Intramembrane Interactions between Neurotensin Receptors and Dopamine

D2 Receptors as a Major Mechanism for the Neuroleptic-like

Action of Neurotensin" K. FUXE,bsC G. VON EULER,b L. F. AGNATI,d

E. MERLO PICH,d W. T. OCONNOR,b S. TANGANELLI,e X. M. LLb B. TINNER,b

A. CINTRA,b C. CARANI,d AND F. BENFENATId b Departments of Histology and Neurobiology, Pharmacology

Karolinska Institutet S-104 01 Stockholm, Sweden

dDepartment of Human Physiology and Endocrinology University of Modena

Modena, Italy eDepartment of Pharmacology

University of Ferrara Ferrara, Italy

INTRODUCTION

Nemeroff and colleagues originally observed that centrally administered neurotensin (NT) exerted a neuroleptic-like action, which probably was related to an interaction with ascending dopamine (DA) pathways, especially the mesolimbic DA systems,' implicating a role of NT also in the pathogenesis and treatment of schizophrenia.2 In 1983 the hypothesis was introduced that a powerful NT-DA interaction in the basal ganglia was due to an intramembrane interaction between NT and DA receptors.3 The NT receptor was later shown in both in vitro and in vivo studies to interact with the DI This hypothesis was based on the observations that in in vitro mem- brane preparations of the basal ganglia, NT in nanomolar concentrations reduced the

a This work has been supported by a grant (04X-715) from the Swedish Medical Research Council and by a grant from the United States Public Health Service (NIMH 44211, Neuroscience Center for Research in Schizophrenia).

C Address for correspondence: Prof. Kjell Fuxe, Department of Histology and Neurobiology, Karo- linska Institutet, Box 6O400, S-104 01 Stockholm, Sweden.

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FUXE et al.: MECHANISM FOR NEUROLEPTIC-LIKE ACTION 187

affinity of the D2 agonist binding sites.3 It was also proposed that the antagonistic NT modulation of D2 receptors explained the neuroleptic-like activity of NT (for re- view, see Refs. 7, 8, and 9). Recently, however, an alternative hypothesis has been introduced by Kalivas and collaborators,I0 namely that antagonism of DA-dependent locomotor activation is related to the formation of a NT-DA complex, in this way reducing the ability of DA to bind to the D2 receptor and thus reducing DA-dependent transmission.

In the present article recent evidence supporting the view that the major mech- anism for interaction between NT and DA in the central nervous system is an intra- membrane interaction between NT and DZ receptors will be presented.

IMMUNOHISTOCHEMICAL CHARACTERIZATION OF NEUROTENSIN-CONTAINING NEURONS IN THE

BASAL GANGLIA

The interaction between NT and DA takes place mainly within the substantia nigra, the neostriatum, and the nucleus accumbens. Available evidence indicates the exis- tence of NT receptors on DA cell bodies of the substantia nigra and the ventral teg- mental area, as shown both by quantitative receptor autoradiography and by means of in situ hybridization techniques. 11-13

We can now report in the present communication that NT IR nerve cell bodies, demonstrated after acute haloperidol treatment within the neostriatum and nucleus accumbens, costore enkephalin (ENK)-like IR based on the analysis of the two im- munoreactivities in thin adjacent sections (5 pm thick) (FIG. 1). In contrast, a similar analysis revealed that hardly any substance P IR nerve cell bodies were associated with NT-like IR. These results strongly support that NT IR is predominantly present within the GABA-ENK efferent output systems of the basal ganglia that innervates the external part of the globus pallidus (for references, see Ariens et al.33). This system is mainly regulated via D2 receptor systems located on the striatal GABA and ENK IR neurons and dendrites. In contrast, the other major output system of the basal gan- glia, namely the GABA-substance P system to the zona reticulata of the substantia nigra and the internal part of the globus pallidus that is mainly regulated by DI re- ceptors, appears not to develop any NT IR following acute haloperidol and colchicine treatment.

