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A great body of evidence has been accumulating in the last 20 years supporting a role for adenosine as a neurotransmitter and neuromodulator in the central nervous system.1 In brain, adenosine acts as a potent depressant of excitatory neurotransmission and is colocalized (either as adenosine per se or as its precursor molecule, adenosine triphosphate (ATP)) with “classic” excitatory transmitters in many presynaptic terminals, whence it is released during physiological neurotransmission. It is now well established that the multiple effects of this nucleoside are mediated by activation of specific cell surface receptors, which, based on biochemical, pharmacological and molecular cloning studies, have been classified into four subtypes, denoted as A1, A2A, A2B and A3.2 All the adenosine receptors are members of the guanine nucleotide-binding protein (G protein)-coupled receptor family and possess seven transmembrane helical regions.3 The functional roles of some of the adenosine receptor subtypes (e.g., the A1 and A2A receptors) are relatively well established (see below), whereas the role(s) of the A2B and the recently cloned A3 receptors are still largely unknown.
The potent depressant, sedative and anticonvulsant actions of adenosine are mainly mediated by the A1 receptor subtype, located to both pre- and postsynapses. The A1 receptor seems to be connected through Gi/Go-proteins to a variety of transduction mechanisms, including inhibition of adenylyl cyclase activity, modulation of phospholipase C-dependent intracellular Ca2+ regulation and modulation of K+, Ca2+ and CI− channels.2,3 In brain presynaptic terminals, a direct inhibition of membrane Ca2+ conductance seems to represent a main mechanism responsible for A1 receptor-mediated inhibition of neurotransmitter release.4,5
The A2 receptors have been defined on the basis of their ability to stimulate adenylyl cyclase activity. Thus, they probably interact with G5 proteins, although stimulation of phosphoinositide-dependent phospholipase C activity has been reported as well.6 Differently from the A1 subtype, which can be detected in all brain areas (although to discrete levels of expression), the A2A receptor subtype is particularly abundant and almost exclusively located to dopamine-rich brain areas. such as corpus striatum and caudatus putamen. This receptor has been involved in regulation of motor functions.2 More recently, the presence of A2A-like receptors in brain areas other than basal ganglia has been reported.7 These receptors have been proposed to play an entirely different role and to be responsible for stimulation of neurotransmitter release. The A2B receptor has low affinity for adenosine and, differently from the A1 and A2A subtypes, which are activated by nM nucleoside concentrations, but similarly to the A3 subtype (see below), requires micromolar concentrations of adenosine to be activated. The A2B receptor can be found in low density in almost all cell types, including brain astrocytes. Recent data indicate that this receptor participates in the regulation of the interleukin 6 gene in astrocytoma cells,8 therefore suggesting a role for this receptor subtype in neuropathological conditions characterized by increased brain levels of this cytokine (e.g., Alzheimer’s disease and brain ischemia).
Very little is known about the A3 receptor, initially identified by molecular cloning and whose pharmacological and molecular characterization has revealed unexpected findings for an adenosine receptor.9 The A3 receptor has a unique structure-activity relationship profile, tissue distribution and effector coupling, being coupled to both inhibition of adenylyl cyclase activity and stimulation of phosphoinositide-dependent phospholipase C.10,11 Although there may be species differences,10,12 actions at this receptor are relatively insensitive to xanthine derivatives,9 which behave as antagonists at the A1 and A2 subtypes.13 Moreover, activation of the A3 receptor requires high (i.e., pathological) concentrations of adenosine; the Ki value of adenosine at this receptor is approximately 1 μM. versus 10 and 30 nM at rat A1 and A2A receptors, respectively.9 Thus, the pathophysiological actions of the A3 receptor may be very different from those of the other adenosine receptors. Insights into the possible role of this receptor in brain have come from recent in vivo studies employing the first really selective A3 receptor agonist N6-(3-iodobenzyl)-5′-(N-methyluronamide)adenosine (IB-MECA).14 In mice, administration of IB-MECA resulted in potent locomotor depression, with >50% reduction in activity. This behavioral effect was not counteracted by xanthine antagonists, therefore confirming the involvement of the A3 receptor subtype.15 More recently, this receptor has also been implicated in the development of ischemic brain damage (see following section) which makes it a novel target for therapeutic interventions in central nervous system diseases characterized by neurodegenerative events.
