The Brains on Adenosine Receptors

Melanie Grably, Ph.D., Scientific Editor, Marketing Division

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 Expression of A1 Adenosine Receptor in Rat DRG Primary Culture.
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 A B C
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 D E F
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 ATP7,40; 3)
Immunocytochemical staining of paraformaldehyde-fixed and permeabilized rat dorsal root ganglion (DRG) primary culture.
extracellular formation following cAMP release11,28.
A. Staining of DRG cells with Anti-A1 Adenosine Receptor antibody (#AAR-006), (1:100), followed by goat anti-rabbit-
The removal of adenosine from the extracellular AlexaFluor-555 secondary antibody. B. Nuclear staining of cells using the cell-permeable dye Hoechst 33342. C. Merged
space occurs through nucleotide transporters, images of A and B. F. Merged images of D and E.
regulated by the activation of adenosine receptors Magnification: A-C: x20, D-F: x100
(ARs) through protein kinase pathways8. Experimental procedure and figure processed at Alomone Labs.

5
GPCR Pathways No.4 Summer 2010 www.alomone.com
Adenosine Receptors One interesting aspect of adenosine receptor
signaling involves their interaction with other
Expression of A2A Adenosine Receptor in

types of G-protein coupled receptors. Synergistic

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; A1, A2A, and A2B
adenosine receptors are well conserved among
mammals, while A3 adenosine 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 A1 and A2A adenosine receptors is
well known16; A1 receptor shows the highest
abundance in the brain, while A2A receptor is
expressed at high levels in only a few regions
of the brain11. Pharmacological tools available
for A2B 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
Rat Lung.
interactions have been reported between low
concentrations of A1 and metabotropic GABA
(B) agonists on inwardly rectifying K+ channels
Br
(IRKs)37. There is also extensive evidence from
studies on direct interactions between A2A and
D1 dopamine receptors, and between A1 and D2
receptors4,22,24 and reviewed in reference 19). In
addition to interaction, dimerization between Br
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 Immunohistochemical staining of paraffin emedded rat
lung sections using Anti-A2A Adenosine Receptor antibody
receptors39 as well as for A1 and mGluR1
(#AAR-002), (1:50). A2A Adenosine Receptor is expressed in
metabotropic glutamate receptor6 and A2A with
the respiratory epithelium of the bronchioli (Br). Note that
mGluR514. smooth muscle and endothelium in blood vessels are negative.
Hematoxilin is used as the counterstain.
Inhibition of Neurotransmitter Release Experimental procedure and figure processed at Alomone Labs.
The most widely recognized effect of adenosine on
nerve activity is the inhibition of neurotransmitter
release and neuronal excitability via the activation
of A1 receptors10,11,18. 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 A1 adenosine receptors
which subsequently inhibits the release of Western blot analysis of RAEC (rat aortic endothelial cells),
glutamate1,3,12. (lanes 1 and 3), rat brain (lanes 2 and 4) and Jurkat cell (lanes
5 and 6) lysates:
1,2,5. Anti-A2A Adenosine Receptor antibody (#AAR-002),
Ischemia, Neurological and (1:200).
3,4,6. Anti-A2A Adenosine Receptor antibody, preincubated with

