Adenosine receptor subtypes

TiPS - October 1993 [Vol. 241

360 tlY89) m-673

Cm.

1. &cchen*. Cell Bid. 67,

11 tndiven, C.. Towazzi, A., P~tias~, G. and Palmieri, F. (1991) Riochinr. Bioplrys. Acta 1%& 231-238 12 Engle, A. G. and Angetini, c. 11973) Sciema 179. 899-902 13 Engle, A. G. (1986) in

14 15

16

17 18

I9 20 21 22

23

Myology@ngk

A. G. and Banker, B. Q.. eds), pp. 1663-1696, McGraw-Hill Coates, P. M+ and Tanaka, K, (1992) i. Lipid Rcs. 33, laQ9-1110 Saggerson, E. D., Ghadimineiad. I. and Awan, M. (1992) A$o. Errzymr Reg. 32, 285-306 Pa&on, D. f., fraxlen, I., Schmidt, M., Noonan, f. and Shug. A. L. I1986) Cardimasr. Res. 20, 536-541 Fen&, R., ed. (1991) Cerdiovasc. Drugs ‘Iltrr. 5 (SuppI. 1). 5-11 Van der Vusse, G. J., Glatz, J. F. C., Slam, H. C. G. and Reneman, R. S. (19%) PIIys~of. Rezl. 72, 881-940 Hut&r, J. F. and Soboll, S. (1992) Itzt. 1. Biocbenr. 24, 399-403 Wu, J. and Corr, P. 8. (1992) Am. [. Physic& 263, H410-H417 Spedding, M. and Mir, A. flQS7) Br. i. Pttarmacof. 92, 457-168 Broderick. T. L., Quinney, W. A. and Lopaschuk, G. D. (1992) J. Bin!. Clrenl. 267,3758-3763 Leip%, f. A. et al. (1991) f. &@. Pltysiol. 71, 1518-1522

24 Barbieri, M. et al. (1991) l?r. [. Pfinrmncoi. 102,7>78 R. 25 Shug, A., Paulson, D., Subramanian, and Regitz, V. (l%l) Cardiouasc. Drclgs Tfrer

5 fSupp1.

I), 77-84

R., Pfehn, 5.. Noonan, j., 26 Subramanian. Schmidt, M. and Shug, A. L. (1987) 2. Knrdiol. 76 (Suppl. 5), 41-45 27 Calucci, W. J. and Gandour, R. D. (1988) Bioorg. Clretn. 16, 307-334 N. et nf. fl993) Cnrdiovasr. 28 corsico, Drugs T&r. 7, 241-251 J. H. C., 29 Halliwell, B. and Gutteridge, eds (1989) Free Radicals in Biology and Medicine (2nd edn), Clarendon Press Bolli, R. &al. (1989) Circ. Res. 6S,607-622 Packers L. et al. (H%) Arch. Biochem. Bioahvs. 288.533-537 132 Ga;&o, H.’ et 01. (1987) in Advnnces in Myockemistn, I (Benri, G., ed.), pp. __ 273-273, John Libbey 33 Bert&i, A., Conte, A., Ronca, G., Segnini, D. and Yu, G. (1991) hf. /. Tiss. React. 13,37-43 34 Schinetti. M. L.. Rossini. D. and Bert&. A. (1989)’ in O&t?t Radlicnls in Biology and Medicke (Simic, M. 6. cf al., eds), cp. 243-247, Ptenlrm Publishing 35 Resnick, A. Z. et al. (1992) Arch. Biochem. Biophys. 296,394-401 36 Arduini, A. et al. (1990) Free Rad. Res. Commu~z. X0.325-332 37 Arduini, A. (1992) At??. Nmrg f. 123, 1726-1727

3”:

Michael G. Collis and Susanna M. 0. Hourani The numerous and widespread effecfs of adenosine provide both WI opportunity for the development of novel therapeutic agents acting via adenosine receptors and the challenge of achieving selectivity of action. The feasibility of achieving selectivity is enhanced if receptor subtypes can be ~den~~ied.~ioc~len~ic~~,f~~nc~~on~~ end receptor-cloning s&dies are beginning fo prouide convergent dnta supporting the existence of Al, AZA,A2s and A3 receptors. However. studies of the functional significance of these receptors in intnct tissues both in vitro and in vivo have iagged behind the biochemical studies. in this article, Michael Cot&s and Susarma Hourani review #he current sfnfus of ~de~ios~ne receptor ci~5sifieu~~onand propose that lig~n~ with greater selectivity need to be evnluated in a wide range of functional preparations if the therapeutic potential of this area is fo be renlized. Adenosine has effects on many mammalian systems (Table I) and is released from cells that are metabolically active or stressed. It moduiates the activity of these cells and of adjacent vascular smooth muscle, resulting in a M. C. CoNis is Mnnager, Cardiooasculnr Research, Ramsgat~ Road, Sandutich, UK CT13 9NJ and S. M. 0.