Treatment of cerebral ischemia with drugs acting at adenosine receptors was proposed over 15 years ago.16 Experimental studies rapidly validated the original concept, showing that, as also expected from the well documented depressant actions of adenosine, the administration of adenosine analogues to experimental animals either before or after the ischemic insult results in substantial improvement of survival, reduction of brain injury and amelioration of associated neurological deficits.1,16,17 With the only exception of A3-selective agents (see below), all adenosine analogues, especially those selective for the A1 receptor, were shown to be neuroprotective (elegantly reviewed in Ref. 1). Consistently, administration of adenosine receptor blockers either immediately before or during the ischemic insult has been shown to worsen the ischemic outcome.1 Conversely, subchronic treatment with xanthine adenosine receptor blockers before induction of ischemia resulted in protection against the subsequent ischemic insult, maybe through an upregulation of neuroprotective A1 receptors.1,17
We now have substantial information on the mechanisms at the basis of adenosine neuroprotective actions. Following trauma and ischemia, brain concentrations of adenosine are strongly and transiently increased from resting values of 50–300 nM to 10–50 μM. Notably, during ischemia, there is a dramatic increase of extracellular adenosine in the affected tissue, mainly as a result of intracellular catabolism of ATP and subsequent transport out of the cells by several specific carrier-mediated membrane transporters.1 There is evidence that during ischemia/hypoxia and trauma, not only decreased energy supply but also enhanced energy demand by the supraphysiological activation of excitatory glutamatergic neurotransmission leads to increased adenosine release, as a result of massively increased release of neurotransmitters from brain terminals.18 Under these conditions, adenosine contributes to limiting the deleterious consequences of sustained activation of glutamate receptors by both reducing further release of neurotoxic mediators via presynaptic A1 receptors, and by counteracting the postsynaptic actions of excitatory transmitters, including the excessive entry of Ca2+ ions, which has been associated to the development of programmed cell death.
The role of the adenosine A3 receptor in ischemia has been recently investigated by von Lubitz and co-workers. These authors showed that the in vivo chronic administration of IB-MECA dramatically improved the histopathological and neurological outcome and preserved short-term memory following cerebral ischemia in gerbils.19 Chronic IB-MECA was also protective in chemically induced (N-methyl-D-aspartate or pentamethylenetetrazole) seizures.20 Significant improvement of seizure latency, neurological impairment and survival was observed. At present, it is unknown whether the protective effects of chronically administered IB-MECA are due to effects on blood flow, neurons and/or other brain cells (see below).
A regimen-dependent inversion of the experimental result was seen after the acute administration of the A3 selective agonist in the stroke model.19 Enhanced mortality and extensive brain damage were observed after a single IB-MECA administration immediately prior to induction of ischemia, suggesting that the A3 receptor may participate to the development of ischemic damage.19 Such regimen-dependent opposite effects raised the hypothesis that, differently from the “neuroprotective” A1 receptor, the brain A3 receptor may mediate induction of cell death, and that its desensitization following chronic exposure to agonists (as may occur as a result of the chronic administration protocol), may play a role in the observed attenuation of ischemic brain damage.
As underlined above, the exact contribution of different brain cells to the in vivo effects of adenosine A3 receptor agonists is not yet established. In brain, adenosine receptors are not only expressed by neuronal cells, but are also abundantly present on endothelial and glial cells, which may therefore significantly contribute to the detected effects.