These morphological results are in line with previous findings that DZ receptor antagonists, but not DI receptor antagonists, increase NT IR within the basal gan- glia, especially those neuroleptics that produce extrapyramidal side-effects. 15 Thus, these results open up the possibility that the development of a D2 receptor supersen- sitivity is, at least in part, related to the ability of this blockade to interfere also with the release of NT costored within the striatal GABA-ENK neurons, operating to re- duce DZ receptor transduction. The NT loop may, therefore, represent a way for stri- atal nerve cells to regulate the sensitivity of their own DZ receptors.

An alternative explanation for the present findings is that the D2 receptors exert a restraining influence on the gene expression of NT and that DZ blockade results in an increased NT synthesis in the GABA-ENK cells associated with an enhancement of NT release. This continuous release may then result in a rapid desensitization of the NT receptors, controlling antagonistically the D2 receptor sensitivity and leading to the development of a DZ receptor supersensitivity phenomenon. I6

188 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 1. Studies on the coexistence of NT/ENK IR in adjacent transverse cryostat sections of neostriatum of male rats after treatment with haloperidol (2 mg/kg, Lp.) and colchicine (100 pg, i.v.t.) 24 hr before killing. Rats were perfused with 4% paraformaldehyde and 0.1% picric acid in 0.1 M phosphate buffer after a short rinse with 0.9% saline. The brains had been dissected and postfixed for 90 min after which the brains were transferred into 10% sucrose in phosphate buffer. Adjacent cryostat sections were cut 5 brn thick and the respective primary antisera applied onto adjacent sec- tions. ENK (kindly supplied by Dr. Elde) or NT (kindly supplied by Dr. Dockray) antisera were both diluted 1:400. The adjacent sections were left at 4°C overnight. The following day a secondary an-

FUXE et al.: MECHANISM FOR NEUROLEPTIC-LIKE ACTION 189

NEWROTENSIN MODULATION OF DOPAMINE-REGULATED BEHAVIORS

Another set of experiments indicate that NT diminishes locomotion and reward behavior in the rat via actions on mesolimbic DA neurotransmission (for review, see Nemeroff et a1.I). Thus, NT injected into the nucleus accumbens antagonizes the be- havioral activation produced by d-amphetamine and cocaine as well as DA-induced locomotion and rearing. w 1 9 These effects may be related to the existence of the dem- onstrated NT-Dz receptor interaction within the dopaminoceptive cells of the nucleus accumbens (see above). Kalivas and collaborators have claimed that the formation of the NT-DA complex is instead the mechanism for the antagonism of DA-dependent locomotor activity by NT.10 In this way the availability of DA in the extracellular fluid for DZ receptor activation is reduced. Thus, NT binds DA directly with a dissocia- tion constant of about 10 nM, the Arg8-Arg9 being the critical position of the NT mole- cule for binding DA. In order to analyze the validity of these interpretations, we have tested the effects of DA-NT microinjected into the nucleus accumbens on locomotion in rats, after the DA nerve terminals within the nucleus accumbens have been degen- erated via local injections of 6-OHDA in this region. The development of the sub- sequent DA receptor supersensitivity within the nucleus accumbens was found to make possible the use of low doses of NT for the antagonistic experiments.

The 6-OHDA lesion selectively reduced to about 20% the levels of DA in the nu- cleus accumbens (nonlesioned rat: 7352 f 232 ng/g tissue; lesioned rats: 1487 f 118 ng/g tissue), the levels of noradrenaline (NA) being unaffected. The DA depletion in striatum was less than 55%. Lesioned rats appeared highly sensitive to DA micro- injections into the nucleus accumbens, the increase in locomotion being still present at the dose of 32.6 fmol. A fixed dose of NT (179.3 pmol) was selected for the an- tagonism experiments. Coadministration of NT and DA did not affect the locomotor activity in control rats while in lesioned rats this dose of NT antagonized the effects of almost all doses of DA (FIG. 2). By using the Kd values indicated by Adachi and coworkers,I5 we calculated the amount of DA complexed by this concentration of NT. The results indicate that NT binds about 2% of DA when DA is administered at a 32.600 pmol dose, and less than 0.5% at the remaining doses (TABLE 1). Even if these calculations give only an approximate estimation of the real values, the quan- tities of DA complexed by NT do not seem to be large enough to explain the difference in locomotor activity observed when NT is coadministered with DA in lesioned rats (see FIG. 2).

