during sustained hypoxia, the central respiratory drive may partially depend on a balance between the excitatory action of ATP and the inhibiting action of adenosine in the ventral respiratory column. It was proposed that modulation of respiratory output induced by adenosine was due both to a decrease in synaptic transmission in respiration-related neurons via presynaptic A1 receptors and inactivation, via membrane hyperpolarization of medullary expiratory neurons by postsynaptic A1 receptors (735). A2A receptor binding studies showed localization in several brain stem regions, including RVLM, nucleus tractus solitarius (NTS), and dorsal vagal motor neurons, involved in autonomic function, consistent with the idea that adenosine acts as a neuromodulator of a variety of cardiorespiratory reflexes (1706). P1(A2A) receptors that are expressed by GABAergic neurons in the ventral and ventrolateral medulla are likely to play a role in mediating adenosine-induced respiratory depression (1946).
Intrathecal application of P2X receptor agonists and antagonists indicate that P2X3 or P2X2/3 receptors on the trigeminal primary afferent terminals in the medullary dorsal horn (trigeminal subnucleus caudalis) enhance trigeminal nociceptive transmission (356, 769), perhaps by increasing glutaminergic neurotransmission (831).
B. Trigeminal Mesencephalic Nucleus
Although the trigeminal mesencephalic nucleus (MNV) is located in the CNS, it contains cell bodies of primary afferent neurons that relay proprioceptive information exclusively. The MNV is known to contain mRNA for P2X2, P2X4, P2X5, and P2X6 subtypes (378, 864). With in situ hybridization studies, higher levels of mRNA for P2X5 were found in this nucleus than in any other brain area (230). ATP-gated ion channels (P2X receptors) were described in rat trigeminal MNV proprioceptive neurons from whole cell and outside-out patch-clamp recording (895, 1318), possibly mediated by P2X5 receptor homomultimers and P2X2/5 heteromultimers (1318). It has been suggested that in the MNV there is ATP receptor-mediated enhancement of fast excitatory glutamate release onto trigeminal mesencephalic motor nucleus neurons (894).
C. Area Postrema
Injection of adenosine into the area postrema produced decreased heart rate and systolic and diastolic blood pressure. Dense areas of P2X2 receptor immunoreactivity were demonstrated in the rat area postrema (69).
D. Locus Coeruleus
There were early reports of modulation of neurons in the locus coeruleus (LC) by adenosine (1548, 1689). The first report of the action (depolarization) of ATP on P2 receptors in LC was by Harms et al. (710), and later papers examined the ionic mechanism and receptor characterization of these responses (794, 1115, 1551). α,β-MeATP and α,β-methylene ADP (α,β-meADP) were shown to increase the firing rate of rat LC neurons (1497). Both P2X and P2Y receptors are present on LC neurons (585, 1497). Intracellular recordings from slices of rat LC led to the suggestion that ATP may be released either as the sole transmitter from purinergic neurons terminating in the LC or as a cotransmitter with NE from recurrent axon collaterals or dendrites of the LC neurons themselves (1243), the latter proposal being supported experimentally in a later paper (1360). Microinjection of ATP or α,β-meATP into LC (and periaqueductal gray matter) led to changes in bladder function and arterial blood pressure (1436). Purinergic modulation of cardiovascular function in the rat LC involves a functional interaction between tonically active purinergic and noradrenergic systems (1928).
E. Nucleus Tractus Solitarius
The NTS (particularly neurons in the caudal NTS) is a central relay station for viscerosensory information to respiratory, cardiovascular, and digestive neuronal networks. Extracellular purines have been claimed to be the primary mediators signaling emergency changes in the internal environment in the CNS. NTS is a major integrative center in the brain stem 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 (923). Injection of adenosine into the NTS produced dose-related decreases in heart rate and systolic and diastolic blood pressures (108, 1729). NTS A2A receptor activation elicits hindlimb vasodilatation (925). ATP and its slowly degradable analog, β,γ-meATP, also produced dose-related potent vasodepressor and bradycardic effects, suggesting that P2 as well as P1 receptors were involved, although P1 receptor antagonists substantially reduced the cardiovascular effects of both ATP and adenosine. The effects of adenosine were shown to be due to its neuromodulatory actions (1187, 1706). Evidence has been presented implicating an interaction between NO and adenosine in NTS cardiovascular regulation (1063). Stimulation of A2A receptors in NTS decreases mean arterial pressure, heart rate, and renal sympathetic nerve activity (1530). A1 and A2A receptors have counteracting effects on hindlimb vasculature (1128).
