Identification and significance of beta-adrenoceptor subtypes

T I P S -.April

"5

1981

ad~-anta~0 o t tht'~e t,c~,hllzquc~ lOT t C O : p h q

Identification and significance of beta-a d re n o c e p t o r subtypes Stefan R. Nahorski Department o~Phamtacok~gy and TherapeutwL tledical .~'tienct'i B*ahlmg. t 'mr ,'r~lt~ o t I I'li+¢'~h" I '~llt ,'rxllt Road. Leicester L E I 7RH. U.K.

At the present time we have little reason to doubt Ahlquist's original propo,~d that catecholamines exert their physiological effects by interacting with alpha or beta adrenoceptors. However. more recent developments have suggested that them receptors may not be homogeneous and the existence of alpfiat, alpha=, betat and beta= adrenoceptors has received considerable experimental support. The possibility that beta-adrenoceptors do not form a homogeneous population was first suggested by Mora# when he drew attention to the fact that betaadrenoceptor antagonists p o s i t i n g alkyl substituents on the alpt >carbon of the ethanolamine side chain display different potencies in blocking vascular and cardiac responses to catecholamines. However, it was Lands et al ~ who first suggested the subclassifieation of beta-adrenoceptors into betm and betat. This division was made on observations that the rank order of potency of cateeholamine agonists fell into two distinct categories d~_pending upon the ti~ue response examined. The receptors in heart and adipose tissuc were classified as betat, whereas those in bronchi and vascular smooth muscle were designated beta=. Further support for this subclassification of beta-adrenoceptors has come from subsequent observations that certain antagonists (e.g. praetolol, atenolol, metoprolol) are more effective in blocking catecholamine responses in the heart, whereas certain agonists such as salbutamol and mterenol were cleady more selective for respirator}' ti,~sue. However, this organ-specific dual receptor hypothesis has not been universally accepted since several authors have reported a spectrum of apparent affinities for agonists and antagonists between tissues of various species (for an excellent review of these pharmacological studies see Ref. 3). It is clear however, that the precise analysis of drug-receptor intcrac-

tions using classical pharmacological approaches in ~ ht~lc tis, uc cain he prone to a number of serious limitation,,, l'hus v, ilh agonists there could be quite different rate,, o f loss by uptake or non-receptor binding bett~een rations tixsues. An apparent selectivity for an agonist could also be manifest hy a different "receptor reser-,c" between tissues. Even with antagonists, the distribution of the coml~3und both bet~veen and ~ithin a tissue could create an apparent selectivity related to the physieo-chemical nature of the drug and be quite independent cfa selective interaction with the receptor. In this respect, it should be noted, for example, that almost all beta.-mlectixe antagonints are more h~.drophilic than non-selective antagonists. The proposal b.~ Carlsson and his colleagues + that both beta, and betaz adrenoceptors ma? co-exist in the same organ and under certain circumstances l|la} mediate the same physiological responm, could also influence the anal}sis of pharmacological responses of u, hole organs. In order to overcome the inherent problems associated with drug-reeeptor interactions in intact pharmacc!ogical preparations, sexentl research groups have recently umd radiolabelled ligand binding techniques to characterise and quantif~ adrenergic receptors. It is probabb because of these ne~ approaches that a clearer picture of the natt+re of betaadrenoceptor subtypes has en'terged.

Identification of bcta.adrenoc~ ptor sub~,pcs using radioligand techniques Over the last few years there haxe been quite dramatic d.-,elopntents in betaadrcnoceptor research ~hich haxe been directly related to the use ofdiiect receptor labelling techniques and the recognition that adenylate e y e l a ~ stimulation is one of the prim:try events that follo,,xs occupation o f the receptor by ,in agonists. +Fheinherent

c h a r d c t c r P , ; t t h + n llc. '~ lit+ thc u,,c o! m++nl b r a n e prcp;tr/~ltlon~. IN x~.|lldl proDh.'nl, o!