In the search for a possible alternative explanation of this effect, we studied the distribution and the density of NT receptors in the limbic areas of the lesioned rats by using receptor autoradiography.

At the rostral level (AP 2.7 mm) the density of 12Wabeled NT binding in the 6-OHDA-lesioned rat was increased in the most rostral nucleus accumbens, in the medial part of the frontal cortex and cingulate cortex, and in the region that vertically crosses the dorsal peduncular cortex and the infralimbic cortex (FIG. 3, left panel).

tibody was put on, namely a donkey anti-rabbit FITC antiserum (Amersham) diluted 1:20. Incubation was performed for 30 min at 37°C. After washing, the sections were coverslipped and examined. Double-labeled cells are indicated by arrows. V = lateral ventricle; asterisk indicates the same vessel.

3000 - 2500 -

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- 6-OHDA+veh - 6-OHDA + NT control + veh control+ NT

Doparnine (nrnoVinjection)

FIGURE 2. Effects of bilateral injections of NT (179.3 pmol/0.5 pl/side) or vehicle (veh) on loco- motor activity induced by various doses of DA administered into the nucleus accumbens of rats le- sioned with 6-OHDA and in control sham-operated rats 14 days after surgery. Data expressed as mean f SEM of photocell interruption during 60 minutes. Six to eight animals were used for each tested dose. Statistical comparison were carried out by means of two-way analysis of variance followed by Duncan test ( * p < 0.05, * * p < 0.01).

At the posterior level and also at intermediate levels, no difference in '25I-labeled NT binding between lesioned and control rats was observed in the neostriatum and nu- cleus accumbens, the dorsal part of lateral septal nucleus being the only structure showing an apparent increase in 1251-labeled NT binding after the 6-OHDA lesion (FIG. 3, right panel).

These results are basically in accordance with previous reports showing that de- nervation induced by intra-VTA administration of 6-OHDA was associated with a lack of change in NT receptor density in the nucleus accumbens20 and an increase in NT receptor density in the prefrontal cortex.z1 It seems possible that the differential sen- sitivity of the supersensitive DA receptors to the antagonistic action of NT is simply

TABLE 1. Calculation of Dopamine-Neurotensin Complex Formation DAI" "TIa [DA] Not Complexeda

32600 pmol 179.3 pmol 31917 pmol 3.26 pmol 179.3 pmol 3.25 pmol 32.60 fmol 179.3 pmol 32.43 fmol 3.26 fmol 179.3 pmol 3.25 fmol

NOTE: Calculations were performed using the apparent dissociation constant of NT-DA binding found by Adachi et al. 10 (& = 7.5 x 10-8). This parameter and the concentration of DA(DAtot) and NT were entered into the following equation:

which was solved for DAhund. (DAhund)' - (NT + Kd + DAtot)DAbund + NT*DAmt = 0

(1 Concentrations of DA and NT in one microinjection (0.5 pl).

FUXE et al.: MECHANISM FOR NEUROLEPTIC-LIKE ACTION 191

Bregma 2.70 rnrn Bregma 0.70 mm

** 6o 1 6o 1

50 0 controt 6-OHDA

40

30

** 20

10

0

Acb IL.-DP Fr-Cg Acb CPu LSD CI i n s

FIGURE 3. Effects of 6-OHDA intra accumbens lesion on ‘**I-labeled NT binding (0.3 nM) in var- ious brain areas of the rat at two rostro-caudal levels (A/P = 0.7 mm and A/P = 2.7 mm, respectively). Nonspecific binding was obtained by incubating an adjacent section with 1 @l unlabeled NT. Re- ceptor densities were analyzed using quantitative microdensitometry.4~ Data are expressed as mean f SEM. Comparison between the values observed in the 6-OHDA lesioned rats and control rats were carried out by means of Mann-Whitney U-test ( *p < 0.05, **p < 0.01). Acb: nucleus accurnbens; IL: infralirnbic cortex; CPu: caudate-putamen; LSD: lateral septa1 nucleus, dorsal part; CI: claustrum; ins: insular cortex: DP: dorsal peduncular cortex; Cg: cingulate cortex.