Patch-clamp studies of neurons dissociated from rat NTS revealed P2 receptor-mediated responses (1749) and microinjection of P2 receptor agonists into the subpostremal NTS in anesthetized rats produced reduction of arterial blood pressure (523) probably via a P2X1 or P2X3 receptor subtype, since α,β-meATP was particularly potent. It was suggested that the actions of ATP and adenosine in the NTS may be functionally linked to selectively coordinate the regulation of regional vasomotor tone (108).
Purines applied in the NTS have been shown to affect baroreceptor and cardiorespiratory functions (1348, 1530). Microinjections into the caudal NTS of anesthetized spontaneously breathing cats showed α,β-meATP elicited a distinct pattern of cardiorespiratory response, namely, dose-related decrease in tidal volume and respiratory minute volume; at higher doses a pronounced apnea was produced (106). This suggested that a P2X receptor was present, perhaps involved in the processing of sensation from pulmonary receptors related to the Breuer-Hering and pulmonary C-fiber reflexes. Impaired arterial baroreflex regulation of heart rate after blockade of P2 receptors in the NTS has been reported (1530). Microinjection of ATP into caudal NTS of awake rats produces respiratory responses, but not the sympathoexcitatory component of the chemoreflex (51). It has been suggested that caudal commissural NTS P2 receptors play a role in the neurotransmission of the parasympathetic (bradycardic) component of the chemoreceptor reflex (1320).
Activation of P2X and P1(A2A) receptors in the NTS elicits differential control of lumbar and renal sympathetic nerve activity, and in a later paper from this group, it was concluded that the fast responses to stimulation of NTS P2X receptors were mediated via glutamatergic ionotropic mechanisms, whereas the slow responses to stimulation of NTS P2X and A2A receptors were not (1530).
The immunohistochemical distribution of P2X receptor subtypes in the NTS of the rat has been described (1925). Colocalization of P2X2 and P2X3 immunoreactivity has been described in the NTS (1811). At the electron microscope level, P2X3 receptor-positive boutons have been shown to synapse on dendrites and cell bodies and have complex synaptic relationships with other axon terminals and dendrites (1062). P2X2 receptors have been localized presynaptically in vagal afferent fibers in rat NTS (70). A whole cell patch-clamp study of neurons in the caudal NTS led to the conclusion that ATP activates 1) presynaptic P1(A1) receptors after breakdown to adenosine, reducing evoked release of glutamate from the primary afferent nerve terminals, and 2) presynaptic P2X receptors on the axon terminals of intrinsic excitatory caudal NTS neurons, facilitating spontaneous release of glutamate (880).
Purinergic and vanilloid receptor activation releases glutamate from separate cranial afferent terminals in the NTS corresponding to myelinated and unmyelinated pathways in the NTS; ATP probably activates P2X3 receptors on vagal afferents (834).
It has been shown that microinjection of ATP into NTS of awake rats produced pressor and bradycardic responses by independent mechanisms: activation of the parasympathetic bradycardic component appears to involve interaction of P2 and excitatory amino acid receptors, whereas the pressor response was not affected by blockade of receptors to ATP or adenosine (437). In a later study by this group with awake rats, they showed that microinjection of ATP into different subregions of the NTS produces a diverse pattern of hemodynamic and respiratory responses (52). With low doses of P2X receptor agonist into the NTS, bradycardia is mediated via sympathetic withdrawal, whereas at high doses, both sympathetic and parasympathetic components contribute similarly to bradycardia; only the sympathetic component of bradycardia contributes significantly to the hypotension induced by NTS P2X receptor stimulation (924).
