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Agonist and Inverse Agonist Efficacy at HumanRecombinant Serotonin 5-HTIA Receptors as a Function

of Receptor: G-protein Stoichiometry

A. NEWMAN-TANCREDI,* C. CONTE, C. CHAPUT, L. VERRIELE and M. J. MILLAN

D eo P s yI d R S 1 C R 7 Cs P F

( 5 J 1

S up rw m f C H O ( c e 1 a4 p o r eh 5 r a d s b w t sa n[ 1 ( [ 3- y lT w c tn o G -a b t f a s ( a 1 p ep rm b [ S s b t i t r G rf N 1 ( t % ( A e w m s [ Sb T s ea e lb a p a ( = 5 Rm eb v a a f a ( = 9 a R m r 5 1 Tl e xa t s t t l i i c oc R c Rm e( 0 W 1 ( N - {}c y c ld n a [ S b f b l e mp rI c s d i a a d [ S b fb l b 1 i R m b b 2 i R m T d i ti nr G r ( a t p f a ( i t e pa a ( i t n e o i a r h 5 rF ut d s t s i s a G b ic oo t r @ 1 E S L

K er [ S b i a s G S 1

Molecular biology techniques have enabled the cloningand sequencingof multiple subtypesof serotonin (5-HT)receptors (5-HT1to 5-HT7;Boess and Martin, 1994),allof which (apart from the 5-HT3receptor) are coupled toguanine nucleotide binding proteins (G-proteins). Thefirst human 5-HT receptor to be cloned was the 5-HTIAreceptor (Fargin e a 1988) which has been hetero-logouslyexpressed in COS7, HeLa, CHO, NIH-3T3, Sf9and E sc cells, enabling the study of itscoupling to G-proteins and second messenger systems(Boessand Martin, 1994;Newman-Tancredie a 1992;Konings e a 1995;Mulherone a 1994;Bertin e a1992). Although the 5-HTIA receptor is capable ofinteractingwith potassiumand calciumchannels,inositol

* w c os b a T ( 14 1F ( 1 4 1E1 l , 2c

phosphate metabolism and arachidonic acid production(Fargin a 1989; Boess and Martin, 1994), its bestcharacterised intracellular functional response is theinhibition of adenylyl cyclase activity which has beenused to differentiate the agonist–antagonistactivity ofserotonergic ligands at this receptor (De Vivo andMaayani, 1986; Pauwels a 1993). More recently,interest has focussed on measuring the first step in theintracellular activation cascade; the activation of the G-protein itself, by measuring its GTPase activity inhippocampaltissue (Odagaki and Fuxe, 1995)or by thestimulationof [35S]-GTPySbinding in recombinantCHOcells expressing the human 5-HTIAreceptor (Newman-Tancredi a 1996a,b).We have previouslyshownthatstimulation by serotonergic agonists of [35S]-GTPySbinding to membranes of CHO-5-HTIAcells is specifi-cally mediatedby 5-HTIAreceptors and can differentiatebetween agonists,partial agonists and antagonists(New-man-Tancredi a 1996a,b). The efficacy of agonists