due to the lower amounts of DA used, since the NT-D2 receptor-receptor interaction is mainly one of an affinity regulation. Also supersensitive D2 receptors in nucleus accumbens may be more prone to regulation by NT receptors due to an enhanced mobility of the D2 receptors, since newly formed D2 receptors may be located out- side the DA synapses and thus not anchored to the cytoplasma skeleton to the same degree as the synaptic DZ receptors. Support for this view was obtained when ana- lyzing the effects of NT on the binding characteristics of supersensitive D2 receptors.z2

NEURmENSIN MODULATION OF DOPAMINE Dz-REGULATED GABA AND DOPAMINE RELEASE AS ANALYZED BY

ZN VZVO MICRODIALYSIS

Effects of Neurotensin on Dz-Regulated GABA Release

We have demonstrated that NT (10 nM) increases extracellular levels of GABA but not of ACh and DA in the dorsal striatum of the halothane-anesthetized male rat (FIG. 4).*3 Thus, NT receptors may exert their neuroleptic activity by antagoniz- ing D2 receptor transduction on crucial subsets of striatal GABA efferent pathways (FIG. 5).

192 ANNALS NEW YORK ACADEMY OF SCIENCES

a

al 1 175-

’ 150-

$ 125-

100-

- a C .- a 0

A 0-0 Control

NTCONC. e-0 1000 nM A-A 10nM A-A 1 n M

* = PcO.05 + NT -NT N = 6 c-

C

0

,O 140- c

a? 120 -- v

2

; loo--

4 0 - .- rl

-80 -60 -40 -20 0 20 40 60 80 100 120

2251 h 200 1 8 v

T

Conlrol - 1 I I I I I 1

0 20 40 60 80 100120 75

Time (min)

FIGURE 4. (A) Effects of NT (1 nM, 10 nM, and lo00 nM) on extracellular GABA levels in the dorsal striatum of halothane anesthetized rats in the presence of 10 pM neostigmine were estimated in 20-min perfusate fractions. The peptide was perfused for 60 min (solid black bur). The changes in GABA levels were expressed as percent of the mean of three basal values. Basal GABA levels mea- sured in 20-min perfusate are 0.32 f 0.03 pmol. The number of animals in each group is represented in parenthesis. Each data point represents the mean f SEM (bars) values. The area (arbitrary units) and the maximum peak values in percent are as follows: Controls (6) = 806 f 395, 18 f 5; 1 nM NT (4) = 687 f 304, 15 f 8; 10 nM NT (7) = 2840 f 457*, 37 f 5*; lo00 nM NT (6) = 2209 * 555*, 49 f 12*. The area and maximum peak values were analyzed by analysis of variance (ANOVA) followed by multiple comparisons ( * p < 0.05).82 (B) Time response curves showing the effects of neurotensin (NT) on extracellular DA levels in the neostriatum of awake, freely moving rats. NT at a dose of loo0 nM (m) or 10 nM (0) in the perfusion medium was perfused locally in the neostriatum for 60 min (solid black bar). Controls (A) were not perfused with peptide. Only the highest dose of NT increased striatal extracellular DA levels. The changes in extracellular DA levels are expressed as percent of three basal values. The values were collected immediately before local perfusion with NT. Mean basal DA levels measured in 30411 perfusate fractions 0.23 f 0.03 pmoles/40 pl perfusate (16 rats). Each data point represent the mean f SEM (burs) values (5-6 rats in each group). The maximum peak values (A) are as follows: 7 f 2, 9 f 4, and 43 f 6 t for the control, 10 nM,

FUXE et al.: MECHANISM FOR NEUROLEPTIC-LIKE ACTION 193

SLsSTANnA STFtlATUM GLOBUS GLOBUS HOFU PALLDUS (ext.) PALLDUS (it . )

+ zone retidata (SN) n

6n NTreceptof

0 Mreceptof 0 D1 receptor

FIGURE 5. Schematic illustration of a possible interaction between postsynaptic DZ receptors and NT receptors located on the soma-denditric region of the medium-sized spiny GABA-ENK efferent neurons projecting to the external part of the globus pallidus. NT may be released from collaterals to activate the NT receptor. The medium-sized spiny GABA-SP striatal efferent neuron, mainly reg- ulated by DI receptors, may not be directly controlled by NT receptors.

