Adenosine A2A Receptors Biology:
(i) modulation of Inflammation:
Jiang-Fan Chen
Department of Neurology
Boston University School of Medicine
Adenosine is a naturally occurring nucleoside that is distributed ubiquitously
throughout the body as a metabolic intermediary
Sitkovsky et al. 2005
Adenosine levels rise markedly in response to ischemia, hypoxia,
inflammation and other brain insults
Ischem ia
Hypoxia
Change in cAMP
( % of maximal
response)
120
Norm al
range
100
A1
80
A2A
60
A2B
40
A3
20
0
10 -10 10 -9
10 -8
10 -7 10 -6
10 -5
Adenosine (M)
10 -4
10 -3
10 -2
Xu et al. 2006
Adenosine Receptors
Code:
A1
A2A
A2B
2.1.ADO.01
2.1.ADO.02A
2.1.ADO.02B 2.1.ADO.03
Human
Human
Human
Rat,Mouse
dog, guinea pig
rat.mouse
chick
rat (alt.splice)
sheep,rabbit
MRS 1754
MRS 1191,
Cloned: Human
Rat.Mouse
dog,bovine
A3
sheep,chick
Antag.
DPCPX, 8-cpt SCH 58261
WRC-0571
Transd. Gi, Go
ZM 241385
KW6002
Gs, Golf
MRS 1220
Gs, Gq ?
Gi
Phylogeny of Adenosine Receptors
Fredholm et al. 2001
CH3
xanthine
CH3
CH3
from Fredholm et al., 1999
Potency of caffeine at adenosine receptors
KD in mM
Receptor subtype
Rat
Human
A1 receptors
A2A receptors
A2B receptors
A3 receptors
20
8.1
17
190
12
2.4
13
80
Bertil Fredholm March 2002
Adenosine inhibition of lymphocyte-mediated cytolysis:
possible role of cyclic adenosine monophosphate
•Wolberg G, Zimmerman TP, Hiemstra K, Winston M, Chu LC.
The in vitro destruction of tumor cells by specifically sensitized mouse lymphocytes was inhibited by
adenosine; this inhibition was markedly potentiated by the presence of an inhibitor of adenosine
deaminase. The inhibition of cytolysis by adenosine was accompanied by a rapid elevation in
lymphocytic adenosine 3',5'-monophosphate (cyclic AMP) concentrations. Both the inhibition of
cytolysis and the elevation of cyclic AMP were reversed by prolonged incubation of the lymphocytes in
the presence of adenosine or, more rapidly, by removal of the adenosine. Low concentrations of
adenosine also caused an elevation of cyclic AMP in human lymphocytes, and this effect of adenosine
may contribute to the lack of immune response associated with adenosine deaminase deficiency.
Science. 1975 Mar 14;187(4180):957-9.
Enhanced and prolonged accumulation of pro-inflammatory cytokines and
exaggerated liver damage in A2A receptor-deficient mice
Enhanced accumulation of pro-inflammatory cytokines and tissue damage
in A2A receptor deficient mice treated with endotoxin
LPS (100 ug/kg)
LPS (5 ug/kg)
J. Clinical Investi. 2006
A Broader Spectrum of Cell Protection by
A2AR Activation in Peripheral Tissues
• Liver injury
– Con-A, CoCl4, endotoxin, ischemia
• Renal injury
– Ischemia, transplantation, antimemberane IgG
• Heart injury
– Ischemia
• Lung injury
– Oxygen intoxication, COPD
PNAS, 2007
Sitkovsky et al. 2005
Ohta & Sitkovsky, 2004
Sitkovsky et al. 2005
Sitkovsky et al. 2005
Extracellular adenosine as a reporter
of excessive tissue damage
• Immediacy of adenosine signaling because it is
triggered by metabolic change.
• Local inhibition of inflammatory cells in injury areas;
• “Metabokines”: adenosine is a metabolite, but also
has cytokine property;
• “Retaliatory” metabolite of tissue protection;
• Indiscriminate protection against tissue damage
Adenosine A2A receptors are enriched in striatum in human (A),
monkey(B), rat (C) and mouse (D) brain.