ffiaiqqy, @i&r Cent&

Hortrarri is it Lecturer at kyica! Sciews, Uuiversily ford,UK CU? 5XH.

the S&al of Bioof Surrey. Guild-

reduction in metabolic demand and/or an increase in nutrient supply. The majority of these effects are mediated via an interaction of the purine with cellsurface receptors, although high concentrations of adenosine can dlS0 inhibit adenylyl cyclase directly via the intracellular ‘P-site’ (a site that requires an intact purine base For activatian but can tolerate compounds with a modified ribosa moiety). Because of the

38 Battelli, D., Bellei, H., A~igoni-Martelli, E.. Muscatello. U. and Bubvleva. V. (l&Z) Biocllint.&opkys. Acfa lil7,3i36 39 Arduini, A. et ai. (1993) Biochim. &i&us. Acta 1146,229-235 40 lM&&en, D. G.. and Jorgensen, K. (1992) BioEssnys 14, 129-136 41 Fink., K. L. and Gross, R. W. (1984) Circ. Res. 55. 585-594 42 Wu, J:, McHowat, J., Saffitz, J, E., Yamada, K. A. and Corr, P. B. (1993) C&c. Rcs. 72.879-889 43 Corr, P. B., &eer, M. H., Yamada, K. A., SafFitz, J. E. and Sobel, B. E. (1989) J. C&x. Inoest. 83, 927-936 44 He&hers, G. i’., kimada, K. A., Kanter, E. M. and Con; P. B. {1987) C&c. Res. 61, 735-746 45 Patmore, L., Duncan, G. P. and Spedding, M. (1989) Br. J. Dharmacol. 97,4&l-450 46 Ramray, R. R. and Arduini, A. (1993) A&. ~i~c~~~~ Biophys. 3@2,307-314 47 Erevetti, G. ei ni. (1992) Erir. Heart I. 13, 251-255 48 Yane. X. F. et al. (1992) I. Cardiovasc. Dh&acoI. 20, 88-98 ’ ’ 49 Uotteriini, R., Samaja, M., Tarantoia, M., Micheletti, Ii. and Bianchi, G. 11992) Mol. Cell. Biocftem. 116, 139-145 BAYK8644: [methyl-l,&dihydro-2,6dimethyl-3-nitro-4-(2” trjBuoromethy~~henyl)-p~dine-5. carboxylatef

widespread nature of these effects, there has been considerable interest in the sub-classification of adenasine receptors, since heterogeneity could provide an opportunity for the development of sdective agonists and antagonists with therapeutic potential. it has not always been easy to reconcile data from biochemical studies with those from functional studies in vitro or in viva, but recent advances including receptor cloning and isolation have resulted in a clearer picture of the heterogeneity of adenosine receptors. Adenosine A1 and A2 receptor subtypes: biochemistry and molecular biology The first proposal that ceil membrane adenosine receptors could be subdivided was made by Van Calker et al.’ based on the abservation that the purine could either inhibit (via the AI subtype) or stimulate (via the A2 subtype) adenylyl cyclase activity in cultured nerve cells. Londos ef a1.2 made similar observations using adipocytes and hepatocytes. They also noted that the order of potency of adenosine anzlogues was different in adipocytes, where adenylyl cyclase was inhibited, from that in hepatocytes, in which adenylyl cyclase was stimulated. Londos

TiPS - October 1993iVoZ. 241 et aL2 named the Inhibitory adenosine receptor Ri and the stimulatory receptor R,, which are equivalent to the A1 and Az receptors described by Van Calker’s group. The ArlAz nomenclature has subsequently become accepted. The classification of receptors based solely on their second messenger systems or on agorist potency is known to be fraught with potential pitfalls3. It is now known that Ai receptors are G protein-linked and can act through effecters other than adenylyl including cyclase, potassium channels, calcium channels, phospholipases AZ or C, and guanylyl cyclase4. The advent of radioactive ligands for adenosine binding sites allowed a more definitive receptor classification to be developed based on agonist affinity. Binding affinity at At sites has commonly been assessed using [31-I]R-N6-phenylisopropyladenosine (R-PIA) or [3H]N%yclohexyladenosine (CHA) (Fig. 1) as the ligand and rat brain membrane preparations as a source of binding sites. Ligand affinity at AZ sites has been assessed by the displacement of [3H]5’-N-ethylcarboxamidoadenosine (NECA) or, more recently, 13H]CGS21680 (Fig. I) from rat striatal membranes or PC-12 cells. In the case of [3H]NECA, it is necessary to treat striatal membranes with the A, receptor-selective ligand, Nb-cyclopentyladenosine (CPA), to prevent the binding of NECA to Ai sites. An example of the different orders of agonist affinity typically observed in these two membrane preparations is given in Table II. In general, substitution in the N6 position of the adenine moiety (Fig. 1) enhances affinity for the Ai binding site (CPA, PIA and CHA) although this is not always the case since the P-substituted compound CI936, exhibits A2 receptor selectivity’. The chirality of the Nt substituent is of greater significance at Ai than at A2 sites in that there is a greater difference in affinity between the diastereoisomers R- and s-PIA at the former. Bulky substituents in the 2 position of the adenine moiety can selectively enhance A2 receptor affinity (CV1808, CGS21680). Few substitution positions on the ribose moiety are tolerated by the adenosine receptor, an exception being the 5’-uronamides such as NECA;

