Proc. Nati. Acad. Sci. USAVol. 86, pp. 7321-7325, October 1989Biochemistry

Antagonists with negative intrinsic activity at 6 opioid receptorscoupled to GTP-binding proteins

(guanine nucleotide-binding regulatory proteins/GTPase/ternary complex)

TOMMASO COSTA* AND ALBERT HERZDepartment of Neuropharmacology, Max-Planck-Institut fuer Psychiatrie, Am Klopferspitz 18a, Martinsried, Federal Republic of Germany

Communicated by H. W. Kosterlitz, June 19, 1989 (received for review November 29, 1988)

ABSTRACT According to classical models of drug-receptor interactions, competitive antagonists share with ago-nists the ability to bind to a common site on the receptormolecule. However, they are different from agonists, as theycannot trigger the "stimulus" that leads to biological re-sponses-i.e., they lack intrinsic activity. For those receptorswhose signals are transduced to effector systems by GTP-binding regulatory proteins (G proteins), a mechanistic equiv-alent of such a stimulus is an increased ability of agonist-boundreceptor to accelerate nucleotide exchange and thus GTPaseactivity on the G-protein molecule. Here we show that for amember of this family of receptors (6 opioid receptors inmembranes of NG108-15 neuroblastoma-glioma cells), twotypes of competitive antagonists can be distinguished. One typehas no intrinsic activity, since it neither stimulates nor inhibitsthe GTPase activity of G proteins and its apparent ainmity forthe receptor is not altered by pertussis toxin-mediated uncou-pling of receptor and G protein. The second type, however, caninhibit GTPase and thus exhibits negative intrinsic activity; itsaffinity for receptors is increased following uncoupling from Gproteins. The existence of antagonists with negative intrinsicactivity may be a general feature of several classes of neuro-transmitters or hormone receptors and calls for a reevaluationof biological effects produced by competitive antagonists.

Although ,u and 8 opioid receptors can be clearly distin-guished on a pharmacological basis (1), recent evidence (2, 3)indicates that these two types of receptors share the ability tointeract with GTP-binding regulatory proteins (G proteins).In this respect, they belong to a large family of hormone andneurotransmitter receptors whose signals are transmitted toenzymes and ion channels across plasma membranes byintervening G proteins (reviews in refs. 4-6). Activation ofone or more G proteins, which results in increase of GTPaseactivity, is the first detectable biochemical event that followsrecognition of this group of receptors by agonists, regardlessof the sort of signal that is actually propagated to effectormolecules (5, 6). Receptor-mediated activation of G proteinsinvolves the establishment of a ternary complex betweenligand-occupied receptor and G protein, as suggested longbefore the isolation ofG proteins. The findings (i) that guaninenucleotides exert negative heterotropic effects on the affinityof the receptor (7) only when the receptor is occupied by anagonist (8, 9), (ii) that a receptor prelabeled by agonists can besolubilized in a higher molecular weight form than whenprelabeled by antagonists (10), and (iii) that agonists but notantagonists display complex binding isotherms in the absenceof guanine nucleotides (11) were all explicable by a generalmodel according to which agonists, but not antagonists, pro-mote the association of receptor to G protein in membranesand that the resulting complex can be destabilized by guanine