In recent years, our laboratory has been particularly interested at characterizing the functional roles of adenosine receptors on astrocytes, a cell type which is now recognized to play important roles in both central nervous system development and in brain repair following trauma and ischemia (for review, see Refs. 21, 22). Not only do astroglial cells provide a metabolic support to neurons, but they also exert a number of highly specific functions. In developing brain, during specification of cortical areas, radial glia offer substratum and guide to migrating neurons and at the end of neurogenesis transforms into type 1 astrocytes. In adult brain, astrocytes express neurotransmitter receptors and uptake systems and directly participate in neurotransmission. Moreover, astroglial cells arc known to respond to various types of injury by rapid and vigorous astrogliosis, a reaction characterized by both increased astroglial cell proliferation and astrocytic hypertrophy, as shown by “stellation” and increased expression of glial fibrillary acidic protein (GFAP), the astrocyte-specific intermediate filament protein. Although there is still debate about whether reactive astrogliosis is beneficial or detrimental to neuronal repair mechanisms, it is known that activated astrocytes are needed for axonal growth and guidance.23
On these bases and in keeping with the well documented protective actions of adenosine analogues, we undertook a study aimed at investigating the effects of the adenosine A1/A3 nonselective agonist 2-chloroadenosine (2-CA) on rat brain primary astrocytes. Choice of this agonist was simply due to the fact that it is relatively hydrolysis-resistant, and therefore seemed to us particularly suited for long-term “trophic” studies. Surprisingly, exposure of cultures to 2-CA resulted, 48–72 hr later, in a marked reduction of astrocytic cell number, as shown by a dramatic decrease of GFAP-positive cells.24 2-CA-induced decrease of astrocytic cell number was not counteracted by either xanthine derivatives or blockers of the adenosine transport system, which suggested the involvement of a xanthine-insensitive extracellular receptor. In an attempt to elucidate the molecular mechanisms responsible for 2-CA-induced reduction of astrocytes, we preincubated cultures with bromodeoxyuridine (BrdU) and then double-labeled cells with both an anti-GFAP antibody (to detect astrocytes) and an anti-BrdU antibody (to quantify the percentage of astrocytes undergoing DNA synthesis). We were surprised to find out that, despite the marked reduction of cell number, 2-CA significantly increased the percentage of cells showing anti-BrdU immunoreactivity in nuclei.24 Other examples had been previously reported in the literature where cell death was shown to be accompanied by a concomitant “paradoxical” increase of DNA synthesis. This phenomenon has been well characterized for secretory epithelial prostate cells that undergo extensive apoptosis following castration. Colombel & co-workers demonstrated that prostatic cells undergoing apoptotic death incorporate BrdU into nuclear DNA prior to DNA fragmentation.25 Based on these data, they concluded that quiescent epithelial cells undergo apoptosis as a result of two sequential events initiated by testosterone depletion. The first event is an active reentry of these cells into the cell cycle. The second event is the apoptotic destruction resulting from the inability of differentiated cells to successfully complete this cycle (“abortive mitosis”).25 Moreover, a number of data has been accumulating in recent years suggesting that two strikingly different cellular programs as cell proliferation and cell death by apoptosis indeed share initial identical patterns of protooncogene expression,26–29 so that the same primary stimulus activates a common pathway that can lead to either event; the timing of a secondary growth stimulus determines the final outcome.30
On this background, we decided to verify whether astroglial cell death induced by 2-CA was apoptosis. Flow cytometric analysis of propidium iodide-stained nuclei showed the appearance of an hypodiploid DNA peak in cultures exposed to 2-CA that was not present in control cultures, suggesting induction of apoptosis, which was also confirmed by light and transmission electron microscopy analysis of cells.11 An example of 2-CA-induced apoptosis is shown in Figure 1. This study represented the first demonstration in favor of a novel action for adenosine (induction of cell death by apoptosis) in the central nervous system.