drug-receptor cqnflibrium

can Ix."mlmmthe dirc<: labclhn,.: ,+I receptors allow s a precise quantltluLdtlorl ot receptor stlbt~,pc density, th,lt r, riot .~'olnpllcared b.', es, ent',, oCctlrrin~ 'd£1~>,n ,,trcarn" ot the receptor it~:lf t)n the other haled it ..hould Ix" appreciated Ihal the lack ol a final bioh+glcal re.,r~n+~c +/dr. be a di'.tlnct tli'++adxanta~c in attempt~ to n.'latc rc
types. [ ' h e lno,,t c o m n l o n l x

tl,,,c++l [l~andx in thi~

field are the non-,,~'lcctlxc bct.+-;idrcno,.cptor a n tagonl,,t, I l-l+llldd~ dr,, alprenolol II+IIIDII.\I and t +_vl,+ll,,,d,, h} drox~, bcn/~. Ipindoh,I t['"al]llt~ Ih .-\hhough the latter Iigand po,,,~',,-c,, the t l d x d n L i g c .if :el con~idcrabl'+ h i ~ h c r sp~.%ttlc :.lCtixit~,. this can be OtllX~.cighcd h} higher

non-,,pcdfic binding and an dpF,m:nt a,,,,ociation x+ith 5-ttT rccognttlon xltc, in some tis,,ut.',,. For thi. rca,,on, the nlAJOllt~ of oth qudicx hast I~'en i'~:rformed xxtth I~H]DH.-\. The fir,,t indication that the d~r¢ct tabcIling of bcta-adrcnoccptof m,ght rcxcaI next intorm;.ltlt',n c o n c c r m n g t h e eXlqcn~.'c o f ret_e.'gtor ~,tlbt.Xl~+,, c d n l c f r o m ",tudlc~ m

r:t~ cerebral cortex and h,.ng~:. IT1 [hc..c cxD:riment. [ a | t I D t | A could Ix.-di.placcd bx other non+,clectixc antagont~,t, ITonl m e m b r a n e . , ot t2it}ler tl:.,:.,ut."In a nl.lllnCT s u g g c q i n g i r l t c r a c t i o n 9, ith a h-,,rnogcncou~

receptor t'~+pulation xxuh binding goxcrncd bx tnelax~ otnlassactiou Hov*.cxcI. cerl,ttll drugs that haxe been rel'~+rted to d+-pi.} be:tat sclecttxit.x m intact prcparatl.,n- ,_'oncrated biphasic displaccillcn'g cup, c. m ~+th tissues.. Our interpretation ot thc..c data x~a.. that torte\ ,rod h l n g tx~'~'~."~.~ ~(+!]: D¢.'t..tt a n d t~'t;.tm a d r e n o c c p t o r , ,

,tnd that t h e

~elccuxc antagonists po.sexs nlarkcdlx dffferen~ aft'initte,, for the txxo pt+ptllation-ot receptor ..ubl.~pes that x~crc being labelled b} laHIDHA. lnitiall.~ an Eadic-|lol),tec transformation of the data allox~ed an e,~tinl,ttlon O; the proD+rtton of receptor .ubtxlx'. and the rclatixe afftnitic., of the contpctmg drug. Using thc,,e techniques it could hc shox~,n that ill cerebral cortc\ ahotlt 0.5 % o f the reccptor~ po..sev;cd high atfillit% tor beta= antagonists, ~hcrea,, m hmg onl~ about 20% ot the ..tics x~erc de.placed x~>th

htgh affinit~ b'. these bcta;-,,clcctix¢ agents. It ,Aas gratif3 mg ~hat dc~pllc different prulxwtions ot high and low ..tffiniD, sites ill lung and brain, the afflnitx con•~tanb, for Imtat ,,electixe agent,, for each

particular subtx+pulation of sites ~+c re tdcn, F '~-,,¢, %,ah tl,,, : ~,, I~,,.m;d,.~+ p e., :~.