nucleotides (11). The correctness of this model was recentlydemonstrated by direct reconstitution of purified ,-adrenergicreceptors and the stimulatory G protein of the adenylatecyclase system (Gs) in liposomes (12, 13). Accordingly, theintrinsic activities of receptor ligands represent their ability tostabilize the ternary complex and range from null values forantagonists, which passively occupy the binding site, to var-ious degrees ofpositive values for partial and full agonists (11).At muscarinic, D2 dopaminergic, and A1 adenosine receptors,however, guanine nucleotides exert more complex effects,since agonist and antagonist binding to these receptors arereciprocally modulated by purine derivatives (14-16). Toaccommodate these observations, an extension ofthe ternary-complex model has been proposed that assumes that antago-nists can be "active" in promoting the dissociation ofreceptorfrom G protein (17). The implicit prediction of this extendedmodel is that an antagonist may have negative intrinsic activitythat should be apparent in its ability to inhibit the tonicactivation of G proteins resulting from the spontaneous asso-ciation between "empty" receptor and G protein (precoupledcomplex). Reconstitution studies in liposomes have shown inat least one case that an antagonist can inhibit the constitutiveactivation ofG proteins by receptors in the absence of agonist(13), but it is not clear whether such effects can occur in intactmembranes. Here we show that for a 6-type opioid receptor innative membranes of NG108-15 cells (mouse neuroblastoma-rat glioma hybrid cell line), two types of antagonists can bedistinguished: those that lack any, and those that have somenegative intrinsic activity.

MATERIALS AND METHODSGTPase Assays. Culturing of NG108-15 cells, membrane

preparation, and GTPase assays were performed as de-scribed (18-20). The GTPase assay mixture (0.1 ml) con-tained 50 mM Hepes/Tris (pH 7.5), 150 mM either NaCI orKCl, 10 mM MgSO4, 1 mM adenosine 5'-[,8,y-imidoltriphos-phate (Li'), 0.5 mM ATP (Tris), 5 mM creatine phosphate(Tris), 2.5 units of creatine kinase, 2.5 mM cyclohexylam-monium phosphate, 0.2 mM dithiothreitol, 0.2 mM EGTA,200 nM [y-32P]GTP (4-7 x 104 cpm per pmol), 5-8 ,ug ofmembrane proteins, and opioid ligands as indicated. Reac-tions were started by the addition of membranes, allowed toproceed at 37°C for 10 min, and arrested by the addition of0.1ml of ice-cold 40 mM H3PO4. The amount of Pi released wasdetermined using activated charcoal as described (18). High-

Abbreviations: G protein, GTP-binding regulatory protein; DADLE,[D-Ala2,D-Leu5]enkephalin; ICI 174864, [NN'-diallyl-Tyr1,Aib2'3]Leu5enkephalin; ICI 154129, [N,N'-diallyl-Tyr1, 4-(CH2S)Phe4,Leu5]enkephalin. The terms "neutral" and "negative"indicate antagonists having null and negative intrinsic activities,respectively. They are used for conciseness and not meant to suggestany new terminology.*Present address: Laboratory of Theoretical and Physical Biology,National Institute of Child Health and Human Development, NIH,Building 10, Room 6 C 101, Bethesda, MD 20892.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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affinity GTPase was determined following the subtraction ofthe cpm of Pi hydrolyzed in the presence of 50 AM GTP(low-affinity GTPase) (19). Low-affinity GTPase was notaffected by any opioid ligand at any concentration.

Pretreatment of Cells or Membranes. In some experimentsmembranes or cells were preincubated with an antagonist tofree receptors from possible contaminating endogenous lig-and. Membranes (1 mg/ml) were preincubated with or with-out 100 ,M MR 2266 in 50 mM Hepes/Tris, pH 7.5/0.5 mMEGTA/0.5 mM dithiothreitol for 30 min at 4°C, diluted 5-foldwith the same buffer, and centrifuged at 25,000 X g for 15 minat 4°C. After three cycles of resuspension and centrifugationin the same buffer they were assayed for GTPase in thepresence and absence of agonist or antagonist (both 10 ,uM)as indicated. Confluent monlayers of NG108-15 cells werepreincubated in growth medium (18) in the absence or pres-ence of either MR 2266 (100 AM) or [D-Ala2,D-Leu5]-enkephalin (DADLE, 100 nM) for 24 hr. Cells were har-vested, washed from the ligand as described (18), and frozenas a pellet (-70°C) prior to membrane preparation andGTPase assay. For N-ethylmaleimide treatment, membraneswere centrifuged, to remove traces of dithiothreitol present instorage buffer (18), and resuspended (1-2 mg/ml) in 50 mMTris/HCl (pH 7.5) containing various concentrations offreshly prepared N-ethylmaleimide. Incubations lasted 30min at 4°C and were stopped by the addition of 10 mMdithiothreitol (final concentration). After centrifugation, themembranes were resuspended and assayed for GTPase. Forpertussis toxin treatment, confluent monolayers ofNG108-15cells were incubated with the indicated concentrations oftoxin for 24 hr. The degree of ADP-ribosylation produced invivo was monitored by the decrease of pertussis toxin-catalyzed incorporation of [32P]ADP-ribose into a 40-kDasubstrate of the membrane (20) and was in good agreementwith the diminution of agonist-stimulated or antagonist-inhibited GTPase (data not shown). Inactivation of opioidreceptors in intact cells with 8-chlornaltrexamine was per-formed as reported elsewhere (20).