451

4 A N ea

corresponds closely to that observed in this cell line forinhibition of adenylyl cyclase activity (Newman-Tancredi e a 1992). In addition, potencies andefficaciescorrespondto those seen in nativehippocampaltissue, for both stimulation of GTPase activity and forinhibitionof adenylylcyclaseactivity,suggestingthat theCHO-5-HTIA cell line, like other such recombinantsystems (eg. GH4ZD1Ocells expressingrecombinantrat5-HTIA receptors, Fowler e a 1992)corresponds to amodel of post-synaptic5-HTIAreceptors. However, it isknown that apparent agonist efficacy can differ depend-ing on the brain region studied. In cortical neurons inprimary culture, for example, ipsapirone and buspironeact as antagonists,whereas in hippocampalneurons theyacted as partial agonists (Dumuis e a 1988).Further-more, ipsapirone acts as a full agonist at presynaptic 5-HTIAautoreceptors(Cox e a 1993).These differencesin efficacy have been attributed to the presence ofsignificant receptor reserve at presynaptic, but not atpostsynaptic, 5-HTIA receptors (Meller e a 1990;Meller and Bohmaker, 1994; Yocca e a 1992).Nevertheless, while several studies have examined theeffect of changes in receptor expression level in clonedcell lines on functional responses (Varrault e a 1992;Bouvier e a 1988), the relationship of receptor toG-protein expression levels has not been quantitativelyevaluated.Hence it is unclear, for example, what level ofreceptor reserve is necessary for a partial agonist toappear “full”. The present study addressed this issue bydeterminingin CHO-5-HTIAcell membranesthe expres-sion level of the recombinant 5-HTIA receptor (bysaturation binding with the agonist [3H]-8-OH-DPATand the antagonist [3H]-S15535,Peglione a 1995)andalso of their coupled G-protein(s) by [35S]-GTPySsaturation binding. This approach allowed the effect ofreceptor:G-protein ratios on agonist efficacy to beinvestigated. Furthermore, although previous studieshave shown that inverse agonistscan reduce intracellularresponses below basal levels (Costa and Hertz, 1989;Barker e a 1994),the influenceof receptor density andreceptor/G-protein stoichimetry on inverse agonist effi-cacy has, so far, not been investigated.The present studyaddressed this issue by determining the negative efficacyof an inverse agonist, spiperone (Sundarame a 1993),in CHO-5-HTIAcell membranes displayingeither a lowor a high receptor:G-protein ratio (RGIOWand RGhighmembranes, respectively).Some of the present data havebeen presented in abstractform (Newman-Tancredie a1996c).

M AA M

C c

Membranes were prepared from recombinant ChineseHamster Ovary (CHO) cells stably expressingthe humanserotonin 5-HT1A receptor (Newman-Tancredi e a1992).Cells were grown in either suspensionor adherentculture. For suspension culture, cells were grown in

“normal” medium containing RPMI-1640 (Sigma, St.Louis, MO, U.S.A.), 2 mM Glutamine, Penicillin/strep-tomycin and 10% foetal bovine serum. Suspensionculture was stopped when the cell density was between0.5 x 106 and 1 x 106 cells/ml (exponential growthphase). Cells were harvested by centrifugation (450g,25 tin, 4“C) and resuspended in ice-cold Earle’sBuffered Saline Solution (EBSS). Cells were re-centri-fuged (450g, 10 rein, 4°C) and the pellet resuspended inbuffer A (HEPES 20 mM, pH 7.5, MgSOd 5 mM) forhomogenisationusing a Kinematic Polytron (maximumspeed, 20 see). The homogenate was centrifuged at48 OOOgfor 25 tin at 4°C and the membrane pelletresuspended in buffer A and frozen at –80”C. Foradherentculture,cells were grown in 225 cmz flasksuntil80% confluentand then treated with cationic liposomes(200 pg/flaskof Lipofectamine@;Gibco, Cergy-Pontoise,France) diluted in 20 ml/flask of Optimem@medium(Gibco). After 5 hr incubation (37”C, 5% C02) themedium was replaced with 150ml ‘normal’medium andthe cells incubatedfor a further 3 days. At the end of thisperiod, the cells in each flask were washed three timeswith 100ml of EBSS before detaching them with arubber policeman. Preparation of membranes was as forsuspension-grown cells. Protein concentration was de-terminedcolourimetricallyusing a bicinchonicacid assaykit (Sigma).

S b C r w [3H]-8 a [3H]-S1

Saturation binding at CHO-5-HTIA receptors wascarried out with 12 concentrations (0.4-10 nM) of [3H]-S 15535(50 Ci/mmol, special synthesis,Amersham, LesUlis, France) or [3H]-8-OH-DPAT(225 Ci/mmol, Amer-sham).Membranes (1W20 pg protein) were incubated inbuffer A at room temperature with radioligand, for 1hr([3H]-S 15535) or 2.5 hr ([3H]-8-OH-DPAT), beforerapid filtrationthroughWhatman GF/B filters (pretreatedwith O.l?iopolyethyleneimine)using a Brandel harvester.Isotherms were analysed by nonlinear regression usingthe program “PRISM” (Graphpad Software Inc., SanDiego, CA, U.S.A.).

