Adenosine receptors and brain diseases: neuroprotection and neurodegeneration

Adenosine acts in parallel as a neuromodulator and as a homeostatic modulator in the central nervous system. Its neuromodulatory role relies on a balanced activation of inhibitory A(1) receptors (A1R) and facilitatory A(2A) receptors (A2AR), mostly controlling excitatory glutamatergic synapses: A1R impose a tonic brake on excitatory transmission, whereas A2AR are selectively engaged to promote synaptic plasticity phenomena. This neuromodulatory role of adenosine is strikingly similar to the role of adenosine in the control of brain disorders; thus, A1R mostly act as a hurdle that needs to be overcame to begin neurodegeneration and, accordingly, A1R only effectively control neurodegeneration if activated in the temporal vicinity of brain insults; in contrast, the blockade of A2AR alleviates the long-term burden of brain disorders in different neurodegenerative conditions such as ischemia, epilepsy, Parkinson's or Alzheimer's disease and also seem to afford benefits in some psychiatric conditions. In spite of this qualitative agreement between neuromodulation and neuroprotection by A1R and A2AR, it is still unclear if the role of A1R and A2AR in the control of neuroprotection is mostly due to the control of glutamatergic transmission, or if it is instead due to the different homeostatic roles of these receptors related with the control of metabolism, of neuron-glia communication, of neuroinflammation, of neurogenesis or of the control of action of growth factors. In spite of this current mechanistic uncertainty, it seems evident that targeting adenosine receptors might indeed constitute a novel strategy to control the demise of different neurological and psychiatric disorders.     

Different cellular sources and different roles of adenosine: A1 receptor-mediated inhibition through astrocytic-driven volume transmission and synapse-restricted A2A receptor-mediated facilitation of plasticity

Adenosine is a prototypical neuromodulator, which mainly controls excitatory transmission through the activation of widespread inhibitory A1 receptors and synaptically located A2A receptors. It was long thought that the predominant A1 receptor-meditated modulation by endogenous adenosine was a homeostatic process intrinsic to the synapse. New studies indicate that endogenous extracellular adenosine is originated as a consequence of the release of gliotransmitters, namely ATP, which sets a global inhibitory tonus in brain circuits rather than in a single synapse. Thus, this neuron-glia long-range communication can be viewed as a form of non-synaptic transmission (a concept introduced by Professor Sylvester Vizi), designed to reduce noise in a circuit. This neuron-glia-induced adenosine release is also responsible for exacerbating salient information through A1 receptor-mediated heterosynaptic depression, whereby the activation of a particular synapse recruits a neuron-glia network to generate extracellular adenosine that inhibits neighbouring non-tetanised synapses. In parallel, the local activation of facilitatory A2A receptors by adenosine, formed from ATP released only at high frequencies from neuronal vesicles, down-regulates A1 receptors and facilitates plasticity selectively in the tetanised synapse. Thus, upon high-frequency firing of a given pathway, the combined exacerbation of global A1 receptor-mediated inhibition in the circuit (heterosynaptic depression) with the local synaptic activation of A2A receptors in the activated synapse, cooperate to maximise salience between the activated and non-tetanised synapses.     

Reverse microdialysis of a dopamine D2 receptor antagonist alters extracellular adenosine levels in the rat nucleus accumbens

Recent evidence suggests that modulation of dopaminergic transmission alters striatal levels of extracellular adenosine. The present study used reverse microdialysis of the selective dopamine D₂ receptor antagonist raclopride to investigate whether a blockade of dopamine D₂ receptors modifies extracellular adenosine concentrations in the nucleus accumbens. Results reveal that perfusion of raclopride produced an increase of dialysate adenosine which was significant with a high (10 mM) and intermediate (1 mM) drug concentration, but not with lower drug concentrations (10 and 100 μM). Thus, the present study demonstrates that a selective blockade of dopamine D₂ receptors in the nucleus accumbens produced a pronounced increase of extracellular adenosine. The cellular mechanisms underlying this effect are yet unknown. It is suggested that the increase of extracellular adenosine might be related to a homeostatic modulatory mechanism proposed to be a key function of adenosine in response to neuronal metabolic challenges.     

