Structure JOEL
and
function
of A1 adenosine
1
LINDEN
Department of Internal Virginia 22908, USA
Medicine
(Cardiology)
and Physiology,
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 (Aia, Aib, 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, NaCa2 exchange, Ca2 channels, C1 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. Linden, J. Structure and function of A1 adenosine receptors. FASEBJ. 5: 2668-2676; 1991. ABSTRACT
Key Words: xanthines GTP-binding coupling purinergic receptor
protein
receptor-ejector
IN RECENT YEARS THERE HAS BEEN AN explosion of new knowledge about purinergic receptors, i.e., receptors for adenosine (P1 receptors) and ATP (P2 receptors) present on most tissues. Certain ligands bind with markedly different affinities to adenosine receptors located on different tissues, or, in some cases, to distinct binding sites that mediate different responses in the same tissue. These differences in structure activity profiles provide part of the basis for dividing adenosine receptors into families referred to as A1 and A2. The A1 receptors found in brain, heart, adipose tissue, and kidney will be the focus of this review. As receptors have been examined more carefully and as new ligands have become available, evidence has emerged suggesting that subtypes of both i and A2 receptors may exist. This hypothesis is generally accepted in the case of A2 receptors, as some ligands have more than 1000-fold selectivity for one subtype over the other (A2a vs. A2b) (1), and recently a canine adenosine receptor with properties that appear to correspond to the A2a subtype has been cloned (2). The existence of A1 subtypes is not clear-cut because structure-activity differences among hypothetical subtypes of these receptors are not as large as differences among subtypes of A2 receptor. However, along with structure-activity differences, there is additional evidence that A1 subtypes may exist. Although it is generally true that activation of A1 receptors has an inhibitory effect on the function of excitable tissues (Table 1), effectors of A1 receptors are remarkably
2668
receptors University
of Virginia,
Charlottesville,
diverse. These effectors, including adenylate cyclase, phospholipase C, and various ion channels, will be discussed. This diversity of responses could be a manifestation of A1 receptor subtypes. However, the possibility that a single receptor or group of similar receptors is capable of activating a variety of G proteins and effectors in various tissues will also be explored.
PHARMACOLOGICAL PROPERTIES PURINERGIC RECEPTOR SUBTYPES
OF
One of the first criteria used to distinguish adenosine receptors from ATP receptors was selective competitive blockade of the former by methylxanthines such as caffeine and theophylline. The xanthine-sensitive adenosine receptors were further subdivided into two subfamilies, A1 (formally also called R) and A2 (Ra), on the basis of the effects of agonists to inhibit or activate, respectively, adenylate cyclase (3). It is now recognized that A1 receptors and possibly some A2 receptors act through effectors other than adenylate cyclase (see below). Current receptor classification is based on the structure-activity profiles of various drugs. (For chemical structures, refer to Fig. 1.) A1 receptors were initially distinguished from A2 receptors based on the potency order of agonists. Prototypical A1 receptors that mediate inhibition of adenylate cyclase in adipose tissue have the potency order: R-phenylisopropyladenosine (R-PIA)2 > 2-chloroadenosine (CADO) 5’-N-ethylcarboxamidoadenosine (NECA). A2 receptors that activate adenylate cyclase in platelets and dilate vascular smooth muscle have the potency order: NECA> CADO > R-PIA. Subtypes
of A1 receptors?
5’-N-Ethylcarboxamidoadenosine has been shown to vary widely in its potency as an A1 agonist. For instance, NECA is 100-fold less potent than R-PIA in rat adipose tissue (3) whereas it is nearly as potent as R-PIA in binding to A1 receptors of rat heart and brain (1, 4). In many studies (Table 1), the potency of NECA to inhibit neurotransmitter release from various nerves is almost as high as R-PIA, resulting in the potency order: R-PIA NECA >CADO. Based initially
1The literature cited is by no means exhaustive. Many citations are taken from previous review articles. The reader should refer to these for additional references. 2Abbreviations: R-PIA, R-phenylisopropyladenosine; CADO, 2-chloroadenosine; NECA, 5 ‘N-ethylcarboxamidoadenosine; CPA, N#{176}-cyclopentyladenosine; CENBA, 5 ‘-chloro-N6-(2-endo-norbornyl)adenosine; DPCPX, l,3-dipropyl-8-cyclopentylxanthine; DPSPX, l,3-dipropyl-8-sulfophenylxanthine; XCC, xanthine carboxylic
cogener;
XAC,
xanthine
amino
cogener;
G protein,
GTP
binding
proteins; NT, neurotransmitter; PLC, phospholipase C; 1P3, 1,4,5 trisphosphate; DAG, diacylglycerol; PKC, protein kinase C; NE,
norepinephrine.
0892-6638/91/0005-2668/$01
.50. © FASEB
w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on September 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
TABLE
1. Some inhibitory effects of A1 adenosine
receptor activation
Tissue
Inhibitory
Adipose tissue, Adipose tissue, Brain Heart Ileum Kidney Motor nerves Neutrophils Pancreas Pituitary (GH Spine Stomach Striatum Vas deferens
white brown
response
References
Lipolysis Thermogenesis Excitatory neurotransmitter release Inotropic, chronotropic, dromotropic Acethycholine, tachykinin release Glomerular filtration, renin, erythropoietin Acetylcholine release Chemotaxis Insulin secretion Prolactin, growth hormone release Nociception Acid secretion Tyrosine hydroxylase activity Acetylcholine release
cells)
on these differences in agonist potency order, Ribeiro and Sebastiao (5) proposed the existence of A3 receptors, distinct from A1 and A2 receptors. More recent examinations of orders of antagonist potency support this classification and have resulted in what appear to be more discrepancies. Gustafsson et al. (6) pointed out that prejunctional A1 receptors at peripheral autonomic ganglia have structure-activity relationships distinct from receptors characterized by radioligand binding to brain membranes. They proposed that central receptors that may originate embryologically from the neural tube be called Aia, that receptors on autonomic nerves that may originate embryologically from the neural crest be called Aib, and that both are distinct from A3 receptors at the neuromuscular junction. Differences in the potency orders of selected antagonists are summarized in Table 2 and Table 3. Additional evidence for a distinction between A1a and Aib receptors is the finding that 2(4 methoxyphenyl)adenosine (CV-1674) selectively activates Aia receptors (Table 3). Despite this evidence, the notion that subtypes of A1 receptors exist has not been universally accepted because some of these apparent discrepancies could result from differences in receptor coupling to effector systems, possible involvement of A2 receptors in some of these responses, generally
or differences hydrophobic
in the compounds
tissue
several
tors,
years
as an A1-selective
has agonist,
widely
but
even
used more
been
used
to
characterize
A1 receptors
on
Figure
1. Ligands
N6 position
that
of agonists
bind (see
aminotriazoloquinoxalines properties
of the
A, ADENOSINE
various
to adenosine arrow,
(e.g., compounds
RECEPTORS
receptors.
adenoine)
CP-68,247) are
and based
the on
that
as
a
selective
compete
radioligand
for
A2a
A
ligands
such
as
with
A1 receptors.
Unfor-
is limited primarily the A2 receptors of
consist mostly of the A2b subtype or beof A2a receptors on most other tissues is Useful radioligands for the A2b recep-
subtype-selective
distributed developed.
in the
central
antagonists
8-Phenylxanthines were developed as potent and somewhat A1-selective antagonists (1). The potency and selectivity for A1 vs. A2 receptors of various antagonists is given in Table 2. General problems with some of these compounds are poor aqueous solubilities and variable activities as cyclic nucleotide phosphodiesterase inhibitors. physiological pH have increased not readily cross cell membranes.
Compounds charged at aqueous solubilities and do They are useful as agents
that act only as receptor blockers and do not block phosphodiesterases or produce other nonreceptor-mediated actions. Polar A5-selective antagonists in this category include the carboxylic acid, oral bioavailability, sulfophenylxanthine
(4).
have
been
3-propyl
group in
used
are known to be diffusely system have not yet been
Receptor
Ci/mmol)
information
been
of highly
of ligands
tors that nervous
with
5-amino
addition
most other tissues cause the density too low to measure.
which
to the
has
tunately, the utility of these radioligands to the A2arich striatum, either because
for
Structures
corresponds
by
absence
selec-
tissues
low receptor density is [‘25I]N6-aminobenzyladenosine, combines the high specific radioactivity of 1251 (2200 with a high ratio of specific to nonspecific binding
[SH]NECA
receptors
tive N6-substituted agonists have recently been developed. These include N6-cyclopentyladenosine (1) (CPA) and 5’chloro-N6-(2-endo-norbornyl)adenosine (7) (CENBA), the most potent and selective agonist yet synthesized. All these compounds have been tritiated for use as A1-selective agonist radioligands. A particularly useful agonist radioligand that has
agonists
the agonist CPA or the antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). Recently, a new ligand with high affinity and selectivity for A2a over A1 receptors has been developed, CGS 21680 (8, 9). [3H]CGS 21680 and similar radioligands (10) can be used to label A2a receptors in the
area will require receptor subtypes.
been
(6)
receptors. However, NECA also binds with high affinity to some A1 receptors, and to achieve selectively for A2a receptors in [3H]NECA binding assays, it is necessary to block A1
agonists
R-N#{176}-Phenylisopropyladenosine
release
The A2 receptor has been further subdivided into A2a and A2b subclasses. Central A2a receptors are localized primarily to the striatum, nucleus accumbens, and olfactory tubercle, and bind adenosine and NECA with higher affinity than A2b receptors (1). Because of its high affinity for A2a recep-
distribution of the used to characterize
receptors. Resolution of this controversial cloning of A1 receptors and hypothetical A1 selective
A2 selective
(42) (30) (31, 53) (26) (6, 54) (55) (50, 51) (56) (57) (28, 58) (45) (59) (39)
drawn group
BWA-1433U (11), which is also notable for its and the sulfonic acid, 1,3-dipropyl-8(DPSPX) (12). BWA-1433U has a longer
to show
a possible
of alkylxanthines
of triazoloquinazolinamines
references
(1, 4,
9,
common
(e.g.,
(e.g.,
DPCPX),
CGS
orientation
such
the
4-amino
15943).
Notes
that group
about
the of
the
11-18).
2669
w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on September 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
RIBOSE
AGONISTS ‘26IABA
A,
Selective
Radioligand
N
Adenosine
N
PIA
NH
A,
SelectIve
126
RIBOSE
/
C2 H,NH.
N
N(
NNH
CENBA
CPA
Very Selective
Very Selective
A1
NECA
A,
Noriselectlve RIBOSE
N
C2,
NNH2
CGS 21680 A20 Selective
CV
1674
A,0
vs
Aib Selective
cH3
ANTAGONISTS KFM.19
Very Selective, A,
OyyN N
N
DPCPX
‘I-BWA-844U
+
Very Selective A,
c.p.c
A1selective
N3
c__c
0 NH.-
xcc
DPSPx Very acidIc
0
Acidic BWA-1433U Acidic,
orally
Potent. prodrug bioavoilable
CGS 15943 Potent norixanthine
cIX
CP.68,247
CP.66,713
A2 Selective potent
2670
Vol. 5
September
nonxar’tliifle
1991
Very Selective A, nonxaflthine
t The FASEB Journal
LINDEN
w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on September 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
TABLE
2. Potency orders and selectivities between A1 and A22 receptors, and putative
of compounds which distinguish subtypes of receptors
(A12, A16, A3f A, Antagonists,
potency
order,
K, nM2 0.43 0.46 0.86 3.9
BWA-844U
DPCPX XAC CGS-15943 BWA-l433U CP-68,247 XCC 8-PT DPSPX CP-66,713 A,
Antagonists,
order
(17).
15
position
both
58
affinity
86
CP-68,247, see arrows in Fig. 1). Based on this observation we have drawn the structures shown in Fig. 1 in a hypothetical common receptor-binding orientation. Peet et al. (19) discuss in detail possible antagonist binding orientations.
273 selectivity
found to have potent adenosine antagonist CP-66,713 belongs to the chemically similar triazoloquinoxaline family and was found to be a potent agent in a screen for antidepressant activity (18). Although CGS 15943 is the most potent A2 antagonist known, CP-66,713 is the only compound that combines high potency and greater than 10-fold selectivity for A2 receptors. Sarges et al. (18) suggested that the amino groups of 4-aminotriazoquinoxalines and adenosine occupy the same properties
28
210
CP-68,247
and unexpectedly
(K, ratio: A2JA,)2 >3000
when
cases
for
A1
1740
PROPERTIES
BWA-l433U
53
G PROTEINS
XCC XAC 8-PT DPSPX CGS-l5943 CP-66,713
41
0.39 7
“Potency orders and K for A, and A2 receptors are based on competition for radioligand binding to rat cortex and striatum, respectively.
serum half-life than another carboxylic acid, xanthine carboxylic cogener (XCC or BWA-79U), apparently because the acrylate residue is more resistant to metabolism than is the oxyacetic acid (S. Daluge, unpublished results). Xanthine amino cogener, XAC, is another polar compound. The aliphatic amino group of XAC assumes a positive charge at neutral pH. Xanthine amino cogener has only moderate A1 selectivity, but can be used to potently block A1 and A2 receptors. It is less potent at A2 receptors than the slightly A2-selective antagonist CGS 15943, but XAC is considerably more soluble in water, 90 vs. 1.74 zM (13). Replacing the phenyl with cyclopentyl on the 8-position of xanthines results in compounds with improved affinity, aqueous solubility, and selectivity for A1 receptors. 1,3-Dipropyl8-cyclopentylxanthine has proved to be a useful compound both as a highly selective A1 antagonist, and in its tritiated form, as a radioligand (14). Another 8-cyclopentylxanthine, ‘251-labeled BWA-844U, has binding characteristics similar to [3H]DPCPX, higher specific radioactivity, and greater A1 selectivity, but it displays greater nonspecific binding (15). A keto derivative of DPCPX, KFMI9, has been reported to have increased aqueous solubility and to have memory improving effects in rats (16). Nonxanthine-potent antagonists have also been developed recently. CGS 15943 is a member of the triazoloquinazoline
TABLE
synthesized
as benzodiazepine
receptors.
groups
receptors
OF A, RECEPTORS
(e.g.,
AND
In
increases CPA
and
ASSOCIATED
antagonists
possibility
that
the
receptors
activate
different
G proteins
to
produce these responses. Direct evidence in support of this concept was provided by Leid et al. (20), who found that subsets of A, receptors on porcine atria are differentially affected by guanine nucleotides. They concluded that the same receptor appears to be coupled to two or more G proteins. Moreover, agonist affinity chromatography of detergentsolubilized bovine brain membranes results in copurification of A1 receptors and at least two different species (G0 and G) of tightly complexed pertussis toxin-sensitive G proteins (21). Receptor-G protein complexes were eluted from agonist columns by GTP or N-ethylmaleimide, agents that uncouple receptors from G proteins and simultaneously lower the affinity of receptors for agonists. When partially purified bovine brain A, receptors were reconstituted with human platelet membranes (which lack A1 receptors), the affinity
functional
reconstitution
of
G
proteins
and
receptors
was
almost completely abolished in membranes treated with pertussis toxin (22). Because platelets contain some G proteins that are not substrates for pertussis toxin, this finding is consistent with the notion that A, receptors couple selectively to the
pertussis toxin-sensitive The A, receptor, free
homogeneity tography were not
from
rat
subset of G proteins. of G proteins, has been
brain
by two
over an antagonist copurified by this
affinity antagonist
cycles
purified
of affinity
to
chroma-
column (23). G proteins affinity chromatogra-
phy procedure, confirming that unlike agonists, antagonists do not stabilize A1 adenosine receptor-G protein complexes. Purified receptors cannot be detected with radioligands unless they are first reconstituted with phospholipids
or
3. Hypothetical A1 receptor subtypes”
Type
Location
Aia
Brain Autonomic
Aib
Potency
nerves
Neuromuscular
A3 “Determinations structures
adenosine
amino
A, receptors are coupled to GTP binding proteins (G proteins). This was initially inferred from characteristic decreases in the binding affinity of agonists upon the addition of GTP or GTP analogs. The existence of various effectors of A1 receptors such as those listed in Table 4 raises the
3.4
originally
to
selectivity
and
740
family,
bind
these
DPCPX
0.07
drugs
on
BWA-844U
31 9.9
these
substitution
and
A, ADENOSINE
of pA2
references.
8PT,
RECEPTORS
junction derived from Schild 8-phenyltheophylline.
were
pA2 for DPCPX
order
DPCPX>XAC> >XCC DPCPX>XAC XCC XAC DPCPX> > XCC analyses
of the effects of antagonists
CV-l674
>9 (rat) 8-9
and
function
of A1 adenosine
1
LINDEN
Department of Internal Virginia 22908, USA
Medicine
(Cardiology)
and Physiology,
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 (Aia, Aib, 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, NaCa2 exchange, Ca2 channels, C1 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. Linden, J. Structure and function of A1 adenosine receptors. FASEBJ. 5: 2668-2676; 1991. ABSTRACT
Key Words: xanthines GTP-binding coupling purinergic receptor
protein
receptor-ejector
IN RECENT YEARS THERE HAS BEEN AN explosion of new knowledge about purinergic receptors, i.e., receptors for adenosine (P1 receptors) and ATP (P2 receptors) present on most tissues. Certain ligands bind with markedly different affinities to adenosine receptors located on different tissues, or, in some cases, to distinct binding sites that mediate different responses in the same tissue. These differences in structure activity profiles provide part of the basis for dividing adenosine receptors into families referred to as A1 and A2. The A1 receptors found in brain, heart, adipose tissue, and kidney will be the focus of this review. As receptors have been examined more carefully and as new ligands have become available, evidence has emerged suggesting that subtypes of both i and A2 receptors may exist. This hypothesis is generally accepted in the case of A2 receptors, as some ligands have more than 1000-fold selectivity for one subtype over the other (A2a vs. A2b) (1), and recently a canine adenosine receptor with properties that appear to correspond to the A2a subtype has been cloned (2). The existence of A1 subtypes is not clear-cut because structure-activity differences among hypothetical subtypes of these receptors are not as large as differences among subtypes of A2 receptor. However, along with structure-activity differences, there is additional evidence that A1 subtypes may exist. Although it is generally true that activation of A1 receptors has an inhibitory effect on the function of excitable tissues (Table 1), effectors of A1 receptors are remarkably
2668
receptors University
of Virginia,
Charlottesville,
diverse. These effectors, including adenylate cyclase, phospholipase C, and various ion channels, will be discussed. This diversity of responses could be a manifestation of A1 receptor subtypes. However, the possibility that a single receptor or group of similar receptors is capable of activating a variety of G proteins and effectors in various tissues will also be explored.
PHARMACOLOGICAL PROPERTIES PURINERGIC RECEPTOR SUBTYPES
OF
One of the first criteria used to distinguish adenosine receptors from ATP receptors was selective competitive blockade of the former by methylxanthines such as caffeine and theophylline. The xanthine-sensitive adenosine receptors were further subdivided into two subfamilies, A1 (formally also called R) and A2 (Ra), on the basis of the effects of agonists to inhibit or activate, respectively, adenylate cyclase (3). It is now recognized that A1 receptors and possibly some A2 receptors act through effectors other than adenylate cyclase (see below). Current receptor classification is based on the structure-activity profiles of various drugs. (For chemical structures, refer to Fig. 1.) A1 receptors were initially distinguished from A2 receptors based on the potency order of agonists. Prototypical A1 receptors that mediate inhibition of adenylate cyclase in adipose tissue have the potency order: R-phenylisopropyladenosine (R-PIA)2 > 2-chloroadenosine (CADO) 5’-N-ethylcarboxamidoadenosine (NECA). A2 receptors that activate adenylate cyclase in platelets and dilate vascular smooth muscle have the potency order: NECA> CADO > R-PIA. Subtypes
of A1 receptors?
5’-N-Ethylcarboxamidoadenosine has been shown to vary widely in its potency as an A1 agonist. For instance, NECA is 100-fold less potent than R-PIA in rat adipose tissue (3) whereas it is nearly as potent as R-PIA in binding to A1 receptors of rat heart and brain (1, 4). In many studies (Table 1), the potency of NECA to inhibit neurotransmitter release from various nerves is almost as high as R-PIA, resulting in the potency order: R-PIA NECA >CADO. Based initially
1The literature cited is by no means exhaustive. Many citations are taken from previous review articles. The reader should refer to these for additional references. 2Abbreviations: R-PIA, R-phenylisopropyladenosine; CADO, 2-chloroadenosine; NECA, 5 ‘N-ethylcarboxamidoadenosine; CPA, N#{176}-cyclopentyladenosine; CENBA, 5 ‘-chloro-N6-(2-endo-norbornyl)adenosine; DPCPX, l,3-dipropyl-8-cyclopentylxanthine; DPSPX, l,3-dipropyl-8-sulfophenylxanthine; XCC, xanthine carboxylic
cogener;
XAC,
xanthine
amino
cogener;
G protein,
GTP
binding
proteins; NT, neurotransmitter; PLC, phospholipase C; 1P3, 1,4,5 trisphosphate; DAG, diacylglycerol; PKC, protein kinase C; NE,
norepinephrine.
0892-6638/91/0005-2668/$01
.50. © FASEB
w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on September 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
TABLE
1. Some inhibitory effects of A1 adenosine
receptor activation
Tissue
Inhibitory
Adipose tissue, Adipose tissue, Brain Heart Ileum Kidney Motor nerves Neutrophils Pancreas Pituitary (GH Spine Stomach Striatum Vas deferens
white brown
response
References
Lipolysis Thermogenesis Excitatory neurotransmitter release Inotropic, chronotropic, dromotropic Acethycholine, tachykinin release Glomerular filtration, renin, erythropoietin Acetylcholine release Chemotaxis Insulin secretion Prolactin, growth hormone release Nociception Acid secretion Tyrosine hydroxylase activity Acetylcholine release
cells)
on these differences in agonist potency order, Ribeiro and Sebastiao (5) proposed the existence of A3 receptors, distinct from A1 and A2 receptors. More recent examinations of orders of antagonist potency support this classification and have resulted in what appear to be more discrepancies. Gustafsson et al. (6) pointed out that prejunctional A1 receptors at peripheral autonomic ganglia have structure-activity relationships distinct from receptors characterized by radioligand binding to brain membranes. They proposed that central receptors that may originate embryologically from the neural tube be called Aia, that receptors on autonomic nerves that may originate embryologically from the neural crest be called Aib, and that both are distinct from A3 receptors at the neuromuscular junction. Differences in the potency orders of selected antagonists are summarized in Table 2 and Table 3. Additional evidence for a distinction between A1a and Aib receptors is the finding that 2(4 methoxyphenyl)adenosine (CV-1674) selectively activates Aia receptors (Table 3). Despite this evidence, the notion that subtypes of A1 receptors exist has not been universally accepted because some of these apparent discrepancies could result from differences in receptor coupling to effector systems, possible involvement of A2 receptors in some of these responses, generally
or differences hydrophobic
in the compounds
tissue
several
tors,
years
as an A1-selective
has agonist,
widely
but
even
used more
been
used
to
characterize
A1 receptors
on
Figure
1. Ligands
N6 position
that
of agonists
bind (see
aminotriazoloquinoxalines properties
of the
A, ADENOSINE
various
to adenosine arrow,
(e.g., compounds
RECEPTORS
receptors.
adenoine)
CP-68,247) are
and based
the on
that
as
a
selective
compete
radioligand
for
A2a
A
ligands
such
as
with
A1 receptors.
Unfor-
is limited primarily the A2 receptors of
consist mostly of the A2b subtype or beof A2a receptors on most other tissues is Useful radioligands for the A2b recep-
subtype-selective
distributed developed.
in the
central
antagonists
8-Phenylxanthines were developed as potent and somewhat A1-selective antagonists (1). The potency and selectivity for A1 vs. A2 receptors of various antagonists is given in Table 2. General problems with some of these compounds are poor aqueous solubilities and variable activities as cyclic nucleotide phosphodiesterase inhibitors. physiological pH have increased not readily cross cell membranes.
Compounds charged at aqueous solubilities and do They are useful as agents
that act only as receptor blockers and do not block phosphodiesterases or produce other nonreceptor-mediated actions. Polar A5-selective antagonists in this category include the carboxylic acid, oral bioavailability, sulfophenylxanthine
(4).
have
been
3-propyl
group in
used
are known to be diffusely system have not yet been
Receptor
Ci/mmol)
information
been
of highly
of ligands
tors that nervous
with
5-amino
addition
most other tissues cause the density too low to measure.
which
to the
has
tunately, the utility of these radioligands to the A2arich striatum, either because
for
Structures
corresponds
by
absence
selec-
tissues
low receptor density is [‘25I]N6-aminobenzyladenosine, combines the high specific radioactivity of 1251 (2200 with a high ratio of specific to nonspecific binding
[SH]NECA
receptors
tive N6-substituted agonists have recently been developed. These include N6-cyclopentyladenosine (1) (CPA) and 5’chloro-N6-(2-endo-norbornyl)adenosine (7) (CENBA), the most potent and selective agonist yet synthesized. All these compounds have been tritiated for use as A1-selective agonist radioligands. A particularly useful agonist radioligand that has
agonists
the agonist CPA or the antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). Recently, a new ligand with high affinity and selectivity for A2a over A1 receptors has been developed, CGS 21680 (8, 9). [3H]CGS 21680 and similar radioligands (10) can be used to label A2a receptors in the
area will require receptor subtypes.
been
(6)
receptors. However, NECA also binds with high affinity to some A1 receptors, and to achieve selectively for A2a receptors in [3H]NECA binding assays, it is necessary to block A1
agonists
R-N#{176}-Phenylisopropyladenosine
release
The A2 receptor has been further subdivided into A2a and A2b subclasses. Central A2a receptors are localized primarily to the striatum, nucleus accumbens, and olfactory tubercle, and bind adenosine and NECA with higher affinity than A2b receptors (1). Because of its high affinity for A2a recep-
distribution of the used to characterize
receptors. Resolution of this controversial cloning of A1 receptors and hypothetical A1 selective
A2 selective
(42) (30) (31, 53) (26) (6, 54) (55) (50, 51) (56) (57) (28, 58) (45) (59) (39)
drawn group
BWA-1433U (11), which is also notable for its and the sulfonic acid, 1,3-dipropyl-8(DPSPX) (12). BWA-1433U has a longer
to show
a possible
of alkylxanthines
of triazoloquinazolinamines
references
(1, 4,
9,
common
(e.g.,
(e.g.,
DPCPX),
CGS
orientation
such
the
4-amino
15943).
Notes
that group
about
the of
the
11-18).
2669
w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on September 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
RIBOSE
AGONISTS ‘26IABA
A,
Selective
Radioligand
N
Adenosine
N
PIA
NH
A,
SelectIve
126
RIBOSE
/
C2 H,NH.
N
N(
NNH
CENBA
CPA
Very Selective
Very Selective
A1
NECA
A,
Noriselectlve RIBOSE
N
C2,
NNH2
CGS 21680 A20 Selective
CV
1674
A,0
vs
Aib Selective
cH3
ANTAGONISTS KFM.19
Very Selective, A,
OyyN N
N
DPCPX
‘I-BWA-844U
+
Very Selective A,
c.p.c
A1selective
N3
c__c
0 NH.-
xcc
DPSPx Very acidIc
0
Acidic BWA-1433U Acidic,
orally
Potent. prodrug bioavoilable
CGS 15943 Potent norixanthine
cIX
CP.68,247
CP.66,713
A2 Selective potent
2670
Vol. 5
September
nonxar’tliifle
1991
Very Selective A, nonxaflthine
t The FASEB Journal
LINDEN
w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on September 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
TABLE
2. Potency orders and selectivities between A1 and A22 receptors, and putative
of compounds which distinguish subtypes of receptors
(A12, A16, A3f A, Antagonists,
potency
order,
K, nM2 0.43 0.46 0.86 3.9
BWA-844U
DPCPX XAC CGS-15943 BWA-l433U CP-68,247 XCC 8-PT DPSPX CP-66,713 A,
Antagonists,
order
(17).
15
position
both
58
affinity
86
CP-68,247, see arrows in Fig. 1). Based on this observation we have drawn the structures shown in Fig. 1 in a hypothetical common receptor-binding orientation. Peet et al. (19) discuss in detail possible antagonist binding orientations.
273 selectivity
found to have potent adenosine antagonist CP-66,713 belongs to the chemically similar triazoloquinoxaline family and was found to be a potent agent in a screen for antidepressant activity (18). Although CGS 15943 is the most potent A2 antagonist known, CP-66,713 is the only compound that combines high potency and greater than 10-fold selectivity for A2 receptors. Sarges et al. (18) suggested that the amino groups of 4-aminotriazoquinoxalines and adenosine occupy the same properties
28
210
CP-68,247
and unexpectedly
(K, ratio: A2JA,)2 >3000
when
cases
for
A1
1740
PROPERTIES
BWA-l433U
53
G PROTEINS
XCC XAC 8-PT DPSPX CGS-l5943 CP-66,713
41
0.39 7
“Potency orders and K for A, and A2 receptors are based on competition for radioligand binding to rat cortex and striatum, respectively.
serum half-life than another carboxylic acid, xanthine carboxylic cogener (XCC or BWA-79U), apparently because the acrylate residue is more resistant to metabolism than is the oxyacetic acid (S. Daluge, unpublished results). Xanthine amino cogener, XAC, is another polar compound. The aliphatic amino group of XAC assumes a positive charge at neutral pH. Xanthine amino cogener has only moderate A1 selectivity, but can be used to potently block A1 and A2 receptors. It is less potent at A2 receptors than the slightly A2-selective antagonist CGS 15943, but XAC is considerably more soluble in water, 90 vs. 1.74 zM (13). Replacing the phenyl with cyclopentyl on the 8-position of xanthines results in compounds with improved affinity, aqueous solubility, and selectivity for A1 receptors. 1,3-Dipropyl8-cyclopentylxanthine has proved to be a useful compound both as a highly selective A1 antagonist, and in its tritiated form, as a radioligand (14). Another 8-cyclopentylxanthine, ‘251-labeled BWA-844U, has binding characteristics similar to [3H]DPCPX, higher specific radioactivity, and greater A1 selectivity, but it displays greater nonspecific binding (15). A keto derivative of DPCPX, KFMI9, has been reported to have increased aqueous solubility and to have memory improving effects in rats (16). Nonxanthine-potent antagonists have also been developed recently. CGS 15943 is a member of the triazoloquinazoline
TABLE
synthesized
as benzodiazepine
receptors.
groups
receptors
OF A, RECEPTORS
(e.g.,
AND
In
increases CPA
and
ASSOCIATED
antagonists
possibility
that
the
receptors
activate
different
G proteins
to
produce these responses. Direct evidence in support of this concept was provided by Leid et al. (20), who found that subsets of A, receptors on porcine atria are differentially affected by guanine nucleotides. They concluded that the same receptor appears to be coupled to two or more G proteins. Moreover, agonist affinity chromatography of detergentsolubilized bovine brain membranes results in copurification of A1 receptors and at least two different species (G0 and G) of tightly complexed pertussis toxin-sensitive G proteins (21). Receptor-G protein complexes were eluted from agonist columns by GTP or N-ethylmaleimide, agents that uncouple receptors from G proteins and simultaneously lower the affinity of receptors for agonists. When partially purified bovine brain A, receptors were reconstituted with human platelet membranes (which lack A1 receptors), the affinity
functional
reconstitution
of
G
proteins
and
receptors
was
almost completely abolished in membranes treated with pertussis toxin (22). Because platelets contain some G proteins that are not substrates for pertussis toxin, this finding is consistent with the notion that A, receptors couple selectively to the
pertussis toxin-sensitive The A, receptor, free
homogeneity tography were not
from
rat
subset of G proteins. of G proteins, has been
brain
by two
over an antagonist copurified by this
affinity antagonist
cycles
purified
of affinity
to
chroma-
column (23). G proteins affinity chromatogra-
phy procedure, confirming that unlike agonists, antagonists do not stabilize A1 adenosine receptor-G protein complexes. Purified receptors cannot be detected with radioligands unless they are first reconstituted with phospholipids
or
3. Hypothetical A1 receptor subtypes”
Type
Location
Aia
Brain Autonomic
Aib
Potency
nerves
Neuromuscular
A3 “Determinations structures
adenosine
amino
A, receptors are coupled to GTP binding proteins (G proteins). This was initially inferred from characteristic decreases in the binding affinity of agonists upon the addition of GTP or GTP analogs. The existence of various effectors of A1 receptors such as those listed in Table 4 raises the
3.4
originally
to
selectivity
and
740
family,
bind
these
DPCPX
0.07
drugs
on
BWA-844U
31 9.9
these
substitution
and
A, ADENOSINE
of pA2
references.
8PT,
RECEPTORS
junction derived from Schild 8-phenyltheophylline.
were
pA2 for DPCPX
order
DPCPX>XAC> >XCC DPCPX>XAC XCC XAC DPCPX> > XCC analyses
of the effects of antagonists
CV-l674
>9 (rat) 8-9