receptors respond to adenosine albeit with

different affinities; A1, A2A, and A3 adenosine
receptors are considered as high affinity while A2B
adenosine 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 Gq thereby also
activating phospholipase C, inositol triphosphate
and intracellular Ca2+ influx21. A2A and A2B activate
adenylyl cyclase through Gs. In addition, A2B
receptors can also bind Gq13. 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.
Psychiatric Disorders
The neuroprotective effect of adenosine against
brain injury is mostly mediated by A1 adenosine
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 A2A may contribute to neuronal
injury in several neurological disease models.
Pharmacological studies using A2A receptor
selective antagonists constantly show reduced
ischemic brain damage20,29. It should also be
noted though, that in some experimental models,
A2A receptor agonists are neuroprotective17.
The fact that both A2A receptor agonists and
antagonists have protective effects may reflect
the complex actions of A2A receptors. A2A 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
the control peptide antigen.
Experimental procedure and figure processed at Alomone Labs.
brain damage while a chronic administration has
protective effects38.
In the past decade, specific A2A receptor
antagonists have emerged as promising
pharmacological agents for the treatment of
Parkinsons disease, primarily because of their
colocalization with D2 dopamine receptors and
the profound antagonistic interaction between
adenosine and dopamine systems30. Indeed,
continuous work on A2A receptor antagonists as
potential treatment for Parkinsons disease is
being done.
Several lines of evidence suggest a potential
role of A2A receptors in the pathogenesis of
Huntingtons disease. A2A receptors are highly
enriched in the striatum, the brain region which
is highly degenerated in Huntingtons disease9.
Neurochemical studies in a Huntingtons disease
mouse model also show that some of the earliest
changes are reduced gene expression including
that of A2A receptor, suggesting a possible