More recently, we extended this study to the investigation of the receptor subtype responsible for adenosine-induced apoptosis. A possible role for the A3 receptor was indirectly suggested by the demonstration that 2-CA effects were not reversed by xanthine antagonists.24 We therefore tested the effects of the selective A3 receptor agonist IB-MECA and its more recent 2-chloro derivative, 2-C1-IB-MECA.32 Exposure of rat astrocytes to high (μM) concentrations of either agonist resulted in development of apoptosis, as shown by both morphological and flow cytometric criteria.33 On both rat astrocytes and human astrocytoma cells (ADF cells),34 at concentrations 2–3 orders of magnitude lower (10–100 nM), these same agonists induced a marked reorganization of the cytoskeleton, with appearance of stress fibers and numerous cell protrusions. These morphological changes were accompanied by a significant reduction of the number of spontaneously detached apoptotic cells in the culture medium.33
The opposing actions induced on astroglial cells by nM and μM A3 agonist concentrations may be, at least in part, the bases of the strikingly different ischemic outcome observed in vivo after either acute or chronic administation of IB-MECA.19 We speculate that a robust and acute activation of these receptors during ischemia as a consequence of massive release of adenosine (see the previous section) may contribute to the development of ischemic damage. It could be hypothesized that A3 receptor-mediated apoptosis of astroglial cells may result in a reduced survival rate of neuronal cells (of course, this does not rule out that possible direct effects of adenosine A3 receptors located on other cell types may contribute to ischemia-induced damage as well). In the von Lubitz et al. study, a marked cerebroprotection associated to increased survival rate was demonstrated if ischemia was induced after a subchronic treatment with low doses of IB-MECA.19 Desensitization of central A3 receptors as a consequence of prolonged agonist exposure was hypothesized to reduce the putative deleterious contribution of this receptor to ischemic damage. Our data on cells of the astroglial lineage show that, under certain experimental conditions, A3 agonists can indeed activate cell protection mechanisms (e.g., changes of the cytoskeletal machinery making cells more resistant to subsequent insults). However, since these beneficial effects are induced at nM agonist concentrations, molecular mechanisms other than agonist-induced receptor desensitization may be involved. A subthreshold stimulation of the A3 receptor prior to the induction of ischemia may result in activation of protective mechanisms that make the brain less sensitive to a subsequent ischemic insult (“ischemic tolerance”). In this respect, it was previously shown that, in gerbils, mild ischemic treatments (e.g., a 2-min carotid occlusion) induce tolerance to a subsequent, and what would be lethal, ischemic stress.35 Alternatively, the differential effects induced by different A3 agonist concentrations may be related to either expression of two distinct astroglial A3 receptor subtypes (endowed with different affinities and mediating cell protection and cell death, respectively) or to coupling of the same receptor to different transduction pathways, simply depending upon the degree of receptor activation. Studies are currently in progress in our laboratory aimed at elucidating this important point.
Adenosine and adenosine analogues have been previously demonstrated to induce apoptosis of cell types other than astroglial cells (summarized in Table 1).
In lymphoid cells, the apoptotic effect of 2-chloro-2′-deoxyadenosine (2-CdA, cladribine, which is utilized as an anticancer agent) has been shown to require intracellular phosphorylation to 2-CdATP (cladribine-5′-triphosphate) which directly competes with dATP for DNA polymerase binding, being incorporated during DNA synthesis and repair to arrest chain extension.36 DNA strand breakage is the immediate consequence of 2-CdA exposure and is more likely the primary lesion for apoptosis in these cells.37–39 Relief from deoxyadenylate stress imbalances was obtained by stably transfecting cells with the antiapoptotic molecule bcl-2.40
In chick embryonic sympathetic neurons, adenosine (1–100 μM) inhibited neurite outgrowth and killed about 80% of cells; also in this case, adenosine toxicity required the nucleoside to enter the cells and to be phosphorylated, as suggested by prevention of toxicity by both inhibitors of adenosine membrane transporter and by inhibitors of adenosine kinase.41
In human thymocytes, adenosine analogues were demonstrated to induce cell death via at least two independent pathways, one requiring the activation of an “atypical” extracellular receptor subtype linked to stimulation of adenylyl cyclase and Ca2+ increases,42 the other one being independent of extracellular receptors for adenosine.43 For example, the effects of 2-CdA were prevented by addition of the nucleoside transport inhibitor dipyridamole, suggesting that 2-CdA has to be taken up by cells in order to trigger cell death.43 On the contrary, but similar to astrocytes,31 the lethal effects of 2-CA were unaffected by dipyridamole, suggesting an extracellular site of action for the nucleoside. This conclusion is in agreement with the recent demonstration that both IB-MECA and CI-IB-MECA can induce apoptosis of HL-60 human promyelocytic leukemia cells.44 This effect, similar to 2-CA-induced astroglial cell death, was not counteracted by xanthine blockers, further confirming an atypical extracellular site of action for these adenosine derivatives.44
In conclusion, it seems that adenosine may utilize different pathways to modulate apoptosis in target cells. Adenosine can either induce cell death by activating a specific extracellular receptor (likely the A3 subtype) linked to some as yet unidentified signaling pathway, or, alternatively, can directly enter cells to exert its toxicity. Some cells apparently express only one of these two pathways leading to cell death; in some other cells, both pathways are operative and the choice of either one of the two may simply depend on specific pathophysiological conditions.
