A<sub>2A</sub> adenosine receptors in human peripheral blood cells (2000)

Adenosine mediates a wide range of physiological functions by activation of at least four cell surface receptors named A1, A2A, A2B and A3. Each receptor subtype has been cloned and shows unique ligand binding properties and a distinct pattern of tissue expression (Fredholm et al., 1994; Linden, 1994; Alexander & Peters, 1997; Ralevic & Burnstock, 1998). Adenosine is a highly active biologic compound with a variety of effects on numerous tissues, including heart muscle, coronary arteries, smooth muscle cells, platelets and cells involved in immune and inflammatory reactions. Through interaction with A2A receptors, adenosine is involved in platelet antiaggregatory effects, neutrophil antiinflammatory responses as well as in modulation of immune cell function (Salmon & Cronstein, 1990; Sullivan et al., 1990; Hoskin et al., 1994; MacKenzie et al., 1994). The presence of A2A receptors on all these different cell types i.e. monocytes, lymphocytes, neutrophils, basophils, mast cells should not be surprising because the cells involved in the immune and inflammatory responses arise from a common stem cell source in the bone marrow. Studies on the effects of adenosine and adenosine analogues on blood cells are important due to the proposed role of adenosine (1) in the pathogenesis of diseases such as severe combined immunodeficiency (ADA SCID) (Giblett et al., 1972; Huang et al., 1997), (2) in the regulation of normal immune processes (Bouma et al., 1997; Apasov et al., 1997), (3) as an endogenous anti-aggregatory and anti-inflammatory agent (Cronstein, 1995; Sullivan & Linden, 1998) and (4) in the use of adenosine analogues as pharmacologic agents (Jacobson et al., 1992; Olah & Stiles, 1995; Poulsen & Quinn, 1998). On this basis the present review summarizes the pharmacological, biochemical and functional data of A2A receptors expressed in human platelets, lymphocytes and neutrophils of peripheral blood and describes the signal transmission mechanisms and the functional responses responsible for adenosine's biological effects. Since 1963 adenosine has been postulated to be an endogenous inhibitor of platelet aggregation and secretion (Born & Cross, 1963). The putative efficacy of dipyridamole as an antiaggregatory agent could be explained at least in part by its effect to block the nucleoside transport system, and thereby increase the extracellular concentration of adenosine and potentiate its effects on membrane receptors. Platelets, though non-nucleated and, thus, strictly speaking, not entitled to be classified as cells, represent a uniform tissue possessing A2 receptors (now known to be the A2A receptor subtype) on the external membrane (Haslam & Cusack, 1981). It is believed that the antiaggregatory action of adenosine depends upon the inhibition, coupled with adenylate cyclase activation, of both calcium influx and mobilization of internal stores (Paul et al., 1990). Considering the relevance of platelet aggregation in a variety of cardiovascular and cerebrovascular disorders, several studies have been performed to characterize A2A adenosine receptors in these blood components. The lack of a selective A2A radioligand had hampered the direct characterization of the A2A receptor subtype. The non-selective agonist NECA (A1, A2A, A2B, A3) was the first pharmacological tool to be used for labelling the A2A receptor in the rat brain but it was also found to bind to different affinity states and subtypes of adenosine receptors (Yeung & Green 1984; Bruns et al., 1986). As in other peripheral tissues, in human platelet membranes the binding properties of [3H]-NECA did not agree with the pharmacology of A2A receptors (Hütteman et al., 1984; Ukena et al., 1984; Lohse et al., 1988; Nakata & Fujisawa, 1988; Keen et al., 1989). Specifically, while only 10% of [3H]-NECA binding to human platelet membranes is displaced by N6-substituted adenosine derivatives, which are known to both activate adenylate cyclase and inhibit platelet aggregation, about 95% of this binding could be displaced with unlabelled NECA suggesting that the majority of the [3H]-NECA binding to human platelet membranes is to non-receptor sites. This ubiquitous adenosine A2-like binding site has been separated chromatographically and distinguished pharmacologically from the A2A adenosine receptor (Lohse et al., 1988). Subsequently, the NECA-binding site has been purified from human placental (Hutchison et al., 1990) and human platelet membranes (Fein et al., 1994) and was named ‘adenotin’, a low affinity binding protein of 98 kDa whose biological significance has not been clarified, and precluded the direct characterization of A2A receptors in platelet membranes. With the aim to overcome the disadvantages observed with NECA, different radioligands have been proposed for the characterization of the human platelet A2A receptor. The A1-selective antagonist [3H]-XAC, having high levels of non-specific binding (Ukena et al., 1986), does not permit satisfactory