Neurotransmitter receptors in the brain-molecular approaches

TINS. November 1978

123

affinity being substantially higher than the affinity of the transmitters themseke.,. Some researchers haxe ~peculated that this higher affinit.,, implle,, a "ph)~iological" actixitx indicating the existence of endogenou~ antagonist~. More probabl.~. the antagoni,t., were di,coxered because of their extremei~ potent pharmacological properties: x~e would hexer haxe heard of them but for their high receptor affinity. Thu.,, atropine, the actixe ingredient of the belladonna plant i~ knoxvn Solomon H. Snyder for its potent medicinal effect.,,, and the i snake to,in~ for their poi,onous action,. The interactions o f the agonists and antag¢~nisla o[ neurotranxmitter.~ with their receptors In labelling neurotran~milter receptor, are helping to elucidate the medtanisms o f action o f ncurotran~mitters m .;).naptic trans. in binding ,tudie, one deai~ onl~ with the mission. In many eases, the agonize "sites" dilYer from the atttagonist3 ".;ites" on a receptor. recognition portion. Some mechani~n) the actual site occupied b.r a molecule affects the "state" o[ tiw receptor and therefore nlu',t Iran,,lale [ran,qllilter o r other agoni,t its function. Local ionic concentratio,t~ can also h,,re.htrth~,r ellec;:, m~ the control ~'xerted recognition into a Iunctioilal neuronal b r the receptor on s.rnapti¢ trmtsmisston. Hence [or research u',,r~er.~ m thi.~ lwhl the alteration. ~hile antagonl,t~ bind xxilh problem is no longer: II'd; a ~ubstance bind to a re~'eptor? A o , it i~: it hat i~ thr cm~equal or exert greater potency but ehc~t sequence o f the substance binding ? $ o l Snrder rerh,,.~ o~r present ~tate o[ progre.~.~ m no change. Hox~ receptor, differentlale answering these questions. agoni,,t,, and antagonist, i, one ",,econd Synoptic transmission by chemical neuro- reversible links to the receptor t~f relatixely generauon" ta,,k of receptor re~earch. transmitters is fundamental to information modest affinity. The opiate receptor. Another I,, to characterize iuq hoxx processing in the brain. Over the past though not first thought of a,. a con- neurotran~,mitter recognition trigger, a 20 years biochemical features of neuro- venlional neurotransmitter recvptor, ha~ change in Ion permeabiht.x or c.xch¢ transmitter synthesis, storage, release, meta- provided the protot)pe for ,ucces~ful nu¢leotlde l\~rmat,on, the ",econd me,,epbolism, and reuptake have been greatly biochemical in~tigation~ of neurotran~- ger," of neurolrallMilP,.~i~fl. clarified. Interaclions of neurotrans- miller receptors in the brain TM. In our mitters with their receptor sites have been laboratory, the potent opiate antagonist Differential intera¢lion.~ of alloni.~t~ the last to yield to biochemical analysis. naloxonc ~a~ u~d labelled ~ith tritium. and anlIgoniq~ ~ith rt,t~plor~ Ik-¢au~¢ of their ~,-on,lderable theraWhy the lag? Whereas neurotrans- R¢lali~c:y high specific acti~it) enabled mitter metabolism could be studied with us to utdize low enough concentration, at peutic II111;)t,rtall¢C. ilIltil~Otll~|, ,Ind IlllXf~d well-characterized enzymological tools, which binding to .~pecilic receptor, would a g o n l , t ,~11t,l[/Onl,,t ftlrlll~, o f o p i a t e tlrtlg,~ there was little precedent for labelling occur with greater probabilit) than non- x~ere d F,l a j o r ~'oflcern in otlr tirol ,ttldIC,~ receptors. Enzyme assays are facilitated specific binding. A ~acuum filter ~),,tenl of the O~lat¢ receptor Ilo~e~er. blnd.ng by the amplifying processofenzymological permitted rapid but thorough ~;t,,hing of experlnlenl, ~ah reel,el-ion.free buffer, catalysis, while neurotransmitter receptor brain membrane~ so that non-,pecilicall) I'Jiled ro dllTerentlate agonl..t, and ,inta~/binding assays involve a single labelled bound pH]naloxone could be ~a~hcd onl~t,. |ha., mt,rphme and it, ~.la,,,l¢ molecule per receptor. The elegant away while receptor bound drug mould antagonl~t nalorphlne had identical polencharacterization of nicotinic acetyl- remain attached. This approach had cle~ in competing for IJII]naloxon¢ bradcholine receptors of the electric organs of earlier permitted characterization of re- ins. Xddlng ,,odium to the incubation invertebrate fish such as the electric ~1 ceptors for peptide hormones such a~ medium merca.,:d the binding of antagonIst., and decreased the binding of agonize,. succeeded in part because this receptor insulin and 81ucagon ~. constituted as much as 20% of membrane With ~arious modifications, the opiate The "sodium" effect ~as I~tenl. t~.'curring protein of the electric organs, and unique receptor techniques facilitated demon- at I m~ N a . and ~¢le¢ti~e. being elicited snake toxins combined with the acetyl- stration of receptor sites for mo~t ncuro- to a hmtted extent b) hlhlum. ~ho~e choline receptor with specificity and transmitter~ in the brain~ including bio- alonuc radiu,, rc,,cnlble~ that of ,.t~llum. affinity bordering on the irreversible. How genie amiaes [noradrenaline (including a- but not at all b) olh:r mono~alenl cations might one detect receptor proteins in the and ~receptors), serolonin, acet)lcholine ~uch a,, ix~ta.,,iur% -ubJdium. or caesium. The ddferentml effect., of .,,odium on brain which we know constitute only about (bolh nicotinic and muscarinic receptors), one-millionth of brain protein? A r'adio- and histamine], amino acid ncurotrans- agonize and antagonist binding Io the labelled neurotransmitter or drug would mittcrs [GABA, glycine, and glutamic opiate receptor favoured a "t~o slate" surely be bound by charged and uncharged acid], and a rapidly gro~ing li~t of pep- model of neurotran~mitter receptor operagroups to tissue membrane constituents un- tide transmitters [lh),rotropin releasing ling in an analogou~ manner to the related to the specific receptors; very little hormone (TRH), angiotcnsin, neuro- alloster;~: model of enz)mes, and propo~d of such'non-specific" binding ought to mask tensin, sub,stam.'e P. bombesin, insulin. for receptors 7 )cars earlier, independently. completely any true receptor interactions. and vasoactive intestinal peptide tVIP)] by Jean-Pierre Changeux ~ and Arthur Karlin'. in his model, ncurolransmitler (Table I). Effective receptor 18bem~ in general, labelled antagonists have receptors can exist in t~o intercomerlible In the brain, investigators h~ve made provided the most efficient routine re- stal~ which favour binding of agonists do with labelled compounds that form ceptor labels because of their nanomolar and antagonists r~pectixely. Functional