Adenosine A2A receptors control the extracellular levels of adenosine through modulation of nucleoside transporters activity in the rat hippocampus

Adenosine, a neuromodulator of the CNS, activates inhibitory-A1 receptors and facilitatory-A2A receptors; its synaptic levels are controlled by the activity of bi-directional equilibrative nucleoside transporters. To study the relationship between the extracellular formation/inactivation of adenosine and the activation of adenosine receptors, we investigated how A1 and A2A receptor activation modifies adenosine transport in hippocampal synaptosomes. The A2A receptor agonist, CGS 21680 (30 nm), facilitated adenosine uptake through a PKC-dependent mechanism, but A1 receptor activation had no effect. CGS 21680 (30 nm) also increased depolarization-induced release of adenosine. Both effects were prevented by A2A receptor blockade. A2A receptor-mediated enhancement of adenosine transport system is important for formatting adenosine neuromodulation according to the stimulation frequency, as: (1) A1 receptor antagonist, DPCPX (250 nm), facilitated the evoked release of [(3)H]acetylcholine under low-frequency stimulation (2 Hz) from CA3 hippocampal slices, but had no effect under high-frequency stimulation (50 Hz); (2) either nucleoside transporter or A2A receptor blockade revealed the facilitatory effect of DPCPX (250 nm) on [3H]acetylcholine evoked-release triggered by high-frequency stimulation. These results indicate that A2A receptor activation facilitates the activity of nucleoside transporters, which have a preponderant role in modulating the extracellular adenosine levels available to activate A1 receptors.     

The role of adenosine A2A and A3 receptors on the differential modulation of norepinephrine and neuropeptide Y release from peripheral sympathetic nerve terminals

The pre-synaptic sympathetic modulator role of adenosine was assessed by studying transmitter release following electrical depolarization of nerve endings from the rat mesenteric artery. Mesentery perfusion with exogenous adenosine exclusively inhibited the release of norepinephrine (NA) but did not affect the overflow of neuropeptide Y (NPY), establishing the basis for a differential pre-synaptic modulator mechanism. Several adenosine structural analogs mimicked adenosine's effect on NA release and their relative order of potency was: 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride = 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-beta-d-ribofuranuronamide = 5'-(N-ethylcarboxamido)adenosine > adenosine > N(6)-cyclopentyladenosine. The use of selective receptor subtype antagonists confirmed the involvement of A(2A) and A(3) adenosine receptors. The modulator role of adenosine is probably due to the activation of both receptors; co-application of 1 nM 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride plus 1 nM 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-beta-D-ribofuranuronamide caused additive reductions in NA released. Furthermore, while 1 nM of an A(2A) or A(3) receptor antagonist only partially reduced the inhibitory action of adenosine, the combined co-application of the two antagonists fully blocked the adenosine-induced inhibition. Only the simultaneous blockade of the adenosine A(2A) plus A(3) receptors with selective antagonists elicited a significant increase in NA overflow. H 89 reduced the release of both NA and NPY. We conclude that pre-synaptic A(2A) and A(3) adenosine receptor activation modulates sympathetic co-transmission by exclusively inhibiting the release of NA without affecting immunoreactive (ir)-NPY and we suggest separate mechanisms for vesicular release modulation.     

Adenosine receptor-mediated modulation of dopamine release in the nucleus accumbens depends on glutamate neurotransmission and N-methyl-D-aspartate receptor stimulation

Adenosine, by acting on adenosine A(1) and A(2A) receptors, exerts opposite modulatory roles on striatal extracellular levels of glutamate and dopamine, with activation of A(1) inhibiting and activation of A(2A) receptors stimulating glutamate and dopamine release. Adenosine-mediated modulation of striatal dopaminergic neurotransmission could be secondary to changes in glutamate neurotransmission, in view of evidence for a preferential colocalization of A(1) and A(2A) receptors in glutamatergic nerve terminals. By using in vivo microdialysis techniques, local perfusion of NMDA (3, 10 microm), the selective A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 3, 10 microm), the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 300, 1000 microm), or the non-selective A(1)-A(2A) receptor antagonist in vitro caffeine (300, 1000 microm) elicited significant increases in extracellular levels of dopamine in the shell of the nucleus accumbens (NAc). Significant glutamate release was also observed with local perfusion of CGS 21680, CPT and caffeine, but not NMDA. Co-perfusion with the competitive NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (APV; 100 microm) counteracted dopamine release induced by NMDA, CGS 21680, CPT and caffeine. Co-perfusion with the selective A(2A) receptor antagonist MSX-3 (1 microm) counteracted dopamine and glutamate release induced by CGS 21680, CPT and caffeine and did not modify dopamine release induced by NMDA. These results indicate that modulation of dopamine release in the shell of the NAc by A(1) and A(2A) receptors is mostly secondary to their opposite modulatory role on glutamatergic neurotransmission and depends on stimulation of NMDA receptors. Furthermore, these results underscore the role of A(1) vs. A(2A) receptor antagonism in the central effects of caffeine.     