Evidence has been presented that the major mechanism of the NTS network excitation by ATP is by way of triggering Ca2+-dependent exocytosis of glutamate, but not that of GABA, by Ca2+ entry through presynaptic P2 receptor activation (1555).
F. Motor and Sensory Nuclei
P1(A1) adenosine receptor agonists presynaptically inhibit both GABAergic and glutamatergic synaptic transmission in periaqueductal gray neurons (77). Adenosine suppresses excitatory glutamatergic inputs to rat hypoglossal motoneurons (128).
The mRNA for P2X4 and P2X6 receptors as well as three P2X2 receptor subunit isoforms has been identified in the hypoglossal nucleus, and this was taken to indicate modulation of inspiratory hypoglossal activity and perhaps a general role in modulatory motor outflow in the CNS (378, 600). A potentially important role for P2 receptor synaptic signaling in respiratory motor control is suggested by the multiple physiological effects of ATP in hypoglossal activity associated with the presence of P2X2, P2X4, and P2X6 receptor mRNA in nucleus ambiguous and the hypoglossal nucleus (378, 600). Presynaptic P2X7-like receptors mediate enhanced excitatory synaptic transmission to hypoglossal motor neurons (808).
Evidence for multiple P2X and P2Y subtypes in the rat medial vestibular nucleus has been presented (352). A P2Y receptor subtype activated by ADP was identified in medulla neurons isolated from neonatal rat brain (791), perhaps, with hindsight, P2Y1, P2Y12, or P2Y13 receptors.
The actions of ATP and ACh were examined with patch-clamp recording on dissociated preganglionic neurons in the dorsal motor nucleus of the vagus (DMV); the results suggested that these neurons functionally colocalized nicotinic and P2X receptors (1198). Over 90% of the preganglionic neurons in this nucleus respond to ATP, and RT-PCR showed mRNA in the DMV encoding P2X2 and P2X4 receptors; it was suggested that the functional receptors expressed in DMV neurons are characterized mainly by P2X2 and P2X2/6 subtypes (1752). Dense areas of P2X2 receptor immunoreactivity were described on the dorsal vagal complex (69). An electromicroscope immunocytochemical study has shown P2X4 receptors expressed by both neurons and glia in the rat dorsal vagal complex (67). The complex effects of ATP on respiratory phrenic motor neuron output, in conjunction with the rich expression of ATP receptors on phrenic motor neurons, suggest that purinergic signaling plays an important role in controlling motor outflow from the CNS (1151).
P2X receptors are expressed in the medial nucleus of the trapezoid body of the auditory brain stem where they act to facilitate transmitter release in the superior olivary complex (1838). Although ATP potentiates release at both excitatory and inhibitory synapses, it does so via different P2X receptor subtypes expressed at different locations: P2X3 receptors on cell bodies or axons of excitatory pathways and P2X1 receptors on the presynaptic terminals of inhibitory pathways. A1 rather than P2X receptors have been implicated during high-frequency glutamatergic synaptic transmission in the calyx of Held (1883). P2 receptors modulate excitability, but do not mediate pH sensitivity of respiratory chemoreceptors in the retrotrapezoid nucleus on the ventral surface of the brain stem (1190).
ATP and α,β-meATP excite neurosecretory vasopressin cells in the SON, an effect blocked by suramin (430). This was claimed to be the first demonstration of a specific physiological role for central purinergic signaling, i.e., regulation of secretion of the neurohormone vasopressin. Suramin did not block the excitatory effect of locally applied NE on vasopressin cells, but did block excitation produced by vagus nerve stimulation. The magnocellular neurons of the supraoptic and paraventricular nuclei receive a dense plexus of fibers originating from noradrenergic neurons in the VLM. Although NE-containing neurons of the caudal medulla provide a direct excitatory input to supraoptic vasopressin cells, they do not use NE as their primary transmitter; ATP was shown to be acting as a cotransmitter with NE in these neurons (235). Corelease of ATP and NE from superfused rat hypothalamic slices was also demonstrated by Sperlágh et al. (1626), although it was questioned whether they were released from the same nerve endings. However, further support for cotransmitter release of ATP with NE at synapses in the hypothalamus comes from evidence that purinergic and adrenergic agonists synergize when stimulating vasopressin and oxytocin release (867). Candidates for coreleased transmitters in NE-containing neurons include SP and NPY as well as ATP, and it has been proposed that SP and NPY differentially potentiate ATP- and NE-stimulated vasopressin and oxytocin release (868). Purinergic and GABAergic cotransmission has also been claimed in the lateral hypothalamus of the chick embryo (835) with cholinergic modulation of the cotransmitter release (836). Evidence has been presented that ATP may be released from magnocellular neurons (1727).