Neurotransmitter receptors in the brain-

molecular approaches

IP.h~vN~qNo~|h.l~lollandIB~mtdcal P~ets 1971;

TINS- Nor~d~r 1978

124 TABLe I. I ~

t ~ p l o ~ Mmliq

Lipmh f~w ~l~ellinll Ilgowm+

AIo~K+s

AmajmU~,

Biogt~i; a m / ~

a-Nmad~-naWc

l+H;L--~mmli,~ [ qll,,~lorzdrenalin¢ PHiAdfenahn¢ i'XlAdtemilm¢

Do~mm¢

l+Xlt~m-,¢

PXlWl~101 I'Hll~hydgetrlocryptin¢

l'XlDi~rml~ l'lllPro~amlol

l'XlH~dop~idol ['XlS~rol,¢naol I'l~-lFlup~l~,ol I'N1LSr) I'HlDil~'~rowlm~p.ne I'HILSD I'nlMcp~ra~m~

i'HlApomorphin¢

St,rmonm H~tamn~.H, Mim:a, ing acclykhohne

|JHl~k'fotonin

Nicotinic acelykholin¢

I'HlA~etylcholinc

[qHI~NB IGuinm.'lidiwflImmla~) I'"IIONB l"q lProl~ll~.n~l~kholm~ mm~rd l'=+l]x-l~.proloxm I'HI+VaJ~~J'+ , o ~ .