6
GPCR Pathways No.4 Summer 2010 www.alomone.com
involvement in the pathogenesis of Huntingtons
disease5.
A2A receptors may also be involved in Alzheimers
disease (AD), since acetylcholine release is
enhanced by A2A receptor activation. Therefore,
increased acetylcholine mediated by A2A
receptors may be beneficial for patients with
Alzheimers disease35. In addition, A1 adenosine
receptor expression has been shown to undergo
a decrease of 40-60% in the hippocampus of
postmortem AD subjects compared to healthy
counterparts25,34. However, some areas in the
brain also show upregulation of A1 receptors in
Alzheimers Disease34. The involvement of A1
adenosine receptor in AD is further strengthened
by the observations that in degenerative
neurons, A1 colocalizes with A-plaques, and
phosphorylated tau protein, two well established
markers for AD. In addition, in a human neural
cell line (SH-SY5Y), A-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-A1 Adenosine Receptor
(#AAR-006), Anti-A2A Adenosine Receptor (#AAR-
002), Anti-A2B Adenosine Receptor (extracellular)
(#AAR-003), and Anti-A3 Adenosine Receptor
(#AAR-004) antibodies. 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).
Expression of A3 Adenosine Receptor in a Human Melanoma Cell Line.
A B
D E
Immunocytochemical staining of paraformaldehyde-fixed and permeabilized human melanoma cells (A2058).
A, D. Cells were stained using Anti-A3 Adenosine Receptor antibody (#AAR-004), (1:100), followed by goat anti-
rabbit-AlexaFluor-555 secondary antibody. B, E. Nuclear fluorescence staining of cells using the membrane-
permeable DNA dye Hoechst 33342. C. Merged image of panels A and B. F. Merged image of panels D and E.
Experimental procedure and figure processed at Alomone Labs.
GPCR Pathways No.4 Summer 2010 www.alomone.com
Expression of A2B Adenosine Receptor in
Rat Lung.
A 6. Ciruela, F. et al. (2001) J. Biol. Chem. 276, 18345.
A 8. Delicado, E.G. et al. (1991) Biochem. J. 279, 651.
Br
A 12. Fastbom, J. and Fredholm, B.B. (1985) Acta Physiol. Scand. 125,
Immunohistochemical staining of rat lung paraffin embedded
sections using Anti-A2B Adenosine Receptor (extracellular)
antibody (#AAR-003), (1:50). Staining is present in the
respiratory epithelium of the bronchiole (Br) as well as in
the pneumonocytes of the alveolar wall (alveoli, (A). Color
reaction was obtained with SuperPicture HRP-conjugated
polymer (Zymed) followed by DAB. Hematoxilin is used as the
counterstain.
Experimental procedure and figure processed at Alomone Labs.
Western blot analysis of human HL-60 (lanes 1 and 2) cells, rat
brain (lanes 3 and 4), and mouse brain (lanes 5 and 6) lysates:
1, 3, 5. Anti-A2B Adenosine Receptor (extracellular) antibody
(#AAR-003), (1:200).
2, 4, 6. Anti-A2B Adenosine Receptor (extracellular) antibody,
preincubated with the control peptide antigen.
Experimental procedure and figure processed at Alomone Labs.
C
F Anti-A2A Adenosine Receptor_ _____________________
References
1. Ambrosio, A.F. et al. (1997) Eur. J. Pharmacol. 340, 301.
2. Angulo, E. et al. (2003) Brain Pathol. 13, 440.
3. Barrie, A.P. and Nicholls, D.G. (1993) J. Neurochem. 60, 1081.
4. Canals, M. et al. (2003) J. Biol. Chem. 278, 46741.
5. Cha, J.H. et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6480.
7. Cunha, R.A. (2001) Neurochem. Res. 26, 979.
9. DiFiglia, M. (1990) Trends Neurosci. 13, 286.
10. Dunwiddie, T.V. (1985) Int. Rev. Neurobiol. 27, 63.
11. Dunwiddie, T.V. and Masino, S.A. (2001) Annu. Rev. Neurosci.
24, 31.
121.
13. Feoktistov, I. and Biaggioni, I. (1997) Pharmacol. Rev. 49, 381.
14. Ferre, S. et al. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11940.
15. Fredholm, B.B. et al. (1994) Pharmacol. Rev. 46, 143.
16. Fredholm, B.B. et al. (2000) Naunyn Schmiedebergs Arch.
Pharmacol. 362, 364.
17. Fredholm, B.B. et al. (2005) Int. Rev. Neurobiol. 63, 191.
18. Fredholm, B.B. and Dunwiddie, T.V. (1988) Trends Pharmacol. Sci.
9, 130.
19. Fuxe, K. et al. (1998) Brain Res. Brain Res. Rev. 26, 258.
20. Gao, Y. and Phillis, J.W. (1994) Life Sci. 55, PL61.
21. Gessi, S. et al. (2008) Pharmacol. Ther. 117, 123.
22. Gines, S. et al. (2000) Proc. Natl. Acad. Sci. U.S. A. 97, 8606.
23. Greene, R.W. and Haas, H.L. (1991) Prog. Neurobiol. 36, 329.
24. Hillion, J. et al. (2002) J. Biol. Chem. 277, 18091.
25. Kalaria, R.N. et al. (1990) Neurosci. Lett. 118, 257.
26. Klinger, M. et al. (2002) Cell Signal. 14, 99.
27. Klishin, A. et al. (1995) Neuroscience 65, 947.
28. Latini, S. and Pedata, F. (2001) J. Neurochem. 79, 463.
29. Monopoli, A. et al. (1998) Neuroreport 9, 3955.
30. Morelli, M. and Pinna, A. (2001) Neurol. Sci. 22, 71.
31. Mozzicato, S. et al. (2004) FASEB J. 18, 406.
32. Newby, A.C. (1991) Adv. Exp. Med. Biol. 309A, 265.
33. Pitcher, J.A. et al. (1998) Annu. Rev. Biochem. 67, 653.
34. Rahman, A. (2009) Curr. Neuropharmacol. 7, 207.
35. Ribeiro, J.A. et al. (2003) Drug News Perspect. 16, 80.
36. Rudolphi, K.A. et al. (1992) Cerebrovasc. Brain Metab. Rev. 4, 346.
37. Sodickson, D.L. and Bean, B.P. (1998) J. Neurosci. 18, 8153.
38. Von Lubitz, D.K. et al. (1994) Eur. J. Pharmacol. 263, 59.
39. Yoshioka, K. et al. (2002) FEBS Lett. 531, 299.
40. Zimmermann, H. (2000) Naunyn Schmiedebergs Arch. Pharmacol.
362, 299.
Related Products
Compound Cat. #
Adenosine Receptor Antibodies
Anti-A1 Adenosine Receptor_______________________ AAR-006
AAR-002
Anti-A2B Adenosine Receptor (extracellular)__________ AAR-003
Anti-A3 Adenosine Receptor_______________________ AAR-004
7