Effects of Neurotensin on Dz-Regulated DA Release

In previous work it was demonstrated that NT antagonizes apomorphine-induced inhibition of DA release, as revealed by intrastriatal microdialysis in the halothane- anesthetized rat.z4 Perfusion with NT alone increases the dialysate levels of DA only at high concentrations (lo00 nM). At low concentrations (10 nM) NT, although in- active by itself, was capable of counteracting the inhibitory effect of apomorphine on dialysate levels of DA.z4

Further studies have been performed in the dorsolateral striatum of the awake, unrestrained male rat, analyzing the effects of intrastriatal coperfusion of NT and per- golide, which is a preferential DZ receptor agonist, or with SKF 38393, which is a preferential DI receptor a g o n i ~ t . ~ ~ As seen in FIGURE 6, a low concentration NT (10 nM) by itself did not affect extracellular levels of DA or its metabolites. However, NT significantly and markedly counteracted the inhibitory effects of pergolide on the extracellular levels of DA, DOPAC, and HVA. In contrast, the DI receptor agonist- mediated reduction of extracellular striatal levels of DA were significantly enhanced by simultaneous perfusion with NT (10 nM). These results may be explained on the basis that NT in vivo, at low concentrations, can reduce the functional effects of pre-

and loo0 nM NT groups, respectively. One-way ANOVA with the JSD test.82 *, significantly different from control (p < 0.05). NT was directly dissolved in the perfusion medium (Ringer solution). * p < 0.05a, significantly different from control. "a" indicates significance for area under the curve.

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NT, PERodtJDE or NT + POPOOUM

NT, SKF or NT+ SKF f 4 0 1

04 i 0 30 60 90 120 150 180 210

8 60t n

FUXE et ol. : MECHANISM FOR NEUROLEPTIC-LIKE ACTION 195

synaptic DZ receptor stimulation. In line with these results, NT was found to signifi- cantly counteract the D2 agonist-induced inhibition of nigral DA cell activity.26

The ability of the DI receptor agonist to inhibit DA release is probably mediated via an increase of striatal GABA release. It seems likely that the enhancement of the action of the DI receptor agonist by NT may involve an enhancement of striatal GABA release23 in view of the fact that D2 receptors reduce GABA release.27 Thus, it seems possible that the enhancement of the D1 receptor agonist action on DA release by NT is due to an antagonistic action of NT receptor stimulation on postsynaptic DZ recep- tors located on the efferent GABA neuron systems, especially the efferent GABA-ENK outflow pathways (see morphological section). Since NT at low concentrations cannot directly modulate the binding characteristics of DI receptors,6 these findings provide indications that NT may have the ability to switch the DA transmission from DZ trans- duction towards a DI receptor-mediated transduction. This putative heteroregulation appears to increase the functional plasticity of the DA synapses of the basal ganglia. We have also recently found synergistic interactions of CCK-8 and NT in the control of D2-regulated release.

NEUROTENSIN MODULATION OF DOPAMINE D2 AGONIST BINDING IN VZVO

Intraventricular injections of NT in the dose range of 0.3 to 3 nM were found to produce a dose-dependent, monophasic reduction of specific [3H]NPA binding within the caudate putamen, the nucleus accumbens, and the olfactory tubercle.

The modulatory effects of NT on DZ agonist binding are apparently unrelated to the coexistence of NT and TH IR5. These results indicate that NT in vivo can reduce the affinity of DZ receptors within the basal ganglia and that the NT involved is released from nondopaminergic nerve terminals.