361 TABLE I. Adenosine receptor subtypes mediating biological effects

Biological effects of adenosine CNSeffects Decreased transmitter release Sedation Decreased locomotor activity Anticonvulsant Chemareceptor stimulation ~yperalgesta

Receptor subtype Al

A..1 ~

AZ.4 A, A2 ?

Cardiovascular effects Vasodilation Vasoconstriction Bradycardia Platelet inhibition Negative cardiac inotropy and dromotropy Angiogenesis

&A. A,B,A3 A, A, A*, AI ?

Renal effects Decreased GFR Mesangial cell contraction Antidiuresis Inhibition of renin release

A,

A, Al

A,

Respiratory effects Broncbodilator B~n~o~nstrictor Mucus secretion Respiratory depression lmcnunological effects lmmunosuppression Neutrophit chemotaxis Neutrophil superoxide generation (inhibition) Mast cell degranulation

A2 A,

f??A A2

Gastro-intestinal effects Inhibition of acid secretion

Al

Metakolfceffects lnhibi~n of fipolysts Stimulation of glucose uptake Increase of insulin sensitivity Stimulat~n of gluconeogenesis

however, this agonist is not selective between the two subtypes. Detailed studies of the structureactivity relationship of Ar and Az receptors have been used to build a model of their binding sites6. The use of agonist ligands in receptor classification can be influenced by changes in the agonist affinity state of the binding site due to factors such as ionic strength and the presence or absence of GTP. There are further theoretical and practical complications associated with the use of agonists in the classification of functional receptors that can be overcome by the use of selective antagonists3. Considerable research effort has therefore been expended in the search for antagonists that show selectivity between the subtypes of adenosine receptors, although this has only been partly successful. The majority of the antagonists developed have been xanthines and a number have been claimed to

A* A’ A:

exhibit selectivity for the Al receptor (Fig. 2). 1,3-dipropyl-8 cyclopentylxanthine (DPCPX), however, is the only currently available antagonist that shows a consistent and marked selectivity for the Ai receptor subtype in preparations from a range of species. Selectivity for the A2 receptor has been claimed for the CGS15943 triazoloquinoxaline, (Ref. 71, and the ~azoIoquinazoline CP66713 (Ref. 8) (Fig. 2). The A2 selectivity of these two compounds however, is not marked (CGS13943: Ai ICsa = 20 nM, Az ICsa = 3nM; CP66713: Ai ICso = 270 nM, Az ICso = 21 nrvr). The xanthine, PD115199, displays poor AZ selectivity in binding studies using rat CNS tissue, however, a .higher degree of As selectivity has been reported in dog and human tissue. The classification of adenosine receptors into AI and AZ subtypes is therefore supported by the marked Ar selectivity of DPCPX, but an antagonist with a

~3’s -October

HO

R,

R/S-PIA

CPA Cl936

OH

R

- -3

R*

CHZOH

H

,.O

CH&%-i

H

-0

CH20H

H

CH20H

H

CH(CH3)CH2Ph

CH2CH(Ph)2

NECA

H

CONH&H5

H

ZCADO

H

CH20H

Cl

CV1608

H

CH*OH

PhNH

CGS21680

H

CONHC2H5

H(, cn”” 2

Fig.

1.

Sfructures of common/y used adenosine recapfor agonisfs.

similar degree of selectivity for the AZ receptor has yet to be identified. The existence of two distinct adenosine receptor subtypes has also been investigated using various molecular probes to detect, isolate and characterize the receptors by physicochemical techniques. These studies have shown that both receptors are glycoproteins but that they have different molecular masses (Al, 34-38 kDa; A?, 45 kDa)%“. Recently, two proteins cloned from dog thyroid with the seven transmembrane domains characteristic of G protein-coupled receptors have been

identified as the A1 and A2 receptors”*‘3. This has led to the rapid cloning of the A, receptor from bovine1’*15, rat16*17 and human’s brain, and of a receptor from rat brain with a sequence closely related to the dog A2 receptor (although no functional studies were performed”). The subdivision of adenosine receptors into At and AZ subtypes is therefore now firmly established with biochemical and genetic evidence, A1 and A2 receptors in isolated tissues In order to assign functional responses to the adenosine re-

TABLE II. Comparison of affinities of adenosine analoguas in rat brain membranes Compound CPA CHA R-PIA ZCADO s-PIA NECA cv16Qa CGS21660