['HlAcetykholme

Am/no ~ M ; GABA (y-Aminobut.~r~ acidl ('H]GABA I'HJMw.ctmol GlulamK aetd GI)¢in¢

PXlBic~:ulim¢ pH|KainK acid pH]~chain¢

Pip,de|

Enlurphalin (Opiate)

Anl~oi¢~,n II TRH Neurolcnun

Sut~¢am:e P rIP Imulin

! JHJt.lOfphin¢ l'HlDll~)dromofphin¢ l'HlLg~orphanol ['fllMorphinc 1:H~3~ymocphof~ PHlMcI.Enk¢lPhalin l'H]Leu-Enkephalin PHl2.f~.ala.
synaptic transmission occurs only when substances bind to the "agonist" state of the receptor. Receptor blockade takes place when drugs bind to the "antagonist" state, making fewer agonist states available for the neurotransmitter. Interconversion of the two states is affected by some ion, whose permeability changes represent the 'second messenger" of synaptic transmission. Thus, in the test tube, added ions change binding of agonists and antagonists, while at the synapse receptor interactions of the neurotransmitter alter binding of the critical ion, which in turn changes the ionic permeability of the membrane. This model implies that sodium is the ion whose permeability underlies neurotransmission by opiate-like substances. Direct neurophysiological studies by Zieglg~insberger~, Herz, and their colleagues support this x.lew. The effects of sodium on the opiate receptor have also provided a means for grading drugs as agonists, antagonists, or mixed agonistantagonists. Since the latter group of drugs offer the greatest promise as relatively non-addicting analgesics, opiate receptor binding has provided a screening

pHJNaloso~ PHi.~hr¢~on¢ iqlJDi~¢norphin~ pfllL~allorphan

l'"ll~a f,-Ic'u.- Anilwlcndn U ~.~

procedure used by the pharmaceutical industry for developing new therapeutic analgesics. Ionic effects upon receptor binding provide a means i'or probing the relation between neurotransmitter recognition and subsequent events. In the case of glycine, a major inhibitory neurotransmitter in the brainstem and spinal cord, its hyperpolarizing effects are known to involve increases in chloride permeability. Binding of [ZH ]strychnine, the glycine antagonist to glycine receptors, is diminished by physiological concentrations of chloride and by other anions in proportion to their ability to mimic the neurophysiological actions of chloride at glycine synapses. Mulfiplici~ of receptors Other neurotransmitter receptors possess distinct agonist and antagonist preferring states, a-Noradrenergic receptors can be labelled by agonists such as the transmitter itself, pH]noradrenaline, as well as by pH]adrenaline and the agonist drug pH]cionidine. ~t-Receptor sites can also be labelled by the potent antagonist pH]WB.4101, a drug related to lihe a-

blocker piperoxane. In general, agonisls have rdativdy higlmr affinity for arecg~ors labelledby the pIt]agonists, while antagonists have relatively higher affinity for the sites labelled by the antagonist PHiWa-4t01. However, a variety of evidence demonstralgs that two distinct z-receptors are laheUed by these ligands ~hich only coincidentally display reciprocal alfinities for agonists and antagonists rffspectively". Thus, the relative numbers of [aH]agonist and pH]WB-4IOI binding sites vary markedly in different regions of the brain and different peripheral orlpms, if these radioactive drugs labelled a single receptor which interconverted between two states, the total number of binding sites should he the same in all tissues, in the rabbit duodenum, which contains exclusively pH]agonist binding with no pH]WB-4101 binding, relative contractile effects of a-adrenergic drugs closely parallel thei; affinities for pHJa-agonists and do not correlate with their potencies in competing for PHIWB-4IOI binding. By contrast, in tissues such as the vas deferens and heart, which contain abundant pH]WB-4101 binding, but no detectable pH]agonist binding, the physiologdcal potencies of a.drenergic drugs fit closely with their affinities for pH ]WB-4101 sites but not with their potencies at pHJagonist sites. This demonstration, both o.n physiological and biochemical grounds, of two very ~eparate a-receptors may explain diver~ actions of noradrenaline in ~arious parts of the brain. Differential hehaviour;d and neurophysiolosical influences might in some cases be due to actions at distinct a-noradrenergic receptors. Both of these ,,-receptors are localized postsynaptically since they are not reduced by destruction of noradrenergic neurones. Interestingly, the postsynaptic a-receptor for which agonists have selective high affinity (referred to as the zz-reeeptor) has a drug specificity resembling that of presynaptic autoreceptors. (Autoreceptors are sites on noradrenergic nerve endings in the brain or periphery where noradrenaline, once released, appears to inhibit further release, and where z-adrenergic blocking drugs enhance noradrenaline release.) Two distinct populations of /3-noradrenergic re,'eptors have been recognized for IO years./3,-Receptors in the heart are responsible for the ability of catecholamines to increase the force and rate of cardiac contraction. O~-Receptors, selectively concentrated in the lung, mediate the bronchodilating effects of catecholamines. Differences" between ~1-and ~z-