A study of the effects of ATP in increasing [Ca2+]i in cultured rat hypothalamic neurons was taken to support the action of ATP as an excitatory neurotransmitter (345). Excitatory effects of ATP via P2X receptors in acutely dissociated ventromedial hypothalamic neurons have also been described (1609). A role for adenosine A1 receptors in mediating cardiovascular changes evoked during stimulation of the hypothalamic defense area has been postulated (429). Application of ATP and UTP (but not adenosine) produced TTX-insensitive depolarizations accompanied by increases in input conductance in supraoptic magnocellular neurosecretory cells; both P2X and P2Y receptors have been suggested to be involved (744).
Ultrastructural localization of both P2X2 (1067) and P2X6 (1066) receptor immunoreactivity at both pre- and postsynaptic sites in the rat hypothalamo-neurohypophysial system has been described. Purinergic regulation of stimulus-secretion coupling in the neurohypophysis has been reviewed (1726). From a study of the expression of P2X receptor subtypes in the SON using RT-PCR, in situ hybridization, Ca2+ imaging, and whole cell patch-clamp techniques, it was concluded that P2X3 and P2X4 receptors were predominant, but that P2X7 receptors were also present (1552). A recent study has shown that P2X5 receptors are expressed on neurons containing vasopressin and NOS in the rat hypothalamus (1905).
It has been suggested that ATP, cosecreted with vasopressin and oxytocin, may play a key role in the regulation of stimulus-secretion coupling in the neurohypophysis (1625) by acting through P2X2 receptors increasing arginine vasopressin (AVP) release, and after breakdown to adenosine, acting via P1(A1) receptors (inhibiting N-type Ca2+ channels) to decrease neuropeptide release (1821). Adenosine was also shown to modulate activity in supraoptic neurons by inhibiting N-type Ca2+ channels via A1 receptors (1254). Evidence for the involvement of purinergic signaling in hypothalamus and brain stem nuclei in body temperature regulation has been presented (669).
Early studies of the roles of adenosine in the hypothalamus have been reviewed (272). Presynaptic P1 receptors mediate inhibition of GABA release in suprachiasmatic and arcuate nucleus neurons. Adenosine-induced presynaptic inhibition of inhibitory postsynaptic currents (IPSCs) and EPSCs in rat hypothalamus SON neurons via A1 receptors indicates inhibition of release of both GABA and glutamate. Release of 3H-labeled nucleosides from [3H]adenine-labeled hypothalamic synaptosomes was first described in 1979. Adenosine deaminase-containing neurons in the posterior hypothalamus innervate mesencephalic primary sensory neurons, perhaps indicating purinergic control of jaw movements.
A hypothalamic role has been suggested for extracellular ATP to facilitate copper uptake and copper stimulation of the release of luteinizing hormone-releasing hormone (LHRH) from medium eminence, via an interaction with purinergic receptors (104). The hypothalamic suprachiasmatic nucleus (SCN) is regarded as the site of the endogenous biological clock controlling mammalian circadian rhythms. Long-term communication between glial cells, reflected by waves of fluorescence indicating Ca2+ movements, probably via gap junctions, can be induced by ATP, as well as by glutamate and 5-HT (1767). ATP releases LHRH from isolated hypothalamic granules (297).
