I. Introduction

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Extracellular purines and pyrimidines have important and diverse effects on many biological processes including smooth muscle contraction, neurotransmission, exocrine and endocrine secretion, the immune response, inflammation, platelet aggregation, pain, and modulation of cardiac function. The concept of purines as extracellular signaling molecules was instigated by Drury and Szent-Györgyi in 1929, in a comprehensive report showing that adenosine and adenosine 5'-monophosphate (AMP), extracted from heart muscle, have pronounced biological effects, including heart block, arterial dilatation, lowering of blood pressure, and inhibition of intestinal contraction. Gillespie, in 1934, drew attention to the structure-activity relationships of adenine compounds, showing that deamination greatly reduces pharmacological activity, and that removal of the phosphates from the molecule influences not only potency, but also the type of response. Removal of phosphates was shown to increase the ability of adenine compounds to cause vasodilatation and hypotension, and ATP caused an increase in rabbit and cat blood pressure that was rarely or never observed with AMP or adenosine. Furthermore, ATP was shown to be more potent than AMP and adenosine in causing contraction of guinea-pig ileum and uterus (Gillespie, 1934). This was the first indication of different actions of adenosine and ATP and, by implication, the first indication of the existence of different purine receptors.

Early investigations into the effects of adenosine and ATP were made on a variety of tissues, but particularly the heart and vasculature (Gaddum and Holtz, 1933; Emmelin and Feldberg, 1948; Folkow, 1949; Green and Stoner, 1950). Initial studies on the effects of extracellular UTP also focused on its cardiovascular effects (Hashimoto et al., 1964; Boyd and Forrester, 1968; Urquilla, 1978; Sakai et al., 1979). Other early lines of purine research concerned the effects of purines on platelet aggregation (Born, 1962) and on mast cells (Cockcroft and Gomperts, 1980). Diverse responses to extracellular purines and pyrimidines have now been documented in a wide range of biological systems, from single cells to whole organisms, and include smooth muscle contraction, neurotransmission in the peripheral and central nervous system, exocrine and endocrine secretion, the immune response, inflammation, platelet aggregation, pain, and modulation of cardiac function (Burnstock and Kennedy, 1986; Gordon, 1986; Seifert and Schultz, 1989; Burnstock, 1990; Olsson and Pearson, 1990; Ralevic and Burnstock, 1991a; Jacobson et al., 1992b; Dubyak and el-Moatassim, 1993; Dalziel and Westfall, 1994; Fredholm, 1995; Burnstock and Wood, 1996; Ongini and Fredholm, 1996; Sebastiâo and Ribeiro, 1996).

Insight into the physiological roles of extracellular purines and pyrimidines comes from studies of their biological sources and the stimuli for their release. In this respect, an important line of research stemmed from studies showing that adenosine is released from the heart during hypoxia to play an important role in reactive hyperemia (Berne, 1963; Gerlach et al., 1963). The general hypothesis of coupling of purine release to metabolic demands via local regulation of blood flow has been applied to other tissues and includes the release of adenine nucleotides, particularly ATP, from skeletal muscle during contraction (Boyd and Forrester, 1968; Forrester and Lind, 1969).

Reports of ATP release from sensory nerves in the rabbit ear (Holton and Holton, 1953; Holton, 1959) were the first in a major line of research concerned with purines as neurotransmitters. ATP was detected in the venous perfusate following antidromic stimulation of the rabbit auricular nerve to elicit vasodilatation of the ear capillary bed, and both antidromic vasodilatation and vasodilatation to arterial infusion of ATP (but not that to other agents) were blocked by cholinesterase inhibitors (Holton and Holton, 1953; Holton, 1959). It is now known that ATP is an important neurotransmitter or cotransmitter and adenosine an important neuromodulator in both the peripheral and central nervous systems (see Stone, 1991; Burnstock, 1990; Edwards and Gibb, 1993; Fredholm, 1995). There are also hints that adenine dinucleotides may play neurotransmitter or neuromodulator roles in the central nervous system (Pintor and Miras-Portugal, 1995b).

Adrenal chromaffin granules (Cena and Rojas, 1990), platelets (Born and Kratzer, 1984; Gordon, 1986), mast cells (Osipchuk and Cahalan, 1992), erythrocytes (Forrester, 1990; Ellsworth et al., 1995), basophilic leukocytes (Osipchuk and Cahalan, 1992), cardiac myocytes (Forrester, 1990), fibroblasts (Grierson and Meldolesi, 1995b), and endothelial (Ralevic et al., 1991a, 1991c, 1995b; Bodin et al., 1992) and epithelial cells (Enomoto et al., 1994; Ferguson et al., 1997) are important sources of purines that can be released under physiological and pathophysiological conditions, which may act on the purine receptors associated with these or neighboring cells. Adenine dinucleotides are released from secretory ganules of thrombocytes, chromaffin cells and neurons, and may represent a novel class of signaling molecules (Hoyle, 1990; Ogilvie, 1992; Ogilvie et al., 1996). Not enough is known about the sources and release of pyrimidines, which limits our understanding of the role played by the widely distributed receptors that are activated by pyrimidines. However, steps toward resolving this are being made with the demonstration that UTP is released by physiologically relevant stimuli from cultured endothelial, epithelial, and astrocytoma cells (Enomoto et al., 1994; Saiag et al., 1995; Lazarowski et al., 1997a).

Purines and pyrimidines mediate their effects by interactions with distinct cell-surface receptors. Early pharmacological evidence for the existence of adenosine receptors has been provided by specific antagonism by methylxanthines of adenosine-mediated accumulation of adenosine 3',5'-cyclic monophosphate (cAMP) in rat brain slices (Sattin and Rall, 1970). "Purinergic" receptors were first formally recognized by Burnstock in 1978, when he proposed that these can be divided into two classes termed "P₁-purinoceptors", at which adenosine is the principal natural ligand, and "P₂-purinoceptors", recognizing ATP and ADP (Burnstock, 1978). This division was based on several criteria, namely the relative potencies of ATP, ADP, AMP, and adenosine, selective antagonism of the effects of adenosine by methylxanthines, activation of adenylate cyclase by adenosine, and stimulation of prostaglandin synthesis by ATP and ADP. This major division remains a fundamental part of purine receptor classification, although adenosine/P1 and P2 receptors are now characterized primarily according to their distinct molecular structures, supported by evidence of distinct effector systems, pharmacological profiles, and tissue distributions. In addition, receptors for pyrimidines are now included within the P2 receptor family (table 1) (Fredholm et al., 1994, 1996). Note that it has been recommended that "P1 receptor" and "P2 receptor" replace the "P₁/P₂-purinoceptor" terminology (Fredholm et al., 1996). The terms "adenosine receptor" and "P1 receptor" are synonymous.

Adenosine/P1 receptors are further divided into four subtypes, A₁, A_2A, A_2B, and A₃, on the basis of their distinct molecular structures and show distinct tissue distributions and pharmacological profiles. All couple to G proteins.

P2 receptors were shown to be ligand-gated cation channels (Benham and Tsien, 1987) or involved G protein activation (Dubyak, 1991), which formed the basis of their subdivision into two main groups termed P2X receptors (ligand-gated cation channels) and P2Y receptors (G protein-coupled receptors) (Abbracchio and Burnstock, 1994; Fredholm et al., 1994). Subtypes are defined according to the molecular structure of cloned mammalian P2 receptors, discriminated by different numerical subscripts (P2X_n or P2Y_n) (Burnstock and King, 1996; Fredholm et al., 1996). This forms the basis of a system of nomenclature that will replace the earlier subtype nomenclature (including P_2X, P_2Y, P_2U, P_2T, and P_2Z receptors) as correlations between cloned and endogenous receptors are established. P3, P4, and P2Y_Ap4A (or P_2D) receptors have been proposed, but evidence for their existence is based solely on the distinct pharmacological profiles exhibited by some biological tissues. As this is no longer tenable for the identification and subclassification of receptors, it remains to be determined, preferably by molecular studies, how these correlate with cloned P2 receptors, and therefore exactly how they will fit within a unifying system of purine and pyrimidine receptor nomenclature.

The main aim of this review is to present the characteristics of receptors for purines and pyrimidines within a framework whereby comparisons can be made between cloned and endogenous receptors. For the P2 receptor family this is in order to promote the conversion from a system of nomenclature that is rapidly losing its value, to a unifying system of classification based on structurally distinct cloned receptors. However, pharmacological characterization of endogenous P2 receptors is often equivocal, largely because of the current lack of selective agonists and antagonists and because of complications introduced by the common and widespread coexpression of different types of P2 receptors. Thus, it will become apparent in the present review that in assigning names to endogenous P2 receptors we have needed to strike a balance between caution (against overinterpretation) and anticipation of the direction in which this field is heading. Signal transduction mechanisms, pharmacological response profiles, receptor desensitization, tissue distribution, and biological effects of receptors for purines and pyrimidines are considered. Because ATP and ADP are rapidly degraded to adenosine, and because most cells and tissues coexpress P1 and P2 receptors, we also consider the interactions that may occur between receptors belonging to these two families. Although modulation of ecto-nucleotidase expression and activity is an important means by which to regulate purine receptor function, this aspect of purinergic signaling is not dealt with in detail in this article; the reader is referred to other reviews on the subject (Ziganshin et al., 1994a; Zimmerman, 1996).

	II. Adenosine/P1 Receptors

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The adenosine/P1 receptor family comprises A₁, A_2A, A_2B, and A₃ adenosine receptors, identified by convergent data from molecular, biochemical, and pharmacological studies (table 2). Receptors from each of these four distinct subtypes have been cloned from a variety of species and characterized following functional expression in mammalian cells or Xenopus oocytes (table 3). A₁ and A₂ receptors were described by Van Calker et al. in 1979, in studies showing that activation of these receptors by adenosine and its derivatives inhibited, via A₁, or stimulated, via A₂, adenylate cyclase activity in cultured mouse brain cells (Van Calker et al., 1979). The effects of adenosine could be antagonized by methylxanthines and the order of potency of adenosine analogs was different for the two receptors (Van Calker et al., 1979). Londos et al. (1980) independently drew similar conclusions using membrane preparations from rat adipocytes, hepatocytes, and Leydig tumor cells; the adenosine receptors coupled to inhibition of adenylate cyclase (those in adipocytes) they named R_i (corresponding to the A₁ subtype) and the adenosine receptors coupled to stimulation of adenylate cyclase (those in hepatocytes and Leydig cells) they termed R_a (synonymous with the A₂ subtype). This alternative system of nomenclature was based on "R" to designate the "ribose" moiety of the nucleoside and "i" and "a" to indicate inhibition and activation of adenylate cyclase respectively (Londos et al., 1980). The A₁/A₂ nomenclature is now used, which is fortunate because A₁ receptors act through a variety of transduction mechanisms in addition to adenylate cyclase. A_1a and A_1b receptors have been proposed (Gustafsson et al., 1990), but this subdivision of the A₁ receptor remains equivocal.

A₂ receptors are further subdivided into types A_2A and A_2B. The suggestion that A₂ receptors could be divided into two classes was originally based on the recognition that adenosine-mediated stimulation of adenylate cyclase in rat brain was effected via distinct high affinity binding sites (localized in striatal membranes) and low affinity binding sites (present throughout the brain) (Daly et al., 1983). This subdivision was supported in a study which compared the high affinity striatal A₂ binding site with a low-affinity A₂ binding site characterized in a human fibroblast cell line; the two sites were termed A_2A and A_2B, respectively (Bruns et al., 1986). Definitive evidence for the existence of these two subtypes comes from the cloning and sequencing of distinct A_2A and A_2B receptors which show distinct pharmacological profiles in transfected cells similar to those of the endogenous receptors.

Consistent with the fact that these are distinct receptors, there is a considerable lack of amino acid sequence homology between cloned A₁, A_2A, A_2B, and A₃ receptors. For example, the homology between rat A₁ and A_2B receptors is only 45% (Stehle et al., 1992), and the human A₃ receptor only shows approximately 50%, 43%, and 40% homology with human A₁, A_2A, and A_2B receptors, respectively (Linden, 1994). The homology between A_2A and A_2B receptors is also slight, being approximately 46% when these subtypes are cloned from rat and 61% when cloned from human (Stehle et al., 1992; Pierce et al., 1992).

An adenosine binding site with high affinity for 2-phenylaminoadenosine (CV 1808) (A_2A-selective agonist) in rat striatal membranes has been suggested to be a novel "A₄" adenosine receptor (Cornfield et al., 1992). The very low affinity of 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680) and Nethylcarboxamidoadenosine (NECA) at this site were taken to indicate that this is not an A₂ receptor. However, the binding studies were carried out at 4°C (Cornfield et al., 1992), and the existence of a distinct A₄ receptor has been challenged by the demonstration that when similar binding studies are carried out at 21°C, the potency order of compounds at the striatal membrane site is characteristic of the A_2A adenosine receptor (Luthin and Linden, 1995). Furthermore, in COS cells transfected with adenosine A_2A receptors, binding studies carried out at 4°C produce an "A₄" binding profile (Luthin and Linden, 1995). This justifies the more rigorous criteria now demanded for classification of receptors, whose identity must be proved by molecular as well as by biochemical or pharmacological means.

There is a vast and rapidly growing literature on adenosine/P1 receptors; it has not been possible to do justice to this in the present review. Out of necessity, therefore, we focus on the more recent literature.

All adenosine receptors couple to G proteins. In common with other G protein-coupled receptors, they have seven putative transmembrane (TM) domains of hydrophobic amino acids, each believed to constitute an helix of approximately 21 to 28 amino acids. The N-terminal of the protein lies on the extracellular side and the C-terminal on the cytoplasmic side of the membrane. A pocket for the ligand binding site is formed by the three-dimensional arrangement of the -helical TM domains, and the agonist is believed to bind within the upper half of this pore. The transmembrane domains are connected by three extracellular and three cytoplasmic hydrophilic loops of unequal size; typically the extracellular loop between TM4 and TM5 and the cytoplasmic loop between TM5 and TM6 is extended. These features are illustrated in a schematic of the A₁ receptor in figure 1.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Schematic of the A1 adenosine receptor. In common with other G protein-coupled receptors, the A1 receptor has seven putative transmembrane domains (I-VII) of hydrophobic amino acids, each believed to constitute an -helix, which are connected by three extracellular and three intracellular hydrophilic loops. The number of amino acids comprising the extra- and intracellular loops and the extracellular N-terminal and intracellular C-terminal regions of the bovine A1 receptor are indicated in parentheses (Olah et al., 1992). The transmembrane regions comprise 23 to 25 amino acids in the bovine A1 receptor (Olah et al., 1992). The arrangement of the transmembrane regions forms a pocket for the ligand binding site. The location of histidine residues (H) in transmembrane regions VI (position 254) and VII (position 278) in the bovine A1 receptor, which are believed to be important in ligand binding (Olah et al., 1992), are indicated. Extracellular and transmembrane regions of the protein believed to be important in agonist and antagonist binding are indicated (Olah et al., 1994b,c). S-S denotes the presence of hypothetical disulfide bridges (Jacobson et al., 1993c). Glycosylation occurs on the second extracellular loop.

N-linked glycosylation often occurs on the second extracellular loop; the roles of the carbohydrate moieties of the glycosylated receptor are not clear, although suggested functions include stabilization of protein conformation, protection of proteins from proteases, and modulation of protein function. Current evidence suggests that glycosylation has no obvious influence on ligand binding (Piersen et al., 1994). The intracellular segment of the receptor interacts with the appropriate G protein with subsequent activation of the intracellular signal transduction mechanism. The third intracellular loop of the adenosine A_2A receptor seems to be the main determinant of its G protein selectivity (Olah, 1997). Phosphorylation by protein kinases of amino acid residues on the cytoplasmic domains seems to be involved in desensitization of A_2A and A₃ receptors (Palmer and Stiles, 1997a, 1997b).

The transmembrane regions are generally highly conserved, with particularly long stretches of amino acid homology being found in TM2, TM3, and TM5. Most sequence differences have been observed in a hypervariable region located at the N-terminal half of the second extracellular loop (Tucker and Linden, 1993). 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 C-terminal and the N-terminal do not seem to be involved in ligand recognition (Olah et al., 1994b, 1995). A number of amino acid residues contribute, in different ways, to ligand specificity within the binding pocket. Sitedirected mutagenesis of the bovine A₁ adenosine receptor suggests that conserved histidine residues in TM6 and TM7 are important in ligand binding. Histidine 278 in TM7 seems to be particularly important because mutation of this amino acid abolishes ligand binding (Olah et al., 1992). Mutagenesis of the human A₁ adenosine receptor has shown that threonine 277 in TM7 is important in binding of the non-selective adenosine receptor agonist NECA, but has little effect on the affinity of binding of the A₁ selective agonist (R)-N⁶-(2-phenyl-1-methylethyl)-adenosine (R-PIA), or of antagonists (Townsend-Nicholson and Schofield, 1994). Modification of Glu 16 in TM1 and Asp 55 in TM2 of the human A₁ receptor alters the affinity of binding for [³H]CCPA (2-chloro-N⁶-cyclopentyladenosine) and other agonists, but does not affect antagonist binding (Barbhaiya et al., 1996). Site-directed mutagenesis of the human A_2A adenosine receptor has identified several residues in TM5-7 that are involved in ligand binding (Kim et al., 1995). Glu 13 in TM1 of the human A_2A receptor seems to be critically involved in agonist, but not antagonist recognition (Ijzerman et al., 1996).

A potential problem inherent in the methodology of site-directed mutagenesis is that changes in primary structure may cause changes in tertiary structure of the molecule. This has been addressed by studies with chimeras constructed from structurally similar, but pharmacologically different receptors. The ligand binding properties of A₁/A₃ chimeric receptors support the concept of a crucial role for histidine residues in TM6 and TM7 in ligand binding (Olah et al., 1995). In addition, a critical role in ligand binding of the distal region of the second extracellular loop has been identified, although its specific interactions are not yet clear (Olah et al., 1994b). Possible roles include direct interaction of an amino acid residue(s) within this region with the ligand, an influence on the conformation of the receptor and/or steric hindrance. Construction of chimeric human A₁ and rat A_2A adenosine receptors was used to show that TM1-4 are important in A₁ receptor agonist and antagonist binding and ligand specificity (Rivkees et al., 1995a).