S b C r w [ SG

The number of receptor-activatedG-proteins in CHO-5-HTIA membranes was determined in binding experi-ments by isotopic dilution of [35S]-GTPyS (1300 Ci/mmol, NEN) with GTPyS in the presence or absence of10PM 5-HT. CHO-5-HTIAmembranes (30 ,ug protein)were incubated (20 rein, 22”C) with ligands in buffer B,containing 20 mM HEPES (pH 7.4), 3 PM GDP, 3 rnMMgClz, 100mM NaCl and 0.25 nM [35S]-GTPyS. Twoconcentration ranges of GTPyS were tested: 0–10 PMand (L45 nM. In the former case, IC50 values werederived by non-linear regression. In the latter case,saturation binding curves were derived from the inhibi-tion curves to determine the number of G-proteins

R G

activated by 5-HT. The total amount of ligand bound toreceptor-activated G-protein (BOUNDTOT)was calcu-lated by:

BOUND~o~= [35S]-GTPyS~ou~~ X GTPyS~o~/[35S]-GTPSco~c where:[35S]-GTPS~ou~~ = the observed 5-HT-dependentbinding in the tubes (fmol/mg);[35S]-GTPSco~c = [35S]-GTPySconcentration in thetubes (= 0.1 nM);GTPyS~oT= [35S]-GTPSco~c + GTPyS concentration.

D eo e a C r

Efficacy at 5-HTIA receptors was determined bymeasuring receptor-linked G-protein activation with[35S]-GTPyS. CHO-5-HTIA membranes (30 pg protein)were incubated (20 tin, 22°C) with ligands in buffer B.Agonist efficacy is expressed relative to that of 5-HT(= 100%) which was tested at a maximally effectiveconcentration(10 MM)in each experiment.All results areexpressed as mean ~ SEM of three or more independentdeterminations. WAY 100,635 and eltoprazine weresynthesised by J.-L. Peglion, Servier. 5-HT was pur-chased from Sigma. Spiperone and ( f )-8-OH-DPATwere purchased from RBI (Natick, MA, U.S.A).

R

5 r s ab

Whereas cells grown in suspension culture grewexponentially and without clumping until the time ofharvesting, cells grown in adherent culture generateddebris after treatment with the cationic liposomes andbegan to detatch from the surface of the flasks when

r a e 4

c w r T 5 r el ( w d s b w[ a [3H]-S15535(Table 1).Isothermsfitted best to a model which assumed the presence of asingle binding site (Fig. 1). Membranes prepared fromadherent-grown liposome-treated cell membranes (de-noted RG~ig~)expressed about 2.6 times more receptorsthan those grown in suspensionculture (denoted RGIOW),whether the ll~m was determinedwith [3H]-8-OH-DPATor [3H]-S15535(Table 1).In each case, the l?~= of [3H]-S 15535 was 1.6-fold higher than that of [3H]-8-OH-DPAT. In contrast,the valuesof the respectiveligandsdid not alter (Table 1) and CHO-5-HTIAcells grown inadherent culture without liposome treatment did notresult in high receptor expression levels and increasedagonist efficacies (data not shown).

[ S i d b

In CHO-5-HTIA (RGIOW)membranes, inhibition ofbasal [35S]-GTPySbinding with GTPyS (0.1 nM to100PM) exhibited biphasic isotherms, With Icso(high)=2.06 t 0.45 nM (3) and IC50(10W)= 141 f 27 nM (3).The percentage of high affinity sites was 50.5 t 10.9%(3). Inhibitionof 5-HT (10 pM)-stimulated [35S]-GTPySbinding also produced biphasic isotherms withIC5whigh)= 1.79 ~ 0.71 nM = and I = 158 t64 nM (3) but the percentage of high affinity sites wasgreater (85.1 ~ 0.8$Z0(3); Fig. 2A). [35S]-GTPyS satura-tion binding isotherms were derived in both RGhighandRGIOWmembranes for the high affinity (agonist-depen-dent) binding component by isotopic dilution withGTPyS (045 nM; Fig. 2B). The values (about1.5~) and B~~Xvalues (about 1100fmol/mg) for[35S]-GTPyS binding were not significantlydifferent inthe two membranes preparations. Thus, the ratio of

