NeurophmwologyVol. 25,No. 6, pp.

563416,1986 Frintcd Great Britain in

0028-3908/86 + 0.00 $3.00

Pergamon Journals Lrd

COMMENTARY PROPOSALS FOR THE CLASSIFICATION AND NOMENCLATURE OF FUNCTIONAL RECEPTORS FOR 5HYDROXYTRYPTAMINE
P. B. BRADLEY, G. ENGEL,* W. FENIUK,~J. R. FOZARD,~ P. P. A. HUMPHREY,~ D. N. MIDDLEMISS,~*E. J. MYLECHARANE,~ P. RICHARDSON* B. and P. R. SAXENA~
Department of Pharmacology, The University of Birmingham, The Medical School, Birmingham Bl5 2TJ, England, rPreclinica1 Research Department, Sandoz Ltd, CH-4002 Basle, Switzerland, Biology Division, Glaxo Group Research Ltd, Ware, Herts SG12 ODJ, England, 4Merrell Dow Research Institute, Strasbourg Center, 16, rue dAnkara, 67084 Strasbourg Cedex, France, 5Department of Pharmacology, University of Sydney, Sydney, NSW 2006, Australia and 6Department of Pharmacology, Erasmus Universiteit Rotterdam, Postbus 1738, 3000 DR Rotterdam, The Netherlands !&mmuy-As a result of controversy in the literature regarding the classification and nomenclature of functional receptors for 5-hydroxytryptamine (5-HT), a framework for classification is proposed. The formulation of these proposals has only been made possible by the recent advent of new drug tools. It is considered that there are three main types of S-HT receptor, two of which have been well character&d pharmacologically, using selective antagonists, and which it is proposed to name S-HT, and S-HT,. These two groups broadly encompass the D and M receptors, respectively, which Gaddum identified in the guinea-pig ileum (Gaddum and Picarelli, 1957). The 5-HT, receptor, which mediates a variety of actions of 5-HT, has been definitively shown to correlate with the S-HT, binding site in the brain. No binding studies in brain tissue have yet been published with radiolabelled ligands specific for S-HT, receptors. A number of other actions of 5-HT appear to be mediated via receptors distinct from 5-HT, or 5-HT, receptors. Since selective antagonists are not yet available, these receptors cannot be definitively characterised, although in many cases they do have some similarities with 5-HT, binding sites, which are a heterogeneous entity. Criteria are proposed for tentatively classifying these receptors as 5-HT,-like (Table 1). Definitive characterisation of these receptors will await the identification of specific antagonists. This classification of 5-HT receptors into three main groups (Table 1) is based largely, but not exclusively, on data from studies in isolated peripheral tissues where definitive classification is possible. However, it is believed that this working classification will be relevant to functional responses to 5-HT in the central nervous system.

Key words: 5-HT, 5-HT, receptors, 5-HT, receptors, 5-HT, receptors, 5-HT agonists, 5-HT antagonists.

It is abundantly clear that significant advances have been made in our knowledge of the pharmacology of 5hydroxytryptamine (5-HT; serotonin) over the last five years or so (see below). However, there is some confusion about the precise classification of types of 5-HT receptors and hence their nomenclature (see Fozard, 1984a; Humphrey, 1984; Peroutka, 1984a, b). The situation is compounded in the literature by a number of speculative claims and extrapolations from ligand binding data made without regard to any functional correlate. The receptors which are classified here are those which mediate, and hence are essential for, the actions of 5-HT in both the periphery and the brain, and unequivocal characterisation of these can only come from functional studies. These proposals attempt to accommodate the current state of knowledge and to provide a unifying concept on which to build and elaborate. The data considered in the present analysis come ,from both functional and ligand binding studies so as to simplify our under*Present address: Continent01 Pharma, Part Scientifique de
Louvain-la-Neuve, Rue Grandbonpre St-Guibert, Belgium.
NP. 2516-A

11, 1348 Mont-

standing wherever possible by correlation of binding sites with pharmacological receptor types. The definitive characterisation and classification of functional 5-HT receptors can only be effected by reliable functional studies carried out by workers who have taken steps to avoid the pitfalls which await the unwary investigator. These pitfalls have been extensively described (e.g. Furchgott, 1972; Kenakin, 1982; Humphrey, 1984) but are often ignored. Major advances in our knowledge have undoubtedly arisen as a result of new drug tools recently identified such as the antagonists ketanserin, MDL 72222 (laH,3c(,5aH-tropan-3yl-3,5dichlorobenzoate) and ICS 205-930 [(3a-tropanyl)-lH-indole-3carboxylic acid ester] and the agonists, 5-carboxamidotryptamine and 2-methyl-5-hydroxytryptamine (see below). However, the situation has been complicated by rapid changes in the state of the art, coupled with the lack of a consensus on how to proceed with the classification and nomenclature of types of 5-HT receptors. The proposals presented here are intended to provide a framework to accommodate current controversies and help to avoid them in the future.
563

564
HJSTORJCAL PERSPECTJVE

P. B.

BRADLEY et al.