ATP injected into the paraventricular nucleus stimulates release of AVP resulting in antidiuretic action through renal AVP (V2) receptors, and in a later study this group showed that ATP (but not ADP, AMP, or adenosine) injected into the SON also decreased urine outflow (1183). Stimulation of the hypothalamic defense area produces autonomic responses that include papillary dilatation, piloerection, tachypnea, tachycardia, and a marked pressor response.
LHRH is released from the hypothalamus in pulses at hourly intervals, which is essential for the maintenance of normal reproductive function. Studies of an in vivo culture preparation of LHRH neurons show that ATP stimulates LHRH release, probably via P2X2 and P2X4 receptor subtypes, and may be involved in synchronization of the Ca2+ oscillations that appear to underlie the pulsatile release of LHRH (1694). The authors also speculate that glial cells expressing P2Y1 and P2Y2 receptors may also participate in this process.
ATP-stimulated vasopressin release from hypothalamo-neurohypophyseal explants is terminated partly by P2 receptor desensitization and partly by ectoenzyme degradation of ATP to adenosine (1604). ATP and the α1-adrenoceptor agonist phenylephrine evoke synergistic stimulation of vasopressin and oxytocin release from the hypothalamo-neurohypophysial systems, and the authors speculate that this allows for a sustained elevation of vasopressin release in response to extended stimuli such as severe hemorrhage, chronic hypotension, or congestive heart failure (1605).
P2X1–6 receptor subunits are present on paraventricular nucleus neurons projecting to the RVLM in the rat, suggesting a role for ATP on the paraventricular nucleus in the regulation of sympathetic nerve activity (327).
H. Spinal Cord
Spinal circuits, spinal afferent influx, and descending influences from brain stem and hypothalamus work together in the integrative activities of the preganglionic sympathetic neurons, which regulate the activity on many organs (824).
There was early identification of dense areas of acid phosphatase and 5′-nucleotidase activity in the substantia gelatinosa of the spinal cords of rats and mice, and the possible implication for purinergic transmission was raised (1672). P1 (A1 and A2) receptors on neurons in the dorsal and ventral spinal cord mediate modulation of neuronal activity by adenosine (358, 628, 1330). Adenosine reduces glutamate release from rat spinal synaptosomes (1040).
Excitation of dorsal horn neurons by ATP was also recognized early (see sect. iiC) and described in more detail later (1039, 1410, 1489). ATP-evoked increases in intracellular calcium were demonstrated in both neurons and glia of the dorsal spinal cord (1491). It was later shown that the ATP-evoked release of Ca2+ from astrocytes was via the PLC-β/IP3 pathway (1492), suggesting mediation via a P2Y receptor. It was proposed that ATP released in synaptic regions acts as a synaptic modulator by augmenting the actions of excitatory amino acids (1037). ATP was also shown to inhibit slow depolarization via P2Y receptors in substantia gelatinosa neurons (1939). Properties of P2Y receptors in Xenopus spinal neurons related to motor pattern generation have been reported (220). A recent study has identified P2Y1 and P2Y4 receptor mRNA in subpopulations of dorsal horn neurons, whereas motor neurons in the ventral horn expressed P2Y4 and P2Y6 receptor mRNA (939). In addition, astrocytes in the gray matter expressed P2Y1 receptor mRNA and microglia throughout the spinal cord expressed P2Y12 receptor mRNA.
mRNA for P2X2, P2X4, and P2X6 receptors have been identified within spinal motor nuclei (378). P2X3 immunoreactivity is apparent on the axon terminals of DRG neurons that extend across the entire mediolateral extent of inner lamina II of the dorsal horn (188, 686, 1208). The immunolabeled nerve profiles in lamina II for P2X3 receptors are located largely on terminals with ultrastructural characteristics of sensory afferent terminals (1062). In contrast, although P2X2 immunoreactivity is most prominent in lamina II, it is also seen in deeper layers, and only rarely overlaps with P2X3 immunoreactivity (1811). Autoradiography of α,β-[3H]meATP showed strong binding in medulla oblongata and spinal cord of the rat (1743). At central terminals of primary afferent neurons, ATP has been shown to act either presynaptically facilitating glutamate release (687, 1039, 1208) or postsynaptically (102, 608, 1037). ATP facilitates spontaneous glycinergic IPSCs in neurons from rat substantia gelatinosa mechanically dissociated from the dorsal horn (1418), and P2X receptors are also expressed on glycinergic presynaptic nerve terminals (823). Distinct subtypes of P2X receptors have been shown to be functionally expressed at pre- and postsynaptic sites in lamina V neurons in rat dorsal spinal cord, and it was suggested that purinergic signaling in deep dorsal horn neurons becomes more important during postnatal development (1561; see sect. xA1).
