I. Introduction

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Adenosine is an endogenous nucleoside that modulates many physiological processes. Its actions are mediated by interaction with specific cell membrane receptors. Four subtypes of adenosine receptors have been cloned: A₁, A_2A, A_2B, and A₃. Significant advancement has been made in the understanding of the molecular pharmacology and physiological relevance of adenosine receptors, but our knowledge of A_2B receptors lags behind that of other receptor subtypes. The lack of selective pharmacological probes has hindered research in this area. Perhaps because of their lower affinity for adenosine compared with other receptors, it is often assumed that A_2B receptors are a low-affinity version of the A_2A receptor and are of lesser physiological relevance. It has been only recently that potentially important functions have been discovered for the A_2B receptor, prompting a renewed interest in this receptor type. It is also recently recognized that A_2B receptors are coupled to intracellular pathways different from those of A_2A receptors, a finding that may provide the basis for their distinct physiological role. A_2B receptors have been implicated in mast cell activation and asthma, vasodilation, regulation of cell growth, intestinal function, and modulation of neurosecretion. We try to review the recent advances made in the study of A_2B receptors and underscore areas in which more progress is needed. We discuss some of the characteristics of A₁, A_2A, and A₃ receptors only to highlight their similarities and differences with A_2B receptors. Recent reviews on specific adenosine receptor subtypes can be found elsewhere (Linden, 1991, 1994; Dalziel and Westfall, 1994; Fredholm, 1995; Palmer and Stiles, 1995; Sebastião and Ribeiro, 1996; Daval et al., 1996; Ongini and Fredholm, 1996).

	II. Classification of Adenosine Receptors

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The properties of extracellular adenosine as a protective autacoid have been known since the study of its cardiovascular effects conducted in 1929 by Drury and Szent-Györgyi (1929). The purinergic receptors that mediate the effects of adenosine were classified as P₁ receptors, whereas the receptors activated by nucleotides like adenosine 5c-triphosphate (ATP) were classified as P₂ receptors (Burnstock, 1978). Adenosine receptors were found to modulate intracellular levels of adenosine 3c,5c-cyclic monophosphate (cAMP) and were initially subdivided into A₁ and A₂ subtypes based on their ability to inhibit or stimulate adenyl cyclase, respectively (van Calker et al., 1979; Londos et al., 1980). The alternative classification of adenosine receptors as R_i and R_a (Londos et al., 1980) was replaced by the A₁ and A₂ terms (van Calker et al., 1979). The further division of A₂ receptors into two subtypes was proposed originally by Daly et al. (1983) based on the finding of high-affinity A₂ receptors in rat striatum and low-affinity A₂ receptors throughout the brain, both of which activated adenyl cyclase. The existence of subtypes of A₂ receptors was also suggested by the finding, independently reported by Elfman et al. (1984), of high-affinity A₂ receptors in cultured neuroblastoma cells and low-affinity A₂ receptors in glioma cells. These high- and low-affinity receptor subtypes were later designated as A_2A and A_2B, respectively (Bruns et al., 1986). The classification of P₁ receptors has been validated by the recent success in molecular cloning and expression of all three anticipated A₁, A_2A, and A_2B adenosine receptors and the previously unrecognized A₃ receptor (Maenhaut et al., 1990; Libert et al., 1991; Zhou et al., 1992; Rivkees and Reppert, 1992; Pierce et al., 1992). This classification has been endorsed by IUPHAR Committee on Receptor Nomenclature and Drug Classification (Fredholm et al., 1994, 1996b, 1997).

	III. Molecular Characterization of A2B Receptors

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Adenosine A_2B receptors were cloned from rat hypothalamus (Rivkees and Reppert, 1992), human hippocampus (Pierce et al., 1992), and mouse mast cells (Marquardt et al., 1994), employing standard polymerase chain reaction techniques with degenerate oligonucleotide primers designed to recognize conserved regions of most G protein-coupled receptors. The human A_2B receptor shares 86 to 87% amino acid sequence homology with the rat and mouse A_2B receptors (Rivkees and Reppert, 1992; Pierce et al., 1992; Marquardt et al., 1994) and 45% amino acid sequence homology with human A₁ and A_2A receptors (fig. 1). As expected for closely related species, the rat and mouse A_2B receptors share 96% amino acid sequence homology. By comparison, the overall amino acid identity between A₁ receptors from various species is 87% (Palmer and Stiles, 1995). A_2A receptors share 90% of homology between species (Ongini and Fredholm, 1996), with most differences occurring in the 2^nd extracellular loop and the long C-terminal domain (Palmer and Stiles, 1995). The lowest (72%) degree of identity between species is observed for A₃ receptor sequences (Palmer and Stiles, 1995). Differences in amino acid sequence of adenosine receptors between species may result in distinct pharmacological characteristics. For example, the rat A₁ receptor has a rank order of potency (R)-N⁶-phenylisopropyladenosine (R-PIA) > 5'-N-ethylcarboxamidoadenosine (NECA) > (S)-N⁶-phenylisopropylaolenesine (S-PIA), and the bovine A₁ receptor has a potency order R-PIA > S-PIA > NECA (Klotz et al., 1991), whereas the canine A₁ receptor binds NECA with a higher affinity than that of R-PIA (Tucker and Linden, 1993). The differences between amino acid sequences of A₃ receptors are reflected in the insensitivity of the rat A₃ receptor to antagonism by methylxanthines (Zhou et al., 1992), a phenomenon that is not observed in the human or sheep A₃ receptor (Linden et al., 1993; Salvatore et al., 1993). These interspecies differences between adenosine receptors explain why the adenosine agonist xanthine amine congener (XAC) is a selective A₁ agonist in the rat, but not in the human or rabbit (Jacobson et al., 1992; Jacobson and Suzuki, 1996). Few comparisons have been made between A_2B receptors from different species. No differences in pharmacological profiles were found between A_2B receptors from fibroblasts of murine and human origin (Bruns, 1981; Brackett and Daly, 1994) or between human A_2B receptor expressed in Chinese hamster ovary (CHO) cells and guinea pig brain A_2B receptors (Alexander et al., 1996).

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Comparison of amino acid sequences of human (M97759) (Pierce et al., 1992), rat (M91466) (Stehle et al., 1992), and mouse (U05673) (Marquardt et al., 1994) A2B receptors. Dashed lines indicate amino acid identity. Predicted transmembrane spanning domains are highlighted and indicated by roman numerals.