The first major iandmark in our understanding of the different types of S-HT receptors came from the work of Gaddum and Picarelli (1957) who presented sound evidence for the existence of two pharmacologically distinct types of receptor for 5-HT in guinea-pig ileum, one mediating contraction of the smooth muscle (D) and the other mediating depolarisation of the cholinergic nerves (M). However, the drug tools at their disposal, phenoxybenzamine (dibenzyfine) and morphine, were not specific 5-HT receptor blocking drugs and are of little or no value as tools for the overall classification of S-HT receptors (see Humphrey, 1984 for refs). Subsequently the term D-receptor was applied to receptors in a variety of other smooth muscle preparations (including that of stomach fundus and uterus of the rat and aorta of the rabbit) where lysergic acid diethylamide and its derivatives (including methysergide) specifically antagonised the contractile actions of 5-HT (see Gyermek, 1966). This extrapolation without comparison of antagonist dissociation constants has, with the benefit of hindsight, been criticised (Humphrey, 1983). Recent studies moreover indicate that the guinea-pig ileum also contains 5-HT receptor types other than those described by Gaddum and Picarelli; one type appears to mediate relaxation of smooth muscle (Feniuk, Humphrey and Watts, 1983) another inhibition of the release of acetylcholine from cholinergic nerves (Kilbinger and Pfeuffer-Friederich, 1985) and another depolarisation of non-cholinergic neurones (Buchheit, Engel, Mutschler and Richardson, 1985). Likewise, some of the effects of 5-HT in other preparations are mediated by receptors which are neither of the D or M type (see Humphrey, 1983, 1984 for Refs). It is clear therefore that any new classification should take account of these new facts. The second major development, which occurred relatively recently, was the identification of various radioligand binding sites for 5-HT in brain tissue. In 1979, Peroutka and Snyder presented evidence for two distinct 5-HT binding sites for which lysergic acid diethylamide has a high affinity. At one site, termed 5-HT,, 5-HT also has a high affinity, whilst at the other, termed 5-HT,, spiperone has a high affinity. Subsequent studies have clearly shown that the affinities of a variety of S-HT antagonists for the 5-HT, site correlate well with those at D receptors, measured in functional studies in vascular and gastrointestinal smooth muscle (Humphrey, Feniuk and Watts, 1982; Engel, Hoyer, Kalkman and Wick, 1984, 1985; Maayani, Wilkinson and Stollak, 1984). There is also a substantial amount of evidence to correlate 5-HT, binding sites with receptors mediating a variety of responses to 5-HT in the brain and on platelets (see Leysen, 1984; Leysen, de Chaffoy de Courcelles, De Clerck, Niemegeers and Van Neuten, 1984). Since the 5-HT, site is so well characterised the

te~inology has already come into common use even for receptors involved in functional studies. The situation regarding the 5-H-f, site is much less clear despite the fact that 5-HT has a high affinity (in the nanomolar range) for this site. Thus, there is still doubt and controversy about its functional significance (e.g. Fozard, 1983a; Leysen, 1984). However, there is evidence for the sub-division of 5-HT, binding sites. Thus, on the basis of atypical displacement curves and the use of spiperone, Pedigo, Yamamura and Nelson (1981) demonstrated two distinct binding sites for [H]5-HT in the brain of the rat; the .%HT,, site shows high affinity for spiperone, whereas the 5-HT,, site has low affinity for spiperone. The tetralin derivative, 8-hydroxy-2-(di-~-propylamino)tetralin (8-OH-DPAT) has been subsequently shown to display both high affinity and selectivity for the 5-HT,, site (Middlemiss and Fozard, 1983). A third subtype of the 5-HT, recognition site, designated 5-HT,c, has recently been proposed based on the high affinity displacement of [HIS-HT by the ergot derivative, mesulergine (Pazos, Hoyer and Palacios, 1985). Despite this evidence for the heterogeneity of the 5-HT, recognition sites, it is considered that developments have not progressed to the point where it would be profitable to consider these in a classification of 5-HT receptors. However, as already mentioned, there is good evidence to show that, in some preparations, the 5-HT receptors are not of the D or M type (e.g. pre-junctional inhibition of neurotransmitter release) and that they have similar characteristics to the 5-HT, site (Feniuk, Humphrey and Watts, 1979; Engel, Gothert, Miiller-Schweinitzer, Schlicker, Sistonen and Stadler, 1983; Kilbinger and PfeufferFriedrich, 1985; Charlton, Bond and Clarke, 1986). The 5-HT receptors which mediate the relaxation of smooth muscle and tachycardia can also be described as 5-HT,-like (Trevethick, Feniuk and Humphrey, 1984; Saxena, Mylecharane and Heiligers, 1985b; Connor, Feniuk, Humphrey and Perren, 1986), though on the basis of agonist data they do not appear identical to those inhibiting transmitter release (see Humphrey, 1984). Definitive characterisation and correlation of such pharmacological receptors will only come from future research and will require the identification of potent and selective antagonists, analogous to the potent, selective S-HT, receptor antagonists such as ketanserin, which have been invaluable for characterisation purposes. Until then this poorly defined and apparently heterogeneous group of receptors can only be referred to as 5-HT,-like.

A CLASSIFICATION AND NOMENCLATURE OF !!-HT RECEPTORS

With the recent discovery of a variety of drugs acting selectively at 5-HT receptors the time is now appropriate to consider a new nomenclature to take

Classification of 5-HT receptors account of Gaddums D and M receptors and to integrate this classification with that of Peroutkas and Snyders binding site classification of 5-HT, and S-HT,. However, neither system of classification is by itself sufficiently comprehensive since the M receptor is not the same as the 5-HTi or 5-HT, binding site and some S-HT receptors are clearly not D or M receptors (see Humphrey, 1984). Furthermore, some drugs known as D receptor antagonists (e.g. lysergic acid diethylamide and methysergide) have high affinities for both S-HT, and 5-HT, binding sites. Despite these apparent complexities, all the evidence to date suggests that there are three main groups of 5-HT receptor type and clearly neither system of classification can eclipse the other. For simplicity, a S-HT, system of nomenclature is suggested, which attempts to correlate receptors mediating functional responses with equivalent radioligand binding sites (Table 1). As discussed above, it is now apparent that the D receptors in the guinea-pig ileum and rabbit aorta are similar to or the same as the 5-H1; binding site and since the latter appellation is now more commonly used, it is proposed that they should be referred to as 5-HT, receptors. The M receptor has no established binding site equivalent (but see Gershon, Takaki, Tamir and Branchek, 1985) and hence it is named the 5-HT, receptor. The nature of the 5-HT1 binding site and its functional correlates are still under investigation but for the foreseeable future it is suggested that receptors which are clearly not 5-HT, or 5-HT, and which have the characteristics defined below (see Table l), should be referred to as 5-HT,-like. Detailed justification for these proposals which integrate a number of similar proposals recently made in the literature (see Humphrey, 1983, 1984; Fozard, 1984a; Kalkman, Engel and Hoyer, 1984; Verdouw, Jennewein, Heiligers, Duncker and Saxena, 1984) are outlined below. Since the functional responses associated with 5HIT,-like receptors are only now being identified, these recep-