Analogs with greater stability than adenosine are produced by modification of the N⁶ and C2 positions of the adenine ring and the 5'-position of the ribose moiety of adenosine, and have been used extensively in the characterization of adenosine/P1 receptors. NECA (Williams, 1989), N-[2-(4-aminophenyl)ethyl] adenosine (APNEA) (Fozard and Carruthers, 1993), and N⁶-(3-[¹²⁵I]iodo-4-aminobenzyl)-5'-N-methylcarboxamidoadenosine (¹²⁵I-AB-MECA) (Olah et al., 1994a) do not discriminate between adenosine receptor subtypes. Agonists with subtype selectivity are detailed in the sections on individual adenosine receptor subtypes and the chemical structure of some of these are illustrated in figure 2.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   The chemical structure of some agonists at adenosine/P1 receptors.

ATP and metabolically stable ATP derivatives, i.e., adenosine 5'-O-(3-thiotriphosphate)(ATPS) and ,-methylene ATP (,-meATP), can act directly as agonists at adenosine/P1 receptors in some tissues where responses are blocked by methylxanthines, but are not affected by adenosine deaminase or by blockade of 5'-nucleotidase. ,-MeATP is approximately equipotent with adenosine at mediating contraction of smooth muscle adenosine/P1 receptors of rat colon (Bailey and Hourani, 1990), and relaxation via adenosine/P1 receptors of rat duodenum (Hourani et al., 1991), and guinea-pig trachealis muscle (Piper and Hollingsworth, 1996). ATP, ATPS, and ,-meATP inhibit [³H]-NA release in a variety of tissues via receptors that are blocked by the A₁ selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) as well as by the P2 receptor antagonist cibacron blue (Von Kügelgen et al., 1992, 1995b, 1996). ATP (Collis and Pettinger, 1982) and diadenosine polyphosphates (Hoyle et al., 1996; Vahlensieck et al., 1996) have been reported to stimulate directly adenosine/P1 receptors in guinea-pig atria, eliciting negative inotropic and chronotropic effects without prior conversion to adenosine. These effects are not consistent with the pharmacological profile of any of the established subtypes of adenosine/P1 receptor, and in some respects are similar to the profile described for the P3 receptor.

Xanthines and xanthine derivatives, including the natural derivatives theophylline and caffeine, are non-selective adenosine/P1 receptor antagonists. They are not universal adenosine/P1 receptor antagonists; xanthine-resistant relaxations to adenosine and its analogs were observed in guinea-pig aorta (Collis and Brown, 1983; Martin, 1992), rat aorta (Prentice and Hourani, 1996), guinea-pig trachea (Brackett and Daly, 1991), porcine coronary artery (Abebe et al., 1994), and guinea-pig taenia cecum (Prentice et al., 1995). Some A₃ receptors, namely those of rat, rabbit, and gerbil, are characteristically insensitive to methylxanthines, thus it is possible that the xanthine-resistant responses to adenosine described in some tissues occur following actions of adenosine at mast cell A₃ receptors and the subsequent release of vasoactive mediators. This hypothesis would predict that guinea-pig and pig A₃ receptors are also xanthine-insensitive, because xanthine-resistant responses to adenosine have been reported in these species. It would be interesting to see if these responses can be blocked by inhibitors of mast cell degranulation.

8-Phenyltheophylline and the more water soluble 8-(p-sulfophenyl)theophylline (8-SPT) (Daly et al., 1985) are more potent than theophylline at adenosine/P1 receptors, but are not subtype-selective. 8-SPT and its derivative 1,3-dipropyl-8-sulfophenylxanthine (DPSPX) do not cross the blood-brain barrier, being purely peripherally acting adenosine/P1 receptor antagonists (Daly et al., 1985) and thus can be used to discriminate between central and peripheral adenosine receptors. A number of xanthines and non-xanthines identified as adenosine receptor antagonists with reasonable subtype selectivity are described below (see Sections III.F., IV.F., and VI.F.) and their chemical structures illustrated in figure 3.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   The chemical structure of some antagonists at adenosine/P1 receptors.

	III. A1 Receptor

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Subdivision of A₁ receptors into high affinity A_1a receptors and low affinity A_1b receptors has been proposed (Gustafsson et al., 1990). This was based on the description of high-affinity binding sites for adenosine agonists and antagonists in rat and guinea-pig brain (A_1a) and low-affinity binding sites in rat vas deferens and guinea-pig ileum (A_1b) (Gustafsson et al., 1990). However, there are no cloned equivalents for these putative subtypes and their existence remains equivocal. It is possible that these reflect high and low affinity states of the same A₁ receptor.

A₁ receptors have been cloned from several species (table 3). The human adenosine A₁ receptor subtype gene (ADORA1) has been localized to chromosome 1q32.1 (Townsend-Nicholson et al., 1995a). The variability in the primary sequence of the A₁ receptor between species is less than 10% for A₁ receptors from dog, rat, and cow, and less than 5% between bovine and human A₁ receptors, but this seems to be sufficient to cause considerable interspecies differences in ligand binding (Tucker and Linden, 1993) and subtle differences in the mechanisms underlying receptor desensitization (Ramkumar et al., 1991; Nie et al., 1997; Palmer and Stiles, 1997b). Species homologs of A₁ receptors have been suggested to differ in their ability to discriminate among the related G_o/G_i protein alpha subunits (Jockers et al., 1994).

The A₁ receptor mediates a broad range of signaling responses, which may be caused by its coupling to different G proteins within the G_i/o family (Freissmuth et al., 1991; Munshi et al., 1991). The G proteins G_i and G_o are substrates for pertussis toxin that ADP-ribosylates the -subunit of G_i/o/t family members, uncoupling them from receptors. Accordingly, effects mediated by A₁ receptors are generally blocked by pertussis toxin. However, presynaptic A₁ receptors seem to be at least partly resistant to pertussis toxin (Fredholm et al., 1989; Hasuo et al., 1992); the reason for this could be the very tight coupling of the presynaptic A₁ receptors to potentially pertussis toxin-sensitive G proteins, rather than coupling to pertussis toxin-insensitive G proteins (Van der Ploeg et al., 1992). A partially-purified protein with selectivity for G protein  subunits has been shown to stabilize the rat brain A₁ receptor-G protein complex, thereby promoting tight coupling of the A₁ receptor with its G protein (Nanoff et al., 1997). Interestingly, this is a feature of the rat brain but not the human brain A₁ receptor; the latter is not under the control of a coupling cofactor, but operates according to the classic ternary complex model of receptor-G protein coupling (Nanoff et al., 1997).

The most widely recognized signaling pathway of A₁ receptors is inhibition of adenylate cyclase causing a decrease in the second-messenger cAMP (Van Calker et al., 1978; Londos et al., 1980). This in turn modulates the activity of cAMP-dependent protein kinase, which phosphorylates diverse protein targets. A₁ coupling to adenylate cyclase has been described in a number of tissues including brain, adipose tissue, and testes. In addition to direct modulation of signaling pathways downstream to cAMP, inhibition of adenylate cyclase via A₁ receptors blocks the effects of other agents which act by stimulating adenylate cyclase activity in cells.

Another signaling mechanism of A₁ receptors is activation of phospholipase C (PLC) leading to membrane phosphoinositide metabolism and increased production of inositol 1,4,5-triphosphate (IP₃) [and diacylglycerol (DAG)] and Ca²⁺ mobilization. This has been described in chinese hamster ovary (CHO)-K1 cells expressing the cloned human A₁ receptor (Iredale et al., 1994; Megson et al., 1995) as well as at endogenous A₁ receptors in a number of tissues including DDT₁ MF-2 smooth muscle cells (Gerwins and Fredholm, 1992a,b; White et al., 1992), heart (Scholz et al., 1993), myometrium (Schiemann et al., 1991a,b), rabbit cortical collecting tubule cells (Arend et al., 1989), renal cells (Weinberg et al., 1989), tracheal epithelial cells (Galietta et al., 1992), cultured mesangial cells (Olivera et al., 1992), and primary astrocytes (Peakman and Hill, 1995). IP₃ stimulates the release of Ca²⁺ from intracellular stores via interactions with specific receptors located on the sarcoplasmic reticulum. Elevation of cytosolic Ca²⁺ by IP₃ can stimulate a variety of signaling pathways, including a family of phosphatidyl serine-dependent serine/threonine-directed kinases collectively called protein kinase C (PKC) (of which there are at least 11 different isoforms), phospholipase A₂ (PLA₂), Ca²⁺-dependent K⁺ channels, and nitric oxide synthase (NOS). Depletion of Ca²⁺ from IP₃-sensitive pools may promote Ca²⁺ influx from extracellular sources.

Activation of phospholipase D (PLD) via A₁ adenosine receptors in DDT₁ MF-2 smooth muscle cells has been described (Gerwins and Fredholm, 1995a, 1995b), although as in the majority of cell systems this may be downstream of phosphoinositide hydrolysis and may require the intermediate activation of PKC or Ca²⁺.

Stimulation of A₁ receptors can activate several types of K⁺ channel, described principally in cardiac muscle and neurons. In supraventricular tissues (sino-atrial and atrioventricular node, and atrium), the A₁ receptor couples directly via pertussis toxin-sensitive G proteins to K⁺ channels (the same K⁺ channels are activated by both adenosine and acetylcholine), and activation causes bradycardia (Belardinelli et al., 1995a; Bünemann and Pott, 1995; Ito et al., 1995). A₁ adenosine receptors also couple to ATP-sensitive K⁺ channels (K_ATP channel); the activity is additionally regulated by metabolic demand (they close when intracellular ATP levels are high). Coupling seems to occur through the G protein in a membrane-delimited manner (Kirsch et al., 1990; Dart and Standen, 1993), although coupling via cytosolic factors is possible given the strong evidence that A₁ receptors, K_ATP channels, and PKC all have a role in ischemic preconditioning. A₁ receptor coupling to K_ATP channels has been described in rat and guinea-pig ventricular myocytes (Kirsch et al., 1990; Ito et al., 1994), porcine coronary arteries (Merkel et al., 1992; Dart and Standen, 1993), rabbit heart (Nakhostine and Lamontagne, 1993), and rat cerebral cells (Heurteaux et al., 1995). Activation of K_ATP channels mediates a reduction in action potential duration, vasodilatation and an increase in blood flow, which is consistent with their having a pivotal role in the coupling of vascular tone to metabolic demand determined both by intracellular purines (ATP/ADP levels) and by the extracellular actions of adenosine (released, for instance, during hypoxia or ischemia).

Neurons express multiple K⁺ channels that A₁ receptors may couple to regulate membrane potential and determine action potential frequency and duration. A₁ receptors reduce neuronal excitability and decrease firing rate by a hyperpolarizing effect mediated mainly by an increase in K⁺ conductance (Trussell and Jackson, 1985; Greene and Haas, 1991; Pan et al., 1995).

A₁ receptors also couple to inhibition of Ca²⁺ currents, which may account for inhibition of neurotransmitter release, although other or multiple mechanisms may be involved in this process (see Fredholm, 1995). Inhibition of Ca²⁺ currents by A₁ receptors has been described in dorsal root ganglion neurons (Dolphin et al., 1986), rat hippocampal pyramidal neurons (Scholz and Miller, 1991), rat sympathetic neurons (N-type Ca²⁺ channels, plus an unidentified Ca²⁺ channel) (Zhu and Ikeda, 1993), rat brainstem (predominantly N-type, but also P-type Ca²⁺ channels) (Umemiya and Berger, 1994), hippocampal CA1 neurons (N-type, plus some unidentified Ca²⁺ channels) (Wu and Saggau, 1994), hippocampal CA3 neurons (N-type Ca²⁺ channel) (Mogul et al., 1993), and mouse motoneurons (N-type Ca²⁺ channel) (Mynlieff and Beam, 1994). In atrial myocytes adenosine has an inhibitory effect on basal L-type Ca²⁺ current, although this is small and may be secondary to a reduction in basal cAMP (Belardinelli et al., 1995a).

Several mechanisms, operational at different levels of the signal transduction cascade, contribute to differential desensitization of G protein-coupled receptors. Rapid desensitization (occuring within a few minutes of agonist exposure) seems to involve phosphorylation of specific residues on the receptor C-terminal or the cytoplasmic loops by G protein-coupled receptor-specific kinases (GRKs) and/or kinases regulated by levels of intracellular second-messengers such as cAMP-dependent protein kinase. The phosphorylated receptor may bind to members of a family of proteins called arrestins, which cause uncoupling of the receptor from its G proteins. Desensitization occuring over a longer time course also involves uncoupling of the receptor-G proteins complex, but phosphorylation does not seem to be a prerequisite. Sequestration of receptors into an intracellular compartment may occur, as described for the increase in A₁ receptors in light vesicle membrane fractions prepared from the hamster vas deferens smooth muscle cell line, DDT₁ MF-2 cells, after chronic exposure to R-PIA (Ramkumar et al., 1991). Prolonged exposure to agonist may additionally lead to down-regulation of receptors and/or of the associated G proteins.

Desensitization of A₁ receptors by exposure to adenosine analogs has consistently been described both in vitro and in vivo, but this usually requires prolonged exposure to agonist (from 15 minutes to several hours or even days) (Parsons and Stiles, 1987; Ramkumar et al., 1991; Abbracchio et al., 1992; Green et al., 1992; Lee et al., 1993; Longabaugh et al., 1989; Casati et al., 1994). This is considerably longer than the time to desensitization of A₃ receptors which typically undergo significant desensitization within several minutes. Interestingly, while an agonist-stimulated increase in phosphorylation has been described for A₁ receptors in hamster DDT₁ MF-2 cells in association with receptor uncoupling from G proteins and desensitization, presumably by GRKs (Ramkumar et al., 1991; Nie et al., 1997), phosphorylation does not occur for the human A₁ receptor expressed in CHO cells at a time when receptor down-regulation is observed (Palmer and Stiles, 1997b). Down-regulation of A₁ receptors and/or of the associated G proteins after prolonged exposure to agonist has been reported in most of the cells and tissues in which this has been studied (Parsons and Stiles, 1987; Longabaugh et al., 1989; Green et al., 1992; Ramkumar et al., 1991, 1993a; Abbracchio et al., 1992).

Down-regulation of G proteins following A₁ receptor activation may lead to heterologous receptor desensitization. Chronic stimulation of A₁ receptors in adipocytes in vivo (Longabaugh et al., 1989) and in isolated adipocytes (Green et al., 1992) with (-)N⁶-phenylisolpropyl adenosine (PIA) for up to 6 and 7 days, respectively, causes down-regulation of A₁ receptors, non-uniform down-regulation of G_i proteins, and heterologous desensitization of other lipolytic hormone responses. In contrast, chronic (7 days) infusion of (R)N⁶-phenylisopropyl adenosine (R-PIA) in guinea-pigs homologously desensitizes the atrioventricular nodal response to adenosine: there is down-regulation of A₁ adenosine receptors, a decrease in high affinity A₁ receptors, and a decrease in G_i and G_o proteins, but no change in responses mediated by muscarinic receptors (Dennis et al., 1995).

Long-term treatment with adenosine/P₁ receptor antagonists generally leads to an increase in the effects of adenosine via a selective increase in the number of A₁ receptors, receptor sensitization and/or altered interaction between the receptor and the associated G proteins (Fredholm, 1982; Murray, 1982; Fredholm et al., 1984; Green and Stiles, 1986; Ramkumar et al., 1991; Fastbom and Fredholm, 1990; Zhang and Wells, 1990; Lupica et al., 1991a, 1991b; Shi et al., 1994). Long-term (12 day) caffeine treatment of rats increases the number of hippocampal A₁ (but not A_2A) receptors, without any changes in A₁ messenger ribonucleic acid (mRNA), suggesting that the adaptive changes are at the posttranslational level (Johansson et al., 1993a). An increase in the density of cortical A₁ receptors has been described after chronic caffeine injestion in mice, but surprisingly, given that striatal adrenergic, cholinergic, GABA, and serotonin receptors and Ca²⁺ channels are also affected by this treatment, there is no change in the density of striatal A_2A receptors (Shi et al., 1993).

Certain N⁶-substituted adenosine derivatives, such as N⁶-cyclopentyladenosine (CPA), N⁶-cyclohexyladenosine (CHA), and R-PIA, are selective agonists at A₁ receptors with K_i values in the range of 0.6 to 1.3 nM (see Jacobson et al., 1992b) (table 2).

Substitutions at both the N⁶- and C2-positions have produced 2-chloro-CPA (CCPA) which is A₁ selective, 1500-fold versus A₂ receptors in binding studies in rat brain, with a K_i of 0.6 nM (Lohse et al., 1988; Thompson et al., 1991; Jacobson et al., 1992b). N-[1S, trans,2hydroxycyclopentyl] adenosine (GR79236) has been reported to be an A₁ selective agonist, which is approximately equipotent with CPA in a variety of isolated tissues and cell types (Reeves et al., 1993; Gurden et al., 1993).

Most of the selective A₁ receptor antagonists described to date are xanthine-based derivatives. The introduction of hydrophobic (particularly phenyl or cycloalkyl) substituents into position 8 of the xanthine ring has yielded potent and A₁-selective antagonists, including 1,3-dipropyl-8-phenyl(2-amino-4-chloro)xanthine (PACPX), DPCPX, and xanthine amine congener (XAC) (Bruns et al., 1987; Martinson et al., 1987; Shimada et al., 1991) (fig. 3). Of these, DPCPX has the greatest affinity (K_i 1.5 nM) for A₁ receptors and the greatest A₁-subtype selectivity (A₂/A₁ affinity ratio 740), as shown in rat brain membranes (Bruns et al., 1987; Lohse et al., 1987). The human A₁ receptor has an approximately lower affinity for DPCPX (Libert et al., 1992; Klotz et al., 1998). A number of other 8-substituted xanthines, including (±)-8-(3-oxocyclopentyl)-1,3-dipropylxanthine (KFM 19) and KW-3902 (8-noradamant-3-yl-1,3-dipropylxanthine), have been shown to be selective antagonists at A₁ receptors (see Williams, 1989; Jacobson et al., 1992b). The alkylxanthine 1,3-dipropyl-8-[2-(5,6-epoxy)norbornyl]xanthine (ENX) is a potent (K_B 3.6 nM) and selective antagonist at A₁ receptors in the guinea- pig heart and brain and in DDT₁ MF-2 cells, with 400-fold greater affinity of binding versus A_2A receptors in guinea-pig brain (Belardinelli et al., 1995b).