Al K OW D.6 1.3 E 49:3 6.3 561 2600

Data taken from Refs 29,31.2CADO,2~hlamadenosi~e~

AZ K +W 462 514 124 62 1820 10 119 15

1993 IVoJ. 141

ceptor subtypes and to map their distribution in different tissues, a number of isolated tissue and cell studies have been performed. From the order of agonist potency observed, the adenosine receptors that mediate cardiac depression, inhibit renin secretion, cause vasoconstriction and bronchoconstriction, inhibit lipolysis and inhibit neurotransmitter release have been classified as A1 receptors (Table I). There has been some controversy over the classification of cardiac and pre-junctional adenosine rece tors, since the potency of the substituted compounds and J NECA are similar. This has led to the proposal of a third subtype of receptor (A3) in these tissues. However, this suggestion has not found widespread support (see below). A2 receptors occur on neutrophils, on airway and vascular smooth muscle (where they mediate relaxation), on liver cells (where they mediate stimulation of gluconeogenesis), and on platelets (where they mediate inhibition of aggregation) (Table I). The above classification must be regarded as preliminary as it is based on agonist potency order. Data obtained with antagonists are more acceptable in the classification of receptors since the complications of receptor number and agonist efficacy can be avoided3. The A, receptor-selective antagonist DPCPX, has been evaluated in guinea-pig smooth muscle and cardiac tissues, and shows high affinity in the latter but not in the former”. In addition, A1 binding sites in CNS tissue and functional receptors in cardiac muscle appear to be similar as there is a strong correlation between the affinities of antagonists in these preparations provided that they are taken from the same specieszl. Thus, there is good evidence that the cardiac adenosine receptor is of the Al subtype. Smooth muscle preparations that relax in the presence of adenosine appear to contain A2 receptors, but structure-activity studies using agonists and antagonists in smooth muscle preparations correlate less well with binding studies at A2 sites. The interpretation of studies using smooth muscle preparations can be complicated by the presence of additional purine-sensitive sites that can also mediate relaxation,

TiPS-

October

2993 IVol. 141

but which are not blocked by xanthines21,22. In addition, both AI and A2 receptors can occur in the same smooth muscle preparation, the former generally mediating contraction and the latter mediating relaxation23-25, although in some tissues they both mediate the same response%zr. In the rat vas deferens and colon, the Al and A2 receptors are clearly at different anatomical locations, although this may not be the case in all other tissues in which the two subtypes coexist. A further complication is the recent discovery of the existence of subtypes of the As receptor, which will require a re-examination of the receptor subtype involved in many tissues. Two types of AZreceptor The suggestion that there could be two types of A2 receptors was made by Daly et al.*s based on the presence of a low-affinity adenosine-sensitive site (AZ*) in intact cells from all brain regions, and a high-affinity site (A& localized in striatal membranes, both of which stimulate adenyiyl cyclase. The low-affinity site also occurs in a human fibroblast cell line. Comparison of ligand potency or affinity order in binding studies using the &i&urn, and in studies of adenylyl cycfase activity using fibrobiasts, has indicated that the AZA site has a relatively higher affinity for agonists and a relatively lower affiniy for antagonists than the Ars site 9, The major differences between the structural requirements of the two sites are that adenosine analogues with bulky 2-position substitutions (such as CWSOS), and the xanthine antagonist PD115199, have very low affinity at the A2e site relative to that at AsA (Refs 29,30). The agonist CGS21680, stimulates adenyfyl cyclase in striatal tissue but not in other brain regions, and shows a similar pattern of binding. Since ADAreceptors are concentrated in the striatum and Azs receptors occur in most brain regions, these results indicate that CGS21680 is also AZA receptor-selective”r*s2, and it is a useful agonist with which to identify this subtype. The cloned dog A2 receptor discussed above’z binds fsH]CGS21690 with high affinity and is therefore presumably of the AZA type. Adenosine receptois with the expected charqcteristies of the A2s

363

Xanthines

Name

R

R*

&Pl-

CH3

Ph

PACPX

C3H7

MC

DPGPX

PDt 15199

Tfi~ol~uiRoxalines

C3H7

-0

W-j?

W7

,-,

OCHpCONH(Cti,),NH,

-0

-cl

,-,

SO2N(GH,t(CH,),NfCX,)*

and Qwj~li~e§

CGSI 5943

CP667 13

Fig.2. Structures of commonly used edenosine receptor antagonisls.

receptor (i.e. NECA was active in stimulating adenylyl cyclase but CGS21680 and A, selective agonists were not) have recently been cloned from rat brain33. This adds weight to the proposed subdivision of A2 receptors. The functional significance of the AZAand A2s subdivision will only become clear when the effects of agonist and antagonists that distinguish between these sites are investigated in a wide range of tissues and cell types. A limited number of studies with the available AzA receptor-selective compounds have been reported in isolated blood vessels. CGS21680

has been shown to be equieffective with NECA as a relaxant of the rabbit aorta, mesenteric and coeliac artery34. In the canine coronary artery, the dog saphenous vein and guinea-pig aorta, both CGS21650 and CV1808 have very weak relaxant effects, whereas NECA is a potent agonist34*35, suggesting that the AZ receptors studied are of the Ars subtype, Further evidence in favour of this hypothesis is provided by the observation that the AZJ, receptorselective antagonist PD115199 has a lower affinity in dog saphenous vein and guinea-pig aorta than at A?,., binding sites in rat striatai