Adenosine mediation of presynaptic feedback inhibition of glutamate release

Conditions of increased metabolic demand relative to metabolite availability are associated with increased extracellular adenosine in CNS tissue. Synaptic activation of postsynaptic NMDA receptors on neurons of the cholinergic brainstem arousal center can increase sufficient extracellular adenosine to act on presynaptic A1 adenosine receptors (A1ADRs) of glutamate terminals, reducing release from the readily releasable pool. The time course of the adenosine response to an increase in glutamate release is slow (tau > 10 min), consistent with the role of adenosine as a fatigue factor that inhibits the activity of cholinergic arousal centers to reduce arousal.     

Adenosine in the central nervous system: release mechanisms and extracellular concentrations

Adenosine has several functions within the CNS that involve an inhibitory tone of neurotransmission and neuroprotective actions in pathological conditions. The understanding of adenosine production and release in the brain is therefore of fundamental importance and has been extensively studied. Conflicting results are often obtained regarding the cellular source of adenosine, the stimulus that induces release and the mechanism for release, in relation to different experimental approaches used to study adenosine production and release. A neuronal origin of adenosine has been demonstrated through electrophysiological approaches showing that neurones can release significant quantities of adenosine, sufficient to activate adenosine receptors and to modulate synaptic functions. Specific actions of adenosine are mediated by different receptor subtypes (A(1), A(2A), A(2B) and A(3)), which are activated by various ranges of adenosine concentrations. Another important issue is the measurement of adenosine concentrations in the extracellular fluid under different conditions in order to know the degree of receptor stimulation and understand adenosine central actions. For this purpose, several experimental approaches have been used both in vivo and in vitro, which provide an estimation of basal adenosine levels in the range of 50-200 nM. The purpose of this review is to describe pathways of adenosine production and metabolism, and to summarize characteristics of adenosine release in the brain in response to different stimuli. Finally, studies performed to evaluate adenosine concentrations under physiological and hypoxic/ischemic conditions will be described to evaluate the degree of adenosine receptor activation.     

Structure and function of A1 adenosine receptors

The A1 adenosine receptor is the best characterized of the widely distributed purinergic receptor family. The purified brain A1 receptor is a monomeric 35- to 36-kDa glycoprotein. A1 receptors can be clearly distinguished from A2 adenosine receptors on the basis of structure activity relationships with selective ligands. Recent structure activity data suggest that subtypes of A1 (A1a, A1b, and A3) and A2 (A2a and A2b) receptors may exist. A1 receptor-mediated responses are coupled via multiple pertussis toxin-sensitive GTP binding proteins (G proteins) to many different effectors in various tissues: adenylate cyclase, phospholipase C, Na+- Ca2+ exchange, Ca2+ channels, Cl- channels, and K+ channels. The formation of calcium-mobilizing inositol phosphates can either be enhanced or inhibited. In general, adenosine has been found to act in concert with other hormones or neurotransmitters in either an inhibitory or a stimulatory way. The myriad modulatory actions of adenosine suggest that: 1) adenosine may simultaneously produce multiple effects within the same cell; and 2) activation of A1 receptors may lead to either a decrease or an increase in the coupling of other receptors to their G proteins.     

The cortico-basal ganglia-thalamocortical circuit with synaptic plasticity. II. Mechanism of synergistic modulation of thalamic activity via the direct and indirect pathways through the basal ganglia

A possible mechanism underlying the modulatory role of dopamine, adenosine and acetylcholine in the modification of corticostriatal synapses, subsequent changes in signal transduction through the "direct" and "indirect" pathways in the basal ganglia and variations in thalamic and neocortical cell activity is proposed. According to this mechanism, simultaneous activation of dopamine D1/D2 receptors as well as inactivation of adenosine A1/A(2A) receptors or muscarinic M4/M1 receptors on striatonigral/striatopallidal inhibitory cells can promote the induction of long-term potentiation/depression in the efficacy of excitatory cortical inputs to these cells. Subsequently augmented inhibition of the activity of inhibitory neurons of the output nuclei of the basal ganglia through the "direct" pathway together with reduced disinhibition of these nuclei through the "indirect" pathway synergistically increase thalamic and neocortical cell firing. The proposed mechanism can underlie such well known effects as "excitatory" and "inhibitory" influence of dopamine on striatonigral and striatopallidal cells, respectively; the opposite action of dopamine and adenosine on these cells; antiparkinsonic effects of dopamine receptor agonists and adenosine or acetylcholine muscarinic receptor antagonists.     