The proposed membrane structure of A_2B receptors is typical of G protein-coupled receptors, with seven transmembrane domains connected by three extracellular and three intracellular loops, and flanked by an extracellular N-terminus and an intracellular C-terminus (Rivkees and Reppert, 1992; Pierce et al., 1992; Marquardt et al., 1994; fig. 2). The highest degree of identity in amino acid sequences between A_2B receptors of different species is found in the transmembrane domains (fig. 1). The 2^nd extracellular loop of the human, mouse, and rat A_2B receptors contains two potential N-glycosylation sites (Rivkees and Reppert, 1992; Pierce et al., 1992; Marquardt et al., 1994). It should be noted that enzymatic treatment failed to demonstrate N-glycosylation of A_2B receptors in T84 epithelial cells (Puffinbarger et al., 1995). However, it is not clear whether A_2B receptors are glycosylated in other cells or glycosylation can alter A_2B function. A_2A receptors were found to be glycosylated in canine striatum and liver membranes (Palmer et al., 1992), but the binding characteristics of A_2A receptors for 4-[(N-ethyl-5'-carbamoyladenos-2-yl)-aminoethyl]-phenylpropionic acid (CGS 21680) appear to be the same in both glycosylated or unglycosylated forms of the receptor expressed in COS M6 cells (Piersen et al., 1994).

View larger version (58K): [in this window] [in a new window]   Fig. 2.   Amino acid sequence of the human A2B receptor. The receptor is drawn according to the seven-membrane spanning motif common to the G protein-coupled receptor superfamily. Possible sites of N-linked glycosylation on the 2nd extracellular loop are highlighted.

The predicted molecular mass of A_2B receptors is similar to that of A₁ and A₃ receptors (36-37 kDa), whereas A_2A receptors have a larger predicted size (45 kDa). The greater molecular mass of the A_2A receptor is explained by the presence of a longer intracellular C-terminus. Together with the 3^rd intracellular loop, the intracellular C-terminus is thought to be involved in the coupling of A_2A receptors to G proteins (Palmer and Stiles, 1995). To date, no mutational analysis of A_2B receptor-G protein coupling has been reported. However, some parallels could be drawn from studies using chimeric A₁/A_2A adenosine receptors (Tucker et al., 1996; Olah, 1997). Using this approach, it has been shown that the amino terminal portion of the 3^rd intracellular loop of the A_2A receptor determines its selective coupling with G_s (Olah, 1997). This 15-mer portion of the A_2A receptor shares 57% amino acid sequence homology with the A_2B receptor, both of which are coupled to G_s, and only 27% with the A₁ receptor, which is not coupled to G_s (fig. 3). In addition, the nature of the amino acids in the 2^nd intracellular loop may indirectly modulate A_2A receptor coupling. In particular, lysine and glutamic acid residues in that portion of the molecule were found to be necessary for efficient A_2A adenosine receptor-G_s coupling (Olah, 1997). These amino acid residues are also present in the A_2B receptor. The long intracellular C-terminal tail of the A_2A receptor, which represents a major structural difference with the A_2B, does not appear to be involved in the determination of receptor coupling to G_s protein. The removal of the C-terminal tail of the A_2A receptor, or its replacement with a cytoplasmic tail of the A₁ receptor, does not impair stimulation of adenyl cyclase when these truncated or chimeric receptors are expressed in CHO cells (Tucker et al., 1996; Palmer and Stiles, 1997; Olah, 1997). The data generated from these studies, however, leave the possibility that this region can still play a role in the modulation of the coupling of A_2A receptors to G proteins. For example, it was suggested that the C-terminal tail confers the A_2A receptors' ability to couple tightly to G_s, a feature considered to be unique for this receptor subtype (Nanoff et al., 1991).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Comparison of amino acid sequences of 2nd and 3rd intracellular loops and C-terminus of human A2A receptors, with corresponding regions of human A1 and A2B receptors. Dashed lines indicate amino acid identity. Asterisks represent gaps in the sequence introduced to highlight amino acid homologies. The amino acid residues discussed in the text are highlighted.

Mutational studies of A_2A receptors revealed that a threonine residue (Thr²⁹⁸) of the C-terminal tail of the A_2A receptor, located in proximity to the seventh transmembrane span (fig. 3), is essential for the development of rapid agonist-mediated desensitization (Palmer and Stiles, 1997). This amino acid residue is also present in the human A_2B receptor (Thr³⁰⁰), but its role in receptor desensitization has not been explored. Although the mechanisms of desensitization are not completely identified, it is of interest that rapid desensitization of A_2A as well as A_2B receptors can be mediated by G protein-coupled receptor kinase 2 (Mundell et al., 1997). It should be noted that A_2B receptors can be coupled to other intracellular signaling pathways in addition to G_s and adenyl cyclase. The similarities and differences in A_2B and A_2A receptor coupling to G proteins warrant studies involving mutational analysis of A_2B receptors, and possibly chimeric A_2A/A_2B receptors, to better understand determinants of A_2B-G protein coupling.

The human A_2B receptor gene was mapped to chromosome 17p11.2-p12 (Jacobson et al., 1995; Townsend-Nicholson et al., 1995). A single intron interrupts the coding sequence of the human A_2B receptor gene in a region corresponding to the 2^nd intracellular loop between Leu¹¹¹and Arg¹¹² (Jacobson et al., 1995). In this respect, the human A_2B receptor gene is similar to the other human adenosine receptor genes in that it also contains a single intron in its coding sequence (Ren and Stiles, 1994; Peterfreund et al., 1994; Murrison et al., 1996). Some G protein-coupled receptors are known to have multiple introns in the coding sequences of their corresponding genes. Alternative splicing of their primary transcripts results in heterogeneity in protein sequences, as observed with EP₃ prostanoid receptors (Neglishi et al., 1995), D₂ dopamine receptors (Giros et al., 1989), lutropin/choriogonadotropin receptors (Aatsinki et al., 1992), and fibroblast growth factor receptors 2 (Dell and Williams, 1992). The presence of only one intron within the coding region of the human A_2B receptor gene precludes structural variations of A_2B receptors by alternative splicing.

In addition to the human A_2B receptor gene, an A_2B pseudogene with 79% identity with the A_2B receptor complementary deoxyribonucleic acid (cDNA), has been localized to chromosome 1q32 (Jacobson et al., 1995; Townsend-Nicholson et al., 1995). When compared with the coding sequence of the A_2B receptor, the pseudogene contained multiple deletions, point mutations, and frame shifts and two in-frame stops (Jacobson et al., 1995). It is doubtful that with all these changes the pseudogene would encode a functional adenosine receptor. However, further studies are needed to determine whether the A_2B pseudogene is transcriptionally competent. For example, dopamine D₅ pseudogene transcripts can be detected in human brain tissues (Nguyen et al., 1991). The same possibility should always be considered in Northern blot analysis or in situ hybridization of A_2B receptor in various tissues, because the use of sequences common between the functional A_2B cDNA and the A_2B pseudogene as probes could potentially lead to misinterpretation of results.