binding studies of A2A receptors. The A2A selective agonist [3H]-CGS 21680, which has been reported to interact with the high affinity A2A adenosine receptor in both binding and adenylyl cyclase assays (Jarvis et al., 1989; Johansson et al., 1992; Mathot et al., 1995), has also been used by Varani et al. (1994) in human platelet membranes. Although saturation experiments showed that [3H]-CGS 21680 interacts with one recognition site only, the affinity of this agonist for the receptor was in the micromolar range, therefore suggesting that this radioligand like NECA, labels also the low affinity non-receptor site (Table 1). Moreover, as previously reported for the platelet [3H]-NECA binding (Hütteman et al., 1984), the displacement of [3H]-CGS 21680 binding by a series of adenosine receptor agonists and antagonists revealed Ki values substantially higher than those obtained for either stimulation or inhibition of cyclic AMP levels (Table 2A,B). Thus, although CGS 21680 interacts with the platelet A2A adenosine receptor, like NECA, it proved to be unsatisfactory for the characterization of A2A human platelet receptors. This fact has led some authors to postulate that the adenosine receptor expressed in platelets may be different from the A2A subtype identified in brain striatum (Cristalli et al., 1994). To overcome the many difficulties associated with the interaction between the A2A adenosine receptors and adenotin sites it was attempted to characterize a purified platelet A2A adenosine receptor preparation by using the selective radioligand [3H]-CGS 21680 (Varani et al., 1996). The method employed (Zolnierowicz et al., 1990) as an alternative to the gel filtration chromatography (Lohse et al., 1988), avoids high non-specific binding by removing most of the adenotin binding protein using extraction of membranes with CHAPS followed by PEG precipitation. In the purified protein preparation [3H]-CGS 21680 interacts with one recognition site only, with an affinity in the nanomolar range (Kd=285 nM) and a binding capacity of 2 pmol mg−1 protein. These values are not far from those obtained for [3H]-NECA in both solubilized A2A receptors (Lohse et al., 1988) and partially purified preparation (Zolnierowicz et al., 1990) and disagree with data obtained for [3H]-CGS 21680 binding in human platelet membranes (Varani et al., 1994) suggesting the presence in this case of a large component of low affinity binding to non-receptor sites (Table 1). Moreover, the affinities of all tested compounds for the purified receptor are systematically higher, by one to three orders of magnitude, than their affinities for platelet membranes and are very similar to those obtained in rat striatum suggesting that, only under these experimental conditions, the agonist CGS 21680 appears to be an adequate radioligand to study purified A2A adenosine receptors in human platelets (Table 2A,B). It is worth nothing that the discrepancies found between the affinity data obtained in cyclic AMP and receptor binding assays for many adenosine receptor ligands in human platelet membranes are markedly reduced in the partially purified platelet preparation in which Ki, EC50 or IC50 values are very similar for all compounds studied (Table 2A,B). These data indicate that in the purified platelet membrane preparation the presence of adenosine receptors is prevalent with respect to the non-receptor component (adenotin site). Agonists at G protein-coupled receptors are not ideal radioligands (Stiles & Jacobson, 1987). Indeed, as in the case of the binding between agonist radioligands and other G protein-linked receptors, the binding of [3H]-CGS 21680 to A2A receptors is influenced by several factors including the state of the G proteins (Johansson et al., 1992). Thus, the availability of antagonist radioligands, having high A2A receptor affinity and selectivity, represents a step forward in the characterization of this adenosine receptor subtype. Important progress has been made with the development of selective A2A adenosine receptor antagonists (Ongini & Fredholm, 1996; Ongini et al., 1999). One of them, the non-xanthine compound SCH 58261 (Baraldi et al., 1994), has been widely characterized in a variety of binding and functional assays (Zocchi et al, 1996a) and has been shown not to interact with the adenotin binding site (Varani et al., 1996). The tritium-labelled form, [3H]-SCH 58261, has been found to label A2A receptors in the rat brain (Zocchi et al., 1996b; Fredholm et al., 1998), in peripheral tissue membranes such as porcine coronary arteries (Belardinelli et al., 1996), and human cloned receptors transfected in mammalian cells (Dionisotti et al., 1997). SCH 58261 has low affinity for A1 receptors measured in rat brain cortex, does not interact with A2B receptors and has low affinity (μM range) for either rat or human A3 receptors (Zocchi et al., 1996a). On the basis of this pharmacological profile [3H]-SCH 58261 has been used to characterize A2A receptors in human platelet membranes. In saturation studies, [3H]-SCH 58261 