NEUROTENSIN MODULATION OF DOPAMINE D2 AGONIST BINDING IN VZTRO

Neurotensin Modulation on Dopamine 0 2 Agonist Aflnity

NT in vitro increases the K d , but not the Bmx [3H]NPA binding in membrane prep- arations of the neostriatum and subcortical limbic areas.3~46~9.28-30 It was shown that [3H]NPA selectively labels the Dz receptor4 and that NT in nanomolar concentrations selectively inhibits Dz agonist binding4$9 without affecting D2 antagonist bindingl~~.~J or DI agonist or antagonist binding.432 The specificity of the action by NT is further underlined by the finding that many other peptides are without effect on [3H]NPA binding.6 In line with these results, NT has been found not to affect either basal or DA-activated adenylyl cyclase activity.

The maximal increase (in the order of 20-40%) of the K,j value of [3H]NPA was observed at 3 nM of NT, in membranes from both the neostriatum and subcortical limbic regions of the rat (FIG. 7)6J and in membranes from postmortem human caudate-putamen.28 Neuromedin N, a neurotensin agonist with no ability to bind DA, was also able to increase the Kd value of [3H]NPA binding sites by 25% at a 10 nM concentration (FIG. 8) supporting the present hypothesis on receptor-receptor inter-

196 ANNALS NEW YORK ACADEMY O F SCIENCES

160 1 A * * * *

80

- 1 1 I 1 ' 1 i

0 0.3 1 3 5 10 30

Neurotensln (nM)

B 110

g 90

CI - s? c loo]

m - 1 1 I I . I 1

0 0.3 1 3 5 10 30

Neurotensin (nM)

FIGURE 7. Effects of neurotensin on (A) Kd values and (B) Bmax values of [3H]N-propylnompo- morphine binding in membranes from the subcortical limbic area. The dotted lines represent the con- trol group mean values. Data are shown as the mean f SEM from nine independent concentration- response experiments in which control curves were performed in duplicate. p < 0.05 within treatments according to single-factor repeated measures ANOVA. * p < 0.05, * * p < 0.01 versus control according to Fisher's PLSD.

actions. Kinetical determination of the Kd value of [3H]NPA revealed a similar mag- nitude of the NT-induced increase, mainly caused by an increase in the dissociation of [3H]NPA (FIG. 9) .6 Recent experiments have shown similar effects of NT on com- petition curves with DA versus [3H]raclopride binding. Under these experimental con- ditions [3H]raclopride selectively binds to the DZ receptor. NT (10 nh4) produced a substantial increase in the KH for DA with no significant change in the KL for DA or the proportion of high- versus low-affinity binding sites (FIG. 10).6 However, con- tinued repetitions of such binding experiments in our laboratory indicate also that the KL may be increased by NT at 10 to 30 nM. Even though the percent change in

FUXE ef 01.: MECHANISM FOR NEUROLEPTIC-LIKE ACTION 197

600

500

400

300

200

100

0

NEUROMEDIN N (1 OnM)

0.0 0 . 5 1 .o 1 . 5 2 .o 2 . 5

Free (3H)NPA (nM)

FIGURE 8. Representative saturation curves showing the effects of neuromedin N47 (10 nM) on [SHINPA binding in rat striatal membranes. The peptide was incubated with [SHINPA and membranes for 30 min at room temperature. The Kd and Bmax values were 408 pM and 398 f m o l h g protein for control (O), and 612 pM and 424 f m o l h g protein for neuromedin N (0) as calculated by nonlinear regression using raclopride (1 w) for the determination of nonspecific binding. Inset: Corresponding Scatchard plots of the saturation binding data.

Dz receptor binding induced by NT is relatively small, it should be emphasized that receptor binding represents the first step in an amplification mechanism33 over the membrane and may reflect a substantial alteration in signal transduction.