	IV. Pharmacology of A2B Receptors

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Highly selective and potent agonists have been designed for A₁, A_2A, and A₃ receptors. These compounds have been important tools in the characterization of adenosine receptors and the determination of their functions. All four subtypes, including A_2B receptors, have a typical order of potency for agonists (table 1; fig. 4). However, no selective agonist for A_2B receptors has been found so far. The adenosine analog NECA remains the most potent A_2B agonist (Bruns, 1981; Feoktistov and Biaggioni, 1993, 1997; Brackett and Daly, 1994), with a concentration producing a half-maximal effect (EC₅₀) for stimulation of adenyl cyclase of approximately 2 µM. It is, however, nonselective and activates other adenosine receptors with even greater affinity, with an EC₅₀ in the low nanomolar (A₁ and A_2A) or high nanomolar (A₃) range (table 1; fig. 4). The characterization of A_2B receptors, therefore, often relies on the lack of effectiveness of compounds that are potent and selective agonists of other receptor types. A_2B receptors have been characterized by a method of exclusion, i.e., by the lack of efficacy of agonists that are specific for other receptors. The A_2A selective agonist CGS 21680 (Webb et al., 1992), for example, has been useful in differentiating between A_2A and A_2B adenosine receptors (Hide et al., 1992; Chern et al., 1993; Feoktistov and Biaggioni, 1995; van der Ploeg et al., 1996). Both receptors are positively coupled to adenyl cyclase and are activated by the nonselective agonist NECA. CGS 21680 is virtually ineffective on A_2B receptors but is as potent as NECA in activating A_2A receptors, with an EC₅₀ in the low nanomolar range for both agonists (Jarvis et al., 1989; Nakane and Chiba, 1990; Webb et al., 1992; Hide et al., 1992; Feoktistov and Biaggioni, 1993; Alexander et al., 1996). A_2B receptors have also a very low affinity for the A₁ selective agonist R-PIA (Feoktistov and Biaggioni, 1993; Brackett and Daly, 1994) as well as for the A₃ selective agonist N⁶-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (IB-MECA) (Feoktistov and Biaggioni, 1997). The agonist profile NECA > R-PIA = IB-MECA > CGS 21680 was determined in human erythroleukemia (HEL) cells for A_2B-mediated cAMP accumulation. The difference between EC₅₀ for NECA and the rest of the agonists is approximately 2 orders of magnitude. Therefore, responses elicited by NECA at concentrations in the low micromolar range (1-10 µM), but not by R-PIA, IB-MECA or CGS 21680, are characteristic of A_2B receptors.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Chemical structure and radioligand binding data on the affinity of adenosine agonists. Ki values (nM) for rat A1/A2A/A2B/A3 receptors are shown, except as indicated (h, human). Numbers in brackets represent EC50 of adenosine agonists for cAMP accumulation in HEL cells. Data compiled from Feoktistov and Biaggioni (1993), van Galen et al. (1994), Jacobson and Suzuki (1996), and Feoktistov and Biaggioni (1997).

Pharmacological characterization of receptors based on apparent agonist potencies, however, is far from ideal, because it depends not only on agonist binding to the receptor but also on multiple processes involved in signal transduction. Selective antagonists are preferable for receptor subtype identification (Kenakin et al., 1992). Highly selective and potent A_2B antagonists are not yet available, but, whereas A_2B receptors have a lower affinity for agonists compared with other receptor subtypes, this is not true for antagonists. The structure-activity relationship of A_2B receptors for adenosine antagonists has not been completely characterized, but at least some xanthines are as potent antagonists at A_2B receptors as at other adenosine receptors (Feoktistov and Biaggioni, 1993; Brackett and Daly, 1994).

The antiasthmatic drug enprofylline (3-n-propylxanthine), is the most selective A_2B antagonist known to date. In early studies, enprofylline was found to be about 20 times more potent in blocking hippocampal A₂ receptors compared with rat fat cell A₁ receptors (Fredholm and Persson, 1982). It was initially proposed, therefore, that enprofylline can selectively block a subtype of A₂ receptors in the hippocampus (Fredholm and Persson, 1982). However, enprofylline was then found to be a poor antagonist of A₂ receptors in thymocytes (Fredholm and Sandberg, 1983) and platelets (Ukena et al., 1985). More recently, enprofylline has also been found to have a low affinity for A₃ receptors (Linden et al., 1993). These findings led to the conclusion that enprofylline was not an adenosine receptor antagonist. These original studies need to be reinterpreted in the light of our current knowledge of adenosine receptor subtypes. It is now known that accumulation of cAMP in hippocampal slices, which was shown to be blocked by enprofylline, is mediated by A_2B receptors (Lupica et al., 1990), and that platelets, found to be insensitive to enprofylline, express mainly A_2A receptors (Feoktistov and Biaggioni, 1993; Dionisotti et al., 1996; Ledent et al., 1997). Therefore, previous contradictory results can now be explained by a selective antagonism of A_2B receptors by enprofylline. Indeed, it was recently demonstrated that enprofylline is equipotent to theophylline as an A_2B receptor antagonist in HEL cells, with a dissociation constant of antagonist-receptor complex (K_B) of 7 µM (Feoktistov and Biaggioni, 1995). An analysis of the original results in the hippocampus (Fredholm and Persson, 1982) reveals an approximate K_B of 6 µM. An identical K_i for enprofylline (7 µM) was found in CHO cells stably transfected with A_2B using radioligand binding with [³H]1,3 diethyl-8-phenylxanthine (Robeva et al., 1996). This value also correlated well with the K_B estimated from inhibition of NECA-induced cAMP generation in a similar cell model (23 µM) (Alexander et al., 1996). Enprofylline is also an effective antagonist of A_2B receptors in human HMC-1 mast cells (Feoktistov and Biaggioni, 1995) and canine BR mastocytoma cells (Auchampach et al., 1996). In comparative radioligand binding studies on all four human adenosine receptors permanently expressed in CHO cells, enprofylline has been shown to be 22-fold selective for A_2B versus A₁, five-fold versus A_2A, and six-fold versus A₃ (Robeva et al., 1996). Enprofylline, therefore, can be considered a relatively selective, though not potent A_2B antagonist.