labels one class of binding sites only, with Kd of 0.85 nM (Dionisotti et al., 1996; Varani et al., 1998b) a value similar to that observed in rat striatal membranes (Kd=0.70 nM) (Zocchi et al., 1996b), but very different from that obtained with [3H]-NECA in human platelet membranes (Table 3). In competition experiments, a series of typical adenosine ligands bound A2A platelet receptors with an affinity and a rank order of potency similar to that found in rat striatal membranes (Table 4). Moreover Ki values of adenosine agonists and antagonists correlate well with results from functional studies such as stimulation of cyclic AMP levels or platelet aggregation inhibition (Dionisotti et al., 1992) (Table 5). Thus, [3H]-SCH 58261 is the first radioligand that has allowed the binding characterization of the A2A receptor subtype in human platelets. Recently, in A2A receptor-knockout mice, it was reported that platelet aggregation was increased, indicating the importance of this receptor subtype in platelet function (Ledent et al., 1997). Activation of A2A receptors in platelets causes an increase in cyclic AMP accumulation and a decrease in platelet aggregation. During ischemia and/or hypoxia, extracellular and plasma levels of adenosine increase to levels suficient to activate A2A receptors and hence decrease platelet aggregability. The availability of [3H]-SCH 58261 should stimulate further the elucidation of the changes, such as up or downregulation, in A2A platelet receptors under a variety of physiologic and pathologic conditions. The interest in the immunomodulatory effects of adenosine arose after the discovery that hereditary deficiency of the enzyme adenosine deaminase (ADA) was associated with severe combined immune deficiency disease (SCID) (Giblett et al., 1972). SCID is a disease characterized by severe lymphocytopenia, affecting both B and T cells and marked susceptibility to infection. ADA SCID has been hypothesized to be due to the accumulation of intracellular products of adenosine metabolism leading to the depletion of lymphocytes (Hirschorn, 1995; Blackburn et al., 1996). However, the studies aimed at elucidating the mechanisms by which absence of ADA leads to immunodeficiency, first suggested the presence of adenosine receptors on lymphocytes to suppress or dampen the immune response (Wolberg et al., 1975). Subsequently, other studies reported that adenosine causes cyclic AMP accumulation in lymphocytes by interacting with a specific external membrane receptor and that the effect of a series of adenosine analogues to inhibit was with the potency of the compounds to increase the of cyclic AMP et al., et al., et al., 1981). Recently, it has been reported that of T lymphocytes to extracellular adenosine causes inhibition of T cell These effects of adenosine in T lymphocytes may be explained by activation of A2A receptors followed by in cyclic AMP that, in T cell receptor including the The effect of the selective A2A agonist CGS 21680 to inhibit the of cells by the of A2A receptor as the expressed receptor in T lymphocytes et al., 1997). the effect of adenosine to inhibit and receptor is by low of and CGS 21680 suggesting the of the A2A subtype et al., 1997). The expression of is an important of activation, because of the of either or its receptor results in a of the immune response 1989). The expression of A2A receptors, which has been in functional assays using selective agonists and antagonists of A2A receptors, was by studies of A1, A2A and A3 expression in A2A receptor but not A1 and A3 receptor was et al., 1997; Huang et al., 1997). Thus, the accumulation of adenosine in ADA SCID or under causes an adenosine receptors on T cells and a for authors that T cell immunodeficiency and could be due to extracellular which in the affecting the and of et al., et al., 1997). This is of interest in of the pharmacologic use of adenosine analogues as and antiinflammatory but the development of that specific adenosine receptors be with a of the role of these receptors in the immune binding study has been performed in membranes to characterize the affinity and the of A2A receptors et al., 1988). The radioligand used was the selective agonist NECA which had a Kd of and a of of protein (Table 3). However, NECA has also been found to interact with non-receptor binding proteins in peripheral (Hutchison et al., 1990) thus, due to the high of non-receptor NECA the A2A of the binding could not be well The availability of [3H]-SCH 58261 has the characterization of A2A receptors. In human lymphocytes, [3H]-SCH 58261 a class of recognition sites with affinity nM) very similar to that observed in rat striatal and platelet membranes and 0.85 (Varani et al., In competition studies, typical adenosine receptor agonists and antagonists bound the A2A receptor with a rank order of potency and affinities similar to those observed in [3H]-SCH 58261 binding to human platelet membranes and in also with those found in rat striatal membranes (Table 4). data from the and of cyclic AMP levels correlate well with