Radioligand Binding Studies. For equilibrium binding stud-ies, membranes were prepared and [3H]diprenorphine (40Ci/mmol, New England Nuclear; 1 Ci = 37 GBq) binding wasassayed (18). The reaction mixture (2 ml) contained 50 mMTris/HCl (pH 7.5), 10 mM MgCl2, 0.1 mM EDTA, 0 or 100mM NaCl (as indicated), and 300 ,g of membrane proteinsand was incubated 60 min at 25°C. Data were analyzed inaccordance with models for the binding to one or two formsof the receptor based on mass action law, by the method ofMunson and Rodbard (21). Statistical comparisons betweenmodels were also performed as described (21), with the levelof significance at P = 0.01. The experiments were done atleast twice, with at least two different batches of membranes.The absolute level of GTPase activity varied as much as2-fold, but the relative proportions of agonist and antagonisteffects on GTPase varied <10% between different batches ofmembranes.

RESULTSEffect of Opioid Antagonists on GTPase. Agonist-mediated

activation of high-affinity GTPase activity in native mem-branes for receptors that interact with the Gi/G0 group of Gproteins is usually amplified in the presence of millimolarconcentrations of NaCl (22, 23). Under these conditions,GTPase in membranes prepared from NG108-15 cells wasstimulated in the presence of the opioid agonist DADLE, andthis stimulation was completely blocked in the presence ofthe peptide opioid antagonist (N,N'-diallyl-Tyr',Aib2'3,Leu5]enkephalin (ICI 174864; Aib, a-aminoisobutyric acid).However, the antagonist also inhibited basal activity. Theinhibition of basal activity was reproducible but small (10%),

and thus not easy to characterize. We performed a detailedstudy on the ionic requirements for this effect (unpublishedwork). The addition of cyclohexylammonium ions (2.5-5mM) to the reaction mixture slightly potentiated the inhibi-tory effect of the antagonist. A pronounced effect wasobserved upon replacement of Na' by K+ (Fig. 1). Substi-tution of K+ for Na' (maintaining Cl- constant at 150 mM)produced an increase in basal GTPase activity, a decrease inthe net activity stimulated by the agonist, and a correspond-ing increase in the inhibition by ICI 174864. Examination ofthe concentration-response curves of several antagonists formodulation of GTPase indicated that [N,N'-diallyl-Tyr1,qi(CH2S)Phe4,Leu5]enkephalin (ICI 154129), a close ana-logue of ICI 174864, was also effective in reducing basalactivity; naloxone produced a much smaller effect; the ben-zomorphan antagonist MR 2266 had no inhibitory effect,whereas the oripavine antagonist diprenorphine was a partialagonist (Fig. 1 a and b). Thus, three types of antagonists canbe distinguished: those exhibiting negative intrinsic activity(ICI analogues), those with a partial negative effect, such asnaloxone, and those with null intrinsic activity, such as MR2266. The relative intrinsic activity of agonists and antago-nists was dependent on the type of cation present in thereaction mixture. For example, diprenorphine was a weakerpartial agonist (30-40% of the maximal effect produced byDADLE) in Na' than in K+ (maximal effect 70-80% of thatof DADLE). Antagonists were affected in an opposite butsymmetrical manner: in K+, ICI 174864 produced a maximalinhibitory effect clearly larger than that of ICI 154129,whereas in Na' there was little difference in the maximal