Several classes of non-xanthine antagonists have been described, some showing reasonable affinity and selectivity for the A₁ receptor (see Jacobson et al., 1992b; Daly et al., 1993). Some of the more active of these are the tricyclic non-xanthine antagonists, including the triazoloquinazolines (Francis et al., 1988), the triazoloquinoxalines (Trivedi and Bruns, 1988; Sarges et al., 1990), and the imidazoquinolines (Van Galen et al., 1991).

The adenine derivative 1,3-dipropyl-8-[2,(5,6-epoxy)norbornyl]xanthine (N 0861) is reasonably selective (10- to 47-fold versus A_2A receptors) and potent at A₁ receptors in a number of tissues (May et al., 1991; Martin et al., 1993a; Belardinelli et al., 1995b). This compound has been superceded by the S-enantiomer 12 (CVT-124) with nanomolar selectivity and 1800- and 2400-fold selectivity at rat and cloned human A₁ receptors, respectively (Pfister et al., 1997), and by 8-(N-methylisopropyl)amino-N⁶-(5'-endohydroxy-endonorbornyl-)9-methyl adenine (WRC 0571) with 62-fold selectivity versus the A_2A receptor and 4670-selectivity versus the A₃ receptor (Martin et al., 1996).

(+)-(R)-[(E)-3-(2-phenylpyrazolo[1,5-]pyridin-3-yl)acryloyl]-2-piperidine ethanol, FK 453, has been reported to be a potent and selective A₁ receptor antagonist with IC₅₀ values of approximately 17 nM at rat cortical A₁ receptors and 11 µM at striatal A₂ receptors (Terai et al., 1995). Chiral pyrolo[2,3-d]pyrimidine and pyrimido[4,5-b]indole derivatives have been shown to be potent and highly stereoselective A₁ adenosine receptor antagonists (Müller et al., 1996a).

A₁ receptors are particularly ubiquitous within the central nervous system (CNS), with high levels being expressed in the cerebral cortex, hippocampus, cerebellum, thalamus, brain stem, and spinal cord (Reppert et al., 1991; Dixon et al., 1996) (fig. 4). Immunohistochemical analysis using polyclonal antisera generated against rat and human A₁ adenosine receptors has identified different labeling densities of individual cells and their processes in selected regions of the brain (Rivkees et al., 1995b). A₁ receptor mRNA is widely distributed in peripheral tissues having been localized in vas deferens, testis, white adipose tissue, stomach, spleen, pituitary, adrenal, heart, aorta, liver, eye, and bladder (Reppert et al., 1991; Dixon et al., 1996). Only very low levels of A₁ mRNA are present in lung, kidney, and small intestine (Reppert et al., 1991; Stehle et al., 1992; Dixon et al., 1996) (fig. 4).

View larger version (69K): [in this window] [in a new window]   Fig. 4.   Tissue distribution of adenosine receptor mRNA expression as examined by RT-PCR. Sizes of PCR products are given in base pairs. (From Dixon et al., 1996, Br J Pharmacol 118:1461-1468; with permission from McMillan Press Limited.)

It is now well established that adenosine is released from biological tissues during hypoxia and ischemic conditions. One of its effects is to reduce neuronal activity and thereby oxygen consumption; thus it acts as a neuroprotective agent. A significant part of these effects seem to be mediated by the A₁ receptor. A₁ receptors are located pre and postsynaptically on cell bodies, and on axons, where they mediate inhibition of neurotransmission by decreasing transmitter release, hyperpolarizing neuronal membranes, reducing excitability and firing rate, and altering axonal transmission. Adenosine can also exert behavioral effects: adenosine actions at A₁ receptors have been implicated in sedative, anticonvulsant, anxiolytic, and locomotor depressant effects (Nikodijevic et al., 1991; Stone, 1991; Jain et al., 1995; Malhotra and Gupta, 1997). Conversely, xanthine antagonists such as caffeine and theophylline have central stimulatory properties ascribed, at least in part, to inhibition of endogenous adenosine, although inhibition of cyclic nucleotide phosphodiesterases may contribute to this effect.

A₁ receptors mediate cardiac depression through negative chronotropic, dromotropic, and inotropic effects (see Olsson and Pearson, 1990). Slowing of the heart rate occurs via A₁ receptors on sinoatrial and atrioventricular nodes causing bradycardia and heart block, respectively, while the inotropic effects include a decrease in atrial contractility and action potential duration (Olsson and Pearson, 1990). This aspect of A₁ receptor-mediated effects has found application in the clinical use of adenosine to treat supraventricular tachycardia, and in the use of adenosine receptor antagonists in the treatment of bradyarrhythmias.

In the kidney, activation of A₁ receptors mediates diverse effects including vasoconstriction (principally of the afferent arteriole), a decrease in glomerular filtration rate, mesangial cell contraction, inhibition of renin secretion, and inhibition of neurotransmitter release (Olivera et al., 1989; Agmon et al., 1993; Barrett and Droppleman, 1993; Munger and Jackson, 1994). Intravenous and intra-aortic administration of adenosine in rats decrease water and sodium excretion via A₁ receptors, while selective antagonism of A₁ receptors causes diuresis and natriuresis (see Mizumoto et al., 1993; Van Beuren et al., 1993). Intrarenal administration of adenosine, but not of the A_2A selective agonist CGS 21680, in dogs also decreases water and sodium excretion (Levens et al., 1991a,b). Furthermore, A₁ receptors increase transepithelial resistance and reduce Na⁺ uptake in inner medullary collecting duct cells in culture (Yagil et al., 1994). On the other hand, intrarenal administration of adenosine and the A₁-selective agonist CHA in rats has been shown to induce marked diuresis and natriuresis which can be inhibited by the A₁-selective antagonist DPCPX (Yagil, 1994).

Direct effects on blood vessel tone via adenosine actions on A₁ receptors are rare. A more significant role of A₁ receptors with regard to regulation of blood vessel tone appears to be prejunctional modulation of neurotransmitter release. Prejunctional inhibition of neurotransmission via A₁ receptors on perivascular sympathetic (Gonçalves and Queiroz, 1996) and capsaicin-sensitive sensory afferents (Rubino et al., 1993) has been shown. However, A₁ receptors have been observed to mediate relaxation of porcine coronary artery (Merkel et al., 1992), and contraction of guinea-pig aorta (Stoggall and Shaw, 1990) and pulmonary artery (Szentmiklósi et al., 1995). A₁ receptors have also been reported to mediate contraction of rat isolated spleen (Fozard and Milavec-Krizman, 1993) and rat vas deferens (Hourani and Jones, 1994), as well as bronchoconstriction and bronchial hyperresponsiveness (Ali et al., 1994a, 1994b; Pauwels and Joos, 1995; el-Hashim et al., 1996). Diverse A₁-mediated effects in the gut have been described, including inhibition of peristalsis of rat jejunum (Hancock and Coupar, 1995b), relaxation of longitudinal muscle of rat duodenum (Nicholls et al., 1992, 1996), and contraction of rat colonic muscularis mucosa (Bailey et al., 1992; Reeves et al., 1993). Interestingly, adenosine mediates contraction of guinea-pig myometrial smooth muscle via A₁ receptors that in non-pregnant animals are coupled to the formation of IP₃, but in pregnant animals are coupled both to IP₃ and negatively to adenylate cyclase (Schiemann and Buxton, 1991; Schiemann et al., 1991a,b).

Selective inhibition of the synthesis of A₁ receptors with antisense oligonucleotides confirmed that these receptors are involved in an animal model of asthma (Nyce and Metzger, 1997). There was a marked reduction in the number of A₁ receptors in the lung and attenuation of airway constriction to adenosine, histamine, and dust-mite allergen (Nyce and Metzger, 1997). Although the site of action remains to be determined, selective antagonism of A₁ receptors offers a possible new approach in asthma therapy.

A₁ receptors on bovine pulmonary artery endothelial cells have been shown to mediate Cl efflux (Arima et al., 1994). In human airway epithelial cells, A₁ receptors have been reported to mobilize intracellular Ca²⁺ and activate K⁺ and Cl conductance (Rugolo et al., 1993), while selective inhibition of A₁ receptors with DPCPX increases cAMP-activated Cl conductance (McCoy et al., 1995).

A₁ adenosine receptors on rat cochleal membranes (Ramkumar et al., 1994), astrocytes (Peakman and Hill, 1994), and epididymal spermatozoa (Minelli et al., 1995) have been described. Release of Ca²⁺ from internal stores in perisynaptic glial cells of the frog neuromuscular junction via A₁ receptors has been described (Robitaille, 1995).

Adenosine acts via A₁ receptors and inhibition of cAMP to inhibit lipolysis and increase insulin sensitivity in adipose tissue (Londos et al., 1985; Green, 1987). Abnormal A₁ receptor function in genetic obesity has been proposed, showing that lipolysis is less active and A₁ receptor signaling more active, which may be caused by changes in receptor phosphorylation, but also possibly by adenylate cyclase activity (LaNoue and Martin, 1994; Berkich et al., 1995). In contrast, insulin sensitivity is decreased by activation of A₁ receptors in skeletal muscle (Challis et al., 1992). A₁ receptors on pancreatic  cells mediate inhibition of insulin secretion (Hillaire-Buys et al., 1989).

A₁ receptors have been widely reported to mediate the protective effects of adenosine in preconditioning and during ischemia or during reperfusion injury in the heart (Tsuchida et al., 1993, 1994; Yao and Gross, 1993; Lee et al., 1995; Lasley and Mentzer, 1995; Strickler et al., 1996; Grover et al., 1992; van Winkle et al., 1994; Sakamoto et al., 1995; Mizumura et al., 1996; Stambaugh et al., 1997), lung (Neely and Keith, 1995), and brain (Heurteaux et al., 1995). Strong evidence for a protective role of A₁ adenosine receptors comes from studies with transgenic mice over expressing the A₁ receptor. Mice over expressing the A₁ receptor have been shown to have an increased myocardial resistance to ischemia (Matherne et al., 1997). The mechanism involved is not yet clear; it may involve A₁ receptor activation of K_ATP channels as infarct size reduction after activation of A₁ receptors has been reported to be completely abolished by the blockade of K_ATP channels (Grover et al., 1992; van Winkle et al., 1994; Mizumura et al., 1996). On the other hand, there seems to be a general consensus that PKC is involved in ischemic preconditioning, and activation of PKC was shown to be the critical factor involved in limitation of myocardial infarct size by A₁ receptors in anaesthetized rabbits (Sakamoto et al., 1995). However, not all researchers are in agreement that adenosine is cardioprotective, or that A₁ receptors mediate ischemic preconditioning (Asimakis et al., 1993; Ganote et al., 1993; Hendrikx et al., 1993; Lasley et al., 1993; Liu et al., 1994). In addition, a protective role for adenosine A₃ receptors has been suggested (see Section VI.G.).

Reperfusion of ischemic tissue results in locally increased permeability and pulmonary edema that is associated with neutrophil accumulation in the microvasculature; neutrophil-endothelial cell interactions are known to be a prerequisite for the associated microvascular injury. Paradoxically, given the protective role of A₁ receptors in ischemia-reperfusion injury, adenosine contributes to inflammatory reactions via effects on neutrophil and/or endothelial A₁ receptors. This is done by augmenting responses to microbial stimuli, promoting chemotaxis, adhesion to endothelium, phagocytosis, and release of reactive oxygen intermediates (Cronstein et al., 1990; Cronstein, 1994; Zahler et al., 1994; Bullough et al., 1995; Felsch et al., 1995). It is possible that the local concentration of adenosine is crucial in determining which type of response predominates. A concentration-dependent dual protective-destructive role has also been described for the A₃ adenosine receptor, but what is even more intriguing is that it involves high and low levels of activation of A₃ receptors on the same cell (in both HL-60 and U 937 cells) (Yao et al., 1997).

A₁ adenosine receptors have been implicated in modulation of nociception in the spinal cord (Reeve and Dickenson, 1995) and in the periphery (Karlsten et al., 1992; Ocana and Baeyens, 1994). This may involve inhibition of sensory neurotransmitter release, because A₁ receptors have been shown to mediate inhibition of calcitonin gene-related peptide (CGRP) release from capsaicin-sensitive sensory neurons in the spinal cord (Santicoli et al., 1993) and in the periphery (Rubino et al., 1993), as well as inhibit GABA currents in dorsal root ganglion neurons (Hu and Li, 1997). Analgesic effects of caffeine have also been described. These effects have been attributed to caffeine's effects on supraspinal A₁ receptors because caffeine's effect is mimicked by the A₁-selective agonist 8-cyclopentyltheophylline (CPT); spinally or peripherally administered caffeine lacks antinociceptive effects (Sawynok and Reid, 1996).

Synergistic interactions between A₁ adenosine receptors and receptors coupled to a different class of G protein, typically pertussis toxin insensitive G_q/11 proteins, have been described, whereby coactivation of the receptors results in an augmented increase in effectors/second-messengers derived from the G_q/11 protein coupled pathway. The intracellular mechanisms underlying this potentiation are not well understood and have been suggested variously to involve intra- and extracellular calcium, second-messengers, and G_i protein subunits. Early evidence for this kind of interaction came with the observation that adenosine enhances ₁-adrenoceptor-induced accumulation of cAMP in rat vas deferens (Häggblad and Fredholm, 1987). Synergistic interactions have since been shown in DDT₁ MF-2 cells for A₁ receptors and ATP receptors (Gerwins and Fredholm, 1992a), histamine H₁ receptors (Dickenson and Hill, 1994), and bradykinin receptors (Gerwins and Fredholm, 1992b). A₁ receptors transfected into CHO cells act synergistically with receptors for thrombin (Dickenson and Hill, 1997), cholecystokinin A (Dickenson and Hill, 1996), and ATP (Megson et al., 1995). A₁ receptors in astrocytes interact synergistically with histamine H₁ receptors (Peakman and Hill, 1995) and glutamate receptors (Ogata et al., 1994) to raise levels of [Ca²⁺]_i. Synergistic interactions between A₁ and ₁-adrenoceptor mediated increases in inositol phosphate accumulation has been shown in mouse striatal astrocytes (el-Etr et al., 1992a,b; Marin et al., 1993). In hippocampal neurons, positive interactions have been described between adenosine A₁ and GABA_A receptors (Akhondzadeh and Stone, 1994), as well as negative interactions between A₁ and metabotropic glutamate receptors (de Mendonça and Ribeiro, 1997). Cross-talk between A₁ and other receptors is clearly widespread; its physiological significance is an important area for future research.

	IV. A2A Receptor

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The A_2A receptor has been cloned from several species (table 3) and has a characteristic pharmacological profile in transfected cells consistent with that of the endogenous receptor. The first cloned adenosine receptor, RDC8, cloned from a canine thyroid cDNA library (Libert et al., 1989), was subsequently identified as an A_2A receptor based on the binding of [³H]NECA and [³H]CGS 21680, and by activation of adenylate cyclase in cells transfected with the receptor (Maenhaut et al., 1990). The exogenous A_2A receptor was shown to have a tissue distribution similar to endogenous A_2A binding sites in brain, that is, limited to the striatum, nucleus accumbens and olfactory tubercule (Schiffmann et al., 1990). Subsequently, A_2A receptors were cloned from rat brain (Chern et al., 1992; Fink et al., 1992), human hippocampus (Furlong et al., 1992), and guinea-pig brain (Meng et al., 1994b). Both A_2A and A_2B receptors have been cloned from mouse bone marrow-derived mast cells (Marquardt et al., 1994). The gene for the A_2A receptor has been mapped to human chromosome 22 (MacCollin et al., 1994; Peterfreund et al., 1996) with reported chromosomal localizations of 22q11.2 (Le et al., 1996) and 22q11.2-q13.1 (Libert et al., 1994).

In common with the other adenosine receptor subtypes, there is significant interspecies differences in the amino acid sequences of cloned A_2A receptors; for example, between rat and human A_2A receptors there is approximately 84% amino acid homology (Chern et al., 1992; Fink et al., 1992; Furlong et al., 1992; Linden, 1994), and between rat and dog A_2A receptors 82% homology (Chern et al., 1992; Fink et al., 1992).

The significantly greater molecular weight of the A_2A receptor (45 kDa) compared with the other adenosine receptor subtypes (36 to 37 kDa) can largely be attributed to its substantially longer carboxy terminal domain. This region is not involved in tight coupling to G_s proteins because this is a function predominantly of the N-terminal segment of the third intracellular loop (Olah, 1997). A truncated mutant of the canine A_2A adenosine receptor was used to show that neither the long carboxy-terminus nor the glycosidic moieties are required for ligand binding (Piersen et al., 1994). Site-directed mutagenesis of the human A_2A adenosine receptor has been used to identify the various residues involved in agonist and antagonist binding (Kim et al., 1995; Ijzerman et al., 1996).

The most commonly recognized signal transduction mechanism for A_2A receptors is activation of adenylate cyclase. This implies coupling with the G protein G_s, although other G proteins may also be involved. Vibrio cholerae (cholera toxin) ADP-ribosylates the -subunit of G_s family members, inhibiting the intrinsic GTPase activity of G_s and thus has been useful in characterizing members of this family. Coupling of the A_2A receptor to its G protein is tight (see Palmer and Stiles, 1995). Hence, there is only slow dissociation of agonist from the receptor and stabilization of the receptor-G protein complex.

cAMP-independent signaling has been suggested for A_2A receptors on striatal GABA nerve terminals (Kirk and Richardson, 1995) and striatal cholinergic nerve terminals (Gubitz et al., 1996). In striatal nerve terminals, A_2A receptors are suggested to mediate dual signaling via P- and N-type Ca²⁺ channels linked to G_s/adenylate cyclase/PKA and cholera toxin-insensitive G protein/PKC, respectively (Gubitz et al., 1996). It has been suggested that A_2A receptor-mediated inhibition of superoxide anion generation in neutrophils may be mediated via cAMP-independent activation of a serine/threonine protein phosphatase (Revan et al., 1996).

A_2A receptor-mediated facilitation of synaptic transmission and transmitter release seems to occur through potentiation of presynaptic P-type Ca²⁺ channels, and probably involves adenylate cyclase and activation of a cAMP-dependent protein kinase (Mogul et al., 1993; Correia-de-Sá and Ribeiro, 1994a; Umemiya and Berger, 1994; Gubitz et al., 1996).