More potent but nonselective A_2B receptor antagonists have been also characterized. These compounds include 1,3-dipropyl-8-(p-sulfophenyl)xanthine (DPSPX), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), and XAC (Feoktistov and Biaggioni, 1993; Brackett and Daly, 1994). The xanthine antagonist DPSPX is 20-fold more potent at A_2B receptors in HEL cells (K_B = 141 nM) compared with platelet A_2A receptors (Feoktistov and Biaggioni, 1993). However, the affinity of A_2B receptors for DPSPX (Feoktistov and Biaggioni, 1993) is similar to those of sheep A₃ (Linden et al., 1993) and rat A₁ (Ukena et al., 1986) receptors. Among nonxanthine compounds, 2,4-dioxobenzo[g]pteridine (alloxazine) was reported to be nine-fold more potent as an antagonist of A_2B receptors in VA13 and NIH 3T3 cells compared with A_2A receptors in PC12 cells (Brackett and Daly, 1994; fig. 5).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Chemical structure and radioligand binding data on the affinity of adenosine antagonists. Ki values (nM) for rat A1/A2A/A2B/A3 receptors are shown, except as indicated (h, human; gp, guinea pig; s, sheep; m, mice). Numbers in brackets represent KB of adenosine antagonists. Data compiled from Feoktistov and Biaggioni (1993), Brackett and Daly (1994), van Galen et al. (1994), Jacobson et al. (1996), Robeva et al. (1996), Jiang et al. (1996), van Rhee et al. (1996), Zocchi et al. (1996a), and Jacobson and Suzuki (1996). *, selective for rat, but not for human or rabbit A1 receptors (Jacobson and Suzuki, 1996).

A_2B receptors are frequently found with other adenosine receptor subtypes in the same tissue, and are even coexpressed in the same cells. Recent advances in the development of selective A₁, A_2A, and A₃ antagonists (table 1; fig. 5) provide a new approach to the study of A_2B receptors; the nonselective agonist NECA can be used in conjunction with highly selective antagonists of other adenosine receptor subtypes to selectively stimulate A_2B receptors. The ability to selectively block other adenosine receptors is particularly useful in situations in which they are present with A_2B receptors.

The first selective A₁ antagonist DPCPX was discovered by two independent groups of investigators (Martinson et al., 1987; Bruns et al., 1987) and has become the reference A₁ receptor antagonist. It is highly selective for A₁ versus A_2A (80- to 500- fold across species) (Jacobson et al., 1992; Robeva et al., 1996). In recent radioligand binding studies involving all four human recombinant adenosine receptors, DPCPX has been confirmed to be 20-fold selective for A₁ versus A_2B (Robeva et al., 1996). Selective blockade of A₁ receptors with DPCPX was successfully used to reveal functional A_2B receptors in tissues coexpressing both A₁ and A_2B receptors (Mogul et al., 1993; Murthy et al., 1995; Nicholls et al., 1996). Other compounds have been identified with even greater selectivity for the A₁ receptor; C⁸-(N-methylisopropyl)-amino-N⁶-(5^'-endohydroxy)-endonorbornan-2-yl-9-methyladenin (WRC-0571) binds to human A₁ receptors with a K_i of 3 nM and to human A_2B receptors with a K_i of 19 µM. This compound, therefore, is approximately 6300-fold selective for A₁ versus A_2B (Robeva et al., 1996).

Among the new generation of A_2A antagonists, 4-(2-[7-amino-2-)2-furyl(triazolo {2,3-a}-[1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) was reported to be 30- to 80-fold selective for A_2A versus A_2B (Poucher et al., 1995). Another antagonist, 5-amino-7-(phenylethyl)-2-(1-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261), has a high affinity (K_i = 0.7-2.2 nM) for A_2A receptors (Belardinelli et al., 1996; Lindström et al., 1996; Dionisotti et al., 1996; Zocchi et al., 1996a,b; Ongini et al., 1996; Ongini and Fredholm, 1996) but was found not to block NECA-induced vasorelaxation of guinea pig aorta, a process thought to be mediated by A_2B receptors (Zocchi et al., 1996a). The selectivity of SCH 58261 for A_2A versus A_2B has been also confirmed in a cellular system; this compound was ineffective on HEL cell A_2B receptors up to a concentration of 100 nM, whereas it inhibited the CGS 21680-induced cAMP accumulation in HMC-1 cell (A_2A receptor) with a K_B of 0.1 nM (Feoktistov and Biaggioni, 1997). SCH 58261, therefore, can be useful in the discrimination of A_2B function in cells also coexpressing A_2A receptors. This approach was applied to the study of adenosine receptors in the human mast cells HMC-1 (fig. 6). The concentration-response relationship of the nonselective adenosine agonist NECA for cAMP accumulation in these cells follows a curve with a Hill slope of 0.64 ± 0.07 best fitted to a two-site model with an apparent pD₂ of 7.69 ± 0.42 and 5.92 ± 0.21 for the high- and low-affinity sites, respectively. Upon complete blockade of A_2A receptors with 100 nM SCH 58261, the concentration-response curve of NECA was transformed into a typical sigmoid curve with a Hill slope of 0.93 ± 0.06 and a pD₂ of 5.68 ± 0.03, consistent with activation of A_2B receptors. Blockade of A_2A receptors in the same cells with SCH 58261 did not affect NECA-induced calcium mobilization, confirming that this process is mediated solely via A_2B receptors (Feoktistov and Biaggioni, 1997), as it has been previously suggested on the basis of the lack of CGS 21680 effectiveness (Feoktistov and Biaggioni, 1995).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Selective activation of A2B receptors with NECA in HMC-1 human mast cells coexpressing A2A and A2B receptors, made possible by blockade of A2A receptors with the selective antagonist SCH 58261 (Feoktistov and Biaggioni, 1997). See text for details.

Recently, several antagonists with A₃ selectivity versus A₁ and A_2A receptors have been introduced. These compounds include the flavonoid derivative 3,6-dichloro-2'-isopropyloxy-4'-methylflavone (MRS 1067) and the dihydropyridine derivatives 3-ethyl 5-benzyl 2-methyl-phenylethynyl-6-phenyl-1,4(±)dihydropyridine-3,5-dicarboxylate (MRS 1191), 3,5-diethyl 2-methyl-4[2-(4-nitrophenyl)-(E)-vinyl-6-phenyl-1,4-(±)-dihydro-pyridine-3,5-dicarboxylate (MRS 1222), and 3,5-diethyl 2-methyl, 6-phenyl-4-[2-[phenyl-(trans)-vinyl]-1,4(±)dihydropyiridine-3,5-dicarboxylate (MRS 1097) (fig. 5), which are selective for the human A₃ receptor by a factor of 45- to 1700-fold, versus rat A₁ and A_2A receptors, as determined from radioligand binding studies (Jiang et al., 1996; Karton et al., 1996; van Rhee et al., 1996). It should be noted, however, that the highest degree of selectivity for these compounds is observed when their effects on human A₃ receptors are compared with their effects on rat A₁ and A_2A receptors. For example, MRS 1191 was selective for the human A₃ receptor by factor of 1300-fold, whereas for the rat A₃ receptor, the selectivity was only 11-fold versus the rat A₁ receptor (Jiang et al., 1996). Among other compounds, the triazolonaphthyridine derivative 6-carboxymethyl-5,9-dihydro-9-methyl-2-phenyl-[1,2,4]-triazolo-{5,1-a}-[2,7]-naphthyridine (L-249313) (fig. 5) is highly potent on human A₃ receptors (K_i =13 nM), but not on rat A₃ receptors (K_i = 58 µM). The thiazolopyrimidine derivative 3-(4-methoxyphenyl)-5-amino-7-oxo-thiazolo-[3,2]-pyrimidine (L-268605) was also shown to be a potent antagonist on human A₃ receptor (K_i = 18 nM). Both compounds are highly selective for the human A₃ receptor versus the human A₁ (>300-fold) and A_2A (>1400-fold) receptors (Jacobson et al., 1996). Unfortunately, A_2B receptors have not been included when characterizing the selectivity of A₃ antagonists. Additional studies of A₃ antagonists with respect to A_2B receptors are required to verify whether they can be useful to discriminate between A₃ and A_2B-mediated effects.