GTPase

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_-A - 0.6* ~~~~~Log(MR2266) Log (MR 2266)

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FIG. 1. Effect of opioid ligands on GTPase activity in membranesof NG108-15 cells. (a and b) High-affinity GTPase was assayed in areaction mixture containing 150 mM NaCl (a) or KCI (b) and variousconcentrations of opioid agonists and antagonists (U, DADLE; o,diprenorphine; o, MR 2266; *, naloxone; A, ICI 154129; A, ICI174864). Basal activity is indicated by a broken line; points are meansof triplicate determinations. Data are representative of three addi-tional experiments performed with different batches of membranes.(c and d) Concentration-response curves for the stimulatory effectof the agonist DADLE, studied in 150 mM NaCl (c), and theinhibitory effect of ICI 174864, studies in 150 mM KCI (d), in thepresence of 0-1 ,uM MR 2266. Fractional response is the ratio ofGTPase activity in the presence of ligand to that in its absence.(Insets) Schild plots (24) of the data. DR, dose ratio.

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inhibition produced by the two antagonists (Fig. 1 a and b).The lack of effect of MR 2266 on GTPase (we observed onlya small stimulation in the presence of K+) is due to absenceof intrinsic activity (either positive or negative) and not lackof affinity for the receptor. In fact, increasing concentrationsofMR 2266 were able to produce parallel rightward shifts inthe concentration-response curves of both the agonist (stud-ied in Na', Fig. ic) and the antagonist (examined in K+, Fig.ld). Schild plots (24) obtained from these experiments (Insetsof Fig. 1 c and d) yielded similar pA2 values (pA2 = negativelogarithm of the dissociation constant of the antagonist) forMR 2266-mediated antagonism of DADLE (8.2 ± 0.1) andantagonism of ICI 174864 (7.9 ± 0.12). The effect ofMR 2266was stereospecific: its inactive enantiomer MR 2267 blockedneither the effect ofDADLE or that of ICI 174864 (data notshown). Thus, a ligand devoid of intrinsic activity can an-tagonize in a similar fashion either the "positive" effect of afull agonist or the "negative" effect of an antagonist.

Reversibility and Dependence of the Effect of Antagonists onReceptor Occupancy and Coupling. The question remained asto whether these findings could be explained in terms of trueability of some antagonists to reduce the probability ofspontaneous interactions between unoccupied receptors andtransduction protein, or whether they simply indicated thatnative membranes were contaminated by an endogenousagonist. Indeed, opioid peptides are synthesized in smallamounts by the NG108-15 cell line (25). Fig. 2 shows that thissecond explanation is unlikely. Preincubation of membraneswith 100 ,uM MR 2266 (sufficient to prevent any binding ofagonists to the receptor), followed by extensive washing, didnot abolish the inhibitory effect of ICI 174864 (Fig. 2 Upper).Likewise, in membranes prepared from cells that had beengrown in the presence ofMR 2266 (100 ,uM, 24 hr), the effectof ICI 174864 was comparable to that measured in controlcells (Fig. 2 Lower). In contrast, in membranes obtained fromcells that had been exposed to the agonist DADLE (100 nM,24 hr) to desensitize and down-regulate opioid receptors (18,26), neither agonists nor antagonists produced any effect onGTPase (Fig. 2 Lower).Exposure of cells to low doses of pertussis toxin, or