K_ATP channels are suggested to be involved in coronary vasodilatation mediated by A₂ receptors in the dog (Akatsuka et al., 1994). Activation of K_ATP channels by A₂ receptors in arterial myocytes is suggested to involve a cAMP-dependent protein kinase (Kleppisch and Nelson, 1995).

Desensitization of A_2A receptors has been reported, which may be more rapid, similar to, or less rapid than that of A₁ receptors. In DDT₁ MF-2 cells, the t_1/2 for desensitization of A_2A receptors (45 min) is more rapid than that for A₁ receptors, and in contrast to A₁ receptors, there is no change in A_2A receptor number or affinity (Ramkumar et al., 1991). A_2A receptor desensitization after exposure to A₂- or A_2A-selective agonists for up to several minutes to 4h has been observed in a number of tissues including porcine coronary artery (Makujina and Mustafa, 1993), rat aortic vascular smooth muscle cells (Anand-Srivastava et al., 1989), DDT₁ MF-2 smooth muscle cells (Ramkumar et al., 1991), rat pheochromocytoma PC12 cells (Chern et al., 1993), and in canine A_2A receptors expressed in CHO cells (Palmer et al., 1994). On the other hand, guinea-pig coronary artery A_2A receptors do not desensitize after more than 2h exposure to 2-[(2-aminoethylamino) carbonylethylphenylethylamino]-5'-N-ethylcarboxamido adenosine (APEC) or 1,4-phenylene-diisothiocyanate, 4-isothiocyanatophenyl aminothiocarbonyl-APEC (DITC-APEC) (Niiya et al., 1993). Furthermore, A_2A receptors seem to be relatively resistant compared with A₁ receptors to desensitization in rat brain slices (Abbracchio et al., 1992) and in spontaneously hypertensive rats after chronic treatment with A₁ and A₂ selective agonists in vivo (Casati et al., 1994). In rat striatum slices, A₂ receptors do not desensitize following exposure to NECA for up to 1h, whereas A₁ receptors desensitize rapidly (Abbracchio et al., 1992).

The mechanism underlying desensitization of A_2A receptors has been studied in some detail in transfected CHO cells, where it has been shown that exposure to agonist causes rapid desensitization and phosphorylation (Palmer et al., 1994; Palmer and Stiles, 1997b). The threonine 298 residue of the carboxy terminal of the A_2A receptor seems to be essential for agonist-stimulated rapid receptor phosphorylation and short-term, but not long-term, desensitization (Palmer and Stiles, 1997a). The majority of the C terminal seems not to be involved in desensitization, because desensitization of a truncated mutant lacking the majority of the A_2A carboxyl-terminal (the last 95 residues) is unchanged (Palmer and Stiles, 1997a). Evidence that desensitization may involve GRKs, implying uncoupling of the receptor-G protein complexes, has been provided by a study in NG108-15 mouse neuroblastoma × rat glioma cells mutants overexpressing GRK2, where the rate of desensitization of endogenous A_2A and A_2B receptors was markedly slowed (Mundell et al., 1997). This effect was selective in that agonist-induced desensitization of secretin and IP-prostanoid receptor stimulated adenylate cyclase were not affected by dominant negative mutant GRK2 overexpression (Mundell et al., 1997). Receptor sequestration, whereby a receptor translocates to a "light membrane" fraction, has been described for A_2A receptors expressed in CHO cells, but this seems to be involved in the recovery of the response of the receptor rather than in desensitization (Palmer et al., 1994).

Studies of long-term desensitization of endogenous A_2A receptors in rat pheochromocytoma PC12 cells showed that whereas a 30 min exposure of A_2A receptors to CGS 21680 is associated with inhibition of adenylate cyclase activity, long-term agonist exposure (12-20h) is associated additionally with down regulation of G_s  proteins and activation of phosphodiesterase (Chern et al., 1993). Long-term (24h) exposure to agonist may additionally lead to down-regulation of receptor number and up-regulation of inhibitory G proteins (Palmer et al., 1994; Palmer and Stiles, 1997a). Approximately 2 weeks of continuous infusion of either NECA or CGS 21680 causes a decrease in the number of A_2A receptor binding sites in rat striatum (Porter et al., 1988; Webb et al., 1993a). A calcium-independent PKC isoenzyme seems to be involved in phosphorylation and inhibition of adenylate cyclase type VI activity after prolonged stimulation and desensitization of the A_2A receptor, at least in rat pheochromocytoma PC12 cells (Lai et al., 1997), providing an additional mechanism by which to regulate A_2A receptor signal transduction.

Striatal A_2A adenosine receptors in rats and mice are up-regulated after chronic caffeine ingestion (Hawkins et al., 1988; Traversa et al., 1994). A_2A receptors seem to be less prone to up-regulation after chronic blockade with non-selective antagonists than are A₁ receptors (Lupica et al., 1991a; Johansson et al., 1993a).

A_2A receptors do not generally bind N⁶-substituted adenosine derivatives and show a preference for derivatives with modifications of the 2nd position of the adenine ring; bulky substituents in this position can selectively enhance A_2A receptor affinity (Jacobson et al., 1992b; Cristalli et al., 1994; Siddiqi et al., 1995). Several synthetic A_2A-selective agonists are modeled according to this structural modification. It should be noted that the agonist studies detailed below have been carried out in species other than humans, and that the human A_2A receptor has a comparatively lower affinity of binding for CGS 21680 and other adenosine receptor agonists (Dionisotti et al., 1997; Klotz et al., 1998).

The C2-substituted NECA derivative, CGS 21680, is 140-fold selective for the A_2A versus the A₁ receptor (Hutchison et al., 1990) (fig. 2). CGS 21680 has only very low affinity at the A_2B receptor, and thus has been used extensively to discriminate between A_2A and A_2B subtypes (Jarvis et al., 1989; Lupica et al., 1990). [³H]CGS 21680 has been reported to bind in rat cortex and hippocampus to adenosine binding sites different to the classic striatal A_2A receptors, which does not seem to be caused by high and low affinity states of the same A_2A receptor, or to binding at A₃ or A₄ receptors (Johansson et al., 1993b; Cunha et al., 1996; Lindström et al., 1996). Amine derivatives of CGS 21680, namely APEC (fig. 2), DITC-APEC and 2-[4-(2-([4-aminophenyl]methylcarbonyl)-ethyl)-phenyl]ethylamino-5'-N-ethylcarboxamido-adenosine (PAPA-APEC), are A_2A-selective agonists (Barrington et al., 1989; Ramkumar et al., 1991; Jacobson et al., 1992a; Niiya et al., 1993). DITC-APEC binds covalently, causing irreversible activation of the A_2A receptor (Niiya et al., 1993).

The C2-substituted adenosine derivative CV 1808 displays poor selectivity (approximately 5-fold) for the A_2A versus the A₁ receptor (Kawazoe et al., 1980; Bruns et al., 1986), but is a valuable precursor for the synthesis of more selective A_2A receptor agonists. N⁶-(2(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl)-adenosine (DPMA) is a selective A_2A receptor agonist (Merkel et al., 1992; Alexander et al., 1994).

A series of 2-aralkynyl and 2-heteroalkynyl derivatives of NECA have been studied for their selectivity at the A_2A receptor (Cristalli et al., 1995). Of these, the 4-formylphenylethynyl derivative shows affinity in the low nanomolar range and approximately 160-fold selectivity. 2-Hexyl-5'-N-ethylcarboxamidoadenosine (2HE-NECA) has been suggested to be selective at A_2A receptors with 60- and 160-fold selectivity in binding studies for A_2A versus A₁ receptors in rat and bovine brain, respectively (Monopoli et al., 1994). Although NECA itself is approximately equipotent at A₁ and A_2A receptors, it can be useful in A_2A receptor characterization provided that A₁-selective ligands are shown not to have equivalent effects.

The 2-hydrazinoadenosine, WRC-0470 (2-cyclohexylmethylidenehydrazinoadenosine) has been shown to be a potent and selective A_2A agonist, with low nanomolar affinity at recombinant A_2A receptors transfected in mammalian cells and in functional assays in a variety of tissues (Martin et al., 1997b).

Several antagonists selective for the A_2A receptor have been synthesized. 8-(3-chlorostyryl)caffeine (CSC) is a potent (K_i 54 nM) and selective A_2A antagonist in radioligand binding assays in rat brain (520-fold selective versus A₁ receptors), in reversing agonist effects on adenylate cyclase in PC12 cells (22-fold selective), and in blocking locomotor depression elicited by the A_2A-selective agonist APEC in vivo (Jacobson et al., 1993a) (fig. 3). 1,3-dialkyl-7-methyl-8-(3,4,5-trimethoxystyryl)xanthine (KF-17837) has been described as a potent and selective A_2A antagonist with 62-fold selectivity for A_2A over A₁ receptors in binding studies in rat brain, and 30-fold selectivity for the A_2A over the A_2B receptor in inhibition of cAMP accumulation (A_2A IC₅₀ = 53 nM; A_2B IC₅₀ = 1500 nM) (Shimada et al., 1992; Kanda et al., 1994; Nonaka et al., 1994). DMPX (3,7-dimethyl-1-propargylxanthine) derivatives have been shown to be potent and selective A_2A antagonists; 8-(m-bromostyryl)-DMPX has a K_i value of 8.2 nM and is 146-fold selective versus A₁ receptors (Müller et al., 1996b).

ZM 241385, (4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo [2,3-] [1,3,5]triazin-5-yl amino]ethyl)phenol) is a potent and selective non-xanthine A_2A adenosine receptor antagonist (Poucher et al., 1995) (fig. 3). It has high affinity for the A_2A receptor (pA₂ value approximately 9), is 1000- and 91-fold selective versus A₁ and A_2B receptors, respectively, and has virtually no effects at A₃ receptors (Poucher et al., 1995).

[³H]SCH 58261 ([³H-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine) is a novel potent and selective A_2A antagonist radioligand which binds with low nanomolar affinity to A_2A receptors in human platelet and rat striatal membranes, and at A_2A receptors transfected into CHO cells (Zocchi et al., 1996; Dionisotti et al., 1997). The analog SCH 63390 (5-amino-7-(3-phenylpropyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) has similar potency at A_2A receptors, but greater selectivity (210-fold) (Baraldi et al., 1996).

A_2A receptors have a wide-ranging but restricted distribution that includes immune tissues, platelets, the CNS, and vascular smooth muscle and endothelium. Functional studies concerned with A_2A receptors in isolated cells and tissues, in the central and peripheral nervous systems, and in isolated blood vessels and vascular beds, are listed in tables 4, 5 and 6, and illustrate the wide distribution and diverse biological effects mediated by this receptor.

Within the brain, the highest levels of A_2A receptors are in the striatum, nucleus accumbens, and olfactory tubercle (regions which are rich in dopamine) (Ongini and Fredholm, 1996). Low levels of A_2A receptor also seem to be expressed in most other brain regions, although for striatal cholinergic neurons this is controversial (Dixon et al., 1996; Peterfreund et al., 1996; Jin and Fredholm, 1997; Svenningsson et al., 1997). Striatal neurons express A_2A receptors in close association with dopamine D₂ receptors and specific negative interactions have been described (Férre et al., 1991, 1992, 1997; Fink et al., 1992; Schiffmann and Vanderhaeghen, 1993). Outside the brain, the most abundant expression of human A_2A mRNA is in immune tissues, eye and skeletal muscle; heart, lung, bladder, and uterus also show strong expression, with less abundant expression in small intestine, kidney, spleen, stomach, testis, skin, kidney, and liver (Dixon et al., 1996; Peterfreund et al., 1996).

A_2A receptors in the CNS and particularly in the peripheral nervous system (PNS) generally facilitate neurotransmitter release (table 5).

The negative interactions that have been observed between A_2A and dopamine D₂ receptors involve a reduced affinity of agonist binding to dopamine D₂ receptors upon stimulation of A_2A receptors in rat striatal membranes (Ferré et al., 1991, 1992, 1997). This raises the possibility of using A_2A receptor antagonists as a novel therapeutic approach in the treatment of Parkinsons disease, to reduce the profound disabling effects arising from degeneration of dopaminergic nigrostriatal neurons of the basal ganglia in this disease (Richardson et al., 1997). Interactions are not observed between A_2A and D₂ receptors transfected into COS-7 cells; it was suggested that the receptors do not interact directly to influence agonist binding (Snaprud et al., 1994). Interestingly, activation of A_2A receptors on rat striatal nerve terminals causes desensitization of coexpressed A₁ receptors by a mechanism which seems to involve PKC (Dixon et al., 1997a). It is noteworthy that both D₂ dopamine and A₁ adenosine receptors couple to G_i proteins to cause inhibition of adenylate cyclase. Thus, with respect to the actions of adenosine at A_2A receptors, negative A_2A-A₁ and A_2A-D₂ interactions will shift the balance of intracellular signaling further toward stimulation of cAMP. Interactions between A_2A receptors and dopamine D₁ receptors, and receptors for CGRP, glutamate, and acetylcholine have also been reported (see Sebastiào and Ribeiro, 1996). Negative interactions whereby activation of the A_2A receptor blocks the protective effects of preconditioning hypoxia, believed to be via A₁ and A₃ receptors, have been described (Strickler et al., 1996).

Behavioral effects of A_2A receptors are evidenced by A_2A-mediated cataleptic activity and antagonism of apomorphine-induced climbing (an animal model of schizophrenia) (Kanda et al., 1994; Kafka and Corbett, 1996).

In the vasculature, A_2A receptors have been described on both the smooth muscle and endothelium, where they are associated with vasodilatation (table 6). There seems to be considerable variation in A_2A receptor expression between blood vessels, although it is possible that vessels unresponsive to A_2A-selective agonists do express the receptor but at very low levels, or that the receptor is not coupled to a functional response. This functional diversity is exemplified by the fact that A_2A receptors mediate relaxation of rat aorta and bovine coronary artery (Conti et al., 1993), whereas in guinea-pig pulmonary artery (Szentmiklósi et al., 1995) and rat mesenteric arterial bed (Rubino et al., 1995), adenosine-mediated relaxation is mediated via the A_2B receptor, and relaxation via A_2A receptors is weak or non existent (fig. 5). Adenosine has a mitogenic effect on endothelial cells, which in human endothelial cells is mediated via the A_2A receptor and subsequent activation of mitogen-activated protein kinase (MAPK) (Sexl et al., 1997). The mitogenic activation seems to be independent of G_s, G_i and typical PKC isoforms, but is associated with activation of p21^ras (Sexl et al., 1997).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Species variation in functional expression of vasodilator A2A and A2B receptors. Note that the agonist potencies suggest the presence of A2A receptors in rat aorta (a) and bovine coronary artery (b), and A2B receptors in rat mesenteric arterial bed, (c) and guinea-pig pulmonary arteries (d).   a., b. Mean dose-response curves for the vasorelaxant activity induced by some adenosine agonists in isolated rat aorta (a) and bovine coronary artery (b). Each response is expressed as the percentage of the maximum contraction induced by PGF2 (3 µM). Vertical bars represent 95% confidence limits. (From Conti et al., 1993).   c. Dose-response curves showing vasodilator responses of the rat mesenteric vascular bed to ATP (), 2-meSATP (), adenosine (), 2-CADO (), NECA (), CPA () and CGS 21680 (). Vasodilator response are shown as percent vasodilatation of the methoxamine sustained tone taken as 100% and are the mean of 4 to 7 preparations. Response are to bolus injections of drugs. Symbols show means ± SEM (From Rubino et al., 1995, Br J Pharmacol 115: 648-652; with permission from McMillam Press Limited).   d. Concentration-dependent relaxation of guinea pig pulmonary arteries by NECA (; n = 5), CADO (; n = 5), adenosine (; n = 16), CGS 21680 (; n = 5), R-PIA (; n = 5) or CPA (; n = 15). Relaxant responses are expressed as a percentage of the noradrenaline-contraction (mean ± SEM). (From Szentmiklósi et al., 1995).

An interesting development in this field is provided by a study of A_2A receptor knockout mice (Ledent et al., 1997). These mice showed reduced exploratory activity. Caffeine, which normally stimulates locomotor activity, substantially depressed activity. The A_2A knockout mice also showed increased aggresiveness, hypoalgesia, an increase in blood pressure and heart rate, and an increase in platelet aggregation (Ledent et al., 1997). It is satisfying that these findings are broadly consistent with those predicted from studies of the endogenous A_2A receptor in isolated cells and tissues, and in whole animals.

	V. A2B Receptor

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A_2B receptors have been cloned from human hippocampus (Pierce et al., 1992), rat brain (Rivkees and Reppert, 1992; Stehle et al., 1992), and mouse bone marrow-derived mast cells (Marquardt et al., 1994) (table 3). The human A_2B adenosine receptor gene (ADORA2B) has been localized to chromosome 17p11.2-p12 (Townsend-Nicholson et al., 1995b) and 17p12 (Jacobson et al., 1995a). A human A_2B receptor pseudogene has been cloned and localized to chromosome 1q32 (Jacobson et al., 1995a). Although the pseudogene is unable to encode a functional receptor, it is 79% identical with the functional A_2B receptor. Thus, it was noted that the existence of the transcript in tissues could lead to misinterpretation of in situ hybridization and northern blot analysis when probes are used to recognize sequences common to these receptors (Jacobson et al., 1995a). As with the other adenosine receptor subtypes, there is considerable species differences in the sequence of the A_2B receptor; for example, 86% amino acid sequence homology between rat and human A_2B receptors (Stehle et al., 1992; Pierce et al., 1992; Linden, 1994).

A_2B receptor coupling to different signaling pathways has been reported, including activation of adenylate cyclase, G_q/G₁₁-mediated coupling to PLC and IP₃-dependent increase in [Ca²⁺]_i (in human mast cells) (Feoktistov and Biaggioni, 1995), and coupling to PLC when expressed in Xenopus oocytes (Yakel et al., 1993).

The lack of A_2B receptor-selective agonists has undoubtedly contributed to the general lack of information on A_2B receptor desensitization. In rat PC12 cells, the A_2B response has been shown to be reduced in A_2A-desensitized cells, possibly through common inhibition of adenylate cyclase (Chern et al., 1993). In mutant NG108-15 cells overexpressing GRK2, desensitization of endogenous A_2B receptors was markedly less than that in normal cells (t_1/2 15-20 min), indicating that receptor phosphorylation and uncoupling from G proteins may be involved in desensitization of A_2B receptors (Mundell et al., 1997). Although it is not yet clear whether there are inherent differences in the rates of desensitization of A_2A and A_2B receptors, the lower affinity of A_2B receptors for adenosine raises the possibility that they may still be fully operational, and thus may act as a backup for adenosine responses, when the higher affinity coexpressed A_2A receptors have been activated and desensitized.