In summary, potent and selective agonists and antagonists are available for all adenosine receptors except for the A_2B subtype. The characterization of A_2B receptors has been based on apparent potencies of agonists selective to other adenosine receptor subtypes. The development of selective A₁, A_2A, and A₃ antagonists provides a new approach when used in conjunction with the nonselective agonist NECA to selectively stimulate A_2B receptors. This approach is particularly useful in tissues or cells expressing more than one adenosine receptor. However, much progress in this field could be achieved by the development of selective A_2B receptor antagonists. Because of the low affinity of this receptor for agonists, the design of selective and potent A_2B antagonists seems to be more promising than the development of selective agonists.

	V. Distribution of A2B Receptors

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The generation of cDNA for A_2B receptors has made possible the identification of the tissue distribution of this receptor subtype. A_2B receptor messenger ribonucleic acid (mRNA) was originally detected in a limited number of rat tissues by Northern blot analysis, with the highest levels found in cecum, bowel, and bladder, followed by brain, spinal cord, lung, epididymis, vas deferens, and pituitary (Stehle et al., 1992). The use of more sensitive reverse transcriptase-polymerase chain reaction techniques revealed a ubiquitous distribution of A_2B receptors. mRNA encoding A_2B receptors was detected at various levels in all rat tissues studied, with the highest levels in the proximal colon and lowest in the liver (Dixon et al., 1996). In situ hybridization of A_2B receptors showed widespread and uniform distribution of A_2B mRNA throughout the brain (Stehle et al., 1992; Dixon et al., 1996). The expression of A_2B receptors in a variety of human and murine tissues has been confirmed by Western blotting and by immunostaining with an anti-A_2B receptor antibody (Puffinbarger et al., 1995).

Pharmacological identification of A_2B receptors, based on their low affinity and characteristic order of potency for agonists, also indicates a widespread distribution of A_2B receptors. In brain, functional A_2B receptors are found in neurons (Mogul et al., 1993; Okada et al., 1996; Kessey et al., 1997) and glial cells (van Calker et al., 1979; Elfman et al., 1984; Hösli and Hösli, 1988; Altiok et al., 1992; Peakman and Hill, 1994, 1996; Fiebich et al., 1996a). Although there is no evidence that A_2B receptor are present in microglia (Fiebich et al., 1996b), there is ample data that show that they are expressed in astrocytes and in different glioma cell lines (Elfman et al., 1984; Hösli and Hösli, 1988; Altiok et al., 1992; Peakman and Hill, 1994, 1996; Fiebich et al., 1996a). The expression of A_2B receptors in glial cells, which represent a majority of the brain cell population, can explain the original observation that slices from all brain areas examined showed an adenosine-stimulated cAMP response (Sattin and Rall, 1970; Daly, 1976).

Functional A_2B receptors have been found in fibroblasts (Brackett and Daly, 1994) and various vascular beds (Martin, 1992; Martin et al., 1993; Chiang et al., 1994; Martin and Potts, 1994; Haynes et al., 1995; Rubino et al., 1995; Prentice and Hourani, 1996; Dubey et al., 1996b). Contamination with these cells may also contribute to the widespread pattern of A_2B receptor distribution in all organs. This possibility should always be considered, especially when data from crude tissue preparations are analyzed. The presence of functional A_2B receptors also has been demonstrated in hematopoietic cells (Feoktistov and Biaggioni, 1993; Porzig et al., 1995), mast cells (Marquardt et al., 1994; Feoktistov and Biaggioni, 1995), myocardial cells (Liang and Haltiwanger, 1995), intestinal epithelial (Strohmeier et al., 1995) and muscle cells (Murthy et al., 1995; Nicholls et al., 1996), retinal pigment epithelium (Blazynski, 1993; Gregory et al., 1994), endothelium (Iwamoto et al., 1994), and neurosecretory cells (Casado et al., 1992; Gharib et al., 1992; Mateo et al., 1995).

Coexpression of A_2B receptors together with other adenosine receptors has been reported in various cell preparations and cell lines. Functionally coupled A_2B and A_2A receptors are coexpressed in rat pheochromocytoma PC12 cells (Hide et al., 1992; Chern et al., 1993; van der Ploeg et al., 1996), T-cell leukemia Jurkat cells (van der Ploeg et al., 1996), mouse bone marrow-derived mast cells (Marquardt et al., 1994), human mast HMC-1 cells (Feoktistov and Biaggioni, 1995), human aortic endothelial cells (Iwamoto et al., 1994), human umbilical vein endothelial cells (Feoktistov and Biaggioni, unpublished observations), and human neutrophil leukocytes (Fredholm et al., 1996c). mRNA encoding A_2A, A_2B, and A₃, but not A₁ receptors, have been found in rat RBL 2H3 mast cells (Ramkumar et al., 1993; Marquardt et al., 1994).

Functional A₁ receptors can also be coexpressed with A_2A and/or A_2B receptors. In most cases, a selective blockade of A₁ receptors is required to unmask functional A_2B receptors. This approach was successfully used in dispersed guinea pig small intestinal muscle cells (Murthy et al., 1995), in rat duodenum longitudinal muscle muscularis mucosae cells (Nicholls et al., 1996), and in guinea pig pyramidal neurons from the hippocampal CA3 region (Mogul et al., 1993). Similarly, uncoupling of A₁ receptor using pertussis toxin unmasks the presence of A_2A and A_2B receptors in ventricular myocytes (Liang and Haltiwanger, 1995). By contrast, it was not necessary to block A₁ receptors in various glial cells to observe either A_2A or A_2B receptor-mediated stimulation of adenyl cyclase (Elfman et al., 1984; Altiok et al., 1992; Peakman and Hill, 1994, 1996; Fiebich et al., 1996a). Also, the balance between A₁- and A₂-mediated responses can be modulated. For example, corticosteroid treatment of DDT₁ MF2 smooth muscle cells increased A₁ receptor number and signaling and decreased A₂ receptor signaling (Gerwins and Fredholm, 1991). A similar decrease in the A_2B signaling upon dexamethasone treatment was also reported in Jurkat cells (Svenningsson and Fredholm, 1997).