binding (Table 5). to the the of A2A human receptors with a selective studies performed and the and to the of the binding The of the for [3H]-SCH 58261 binding in human lymphocytes that the values of the is which that and values not by at least the range et al., It is that such a of in a range of appears to be a common of all membrane receptor ligands far from a of et al., 1994). data obtained from the indicate that [3H]-SCH 58261 binding is and a found to be typical of the interaction of antagonists with A2A receptors in rat striatum et al., all these data that adenosine receptors present on lymphocytes have a pharmacological and biochemical profile typical of the A2A receptor subtype. about the expression of adenosine receptors be but it has been to of cells to expression of adenosine receptors in biochemical or radioligand binding in the of The role of A2A adenosine receptors in the regulation of immune response has been by the expression levels of this receptor in different of functional lymphocytes et al., 1999). receptor was used to a The levels of expression of A2A receptors are higher T than B T cells are distinguished by the expression of and involved in recognition of class and class than T cells A2A adenosine receptors, but activation of T cells A2A expression in T T cells are cells, cells have been in T cell Studies of T cell and that cells are to A2A receptors than are cells that not is that A2A receptors are in cells that as a of et al., 1999). on the basis that a deficiency of ADA combined immunodeficiency disease in the observed in has been to the effects of Thus, which is an adenosine of has been proposed for the of and et al., 1994). is an non-selective receptor agonist and it has been in [3H]-SCH 58261 binding to its of action the is due to the interaction with A2A receptors. However, showed a low affinity in the binding and was to stimulate cyclic AMP accumulation suggesting that the interaction with A2A receptors is not the only of action of this Recently, the of adenosine in the of development has been reported et al., 1998). In order to characterize the role of the effect of both and to of has been The data that, for is partially by the activation of the A2A receptor role has for adenosine receptors in Moreover, in these cells, could also be the activation of the A3 receptor using selective but this is not involved in either or Thus, are at least three different by which adenosine may including the extracellular membrane receptor or the activation of the A3 receptors, or the of nucleoside cells and direct activation of intracellular involved in the and this is the proposed of action of In the it be of interest to study to in the that these agents and are the most cells in the peripheral blood and are the first cells to at an or than a by interacting with specific receptors on the surface of neutrophils, has been as an endogenous antiinflammatory agent & et al., et al., 1992; Sullivan et al., in other physiological the effects of adenosine on inflammatory are not all in one have been found to both A1 and A2A adenosine subtypes and the antiinflammatory of adenosine to A2A receptors are by the effects the A1 receptor subtype (Salmon & Cronstein, 1990; Fredholm et al., 1996). Thus, the of an inflammatory response low of adenosine neutrophil et al., 1990; & 1990) and cell et al., 1992; et al., at the higher present in tissues, A2A receptors, adenosine and & 1992; Sullivan & Linden, 1998). by in part from the of neutrophils to the site of followed by the of Adenosine both and therefore could be important in and inflammatory (Cronstein, 1994). other antiinflammatory effects of adenosine at its receptors, have been activation of A2A receptors to be associated with inhibition of and by and other are important in the pathogenesis of & 1990) and et al., and the effects of adenosine on have been to direct inhibition of neutrophil function et al., The antiinflammatory effects of adenosine and its receptor analogues first suggested in et al. that from neutrophils in by and the calcium This effect was by dipyridamole which adenosine the The of the by adenosine results from its interaction with a specific membrane receptor that has been identified as the A2 subtype (now known to be the A2A subtype) on the basis of some important such as the IC50 of adenosine for inhibition of the order of potency of adenosine the effect of to the of adenosine and the binding of a adenosine to cells et al., In of A2A adenosine receptors accumulation of intracellular cyclic which as a to function et al., et al., Fredholm et al., 1994; Sullivan & Linden, 1998). However, is to that the of adenosine in the inhibition of not be due to cyclic This is on the that a inhibitor intracellular cyclic AMP levels and et al., 1988). and NECA inhibit in an but does not potentiate the functional effect of in it has been reported that an agent which cyclic protein the effects of analogues of cyclic AMP cyclic on but not of NECA et al., the that cyclic AMP does not as the intracellular for adenosine inhibition