The demonstration that adenosine can regulate the viability of various cell types has important pathophysiological implications.
Adenosine-induced apoptosis of thymocytes may play a critical role in the intrathymic cell selection process which functions to prevent autoimmunity and to remove thymocytes expressing functionally inactive T cell receptors. It may be hypothesized that a disregulation of adenosinergic mechanisms in this crucial phase of T cell development may lead to serious immune system dysfunctions, as also documented by the adenosine-deaminase immunodeficiency syndrome.45,46 Moreover, confirmation of an active role of the adenosine A3 receptor in regulating apoptosis in lymphoid cells44 may lead to the development of novel anticancer agents of potential value in the treatment of leukemias and other tumors.
Adenosine-regulated apoptosis of nervous system cells may have important pathophysiological implications as well. Purine-induced cell death could play a role in naturally occurring apoptosis during brain development.47 Additionally, this mechanism could play a role in the remodeling of the brain circuitries following trauma and ischemia, by favoring the elimination of irreversibly damaged cells in order to spare energy and space for recovering ones.22 Moreover, in contrast to necrosis, where cellular contents (including neurotoxic mediators) are massively released in the extracellular space, cell death by apoptosis occurs with preservation of cell membrane integrity; cellular contents are safely sealed until phagocytosis intervenes and death occurs with no inflammation.48 It might be speculated that adenosine-induced apoptosis in infarcted brain may function as a signal to limit the spreading of cell death following ischemic stroke.
Further possible pathophysiological implications come from a recent revaluation of the significance of apoptosis in the central nervous system. For many years apoptosis has been regarded simply as a physiological form of cell death.49 It was believed that physiologically appropriate death is due to apoptosis, and that pathological mechanisms involve necrosis.49–51 However, recent evidence suggests that in a number of nervous system diseases characterized by neurodegenerative events. pathological cell death may also occur by apoptosis. Apoptotic cell death has been demonstrated in ischemia (but see also above), status epilepticus, HIV-1 encephalopathy and neurodegenerative diseases such as amyotrophic lateral sclerosis. Parkinson’s and Alzheimer’s disease.52 On these bases, the pharmacological modulation of apoptosis would open new avenues to the therapy of invalidating diseases which are also leading causes of death. A direct confirmation of the involvement of the adenosine A3 receptor subtype in induction of cell death in the brain may therefore lead to the discovery of a novel class of potent modulators of apoptosis of potential use in neurological disorders characterized by neurodegenerative events.
aThe authors are involved in the concerted action ADEURO (EU, BIOMED1) (ADEURO is the logo for the concerted action “Physiology and pharmacology of brain adenosine receptors—implications for the rational design of neuroactive drugs” supported by the European Union within the Biomedical and Health Research Programme). Part of the research presented here was supported by the Italian Ministero dell’Università e Ricerca Scientifica e Tecnologica (40%).