Despite intensive efforts in this area, there are no A_2B-selective agonists. Thus, at present, activation of adenylate cyclase in membranes and accumulation of cAMP in cells is used to characterize A_2B receptors, provided a lack of activity/binding of A₁-, A_2A-, and A₃-selective agonists is confirmed. As with A_2A receptors, A_2B receptors show a preference for adenosine derivatives with modifications of the C2 position of the adenine ring. NECA is currently the most potent agonist at A_2B receptors, having low micromolar affinity (Brackett and Daly, 1994; Alexander et al., 1996; Klotz et al., 1998), but is less useful in characterization of A_2B receptors in cells or tissues in which A_2A receptors are coexpressed because it is non-selective. 2-ClADO, N⁶-(3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine (IB-MECA), and R-PIA are among the more potent of other conventional adenosine-receptor agonists that act also at A_2B receptors, but their affinity for the A_2B receptor is relatively low (EC₅₀ values 9 to 11 µM) (Brackett and Daly, 1994; Klotz et al., 1998).

Enprofylline blocks A_2B receptors in human mast cells HMC-1 (K_i 7 µM) and canine BR mastocytoma cells and is inactive at A₁, A_2A, and A₃ receptors. It may, therefore, be a valuable starting compound from which to develop more potent selective A_2B receptor antagonists (Feoktistov and Biagionni, 1996). The non-xanthine alloxazine has been reported as having approximately 9-fold selectivity for the A_2B compared with the A_2A receptor (Brackett and Daly, 1994). XAC and CGS 15943 are antagonists with low nanomolar affinity at A_2B receptors, but are non-selective versus other subtypes of adenosine receptor (Alexander et al., 1996; Klotz et al., 1998).

A_2B receptors are found on practically every cell in most species; however, the number of receptors is small and relatively high concentrations of adenosine are generally needed to evoke a response. The sensitive technique of reverse transcription-polymerase chain reaction (RT-PCR) showed low levels of A_2B receptors in all rat brain regions tested (Dixon et al., 1996). Northern blot analysis showed relatively high expression of A_2B receptors in the caecum, large intestine, and urinary bladder, with lower levels in the brain, spinal cord, lung, vas deferens, and pituitary (Stehle et al., 1992). RT-PCR revealed the highest expression of A_2B receptors in the proximal colon, with lower levels in the eye, lung, uterus, and bladder; still lower levels in the aorta, stomach, testis, and skeletal muscle; and the lowest levels in the jejunum, kidney, heart, skin, spleen, and liver (Dixon et al., 1996).

Selected distributions and biological effects mediated by A_2B receptors in isolated cells and tissues are listed in tables 4 and 6. Functional studies have identified A_2B receptors in airway smooth muscle, fibroblasts, glial cells, the gastrointestinal tract, and the vasculature. A_2B receptors have been cloned from, and immunolocalized on, mouse bone marrow-derived mast cells (Marquardt et al., 1994), and shown to mediate degranulation of canine BR mastocytoma cells (Auchampach et al., 1997a). They have also immunolocalized and been shown to activate human mast cells (Feoktistov and Biagionni, 1996). This implies a possible role in allergic and inflammatory disorders. The antiasthmatic effects of enprofylline, a potential A_2B receptor antagonist, are consistent with this hypothesis (Feoktistov and Biaggioni, 1996).

Vascular A_2B receptors identified by pharmacological and biochemical studies are listed in table 6, which shows that these receptors may couple to a functional response (vasodilatation) in both smooth muscle and endothelium. Interestingly, A_2B receptors seem to be important in mediating vasodilatation in some vessels, including the rat mesenteric arterial bed (Rubino et al., 1995) and guinea-pig pulmonary arteries (Szentmiklósi et al., 1995), but not in others where the A_2A subtype predominates (table 6, fig. 5). Rat aortic smooth muscle A_2B receptors have been implicated in inhibition of growth (Dubey et al., 1996), identifying a possible long-term trophic role for these receptors.

	VI. A3 Receptor

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A₃, the fourth distinct adenosine receptor, was identified relatively late in the history of adenosine/P1 receptors with the cloning, expression, and functional characterization of a novel adenosine receptor from rat striatum (Zhou et al., 1992). This was identical with a clone previously isolated from a rat testis cDNA library encoding a G protein-coupled receptor with greater than 40% sequence homology with canine A₁ and A_2A adenosine receptors, although its ligand had not then been identified (Meyerhof et al., 1991). The recombinant striatal A₃ receptor does not resemble any other adenosine/P1 subtypes in agonist or antagonist binding; it binds ligands with a potency order of R-PIA = NECA > S-PIA and is coupled to inhibition of adenylate cyclase activity in a pertussis toxin-sensitive manner; it binds with high affinity to the radioligand N⁶-2-(3-iodo-4-aminophenyl)ethyladenosine but not to the A_2A-selective adenosine ligand [³H]CGS 21680 or the alkylxanthine antagonists XAC, IBMX, or the A₁-selective antagonist DPCPX.

Homologs of the rat striatal A₃ receptor have been cloned from sheep pars tuberalis (pituitary tissue) (Linden et al., 1993), human heart (Sajjadi and Firestein, 1993, and striatum (Salvatore et al., 1993) (see also Linden, 1994) (table 3). Interspecies differences in A₃ receptor structure are large; the rat A₃ receptor shows only approximately 74% sequence homology with sheep and human A₃ receptors each, although there is 85% homology of sheep and human A₃ receptors. This is reflected in the very different pharmacological profiles of the species homologs, particularly with respect to antagonist binding, and this has caused considerable complications in the characterization of this receptor. The human A₃ receptor has been localized to chromosome 1 p13.3 (Monitto et al., 1995).

The rat, but not the human, A₃ receptor transcript may be subject to extensive alternative splicing, further evidence of the profound interspecies differences involving the A₃ receptor. A splice variant of the rat A₃ receptor (A_3i), having a 17 amino acid insertion within the second intracellular loop, has been cloned and characterized (Sajjadi et al., 1996). There was no evidence for alternative splicing of the human A₃ receptor transcript (Sajjadi et al., 1996).

This A₃ receptor has taken precedence over the controversial A₃ receptor defined principally according to its pharmacological profile by Ribeiro and Sebastiào (1986), which probably represents an A₁ receptor (Carruthers and Fozard, 1993; Ribeiro and Sebastiào, 1994).

The A₃ receptor is G protein-linked, coupling to G_i2-, G_i3- and, to a lesser extent, to G_q/11 proteins (Palmer et al., 1995b). In rat basophilic leukemia cells (RBL-2H3; a cultured mast cell line) (Ali et al., 1990; Ramkumar et al., 1993b) and in rat brain (Abbracchio et al., 1995a), the A₃ receptor stimulates PLC and elevates IP₃ levels and intracellular Ca²⁺. PKC has been suggested to be involved in A₃ receptor-mediated preconditioning in rabbit cardiomyocytes (Armstrong and Ganote, 1994). The A₃ receptor has also been shown to inhibit adenylate cyclase activity (Zhou et al., 1992; Abbracchio et al., 1995b).

Recombinant rat and human A₃ receptors have been shown to desensitize within minutes in response to agonist exposure; this is associated with uncoupling of the receptor-G protein complex, as indicated by a reduction in the number of high affinity binding sites (Palmer et al., 1995a; Palmer et al., 1997). Desensitization of the rat A₃ receptor is rapid (within a few minutes), homologous, and is associated with rapid phosphorylation by a G protein-coupled receptor kinase similar to, or identical with, GRK2 (Palmer et al., 1995a; Palmer and Stiles, 1997b). Rapid, homologous functional desensitization of A₃ receptors has also been described in RBL-2H3 cells (Ali et al., 1990; Ramkumar et al., 1993b). A chimeric A₁-A₃ receptor constructed from an A₁ receptor (non-desensitizing under the conditions of the study) and the C-terminal domain of an A₃ receptor was expressed in CHO cells and shown to undergo rapid desensitization. This indicates that the C-terminal domain of the A₃ receptor is the site for phosphorylation by the G protein-coupled receptor kinases involved in desensitization (Palmer et al., 1996).

The effects of long-term agonist exposure on interaction of the rat A₃ receptor with G proteins was assessed using a transfected CHO cell system (Palmer et al., 1995b). Chronic exposure of A₃ receptors to the non-selective agonist NECA (for up to 24h) causes selective down-regulation of G_i3- and -subunits, without changing levels of G_i2 or G_q-like proteins (Palmer et al., 1995b).

In situ hybridization identified the A₃ receptor in mesenchymal cells and eosinophils within the lamina propria of the airways and the adventitia of blood vessels in the lung, as well as in peripheral eosinophils, but interestingly, not in mast cells (Walker et al., 1997). It was found that the A₃ receptor transcript was greater in lung tissue from subjects with airway inflammation than in normal lung. This is consistent with the hypothesis that there is a distinct distribution of the A₃ receptor in inflammatory cells and that this is up-regulated in airway inflammation (Walker et al., 1997).

The main class of selective A₃ receptor agonists is the N⁶-substituted adenosine-5'-uronamides. N⁶-benzylNECA is potent (K_i 6.8 nM) and moderately selective (13- and 14-fold versus A₁ and A_2A) at rat A₃ receptors transfected into CHO cells (van Galen et al., 1994). N⁶-(3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine (IB-MECA) (K_i 1.1 nM) is 50-fold selective for rat brain A₃ receptors versus A_2A or A₁ receptors (Gallo-Rodriguez et al., 1994) (fig. 2). The iodinated radioligand [¹²⁵I]AB-MECA binds with approximately nanomolar affinity to rat brain A₃ adenosine receptors expressed in CHO cells, but also binds to native A₁ receptors. Selectivity is increased by 2-substitution of N⁶-benzyladenosine-5'-uronamides; 2-chloro-IB-MECA (2Cl-IB-MECA, K_i = 0.33 nM) is highly selective for A₃ versus A₁ and A_2A receptors, by 2500- and 1400-fold, respectively (Kim et al., 1994) (fig. 2). There is pronounced interspecies differences in the relative affinities of agonist binding at A₃ receptors (Ji et al., 1994; Linden, 1994).

Several classes of compounds have been developed as A₃ antagonists. One class comprises xanthines and their derivatives. Rat, rabbit, and gerbil brain A₃ receptors bind only weakly to xanthine derivatives compared with human and sheep A₃ receptors, which exhibit high affinity (Zhou et al., 1992; Linden et al., 1993; Salvatore et al., 1993; Ji et al., 1994). The most potent of the 8-phenyl-substituted xanthines, I-ABOPX (3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine, or BW-A522) binds with nanomolar affinity to human and sheep A₃ receptors (Linden et al., 1993; Salvatore et al., 1993), but by contrast with micromolar affinity at rabbit, gerbil, and rat A₃ receptors (Ji et al., 1994).

Five chemical classes of non-xanthine antagonists have been reported. L-268605 (3-(4-methoxyphenyl)-5-amino-7-oxo-thiazolo [3, 2]pyrimidine) is a potent and selective A₃ antagonist with a K_i value of 18 nM and no appreciable affinity for human A₁ and A_2A receptors (Jacobson et al., 1996) (fig. 3). Another class is represented by L-249313 (6-carboxymethyl-5,9-dihydro-9-methyl-2-phenyl-[1, 2, 4]-triazolo[5,1-a][2, 7]naphthyridine) with high affinity at cloned human A₃ receptors, K_i value of 13 nM, but low affinity at native rat brain A₃ receptors, K_i 58 µM, and selectivity of approximately 300- and 1460-fold over A₁ and A_2A receptors, respectively (Jacobson et al., 1996) (fig. 3).

The three other categories of molecules with promise as A₃ receptor antagonists are the flavonoid MRS 1067 (3,6-dichloro-2'isopropyloxy-4'-methyl-flavone), the 6-phenyl-1,4-dihydropyridines MRS 1097 (3,5-diethyl[2-methyl-6-phenyl-4-(2-phenyl-(E)-vinyl]-1,4-(±)-dihydropyridine-3,5-dicarboxylate) and MRS 1191 (3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate) and the triazoloquinazolene MRS 1220 (9-chloro-2-(2-furyl)-5-phenylacetylamino[1, 2, 4]triazolo[1,5-c]quinazoline). Of these, MRS 1220 and MRS 1197 show promise as potent and selective competitive antagonists, with K_i values of 0.6 and 31 nM, respectively, for inhibition of [¹²⁵I]AB-MECA binding and K_B values of 1.7 and 92 nM at human recombinant A₃ receptors (Jacobson et al., 1997). A much lower affinity was observed at the rat A₃ receptor: >2000-fold for MRS1220 and 112-fold for MRS 1197 (Jacobson et al., 1997) as has been noted with xanthine-based antagonists.

The A₃ receptor is widely distributed, but its physiological role is still largely unknown. A₃ mRNA is expressed in testis, lung, kidneys, placenta, heart, brain, spleen, liver, uterus, bladder, jejunum, proximal colon, and eye of rat, sheep, and humans (Zhou et al., 1992; Linden et al., 1993; Salvatore et al., 1993; Linden, 1994; Rivkees, 1994; Dixon et al., 1996) (fig. 4). A₃ mRNA was not detected in rat skin or skeletal muscle (Dixon et al., 1996) (fig. 4). Rat testis seems to have particularly high concentrations of A₃ mRNA (in spermatocytes and spermatids), compared with rather lower levels in most other rat tissues (Linden et al., 1993; Salvatore et al., 1993). The highest levels of human A₃ mRNA are found in lung and liver, with lower levels in aorta and brain (Salvatore et al., 1993). In sheep, the highest levels of A₃ mRNA are found in lung, spleen, pars tuberalis, and pineal gland (Linden et al., 1993). PCR was used to establish the presence of A₃ receptors in rabbit cardiac myocytes (Wang et al., 1997).

The A₃ receptor on mast cells facilitates the release of allergic mediators including histamine, suggesting a role in inflammation (Ramkumar et al., 1993b). Systemic administration of 3-IB-MECA causes scratching in mice that is prevented by coadministration of a histamine antagonist (Jacobson et al., 1993b). APNEA has been shown to be a bronchoconstrictor in rats in vivo, an effect that may be mediated by mast cells (Pauwels and Joos, 1995), but it does not elicit bronchoconstriction in rabbits (el-Hashim et al., 1996). Constriction mediated by adenosine in isolated arterioles of golden hamster cheek pouches is blocked by an inhibitor of mast cell degranulation, which suggests a role for A₃ receptors on mast cells in this response (Doyle et al., 1994).

The A₃ receptor has been implicated in the 8-SPT-resistant hypotensive response to APNEA in the pithed rat (Fozard and Carruthers, 1993). The response is pertussis toxin-sensitive and is blocked by the A₃ receptor antagonist BW-A522 (Fozard and Hannon, 1994). However, it seems that the hypotensive response may be caused by the secondary action of histamine released after activation of mast cell A₃ receptors (Hannon et al., 1995).

Systemic administration of 3-IB-MECA depresses locomotor activity in mice, which may suggest a role for brain A₃ adenosine receptors in modulation of behavior (Jacobson et al., 1993b). Interestingly, activation of rat hippocampal A₃ receptors has been shown to desensitize A₁ receptor-mediated inhibition of excitatory neurotransmission in this brain region, indicating cross-talk between these two receptors (Dunwiddie et al., 1997).

A₃ receptors on human eosinophils (Kohno et al., 1996a) and human promyelocytic HL-60 cells (Kohno et al., 1996b; Yao et al., 1997) seem to be involved in apoptosis, an active self-destructive process caused by a genetically programmed cascade of molecular events involving DNA degradation and death of the cell by nuclear and cytoplasmic breakup. This seems to require high concentrations of agonist or chronic activation of the A₃ receptor in a manner that mimicks the requirement of high levels of ATP to activate the non-specific pore-formation of the P2X₇ receptor and apoptosis, and suggests that this potentially autocatalytic process may occur during pathological conditions resulting in cell damage and release of high levels of purines. Apoptotic effects are caused by high concentrations (micromolar) of A₃ receptor agonist in HL-60 leukemia and U-937 lymphoma cells, but paradoxically, A₃ receptor antagonists also induce apoptotic cell death, and this is opposed by low (nanomolar) concentrations of Cl-IB-MECA (Yao et al., 1997). This indicates that low-level activation of A₃ receptors may result in cell protection, and furthermore that this may occur as a consequence of endogenously released adenosine (Yao et al., 1997). Acute stimulation of A₃ receptors with micromolar concentrations of Cl-IB-MECA has also been shown to cause lysis of granular hippocampal neurons in culture (Von Lubitz et al., 1996).

A₃ receptors may be involved in the cardioprotective effect of adenosine in ischemia and preconditioning during ischemia reperfusion injury (Liu et al., 1994; Armstrong and Ganote, 1994, 1995; Auchampach et al., 1997b; Stambaugh et al., 1997). Preconditioning is blocked by A₃ receptor antagonists, whereas APNEA (A₁/A₃ selective), but not R-PIA (A₁ selective), protect against ischemia in rabbit cardiomyocytes (Armstrong and Ganote, 1995). A₃ receptors have been shown to mediate preconditioning and to reduce myocardial injury (Strickler et al., 1996; Tracey et al., 1997). In isolated cardiac myocytes, maximal preconditioning-induced cardioprotection was shown to require activation of both A₁ and A₃ receptors (Wang et al., 1997). Acute IB-MECA has a detrimental effect on ischemic brain injury, whereas chronic IB-MECA has a protective effect (Von Lubitz et al., 1994). This dual effect mimicks the effects of Cl-IB-MECA on leukemia and lymphoma cell lines (Yao et al., 1997). Activation of an A₃ receptor in basophilic leukemia cells (RBL-2H3), endothelial cells, cardiac myocytes, and smooth muscle cells activates the cellular antioxidant defense system by increasing the activity of superoxide dismutase, catalase, and glutathione reductase, thereby providing a means by which adenosine may have a cytoprotective action in ischemia (Maggirwar et al., 1994).