membranes to low concentrations of N-ethylamaleimide, is

an effective way to covalently modify a critical cysteineresidue in the Gj/GO group of G proteins (27). These modi-fications result in loss of their ability to interact with recep-tors (28, 29). Either N-ethymaleimide treatment (Fig. 3a) orexposure to pertussis toxin (Fig. 3b) reduced in parallelagonist-mediated stimulation and antagonist-mediated inhi-bition of GTPase. Both manipulations also reduced "basal"activity, indicating that a substantial amount of this activityreflects an activated state of the G protein, inasmuch as theintrinsic basal activity of purified G proteins is not altered bypertussis toxin (29). Alkylation of opioid receptors in intactcells with the irreversible antagonist 13-chlornaltrexaminealso diminished the effects ofboth agonist and antagonist andresulted in 20-25% reduction of basal activity (Fig. 3c).

Multiple Affinity Forms of the Receptor for Agonist andAntagonist. Antagonists that neither promote nor oppose theformation of the ternary complex between ligand-occupiedreceptor and G protein do not discriminate between high- andlow-affinity forms of receptor (11, 17) and their affinity is notchanged by pertussis toxin treatment (29, 30). However, ifanantagonist had greater affinity for the uncoupled form of thereceptor than for the coupled form (17), its apparent bindingaffinity would conceivably be increased by toxin treatment.The binding isotherms ofDADLE, ICI 174864, and MR 2266(Fig. 4 a-c) were obtained from competition for the bindingsites labeled by [3H]diprenorphine and compared in mem-branes prepared from cells that had been treated with per-tussis toxin. Following toxin-mediated uncoupling, the ap-parent affinity of the receptor was diminished for the agonist,increased for the "negative" antagonist, and unchanged for

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FIG. 2. Effect of preexposure of opioid receptors to MR 2266 inisolated membranes (Upper) or intact cells (Lower) on the ability ofICI 174864 to inhibit basal GTPase. Assays were performed in thepresence of 150 mM NaCl.

Log (N-ethylmaleimidel Mb

Log tPTX] ng ml-1c

41L-8 -7 -6

Log tCNA] M-5

FIG. 3. Effect ofN-ethylmaleimide (a), pertussis toxin (PTX) (b),or j3-chlornaltrexamine (CNA) (c) on GTPase measured in theabsence (basal) or presence of agonist (DADLE) or antagonist (ICI174864). GTPase was assayed in 150 mM KCI (a and b) or NaCl (c).Data are means of triplicate determinations (± SEM).

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1.0

0.6

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FIG. 4. Binding isotherms of DADLE, ICI 174864, and MR 2266for opioid receptors in membranes prepared from NG108-15 cellspreincubated for 24 hr in the presence (A) or absence (i) of pertussistoxin (10 ng/ml). Binding was measured (in the presence of 100 mMNaCl) as competition for the binding sites labeled by [3H]-diprenorphine (0.25 nM). Nonspecific binding (measured in thepresence of 10 gM diprenorphine) was subtracted. B/Bo is the ratioof specific binding in the presence of competing ligand to that in itsabsence. Bo was 0.29 and 0.11 pmol/mg of protein in control andtoxin-treated membranes, respectively.

the "neutral" antagonist. The leftward shift in the competi-tion curve of ICI 174864 produced by pertussis toxin wasmore pronounced when binding was assayed in the absenceofNa' (see also Fig. 5), because, as reported previously (31),this ion itself increases the affinity of this antagonist. Whenthe binding isotherms of ICI 174864 obtained in untreatedmembrane were analyzed sequentially by models assumingone or two distinct affinity forms of the receptor, the modelinvolving two classes of sites provided a consistently betterfit (P < 0.005; Fig. 5). In contrast, the two-site model did notimprove significantly (P = 0.12) the goodness ofthe fit for thebinding isotherms obtained in membranes that had beenADP-ribosylated by pertussis toxin in vivo (Fig. 5). Thecompetition curves of MR 2266 were satisfactorily modeledby assuming a simple bimolecular interaction with a singleclass of sites for both the control and the toxin-treatedmembranes (data not shown).