565

tors will be discussed in more detail than the more fully characterised 5-HT, and 5-HT, receptors.
S-HT,-LIKE RECEPTORS

The principal characteristics of the 5-HT, recognition sites defined by Peroutka and Snyder were the high affinity of recognised 5-HT receptor agonists and the relatively low affinity of classical S-HT receptor antagonists (now generally considered as 5-HT, receptor antagonists) such as methysergide, cyproheptadine and cinanserin. It is a considerable handicap to the definition of this site that no selective antagonists have yet been described. Moreover, as discussed above, the 5-HT, radioligand binding site is clearly not a homogeneous entity. Notwithstanding these obvious disadvantages it is thought to be appropriate to adopt the term 5-HT,-like at this stage, However, two important points must bc made: firstly, the criteria suggested for the identi~cation of 5-HT,-like receptors have deliberately been kept as broad as possible to allow scope for modification as knowledge advances; secondly, although the 5-HT,-like receptor has pharmacological similarity to the 5-HT, recognition site it may not necessarily be identical to it. In other words, it is the criteria defined below which, at this stage, define a 5-HT,-like receptor and not the necessity for identity with the 5-HT, binding site.
Criteria for characterisation ofS-HT,-like receptors

The first and most important criterion for the definition of any receptor, that the agonist response should be blocked by antagonists with high potency and selectivity for that site, cannot be satisfied in the case of the 5-HT,-like receptor, since no such antagonist for this site has yet been described. Several compounds do, however, have appreciable affinities for the 5-HT, binding site, including methiothepin and methysergide (which have even higher affinities at 5-HT, receptors), and a number of stereoselective

Table 1. Sunnnary of proposals for classification and nomenchuure of functional 5-HT xeptors
Proposed receptor nomenclature 5-HT,-like Typical responses Prejunctional inhibition of neuronal transmitter release, smooth muscle relaxation, contraction of some vascular smooth muscles and tachycardia in the cat Gastrointestinal and vascular smooth muscle contraction, platelet aggregation, neuronal depolarisation. ~la~~t~on of neriuheral neurones. . . SeIective agonists 5Carboxamidotryptamine Selective antagonists Mcthysergide* Methiothepint Equivalent binding site 5-HT,?

S-HT,

Ketanserin Cyproheptadine Methysergide

S-HT,

5-HT,

2-Methyl-S-hydroxy~p~ine

Cocaine MDL 72222 ICS 205-930

None

*Weak antagonist (or partial agonist) at some $HT,-like receptors. tLess potent antagonist than at 5-HT, receptors but inactive at 5-HT, receptors.

566

P. B. BRADLEY et al.

Table 2. Functional responses which appear Antagonism by methiothepin or methysergide* Schlicker and Giithert (1981); Cox and Ennis (1982); Engel et al. (1983, 1986) Feniuk et al. (1979) Charlton et al. (1986) North et al. (1980); Kilbinger and Pfeuffer-Friederich Apperley et al. (1980); Apperley and Humphrey (1986) Feniuk et al. (1983) Frankhuyxen and Bonta (1974); Cohen and Wittenauer (1985) Feniuk et al. (1983)

Responses to 5-HT In vitro Neuronal inhibition

Species/location Rat brain

Neuronal inhibition Neuronal inhibition Neuronal inhibition Vascular smooth muscle contraction Vascular smooth muscle relaxation Smooth mucle contraction Smooth muscle relaxation In via0 Tachycardia Hypotension

Dog saphenous vein Rat kidney Guinea pig ileum Dog saphenous vein Cat saphenous vein Rat stomach fundus Guinea pig ileum Cat Rat

(1985)

Vasodilation, hypotension Constriction of carotid artetiovenous anastomoses and dilatation of arterioles *No

Cat Pig

Saxena et al. (1985b); Connor et al. (1986) Fozard and Leach (1968); Dalton, Feniuk and Humphrey (1985); Saxena and Lawang (1985) Connor et al. (1986) Saxena et al. (unpublished) sometimes displays partial

potent sr,l&ive5-HT,-like

receptor blocking drugs have yet been described. Note that methysergide

/I-adrenoceptor antagonists such as propranolol, pindolol and cyanopindolol (Middlemiss, Blakeborough and Leather, 1977; Leysen, Awouters, Kennis, Laduron, Vandenberk and Janssen, 1981; Nahorski and Willcocks, 1983; Middlemiss, 1985a; Engel, Gdthert, Hoyer, Schlicker and Hillenbrand, 1986). In line with the intention to keep the definition of the 5-HT,-like receptor as broad as possible at this stage, it is proposed as the first criterion for identification of 5-HT,-like receptors that the response mediated should be antagonised by methiothepin and/or methysergide. Stereoselective blockade by certain P-adrenoceptor antagonists cannot be a criterion since many 5-HT,-like receptors mediating functional responses are not blocked by these agents. The second criterion for a pharmacological response to be mediated through 5-HT,-like receptors is that antagonists with high selectivity for other recognised 5-HT receptor sites should be inactive. Thus, compounds like ketanserin, which has affinity for the 5-HT, recognition site and negligible affinity for the 5-HT, site (Leysen et al., 1981), should not be antagonists at the putative 5-HT,-like receptor. Other classical 5-HT receptor blocking agents which are similarly able to discriminate between 5-HT, and 5-HT, recognition sites include cyproheptadine and cinanserin (Leysen et al., 1981). By similar reasoning, compounds with high selectivity for the 5-HT, receptor yet with essentially no affinity for the 5-HT, recognition site, such as MDL 72222 (Fozard, 1984b), ICS 205-930 (Richardson, Engel, Donatsch and Stadler, 1985), metoclopramide (Fozard and Mobarok Ali, 1978) (-)cocaine (Fozard, Mobarok Ali and Newgrosh, 1979) and