T 1 S ab t C c m w l ( h @ $HTIA

r e l

RGIOW(n) R ( R

[3H]-S 1B ( 1 t 1 ( 4 ~ 6 ( 2

( 1 * 0.07 1.06 ~ 0.23’f

[3H]-8-OH-DPAT11~=(fmot/mg)

(1023 + 80 (5) 2654 t 278 (3)*** 2.590.65 ~ 0.07 0.46 t 0.05

[35S]-GTPYSB ( 1 f 1 ( 1 t ( 0.90

(nM) 1.29 t 0.13 1.54 i 0.14

R/G ratio[3H]-S 15535 1.39 3.98 2.86[3H]-8-OH-DPAT 0.87 2.50 2.87

Saturation binding with [ 15535and [ 3w c o R Rm eS b i f [ S b w d id a d i t M a M T R r c d tB f e r ab t B for [ S b T R rc fb d R v b t r R v D m *( i nd e7 > 0 O * O c wc oR d ( S t

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[[3H]-S 15535] ( [ [ (

F 1 C Hs ab w [ 3a [ 1 S b w [O a [ 1 w c o b i r w C c m ab c o2 m H ( 7 a 5 m M a r t f 2 a 1 h rI nw t eb r f a n b d w 1 5 Ps a m o d d ef r ee P S b w[ 1 P B S ab w [ 3( R m ( R

m

receptors to G-proteins was increased almost three-foldin RG~i~~versus RGIOWmembranes.

E fa C Hr

The degree of stimulation by 5-HT of [35S]-GTPYSbinding in RGIOWand R membraneswas compared.In RGIOWmembranes 5-HT (10 PM) increased bindingfrom 111 t 17 to 268332 fmol/mg (n= 6) whereas inRG~ig~membranes binding increased from 107 t 18 to239 t 50 fmol/mg (n = 5). There was no significantdifference in basal or agonist-stimulated binding(P> 0.05, two-tailed Student’s t-test). The efficacy of5-HT, eltoprazine, WAY 100,635 and spiperone forstimulation of [35S]-GTPyS binding was determined inRG~ig~and RGIOWmembranes (Table2). The full agonist,5-HT, displayeda two-fold increase in potency in RGhl~hcompared to RGIOWmembranes. Eltoprazine showed asignificant increase in efficacy whereas WAY 100,635did not alter [35S]-GTPyS binding from basal levels ineither membrane preparation. In contrast, spiperonedisplayed negative efficacy, inhibiting [35S]-GTPYSbinding about twice as much in RGhighas in RG1OWmembranes without a change in IC50(Fig. 3).

D I

5 r e xl i C c

The present study characterised the effect of receptor-G-protein ratios on agonist and inverse agonist efficacy.Membranes from cationic liposome-treated adherent-grown cell membranes (RGhigh)displayed a receptorexpression level 2.6-fold higher than membranes fromsuspension-grown cells (RGIOW),in accordance withreports that receptor expression levels in this cell lineand others can be modulated by a variety of cell culture

conditions including serum deprivation, high levels ofconfluence and the presence of cationic liposomesthemselves (H. Sundaram, personal communication;Lewis a 1996; Seidman a 1996). Thisemphasises the need to define cell culture conditionswhen using recombinantcell lines. In any case, althoughexpressionlevels (B~,X)in CHO-5-HTIAcells, measuredby [3H]-8-OH-DPATand by [3H]-S 15535 (Pegliona 1995), were higher in membranes from adherent-grown cells, there was no change in their dissociationconstants (K&), indicating that no akeration in reCeptOr/ligand binding affinityhad taken place but only a changein the number of sites labelled. However, in bothmembrane preparations, [3H]-S 15535 yielded a B~,Xvalue 1.6-fold higher than that of [3H]-8-OH-DPATprobably reflectingthe capacities of the two radioligandsto bind to different coupling states of the recombinant5-HTIA receptor. The agonist, [3H]-8-OH-DPATlabelspreferentially 5-HTIA receptors which are coupled toG-proteins (Newman-Tancredi a 1992;Sundarama 1993). In contrast, [3H]-S 15535 binding is onlyslightlyinhibitedby guaninenucleotides,suggestingthatit acts as a weak partial agonist/antagonist(Peglionet a1995).Radioligandsof this type are capable of recogniz-ing both coupled and uncoupled receptors and it isthereforeconcludedthat [3H]-S15535occupies a greaterproportion of available 5-HT1~ receptors than [3H]-8-OH-DPAT. Nevertheless, the fold change in 1l~,.between the two types of membranes was virtuallyidentical regardless of the radioligand used (2.58 and2.59-fold, Table 1) indicating that the receptor numberhas increased and not just shifted from an uncoupled to acoupled conformation,as could be hypothesisedfrom anincrease in [3H]-8-OH-DPAT Z3~,Xalone.