DISCUSSIONThe major finding of this study is that antogonists for aG-protein-coupled 6 opioid receptor can display a spectrumof intrinsic activities that ranges from null to negative values,as determined from their abilities to inhibit basal GTPaseactivity in intact membranes. This inhibitory effect of antag-onists is neither due to contamination of membranes byendogenous opioid agonists nor due to the putative existence

B/Bo1.0

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FIG. 5. Competition of ICI 174864 for the binding sites of[3H]diprenorphine in membranes obtained from cells treated witheither pertussis toxin (PTX, A) or its diluent (control, *). Membraneswere prepared and assayed simultaneously. Binding was measured in10mM MgCl2 in the absence of NaCl. Specific binding in the absenceof ICI 174864 (BO) was 0.58 and 0.49 pmol/mg in control andtoxin-treated membranes, respectively. In control membranes therewas a considerable improvement (P < 0.005) in goodness of fit witha model involving two classes of sites (solid line) as compared to amodel with one class of sites (dotted line). In toxin-treated mem-branes a model involving one class of sites could not be rejected (P= 0.12). Estimates (± SEM) of the percentage of receptors in thehigh-affinity (RH) and low-affinity (RL) forms and the correspondingdissociation constants (KH and KL) were as follows: RH = 53 9%,RL = 47 8%; KH = 75 + 22 nM; KL = 1.05 0.23 ktM. Intoxin-treated membranes the curve was consistent with a singleaffinity form with K = 63 + 5 nM.

in the membrane of a form of receptor tightly bound to anagonist. In fact, the neutral antagonist MR 2266, which lacksintrinsic activity, could reverse the effects on GTPase ofboththe agonist and the negative antagonist ICI 174864; fromSchild plots it is apparent that MR 2266 has similar affinitiesin each instance. This observation is incompatible with theidea that basal GTPase activity results from a quasi-irre-versible agonist-bound form of the receptor. Moreover,washing of intact cells or membranes after they were exposedto a saturating concentration of neutral antagonist did notprevent inhibition of GTPase by the negative antagonist. Theeffect of the negative antagonist was abolished in parallel tothat of the agonist when loss of receptor responsiveness wascaused either (i) by interfering with the receptor itself byagonist-mediated down-regulation or alkylation by an irre-versible antagonist or (ii) by interfering with the ability of thereceptor to couple to G proteins by ADP-ribosylation bypertussis toxin or alkylation by N-ethylmaleimide. Collec-tively, these data clearly indicate that the inhibitory effect ofICI 174864 on GTPase does not result from reversal ofstimulation by contaminating endogenous agonists, but thatit requires a functional G protein able to interact with thereceptor and that it depends on receptor occupancy by theantagonist. In addition, the degree of negative intrinsic ac-tivity of antagonists for GTPase is correlated with an abilityof the antagonist to discriminate between the free and theG-protein-bound form of the receptor by having high and lowaffinities. Thus the data are best explained by, and provideexperimental support for, the model of Wregget and De Lean(17), which predicts that antagonists may be "active" byhindering the ability of receptors to associate spontaneouslywith G proteins in membranes. The negative intrinsic activityof ICI 174864 can also explain the notable discrepancy inaffinity for binding and potency in bioassays noted previouslyfor this antagonist (31-33).A further suggestion arising from this study is that basal

GTPase activity in NG108-15 cell membranes is due tostimulated activity, although it is not possible to decide