TiPS - October 2993 lIJol. 241 TABLE 111. A basis for classification of adenosine receptors in functional studies, Using agonists and antagonists A, receptor Agonist potency CPA>R-PIA=CHA=>NECA>2CADO>s-PIA>CVl808>=CGS21680 order Antagonist affinity order

DPCPXrPDl15199MPT

AZ’, receptor Agonist potency order Antagonist affinity order

PDI 15199>DPCPX=8PT

AZBreceptor Agonist potency order Antagonist affinity order A3 receptor Agonist potency order Antagonist affinity order

DPCPX=BPT>=PDl15199

APNEA%-PIA=NECA>CGS21680

None identified, all xanthines tested to date have low affinity

‘Specuiatiieaffinity order: PD115199 has low affinity at A2e compared with A= receptors. Comparative binding data in a CNS preparation of A2e receptors for these three antagonists are iot available. 8pT,I-phenyltheophylline In fact, the affinity of I’D115199 in these two vascular preparations is no different from that of the A, selective xanthine, DPCPX. In nonvascular tissues, CGS21680 has been shown to be almost inactive as an agonist at the AZ receptors in the rat duodenum and bladder, indicating that they are of the AZBsubtype26. Interestingly, the cloned A2s receptor has been shown to be highly expressed in the rat intestine and bladdes3, which correlates well with the functional studies in this species.

membranes35.

A3 receptors The A, adenosine receptor subtype has been classified partly on the basis of the higher affinity or potency of M-substituted adenosine analogues than of the 5’substituted compound NECA, and by an inhibitory effect on adenylyl cyclase. Ribeiro and Sebastilo36 have observed that in atrial tissue, and at pre-junctional sites on neuronal tissue, the N6and the 5’-substituted analogues of adenosine are often equipotent. There is also little evidence for an effect of adenosine receptors in these tissues on adenylyl cyclase. This prompted the proposition that receptors with these characteristics represent a third subclass: the A3 receptor. This proposal has

not been widely accepted and is still a matter of active scientific debate; a recent review concluded that such a receptor did not exist37. The variable potency order of the N6- and 5’-substituted analogues in cardiac and neuronal tissue may be a result of agonist- or tissuerelated factors such as efficacy, receptor number or of inadequate equilibration with the biophase. In addition, where both A1 and A2 receptors exist in a tissue, and mediate the same functional response, I@- and 5’-substituted analogues may be equipoten@27 and this could be mistaken for the presence of an A3 receptor. A G protein-coupled receptor that binds both 13H]NECA and the Al-selective agonist [‘251](N6(4-aminophenyl)-ethyladenosine (APNEA) but not the Al-selective antagonists [3H]DPCPX, or [3H]xanthine amine congener (XAC), or the AzA-selective [3H]CGS21680, has recentl;gEz cloned and expressed%. This receptor, which can inhibit adenylyl cyclase, has been termed the A3 receptor as it is clearly different from the A1 and A2 receptors. However, this nomenclature is not meant to imply identity with the putative A3 receptor of Ribeiro and SebastiZo, at which the alkylxanthines are antagonists. This novel A3 receptor is expressed in

high levels in the testes, and coincides temporarily and spatially with developing sperm38,39.Recent studies in the pithed rat have demonstrated that APNEA lowers blood pressure (which has been supported by angiotensin II) and that this response is resistant to blockade by the xanthine adenosine receptor antagonist 8-(ptheophylline4’. sulphophenyl) These exciting observations provide the first evidence for a functional role of the A3 receptor4’, although their precise location remains to be determined. Effects of subtype-selective ligands in vivo The demonstration of receptor subtype selectivity in viva is particularly difficult because of the of achieving equiproblems librium, the influence of metabolism, excretion, protein binding and the tissue distribution of novel agonists and antagonists. Studies in the anaesthetized rat have shown that CGS21680 causes a lowering of blood pressure that is accompanied by a reflex tachycardia42. This response differs from that seen with NECA, which lowers both blood pressure and heart rate. It has also been demonstrated that DPCPX selectively antagonizes bradycardic responses to adenosine in the anaesthetized rat43. Both of these studies support the idea that A1 receptors predominate in rat cardiac tissue. The receptor mediating decreases in blood pressure appears to be A2A, however the recent work on A3 receptors in the rat indicates that these may also be involved in the depressor response to some adenosine analogues40. The use of variables, such as heart rate and blood pressure in reflexic animals, is not ideal for the evaluation of ligand selectivity in viva, and preparations have been designed to allow for the evaluation of adenosine receptor ligands without the complication of the autonomic reflexes44*45. In the anaesthetized areflexic dog, decreases in heart rate and hindlimb perfusion pressure (analogous to resistance) can be measured after the administration of adenosine or its analogues. The order of agonist potency and the high potency of I’D115199 (30 times that of DPCPX) on hind-limb vascular resistance indicates that