Parallel modification of adenosine extracellular metabolism and modulatory action in the hippocampus of aged rats

The neuromodulator adenosine can be released as such, mainly activating inhibitory A1 receptors, or formed from released ATP, preferentially activating facilitatory A2A receptors. We tested if changes in extracellular adenosine metabolism paralleled changes in A1/A2A receptor neuromodulation in the aged rat hippocampus. The evoked release and extracellular catabolism of ATP were 49-55% lower in aged rats, but ecto-5'-nucleotidase activity, which forms adenosine, was 5-fold higher whereas adenosine uptake was decreased by 50% in aged rats. The evoked extracellular adenosine accumulation was 30% greater in aged rats and there was a greater contribution of the ecto-nucleotidase pathway and a lower contribution of adenosine transporters for extracellular adenosine formation in nerve terminals. Interestingly, a supramaximal concentration of an A1 receptor agonist, N6-cyclopentyladenosine (250 nM) was less efficient in inhibiting (17% in old versus 34% in young) and A2A receptor activation with 30 nM CGS21680 was more efficient in facilitating (63% in old versus no effect in young) acetylcholine release from hippocampal slices of aged compared with young rats. The parallel changes in the metabolic sources of extracellular adenosine and A1/A2A receptor neuromodulation in aged rats further strengthens the idea that different metabolic sources of extracellular adenosine are designed to preferentially activate different adenosine receptor subtypes.     

A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase

A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.     

Modulation of NMDA-receptor efficiency by intracellular agents

Glutamate in one of the principle transmitters in the CNS. Ionotropic receptors of glutamate selectively activated by N-methyl-d-aspartate (NMDA) play an important role in the processes of development, learning, memory etc. Hyperactivation of these receptors is responsible for a number of pathological processes. Due to their importance, the NMDA receptors are subjected to strong modulatory influences of different modulatory systems of the brain. Modulation of the NMDA receptor efficiency by extracellular factors is well known and described in a number of reviews, while their modulation by intracellular factors is less known and has not yet been reviewed. This review presents the experimental data concerning a modulatory control of the NMDA receptors by intracellular factors. Some of these factors are: phosphorylation by protein kinases (PK) C, A, Ca2+/calmodulin-dependent PK II, tyrosine kinases; dephosphorylation by protein phosphatases 1, 2A, 2B; interaction with regulatory peptides and cytoskeleton; influence of surrounding lipids etc. Interaction between these factors creates a labile intracellular system, which efficiently modulates activity of the NMDA receptors mediating the activity of different extracellular active compounds (neurotransmitters, neurotoxins, drugs etc.). A cheme summarizing different intracellular pathways of modulation of the NMDA receptor efficiency is described.     

锌离子对配体门控型离子通道的调节作用

在中枢神经系统(central nervous system,CNS)中,锌离子对配体门控型离子通道具有重要的调节作用。锌离子随着神经元的活动从突触前膜的囊泡中释放到突触间隙,对突触内受体进行调控。锌离子抑制N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)型谷氨酸受体的活性,而对非NMDA型谷氨酸受体的调控具有多样性。由γ氨基丁酸(γ-aminobutyric acid,GABA)受体所介导的抑制性突触传递活动也受到锌离子的抑制;而锌离子对glycine受体则呈现出浓度依赖的双向调节效应。病理条件下,锌离子参与了兴奋性细胞毒作用所触发的神经元凋亡过程。本文主要阐述了在CNS中,锌离子对配体门控型离子通道所介导的突触传递活动的调控作用,以及这些调控作用的生理功能和病理意义。     

Physiological implications of adenosine receptor‐mediated platelet aggregation

Adenosine is an important mediator of inhibition of platelet activation. This metabolite is released from various cells, as well as generated via activity of ecto‐enzymes on the cell surface. Binding of adenosine to A₂ subtypes (A_2A or A_2B), G‐protein coupled adenosine receptors, results in increased levels of intracellular cyclic adenosine monophosphate (cAMP), a strong inhibitor of platelet activation. The role and importance of adenosine and its receptors in platelet physiology are addressed in this review, including recently identified roles for the A_2B adenosine receptor as a modulator of platelet activation through its newly described role in the control of expression of adenosine diphosphate (ADP) receptors. J. Cell. Physiol. 226: 46–51, 2010. © 2010 Wiley‐Liss, Inc.     