R G r a e 4

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3-

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1-

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O Basal

-io -9 -8 -7 -6 -5

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1 1 I I Io 2 4 6 8 1

[ G ] (

Fig. 2 I no [ S b b i dw G [ S b e xw co o m eo C c e l o hr eG -r ( a R mr eR ec a s i w ep i t m o d d eS rw o i a l t i ne xPA B ( s a 5 ( p M(s [ S b a R m wd ei t p o c oo Gb O a 1 p P B B a 5 ( #s t[ S b t R ( s aR m e( s w d i tp o c oo G b O a 1 nT d w t ra d i t M aM t g a s ab i f ad eG b I S p o t

s ab i

D o 5 - HG iC Hc m

In common with [35S]-GTPYS binding in humanneuroblastoma cells (Traynor and Nahorski, 1995),unlabeled GTPyS inhibited agonist-stimulatedbindingbiphasically,consistentwith the presence of high affinityand low affinity binding components in CHO-5-HTIAmembranes. The low affinity site most likely representsendogenous (agonist-independent)GDP/GTP exchangeby non-receptor-activated G-proteins. This does notdifferentiate between those G-proteins which can be

1oo- A- 0

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F4 0 5-HT RGIow- 50- . 5-HT RGhigh

5; WAY RGIow

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F E 5 e W 1 as [ S b R a RC c m [ S b em w c o m C c el h r G r (RGI.~ and RGhighm r R c a si w e p t m t dS r w o l t ie O s = R m F sb = R m P S 5( a W 1 ( [ S ba R a R m D a e ap m s i 5 (P S e [ S bR a R m D a e ap m s i 5( O P I s [ Sb ia R a RGhighm D a e

a p b [ S b

4 A N ea

Table 2. Stimulation of [35S]-GTPySbinding to CHO cell membranes with low (RGIOW)or high(RGhi,J 5-HTIAreceptor expression ]evek

RGIOW RGhigh

Em= (%) (.) EC50(nM) Em= (%) (n) EC50(nM)

5-HT 100 (6) 18.7 f 2.0 100 (4) 8.6 f 2.0**Eltoprazine 52.7 t 2.2 (4) 242 & 79 93.2 ~ 4.3 (5)*** 178 t 70WAY 100635 3.8 t 3.7 (3) n.d. 4.1 + 4.0 (4) n.d.Spiperone~ –17.0 t 2.5 (6) 86.6 f 11.5 –28.1 t 3.8 (4)* 126 f 34

Efficacy at CHO-5-HT1~receptors was determined by stimulationof [35S]-GTPySbinding, yieldingestimates of maximat efficacy (E&J and effective concentration 50 (EC50).Data shown aremeanst SEMof (n) independentdeterminations.For eltoprazine and WAY 100,635,I&a valuesare expressed as a percentage of the maximat stimulation given by 5-HT.J5Forspiperone, Evalues for negative efficacy are expressed as a percentage of basal [ S]-GTPYSbinding.*P < 0.05; **P < 0.01; ***P<0.001 compared with corresponding RGIOWdata (two-tailedStudent’s t-test). n.d. = not determined.