TIPS - April 1981

96

data has yielded v~rlu~.,.llyidentical results. Using these techniques, a~mlysis of the proportions of beta~ and beta, adrenocepO _z ~0 tots can be made in a variety of mammalian tissues and a summary of studies using m / / ~ J RATLUNG.2 ~ HIGHAFFINITY(BETA,) [+H]DtlA are shown in Table I. These results suggest that (a) there are only two -r beta-adrenoceptor subtypes in mammalian u. tissues and that they correspond to the O betal and beta, subclasses suggested previously from classical pharmacological approaches, and (b) in most tissues both "I" _z 2o beta+ and beta, adrenoceptors co-exist but ~e the proportion of each subtype depends upon the tissue being examined. Not only .I.AI + # . . . ~ . . . . are there differences between various tis7 6 5 4 3 2 sues of the same species, but even in the - LOG [A'I~NOLOLI same tissue of different species. Thus, unlike other mammalian species, betat rather than beta, receptors predominate in rabbit lung8. More recently, using the radioligand [mI]IHYP and techniques virtually identie-. cal to those above, Minneman and his colz_ leagues have been able to confirm the copresence of betat and bet,at adrenoceptors in several tissues* Overall, therefore, the T use of receptor labelling techniques has provided direct evidence for Carlsson's 40. original suggestion that betai and beta, _m adrenoceptors can co-exist in the same tis"1" sue. These findin~.,s may provide some _z 20. explanation for data from classical pharmacological approaches that could not be easily reconciled with a simple organS 5 e g specific dual beta-adrenoceptor concept. Thus, if most tissues possess both receptor -4.C:G [IG1118.551 l subtypes, and if (and this is a critical Fig. I. inhibitzo~ of[JHJDHA l~mdi~g to rat lung end erythroeyle membranes by ( A ) ate betat selective antagonisl assumption) both populations mediate the menolol and (BJ the bera~ sf'+ecuve ant~goms! ICI l l & J S l [ery~ro-l-(7-methfli~dan-4-ylozy.3.iaopropylaminobman-2-of)[ Contpuler-~;uL~ted crave fitdng revealed thai the erythrocyW membranes possess a homosame physiological function, then the affingeneoua popu~atJon of sues dmt have low al.~}inizyfor atenolol and high affinity for I CI 118. 551. On the other hand. ity of an antagonist or the potency of an the lung curves are best luted by a two-si*e model in which 20% ~ f the sites have high affmily for caenotol and ate agonist will depend i.n part on the relative remaining 80% high a)~mity for 1(7111&551. proportion of each subtype in the tissue in question. tical for the two tissues. However, conclu- and beta, adrenoceptors can co-exist in tissive evidence that the:,e sites represented sues in a non-interacting manner and are Cellular localization of beta-adrenoceptor beta+ and bctaz adrenoceptors only came probably quite separate entities. when it could be shown that highly selecSubsequent to these initial studies, it has subtypes tive beta. adrenoceptor drugs such as pro- been correctly pointed out that the linear It is far from clear whether we can caterol and ICI 118.551 generated curves transformation methods of analysing the extend the findings using ligand binding that were the precise 'mirror image' of complex curves generated by betat or betat studies described above to conclude that those ~ c n with beta+ selective drugs. The selective agents can be inaccurate, particu- both subtypes co-exist on the same cell. It curves generated by ,~he beta,-selective larly with drugs that display less than 30 must be stressed that the tissues in which antagonist ltenolol and the betas-selective fold selectivity for the receptor subtypes. both beta-receptor subtypes apparently antagonist ;C! 1118.551 in rat lung and The advantages of using non-linear curve co-exist possess several different cell types. eq!throcym are illustrated in Fig. 1. It could fitting techniques have been detailed Thus, the presence of both subtypes within also be demon;trated that the prior selec- recentlys and this form of analysis is a tissue may merely reflect this cellular tive occupation of only one c~ass of recep- routinely used in our laboratory at the pre- heterogeneity. A recent examination of tour subtype c~nverted the biphasic dis- sent time. However, in view of the fact that membranes prepared from rat erythrocytes pl:lcement curves of agents that were selec- only highly selective agents were used in and reficulocytes has revealed that these tise for t.he. other subtype into uniphasic our own studies, direct comparison of homogeneous populations of ce0s only mass action curves. This suggests that betat linear and non-linear handling of the possess beta. adrenoceptors. On the other 100

A.