Coexistence of different adenosine receptor types in cells obtained from primary tissue cultures (Iwamoto et al., 1994; Peakman and Hill, 1994, 1996) may be attributed to the presence of different subpopulations of cells, each one expressing a single type of adenosine receptor. However, studies on established cell lines (Hide et al., 1992; Feoktistov and Biaggioni, 1995; van der Ploeg et al., 1996) have confirmed the coexpression of adenosine receptors in a single target cell. Moreover, studies performed on single cells have also demonstrated the presence of more than one adenosine receptor subtype (Liang and Morley, 1996; Strickler et al., 1996), including A_2B receptors (Liang and Haltiwanger, 1995).

Coexpression of A_2B and A_2A receptors has been demonstrated even in clonal cell lines originally used to describe prototypic A_2A (PC12 cells) and A_2B receptors (Jurkat cells) (van der Ploeg et al., 1996). These cells predominantly express A_2A and A_2B receptors, respectively, and the presence of the other receptor type was recognized only after carefully conducted studies using differential responses to a series of 2-substituted adenosine analogs (Hide et al., 1992; van der Ploeg et al., 1996). It is entirely possible, therefore, that more examples of cells coexpressing adenosine receptors may become apparent after selective adenosine antagonists are applied in the characterization of these cells.

The functional meaning of this simultaneous expression of multiple adenosine receptor subtypes in a single target cell is not known. Because A₁ and A_2A receptors have a higher affinity for adenosine, in many cellular systems, these receptors need to be blocked before A_2B-mediated effects are apparent (Mogul et al., 1993; Liang and Haltiwanger, 1995; Murthy et al., 1995; Nicholls et al., 1996; Kessey et al., 1997; Feokstistov and Biaggioni, 1997). This, however, is not always the case. Both A₁ and A_2B receptors are present in glial cells of rat astrocytes, and stimulation of A_2B receptors with the nonselective agonist NECA induces cAMP accumulation that is evident even in the presence of A₁ receptors (Elfman et al., 1984; Altiok et al., 1992; Peakman and Hill, 1994, 1996; Fiebich et al., 1996a). It is possible that the relative importance of A_2B receptors is greater in situations in which high interstitial levels of adenosine are reached, e.g., in tissues in which metabolic demands are increased or oxygen supply is decreased, whereas the high affinity A₁ and A_2A receptors may modulate cellular functions in response to lower concentrations of this autacoid. The recent recognition that in cells coexpressing other adenosine receptors, A_2B receptors can be coupled to distinct intracellular pathways (Feoktistov and Biaggioni, 1995), may also provide the basis for a differential physiological role.

	VI. Intracellular Pathways Regulated by A2B Receptors

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It is generally accepted that A_2A and A_2B receptors are coupled to G_s proteins, because both activate adenyl cyclase in virtually every cell in which they are expressed. Although activation of adenyl cyclase is arguably an important signaling mechanism for A_2A receptors, this is not necessarily the case for A_2B receptors, as other intracellular signaling pathways have been found to be functionally coupled to A_2B receptors in addition to adenyl cyclase (fig. 7).

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Schematic representation of intracellular pathways coupled to adenosine A2B receptors in various cells. A2B receptors are coupled to adenyl cyclase (AC) in all cells shown. Activation of this pathway results in accumulation of cAMP and stimulation of protein kinase A (PKA). (A) A2B receptors are coupled to phosphatidylinositol-specific phospholipase C (PI-PLC) via a G protein of the Gq family [G(q)] in mast cells (Marquardt et al., 1994; Feoktistov and Biaggioni, 1995; Auchampach et al., 1996). Activation of this pathway results in increase in diacylglycerol (DAG) and inositol trisphosphate (IP3). Diacylglycerol stimulates protein kinase C (PKC). Inositol trisphosphate activates mobilization of calcium from intracellular stores. (B) A2B receptors potentiate calcium influx directly by coupling with Gs protein in HEL cells (Feoktistov et al., 1994). (C) In contrast, A2B receptors potentiate calcium influx via cAMP and activation of protein kinase A in pyramidal neurons from the CA3 region of guinea pig hippocampus (Mogul et al., 1993).

Recombinant rat A_2B receptors expressed in Xenopus oocytes activate calcium-dependent chloride conductance presumably by stimulation of phospholipase C (Yakel et al., 1993). Likewise, it has been proposed that A_2B receptors stimulate phospholipase C in mouse bone marrow-derived mast cells (Marquardt et al., 1994). Regulatory proteins of the G_q family are thought to play a role in the coupling of A_2B receptors to -phospholipase C in human mast HMC-1 cells (Feoktistov and Biaggioni, 1995) and canine BR mastocytoma cells (Auchampach et al., 1996), because this process is unaffected by treatment with pertussis or cholera toxins. A_2B receptor-mediated stimulation of -phospholipase C results in mobilization of intracellular calcium in HMC-1 cells and eventually promotes synthesis of interleukin-8 (IL-8) (Feoktistov and Biaggioni, 1995; fig. 7a). In contrast to A_2B receptors, there is no evidence that A_2A receptors can stimulate phospholipase C under physiological conditions, even though cotransfection of human A_2A receptors with murine G₁₅ and human G₁₆, but not with G_q, G₁₁ or G₁₄, results in A_2A-mediated stimulation of phospholipase C in COS-7 cells (Offermanns and Simon, 1995). However, promiscuous coupling of G₁₅ and G₁₆ has been observed when these G proteins are coexpressed with receptors which are otherwise not normally coupled to phospholipase C (Milligan et al., 1996). Also, expression of G₁₅ and G₁₆ is limited only to a subset of hematopoietic cells (Amatruda et al., 1991; Wilkie et al., 1991).

Stimulation of A_2B receptors also increases intracellular calcium in HEL cells but not through a mechanism involving phospholipase C activation (fig. 7b). In contrast to the cholera toxin- and pertussis toxin-insensitive mobilization of intracellular calcium observed in HMC-1 mast cells, A_2B receptors facilitate calcium influx through a cholera toxin-sensitive mechanism in HEL cells. This effect was observed only when intracellular calcium levels were elevated, either by receptor-dependent (e.g., by thrombin) or -independent (e.g., thapsigargin) mechanisms. Even though this process is coupled to G_s-proteins, it is cAMP-independent. It has been suggested that G_s, coupled to A_2B receptors, can directly stimulate a putative calcium channel (Feoktistov et al., 1994), as proposed for other G_s-coupled receptors (Imoto et al., 1988; Scamps et al., 1992).