	VII. Integrated Effects of Adenosine/P1 Receptors

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A₁, A_2A, A_2B, and A₃ adenosine receptors have distinct but frequently overlapping tissue distributions. The fact that more than one adenosine/P1 receptor subtype may be expressed by the same cell raises questions about the functional significance of this colocalization. Because the different adenosine/P1 receptor subtypes have quite different affinities for the endogenous agonist, the local concentration of adenosine in physiological and pathophysiological conditions is likely to be extremely important. EC₅₀ values for adenosine at rat A₁, A_2A, A_2B, and A₃ receptors of 73 (Daly and Padgett, 1992), 150 (Daly and Padgett, 1992), 5100 (Peakman and Hill, 1994), and 6500 (Zhou et al., 1992), respectively, have been reported. At rat phrenic motor nerve terminals (Correia-de-Sá et al., 1996) and prejunctional receptors in rat vas deferens (Gonçalves and Queiroz, 1993), the concentration of adenosine needed to increase transmitter release via activation of A_2A receptors seems to be higher than that required to inhibit transmitter release via A₁ receptors. Because adenosine is formed as a breakdown product of ATP released from nerves, this implies that the adenosine concentration is crucially linked to the ongoing neuronal activity, which therefore may be an important determinant of the subtype of autoregulatory adenosine receptor that is activated. In rat hemidiaphragm, the frequency and intensity of stimulation of motor nerves and subsequent formation of endogenous adenosine was shown to be critical, with high-intensity, high-frequency nerve stimulation favoring A_2A receptor-mediated facilitation of [³H]acetylcholine (ACh) release (Correia-de-Sá et al., 1996). Thus, adenosine concentration and receptor affinity may determine the pattern of differential activation of coexpressed A₁ and A_2A receptors (and other adenosine receptors).

Expression of more than one type of adenosine/P1 receptor on the same cell may allow the common agonist adenosine to activate multiple signaling pathways. Adenylate cyclase is a common effector, which is negatively coupled to A₁ and A₃ receptors and positively coupled to A₂ receptors, affording the opportunity for reciprocal control and, therefore, fine tuning of this signaling pathway. Coexisting A₁ and A₂ adenosine receptors with opposite actions on adenylate cyclase activity have been described in a number of cells, including the smooth muscle cell line DDT₁ MF-2 (Ramkumar et al., 1991), cultured porcine coronary artery smooth muscle cells (Mills and Gewirtz, 1990), and glomeruli and mesangial cells (Olivera and Lopez-Novoa, 1992). A₁ and A_2B receptors on primary rat astrocytes each regulate adenylate cyclase activity, but independently (Peakman and Hill, 1994).

The extracellular adenosine concentration may be a crucial determinant of the differential activation of coexisting adenosine/P1 receptors under pathophysiological as well as physiological conditions. Induction and inhibition of the inflammatory response by neutrophil A₁ and A₂ receptors, respectively, has been reported (Cronstein, 1994; Bullough et al., 1995). Low concentrations of adenosine caused activation of the A₁ receptor and induced superoxide anion generation, phagocytosis via Fc receptors, and adhesion to endothelial cells, whereas higher concentrations of adenosine (>500 nM) required to saturate A₂ receptors lead to inhibition of these effects. A_2A and A_2B receptors coexist on fetal chick heart cells; the high affinity A_2A receptor has been suggested to be an important modulator of myocyte contractility under physiological conditions, whereas under pathophysiological conditions, such as cardiac ischemia resulting in release of large amounts of adenosine, the low affinity A_2B receptor may assume functional significance (Liang and Haltiwanger, 1995). Such studies are helping to expand on the established link between adenosine release and the metabolic demands of tissues by building in specific actions on identified cell-surface adenosine/P1 receptors.

Stimulation of the A_2A receptor on rat striatal synaptosomes causes desensitization of coexpressed A₁ receptors, favoring A_2A receptor-mediated signaling (Dixon et al., 1997a). This has important implications for other coexpressed adenosine receptors, and it would be interesting to see if this is a general phenomenon for these subtypes.

There is an interesting sidedness to the opposite responses evoked by A₁-like and A_2A-like adenosine receptors colocalized on monolayers of renal epithelial cells (Casavola et al., 1997). The A₁-like receptors are located on the apical surface and mediate inhibition of transepithelial Na⁺ transport by (a) inhibition of the basolaterally located Na⁺/H⁺ exchanger and (b) an increase in intracellular H⁺, probably via Ca²⁺/PKC. The A_2A-like receptors are located on the basolateral side and stimulate transepithelial Na⁺ transport, suggested to be via stimulation of Na⁺/H⁺ exchange and thereby cellular alkalinization, probably via an increase in cAMP/PKA (Casavola et al., 1997). The same adenosine receptor can elicit a different functional response in different tissues. In rat duodenum, A_2B (and A₁) adenosine receptors on the longitudinal muscle mediate relaxation, whereas A_2B receptors on the muscularis mucosae mediate contraction (Nicholls et al., 1996).

Integrated effects of adenosine/P1 receptors in whole tissue responses are considered, together with P2 receptors, in Section XXII.

	VIII. P2 Receptors

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P2 receptors are divided into two main classes based on whether they are ligand-gated ion channels (P2X receptors) or are coupled to G proteins (P2Y receptors) (Abbracchio and Burnstock, 1994; Fredholm et al., 1994) (table 7).

The P2X/P2Y nomenclature was adopted from that originally used in a subdivision of P2 receptors proposed in 1985 by Burnstock and Kennedy, who described "P_2X-" and "P_2Y-purinoceptors" with distinct pharmacological profiles and tissue distributions: the "P_2X-purinoceptor" was shown to be most potently activated by the stable analogs of ATP, ,-methylene ATP (,-meATP), and ,-meATP. At the "P_2Y-purinoceptor" 2-methylthio ATP (2MeSATP) was the most potent agonist and ,-meATP and ,-meATP were weak or inactive. Furthermore, the "P_2X-purinoceptor" was shown to be selectively desensitized by ,-meATP and to be antagonized by 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]-propionyl)ATP (ANAPP₃) (Burnstock and Kennedy, 1985). Distinct tissue distributions and functions reinforced this subdivision: "P_2X-purinoceptors" were shown to be present in vas deferens, urinary bladder, and vascular smooth muscle, and to mediate contraction; "P_2Y-purinoceptors" were shown to be present in guinea-pig taenia coli and on vascular endothelial cells, as well as to mediate relaxation. P2 receptors have since been cloned from smooth muscle and endothelium; the pharmacological profiles originally attributed to "P_2X-" and "P_2Y-purinoceptors" seem to correspond most closely to activation of P2X₁-like and P2Y₁-like receptors, respectively. However, it is now apparent that there is heterogeneity of P2X responses among different smooth muscles, and of P2Y responses between taenia coli and endothelium, which may be caused by different receptor subtypes or small differences in structure of the same receptor.

Other P2 receptors that have been identified in biological tissue principally according to their different pharmacological profiles are the P_2U receptor (activated equally by ATP and UTP; widely distributed), the P_2T receptor (platelet ADP receptor; mediates aggregation), and the P_2Z receptor (found on mast cells and lyphocytes; mediates cytotoxicity and degranulation) (Gordon, 1986; O'Connor et al., 1991). P_2S (Wiklund and Gustafsson, 1988), P_2R (Von Kügelgen and Starke, 1990), P_2D (Pintor et al., 1993), uridine nucleotide-specific receptors ("pyrimidinoceptors") (Seifert and Schultz, 1989; Von Kügelgen and Starke, 1990), P3 (Shinozuka et al., 1988; Forsythe et al., 1991), and P4 (Pintor and Miras-Portugal, 1995a) receptors have also been proposed. Of these the P_2U, P_2Z, and uridine nucleotide-specific receptors have been cloned. Because receptor subclassification based on pharmacological criteria alone is no longer tenable, the separate identity of the other proposed subtypes remains to be proved.

The revision of P2 receptor nomenclature was prompted by evidence that extracellular ATP works through two different transduction mechanisms, namely intrinsic ion channels and G protein-coupled receptors (Benhan and Tsien, 1987; Dubyak, 1991), and by the cloning of the first two P2 receptors, P2Y₁ (a "P_2Y-purinoceptor") (Webb et al., 1993b) and P2Y₂ (a "P_2U-purinoceptor") (Lustig et al., 1993). It was also becoming increasingly apparent that there was significant heterogeneity among native P2 receptors, reflected in an increasing diversity of pharmacological response profiles that could not easily be accommodated within the existing system of receptor subclassification. Thus, in 1994 it was formally suggested that P2 receptors should be divided into two broad groups termed P2X and P2Y according to whether they are ligand-gated ion channels or are coupled to G proteins, respectively, with subtypes defined by the different structure of mammalian P2 receptors (Abbracchio and Burnstock, 1994; Barnard et al., 1994; Fredholm et al., 1994).

To date seven mammalian P2X receptors, P2X_1-7, and five P2Y receptors, P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ have been cloned, characterized pharmacologically and accepted as valid members of the P2 receptor family. The use of lower case to define the cloned p2y3 receptor reflects the possibility that this may be the avian homolog of the human P2Y₆ receptor, although this has not yet been confirmed. The jump in sequence in the numbering of the P2Y receptor family is caused by the recognition that certain receptors had been erroneously identified as belonging to this family, leading to the subsequent withdrawal of P2Y₅ (Webb et al., 1996b) and P2Y₇ (Akbar et al., 1996). The cloned receptors P2Y₉ and P2Y₁₀ are also not nucleotide receptors. A P2Y receptor cloned from Xenopus neural plate (provisionally called P2Y₈) is not included in the definitive P2Y receptor family recognized by the IUPHAR committee, based largely on the rationale that this is a non-mammalian receptor. The platelet ADP receptor P2Y_ADP (or P_2T receptor) has not yet been cloned and, therefore, as recommended by the IUPHAR committee, the name of this receptor is given in italics.

P2Y₄ (human but not rat receptor) and P2Y₆ are uridine nucleotide-specific receptors (receptors not activated or only weakly activated by purines) that have been cloned and shown to be sensitive preferentially to UTP and UDP, respectively (the rat P2Y₄ receptor is also activated potently by ATP; see Section XV). Their identification complements earlier suggestions of the existence of endogenous uridine nucleotide-specific receptors based on distinct pharmacology of some biological tissue. Before the cloning of these receptors, the possibility that there were subtypes of endogenous uridine nucleotide-specific receptors was not considered, and by implication the possibility of different UTP/UDP selectivities for members of this family was not appreciated. Thus, in much of the literature to date, the agonist potency profiles documented for endogenous uridine nucleotide-specific receptors are incomplete, leaving open the possibility that these are P2Y₄ or P2Y₆ receptors, or some other subtype not yet cloned. The lack of selective agonists and antagonists, and complications introduced by receptor coexpression and agonist interconversion, means that the subtype identity of most endogenous uridine nucleotide-specific receptors is currently unclear. Because of this, a separate section in this review is devoted to endogenous uridine nucleotide-specific receptors (see Section XVIII.). Interestingly, the P2Y₁₁ receptor is so far the only P2Y receptor selective for ATP versus other purine and pyrimidine nucleotides.

For researchers in this field, important discoveries made in the last 10 years have been the source of insight, and in some cases frustration, because these demand a reevaluation of conclusions drawn from earlier studies on P2 receptors. These include the discovery that: (a) multiple P2X receptor proteins are often coexpressed in different proportions in different tissues; (b) P2X receptors are multisubunit receptors that may exist as heteromers with different pharmacology compared with the homomers; (c) cations can profoundly affect P2X channel activity; (d) 2MeSATP, previously widely regarded as a selective "P_2Y-purinoceptor" agonist, is also a potent agonist at P2X receptors; (e) ecto-nucleotidases can profoundly alter agonist potencies; and (f) antagonists previously used with some confidence as P2 receptor blockers are non-selective, can modulate ecto-nucleotidase activity and may have allosteric effects on P2 receptors. The general lack of selective agonists and antagonists, together with complications introduced by coexistence of different P2 receptors and impure solutions caused by purine and pyrimidine degradation and interconversion, also has significantly hindered advances in P2 receptor characterization.

Although much valuable information can be derived from studies of populations of cells in culture, there are potential pitfalls associated with this technique. Thus, emerging evidence that the expression of P2 receptors may alter in culture conditions adds another potential complication to the study of purine receptors. For example, astrocytes studied in situ, or after acute isolation from rat brain, are insensitive or only a few cells respond to ATP, whereas in primary cultures, there is a profound increase in the number of cells responding to ATP (Jabs et al., 1997; Kimelberg et al., 1997). Similarly, up-regulation of the P2Y₂ receptor in rat salivary gland cells in culture compared with acutely isolated cells has been reported (Turner et al., 1997). Thus, caution is needed in the interpretation of studies of P2 receptors on cells in culture.

Autocatalytic release of ATP has been shown from endothelial cells (Yang et al., 1994) and it is possible that this phenomenon will be described for other cell types as well as for other purines and pyrimidines. In addition, ATP is released from many different cells in response to stimuli such as shear stress and hypoxia, which may be relevant for the ongoing level of activation of purine receptors expressed by the same or neighboring cells. This may be particularly important with respect to the activity of P2X₁ and P2X₃ receptors, as these receptors desensitize rapidly.

Because of the diverse reasons discussed above, it is currently a considerable challenge to dissect out and characterize endogenous receptors for purines and pyrimidines in different biological systems, and even more of a challenge to identify for each of these a physiological or pathophysiological role. However, endogenous receptor counterparts have been shown for some cloned P2 receptors, matching both in terms of receptor distribution, signaling mechanisms, and pharmacology. In this review, we use the name of the clone in preference to the classical nomenclature where possible to promote the conversion from the older system to the newer terminology. However, because for the majority of cases this characterization is currently equivocal, we qualify this with the term "-like". Thus, "P2X₁-like receptor" replaces the classical "P_2X-purinoceptor" of smooth muscle, "P2X₇-like receptor" is used for the "P_2Z-purinoceptor", "P2Y₁-like receptor" is used in preference to the classical "P_2Y-purinoceptor," and "P2Y₂-like receptor" replaces "P_2U-purinoceptor". Unequivocal characterization of endogenous P2 receptors awaits the development and use of subtype-selective agonists and antagonists.

P2 receptors have broad natural ligand specificity, recognizing ATP, ADP, UTP, UDP, and the diadenosine polyphosphates (table 7). The chemical structures of some principal P2 receptor agonists are illustrated in figure 6. At present there are no agonists or antagonists that discriminate adequately between families of P2X and P2Y receptors, or between subtypes of receptors within each of these groups (table 7). Some of the most useful agonists are the stable ATP analogs ,-meATP and ,-meATP, which if effective, strongly imply actions at P2X receptors (specifically at P2X₁ and P2X₃ subtypes) and are generally inactive at P2Y receptors. Also useful are ADP, adenosine 5'-O-(2-thiodiphosphate)(ADPS,) and UTP, as these are agonists at some P2Y receptors, but are weak or inactive at P2X receptors.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   The chemical structure of some key agonists at P2 receptors. (Adapted from Windscheif, 1996).

Agonist potency orders, important in the characterization of cloned and native P2 receptors, are profoundly influenced by the different stabilities of P2 receptor ligands in the presence of ecto-nucleotidases. ,-MeATP is considerably more stable than ATP and 2MeSATP when ecto-nucleotidase activity is not suppressed, which contributes significantly to its greater potency (up to 100-fold more potent) at native P2X₁ receptors in vascular smooth muscle, bladder, and vas deferens. However, when ecto-ATPase effects are controlled by use of single cells and rapid concentration-clamp applications of agonist, or by inhibition of ecto-ATPase activity [for instance using 6-N,N-diethyl-D-,-dibromomethylene ATP (ARL 67156) or removal of divalent cations], ,-meATP is less potent than 2MeSATP and ATP at native and cloned P2X₁ receptors (Crack et al., 1994; Evans and Kennedy, 1994; Humphrey et al., 1995; Khakh et al., 1995b). Thus, greater caution is now advised in the interpretation of the order of agonist potency where ecto-nucleotidase activity has not been suppressed. This is a particularly important consideration in the pharmacology of P2X receptors because of the wide range of stabilities of commonly-used P2X agonists, but seems to have had less of an impact on P2Y receptor profiles, probably because many of the commonly used P2Y agonists are similarly unstable. An additional consideration is that many P2 receptor antagonists inhibit ecto-nucleotidase activity, which may reduce their effectiveness against biologically unstable P2 agonists.

Antagonists selective for subtypes of P2X and P2Y receptors are considered in later sections on individual receptors (see Sections X.F., XII.E., XVIV.D.). This section considers other established and putative P2 receptor antagonists, which, unfortunately, do not discriminate well, if at all, between P2X or P2Y receptors, let alone for subtypes within these families (table 7). Many of these also inhibit ecto-nucleotidases and may have allosteric effects on the receptor (Michel et al., 1997). Table 8 summarizes the potencies of some of the most commonly used antagonists at recombinant and endogenous P2 receptors. The general lack of selective antagonists highlights the problems currently encountered in subtype-identification of P2 receptors using ligand binding. The chemical structures of some P2 receptor antagonists are illustrated in figure 7.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   The chemical structures of some P2 receptor antagonists. (Adapted from Windscheif, 1996).

In principle, any P2 receptor antagonist should be tested for its selectivity against all known subtypes of this family. Evaluation of antagonist selectivity at heteromeric P2X receptors is also important because of its relevance for biological tissue where P2X receptor proteins are typically coexpressed; such studies might additionally provide useful information about the specific contribution of the different subunits to the pharmacology of the receptor heteromer. A commonly used biological assay is antagonism of constriction by ,-meATP of vas deferens and vascular smooth muscle. This is generally taken as an indication of actions at endogenous P2X₁-like receptors for a number of reasons: (a) the P2X₁ receptor has been cloned from smooth muscle; (b) immunohistochemical studies have shown that it is the predominant P2X receptor protein expressed by smooth muscle; (c) ,-meATP is selective for P2X₁ and P2X₃ receptors, but the latter is not expressed by smooth muscle; and (d) the smooth muscle P2X response shows a similar pharmacology to the recombinant P2X₁ receptor, and as with the P2X₁ receptor, undergoes rapid desensitization. Relaxant effects of 2MeSATP or ADPS at guinea-pig taenia coli and via the vascular endothelium have been used to examine antagonist potencies at endogenous P2Y₁-like receptors. The potencies of antagonists at endogenous P2 receptors in these and other biological assays are reported in table 8b.