TINS- November 1978 rex'plots permitted the design of catecholamine agonist drugs which selectively stimulate the fl=-regeptors of the lung, relieving the symptoms of asthma, with a lesser tendency to cause tachycardia via cardiac flt-receplors. Unlike most other neurotransmitter receptors, agonists and antagonis~ compete with the same affinity for p-receptor binding of either radioactive agonists or antagonists, indicating that ~receptors do not exist in distinct agonist or antagonist preferring states~. Dopamine receptors can be labelled with both [q4]agonists and [ZH]antagonists; algonists have preference for sites labelled by [=H]agonists while the reverse is true for antagonists. In part [~H]agonists and [~H]antagonists appear to label different interconvertible states of one dopamine receptor. However, in addition, they also may label physically distinct receptors. The synaptic effects of dopamine involve a dopamine-sensitive adenylate cyclase which increases the formation of cyclic AMP when stimu. lated by dopamine. Relative drug poten. ties at the dopamine cyclase resemble their effects at dopamine receptors labelled by [zH ]agonists and differ from potencies at sites labelled by [~H]butyrophenone antagonists such as haloperidol and spiroperidol. Brain lesion studies also show that the doimmine r~,nsitive adenylate cyclase and a major portion ofdopamine receptors labelled with [=H]agonists are localized to physically distinct entities from the s i ~ labelled by pH ]butyropbenones =. it is fairly well established that the anti. ~¢hizophrenic effects of the major anti. psychotic drugs, comprising phenothiazincs and butyrophenones, involve blockaxle of dopamine receptors. Which of the two populations of dopamine receptors are involved in these therapeutic effects, those associated with the dopaminesensitive adenylate cyclase or those labelled by [tH]butyropbenones? The most potent of all, antischizophrenic drugs are the butyrophenones. For instance, one hatyrophenone, spiroperidol, is hundreds of times more potent than the classic neuroleptic cldmwomazine. The potencies of a large series of neuroleptic drugs do not correlate with their ability to block the dopamine-sensitive adenylate cyclase but correlate very closely with their affinities for dopamine re~ptors labelled by [~H]butyrophenones. Thus, while ~iroperidol is no more potem than chiorpromazine at the dopamine sensitive adenylate cydase, it is up to 50 times more potent than chlorpromazine at sites labelled by [ZH]butyrophenones. Thus, of the two