TIPS -April 1981

97

hand, investigation of the ontogeny of rat lung beta-adrenoceptors indicated that the proportion of beta, and betas sites remained constant, despite the markedly different ratio of cell types found at various stages of development. This could indicate that both receptor subtypes are present on the same cell type and would support pharmacological evidence that catecholamines mediate smooth muscle relaxation in guinea-pig trachea by interacting with both beta, and beta~ adrenoceptors m. An attractive concept is that betaadrenoceptor heterogeneity could reflect a differential pre- and post-synaptie distribution of beta, and betas receptors. Since presynaptic beta-adrenoceptors may be involved in the regulation of noradrenaline release, the therapeutic consequences of such a phenomenon could be particularly important. However, chemical sympathectomy with 6-hydroxydopamine has failed to indicate any significant proportion of presynaptic beta receptors in spleen, and has revealed that both be,am and be,an subtype~ co-exist postsynaptically in this tissue ~.. In the central nervous system it is probable that glial cells and possibly cerebral blood vessels as well as neurones, possess beta-adrenoceptors. In support of this it has been recently shown that specific lesions of nerve cell bodies in the stria,urn using the neurotoxin kainic acid leads to only a small (30%) loss of betaadrenoceptor binding sites ~. However, it is of interest that the sites lost are almost exclusively from the beta, population. A better indication of the precise cellular localization of beta~ and betaa adrenocep-

tots is clearly required. It seems probable that refinement of the autoradiographic studies available nov,- for ~veral neurotransmitter receptors may provide better clues in the near future. The question of selective b e t a - a d r e n t z e p ~ agonists

One of the corner-stones of the original suggestion of beta-adrenoceptor heterogeneity is that certain agonists such as salbutamol, soterenol and terbutaline are clearly selective in their ability to produce bronchodilation rather than cardiac stimulation. This property forms the basis of their therapeutic usefulness. It is therefore particularly surprising to find that these agents do not demonstrate at~y significant selective affiniO' for beta~ adrenoceptors when examined in ligand binding studies~U. Perhaps a partial explanation for this paradox lies in the finding that these agonists may have a greater efficacy at beta, adrenoceptor= than at beta~ sites. Thus, in rat and rabbit lung that possess a predominance of beta, and beta~ receptors respectively, although salbutamol and soterenol displace [SH]DHA binding with equal affinity in both preparations, these agonists stimulate adenylate eyclase to a greater extent ie betas-rich rat lung membranes ~. If one considers the large amplification of the signal from receptor to the final biological response, these findings could provide an answer to the tissue selectivity of these beta~ agonists. Several years ago, Jenkinson" proposed a related explanation for the selectivity of salbutamol-like compounds. He argued that if there was a

TABLE i. Tissta:distributionof beta-adrenoceptorsubPypes

Tissue Rat lung Rabbit lung Bovinelung Rat ventricle Rat spleen Rat uterus (oestrugendominated) Rat uterus (progesteronedominated) Rat erythrocyte Rat reticulocyte Rat cerebralcortex Rat cerebellum Rat striatum Rat limbicforebrain

Total beta-adreno.'eptor density (fmol x mg pro,. -L)

Beta~

Beta~

400 350 250 50 250 100

2O 80 25 65 35 20

so 20 "5 35 ~5 Stl

100

0

100

10a ¢~00 120 50 tO0 70

0 0

l~'J It~

~,5 0 65 55

35 100 35 45

%

Allstudieswere performedusingthe ligand( - )-pHIdihydtoalprenolol.Proportionsof beta~and beta, siteswere estimated by computer-assistedcu~e fittingof the atypicaldisplacementcurves generated by highly~lecti~e bet,ator beta- agents (see text).