Of interest, a similar mechanism has been suggested for A_2A receptors in fetal chicken ventricular myocardium cells. These cells coexpress A_2A and A_2B receptors, and both are positively coupled to stimulation of adenyl cyclase and myocyte contractility (Liang and Haltiwanger, 1995). Selective activation of A_2A receptors with CGS 21680 results in cAMP-independent calcium entry in pertussis toxin-treated cells. This effect does not involve simulation of phospholipase C and was blocked by the selective A_2A antagonist 8-(3-chlorostyryl)caffeine (Liang and Morley, 1996). This study did not explore the possibility that A_2B receptors share a common mechanism of G_s-mediated stimulation of calcium entry with A_2A receptors. This could be tested by using the nonspecific A₂ agonist NECA in the presence of a selective A_2A antagonist such as SCH 58261.

In another example of positive modulation of intracellular calcium, it has been reported that activation of A_2B receptors results in significant potentiation of P-type, but not N-type, calcium currents in pyramidal neurons from the CA3 region of guinea pig hippocampus. This mechanism was thought to be mediated by adenyl cyclase, because this potentiation could be inhibited by blocking the cAMP-dependent protein kinase (Mogul et al., 1993; fig. 7c).

It has recently been recognized that intracellular signaling of A_2B receptors can be modulated by interaction with other receptor systems (Fredholm, 1995; Fredholm et al., 1996a; fig. 8). For example, agents that increase intracellular calcium or activate protein kinase C significantly potentiate A_2B-mediated cAMP production in various cells (Hollingsworth et al., 1985; Norstedt and Fredholm, 1987; Fredholm et al., 1987; Norstedt et al., 1989; Kvanta et al., 1989, 1990; Altiok et al., 1992; fig. 8a). On the other hand, bradykinin-stimulated calcium entry caused inhibition of A_2B receptor-stimulated adenyl cyclase in astrocytoma D384 cells (fig. 8b), but direct stimulation of protein kinase C enhanced the A_2B response (Altiok et al., 1992; Altiok and Fredholm, 1993). The exact mechanism of the interaction between protein kinase C and A_2B-mediated pathways is not known, but it cannot be considered a unique feature of A_2B receptors. For instance, activation of thrombin-induced phospholipase C pathways potentiate cAMP accumulation stimulated by IP prostanoid receptors in HEL cells (Turner et al., 1992; Feoktistov et al., 1997). It has been suggested that protein kinase C does not exert its effect at the level of the receptor but rather affects the coupling of the stimulated G_s protein with adenyl cyclase (Fredholm, 1995). The synergistic interaction between the A_2B receptors and the calcium/protein kinase C pathway can occur further down in the signaling cascade. Thus, A_2B receptors greatly potentiate the phorbol 12-myristate 13-acetate-induced synthesis of IL-8 in human mast cells (Feoktistov and Biaggioni, 1995). In T-lymphocytes, the T-receptor is known to activate immediate early gene transcription, leading to the activation of the AP-1 transcription factor (Kvanta et al., 1992). A_2B receptors significantly potentiate this response, implying that cAMP, and calcium/protein kinase C pathways, may act in concert in the regulation of gene transcription (Kontny et al., 1992; Kvanta and Fredholm, 1994).

View larger version (42K): [in this window] [in a new window]   Fig. 8.   Modulation of A2B receptor signaling. (A) In Jurkat cells, stimulation of calcium entry and of protein kinase C (PKC) by T-cell receptor activation increases the magnitude of the cAMP response to stimulation of A2B receptors (Kvanta et al., 1989; Fredholm et al., 1996a). (B) In human astrocytoma cells, D384 activation of protein kinase C is also able to enhance A2B receptor-mediated cAMP accumulation, but the stimulation of calcium entry via the bradykinin B2 receptor leads to inhibition of A2B receptor-mediated cAMP accumulation (Altiok and Fredholm, 1993; Fredholm et al., 1996a).

In summary, A_2B receptors are coupled to adenyl cyclase through G_s proteins in every cell studied. Current evidence suggests that the actions of A_2B receptors can be mediated not only by cAMP, but also by other intracellular pathways that may vary between cells. A_2B receptors can couple to calcium channels through G_s, but additional studies are needed to determine the type of channel involved. Similarly, it remains to be determined which member of the G_q family is responsible for A_2B receptor coupling to phospholipase C. It is of interest that, as far as intracellular pathways are concerned, A_2B receptors have as much in common with A₁ or A₃ receptors (activation of phospholipase C), as with A_2A receptors (activation of adenyl cyclase). It would be important to determine which domain of the A_2B receptor defines the differences in G protein coupling between A_2A and A_2B receptors.

	VII. Physiological Functions of A2B Receptors

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Adenosine-induced vasodilation has been traditionally attributed to activation of A_2A receptors. However, the recent finding of the presence of A_2B receptors in some vascular beds raised the possibility that they participate in the regulation of vascular tone. Indeed, there are vascular beds in which the nonselective agonist NECA produces profound vasodilation, but the selective A_2A agonist CGS 21680 has little effect, suggesting that adenosine-induced vasodilation is mediated via A_2B receptors (for review, see Webb et al., 1992). This phenomenon is observed in guinea pig aorta and dog saphenous vein (Hargreaves et al., 1991), and in dog coronary arteries (Balwierczak et al., 1991). This effect is not due to species differences, because both A_2A and A_2B receptors may mediate vasodilation in the same species. In guinea pig, for instance, A_2A receptors mediate relaxation of coronary vessels, whereas A_2B receptors produce vasodilation of the aorta (Martin, 1992; Martin et al., 1993). Likewise, the A_2A agonist CGS 21680 lowers blood pressure in the intact dog (Levens et al., 1991), presumably by inducing vasodilation, despite its lack of efficacy in the coronary arteries of this species.

The vasodilatory effects of adenosine can be accounted for by a direct relaxing action on vascular smooth muscle cells. However, recent studies have suggested that the endothelium contributes to, or is even essential for, the vasodilatory effects of intravascular adenosine. It has been shown that most of the labeled adenosine administered intra-arterially is contained within endothelial cells, and very little escapes this endothelium trap to reach the underlying vascular smooth muscle (Nees et al., 1985). Similarly, intravascular administration of adenosine linked to macromolecules, and therefore less likely to cross the endothelium, is still able to produce vasodilation (Olsson et al., 1977).