nor( -)cocaine (Fozard, 1983b), should not antagonise responses mediated through the putative 5-HT,-like receptor. The third criterion is based on agonists. Several compounds display a high affinity for the 5-HT, recognition site. These include 5-methoxy-3( 1,2,3,6tetrahydropyridin-4-yl) 1H indole succinate (RU 24969; Hunt and Oberlander, 1981; Middlemiss, 1985b) and 5-carboxamidotryptamine (Feniuk, Humphrey and Watts, 1981) which displaces 13H]5-HT binding with a potency (-log K, = 8.64) somewhat greater than that of 5-HT (-log K, = 8.51; Engel et al., 1983). In contrast, 5-carboxamidotryptamine is 26 times less active than 5-HT in activating the 5-HT, receptor of the rabbit aorta (Feniuk et al., 1981) and more than 50 times less active as a stimulant of 5-HT, receptors of the guinea-pig ileum (Humphrey, 1984). Thus, the actions of 5-HT at 5-HT,-like receptors should be mimicked by 5carboxamidotryptamine (with a potency at least equal to that of 5-HT; e.g. see Feniuk, Humphrey, Perren and Watts, 1985a). However, RU 24969 does not exhibit such selectivity (Feniuk, unpublished observations), being of similar potency relative to 5-HT in both the rabbit aorta (containing 5-HT, receptors) and dog saphenous vein (containing 5-HT,-like receptors). This highlights the danger of putting too much emphasis on agonist data. It is evident that 5-HT itself has a low affinity for the 5-HT, binding site but a high affinity for the 5-HT, binding site and that most 5-HT receptor agonists appear to be 5-HT, selective as judged from binding studies alone (e.g. see data in Martin and SandersBush, 1982). Thus, the following criteria are proposed for a

Classification of 5-HT receptors

to be mediated by S-HT,-like receptors

567

Resistance antagonism to
by 5-HT, receptor blocking drugs Engel ef al. (1983) Resistance to antagonism by S-HT, receptor btockina drum Engel et al. (1983); Middlemiss (unpublished) McGrath (1977); Feniuk et al. (1979) Charhon ef al. (1986) Kilbinger and Pfeuffer-Friderich (1985) Curro, Greenberg, Verbeuren and Vanhoutte (1978) No data Fczard (1984b) No data Saxena er al. (1985b) Forard (1984b); Dalton et a/. (1985) Saxena er al. (1985b) Saxena er al. (unpublished) Action of 5-HT mimicked bv 5-carboxamidotrwtaminet Engel e( al. (1983, 1986)

Van Nueten et al. (1981); Watts et al. (1981) Char&on ef 01. (1986) North ef al. (1980); Kilbinger and PfeufTer-Friederich (1985) Apperley et al. (1980); Feniuk et al. (1985a) Fen&k et al. (1983) Van Nueten, Leysen, Schuurkes and Vanhoutte (1983); Cohen and Wittenauer (1985) Feniuk et al. (1983)
Saxena et al. (1985b); Connor ef al. (1986) Kalkman, Boddeke, Doods, Timmennans and Van Zwieten (1983); Saxena and Lawang (1985) Connor et al. (1986) Saxena and Verdouw (1982); Verdouw et al. (1984)

Feniuk er al. (1981); Engel et al. (1983) Charlton et al. (1986) No data Feniuk et of. (1985a) Feniuk et al. (1984) Feniuk (unpubjis~d) Feniuk er ui. (1984) Connor ef al. (1986); Saxena er al. (1985b) Saxena and Lawang (1985)

Connor et al. (1986) Saxena and Verdouw (1985)

agonisticactivity.@electivity only relative (see Humphrey, 1984).

response to be defined as being mediated through 5-HT,-like receptors: 1. Susceptible to antagonism by methiothepin and/or methysergide (which sometimes displays partial agonistic activity) usually at doses or concentrations greater than those needed to block S-HT, receptors. 2. Resistant to antagonism by selective S-HT, receptor antagonists such as ketanserin and cyproheptadine and by S-HT, receptor antagonists such as cocaine, MDL 72222 or ICS 205-930. 3. Mi~cked by the selective 5-HT, agonist 5~r~xa~do~pta~ne at ~ncentratio~ equal to or less than those of 5-HT.
Distribution and function of putative S-HT,-like receptors

A number of responses has been claimed in the literature to be mediated through 5-HT, receptors; the details of those for which at least two out of the above three criteria have been satisfied are presented in Table 2. The most studied of these are discussed below.
The 5-HT autoreceptor of the ventral neruous sys-

tem of the rat. Agonist activity at the S-HT autoreceptor causes inhibition of transmitter release evoked by depolarising stimuli. As is clear from Table 2, the properties of the autoreceptor present on the terminals of S-HT-containing neurones satisfy in large part the three criteria laid down above for classification of a receptor as 5-HT,-like.
The inhibitory receptor for 5-NT on postganglionic sympathetic neurones. Two tissues have been particu-

