The Brains on Adenosine Receptors

Adenosine is a small ubiquitous molecule known for its role as an energy and genetic code building block. Adenosine also serves as a ligand for Adenosine receptors, members of the G-protein coupled receptor superfamily and is involved in many physiological processes including regulation of sleep and the level of arousal, neuroprotection, regulation of seizure susceptibility, locomotor effects as well as involved in many pathophysiological conditions. Alomone Labs offers antibodies to all Adenosine receptors, providing an essential tool in the understanding of this system in normal and disease states. Although this system is clearly functional in the periphery, this short review mainly focuses on the role of adenosine and its receptors in the brain.

Introduction

Adenosine is an endogenous nucleoside involved in many biochemical processes and best known for its role in energy transfer in the form of ATP, and the secondary messenger molecule, cAMP, involved in cell signaling pathways. Adenosine in its pure nucleosidic form also influences many functions in the central nervous system (CNS); its levels are determined by the ratio between energy delivery and energy use; thus, increased neuronal activity, particularly hypoxia or ischemia, results in noticeably elevated levels of adenosine³². Adenosine is not stored in vesicles, is not released by exocytosis and does not act only in synapses¹⁷, and therefore is not a classical neurotransmitter. As adenosine has a very short half-life and is quite unstable, its effects are usually local²⁶. Adenosine can appear in the extracellular area via three different mechanisms: 1) through nucleoside transporters following an increase in the intracellular levels of adenosine or a reversal of the Na⁺ gradient; 2) extracellular formation following the release of ATP^7,40; 3) extracellular formation following cAMP release^11,28. The removal of adenosine from the extracellular space occurs through nucleotide transporters, regulated by the activation of adenosine receptors (ARs) through protein kinase pathways⁸.

Adenosine Receptors

Adenosine receptors were defined in the 1970s, partly based on the antagonistic effects of caffeine¹⁵. To date, four adenosine receptors have been identified and cloned; Adenosine A₁, A_2A, and A_2B receptors are well conserved among mammals, while adenosine A₃ receptor shows considerable structural variability within different species and compared to the other adenosine receptors. Splice variants of all four receptors are also expressed¹⁷. The brain expresses high concentrations of adenosine receptors, where adenosine has been shown to be involved in both normal and pathophysiological processes. Due to good pharmacological tools, the distribution of both adenosine A₁ and A_2A receptors is well known¹⁶; A₁ receptor shows the highest abundance in the brain, while A_2A receptor is expressed at high levels in only a few regions of the brain¹¹. Pharmacological tools available for A_2B and A₃ receptors are not so extensive and their distribution is mostly based on their corresponding mRNA levels¹⁶; both receptors are also expressed in the brain. In fact, the A₃ receptor is the only adenosine receptor subtype which was cloned before any pharmacological identification²¹. Although this short review focuses on adenosine receptors mainly in the brain, it is important to note that they are also distributed in the periphery.

Cell Signaling

The four subtypes of adenosine receptors are G-protein coupled receptors (GPCRs). All four receptors respond to adenosine albeit with different affinities; adenosine A₁, A_2A, and A₃ receptors are considered as high affinity while adenosine A_2B receptor requires higher concentrations of adenosine to be activated. Adenosine receptors could also be differentiated on the basis of the signaling pathway(s) they activate or inhibit. A₁ and A₃ receptors interact with the pertussis toxin-sensitive G-proteins of the Gi/o family¹³. A₃ receptor can also couple G_q thereby also activating phospholipase C, inositol triphosphate and intracellular Ca²⁺ influx²¹. A_2A and A_2B activate adenylyl cyclase through G_s. In addition, A_2B receptors can also bind G_q¹³. While the basic components of the activation of these receptors are the G-protein heteromer, many additional proteins affect the signaling pathways activated in response to adenosine receptor stimulation by direct interaction with the G-proteins and the receptors³³. Following activation of the G-proteins, enzymes and some ion channels are affected; for example A₁ receptors are thought to activate several types of K⁺ channels, leading to membrane hyperpolarization²³ and inactivate N, P, and Q-type Ca²⁺ channels¹, as well as modulate NMDA receptor function²⁷. On the other hand, A₃ receptors activate ATP-sensitive K⁺ channels (KATP)³¹. One interesting aspect of adenosine receptor signaling involves their interaction with other types of G-protein coupled receptors. Synergistic interactions have been reported between low concentrations of A₁ and metabotropic GABA (B) agonists on inwardly rectifying K⁺ channels (I_RKs)³⁷. There is also extensive evidence from studies on direct interactions between A_2A and D₁ dopamine receptors, and between A₁ and D₂ receptors^4,22,24 and reviewed in reference 19). In addition to interaction, dimerization between different GPCRs is being increasingly proposed as a common molecular mechanism for cross-talk among various G-protein signaling pathways, and has been observed for many neurotransmitter receptors¹⁷. Heteromeric associations were reported for A₁ receptor and P2Y1 purinergic receptors³⁹ as well as for A₁ and mGluR1 metabotropic glutamate receptor⁶ and A_2A with mGluR5¹⁴.