Another aspect to be discussed is the inverted, U-shaped concentration-response curves observed when analyzing the NT action on Dz agonist binding. At 30 to 100 nM of NT, the modulatory action on the Kd value of the [3H]NPA binding has dis- appeared.6.28 Several explanations may exist for this phenomenon, such as the elic- itation of feedback mechanisms or a rapid desensitization of the NT receptor.1634 How- ever, the discrepancy against the monophasic concentration-effect curves seen after in vivo treatment with NT suggests that the biphasic concentration-response curves

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* I 1 I 1

0 1 0 2 0 30 4 0

Time (minutes) FIGURE 9. Effects of 3 nM neurotensin (W) on the association and dissociation (arrow) of 1 nM [3H]N-propylnorapomorphine (PHINPA) binding in rat neostriatal membranes. The dissociation was induced by 1 pM of LY 171555. Data are expressed in percent of equilibrium binding (about 150 fmollmg protein) from three independent experiments (s.e. about 10% of the mean in each experiment), per- formed in quadruplicate. The K-1 values were increased by 39 f 3%*, whereas &bs and KI values were unaffected. The kinetic Kd values of [SHINPA were calculated 90 f 3 pM for control and 131 f 2 pM* for neurotensin using linear fitting of transformed data. Nonlinear fitting of nontrans- formed data yielded similar results (* p < 0.05 versus control [O], Student’s unpaired t-test). * shows the significance (p < 0.05) between the two dissociation curves (Student’s t-test).

t . l . r . l - l . I ’ l ’ r . 1

, 1 2 - 1 1 - 1 0 - 9 - 8 - 7 - 6 - 5 - 4

Dopamine (log M)

FIGURE 10. Representative competitive-inhibition curves illustrating the effect of neurotensin (10 nM) on [3H]raclopride binding in rat striatal membranes. The dissociation constants of high- and low- affinity Dz agonist binding (KH and KL) were estimated to 1.46 nM and 76 nM under control con- ditions (0) and 5.79 nM and 130 nM in the presence of neurotensin (.). The percentage of D2 agonist binding sites in the high-affinity state (RH) was 42.2% and 48.7%, respectively.

FUXE et al. : MECHANISM FOR NEUROLEPTIC-LIKE ACTION 199

seen after treatment with NT in vitro may reflect the depletion of a membrane factor essential for mediating the action of NT. Supporting this hypothesis is the finding that adenosine AZa agonists and the trypsin inhibitor-like peptide PEC-60 modulate DZ agonist binding sites in a similar biphasic f a ~ h i o n . ~ ~ . ~ ~ In addition, these results suggest that the increase in the Kd value observed at the DZ receptors is in fact related to a phenomenon of receptor-receptor interaction first described by Agnati, Fuxe, and colleagues in 198037 since the biphasic dose-response curve should not have been obtained, based on the hypothesis that NT and DA bind to one another.'O

Possible Lack of Involvement of G Proteins in the lntramembrane NT- DZ Receptor Interaction

Studies using pertussis toxin, N-ethylmaleimide (NEM), and GTP indicate that the NT-induced modulation of DZ agonist affinity is not mediated by G proteins, but may nevertheless be enhanced in the presence of GTP.6.30

It is well known that the DZ receptors are coupled to the pertussis toxin sensitive G proteins Gi and G,.38 It is also known that the G proteins, by binding to the DZ receptor, can regulate the binding characteristics of the DZ recept0rs.~9 Pretreatment with NEM was also found to cause an almost complete blockade of the subsequent pertussis toxin-induced back ADP ribosylation that led to an increase in the Kd value and a decrease in the Bmax value of Dz-agonist binding, without affecting D2 antag- onist binding. Thus, this treatment selectively interfered with the G proteins without a direct action on the D2 receptor.30 In spite of the complete inactivation of NEM- sensitive G proteins, NEM could not block the ability of NT to increase the affinity of the D2 agonist binding ~ i tes .3~ Thus, these results indicate that G proteins may not be involved in mediating the action of NT on DZ agonist binding.

The action of NT was even enhanced in the presence of GTP (50 pM), in which case a significant increase in both KH and KL of DA was observed, being on the order of 200 to 300% and 50 to loo%, respectively.6 The proportion of high-affinity sites in relation to the total number of binding sites was lowered by GTP only in the absence of NT. These results suggest that NT may interfere with the dissociation of the G protein from the DZ receptor. In this way, an uncoupling of the DZ receptor to its bio- logical effector, for example, to the adenylate cyclase, to the phospholipase C, and/or to potassium channels, may be obtained and may provide an explanation for the fact that although the Kd changes observed are not very marked, they may reflect pow- erful alterations in the D2 receptor transduction.33 Similar observations have been made for the Aza/Dz receptor interactions, where AZ receptor activation blocks the reduction in the proportion of high-affinity DZ receptors induced by GTP (FerrC et al., unpublished data).