of the dog (McGrath, 1977; Feniuk et al., 1979; Watts, Feniuk and Humphrey, 1981) and the perfused kidney,. of the rat (Charlton et al., 1986). Activation of the pre-junctional S-HT receptor results in inhibition of depolarisation-induced release of noradrenaline from the sympathetic nerves of these tissues. From Table 2, it is clear that the principal criteria for mediation of the response by 5-HT,-like receptors have been satisfied. Furthermore, it should be noted that in the saphenous vein of the dog, methysergide has marked partial agonist activity at the pm-junctional S-HT receptor (Watts et al., 1981) and an estimate of its affinity based on the concentration required to produce 50% of maximal inhibition of release of noradrenaline is very similar to its known affinity at 5-HT1 binding sites (Peroutka, Lebovitz and Snyder, 1981). It is important to note that even large concentrations of propranolol, pindolol or cyanopindolol were ineffective in antagonising this pm-junctional effect of 5-HT (Watts et al., 1981; Charlton et al., 1986) and that the potency of some putative agonists of 5-HT,-like receptors did not correlate with the affinities for 5-HT, binding sites (Charlton et ul., 1986). This provides a good example where the criteria for a 5-HT,-like receptor have been largely satisfied yet a clear difference from the 5-HT, recognition site can be demonstrated.
The inhibitory receptor for 5-HT on enteric cholinergic neurones. Both electrophysiological and bio-

larly well studied in this context, the saphenous vein

chemical evidence indicates the presence of a 5-HT receptor within the guinea-pig myenteric plexus, activation of which results in inhibition of the release of acetylcholine from the enteric cholinergic neurones (North, Henderson, Katayama and Johnson, 1980;

568

P. B. BRADLEY et al. receptor antagonists and that methiothepin is an effective, though rather weak antagonist (Table 2) the receptor of the rat fundus can be said to be 5-HT,-like. This interpretation is supported by the demonstration of a significant correlation between the potency of 19 antagonists of 5-HT on the fundus and their affinities for the 5-HT, recognition site (Leysen and Tollenaere, 1982). However, certain other pieces of evidence, apparently not supportive of this conclusion (Table 2) deserve comment. Thus, 5-carboxyamidotryptamine, although mimicking the effects of 5-HT on this tissue, is some 10 times less active than 5-HT in this respect (Feniuk, unpublished data) and hence significantly less active than would be predicted from its relative affinity for the 5-HT, recognition site. Moreover, methysergide is at least as active as an antagonist of 5-HT on the fundus (although it does not behave competitively) as it is against responses mediated through the 5-HT, receptor (Frankhuyzen and Bonta, 1974; Cohen and Wittenauer, 1985). In conclusion, whilst the receptor mediating the contractile responses to 5-HT in rat fundic strip (which has been referred to as a D receptor in the past) clearly cannot be classified as either a 5-HT, or 5-HT, receptor, it may be classified as 5-HT,-like only subject to the anomalies outlined above.
The receptor mediating tachycardia in the cat heart.

Kilbinger and Pfeuffer-Friederich, 1985). Despite the fact that no selective 5-HT,-like receptor agonist has yet been described as mimicking the effect of 5-HT at this site, other evidence strongly implicates a 5-HT,-like receptor as the mediator of the response (Table 2).
The receptor for S-HT mediating contraction of certain blood vessels. Certain blood vessels, notably

the saphenous vein of the dog and basilar artery from rabbit and man, respond to small concentrations of 5-HT with contractile responses which are resistant to blockade by selective 5-HT, receptor blocking agents (Apperley, Feniuk, Humphrey and Levy, 1980; Forster and Whalley, 1982; Peroutka, 1984a; Feniuk et al., 1985a; Bradley, Humphrey and Williams, 1986). The majority of the criteria for mediation of these 5-HT,-like receptors are through responses satisfied (Table 2). The evidence is thus consistent with a close similarity between the pre-junctional receptor in the saphenous vein of the dog (see above) and these post-junctional receptors, a conclusion reinforced by the highly significant correlation between the potency of a number of agonists at both sites (Feniuk et al., 1981; Miiller-Schweinitzer, 1981). The similarity extends, too, to the fact that methysergide is a partial agonist of similar potency both pre- and post-junctionally (Apperley et al., 1980; Watts et al., 1981; Peroutka, 1984a). There is also evidence that such receptors mediate vasoconstriction in the arteriovenous anastomoses located in the carotid circulation in vivo (Saxena, 1974; Saxena and Verdouw, 1982, 1985; Verdouw, Jennewein, Mierau and Saxena, 1985).
The receptor for S-HT mediating relaxation of smooth muscle. S-Hydroxytryptamine induces relax-

ation of some vascular and gastrointestinal smooth muscle by a direct effect on smooth muscle (e.g. Feniuk et al., 1983; Trevethick et al., 1984). This effect is selectively mimicked by %carboxamidotryptamine (Feniuk, Humphrey and Watts, 1984; Trevethick, Feniuk and Humphrey, 1986) which is a potent vasodilator and hypotensive agent in anaesthetised and conscious animals (Feniuk, Humphrey and Hunt, 1985b; Saxena and Lawang, 1985; Connor et al., 1986). In addition, 5-HT-induced relaxation of vascular smooth muscle appears to be resistant to blockade by the 5-HT, receptor blocking drug ketanserin and also propranolol (Van Nueten, Janssen, Van Beek, Xhonneux, Verbeuren and Vanhoutte, 1981; Cocks and Angus, 1983; Feniuk et al., 1983) and yet can be blocked, albeit weakly, by methysergide (Feniuk et aI., 1983; Cohen, Shepherd and Vanhoutte, 1983b). The evidence strongly implicates a 5-HT,-like receptor in the mediation of these responses (Table 2).
The receptor for 5-HT mediating contraction of the stomach fundus of the rat. 5-Hydroxytryptamine

Intravenous injection of 5-HT to anaesthetised or spinal cats induces tachycardia which is not mediated through /3-adrenoceptors (Saxena et al., 1985b). The 5-HT receptor involved may be the same as the 5-HT receptor which mediates relaxation of smooth muscle (Connor et al., 1986). Reference to Table 2 shows all the criteria to have been satisfied allowing the conclusion that 5-HT,-like receptors mediate the response. As in the stomach fundus of the rat, this response has previously been regarded as a D receptor effect (see Saxena et al., 1985b).
Further class$cation of dHT,-like receptors

causes powerful contractile responses on the rat fundic strip (Vane, 1957). To the extent that responses are not antagonised by ketanserin or 5-HT,