ATP has been shown to be released from dorsal and ventral spinal cord synaptosomes (1506, 1856). Morphine and capsaicin release purines from capsaicin-sensitive primary afferent nerve terminals in the spinal cord (1676).
Within the spinal cord, P2X receptors are present in a subpopulation of dorsal horn neurons (102). ATP is coreleased with GABA (837). In addition to acting as a fast excitatory synaptic transmitter, ATP facilitates excitatory transmission by increasing glutamate release and enhances inhibitory neurotransmission mediated by both GABA and glycine (779, 1418). P2X3 receptors are involved in transient modulation of glutamate release in lamina II of the spinal cord, but a different P2X receptor subtype (perhaps P2X1/5 or P2X4/6) was involved in long-lasting modulation in lamina V (1211). The authors concluded that differential modulation of sensory inputs into different sensory regions by P2X receptor subtypes represents an important mechanism of sensory processing in the spinal cord dorsal horn. There is potentiation of inhibitory glycinergic neurotransmission by Zn2+ and a synergistic interplay between presynaptic P2X2 and postsynaptic glycine receptors (1003).
In the ventral horn, almost all large cholinergic COOH terminals contacting motoneurons (91%) show P2X7 receptor immunoreactivity, while only ∼32% of the motor axon terminals in the ventral horn are P2X7 receptor immunoreactive (449). This suggests that distinct populations of synapses involved in spinal cord motor control circuits may be differentially regulated by the activation of P2X7 receptors. Blockade of P2X receptors in the dorsal horn with PPADS attenuates the cardiovascular “exercise pressor reflex” to activation of muscle afferents, while stimulation of P2X receptors enhances the reflex response (619).
VIII. NEURON-GLIA INTERACTIONS
Purinergic signaling is emerging as a major means of integrating functional activity between neurons, glial, and vascular cells in the nervous system. These interactions mediate effects of neural activity in development and in association with neurodegeneration, myelination, inflammation, and cancer (see Refs. 4, 272, 552, 1226 and the recent Novartis Foundation Symposium 276 devoted to “Purinergic Signaling in Neuron-Glial Interactions,” 2006).
New findings from purinergic research began to converge with glial research as it became more widely appreciated that ATP was coreleased from synaptic vesicles and thus accessible to perisynaptic glia. Receptor expression and pharmacological studies revealed a broad range of purinergic receptors in all major classes of glia, including Schwann cells in the PNS, oligodendrocytes, astrocytes, and microglia in the CNS. This common currency for cell-cell communication opened the possibility of an intercellular signaling system that could unite glia and neurons functionally.
ATP release from axons, dendrites, cell bodies of neurons, and from glia, by membrane channels and vesicular exocytosis, further expands the potential functional significance of purinergic signaling in the brain. It has also been suggested recently that P2X7 receptor pores may directly mediate efflux of cytosolic ATP, glutamate, and GABA from glial cells in the CNS (483). In addition to its rapid neurotransmitter-like action in intracellular signaling for neurons and glia, it became evident that ATP could also act as a growth and trophic factor, altering the development of neurons (1158) and glia (1226) by regulating the two most important second messengers: cytoplasmic calcium and cAMP. Moreover, the release of ATP by neural impulse activity provides a mechanism linking functional activity in neural circuits to growth and differentiation of nervous system cells. How development is regulated by changes in expression of purinergic receptors and ectoenzymes controlling ATP availability is only beginning to be