In line with this hypothesis are also the results obtained following adrenalect~my.~~ Thus, adrenalectomy alone increases the Kd value of the DZ agonist binding sites, an action which possibly is produced via an increase in Gi mRNA levels and in Gi IR.40 Nevertheless, the ability of NT to increase the Kd value is maintained in adrenal- ectomized rats, suggesting that the mechanism mediating the action of the peptide is different from the one of adrenalectomy.

Taken together, the present evidence would be compatible with the hypothesis that the modulation by NT receptors of D2 receptors involves an allosteric interaction4' between adjacent transmembrane regions holding the respective transmitter binding

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FUXE et al.: MECHANISM FOR NEUROLEPTIC-LIKE ACTION 201

TRANSMISSION PATHWAY

FIGURE 12. Possible pre- and postsynaptic intramembrane interactions between neurotensin recep- tors (NTR) and dopamine DZ receptors in the medium-sized spiny neuron of the neostriatum. Acti- vation of neurotensin receptors causes a reduced affinity in D2 agonist binding sites and enhanced effects of DI agonists. The broken arrow illustrates the reduced transmission of the Dz-mediated DA signal, and the two uninterrupted arrows the increased transmission of the DI mediated DA signal. Some DI receptors may be located also on the DZ receptor-containing GABA-ENK striatal efferent neurons, although most of them are located in the separate GABA-SP efferent striatal pathways. Only the interactions demonstrated in the present paper are indicated.

sites of the NT and DZ receptors, which both are G protein-coupled receptors. How- ever, it must be emphasized that NT has been shown to activate the phosphorylation of caudate nucleus synaptosomal proteins.4z Therefore, it is also possible that the NT receptor activation may also lead to an activation of membrane-bound kinases or phos- phatases that could affect the phosphorylation of serine, threonine, or tyrosine resi- dues of the third intracytoplasmic loop of the Dz receptor, although it should be noted that ATP and CAz+ ions were not present in the experiments during which the NT-DZ interaction has been demonstrated.

It has been demonstrated that NT can stimulate the formation of cyclic GMP (cGMP) in the neuroblastoma cell line NIE-11543 and that NT can stimulate inositol phospho- lipid hydrolysis in rat brain slices44 and increase intracellular calcium levels.45 Thus, NT may activate a number of second messenger systems which possibly may affect the DZ receptor inter alia via phosphorylation, but this possibility has yet to be in- vestigated (FIG. 11).

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DOPAMINE MODULATION OF NEUROTENSIN BINDING SITES

With ['HINT as a radioligand for NT receptors, DA has been shown to reduce the affinity and increase the number of [3H]NT binding sites in rat neostriatal mem- brane preparations,*2-& possibly operating via D1 receptors. This action was found to be enhanced by the development of 6-hydroxydopamine (6-OHDA)-induced DA receptor supersensitivity.Z2 These changes in the binding characteristics lead to an increase of ['HINT binding. It was suggested that in this way DA receptor activation may lead to an activation of the NT receptor mechanism, operating as an inhibitory feedback loop to reduce D2 receptor transduction.

SUMMARY

Evidence has been presented that behavioral actions of NT, inducing its neuroleptic- like action, can be explained on the basis of NT-D2 intramembrane receptor-receptor interactions in the basal ganglia, unrelated to the coexistence phenomenon, leading to reduced affinity and transduction of the D2 agonist binding site. By reducing se- lectively D2 receptor transduction at the pre- and postsynaptic level, the NT receptor appears capable of switching the DA synapses towards a DI receptor-mediated trans- duction, illustrating how receptor-receptor interactions can increase the functional plasticity of central synapses (FIG. 12).

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