It bears emphasis that the category 5-HT,-like as defined here is intended as an interim classification to serve until new developments force its modification. Such developments could come as a result of the discovery of compounds with improved selectivity for the 5-HT, binding site and its subtypes and the use of such agents in functional tests. Increasing numbers of such drugs are now being described including buspirone (Glaser and Traber, 1983), 8-OH-DPAT (Middlemiss and Fozard, 1983), TVX Q 7821 [2 - (4 - (4 - (2 - pyrimidinyl) - 1- piperazinyl) - butyl - I,2 benzisoltriazol- 3(2H)one - 1,l - dioxidehydrochloride] (Dompert, Glaser and Traber, 1985), BEA 1654 [N-(3-acetylaminophenyl)piperazine] (Verdouw et al., 1985). Appropriate functional analyses with such agents should facilitate attempts to arrive at a comprehensive classification of 5-HT,-like receptors. In this context, evidence for the inhibitory receptor for 5-HT on enteric cholinergic neurones of the guinea-

Classification of 5-I-IT receptors pig being of the 5-HTrA subtype has appeared (Fozard and Kilbinger, 1985), while it has been suggested that the S-HT autoreceptor in the brain is of the S-HT,, subtype (Engel et al., 1986). However, definitive characterisation of all such responses will await the identification of potent, selective, competitively-acting blocking agents. 5.HT, RECEPTORS
The introduction of the term 5-I-IT, receptor by Peroutka and Snyder (1979) to describe a 5-HT recognition site in the brain with a low affinity for 5-HT but with a high affinity for spiperone and other classical (so called D) 5-HT receptor blocking drugs, such as methysergide and ~~rohep~dine, has led many to investigate the correlates of this binding site with functional responses. The fact that some of these antagonists also have appreciable affinity for the 5-HT, binding site (e.g. methysergide and methiothepin) or are potent calcium antagonists (e.g. cyprohep~dine) has led to some confusion in attempts to correlate the 5-HT, binding site with the functional effects of 5-HT (see Leysen et al., 1981; Lowe, Mathews and Richardson, 1981). However, the identification of the 5-HT antagonist, ketanserin, which has a marked affinity for 5-HT, recognition sites and a negligible affinity for 5-HT, recognition sites {Leysen et al., 1981) provides a particularly useful tool with which to characterise these receptors pharmacologically. Although certain agonists, such as S-carboxamidotryptamine, appear to possess a degree of selectivity for 5-HT,-like receptors, as far as is known, no compound has yet been identified which shows markedly selective agonistic activity in preparations containing 5-HT, receptors.

569

of at least these two 5-HT receptor blocking drugs before the receptor subserving a given response can be character&d as being of the 5-HT, type. 2. The second criterion is that compounds with a high selectivity for other 5-HT receptor types should not antagonise responses mediated through the putative 5-HT, receptor. Thus, antagonists with a high selectivity for the 5-HT, receptor, e.g. cocaine, MDL 72222 and ICS 205-930 are inactive at 5-HT, receptors. Unfort~ately, since no potent, selective, j-HT,-like receptor blocking drugs have yet been de&bed this particular criterion cannot be tested fully at this time. 3. No suitably selective agonist has been identified.
Distribution and function of S-NT, receptors

Criteria for the characterisation of .5-HT, receptors

1. Antagonists with a high affinity for 5-HT, recognition sites should show a similarly high antagonistic potency at the putative 5-HT, receptor. Thus, compounds such as ketanserin, spiperone, methysergide, pizotifen and cyproheptadine are all potent antagonists at S-HT, receptors with pA, values (between 8.5 and 9.4) which are similar to their pKn values determined from ligand binding studies (see Humphrey et al., 1982). Experiments with ketanserin are particularly important since this compound shows negligible affinity for 5-HT, binding sites and is not a potent calcium antagonist (Van Nueten and Vanhoutte, 1981). It should however be noted that ketanserin does show appreciable affinity for a-adrenoceptors and it is imperative to dissociate those effects of S-HT which are mediated via stimulation of specific 5-HT receptors from those effects which may be mediated via a-adrenoceptors (see Feniuk, 1984). In contrast, methysergide has negligible affinity for a-adrenoceptors. It should therefore be necessary to determine the antagonistic potencies

Table 3 summarises functional responses to S-HT both in vivo and in vitro which appear to be mediated by stimulation of 5-HT, receptors. This diverse list of actions includes not only contraction of a variety of types of smooth muscle but also platelet aggregation and neuronal depolarisation (Fozard, 1984a). In many cases the classification as a 5-HT, receptormediated mechanism is tentative because strict measures of the dissociation constants of 5-HT receptor blocking drugs and hence a comparison of equivalent values in ligand binding studies have only been carried out in some smooth muscle preparations. Another complication is that while many potent 5-HT, receptor blocking drugs (e.g. ketanserin) appear to be competitive antagonists of 5-HT-induced contraction in vascular smooth muscle, they appear to be potent but unsurmountable antagonists in other preparations, e.g. the rat uterus (Ichida, Hayashi and Terao, 1983). Precisely why this is so is not yet clear. Interestingly, trazodone is a potent competitive antagonist of S-HT in the uterus (Wrigglesworth, 1983) and jugular vein of the rat (Cohen, Mason, Wiley and Fuller, 1983a) but it is approx. 20-30 times weaker as a competitive antagonist in the rabbit aorta (Black, Brazenor, Gerskowitch and Leff, 1983). One possible explanation therefore is that S-HT, receptors are heterogeneous but it would be unwise to reach this conclusion without considering the potential involvement of complicating experimental factors or mixed receptor populations (e.g. see Humphrey, 1984). It is evident that ketanserin and methyser~de are particularly useful in characterising 5-HT, receptors. Ketanserin has the advantage of not only being inactive at !I-HT, receptors but also having little affinity for the 5-HT,-like receptor.
5_HT, RECEPTORS