725 - Ocf,rber 2993 IVol. 141 an AzA receptor is mediating the irasodilator response. The bradycardie response, however, exhibits unusual characteristics with NECA being more potent than CPA, but with the ADA selective agonist CV1808, having little effect. The potency of the A1 selective antagonist DPCPX, ta block the bradycardic response to adenosine is low in this preparation and equivalent to that of PDll5199. These results are in agreement with those of Belloni et af.46 who concluded that the cardiac receptor in the dog is not a typical Al subtype, and this finding is further supported by studies on isolated dog left atrium, which suggest that the receptor may be of the Aas subtype*‘. Additional in vim preparations, in which the adenosine receptor subtype selectivity of novel agonists and antagonists can be confirmed, need to be developed. A recent publication suggests that adenosine receptors in the anaesthetized pig heart are of the AI type and this species may therefore provide a more typical animal preparation than the dog4s. Therapeutic impli~tions Currently, the major clinical use of adenosine is in the acute treatment and diagnosis of supraventricular arrhythmia. Given the wide variety of effects elicited by the purine, many more therapeutic applications can be envisaged. Agonists with the appropriate selectivity for Alt AsAI Aas or As receptors could be beneficial in a variety of metabolic, CNS and cardiovascular disorders. The therapeutic use of selective antagonists is even more attractive, since their range of effects will depend on the level of endogenous purinergic tone rather than the absolute dist~bution of adenosine receptors. Ai selective antagonists have been proposed to be useful for renal disease43***, for ischaemic bradyarrhythmias~~~ for sleep apnoea and for cognitive disorden?‘. A2 selective antagonists could be useful in the treatment of ischaemic conditions by a reversal of the ‘vascular steal phenomenon5* and in erythrocytosis following renal transpiantation52. The major therapeutic use of the non-selective adenosine receptor antagonist, theophylline, is in the treatment of asthma, al-

3b5

though it is still unclear what role adenosine plays in the asthmatic condition. IJ

q

D

Despite the potential for therapeutic application and the large number of analogues synthesized, no new drugs interacting directly with adenosine receptors have yet emerged=. This is due, in part, to the difficulty of separating therapeutically desirable effects of purine receptor ligands from their side-effects. With the evidence from biochemical and molecular studies now demonstrating that adenosine receptors are heterogeneous and are divisible into Al, AZ&,A2s and As subtypes, there is the potential for greater selectivity of action for novel.ligands. However, the systematic assignment of functional roles to these receptors by studies of the effects of seleetive ligands in vitro and in viva is now needed to elucidate their physiological and pathophysiological roles. A proposed framework to facilitate the classification of functional adenosine receptors is given in Table III. The development of new ligands with greater selectivity for these subtypes would aid this classification and could realize the potential for novel drugs acting via adenosine receptors. References 1 Van Calker, D., Muller, M. and Hamprecht, B. (1979) J. Neurocirem. 33,

999-im 2 Londos, C., Cooper, D. M. and Wolff, J. (1900) Proc. Nat1 Acad. Sci. WA 77, 2551-2554 3 Collis, M. G. (1985) in Purines: Pharmacotogy and physjolog~ca~ Roles (Stone, T. W., ed.), pp. E-84, Macmillan 4 Olsson. R. A. and Pearson. 1. D. (1990) ’ ’ Dhysiol: Rev. 70, 761-845 .’ 5 Bridges, A. J. et al. (1987) J. Med. Chem. 30,1?09-1711 6 Jacobson, K. A., Van Galen, P. J. M. and Williams, M. (1992) f, Med. Chem. 35, 407-422 7 Williams, M. ef al. (1987) J. Pharmacot. Exp. Ther. 241,415-420 8 Sarges, R. cf al. (19901 J. iwed, Chem. 33, 2240-2254 9 Stiles, G. L., Daly, J. W. and Olsson, R. A. (1985) f. Biof. Chem. 260. 10806-10811 10 Barrington, W. W., Jacobson, K. A., Williams, M., Hutchinson, A. J. and Stiles, C. L. (1989) Proc. Nut/ Acad. Sci. USA 86.4572-6576 11 Nakata. H. (1992) Eur. 1. Biochem. 206. 171-177 ’ 12 Maenhaut, C, ef al, (1990) Biochem. Biopkys. Res. Commun. 173,116~1178 13 Libert, F. ef al. (1991) EMEO J, 10,