1. Suramin. The trypanoside suramin (8-(3-benzamido-4-methylbenzamido)-naphthalene-1,3,5-trisulfonic acid) is generally selective as an antagonist at P2 receptors versus other types of receptors (Dunn and Blakeley, 1988) (but see later this section), but is not a universal P2 receptor antagonist, and does not discriminate between P2X and P2Y receptors (table 8). Furthermore, suramin inhibits ecto-nucleotidase (Crack et al., 1994; Beukers et al., 1995; Ziganshin et al., 1995; Bültmann et al., 1996b; Chen et al., 1996c) and neural ecto-diadenosine polyphosphate hydrolase (Mateo et al., 1996) activity, which may complicate interpretation of antagonist activity when it is used against ligands which are biologically unstable.

2. NF023. NF023 (symmetrical 3'-urea of 8-(benzamido)naphthalene-1,3,5-trisulfonic acid) is a suramin-based compound which is moderately selective as an antagonist of P2X receptors. NF023 is about 30-fold selective for P2X₁-like receptors in the rat vas deferens versus P2Y₁-like receptors in the guinea-pig taenia coli (Bültmann et al., 1996b). It has 79-fold selectivity for endogenous P2X₁-like receptors in rabbit vas deferens versus P2Y₁-like receptors in turkey erythrocytes; pA₂ values of 5.5 to 5.7 at P2X₁-like receptors in rabbit isolated blood vessels, rabbit vas deferens, and rat and hamster mesenteric arterial beds; and pA₂ values of 4.6 to 5.5 at vascular and nonvascular smooth muscle P2Y₁-like receptors (Lambrecht et al., 1996; Ziyal, 1997; Ziyal et al., 1997). Its effects at the other P2X (and P2Y) receptor subtypes have not been reported. Antagonism is competitive and reversible. Like the parent compound suramin, NF023 inhibits ecto-nucleotidase activity, but unlike suramin, it has high P2X₁-like versus ecto-nucleotidase-selectivity (Beukers et al., 1995; Bültmann et al., 1996b).

3. NF279. NF279 (8, 8'-(carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3,5-naphthalenetrisulfonic acid) is a suramin analog that is about 10-fold more potent than NF023 in blocking ,-meATP-mediated contractions at P2X₁-like receptors in rat vas deferens, pIC₅₀ 5.7 (Damer et al., 1998). With a pA₂ value of 4.3 at P2Y₁-like receptors in the guinea-pig taenia coli, it has the highest P2X- versus P2Y- and ecto-nucleotidase-selectivity so far reported (Damer et al., 1998).

4. Pyridoxal-5-phosphate (P5P). P5P is a non-selective P2 receptor antagonist, but has proved useful as a starting compound for the synthesis of more P2X-selective antagonists (Lambrecht et al., 1996). Antagonism by P5P is, however, selective versus non-purine receptors and seems to be competitive at P2X₁-like receptors in vas deferens of rabbit (Lambrecht et al., 1996) and rat (Trezise et al., 1994b), and at ,-meATP-mediated depolarization of rat vagus nerve (Trezise et al., 1994b). P5P non-competitively inhibits responses mediated by recombinant receptors P2X₁ and P2X₂ but is less potent than its derivative pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (Evans et al., 1995). P5P inhibits ,-meATP-induced depolarization of rat superior cervical ganglion (Connolly, 1995).

5. PPADS. Although originally put forward as a P2X-selective antagonist, unfortunately it must now be accepted that PPADS is a non-selective (but non-universal) P2 receptor antagonist. PPADS is a slowly-equilibrating and slowly-reversible antagonist with pA₂ values of approximately 6 to 6.7 at endogenous P2X₁-like receptors in a variety of smooth muscle preparations (table 8; Lambrecht et al., 1996; Ziganshin et al., 1993, 1994b; Bültmann and Starke, 1994a; McLaren et al., 1994; Windscheif et al., 1994; Galligan et al., 1995; Von Kügelgen et al., 1995a; Eltze and Ullrich, 1996; Ralevic and Burnstock, 1996b; Usune et al., 1996). It also blocks recombinant P2X₁, P2X₂, P2X₃, and P2X₅ receptors with IC₅₀ values of 1 to 2.6 µM (Collo et al., 1996). A lysine residue in receptors P2X₁, P2X₂, and P2X₅ (amino acid 249 in P2X₁) seems to be involved in the slowly reversible component of block by PPADS, probably involving formation of a Schiff's base (Buell et al., 1996b). Rat recombinant P2X₄ and P2X₆ receptors are not blocked by PPADS (Buell et al., 1996b; Collo et al., 1996; Soto et al., 1996a,b; Garcia-Guzman et al., 1997a), but interestingly, the human homolog of the P2X₄ receptor is blocked by PPADS with an IC₅₀ of 28 µM (Garcia-Guzman et al., 1997a). PPADS antagonizes depolarizations induced by ,-meATP in rat superior cervical ganglion (Connolly, 1995).

6. Iso-PPADS. An isomer of PPADS, pyridoxalphosphate-6-azophenyl-2',5'-disulfonic acid (iso-PPADS) is a slowly-equilibrating and slowly-reversible antagonist of responses at P2X receptors with similar potency to PPADS (Trezise et al., 1994c) and competes for [³H],-meATP binding sites in the rat vas deferens (Khakh et al., 1994). Iso-PPADS blocks depolarizations evoked by ,-meATP, but not those to UTP in rat superior cervical ganglion, but in contrast to PPADS also blocks depolarizations to muscarine (Connolly, 1995).

7. Reactive blue 2. The anthraquinone-sulfonic acid derivative reactive blue 2 (synonymous with cibacron blue) is a non-competitive P2 receptor antagonist which does not discriminate adequately between P2X and P2Y subtypes. In the vasculature, it has micromolar affinity and some selectivity for endothelial P2Y₁ and smooth muscle P2Y₁-like receptors versus other vascular P2X and P2Y receptors; however, selectivity versus the smooth muscle P2X₁-like receptor is low, and its use is limited by a narrow effective concentration range and time of exposure (Burnstock and Warland, 1987a; Hopwood and Burnstock, 1987; Houston et al., 1987). Reactive blue 2 antagonism of P2Y receptors includes block of the recombinant P2Y₆ receptor (Chang et al., 1995) and some endogenous P2Y₂-like and uridine nucleotide-specific receptors (Nakaoka and Yamashita, 1995; Chen et al., 1996c). Reactive blue 2 blocks selectively contractile responses to ADPS at a P2Y-like receptor, but enhances P2X receptor-mediated contractions to ,-meATP and ATP in rat anococcygeus smooth muscle (Najbar et al., 1996)

8. Reactive red. Reactive red is at least 350 times more potent than reactive blue 2 as a competitive antagonist at the P2Y₁-like receptor of guinea-pig taenia coli (K_d, 28 nM); however, it is only 15-fold selective versus the P2X₁-like receptor in rat vas deferens, and has ecto-nucleotidase activity (Bültmann and Starke, 1995). Its effects at other P2X and P2Y subtypes are largely unknown.

9. Trypan blue. Trypan blue blocks selectively (versus K⁺ and noradrenaline) ,-meATP-mediated contractions at the P2X₁-like receptor in rat vas deferens but is also an inhibitor of ADPS-mediated relaxations via P2Y₁-like receptors in guinea-pig taenia coli and an inhibitor of ecto-nucleotidase activity (Bültmann et al., 1994; Wittenburg et al., 1996).

10. Evans blue. Evans blue blocks selectively responses to ,-meATP in the rat vas deferens versus those mediated by ADPS in the guinea-pig taenia coli, but potentiates contraction to ATP, ADP, and 2MeSATP in a manner attributable in part to ecto-nucleotidase inhibition; it also has non-specific potentiating effects (Bültmann and Starke, 1993; Bültmann et al., 1995; Wittenburg et al., 1996). The desmethyl derivative of Evans blue, NH01, is highly selective for the P2X₁-like receptor in vas deferens versus the P2Y₁-like receptor in guinea-pig taenia coli (K_d values 0.8 and > 100 µM, respectively), but is only moderately selective for the P2X₁ receptor versus inhibition of ecto-nucleotidase activity (Wittenburg et al., 1996).

11. DIDS. The Cl transport blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) is a noncompetitive, pseudo-irreversible antagonist of P2X₁-like receptor-mediated contractions to ,-meATP and of the purinergic component of the neurogenic contractile response in guinea-pig and rat vas deferens, and is selective versus the P2Y₁-like receptor of guinea-pig taenia coli (Fedan and Lamport, 1990; Bültmann and Starke, 1994b; Bültmann et al., 1996a). However, it is nonselective versus inhibition of ecto-nucleotidase activity (Bültmann et al., 1996a). DIDS discriminates between subtypes of P2X receptors, being a potent inhibitor of responses mediated at the P2X₁ receptor cloned from human bladder (IC₅₀ 3 µM), but less than 40% effective at recombinant P2X₂ receptors from PC12 cells at concentrations of up to 300 µM (Evans et al., 1995). DIDS blocks depolarization to ,-meATP in rat superior cervical ganglia, but has no effect on depolarization to UTP or potassium, or hyperpolarization to adenosine (Connolly and Harrison, 1995a). DIDS and some analogs of DIDS also block endogenous P2X₇-like receptors (el-Moatassim and Dubyak, 1993; McMillian et al., 1993; Soltoff et al., 1993). DIDS, PPADS, and dextran sulfate discriminate between recombinant human P2X₁ and rat P2X₂ receptors in displacement of binding studies, having 7- to 33-fold higher affinity for P2X₁ receptors (Michel et al., 1996).

12. Arylazidoaminopropionyl ATP (ANAPP₃). ANAPP₃, a photo-affinity analog of ATP, activates and desensitizes endogenous smooth muscle P2X₁-like receptors, irreversibly so after exposure to light, and selectively versus non-purine receptors (Hogaboom et al., 1980; Fedan et al., 1985; Venkova and Krier, 1993). Its effects at other P2X receptor subtypes have not been determined. However, ANAPP₃ also weakly antagonizes relaxations to ATP, ADP, and adenosine in the guinea-pig taenia coli (Westfall et al., 1982).

13. 2-Alkylthio derivatives of ATP. 2-Alkylthio derivatives of ATP are potent P2Y₁ receptor antagonists: both base modifications, leading to 8-(6-aminohexylamino)ATP and N-oxide ATP, and ribose modifications, leading to 2',3'-isopropylidene-AMP, result in derivatives that display selectivity for endothelial P2Y₁-like receptors and are virtually inactive at smooth muscle P2Y₁-like and P2X₁-like receptors (Burnstock et al., 1994).

14. 5'-p-Fluorosulfonyl benzoyladenosine. This is an irreversible inhibitor of ATP-induced Ba²⁺ influx via the P2X₇ receptor in human lymphocytes, although maximal inhibition does not exceed 90% (Wiley et al., 1994).

	IX. P2X Receptors

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P2X receptors are ATP-gated ion channels which mediate rapid (within 10 ms) and selective permeability to cations (Na⁺, K⁺ and Ca²⁺)(Bean, 1992; Dubyak and el-Moatassim, 1993; North, 1996). This is appropriate given their distribution on excitable cells (smooth muscle cells, neurons, and glial cells) and role as mediators of fast excitatory neurotransmission to ATP in both the central and peripheral nervous systems. This contrasts with the slower onset of response (less than 100 ms) to ATP acting at metabotropic P_2Y receptors, which involves coupling to G proteins and second-messenger systems. Seven P2X receptor proteins (P2X₁ to P2X₇) have been cloned and the ion channels formed from homomeric association of the subunits when expressed in Xenopus oocytes or in mammalian cells have been functionally characterized and show distinct pharmacological profiles (table 9). The P2X₇ receptor is considered separately below (see Section X.) because it is functionally unique among P2X receptors in being able to act as a non-selective pore.

Structural features of P2X receptors have been predicted from the amino acid sequences of cloned P2X receptor subunits. It is important to bear in mind that the P2X proteins that have been cloned are receptor subunits, not actual receptors; a single 2 transmembrane subunit alone cannot form an ion channel. The proteins have 379 to 472 amino acids and are believed to insert into the cell membrane to form a pore comprising two hydrophobic transmembrane domains, with much of the protein occuring extracellularly as an intervening hydrophilic loop (fig. 8). The overall structure of the receptor most closely resembles that of amiloride-sensitive epithelial Na⁺ channels. The putative extracellular loop of cloned receptors P2X₁ to P2X₇ has 10 conserved cysteine residues, 14 conserved glycine residues and 2 to 6 potential N-linked glycosylation sites. It is believed that disulfide bridges may form the structural constraints needed to couple the ATP-binding site to the ion pore. Most of the conserved regions are in the extracellular loop, with the transmembrane domains being less well-conserved.

View larger version (44K): [in this window] [in a new window]   Fig. 8.   Diagram depicting a proposed transmembrane topology for P2X2 protein showing both N- and C-terminals in the cytoplasm. Two putative membrane spanning segments (M1 and M2) traverse the lipid bilayer of the plasma membrane and are connected by a hydrophilic segment of 270 amino acids. This putative extracellular domain is shown containing two disulfide-bonded loops (S-S) and three N-linked glycosyl chains (triangles). The P2X2 cDNA was sequenced on both strands using Sequanase. (From Brake et al., 1994).

The quaternary structures of classical ligand-gated channels, for example, those of the nicotinic ACh receptor and the epithelial Na⁺ channel, generally take the form of heteromeric complexes of structurally related subunits. P2X receptors are believed to complex in a similar way in biological tissues. Their subunit stoichiometry is unknown, but may involve three subunits (or multiples of three subunits) based on SDS polyacrylamide gel electrophoresis estimates of the relative molecular mass of the recombinant P2X₁ and P2X₃ receptors determined under non-denaturing conditions (Nicke et al., 1998).

The pharmacological properties of endogenous P2X receptors in smooth muscle and PC12 cells correlate well with those of the recombinant receptors cloned from these tissues, P2X₁ and P2X₂ receptors, respectively; both native and recombinant P2X₁ receptors are sensitive to ,-meATP and rapidly desensitize, whereas P2X₂ receptors are insensitive to ,-meATP and do not desensitize. A good correlation is also seen between the properties of endogenous P2X receptors in neonatal dorsal root ganglion and the recombinant P2X₃ receptor (cloned from and expressed predominantly or exclusively in sensory neurons); both are sensitive to ,-meATP and rapidly desensitize (Evans and Surprenant, 1996). Thus, there is good reason to believe that the native P2X receptors in these tissues are predominantly homomers formed by the association of a single type of subunit.

However, this is not always the case. ATP-gated currents at endogenous P2X receptors in rat nodose neurons are mimicked by ,-meATP and do not desensitize (Lewis et al., 1995), a pharmacological profile that does not correspond to any of the homomeric P2X receptors cloned to date; all are expressed in sensory ganglia except P2X₇. Although P2X₃ is expressed preferentially in sensory neurons, currents evoked by ATP and ,-meATP at the recombinant P2X₃ receptor rapidly desensitize. However, when P2X₃ is coexpressed in HEK293 cells with P2X₂ (but not with other subtypes), a nondesensitizing response to ATP is observed which mimicks that seen in rat nodose neurons and which cannot be explained by additive effects of the two homomeric channels (Lewis et al., 1995). It was suggested that a new heteromeric receptor, P2X₂P2X₃, is formed from the P2X₃ and P2X₂ subunits (Lewis et al., 1995). This hypothesis is supported by the observation of a high level of colocalization of P2X₂- and P2X₃-immunoreactivity in rat nodose and dorsal root ganglia (Vulchanova et al., 1997). Direct evidence for the formation of a P2X₂P2X₃ heteromer comes from a study showing that in cells coinfected with P2X₂ and P2X₃ receptors, the two proteins can be cross-immunoprecipitated with antibodies specific for either of the epitope tags introduced at the C terminal of the proteins (Radford et al., 1997). Electrophysiological studies showing sensitivity to ,-meATP and a slowly desensitizing response is consistent with formation of heteromeric receptors because this is distinct from responses mediated by homomeric P2X₂ and P2X₃ receptors (Radford et al., 1997).

Further evidence for the existence of P2X₂P2X₃ heteromers in sensory neurons is suggested by electrophysiological studies in cultured neurons of adult rat dorsal root (Robertson et al., 1996) and trigeminal ganglion neurons (Cook et al., 1997). However, heterogeneity within populations of sensory neurons has been identified in the form of single labeling for P2X₂ or P2X₃ of some rat nodose and dorsal root neurons (possibly coexisting with other P2X proteins) (Vulchanova et al., 1997), and by the demonstration of two types of inward current to ATP (transient and slowly desensitizing) in tooth-pulp nociceptors (Cook and McCleskey, 1997). This raises interesting questions about the patterns of P2X receptor subtype expression and the physiological properties of different neurons.

The likely formation of P2X₂P2X₃ heteromers in sensory neurons has important implications for the subunit organization of P2X receptors in other biological tissues, because the different P2X proteins have widespread and overlapping distributions. However, it seems that not all combinations are possible; for example, cotransfected P2X₁ and P2X₂ subunits do not combine to form heteromeric receptors (Surprenant, 1996). Figure 9 shows examples of ATP-gated currents in native cells and how these correlate with recombinant P2X receptors.

View larger version (25K): [in this window] [in a new window]   Fig. 9.   Examples of ATP-gated currents evoked in native cells (A-D) and in HEK293 cells expressing homomeric (E-G) or heteromeric (H) P2X receptors. Bars above each trace refer to the duration of agonist application. All recordings are at holding potential of 70 mV. Traces shown in C from neonatal dorsal root ganglion neurons are unpublished records kindly supplied by M. Rae, S. Robertson, E. Rowan, and C. Kennedy, University of Strathclyde; all other traces from authors unpublished records. (From Evans and Surprenant, 1996.)

Alternative splicing of P2X pre-messenger RNA has been shown for the P2X₂ receptor (Brändle et al., 1997; Simon et al., 1997). The splice variant exhibits a different pharmacology to the native receptor, suggesting that there may be heterogeneity in responses of tissues expressing the different proteins.