For many years the identification of 5-HT, receptors was hampered by the lack of specific antagonists as drug tools. However, in 1979 it was shown that M receptors, similar to those in the guinea-pig ileum, mediated the release of noradrenaline from

570

P. B. BRADLEY et al. Table 3. Functional responses which

Responses to 5-HT
In vitro

Species/location Rabbit aorta

Specific antagonism by ketanserin or methysergide Apperley, Humphrey and Levy, (1976); Feniuk et al. (1985a) Bradley, Humphrey and Williams (1985); Van Nueten Van Nueten et al. (1981) Van Nueten, Leysen, Vanhoutte and Janssen (1982) Ichida et al. (1983) Engel et al. (1985) De Clerck, Xhonneux, Leysen and Jannsen (1984) No data rat Van Nueten er nf. (1981); Feniuk, Humphrey and Perren (1982); Fozard (1982) Saxena and Lawang (1985); Dalton

Contraction of vascular smooth muscle

Contraction of extra-vascular smooth muscle Platelet aggregation

In viva

Rat tail artery Dog gastrosplenic v Guinea-pig trachea Rat uterus Guinea-pig ileum (atropine treated) Cat Rabbit and man Pithed/anaesthetised

etal. (1981)

Vasopressor responses

Conscious rat Anaesthetised cat Anaesthetised guinea-pig Conscious rat/mouse Conscious rat Rat paw Anaesthetised cat

et al. (1985)

5_Hydroxytryptophaninduced head twitch 5_Hydroxytryptophaninduced wet dog shake Oedema Urinary bladder contraction (second phase)

Reiche and Frey (1983); Connor et al. (1986) Saxena and Lawang (1985) Malick, Dose and Barnett (1977) Colpaert and Janssen (1983); Yap and Taylor (1983) Ortmann, BischotT, Radeke Buech and Delini-Stula (1982) Saxena, Heiligers, Mylecharane and Tio (1985a)

sympathetic nerves innervating the rabbit heart and cocaine was shown to be a specific, albeit weak, antagonist at these receptors (Fozard et al., 1979). Such studies led to the development of a potent and selective 5-HT, (M) receptor blocking drug, MDL 72222 (Fozard, 198413). Work in parallel led to the identification of even more potent 5-HT, antagonists including ICS 205-930 (Richardson et al., 1985). With the availability of these specific antagonists, 5-HT, receptors have been identified in a variety of locations on peripheral efferent and afferent neurones (Fozard, 1984a). A useful selective agonist is also available for the study of 5-HT, receptors. Thus, of a series of tryptamine analogues tested as agonists in a number of test systems, 2-methyl-5-HT proved to have selective stimulant effects at peripheral neuronal 5-HT, receptors (Richardson et al., 1985; see also Humphrey, 1984).
Criteria for the characterisation of 5-HT, receptors

3. Mimicked by the selective 5-HT, agonist 2-methyl-5-HT, which has a potency similar to that of 5-HT itself.
Distribution and function of 5-HT, receptors

The distribution and functions of receptors which fulfil some, or all, of the criteria listed above for 5-HT, receptors are summarised in Table 4. It is apparent from this table that these receptors for 5-HT are invariably located on peripheral neurones where they are responsible for eliciting the depolarising action of 5-HT. Nevertheless, this does not preclude the possibility that receptors fulfilling the criteria for designation as 5-HT, receptors may eventually be found in the central nervous system or in non-neuronal locations.
Heterogeneity of 5-HT, receptors

For a response to be defined as being mediated through 5-HT, receptors it should be: 1. Susceptible to antagonism by (-)cocaine, MDL 72222 or ICS 205-930 which are selective antagonists with pA, values in the order of 6.0-7.0, 7.5-9.5 and 8.0-l 1.O respectively. 2. Resistant to antagonism by antagonists that are active at 5-HT, receptors (e.g. ketanserin or methysergide) or 5-HT,-like receptors (e.g. methiothepin).

Evidence for the existence of 5-HT, receptor subtypes stems from the recent observation that significantly different pA, values for the same competitive antagonists have been obtained in different bioassay systems. Thus, MDL 72222 yields a pA, value of 8.9 for 5-HT, receptors on post-ganglionic sympathetic nerve fibres in the rabbit heart, and a pA, value of 7.9 on primary afferent neurones in the vagus nerve of the rabbit, but is a weak non-specific antagonist at 5-HT, receptors on enteric neurones in the guinea-pig ileum (Donatsch, Engel, Richardson and Stadler, 1984). Similarly, ICS 205-930 has pA, values on these systems of 10.6, 10.2 and 7.9 respectively (Richardson et nl., 1985). Moreover, ana-

Classification of 5-HT receptors

appear to be mediated by 5-HT, receptors Resistance to antagonism by 5-HT, recentor blocking drugs MDL 72222 inactive at 10 pM (Feniuk, unpublished observations) No data No data No data Fozard (1984b); Richardson et al. (1985) Engel et al. (1985) No data Taparelli (unpublished observations) Kalk;nan et al. (1984) Comments

571

Comparison of dissociation constants at the 5-HT, binding site with equivalent values from pharmacological studies suggests that the binding site and pharmacological receptors are similar (see Humphrey, 1984).

Receptor in guinea-pig ileum is Gaddum and Picarellis original D-receptor (see Engel er al., 1984, 1985). 5-HTs-binding sites have been identified on cat platelets. Inhibition of 5-HT-induced [sP]phosphatidic acid formation in human platelets also appears to be 5-HT, mediated (see Leysen et al., 1984).