AF MR 2266

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whether this apparent activation results from a truly spon-

taneous interaction between empty receptors and G proteinsor from another mechanism. Spontaneous activation of Gprotein by the receptor in the absence of agonist occurs whenP-adrenergic receptors and G. are reconstituted in liposomes;this tonic activity is stereospecifically reduced by the antag-onist alprenolol (13). Furthermore, D2 dopaminergic anda2-adrenergic receptors can be purified as tight complexeswith a G protein, and the GTPase activity of the complexescan be stimulated by agonists (34, 35). However, the spon-

taneous activity that results from the insertion of P-adren-ergic receptors and G protein in phospholipid vesicles largelydepends on the ability of thiols to activate this receptordirectly (36). We have shown that the inhibitory effect ofantagonists is clearly detectable only when basal GTPaseactivity is enhanced by replacing Na' with K+ in the reactionmedium. It is noteworthy that under physiological conditionsG proteins are exposed to the intracellular milieu, where theconcentration of K+ is high and that of Na' is low. Appel-mans et al. (31) showed that Na' enhances the apparentaffinity of ICI 174864 for 8 opioid receptors both in rat brainand in NG108-15 cell membranes. The magnitude of theNa'-induced reduction of IC50 described (31) was larger forICI 174864 than for ICI 154129 and thus correlates with thedegree of negative intrinsic activity observed for these an-

tagonists in the present study. This finding suggests that Na'selectively regulates the spontaneous association betweenreceptor and regulatory G protein in native membranes andthat a larger proportion ofthe uncoupled form ofthe receptor,which has high affinity for the negative antagonist, is avail-able in the membrane in the presence of Na'.The existence of antagonists with negative intrinsic activity

is likely to be a general phenomenon that is not limited to theopioid receptors in this particular cell line. Guanine nucle-otide-mediated effects on antagonist binding have been re-

ported for muscarinic (14), D2 dopaminergic (15), and Aladenosine (16) receptors. These observations strongly sug-

gest that antagonists with negative efficacy may exist formost, if not all, G-protein-coupled receptors. This hypothesishas several interesting implications. (i) The interpretation ofantagonist affinities and their use for receptor classificationare of great importance. If an antagonist has higher affinityfor the form of the receptor that is not associated with Gprotein, its apparent dissociation constant would be expectedto vary depending on the proportion of preformed receptor-Gprotein complexes in different membranes. Moreover, thecompetition of a negative antagonist with a full agonist maybe not a simply linear, for the two ligands will induce twodistinct, although interconvertible, forms of the receptors(17). Hence, if an antagonist has negative intrinsic activity,differences in pA2 values of that antagonist determined indifferent tissues and with different agonists would not un-

equivocally reflect receptor heterogeneity.(ii) The interpretation of biological effects observed in the

presence of an antagonist may be affected. The concept thatan antagonist may have biological effects opposite to those ofagonists because of an ability to stabilize an antipodal con-formation of the receptor is well established in the area ofy-aminobutyrate/benzodiazepine receptor research; theterm "inverse agonism" has been proposed to describe suchan interaction (37). At G-protein-coupled receptors, biolog-ical effects observed with antagonists are usually interpretedas the result of their ability to block receptor activationproduced by an endogenous agonist. However, some of thebiological effects of antagonists may possibly be due tonegative rather than to zero efficacy. For instance, muscar-inic receptors in atrial cell membranes are coupled to K+channels via a pertussis toxin-sensitive G protein (38) and can

depress basal K+-channel activity when occupied by theantagonist scopolamine (39). Should the occurrence of neg-

ative antagonists become a general finding for these type ofreceptors, the distinction among antagonists that have nointrinsic activity and those that have negative intrinsic ac-tivity will not be ofpurely academic interest. Some biologicaleffects produced by these antagonists in vivo or in vitro maynot necessarily indicate tonic receptor stimulation by endog-enous neurotransmitters. Similarly, the receptor hypersen-sitivity that often follows chronic exposure to antagonistsmight not simply be the result of hindering endogenousagonistic imput to the receptor.

Christine Gless performed these experiments with superb skill. Weare also grateful to Dr. R. Alan North (Oregon Health SciencesUniversity) for stimulating discussions, Dr. David Rodbard (Nation-al Institutes of Health) for helpful reviews of the manuscript, andUrsula Bauerle for cell culture and artwork. This work was sup-ported by the Deutsche Forschungsgemeinschaft, F.R.G.

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