Inhibition of Neurotransmitter Release

The most widely recognized effect of adenosine on nerve activity is the inhibition of neurotransmitter release and neuronal excitability via the activation of A₁ receptors^10,11,18. A₁ receptors are more effective in depressing excitatory than inhibitory transmission in the CNS. This inhibition of excitatory transmission largely depends on the activation of presynaptic adenosine A₁ receptors which subsequently inhibits the release of glutamate^1,3,12.

Ischemia, Neurological and Psychiatric Disorders

The neuroprotective effect of adenosine against brain injury is mostly mediated by adenosine A₁ receptor stimulation, as A₁ receptor agonists diminish brain damage and the opposite effect is observed using A₁ receptor antagonists. Also, activation of A₁ has been well documented to protect against ischemic brain injury in adult animal models³⁶.

While A₁ activation leads to neuroprotection, activation of A_2A may contribute to neuronal injury in several neurological disease models. Pharmacological studies using A_2A receptor selective antagonists constantly show reduced ischemic brain damage^20,29. It should also be noted though, that in some experimental models, A_2A receptor agonists are neuroprotective¹⁷. The fact that both A_2A receptor agonists and antagonists have protective effects may reflect the complex actions of A_2A receptors. A_2A receptor antagonists may impose their neuroprotective effect at an early phase of injury (perhaps by decreasing glutamate release) and have opposite effects at the later phase of injury¹⁷. The ischemic scenario is similar for that of A₃; the acute administration of A₃ agonists (before imposing ischemia) demonstrates an increase in ischemic brain damage while a chronic administration has protective effects³⁸.

In the past decade, specific A_2A receptor antagonists have emerged as promising pharmacological agents for the treatment of Parkinson’s disease, primarily because of their colocalization with D₂ dopamine receptors and the profound antagonistic interaction between adenosine and dopamine systems³⁰. Indeed, continuous work on A_2A receptor antagonists as potential treatment for Parkinson’s disease is being done.

Several lines of evidence suggest a potential role of A_2A receptors in the pathogenesis of Huntington’s disease. A_2A receptors are highly enriched in the striatum, the brain region which is highly degenerated in Huntington’s disease⁹. Neurochemical studies in a Huntington’s disease mouse model also show that some of the earliest changes are reduced gene expression including that of A_2A receptor, suggesting a possible involvement in the pathogenesis of Huntington’s disease⁵.

A_2A receptors may also be involved in Alzheimer’s disease (AD), since acetylcholine release is enhanced by A_2A receptor activation. Therefore, increased acetylcholine mediated by A_2A receptors may be beneficial for patients with Alzheimer’s disease³⁵. In addition, adenosine A₁ receptor expression has been shown to undergo a decrease of 40-60% in the hippocampus of postmortem AD subjects compared to healthy counterparts^25,34. However, some areas in the brain also show upregulation of A₁ receptors in Alzheimer’s Disease³⁴. The involvement of adenosine A₁ receptor in AD is further strengthened by the observations that in degenerative neurons, A₁ colocalizes with Aβ-plaques, and phosphorylated tau protein, two well established markers for AD. In addition, in a human neural cell line (SH-SY5Y), Aβ-plaque formation and tau phosphorylation are both mediated by A₁ receptor².

In addition to neurological disorders, adenosine receptors are also marked targets for the treatment of some psychiatric disorders such as schizophrenia as well as some addictive behaviors¹⁷.

Alomone Labs has extensively expanded its GPCR portfolio. We offer antibodies to all four adenosine receptors: Anti-Adenosine A₁ Receptor Antibody (#AAR-006), Anti-Adenosine A_2A Receptor Antibody (#AAR-002), Anti-Adenosine A_2B Receptor (extracellular) Antibody (#AAR-003), and Anti-Adenosine A₃ Receptor Antibody (#AAR-004). Alomone Labs Adenosine Receptor antibodies enable the detection of their respective receptors using various applications such as western blot, immunohistochemistry, and immunocytochemistry (see the adjoined figures).

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