Dalton el al. (1985) No data Dhasmana e( al. (unpublished) Shearman (unpublished observations) No data Not tested Saxena et al. (1985a) Selective 5-HT, antagonist, pirenperone, potent antagonist of 5-hydroxytryptophan-induced head twitch in mice (Green, OShaughnessy, Hammond, Schachter and Grahame-Smith, 1983). Effects of ketanserin not studied. Although ketanserin not studied many classical 5-HT antagonists are potent inhibitors of 5-HT-induced rat paw oedema (Ortmann e( al., 1982). Second prolonged phase of contraction (first transient phase is 5-HT, mediated).

logues of ICS 205-930 have now been described which discriminate between 5-HT, receptors in the rabbit heart and those in the vagus nerve (Richardson et al., 1985). On the other hand, (-)cocaine is a non-selective antagonist which has a pA, value of 6.0-6.5 on all three systems (Fozard et al., 1979; Engel and Richardson, unpublished observations). The foregoing suggests that subtypes of the 5-HT, receptor exist, but more rigorous studies with the selective antagonists are needed for their proper characterisation.
CONCLUSIONS

This paper has reviewed the recent progress made in our understanding of the pharmacology of 5-HT with a view to defining the broad criteria necessary for the characterisation and classification of 5-HT receptors. Using these criteria, three distinct groups of 5-HT receptors can be identified which have been named 5-HT,-like, 5-HT, and 5-HT,. As emphasised at the outset, the proposed classification of 5-HT receptors is intended to provide a general framework which can be built upon as new knowledge accumulates. It is recognised that, with the discovery of selective antagonists (particularly for the 5-HT,-like receptors), the proposed classification will undergo modification and/or extension. However, a plea must be made for investigators to exercise extreme caution before attempting to define yet another 5-HT, receptor. Erroneous interpretation can readily result from, for example, mixed receptor types mediating a common function.

Definitive characterisation (and hence naming) must await well controlled quantitative studies with potent, selective, competitively acting receptor blocking drugs. This classification is based largely, but not exclusively, on data from studies in isolated peripheral tissues where definitive characterisation is possible. Nevertheless, specific drug tools are now available to characterise. 5-HTr and 5-HT, receptor mechanisms wherever they exist, including the brain. There is evidence for the involvement of 5-HT, receptors in the depolarising action of 5-HT in neurones of the central nervous system (McCall and Aghajanian, 1980) as well as in 5-HT-stimulated inositol phospholipid hydrolysis (Conn and Sanders-Bush, 1985; Kendall and Nahorski, 1985). Moreover, 5-HT,like receptors may mediate the hyperpolarising action of 5-HT on neurones in the brain, as well as its pre-junctional inhibitory action on neuronal transmitter release (Haigler and Aghajanian, 1977; Ennis, Kemp and Cox, 1981; Davies, Roberts and Wilkinson, 1985). However, without good selective antagonists, characterisation of 5-HT,-like receptor mechanisms in both brain and periphery will be slow. As yet, 5-HT, receptors have not been identified in the brain. Nevertheless, it is believed that the proposed classification of 5-HT-receptors will ultimately be shown to be relevant to studies in the central nervous system. Authors Footnote-This
publication has come about following a Symposium on SHT, Peripheral and Central

Table 4. Functional responses which appear to be mediated by S-HT, receptors Specific antagonism by ICS 205-930 or MDL 72222 Resistance to antagonism by 5-HTz or 5-HT,-like receptor blocking drugs Action of S-MT mimicked by 2-methyl-S-HT

Response lo S-HT

Species/location

Guinea-pig ileum

In vitro Transmitter release from emeric neumnes Depolarisation Depolarisation Buchheit et al. (19%); Richardson et at. (1985) Richardson (unpublished) No data

Rabbit vagus nerve Rabbit nodose ganglion

Ruchheit er at. (unpublished) Richardson N al. (1985) No data No data Richardson el al. (1985)

71 pl

Depolarisation

Rabbit superior cervical ganglion

Nash, Wallis and Ash (1984) Fozard et al. ( i 979); Fozard (f984b)

Release of noradrenaline by ~la~~tjo~ of ~~thetio nerves In vhro Elicitation of mIlex bradycardia (v. RezoldJariseh reflex) Fozard (1984b); Kalkman er al. (1984); Saxeaa et al. (1985b) Richardson ef al. (1985) Richardson er al. (1985) Saxena et al. (198%)

Rabbit hear6

Ruchheit et al. (1985); Riizhardson ef aL. (1985) Richardson er al. (1985) Fozard, Humphreys, Round and Wallis (1985); Azami, Fozard, Round and Wallis (1985) Foaard ef al. (1985); Azami ef al. (19851 Fozard (19&4b~ ~char~on et al. (1985)

F P 2 z c .?Fozard (1984b); No data Richardson et al. (1985) Richardson et al. (1985) Saxena et al, (1985a) Richardson et al. (1985) No data Richardson er ol. (1985) No data

Man

Provocation of pain on a blister base Urinary bladder (Ilrst phase; neuronally mediated ~ntr~tion~

Cat

ClassifiMrion of 5-H-F receptors

Receptors, and Function, which was held at Birmingham University on April 1I, 1984, as mu+tof the Spring Meeting of the British P~~~a~l~~~~ Society. The Proceedings of the S~~sium were Fub~~sh~ as a Special fssue of this Journal (Vol. 23, No. 12B, 1984). Following discussions among the participants, together with a number of those attending the Symposium, it was decided to set up a Working Party to see if something oould be done about the unsatisfactory state concerning the classification of 5-HT receptors. The present authors comprised the core of the Working Party, although others participated from time to time and we should like to thank them for their contributions. A number of meetings was held and this Yommentary is the end result of the Working Partys dehberations.

s73

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116-124~ .

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36-44.

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