B. Cloned P2X Receptors

1. P2X₁ receptor. The P2X₁ receptor has been cloned from rat vas deferens and human and mouse urinary bladder (Valera et al., 1994, 1995, 1996) (table 9). The recombinant receptor is activated by 2MeSATP  ATP > ,-meATP  ADP, and inward currents evoked by these compounds are reversibly blocked by suramin and PPADS (Valera et al., 1994). The receptor desensitizes very rapidly (in hundreds of milliseconds).

2. P2X₂ receptor. The P2X₂ receptor first cloned from rat pheochromocytoma PC12 cells (originally called P2XR1) (Brake et al., 1994) displays only 41% amino acid homology with the rat vas deferens P2X₁ receptor. At the recombinant P2X₂ receptor ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPS) and 2MeSATP are approximately equipotent at eliciting non-selective inward cation currents, whereas ,-meATP and ,-meATP are inactive as agonists or antagonists (Brake et al., 1994). This receptor undergoes little or no desensitization. It also differs from the P2X₁ receptor in that it is less permeable to Ca²⁺ and shows much higher sensitivity to inhibition by extracellular Ca²⁺ (Evans et al., 1996).

3. P2X₃ receptor. The P2X₃ receptor cloned from rat dorsal root ganglion (Chen et al., 1995a; Lewis et al., 1995) shows only 43% amino acid sequence homology with the P2X₁ receptor and 47% identity to the P2X₂ receptor. The P2X₃ receptor is activated by agonists with a potency order of 2MeSATP  ATP > ,-meATP and undergoes rapid desensitization (in less than 100 ms).

4. P2X₄ receptor. The P2X₄ receptor protein has been cloned from rat hippocampus (Bo et al., 1995), DRG cells (Buell et al., 1996b), rat (Séguéla et al., 1996; Garcia-Guzman et al., 1997a) and human brain (Soto et al., 1996a; Garcia-Guzman et al., 1997a), as well as rat endocrine tissue (Wang et al., 1996). The P2X receptor cloned from rat brain by Séguéla et al. (1996) was refered to as P_2x3 in their paper, but a comparison of the receptor sequence with known subtypes identifies it as P2X₄. A sequence homology of 87% between the human and rat P2X₄ receptors is sufficiently different to produce subtle differences in antagonist binding and desensitization. The recombinant P2X₄ receptor is most potently activated by 2MeSATP, but ,-meATP is weak or inactive (Bo et al., 1995; Séguéla et al., 1996). P2X₄ is relatively insensitive to the antagonists suramin and PPADS; high concentrations (>100 µM) are required to block ATP-evoked currents (Bo et al., 1995; Séguéla et al., 1996), although the human receptor shows a higher sensitivity for suramin and PPADS (Garcia-Guzman et al., 1997a). A lysine residue present in the P2X₁ and P2X₂ receptors, but absent in the P2X₄ receptor, is critical for the binding of antagonists but not agonists (Buell et al., 1996a). The P2X₄ receptor does not desensitize rapidly, although reversible rundown of the current occurs during prolonged exposure to ATP (Séguéla et al., 1996). More rapid desensitization of the human P2X₄ receptor (Garcia-Guzman et al., 1997a) compared with the rat P2X₄ receptor (Buell et al., 1996a) has been described. P2X₄ ATP-gated currents are potentiated by coapplication of Zn²⁺ (Séguéla et al., 1996; Garcia-Guzman et al., 1997a).

5. P2X₅ receptor. This P2X receptor was first cloned from rat coeliac ganglia (Collo et al., 1996). Human homologs of the P2X₅ receptor have tentatively been identified (Tokuyama et al., 1996a, 1996b). Rapid inward currents are activated by ATP > 2MeSATP > ADP, whereas ,-meATP is ineffective as an agonist. The receptor does not readily desensitize. Currents are readily inhibited by suramin and PPADS. In situ hybridization shows P2X₅ mRNA in motoneurons of the ventral horn of the cervical spinal cord, and in neurons in the trigeminal and dorsal root ganglia. With the exception of the mesencephalic nucleus of the trigeminal nerve, the brain does not express P2X₅ mRNA (Collo et al., 1996). Appropriately, functional studies have identified P2X receptors in rat trigeminal mesencephalic nucleus neurons with a profile most similar to that of P2X₅ receptors (Khakh et al., 1997)

6. P2X₆ receptor. This clone was isolated from a rat superior cervical ganglion cDNA library (Collo et al., 1996). Rapid currents are mediated by ATP > 2MeSATP > ADP, but ,-meATP has no effect. Currents are only partially inhibited by suramin or PPADS. P2X₆ mRNA is heavily expressed in the CNS, with heaviest staining in cerebellar Purkinje cells and ependyma (Collo et al., 1996). Staining is also detected in the cervical spinal cord, notably in spinal motoneurons of lamina IX, and the superficial dorsal horn neurons of lamina II. P2X₆ mRNA is also present in trigeminal, dorsal root, and coeliac ganglia; and in gland cells of the uterus, granulosa cells of the ovary, and bronchial epithelia, but is absent from salivary epithelia, adrenal medulla, and bladder smooth muscle (Collo et al., 1996).

P2X receptors mediate the rapid (onset within 10 ms) non-selective passage of cations (Na⁺, K⁺, Ca²⁺) across the cell membrane resulting in an increase in intracellular Ca²⁺ and depolarization (Bean, 1992; Dubyak and el-Moatassim, 1993). The direct flux of extracellular Ca²⁺ through the channel constitutes a significant source of the increase in intracellular Ca²⁺. However, membrane depolarization leads to the secondary activation of voltage-dependent Ca²⁺ channels, which probably make the primary contribution to Ca²⁺ influx and to the increase in intracellular Ca²⁺. Because this transduction mechanism does not depend on the production and diffusion of second-messengers within the cytosol or cell membrane, the response time is very rapid, and appropriately plays an important role in fast neuronal signaling and regulation of muscle contractility. P2X channels often show considerable current fluctuation, or "flickery bursts," in the open state that may represent unresolved closures or rapid transition between states (Evans and Surprenant, 1996). Selectivity for Ca²⁺ permeability between P2X receptors on sensory versus autonomic nerves and smooth muscle has been suggested, but the patterns are not entirely clear (see Evans and Surprenant, 1996). The kinetics of ATP-gated currents have been reviewed (Surprenant, 1996).

Cations can modulate ATP-activated currents in native and endogenous P2X receptors. Mg²⁺ and Ca²⁺ generally inhibit P2X receptor currents, probably by decreasing the affinity of the ATP binding site by an allosteric change in the receptor (Honoré et al., 1989; Nakazawa et al., 1990; Li et al., 1997a). However, an increase in the transient ATP response (but not the slowly-desensitizing ATP response) has been observed when Ca²⁺ replaces Na⁺ in the extracellular solution in rat trigeminal sensory neurons (Cook and McCleskey, 1997). Interestingly, the recombinant P2X₂ receptor seems to be more susceptible than the P2X₁ receptor to inhibition by increases in extracellular Ca²⁺ (Evans et al., 1996). Allosteric interactions may also be responsible for the ability of monovalent cations to negatively modulate binding to recombinant P2X₄ receptors (Michel et al., 1997), and trivalent cations to negatively modulate the binding site of recombinant P2X₁ and P2X₂ receptors and the endogenous receptor of PC12 cells (Nakazawa et al., 1997).

Zn²⁺ potentiates the cation conductance induced by ATP at most P2X receptors, including those in rat superior cervical ganglion (Cloues et al., 1993; Cloues, 1995), nodose and coeliac ganglion neurons (Li et al., 1993, 1996), PC12 cells (Koizumi et al., 1995a), and recombinant P2X₁ (Brake et al., 1994) and P2X₄ receptors (Séguéla et al., 1996). The P2X₇ receptor is an exception in this respect because it is inhibited by Zn²⁺ and Cu²⁺ (Virginio et al., 1997). Ni²⁺ enhances ATP-activated currents in rat superior cervical ganglia (Cloues et al., 1993) and Cd²⁺ potentiates ATP-evoked inward currents and dopamine release in rat phaeochromocytoma cells (Ikeda et al., 1996).

Modulation of the affinity of the ATP-binding site occurs by extracellular protons; acid pH causes an increase, and alkaline pH causes a decrease in currents, as shown for the recombinant P2X₂ receptor and endogenous P2X receptors in rat dorsal root and nodose ganglion cells (King et al., 1996b; Li et al., 1996, 1997b; Wildman et al., 1997). This may be particularly significant for P2X receptor-mediated signaling in pathophysiological conditions where injury or inflammation can profoundly alter extracellular pH.

P2X receptors can be divided into two broad groups according to whether they desensitize rapidly, that is, within 100 to 300 ms, or slowly if at all (table 10). This subdivision hinges critically on the time to desensitization; "rapid" desensitization should not be confused with desensitization which occurs over a few seconds, and thus is a phenomenon which is difficult to identify in other than studies of single channel activity. As a general rule, all rapidly desensitizing P2X receptors are activated by ,-meATP as well as by 2MeSATP and ATP. These include: recombinant P2X₁ and P2X₃ receptors; their endogenous counterparts, namely P2X₁-like receptors of smooth muscle (with some exceptions, indicated below); P2X₁-like receptors of promyelocyte HL60 cells (Buell et al., 1996b); and platelets (MacKenzie et al., 1996) and P2X₃-like receptors of neonatal sensory neurons (dorsal root ganglion and nodose ganglion) (Krishtal et al., 1988a,b; Li et al., 1993; Robertson et al., 1996). Desensitization of P2X₃-like receptors of neonatal sensory neurons, but not P2X₁-like receptors of smooth muscle, is concentration-dependent (Evans and Surprenant, 1996; Robertson et al., 1996). Desensitization will clearly serve to terminate the purinergic response even though ATP release may still be ongoing, but exactly why this is more important in some tissues remains to be determined.

P2X receptors which do not desensitize rapidly, desensitize slowly or not at all. These "non-desensitizing" P2X receptors are defined as receptors for which the currents are maintained for at least a few seconds in the continuous presence of agonist. Non-desensitizing P2X receptors can be further subdivided into two groups: 1) those that are sensitive to ,-meATP, and 2) those that are insensitive or only weakly sensitive to ,-meATP (Evans and Surprenant, 1996). Non-desensitizing ,-meATP-sensitive P2X receptors are those in adult sensory ganglia (nodose and dorsal root ganglion) (Krishtal et al., 1988a, 1988b; Li et al., 1993; Khakh et al., 1995a; Wright and Li, 1995), and guinea-pig coeliac ganglion (Evans et al., 1992; Khakh et al., 1995a). It has been suggested that these receptors may be heteromers of P2X₂ and P2X₃ subunits (P2X₂P2X₃ receptors) (Lewis et al., 1995) (fig. 9). Non-desensitizing ,-meATP-sensitive responses have also been shown in some smooth muscle, namely in the arterial vasculature of human placenta (Dobronyi et al., 1997; Ralevic et al., 1997), and intestine of the three-spined stickleback Gasterosteus aculeatus L (Knight and Burnstock, 1993), and similarly may be caused by actions at P2X heteromers. Non-desensitizing ,-meATP-sensitive P2X receptors have also been described in the CNS, on rat locus coeruleus neurons (Tschöpl et al., 1992; Shen and North, 1993), and some rostral ventrolateral medulla neurons (Ralevic et al., 1996).

Non-desensitizing ,-meATP-insensitive P2X receptors are cloned P2X₂, P2X₄, P2X₅, and P2X₆ receptors (table 10a), as well as native P2X receptors on most autonomic neurons, including rat superior cervical ganglia (Cloues et al., 1993; Nakazawa and Inoue, 1993; Khakh et al., 1995a), guinea-pig submucosal enteric neurons (Barajas-Lopez et al., 1994), PC12 cells (Nakazawa et al., 1990; Nakazawa and Hess, 1993; Kim and Rabin, 1994), rat cardiac parasympathetic ganglia (Fieber and Adams, 1991), and chick ciliary ganglion neurons (Abe et al., 1995). Non-desensitizing ,-meATP-insensitive receptors have also been described in the CNS in nucleus tractus solitarius neurons (Ueno et al., 1992; Nabekura et al., 1995) and trigeminal mesencephalic nucleus neurons (Khakh et al., 1997); these may correspond to P2X₄, P2X₅, or P2X₆ receptors, or to combinations of these subunits, given the rich expression of these proteins in the brain. ATP-gated ,-meATP-insensitive currents in myometrial smooth muscle cells from pregnant rats have been reported to be resistant to desensitization (Honoré et al., 1989).

The mechanism of P2X receptor desensitization is not well understood. For the rapidly desensitizing P2X₁ receptor, this may involve the hydrophobic domains of the receptor because transfer to the P2X₂ receptor of both of the hydrophobic domains, but not the extracellular loop, of the P2X₁ receptor changes the phenotype of the P2X₂ receptor from non-desensitizing to rapidly-desensitizing (Werner et al., 1996). Amino acid deletions of the carboxyl terminal of the P2X₂ receptor produces splice variants that desensitize more rapidly than the original receptor (Brändle et al., 1997; Simon et al., 1997). On the other hand, the N-terminal region of the receptor has been suggested to be important in desensitization of the P2X₃ receptor (King et al., 1997). Desensitization of the P2X₃ receptor seems to involve the activation of calcineurin through the entry of extracellular calcium (King et al., 1997).

There are no universal or subtype-selective P2X receptor agonists. ATP and diadenosine polyphosphates with a phosphate chain length greater than or equal to three are naturally-occuring agonists at P2X receptors (Hoyle et al., 1989; Hoyle, 1990; Bo et al., 1994; Schlüter et al., 1994; Bailey and Hourani, 1995; Ralevic et al., 1995a; Usune et al., 1996). The greater potency of the longer chain diadenosine polyphosphates (Ap₄A-Ap₆A) compared with ATP at endogenous P2X₁-like receptors may be caused by their greater resistance to breakdown (Hoyle, 1990; Ogilvie, 1992; Ralevic et al., 1995a). UTP is a weak agonist of P2X₃ receptors (Chen et al., 1995a; Robertson et al., 1996) and may interact with P2X₁-like receptors in rat urinary bladder (Hashimoto and Kokubun, 1995) as well as mouse vas deferens (Von Kügelgen et al., 1990).

In physiological solution, Ca²⁺ and Mg²⁺ ions form complexes with the free acid ATP⁴, such that the solution contains a mixture of ATP⁴, MgATP², and CaATP² (together with lower concentrations of the species variants MgHATP, CaHATP, and Ca₂ATP). Under physiological conditions, ATP⁴ is a minor component of the total ATP concentration (approximately 1 to 10% depending on temperature, pH, and divalent cation concentration). The concentration of ATP⁴ decreases with increasing cation concentration and with acidic pH (that results in conversion of ATP⁴ to HATP³, which has proved useful in studies aimed at investigating the identity of the active form of ATP). Cockroft and Gomperts (1980) raised the question of which was the active form of ATP with their suggestion that ATP⁴ causes an increase in mast cell plasma membrane permeability. It has since been shown that this form of the ligand is likely to be responsible for pore-forming actions in mast cells, macrophages, and lymphocytes as well as a number of other cell types expressing a receptor termed the P_2Z or P2X₇ receptor. Addition of Mg²⁺ forms the inactive species MaATP² and thereby reduces the concentration of ATP⁴, rapidly closing the cation channel (Greenberg et al., 1988; el-Moatassim and Dubyak, 1993; Gargett et al., 1996; Lin and Lee, 1996). Similarly, 3'-O-(4-benzoyl)benzoyl ATP (BzATP⁴), and not the complex MgBzATP², seems to be the active species in P_2Z or P2X₇-mediated pore formation.

The idea that ATP⁴ is the active form of ATP has been extended to P2X receptors other than the P_2Z or P2X₇ receptor. Hence, ATP⁴ has been suggested to be the ligand that activates P2X receptors in guinea-pig vas deferens smooth muscle (Fedan et al., 1990), rat parotid acinar cells (McMillian et al., 1993), and PC12 cells (Kim and Rabin, 1994; Choi and Kim, 1996); it also mediates ATP-gated currents in pregnant rat myometrial smooth muscle cells (Honoré et al., 1989). The P2X receptors expressed by these tissues do not form nonspecific membrane pores. In these studies, suggestion of a role for ATP⁴ as the active ligand is based primarily on the fact that responses are inhibited by elevation of extracellular Mg²⁺ or other cations which chelate with ATP, and because responses correlate well with the calculated ATP⁴ concentration and not with the total ATP concentration or with the concentration of Mg²⁺ in solution. However, this alone does not seem to be sufficient evidence in light of more recent studies which show that divalent cations can influence agonist potency by effects other than by changes in the relative concentrations of the ATP species in solution.

It is now apparent that interpretation of the effects of removal of Mg²⁺ and Ca²⁺ from solution on agonist potency is complicated by additional inhibition of ecto-nucleotidase activity, disinhibition of single channel conductance of P2X receptors, and possibly membrane depolarization. These effects seem to have a greater influence on the end response than does a shift in the concentration of the active species of ATP. Inhibition of ecto-nucleotidase activity seems to be the overriding effect of Ca²⁺ and Mg²⁺ removal on agonist potency in the rat isolated vagus nerve, where the potency of responses to ATP and 2MeSATP was increased, but that of the stable analog ,-meATP was unchanged (Trezise et al., 1994a). Studies on single channel conductance of native P2 receptors in rat nodose ganglion, PC12 cells, and recombinant P2X₁ and P2X₂ receptors, in which consideration of ecto-nucleotidase activity is effectively bypassed in conditions of concentration clamp, have confirmed that raising Ca²⁺ or Mg²⁺ decreases the potency of ATP (Nakazawa and Hess, 1993; Evans et al., 1996; Li et al., 1997a; Virginio et al., 1997). However, the mechanism seems to involve a decrease in the affinity of the agonist binding site by allosteric effects on the receptor (although direct cation block of the channels is also possible) (Nakazawa and Hess, 1993; Evans et al., 1996; Li et al., 1997a). The fact that recombinant P2X₂ receptors show a higher sensitivity than P2X₁ receptors to inhibition by extracellular Ca²⁺ (Evans et al., 1996) is further consistent with the hypothesis that cation modulation of P2X receptors is due to changes occuring at the level of the receptor, and can be influenced by the intrinsic properties of that receptor, rather than a change in the relative concentrations of ATP species in the extracellular solution. Because of these complicating factors, the identity of the active species of ATP acting at P2X receptors is currently unclear.