Purinergic mechanisms in the control of gastrointestinal motility

For many years, ATP and adenosine have been implicated in movement regulation of the gastrointestinal tract. They act through three major receptor subtypes: adenosine or P1 receptors, P2X receptors and P2Y receptors. Each of these major receptor types can be subdivided into several different classes and is widely distributed amongst various neurons, muscle types, glia and interstitial cells that regulate intestinal functions. Several key roles for the different receptors and their endogenous ligands have been identified in physiological and pharmacological studies. For example, adenosine acting at A(1) receptors appears to inhibit intestinal motility in various pathological conditions. Similarly, ATP acting at P2Y receptors is an important component of inhibitory neuromuscular transmission, acting as a cotransmitter with nitric oxide. ATP acting at P2X and P2Y(1) receptors is important for synaptic transmission in simple descending excitatory and inhibitory reflex pathways. Some P2Y receptor subtypes prefer uridine nucleotides over purine nucleotides. Thus, roles for UTP and UDP as enteric transmitters in place of ATP cannot be excluded. ATP also appears to be important for sensory transduction, especially in chemosensitive pathways that initiate local inhibitory reflexes. Despite this evidence, data are lacking about the roles of either adenosine or ATP in more complex motility patterns such as segmentation or the interdigestive migrating motor complex. Clarification of roles for purinergic transmission in these common, but understudied, motility patterns will depend on the use of subtype-specific antagonists that in some cases have not yet been developed.     

Adenosine modulates cell growth in human epidermoid carcinoma (A431) cells.

Adenosine mediates many physiological functions via activation of extracellular receptors. The modulation of cell growth by adenosine was found to be receptor-mediated. In A431 cells adenosine evoked a biphasic response in which a low concentration (approximately 10 microM) produced inhibition of colony formation but at higher concentrations (up to 100 microM) this inhibition was progressively reversed. Evidence for the involvement of A1 (inhibitory) and A2 (stimulatory) adenosine receptors in regulating cell growth of these tumor cells was obtained through plating efficiency studies based on the relative potency of adenosine agonists and antagonists. When both A1 and A2 receptors were blocked, colony formation or growth was not inhibited at low concentrations of adenosine but was inhibited at high adenosine concentrations.     

Studies on a modulatory role for adenosine in antigen and arachidonic acid induced contractions of guinea pig airways

The modulatory effects of adenosine and selected derivatives were examined on antigen and arachidonic acid (AA) induced contractions of indomethacin-treated tracheal spirals and lung parenchymal strips from actively sensitized guinea pigs. Adenosine (up to 2 X 10(-4) M) had no effect on antigen-induced contractions, but inhibited AA-induced contractions by 30-40% if added 30 min prior to challenge. The weak effect of adenosine suggests that endogenous adenosine may only have a limited modulatory role in allergic bronchospasm. 2-Chloroadenosine (10(-6)-10(-4) M) dose-dependently inhibited antigen- and AA-induced contractions of trachea, but was considerably less effective on parenchyma. The substituted adenosine derivatives, R-phenylisopropyladenosine (R-PIA) and 5'-(N-ethylcarboxamido)-adenosine (NECA), and the adenosine transport inhibitor, 6-[p-nitrobenzyl)thio]-9-beta-D-ribofuranosyl purine, were also active as modulators, but their activity was relatively weak and varied with the stimulus and the tissue. An order of potency for R-PIA, NECA, and 2-chloroadenosine could not be determined and 8-phenyltheophylline (10(-5) M) was not an effective inhibitor of the effects of adenosine or the adenosine derivatives. This suggests that adenosine and its derivatives may modulate cells through mechanisms other than activation of conventional A1 and A2 receptors. A lack of specificity for the adenosine derivatives must also be considered.     

AMP affects intracellular Ca2+ signaling, migration, cytokine secretion and T cell priming capacity of dendritic cells

The nucleotide adenosine-5'-monophosphate (AMP) can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A(1) and A(2a) receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC) function. AMP increased intracellular Ca(2+) concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A(1) receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A(2a) receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4(+)CD45RA(+) T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic na?ve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5'-(alpha,beta-methylene) diphosphate (APCP). Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA) was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders.