Gen. Pharmac. Vol. 22, No. 1, pp. 25-31, 1991 Printed in Great Britain. All rights reserved
0306-3623/91 $3.00+ 0.00 Copyright © 1991 PergamonPress pie
MINIREVIEW RECEPTORS FOR ADENOSINE AND ADENINE NUCLEOTIDES TREVOR W. STONE Department of Pharmacology, University of Glasgow, Glasgow (312 8QQ, Scotland, U.K. [Tel. (041)330-4481; Fax (041)330-4100] (Received 23 April 1990) Abstract--1. A summary is provided of current classifications of receptors for the adenine nucleosides and
nucleotides. 2. The origin of the division of nucleoside (PI) and nucleotide (P2) receptors is discussed as well as the subdivisions of these into A1 and A2, determined by structure activity considerations, and P2x and P2y. 3. Further suggestions have been made recently for identifying Ala and Alb, A2a and A2b subtypes of the PI receptor family, and for proposing distinctive nucleotide receptors on platelets (P2t) and mast cells (P2z). 4. In addition, an A3 site may exist with properties intermediate between A1 and A2, and a novel P2s receptor on some smooth muscle systems and a P3 site.
INTRODUCTION
concentrations 2 or 3 orders of magnitude less than are required to inhibit cyclic nucleotide phosphodiesterase. Indeed it is now widely recognised that inhibition of this enzyme only occurs at toxic levels of the common methylxanthines. The difficulties presented by other actions of the methylxanthines (such as inhibition of alkaline phosphatase, phosphodiesterase or nucleotidase (Fredholm et al., 1978) have been surmounted by the development of more potent and selective agents, notably xanthines possessing substituted 8-phenyl or 8-cyclopentyl groupings. These will be discussed again below in relation to subtypes of the P1 receptor, although 8-phenyltheophylline and the more water soluble, though less potent analogue, 8-(p-sulphophenyl)theophylline remain widely used in physiological studies of P1 function. Nevertheless this classification of P1/P2 is not without its problems. In particular there is now evidence from several sources that nucleotides including analogues of ATP which are not as susceptible to metabolism such as ~,fl-methylene ATP (APCPP) and fl,y-methylene ATP (APPCP) are able to stimulate in some tissues receptors which can be antagonised by xanthines (Wiklund and Gustaffson, 1986; Westfall et al., 1990). The implication of this is that nucleotides can in some cases directly activate nucleoside receptors or a distinct population of sites. If so this may represent a new species of "P3" receptor unless the P1 site is redefined in terms of xanthine sensitivity rather than agonist potency. Another feature of the P1 receptors is their linkage in some cases to adenylate cyclase (Londos and Wolff, 1977; Londos et al., 1980) (Fig. 2), though it cannot be emphasised too strongly that many cases are now known of adenosine effects for which no change of cyclase activity has been demonstrated. Many of these are situations in which adenosine
Following the first demonstrations by Sattin and Rail (1970) that adenosine had marked effects in the cerebral cortex which were independent of its role in intermediary metabolism, and that those effects could be antagonised by methylxanthines, the study of extracellular receptors for the nucleoside has developed considerably. This has been fuelled by the synthesis of adenosine analogues by academic and industrial laboratories for the study of structure activity relationships and the growing realisation that other purines, notably ATP may also play a critical role in cell function (Burnstock, 1972; Stone, 1989). The purine field appears now to have gone through an era in which attempts have been made to classify receptors into subtypes and to consolidate receptor classes into meaningful groups. It is therefore timely to summarise the present state of purine receptor nomenclature and to define areas of uncertainty and potential development. NUCLEOSIDES AND NUCLEOTIDES One of the most fundamental divisions of purine receptors is between those for nucleosides such as adenosine and those for nucleotides such as ATP (Fig. 1). This classification was introduced by Burnstock in 1978 when the terms Pl for the nucleoside receptor and P2 for the nucleotide receptors were first introduced. In spite of some arguments that the terms Pl and P2 are superfluous the nomenclature has gained wide acceptance and is generally a useful and valuable nomenclature (Table 1; Fig. 2). The P1 receptors are most readily characterised as sites at which xanthines such as theophylline (1,3dimethylxanthine) and caffeine (l,3,7-trimethylxanthine) act as competitive antagonists, generally at 25
TREVORW. STONE
26
0
0
II
II
0
II
q,
H0~ P m 0 ~ P ~ 0 ~ P~ 0 ~ I I I 000[ A d e n o s i n eI ATP
0 0 0 II II II ~' HO~P~CmP~O~P~O---~ I H2 I I \ O" O" O- | A d e n o s l n e l APPCP NH2 0
7 ~~l ~N ~a
H0
s'
0
/CH3
0
I
CH3 OH OH Adenosine
H5C2HNOC~oH OH
Caffeine
HO
OH 5'N-Ethylcarboxamide adenosine
(NECA)
OH
NS-Phenylisopropyladenosine (PIA)
Fig. 1. Structures of purine molecules important in the development of receptor classification. The structure of the nucleoside adenosine (adenine base plus ribose sugar) is illustrated with its atomic numbering system. The addition of a triphosphate side chain on the 5' oxygen converts adenosine into the nucleotide ATP, and for comparison is shown the analogue ~/,7-methyleneATP(APPCP) in which one of the bridging oxygen atoms is replaced by a methylene group. This confers a degree of resistance to metabolic degradation. The adenosine analogue R-PIA [phenylisopropyladenosineor N6-(R)-(l-methyl-2phenyl)ethyl-adenosine]was one of the first derivatives with selectivity for the A1 subtype of adenosine (P1) receptor. NECA has been used in the study of A2 receptors, often on the basis of presumed selectivity, though it actually has the same affinity for AI and A2 sites. Caffeine is 1,3,7-trimethylxanthine,and, as the first adenosine antagonist, is the compound from which many of the currently available potent xanthines were derived.
appears to act directly upon ion channels, usually decreasing calcium conductance or increasing potassium conductance and thus causing hyperpolarisation, inhibition or relaxation. It is interesting to note that in presynaptic terminals there is evidence that the inhibitory effects of adenosine on transmitter release can be prevented by pertussis toxin, but are not mediated by cyclic AMP, implying the mediation of these effects by G-proteins which are not coupled to
cyclase (Fredholm et al., 1986). The receptor still shows the structure activity profile expected for an AI site. Pl SUBDIVISIONS
Adenosine receptors can be further subdivided. In their original work on adipocytes Londos and Wolff (1977) considered that adenosine would activate two
Purine receptors
27
Table I. Properties of PI and P2 receptors for purines (it should be emphasized that there is significant overlap betw¢en PI and P2 receptors with respect to several of the following parameters, as discussed in more detail in the text) PI
P2
Adenosine > AMP > ATP
ATP > AMP > adenosine
Transmitter release Direct on smooth muscle
Reduced Relaxation
Adenylate cylase activity K + Permeability
Increased or decreased in some tissues Increased in some tissues Decreased No
Little effect Relaxation or contraction No effect
Agonist potencies Function:
Ca 2+ Permeability Activation of cyclo-oxygenase Cardiac activity Localisation Xanthine antagonism ANAPP3 antagonism Activity of derivatives
Negative inotropy (atria) Presynaptic and postsynaptic Yes No L-Adenosine inactive 8-Bromoadenosine inactive 2'-Deoxyadenosine inactive
distinct receptor forms, one of which was responsible for the inhibition of adenylate cyclase and was therefore referred to as Ri, whereas the other form would stimulate adenylate cyclase and was referred to as Ra. The abbreviation R itself arose from the fact that limited structure activity studies indicated that the ribose component of the adenosine molecule was the most crucial in modifying adenylate cyclase activity; the enzyme stimulation was much more tolerant to changes in the adenine portion of the molecule.
R-PIA t--.~.m,,f CHA CPA P1 (xar~entagm~m) CGS21680~~ 2PAA J
ZS'-dtdooxy-adenoslno
I I=
~mATP P2 ATP 2-M~TP
Fig. 2. Schematic drawing of a cell membrane to summarise some of the major divisions of adenine derivative receptor. Gi and Gs refer to the regulatory G-proteins which couple the adenosine AI (inhibitory) and A2 (stimulatory) adenosine receptors to adenylate cyclase in some tissues. It is important to note that cyclase involvement is not an invariant feature of adenosine receptor activation: many responses are known where cyclase has no apparent role whatever, but the receptors still show structure-activity relationships characterising them as AI or A2. Cyclase modulation should therefore be regarded as merely one example of many possible cellular responses to A I or A2 activation.
Increased in some tissues Increased Yes Positive or negative inotropy (ventricle) Mainly postsynaptic No Yes L-ATP active 8-BromoATP active 2'-DeoxyATP active
The distinction 'R' was from a different site for which the purine moiety was the most critical; this appears to be an intracellular site again linked to adenylate cyclase but for which no cellular function has been discovered. It was named the 'P' site. It is receiving relatively little attention from pharmacologists since it presents the dual problems of studying functionally compounds that have to be selective structurally for the P site but which are also able to pass into the intracellular environment. At the same time as the original work by Londos and Wolff (1977) was being performed Van Calker et al. (1979) also reported a dual modulation of adenylate cyclase activity by adenosine analogues, but in this case the inhibitory receptor was referred to as AI and the stimulatory site as A2. There has been much discussion over the intervening years about the nomenclature to be generally adopted for these two sites. The A1/A2 classification does not inherently imply any activation or inhibition for adenylate cyclase and many examples are now known where adenosine analogues have biological effects with potency orders suggesting an A1 or A2 site but without any apparent involvement of adenylate cyclase. The A I/A2 nomenclature has therefore become widely adopted and is recommended for general use (Stone, 1985). The overall organisation of adenosine receptors is illustrated schematically in Fig. 2 and the differences between PI and t)2 receptors are summarised in Table 1. The differences between AI and A2 sites are summarised in Table 2. An additional proposal, for an A3 site, has been made by Ribeiro and Sebastiao (1986) on the basis that many of the presynaptic effects of purines exhibited potency orders of analogues which were not consistent with a simple classification as AI or A2. It was suggested that the A3 receptor might be coupled to calcium channels. This remains an interesting hypothesis but lacks the support of selective agonists or antagonists. In addition, the entire field has experienced recent confusion with the inability of Gurden
28
TREVOR W. STONE
Table 2. Properties of A1, A2 and A3 subtypes of the adenosine PI receptor (as in the other tables these distinctions should not be regarded as clearcut, only as a guide to recent attempts to devise a receptor classification; there is much overlap between these properties as noted in the text and it is probably wisest to regard the proposed A3 site as only one variant of the AI receptor population) AI
A2
A3
Potency series R:S-PIA potency ratio Xanthine block Affinity of adenosine Preferred agonist Preferred antagonist Localisation
R-PIA > NECA > 2-ehloro-adenosine >10 Yes Micromolar N6-cyclopentyladenosine XAC; DPXPX [PD116,948] Presynaptic on neurones; cardiac muscle
R-PIA = NECA About 10 Yes Micromolar
Effects of N-ethylmaleimide Effect of cooling
Blocks
NECA > 2-chloro-adenosine > R-PIA
0306-3623/91 $3.00+ 0.00 Copyright © 1991 PergamonPress pie
MINIREVIEW RECEPTORS FOR ADENOSINE AND ADENINE NUCLEOTIDES TREVOR W. STONE Department of Pharmacology, University of Glasgow, Glasgow (312 8QQ, Scotland, U.K. [Tel. (041)330-4481; Fax (041)330-4100] (Received 23 April 1990) Abstract--1. A summary is provided of current classifications of receptors for the adenine nucleosides and
nucleotides. 2. The origin of the division of nucleoside (PI) and nucleotide (P2) receptors is discussed as well as the subdivisions of these into A1 and A2, determined by structure activity considerations, and P2x and P2y. 3. Further suggestions have been made recently for identifying Ala and Alb, A2a and A2b subtypes of the PI receptor family, and for proposing distinctive nucleotide receptors on platelets (P2t) and mast cells (P2z). 4. In addition, an A3 site may exist with properties intermediate between A1 and A2, and a novel P2s receptor on some smooth muscle systems and a P3 site.
INTRODUCTION
concentrations 2 or 3 orders of magnitude less than are required to inhibit cyclic nucleotide phosphodiesterase. Indeed it is now widely recognised that inhibition of this enzyme only occurs at toxic levels of the common methylxanthines. The difficulties presented by other actions of the methylxanthines (such as inhibition of alkaline phosphatase, phosphodiesterase or nucleotidase (Fredholm et al., 1978) have been surmounted by the development of more potent and selective agents, notably xanthines possessing substituted 8-phenyl or 8-cyclopentyl groupings. These will be discussed again below in relation to subtypes of the P1 receptor, although 8-phenyltheophylline and the more water soluble, though less potent analogue, 8-(p-sulphophenyl)theophylline remain widely used in physiological studies of P1 function. Nevertheless this classification of P1/P2 is not without its problems. In particular there is now evidence from several sources that nucleotides including analogues of ATP which are not as susceptible to metabolism such as ~,fl-methylene ATP (APCPP) and fl,y-methylene ATP (APPCP) are able to stimulate in some tissues receptors which can be antagonised by xanthines (Wiklund and Gustaffson, 1986; Westfall et al., 1990). The implication of this is that nucleotides can in some cases directly activate nucleoside receptors or a distinct population of sites. If so this may represent a new species of "P3" receptor unless the P1 site is redefined in terms of xanthine sensitivity rather than agonist potency. Another feature of the P1 receptors is their linkage in some cases to adenylate cyclase (Londos and Wolff, 1977; Londos et al., 1980) (Fig. 2), though it cannot be emphasised too strongly that many cases are now known of adenosine effects for which no change of cyclase activity has been demonstrated. Many of these are situations in which adenosine
Following the first demonstrations by Sattin and Rail (1970) that adenosine had marked effects in the cerebral cortex which were independent of its role in intermediary metabolism, and that those effects could be antagonised by methylxanthines, the study of extracellular receptors for the nucleoside has developed considerably. This has been fuelled by the synthesis of adenosine analogues by academic and industrial laboratories for the study of structure activity relationships and the growing realisation that other purines, notably ATP may also play a critical role in cell function (Burnstock, 1972; Stone, 1989). The purine field appears now to have gone through an era in which attempts have been made to classify receptors into subtypes and to consolidate receptor classes into meaningful groups. It is therefore timely to summarise the present state of purine receptor nomenclature and to define areas of uncertainty and potential development. NUCLEOSIDES AND NUCLEOTIDES One of the most fundamental divisions of purine receptors is between those for nucleosides such as adenosine and those for nucleotides such as ATP (Fig. 1). This classification was introduced by Burnstock in 1978 when the terms Pl for the nucleoside receptor and P2 for the nucleotide receptors were first introduced. In spite of some arguments that the terms Pl and P2 are superfluous the nomenclature has gained wide acceptance and is generally a useful and valuable nomenclature (Table 1; Fig. 2). The P1 receptors are most readily characterised as sites at which xanthines such as theophylline (1,3dimethylxanthine) and caffeine (l,3,7-trimethylxanthine) act as competitive antagonists, generally at 25
TREVORW. STONE
26
0
0
II
II
0
II
q,
H0~ P m 0 ~ P ~ 0 ~ P~ 0 ~ I I I 000[ A d e n o s i n eI ATP
0 0 0 II II II ~' HO~P~CmP~O~P~O---~ I H2 I I \ O" O" O- | A d e n o s l n e l APPCP NH2 0
7 ~~l ~N ~a
H0
s'
0
/CH3
0
I
CH3 OH OH Adenosine
H5C2HNOC~oH OH
Caffeine
HO
OH 5'N-Ethylcarboxamide adenosine
(NECA)
OH
NS-Phenylisopropyladenosine (PIA)
Fig. 1. Structures of purine molecules important in the development of receptor classification. The structure of the nucleoside adenosine (adenine base plus ribose sugar) is illustrated with its atomic numbering system. The addition of a triphosphate side chain on the 5' oxygen converts adenosine into the nucleotide ATP, and for comparison is shown the analogue ~/,7-methyleneATP(APPCP) in which one of the bridging oxygen atoms is replaced by a methylene group. This confers a degree of resistance to metabolic degradation. The adenosine analogue R-PIA [phenylisopropyladenosineor N6-(R)-(l-methyl-2phenyl)ethyl-adenosine]was one of the first derivatives with selectivity for the A1 subtype of adenosine (P1) receptor. NECA has been used in the study of A2 receptors, often on the basis of presumed selectivity, though it actually has the same affinity for AI and A2 sites. Caffeine is 1,3,7-trimethylxanthine,and, as the first adenosine antagonist, is the compound from which many of the currently available potent xanthines were derived.
appears to act directly upon ion channels, usually decreasing calcium conductance or increasing potassium conductance and thus causing hyperpolarisation, inhibition or relaxation. It is interesting to note that in presynaptic terminals there is evidence that the inhibitory effects of adenosine on transmitter release can be prevented by pertussis toxin, but are not mediated by cyclic AMP, implying the mediation of these effects by G-proteins which are not coupled to
cyclase (Fredholm et al., 1986). The receptor still shows the structure activity profile expected for an AI site. Pl SUBDIVISIONS
Adenosine receptors can be further subdivided. In their original work on adipocytes Londos and Wolff (1977) considered that adenosine would activate two
Purine receptors
27
Table I. Properties of PI and P2 receptors for purines (it should be emphasized that there is significant overlap betw¢en PI and P2 receptors with respect to several of the following parameters, as discussed in more detail in the text) PI
P2
Adenosine > AMP > ATP
ATP > AMP > adenosine
Transmitter release Direct on smooth muscle
Reduced Relaxation
Adenylate cylase activity K + Permeability
Increased or decreased in some tissues Increased in some tissues Decreased No
Little effect Relaxation or contraction No effect
Agonist potencies Function:
Ca 2+ Permeability Activation of cyclo-oxygenase Cardiac activity Localisation Xanthine antagonism ANAPP3 antagonism Activity of derivatives
Negative inotropy (atria) Presynaptic and postsynaptic Yes No L-Adenosine inactive 8-Bromoadenosine inactive 2'-Deoxyadenosine inactive
distinct receptor forms, one of which was responsible for the inhibition of adenylate cyclase and was therefore referred to as Ri, whereas the other form would stimulate adenylate cyclase and was referred to as Ra. The abbreviation R itself arose from the fact that limited structure activity studies indicated that the ribose component of the adenosine molecule was the most crucial in modifying adenylate cyclase activity; the enzyme stimulation was much more tolerant to changes in the adenine portion of the molecule.
R-PIA t--.~.m,,f CHA CPA P1 (xar~entagm~m) CGS21680~~ 2PAA J
ZS'-dtdooxy-adenoslno
I I=
~mATP P2 ATP 2-M~TP
Fig. 2. Schematic drawing of a cell membrane to summarise some of the major divisions of adenine derivative receptor. Gi and Gs refer to the regulatory G-proteins which couple the adenosine AI (inhibitory) and A2 (stimulatory) adenosine receptors to adenylate cyclase in some tissues. It is important to note that cyclase involvement is not an invariant feature of adenosine receptor activation: many responses are known where cyclase has no apparent role whatever, but the receptors still show structure-activity relationships characterising them as AI or A2. Cyclase modulation should therefore be regarded as merely one example of many possible cellular responses to A I or A2 activation.
Increased in some tissues Increased Yes Positive or negative inotropy (ventricle) Mainly postsynaptic No Yes L-ATP active 8-BromoATP active 2'-DeoxyATP active
The distinction 'R' was from a different site for which the purine moiety was the most critical; this appears to be an intracellular site again linked to adenylate cyclase but for which no cellular function has been discovered. It was named the 'P' site. It is receiving relatively little attention from pharmacologists since it presents the dual problems of studying functionally compounds that have to be selective structurally for the P site but which are also able to pass into the intracellular environment. At the same time as the original work by Londos and Wolff (1977) was being performed Van Calker et al. (1979) also reported a dual modulation of adenylate cyclase activity by adenosine analogues, but in this case the inhibitory receptor was referred to as AI and the stimulatory site as A2. There has been much discussion over the intervening years about the nomenclature to be generally adopted for these two sites. The A1/A2 classification does not inherently imply any activation or inhibition for adenylate cyclase and many examples are now known where adenosine analogues have biological effects with potency orders suggesting an A1 or A2 site but without any apparent involvement of adenylate cyclase. The A I/A2 nomenclature has therefore become widely adopted and is recommended for general use (Stone, 1985). The overall organisation of adenosine receptors is illustrated schematically in Fig. 2 and the differences between PI and t)2 receptors are summarised in Table 1. The differences between AI and A2 sites are summarised in Table 2. An additional proposal, for an A3 site, has been made by Ribeiro and Sebastiao (1986) on the basis that many of the presynaptic effects of purines exhibited potency orders of analogues which were not consistent with a simple classification as AI or A2. It was suggested that the A3 receptor might be coupled to calcium channels. This remains an interesting hypothesis but lacks the support of selective agonists or antagonists. In addition, the entire field has experienced recent confusion with the inability of Gurden
28
TREVOR W. STONE
Table 2. Properties of A1, A2 and A3 subtypes of the adenosine PI receptor (as in the other tables these distinctions should not be regarded as clearcut, only as a guide to recent attempts to devise a receptor classification; there is much overlap between these properties as noted in the text and it is probably wisest to regard the proposed A3 site as only one variant of the AI receptor population) AI
A2
A3
Potency series R:S-PIA potency ratio Xanthine block Affinity of adenosine Preferred agonist Preferred antagonist Localisation
R-PIA > NECA > 2-ehloro-adenosine >10 Yes Micromolar N6-cyclopentyladenosine XAC; DPXPX [PD116,948] Presynaptic on neurones; cardiac muscle
R-PIA = NECA About 10 Yes Micromolar
Effects of N-ethylmaleimide Effect of cooling
Blocks
NECA > 2-chloro-adenosine > R-PIA