Svenningsson et al. 1999
(A2A)
(A2A)
Traumaticbrain injury (A2A)
Neuroprotection
A2A antagonists
Multiple sclerosis (A1, A2A)
Neuroprotection
A1 agonists & A2A antagonist
Modified from Boison, TINS (2007)
100
2
A2a +/+
A2a +/+
A2a -/-
*
1.5
*
50
25
0
Neurological Deficit Score
Infarct Volume (mm3)
75
A2a -/-
1
*
*
0.5
0
C57BL/6 x 129-Steel
C57BL/6 x 129-Steel
Pure 129-Steel
Pure 129-Steel
J. Neurosci. 1999
A2AR inactivation attenuates dopaminergic
neurotoxicity in the MPTP model of PD
irradiation
Bone marrow transplantation
donor
recipient
chimeric mice
Selective reconstitution of A2AR in bone marrow derived
cells (BMDCs) reinstate ischemic brain injury
120
20
WT  WT
100
60
*#
40
Infarct Area (mm2)
80
Infarct Volume (mm3)
WT  KO
16
KO  KO
12
8
20
4
0
0
WTWT
WTKO
KOKO
1.60
0.97
0.33
-0.30
-0.93
-1.57 -2.20
-2.83 -3.47
Distance from Bregma (mm)
-4.10
Selective deletion of A2AR in bone marrow derived cells
attenuate ischemic brain injury
A Broader Spectrum of Neuroprotection by
A2AR Inactivation in Brain
• Ischemic brain injury models
– focal and global ischemia model
• Parkinson’s disease models
– MPTP, 6-OHDA
• Traumatic brain injury models
– Control cortical impact
• Huntington’s disease models
– Excitotoxins or mitochondrial toxins
Modulation of brain injury by A2AR ligand is associated with the
local brain glutamate levels
Modulation of brain injury by A2AR agonists and antagonists depends on
the time of drug administration post-TBI
(s)-4c3HPG dose-dependently decreased the glutamate level in
CSF at 15 min post TBI in WT and A2AR KO mice
Blocking glutamate release converts the effect of CGS21680 on TBI
from exacerbation to attenuation
Summary
1. A2AR activation protect peripheral tissues from
excessive inflammatory damage by direct action in
inflammatory cells
2. A2AR inactivation protect brain from a variety of
insults partly by control of neuroinflammation,
possibly through a neuronal mechanism
3. Local glutamate level dictates the A2AR modulation
of neuroinflammation and brain injury
Experimental approaches for increasing BBB permeability
A. Chemical: mannitol (epilepsy)
vasoactive peptide bradykinin (clinical trial failed)
B. Physical: intraventricular injection
C. Drug modifications:
i) chemical addition of moieties to increase drug
lipophilicity
ii) Vector-based: attaching a drug to receptor-mediated
endocytosis (transferrin receptor: limited to receptor
expression level and endothelium)
NECA increases BBB permeability in a temporally discrete and reversible manner
Increased BBB permeability depends on selective agonism of A1R and A2AR activation
The A2AR agonist Lexiscan increase BBB permeability in mouse model
Anti-B-amyloid antibody administered intravenously cross BBB and label
B-amyloid Plaques after treatment with NECA in transgenic APP mice
Anti-B-amyloid antibody administered intravenously cross BBB and label
B-amyloid Plaques after treatment with NECA in transgenic APP mice
AR signaling affect paracellular but not transcellular pathway in cultured BECs
Transendothelium electric resistance
AR agonists increases actinomysin stress fiber formation in cultured BECs (i-p)
AR agonists increases expression of tight junction adhesion molecules in BECs (q-y)
Drugs like Lexiscan, the FDA-approved A2A AR agonist, which
increases BBB permeability and facilitates CNS entry of
macromolecules like dextrans, represent a possible pathway
toward future therapeutic applications in humans.
The time course of BBB opening and closing after treatment with
NECA (half-life 5 h) is much longer than the time course after
treatment with Lexiscan [half-life 3 min.
Importantly, our findings suggest this technique could
be used for CNS delivery of macromolecular therapeutics like
antibodies, which traditionally have been limited in their use in
treating neurological diseases because they required invasive
delivery technologies.
“ These results suggest that AR signaling can be used to
modulate BBB permeability in vivo to facilitate the entry
of potentially therapeutic compounds into the CNS. AR
signaling at brain endothelial cells represents a novel
endogenous mechanism of modulating BBB permeability.
We anticipate these results will aid in drug design, drug
delivery and treatment options for neurological diseases
such as AD, Parkinson’s disease, multiple sclerosis and
cancers of the CNS”