larger "receptor reserxe" fi,r pulmonar 3 over cardiac beta-adrenoceptors, then partial agonists such as .,albuu-mol could di~pla~ tissue ~lecti~ it) w ithout possessing a ~lcctivc affinity for betas adrent~:eptor~. In any case. the demonstration that certain agonists apparentl.v bind to beta, and beta, adrenoceptors with equal affinity hut induce a more efficient cot:piing of betzu adrenoceptors to adenylate cyclase, could suggest that there is a different interaction between the two beta-adrenoceptor recognition sites with guanine nuclcotide binding proteins and/o:r catabtic sites of aden xlate cyclase. Pre:~minaD experiments in this laborato D a l ~ sug,.~,est that there may be a different int]uence of guanine nudeotides on the interaction of agonists (see Ref. 5) ~'ith beta. and bema adrenoceptors in lung. perhaps a[:ain suggesting that there are different receptor--effector coupling relationships between the t~-o subtypes. At "pical beta-adreno~ptor subtypes As discussed above, information from ligand binding studies suggests that mammalian tissues contain only t~o betaadrenoceptor subtypes, since the pharmacological properties of beta, or betaz binding sites are virtually identical in all mammalian tissues examined to date. However, the recent extension of these techniques to non-mammalian tissue,,, has revealed significant differences in the characteristics of the beta-ad,'ent~.~eptors. particularly in certair_ non-mammalian et3throcytes. A~ian and f.t~g erythrt~.-xtes hase al~vavs been considered to possc~s beta~ and beta2 adn: noceptors rcspecti,.el), and indeed have been used as mt~el sssterns for these receptor subtypes. Minneman and colleagues first suggested that the affinity of certain beta-adrene~ic agents for the turkey e~throcyte betaadrenoceptor differed significantly from either the beta~ or beta2 mammalian receptors is. We have recently a l ~ noted a similar atypical site on chick eDthtoc)tc, and ha~e further shown that the Ix,taadrenoceptor on frog ~r)throc.~te t, al.~ different to mammalim bcta~ or bet:a receptors. Since the d~ffe'.-ences in these eD+throcyte preparations are particularly es ident in respect to the affinities of certain beta-adrenoceptor antagonists but the rank order of catecholamines still re,.emhies beta, or beta, receptors, it is debatable whether one should assign a new clarification to these beta-adrenoceptors. Hobeve;. in view' of other differences relating to receptor-adenylate c.vcla~ coupling, one should be cautious in any extrapolation of

TIPS -April 1981

9g results from these model systems to the situation at mammaRian betat and betat adrenoceptors. Conclusion It is quite evident that the use of ligand binding ha:; provided new information concerning the precise pharmacological characterizat/on and quantification of betaadrenoceptor subtypes. This bi~bemical approach suggests strongly that., in mammalian tismes at least, there are only two subtypes that ~orrespond pharmacologically to betat and beta= subclasses, and further that most tissues possess b o t h subt)pes. Although it can be shown that betat and betat adrenoceptors can co-exist in tig~ues in a non-interacting manner, the definitive identification of distinct receptor subtypes requires the physical separation of the subtypes from solubilised membrane preparation,~, It is possible that a single betaadrenoceptor p~otein could display betat or beta, characteristics, depending upon which acces~ry binding sites are revealed in the intact membranes. We t~cel this is unlikely in view of preliminary studies from this laboratory which indicate that solubili~ed prcvarations, free of the restraint of the membranes, still retain betat and betas pharmacological characteristics. Moreover, the individual recovery of the subtypes from membranes containing both subclasses is very different and it seems

probable that the receptor subtypes are separate proteins. Conclu:,ive evidence on this point, however, awaits the physical separation of betat ~md betat adrenoceptot's from solubilized preparations. There has been considerable debate concerning the 'physiological' or "pharmacological' significance of betat and betam adrenoceptors and the relative rotes of adrenaline and noradrenaline in the initiation of responses at these sitess. It seems probable that there will soon be new information on the individual receptoreffector coupling characteristics and precise Iocalisation of beta~ and betas subtypes in tissues. A*. that time we should be in a better position to discuss the relative importance of these receptors ia normal and disea ~ed tissues. Acknowledgements it is a pleasure to acknowledge the important contributions of a number of coUeagues - D. Barnett, K. Dickinson. A. Richardson. E. Rugg and A. Willcocks - to different aspects of the work described in this revtew. Reading list I Moran. N. C. (19661 Pharmacol. Rev. 18, 503-512 2 Lands. A. M.. Arnold, A.. McAuliff. J. P.. Ladttena. F. P. and Brown, T G. (19671 Nature fLondon) 214. 597-598 3 Daly. M. J. and Levy, G. P. (19791 in Trends in Autonomic PharmaeoloKv (Kalsner. D.ed.), VoL 1. pp. 347-385. Urban and Schwarmntx:rg, Baltimore and Munich