of In are studies in which and other protein such as adenosine are to the potency of adenosine et al., Fredholm et al., 1996). Thus, although the role of cyclic AMP in the effects of adenosine analogues is in this that cyclic AMP may at least and is in with data reported by Varani et al. The effects of adenosine on other in the activation of the neutrophils have also been of leads to mobilization of intracellular in Adenosine does not the and increase in in neutrophils but the increase in intracellular that stimulation by using suggesting that adenosine does not with the in cell activation & 1992). et al. have reported that adenosine may as a in neutrophils because it in response to the calcium In other authors observed that adenosine in the absence of extracellular and, as a the role of adenosine on step in neutrophils activation et al., 1997). studies indicate that adenosine receptor activation could also with a step in signal for receptors. It has been that the direct activation of G proteins with in neutrophils and the adenosine receptor does not by suggesting that adenosine the interaction between receptors and G proteins et al., 1990; & the activation of adenosine receptors may the of receptors from the signal and the of adenosine receptors the of receptors with the their et al., 1989; & 1992; et al., 1996). pharmacological studies [3H]-SCH 58261 binding with functional data in human neutrophils have some on the signal responsible for the inhibition of and the role of cyclic AMP (Varani et al., In the radioligand binding studies in neutrophils have been performed with the non-selective agonist NECA et al., that showed a high interacting also with the adenotin site (Table 3). [3H]-SCH 58261 has been found to label in human neutrophils a class of recognition sites with Kd and values nM and mg−1 with the order of as that in human platelet and membranes (Table 3). In competition studies, typical adenosine agonists and antagonists bound the A2A receptor with a rank order of potency and affinity range similar to that observed in [3H]-SCH 58261 binding to human platelet and membranes (Table 4). in the stimulation of cyclic AMP the compounds studied a rank order of potency similar to that observed in binding experiments (Table 5). Thus, like A2A receptors in other mammalian tissues, those present on neutrophils are coupled with adenylate cyclase Moreover, the studies of inhibition of revealed that the receptor on neutrophils is of the A2A subtype. Adenosine analogues similar in both their effects to inhibit and to inhibit the binding of [3H]-SCH 58261 and 5). the data on antagonists of in human neutrophils in with those obtained in binding was found also between cyclic AMP accumulation data and inhibition of by adenosine receptor agonists in this reported for in rank suggesting that cyclic AMP could be involved in the action of A2A receptors to inhibit of [3H]-SCH 58261 binding was performed and the and to the of the binding obtained from this study indicate that [3H]-SCH 58261 binding to human neutrophils is and a similar to that found in rat which is typical of A2A receptor antagonists et al., and in with that found in human In the use of the antiaggregatory and antiinflammatory properties of adenosine as well as of its effects a satisfactory characterization of the receptor subtype involved and the development of ligands selective for this receptor. it be that [3H]-SCH 58261 has to characterize A2A receptors which are present in have been known to have a functional role such as platelets, lymphocytes and Thus, due to labelling of the adenotin site by [3H]-NECA or [3H]-CGS 21680 have been from the most binding and functional data is that A2A receptors have similar in different and This is in with the data that the A2A receptor is a highly in The studies the presence of A2A adenosine receptors in human blood cells, that adenosine could an important role in immune and inflammatory processes and that the activation of A2A receptors may have adenosine has been reported as a of cells in tissue and although several mechanisms are the activation of extracellular adenosine receptors is the most The activation of A2A receptors platelet aggregation, the immune response in and diseases and the inflammatory reactions. This is important on suggesting the presence of an inflammatory component in a variety of disorders, including and of & Burnstock, 1998). The of adenosine and its analogues are by severe effects like and that are due to the ubiquitous of the adenosine receptors as well as a (Belardinelli et al., 1989). As an alternative agents that endogenous adenosine at sites of be studies indicate that of adenosine mediates the antiinflammatory effects of both and the most widely used agents for the of & et al., 1996). and inhibit with intracellular of its accumulation the of adenosine from the by the presence of and activate and the of this biochemical is a reduced in the site and receptor selective are to pharmacologic agents having efficacy with effects for the of a variety of diseases with