In vitro studies, however, have yielded conflicting results as to whether the vasodilatory actions of adenosine are different in vascular preparation with intact or denuded endothelium (Rubanyi and Vanhoutte, 1985; Yen et al., 1988; Falcone et al., 1993; Maekawa et al., 1994). Evaluation of a putative endothelium-dependent vasodilation by adenosine is challenging in ring preparation, because adenosine will produce vasodilation in preparations with or without endothelium. This is particularly true when stable agonists are used, because they are not trapped by the endothelium in the way adenosine is and have more ready access to the underlying vascular smooth muscle. Other endothelium-dependent vasodilators will constrict vascular smooth muscle in the absence of endothelium (Furchgott, 1984), making their distinction easier. Conversely, adenosine-induced vasodilation could conceivably produce flow-related release of nitric oxide (NO), giving the appearance of NO-mediated vasodilation (Olsson, 1996).

Methodological difficulties notwithstanding, more fundamental differences may explain the apparent discrepancies regarding the role of the endothelium on adenosine-induced vasodilation. Given the diversity of endothelial cell types, it is possible that endothelial vasodilatory responses to adenosine vary between species, and within the same species depending on the vascular bed being studied. It is unclear to what degree endothelial A_2A or A_2B receptors may contribute to these differences. This issue will be resolved only if studies that examine the endothelium-dependency of adenosine-induced vasodilation also define the adenosine receptor subtype involved.

A_2B receptors have been shown in endothelial cells. Both A_2B and A_2A receptors regulate cAMP production in human aortic (Iwamoto et al., 1994) and human umbilical vein (Feoktistov and Biaggioni, unpublished observations) endothelial cells, and A_2B receptor mRNA has been detected in human aortic endothelial cells (Iwamoto et al., 1994). Few studies have directly examined the possible interaction between A_2B receptors and endothelium-derived vasodilation, and results vary depending on the vascular bed studied. A_2B receptors mediate vasodilation in the rat mesenteric arterial bed (Rubino et al., 1995) and in the isolated blood-perfused rat lung preparation (Haynes et al., 1995). In both cases, A_2B-mediated vasodilation seems to be independent of NO generation, because they were not reversed by inhibition of NO synthase by N^G-nitro-L-arginine methyl ester (L-NAME) (Haynes et al., 1995; Rubino et al., 1995). On the contrary, isolated rat renal artery rings contain A_2B receptors that are located exclusively on the endothelium and cause NO release and vasodilation, because this vasodilation can be blocked with L-NAME and prevented by removal of the endothelium (Martin and Potts, 1994). Similarly, A_2B receptors also appear to vasodilate the rabbit corpus cavernosum, and this effect is reduced by removal of the endothelium (Chiang et al., 1994).

In summary, both A_2A and A_2B receptors mediate vasodilation. The relative contribution of A_2B receptors to adenosine-induced vasodilation is not defined. There are also conflicting results as to the importance of NO generation in adenosine-induced and A_2B-induced vasodilation, but there are some examples in which the endothelium contributes to A_2B-mediated vasodilation. To complicate things further, in some vascular beds, adenosine-induced vasodilation is endothelium-dependent but does not appear to be mediated by NO because it is not blocked by inhibition of NO synthase, raising the possibility that other endothelial factors, such as endothelium-dependent hyperpolarizing factor, may be involved (Headrick and Berne, 1990). The precise nature of the interaction between A_2B receptors and endothelial cells and their role in the regulation of vascular tone are areas where more research is needed.

Adenosine has important protective effects against ischemia in the myocardium, but these effects are largely attributed to A₁ receptors (for review, see Olsson and Pearson, 1990). It has been reported recently that myocytes isolated from fetal chick ventricles, but not from the atria, possess functional A_2B and A_2A receptors. Both receptors are capable of augmenting myocardial contractility in this model. These adenosine effects, however, become evident only after inhibitory A₁ receptor pathways are inactivated with pertussis toxin (Liang and Haltiwanger, 1995). Presence of A₂ receptors, capable of stimulating cAMP accumulation, was demonstrated in cultured adult rodent myocardial cells after A₁ receptor blockade (Romano et al., 1989; Stein et al., 1993; Xu et al., 1996). These results could be explained by a possible contamination of myocardial preparations with fibroblasts and endothelial cells expressing A_2B receptors. However, studies performed on single cells argue against this possibility. A positive inotropic response mediated via A₂ receptors was demonstrated in cultured rat and guinea pig ventricular myocytes (Stein et al., 1993; Xu et al., 1996; Dobson and Fenton, 1997). The role of myocardial A₂ receptors in mediating a positive inotropic effect remains a controversial issue (Olsson, 1996), and their physiological significance is unclear, given that their effects become evident only under blockade of A₁ receptors.

Adenosine is in general considered to be a depressor of neurons, inhibiting neurotransmitter release and other neuronal functions (Phillis et al., 1993a) and acts as a neuroprotective against ischemia (Dragunow and Faull, 1988). Many of these inhibitory actions are mediated by A₁ receptors (Dunwiddie and Fredholm, 1989). A₂ receptors, on the other hand, have been shown to mediate excitatory actions on the nervous system (Sebastião and Ribeiro, 1996). Earlier studies did not use specific agonists or antagonists to allow a precise identification of the A₂ receptor subtype involved, and relatively little information is available for the A_2B receptor. More recently, several excitatory actions have been linked to the A_2A receptor, including enhancement of the release of several neurotransmitters, including acetylcholine, the excitatory amino acids glutamate and aspartate, dopamine, and norepinephrine (for review, see Sebastião and Ribeiro, 1996). However, gene knockout mice lacking A_2A receptors exhibit aggressive behavior and lack the stimulant effect of caffeine (Ledent et al., 1997), suggesting that A_2A receptors normally exert a tonic central depressant action. This is in agreement with several observations indicating a depressant effect of A_2A agonists on locomotor activity (Sebastião and Ribeiro, 1996). It should be noted, however, that comparisons between molecular mechanisms of excitation and integrated physiological responses need to be done with care. For example, adenosine depresses sympathetic nerve activity and blood pressure when injected into the nucleus tractus solitarii (Tseng et al., 1988) via activation of A_2A receptors (Barraco et al., 1991). This apparent depressant action, however, is mediated by local stimulation of the release of the excitatory amino acid glutamate (Mosqueda-Garcia et al., 1989, 1991), mediated by A_2A receptors (Castillo-Melendez et al., 1994).

A_2B receptors are widespread in the brain, but little is known about their function. There are, however, several examples of neuroexcitatory actions. Adenosine agonists increase the release of the excitatory amino acid aspartate in rat cerebral cortex cup superf