4 Cadsson. E., Ablad, B., Brandstrnm, A. and Carkson, B. ( It172)I,i.~. gci. I I, q53-958 5 Lcfkowitz, R. J. and Hoffman. B. B. (19801 Trend~ Pharm,*col. Sci. I, 314-318 6 Nahorski, S. R. (19781 Ear. J. Pharmacol. 51, 199-2(19 7 Barnett, D. I1., Rugg, E. and Nahorski. S. R. (19781Nature (London) 273, 166-d68 8 Rugg. E.. Barnett, D. B, and Nahorski, S. R. (1978) Mol. Pharmacol. 14,996--1005 9 Minneman,K. P., Hegstrand, L. R. and Molinoff, P. B. ( 19791Mol. Pharmacol, 16, 34-46 10 Zaagsma.J., Oudhof, R,, van der Heijden, P. J. and Plamjc,J. F. (1979) in Catecholamines, Basic and Clinical Frontiers" (Usdin. E., Kopin. I. J. and Barchas.J. eds), Vol. i. pp. 435-437 [ I Nahorski.S. R,. Barnett. D. B. Howlett, D. R. and Rugg, E. (19791 Naunyn.Schmiedebergs Arch, Pharmacol. 307. 227-233 12 Nahorski.S. IL, Howlcn, D. R. and Redgrave. P. (19791 Eur. 1. Pharmacol. 60. 249-252 13 NahorskLS. R. (1979) in Meda'ina/Chemisto,VI (Simkins, M.ed.), pp. 147-156. Cotswold Press Limited.Oxford 14 Jenkinson. D. H. (19731 Br. Med. Bull. 29, 142-147 15 Minneman.K. P.. Weiland, G. A and Molinoff. P. B. (19801Mol. Pharmucol, 17. ]-7

The author graduated in Physiology and Biochemistry from the University o f Soulhampton in 1968 and was awarded a Ph.D. in Pharmacology from Portsmolah School o f Pharmacv m 1971. Following four years as a research fellow in the Department o f Pharmacology, University of Sheffield, the was appoinled Lecturer in Pharmacology and Therapeutics at the University o f Leicester Medical School in 1976. He is c~trrendy Reader in Pharmacology at Leicester and his main research interests relate to Ire characterization and regulation of neurotrattsmitter receptors.

II

Antiestrogens Venkat R. Mukku*, John L. Kirkland* and George M. Stancel ° • Department ofPharmacologg. , The Univ,erxiW o f Te±asMedical 2;choolat Houston, P.O. Box 20708, Houston, Teta~ 77025. U.S.A. and ¢ Division o f Er,doc rinology, Depamru'nl o f Pediatrics, Baylor College o f Medicine, Houston, Texas 770.70. U.S.A.

Antiestrogeas are potentially t~ieful for a variety of purposes. Such drugs could be used to regulate fertility, to treat menstrual and reproductive disorders and ¢o retard the growth of estrogen .~,-nsitive tumors. Experimentally, antiestrogens are valuable tools for analysing the mechanism of estrogen action. Mechanism of ¢sUog, n action Estrogens are thought to enter cells by diffusion (Fig, 1, step a) and combine with a receptor prot,:in present in the cytoplasm (step b). The hormone-receptor complex then migrates to the nucleus (step c) and interacts with nuclear acceptor sites (step EUc*~r/Nmlb-HollandBg~l~ed/ca]Pr¢~. 1~81

d) to initiate changes in transcription which lead to specific target cell responses. Since the ~,3'toplasm is depleted of receptor by nuclear translocation, receptor levels must be increased in the cytoplasmic portion of the cell. This is thought to occur by ~he synthesis of new receptor and the "recycling' of receptor from the nucleus back to the cytoplasm (step e). As a theoretical point we f¢¢1 that a discussion of estrogen and antiestrogen action is best understood from the pharmacological point of view if one considers what is commonly referred to as the 'receptor-hormone complex' as the true 'agonist', and the 'nuclear "cceptor site' as the

true "pharmacological receptor', since it is thought that the interaction of these two elements is Lhe step which actuall) elicits the initial biological response (i.e. an alteration in transcription) of the target cell. To be consistent with the literature, however, we shall continue to refer to the cytoplasmic protein in Fig. I which initially. binds the estrogen as the 'estrogen receptor'. Classes of antiestrogens Most pharmacology texts use the term antiestrogens rather loosely to refer to agents that fall into one of three major classes: triphenylethylene derivatives and related compounds, e.g. clomiphene; 'weak' or 'impeded' estrogens, e.g. ¢striol; and other naturally occurring hormones, e.g. progesterone and androgens. While this article will focus primarily on the first group of drugs, the other two groups are mentioned briefly below. Compounds such as estriol have some estrogenic activity, yet also display antiestrogenic effects when administered jointly with estradiol under certain conditions.