activatedby 5-HTIAreceptorsand thosewhich cannot.Incontrast, only a small amount of the high affinity site(IC50(~ig~)= 1.79nM) was detected in the basal bindingcurve, indicating that it constitutesthe agonist-dependentbinding state (Fig. 2A). The ‘K~’ of [35S]-GTPyS foragonist-activated G-protein was determined in isotopicdilution saturation binding experiments and yielded avalue in RGIOWmembranes (1.3 nM; Table 1) similar tothat of the IC~~(hi~h)above. The B~a value f 5stimulated [35S]-GTPyS binding in RGIOWmembranes(about 1.1pmol/mg) does not differentiate between thesubtypesof G-proteinsactivatedby the 5-HTIAreceptorsin the cell line, providing, rather, a global determinationof all the G-proteinsactivated by 5-HT-boundreceptors.Indeed, 5-HTIA receptors can couple to at least two G-proteins in CHO cells (Giaz and Giu3;Raymond e a1993).[35S]-GTPySsaturationbindingof 5-HT-activatedG-proteins in RGhig~membranes yielded no significantdifference in B~~ or Kd values from those of RGIOWmembranes. It therefore appears that, in contrast to otherstudies of G-protein levels (Milligan, 1993; Shin e a1995), in the present system the change in cultureconditionsdoes not affect the number of 5-HT-activatedG-proteins but only the number of recombinant 5-HT1~r eT i nr n i Rv R m et w t a oc i G -n r i a ti ni r eG r f a p1 r e ct a pf rp G -( [ 1 b T 1

A a i a e o [ Sb t R c m

Eltoprazine, stimulated [35S]-GTPySbinding to RGIOWmembranes with a relative efficacy of 52.7Y0.Thisagonistaction is consistentwith (a) the inhibitionof [3H]-eltoprazine binding to rat brain membranes by GTP andGppNHp (Sijbesma e a 1990), and (b) the partialagonist action of eltoprazine for inhibition of forskolin-stimulatedcAMP accumulationin rat hippocampalslices(Schipper e a 1990).Eltoprazine also acts as a partialagonist i v where it elicits spontaneous tail flicks,

flat-bodyposture and hypothermia (Millan a 1994).These responses are mediated by post-synaptic 5-HTIAreceptors, which lack receptor reserve (Yocca a1992) and therefore support the contention that RGIOWmembranes are a model of post-synaptic 5-HTIAreceptors (Introduction; Newman-Tancredi et a1996b). In contrast to eltoprazine, spiperone exhibitednegative efficacy in RGIOWmembranes, inhibiting [35S]-GTPyS binding below basal levels. As discussed else-where (Newman-Tancredi a 1997),this is consistentwith the observation that [3H]-spiperone binds withhigher affinity to CHO-5-HTl~ receptors which are notcoupled to G-protein (Sundaram a 1993).Inhibitionof basal activity is characteristic of inverse agonists (forexample Freissmuth a 1991;Costa and Hertz, 1989;Barker a 1994)and maybe due to an induction andfor stabilisation of a receptor conformation which is notcapable of activating G-proteins (Samama a 1994).

E i r G si a a

The present study quantified the receptor/G-proteinstoichiometry in CHO-5-HTIA cells, showing that athree-fold increase in receptor:G-protein ratio inducedalmost a doubling of the relative efficacy of eltoprazine(from 52.7 to 93.2%; Table 2), without a change in itspotency. It may be expected that further increases in thereceptor:G-protein ratio would result in a left-shift of theeltoprazine stimulationcurve, as observed in the case ofthe full agonist, 5-HT, which exhibited a two-folddecrease in its ECS~value c 0.01; Table 2). Increasesin partial agonist efficacy at 5-HTIAreceptors have beenreported by Varrault a (1992) whereas others (Fargine a 1989;Hoyer and Boddeke, 1993)found no changein the potency of 5-HT for adenylyl cyclase inhibitionbetween two HeLa cell lines expressing different levelsof recombinanthuman 5-HTIAreceptor. However, noneof these studies determined the number of receptor-activated G-proteins, so the receptor:G-protein ratio isnot known and receptor reserve may be absent. Incontrast, an increase in apparent efficacy and potency istypical of physiological systems which display receptor

R G r a e 4

reserve, such as control of Raphe neuronal firing by5-HTIAautoreceptors(Cox e a 1993).Indeed, a recentstudy compared the isomers of 8-OH-DPAT for theirelectrophysiological inhibition of Raphe neuron firing.The (+) isomer (an efficaciousagonist in vitro)was morepotent than the (-) isomer (partial agonist in vitro) butboth fully inhibited neuronal firing (Lejeune e a inpress). Accordingly,eltoprazinealso acts as a full agonistin this system (Millan e a 1994). Future studies willaddress the issue of receptor reserve in CHO-5-HTIAcells by classical receptor inactivation experimentsutilising N-ethoxycarbonyl-2-ethoxyl,2-dihydroquino-line (EEDQ; Pinto and Battaglia, 1994).