1677-l&32 14 Tucker, A. L., Linden, J., Robeva, A. s., R’Angeio, D. D. and Lynch, K. R (1992) FEBS Left. 297, 107-111 15 Blah, M. E., Ren, H., Ostrowski, J.. Jacobson. K. A. and Stiles, G. L. (1992) J. &of. Ckem. 267,10764-10770 16 Mahan, L. C. et al. j1991) Mol. P~a~acol. 40,1-7 17 Reppert, S. M., Weaver, D. R., Stehie, J. H. and Rivkees, S. A. (1991) Mol. Endocrinol. 5. 1037-1848 18 Libert, F. ef hi. (1992) B~5chern. Biophys. Res. Common. 187.919-926 19 Chem, Y., King, K., Lai, H-L. and Lai, H. T. (1992) Biochem. Biephys. I&s. Cornrn~~. 185,304-309 20 Coilis, M, G., Stoggall, S. M. and Martin, F. M. (1989) BT. J. P~a~arol. 97, 1274-1278 21 CoIIis, M. G. (1991) Nucieosides and Nucfeotides 10,1057-1066 22 Martin, I’. (1992) Eur. J. Pharmacol. 216, 235-242 23 Stoggail, S. M. and Shaw, J. S. (1990) Eur. I. Pharmacol. 190, 329-335 24 Bailey, S. J., Hickman, D. and Hourani, S. M. 0. (1992) Br. J. Pharmncol. 105, 400-404 25 Bailey, S. J. and Hourani, S. M. 0. (1992) Br. f. Pharmacol. 105. 885-892 26 Nicholls, J., Hourani, S. M. 0. and Kitchen, I. (1992) Br. 1. P~ar~aacu~. 105, 639-642 27 Hourani, S. M. O., NichoIis, J., Lee, B. S. S., Ha&bide, E. J. and Kitchen, I. (1993) Br. J. ~~armacof. 108,754-758 28 Daly, J. W., Butts-Lamb, P. and Padgett, W. (1983) Cetf. Mol. Neurobiol. 3,69-30 29 Brims, R. F., Lu, G. H. and Pugsley, T. A. (1986) Mol. Pha~acut. 29,331~346 30 Brims, R. F. et al. (1987) NaununSchmiedebergs Arch. fihar&cal. $35, 64-69 31 Jarvis, M. F. ef al. (1989) J. Pkarmacof. Exp. 7%~:: 251,888-893 32 Lupica, C. R.. Cass, W. A., Zahniser, N. R. and Dunwiddie, T. V. (1990) J. Pliarmacol. Exp. Ther. 252, 1X34-1141 33 Stehle, J. H. et al. 11992) Mot. Endocrinol. 6384-393 34 Baiwierczak, J. t. et ai. (1991) Eur. f. Pharma~o~. 196,317-123 35 Hargreaves, M. B., Stoggail, S. M. and Collis, M. G. (1991) fir. J. Pkarmacol. 102, 198P A, M. 36 Ribeiro, J. A. and Sebastiao, (1986) Prog. ~e~rob~of. 26, 179-209 37 Kennedy, I., Gurden, M. and Strong, P. (1992) Gem Pharmacol. 23,303-307 38 Zhou, Q-Y. et al. (1992) Droc. Nut1 Acad. Sri. USA 89,7432-7436 39 Meyerhof, W., Miller-Br~hlin, R. and Richter, D. (1991) FEBS Left. 284, 155-160 A. M. 40 Fozard, J. R. and Cam&hers, (1993) Br. I. Pharmacoi. IO?, 3-5 41 Car&he&, A. MA. and Fozard, J. R. (1993) Trends Pkarmacol. Sci. 14,290-291 42 Hutchison, A. J. et af. (1989) J. Pka~aco~. Exp. Tker. 251,47-55 43 Kellet, R., Bowmer, C. J., Coliis, M. G. and Yates, M. S. (1989) Br. J. Pharmacol. 98,X%6-1074 44 Nally, J. E., Keddie, J. R., Shaw, G. and Coilis, M. G. (1991) Br. f. Pharmacol. 102, 34OP 4.5 Wesley, R. C., Porzio, D. and Sadeghi, M. (1990) Circulation 82 (Suppi, 3), 150 46 Belloni, F. L., Belardinelli, L., Halperin, C. and Hintze, T. I-I. (1983) Am. 1. Physiof. 256H, 1553-1564

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.S Collis, M. G., Shaw, C. and Keddie, I. (1991) J. r%errrr. Phnnnn:XIL 43, 135-139 49 Wesley, R. C.. Lerman, 8. B., DiMarco, J. P., Beme. R. M. and Bdardinelli, L. (198b) J_ Atu. Coli. Cardioi. 8, 1232-1234 50 Bednarek, F. J. and Rofoff, D. W. (1976) Pediatrics 5%. 335-339 51 Picano, E., Pogiiani, M., Lattanzi, F., Distante, A. and L’Abbate, A. (1989)