125 TABLE li. Ro~.ptors whose binding is regulated by guanine nudeotides

logical responses, a notion propos,--d for the neuromuscular junction 20 )ears ago by Bernard Katz and Stephen Thesleffs. Chanl~'s associated ~ith Receptor physiolo~c~l effects and directly demonstrated in mcotinic cholinergic receptors of invertebrat~ elecGlucagon Cyclic AMP ira"tease tric organs t=. In any event, gaanine ~Noradrcneqlic Ckclic AMP increase nucleotides seem to "rcscnsitize" the reDopamine Cyclic A M P increase a-Noradrcnergic Cyclic AMP decrease~ ceptor by decreasing its affinity for the Calcium Igrmeability neurotransmitter and putting it into increase? a state which will function optimally. Opiate Cydic A M P decrease" Whether such "optimal functioning" in all Cyclic GM P increase" cases involves a coupling to adcnylate Calcium effects? Angiolemin It None known cyclase is not clear. Thus, angk, tensin receptors, which are not thought to be types ofdopamine receptors, those labelled associated with adenylate cycla.~, are by pH]butyrophenones mediate the regulated by GTP in the same wa.v as antischizophrenic effects of neuroleptic f/-adrenergic receptors 3. Moreover. it is drugs. not at all clear whether aden.,.late c.~clase represents the physiological ~¢cond G ~ i ' l e o t i d ~ Md mes~nger for opiate or a-noradrenergi¢ ..m~r=mmilter receptors receptors. Cyclic AMP is thought to be a second In summary, relatively simple binding messenger at ~-noradrenergic and dop- techniques ha~e provided rapid and efli. amine receptors where synaptic effects are cient assays for a wide range of neuro. associated with an increase in ~'clic transmitter receptors in the brain. These AMP formation. While the situation is techniques are now providing a means to less clear with a-adrenergic receptors and reach beyond the recognition site of the opiate receptors, in some situations stimu- receptor to elucidate way.,, in which s)naplation of these sites pros'okes a decrease tic transmission alters nemonal function. in cyclic AMP. Guanine nucl¢otides have been shown by Martin RodbelF. R~lhl= I ~ Robert LcfkowitP, and their collaborator~ I. Chanlcux. J.-P. (1966~ .qfo/. Pburmacol. 2, .tbq 392. to play a major role.in linking the recog2. Cualre~a~,b. P. and tlollenberg, M (lg"h) v.ition site of hormone and ~,adrenergic Adr. Protein Chem..10, 2~ I 4~ I. receptor~ to the adenylate cyclas¢ system, 3. OIo~sman. ll., Baukal, A. and ('at,, K. J. Effects of GTP and its analogues upon /1974)J. Bml. Chem. 249. 664 666. receptor binding of variou.~ neurotran~4. Karhn, A. J. I19~'/) J. l heor. B,,! I~,, 3O6 320. mitter~ provides another probe whereby ~, Kale, B. and The~l¢ff, S. (195"/) J. Pl#l~iol transmitter recognition may be related I.t8.6.t 80. to subsequent events. GTP is thought to o. Lefko~itz. R. J, and Wdham,,. L I. (19"~ activate receptors by linking them to the Adr. Crclic ~uel. Res. 9. I ! q.. adenylate cyclase. Thus. in binding studies 7. Rodb¢ll, M. (1978) In: O. Fol¢o and R Paolclti (eds). :$lol¢cular Biolmt~ and Phur. one would anticipate that GTP should ma¢ologr of Cyclic Nucleotides. Flsc~ier affect only agonist binding interactions, North-Holland Biomedical Press. Amblerwhich is indeed the case for all neurodam. pp. 1-12. transmitter receptors so far studied. At 8. Schwarcz. R.. Creche. !.. ,'o.~le. J. T. and Snydcr, S. H. (1978) Nature (London) fl-adrenergic, a-noradrenergic, dopamine, 271,866- 878. and opiate receptor. GTP decreas~ the affinity of agonists for receptor sites while 9. gnyder, S. H. and BcnnctL J. P.. Jr. ~,1976) Annu. Rev. Physiol. 38. 153-175. having no effect on antagonists (Table !!). I0. gnydero $. H. tl9781 Am. J. P~lehlat. 1~5. Why should a decrease in the affinity of 645-652. the neurotransmitter be associated with I I . U'Prichard. D. C. and Snydcr. S. H., Proc. ,Vat. Acad. $ci. U.S.A. (in press). more efficient coupling of the receptor to VVcber. M.. Pfeuly. T.-D. and Changewt. the cyclase? One possibility is that under 12. J..P. |!975) Proc. Nut. Acad. $ci. U.S.A. 7.'. the binding conditions employed the re3443.3447. ceptors exist in an abnormal state which 13. ZiqllB/msbcrger, K. W. and Ba)erl, H. (197b) Brui.~ Res. ! 15, I I ! -128. binds the neurotransmitters too tightly for physiological functioning. Excessively avid binding of a neurotransmitter by $. H. Snyder is Distinguished Service Peoftssor of its receptor in intact animals is thought Pharmacology and Psrehiatry of the Departments of Pharmacology and Experimental Therapeutics to be respoja~,.'ble for the process of and PJ)'ckiatry and Behavioral gtiences, Johns desensitization in which chron;c exposure Hopkins University School of Medicine, Baltimore. to the neurotra,:smitter decreases physic,- MD 21205, U.S.A.