E o i nr G s toi na o i a

N previous studies have systematically investigatedthe effect of R:G ratios on inverse agonist efficacy. Incontrast, the present data showed that a three-foldincrease in 5-HTIA receptor:G-protein ratio roughlydoubled the negative efficacy of s a RGhighv R m ( 2 T i s t tt i i p e o t ae lT c om b d f to bF g t b G at d n r t p o a i m be t a h r e l ( iR m em i b Ga cH i t p s t tf i i r n d n a b[ S b T s t b Ga ci a a i m ( t n oG -i l iT i s b to bt i RG1OWmembranes, the [%1-S15535 Bin,. is 40% greater than the ll~u for 5-HT-stimulated [35S]-GTPyS saturation binding, suggestingthat equilibrium may already lie towards a receptor-coupled conformation of the cellular G-proteins. Underthese conditions,increasesin basal activitywoulddependon an increase in G-proteinexpressionlevel (for exampleBurstein e a 1995).Secondly,it appears insufficienttopostulate (cf. Schiitz and Freissmuth, 1992), that inverseagonists such as spiperone simply bind to/stabilise G-protein uncoupled receptors. The present data shows thatan increase in receptor expression level aspiperone’s negative efficacy. It therefore appears thatspiperone also stabilises c receptors in a con-formation which is n capable of G-protein activation.Therefore, a higher density of spiperone-occupiedreceptors, which “traps” G-proteins in an inactive state,reduces the pool of G-proteinsavailablefor activationbynon-spiperone-occupied receptors. This hypothesis isconsistent with the “cubic” or “general” ternarycomplex model, described by Kenakin (1996) and Fong(1996), in which inactive receptor can existnot only in anuncoupled state but also in a state which is coupled toG-protein.

E i r G sn a

WAY 1000,635(Fletcher a 1996),did not exhibitany positiveor negative intrinsicefficacy in either RGIOWor RGhighmembranes.Indeed, we have shown that WAY100,635antagonisesboth 5-CT-induced stimulation andspiperone-inducedinhibition of [35S]-GTPyS binding toCHO-5-HTIARGIOWcell membranes (Newman-Tancredie a 1997),demonstratingits abilityto block the actionsof both agonists and inverse agonists. The present studyfurther confirms that WAY 100,635 is a neutralantagonist because it remains “silent” even underconditionswhich ‘amplify’any partial agonistor inverseagonist effects, such as those of eltoprazine andspiperone. Other compounds which were claimed asantagonistsat 5-HTIAreceptors, such as NAN 190,BMY7378, SDZ 216,525 and WAY 100,135, have subse-quently been found to display partial agonist propertieswhen tested in physiologicalsystemswhich exhibit highdegrees of receptor reserve (Greuel and Glaser, 1992;Routledge, 1996).In contrast, the present study providesfurther evidence that WAY 100,635is indeed devoid ofintrinsic agonist i a activity.

C

The influence of a three-fold increase in receptorexpressionlevel on both agonist (eltoprazine)and inverseagonist (spiperone) efficacy at recombinant human5-HTIA receptors was quantitatively demonstrated. Un-like previous reports, the present study quantitativelydetermined the number of receptor-activatedG-proteins,by [35S]-GTPyS saturation binding. This revealed thatthere was no alteration in maximal G-protein labellingbetween R and RGhighmembranes. Hence, thealmost two-fold increase in the positive efficacy ofeltoprazine and the negative efficacy of spiperone isattributableto the higher receptor/G-proteinstoichiome-try. Further, since there was no effect of increasedreceptor density on basal [35S]-GTPySbinding, we con-clude that spiperone is able to induce/stabilise 5-HTIAreceptors in a G-protein-coupledbut inactive conforma-tion.

R

B L W S S a S( C a 5 rt r n i a a r lJ B C 2 1

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