A,,I. J, Cardroi. 63, 14-16 52 Bakris, G. L. et nl. (1990) New Eugl. /. Med. 232, 86-90 53 Williams, M. (1993) Drug Der. Res. 28, 438-444 CGSl5943:9-chioro-Z-(2-fu~I)-5,6dihydro[l,2,4J-triazolo[l,S-cJ quinazolineS-imine CGSZ1680: Z-(p-(-carboxyethyl)phenylethylamino)-5’-N-

Carlo Tapparelli, Rainer Metternich, Claus Ehrhardt and Nigel S. Cook Thrombin is a multifuncfional protein: in addition to ifs role in coagulation, thrombin has important biological effects on platelets, endothelial and smooth musde cells, leukocytes, the heart and neurones. A defailed understanding of the structure of thrombin, of related serine proteases and of enzyme-inhibitor complexes has aided in the discovery of potent and selective new inhibitor molecules. Some of these novel thrombi?z inh~bifors are acfive when administered orally and have shown remarkable efficacy as anfifhrombotic agents in animal models, offering a greater therapeutic potential fhan presently available drugs. This potential extends also to non-thrombotic indications where thrombin may be involved, namely inflammation, cancer and neurodegenerative diseases. The recent identification of specific thrombin receptors on different cells provides an alternative strategy for inhibiting fhrombin*s celiufar actions, ~it?louf necessa~~~ com~romjsing its role in haemosfasis. In this review, Carlo Tapparelli and colleagues present a comprehensive update of these recent developments in the field of fhrombin biology and pharmacology suggesting a new era oftherapeutic drugs is on the

Thrombin is a trypsin-like serine protease fulfilling a central role in both haemostasis and thrombosis. In the coagulation cascade, thrombin is the final key enzyme, proteolytically cleaving fibrinnsen to release fibrinopeptides A and B and generate fibrin, which can then polymerize to form a haemostatic plug. Thrombin generation is the final result after activation of both the intrinsic (‘contact activation’) and extrinsic (activation by exposure of plasma to a nonendothelial surface, or damage to C. Tapparelli is Head of the Thrombin Project, R. Metternich is Head of Chemistry and N. S. Cook is Head of Biology in the Vascular Biology and Haemostasis Group, and C. Ehrhardt is in tke Drug Design Group, Preciinical Research, Sandoz Pharma lid, CH-4002 Busel, Switzerland. @ 1993, Elsevier Science Publishersttd (UK)

vessel walls and tissue factor release coagulation pathways). In addition to fibrinogen cleavage, thrombin exerts a positive feedback on its own production by activating coagulation factors V and VIII. It also activates factor XIII, which cross-links and stabilizes the fibrin polymer. Natural anticoagulant mechanisms limit these processes, notably via antithrombin III (which binds to and inactivates thrombin) and protein C, which itself is activated by thrombin. Thus, under physiological conditions, this baIance between pro- and anti-coagulant mechanisms allows the local generation of thrombin, whilst prevenfing it from becoming a systemic or potentially dangerous process.

0165 - 6147/93/$06.00

ethykarboxamido adenosine CI936: Nh-(2,2-diphenylethyl) adenosine CP66713: 4-amino-8-chloro-l-phenyl(1,2,4)triazolo(4.3-a)-quinoxaline CVlSOB: 2-phenylaminoadenosine PACPX: I,3 dipropyl-8-(2amino-4chlorophenyi)xanthine PD115199: 1,3-dipropyl-S-N-[2(dimethylamino)ethyl]-N-methyl-4(2,3,6,7-tetrahydro-2,6-dioxo)benzenesulphonamidexanthine

Thrombin has many varied biological roles Thrombin acts on many different cells in addition to its involvement in coagulation (Fig. 1). It activates platelets and endothelial cells via a unique proteoIytic cleavage reaction at a cell surface receptor*. The activation of platelets by thrombin causes shape change, aggregation, the release of storage granule contents (e.g. platelet factor-$, adenosine ~-hydrox~~ptdiphosphate, amine) and the synthesis and secretion of thromboxane AZ, platelet activating factor (PAF) and lysosomal enzymes’. The interaction of thrombin with endothelial cells also results in the secretion of various agents (tissue plasminogen activator, piasminogen activator inhibitor-l, thromboplastin/tissue factor, PAF, PGIZ, PDGF, endothelin, nitric oxide), as well as the activation of protein C3 binding to thrombomodulin, A further consequence of the activation of endothelial cells by thrombin is an increased vascular permeability and an increased endothelial adhesiveness for mononuclear cells4s5. When combined with the chemoattractant effect of th~mbin on monocytes and macrophages and an ability to enhance cytokine production by such cells6r7, the net result is an extravasation of leukocytes at sites of thrombin generation. The potent&I therapeutic use of thrombin inhibitors as ~ti-~~ato~ agents is discussed later (Box). The induction of fibroblast and smooth muscle cell proliferation and migration is another important vascular response to thrombin8*g. This has prompted studies suggesting that thrombin contributes to lesion development following vascular damage and wound healing9*10. The receptormediated proliferative effect of thrombin on both smooth muscle cells and leukocytes, despite being