Haloperidol-mediated phosphoinositidehydrolysis via direct activation ofa1-adrenoceptors in frontal cerebral rat cortex

Tania Gabriela Borda, Graciela Cremaschi, and Leonor Sterin-Borda

Abstract: In addition to its effect on D2 dopamine receptor blockades, haloperidol is able to interact with multipleneurotransmitters (NTs). Its action on phosphoinositide (PI) turnover was studied on cerebral cortex preparations. Itinduces an increase in inositol phosphate (IP) accumulation, which was only blunted by the1-adrenoceptor blockerprazosin. Haloperidol maximal effect (Emax) was less than the effect of the full agonist norepinephrine (NE), anddoseresponse curves for both NE in the presence of submaximal doses of haloperidol and haloperidol in the presenceof Emax doses of NE showed that haloperidol behaves as a partial agonist of cerebral1-adrenoceptors. Its effect on PIhydrolysis is mediated through phospholipase C activation, as 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC)and 1-[6-([(17)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione) (U-73122) were able toabrogate both haloperidol and NE actions. Further, the typical neuroleptic exerts a direct activation of1-adrenoceptorsas its actions were not modified by cocaine and persisted in spite of chemical rat cerebral denervation with6-hydroxydopamine (6-OHDA). The possibility that this agonistic action on1-adrenoceptors would be involved inhaloperidol side effects is also discussed.

Key words: haloperidol, neuroleptics,1-adrenoceptor, phosphoinositide hydrolysis, cerebral frontal cortex.

Rsum: Lhalopridol influe sur le blocage des rcepteurs D2 de la dopamine, mais il peut aussi interagir avec demultiples neurotransmetteurs (NT). Son action sur le renouvellement des phosphoinositides (PI) a t examine sur desprparations de cortex crbral. Lhalopridol a induit une augmentation du taux dinositol phosphates (IP), qui na tdiminue que par le bloqueur des rcepteurs adrnergiques1, prazosine. Leffet maximal de lhalopridol (Emax) a tplus faible que celui de lagoniste complet norpinphrine (NE). Les courbes doserponse tant pour la NE en prsencede doses sous-maximales dhalopridol que pour lhalopridol en prsence de dosesEmax de NE ont montr quelhalopridol agit comme un agoniste partiel des rcepteurs adrnergique1 crbraux. Leffet de lhalopridol surlhydrolyse des PI est vhicul par lactivation de la phospholipase C, tant donn que NCDC et U-73122 ont puinhiber tant les actions de la NE que celles de lhalopridol. De plus, le neuroleptique induit une activation directe desrcepteurs adrnergiques1, tant donn que ses actions nont pas t modifies par la cocane et ont persist malgrune dnervation crbrale chimique des rats au moyen de 6-hydroxytryptamine (6-OHDA). La possibilit que cetteaction agoniste sur les rcepteurs adrnergiques1 fasse partie des effets secondaires de lhalopridol est aussidiscute.

Mots cls: halopridol, neuroleptiques, rcepteurs adrnergiques1, hydrolyse des phosphoinositides, cortex frontalcrbral.

[Traduit par la Rdaction] Borda et al. 28

Neurotransmitter (NT) interactions have been demonstratedanatomically, physiologically, and behaviorally (Kinon andLieberman 1996; Memo et al. 1988;Gomez-Vargas and Ogawa1996). Incontrast, however, neuropsychiatric disorders such asschizophrenia have been classically attributed to deficits

within a single NT system. Although this could be the causefor the initial defect in these disorders, it is likely that diseaseprogression is related to the inability of the initially affectedsystem to modulate or be modulated by other NTs (Deweyet al. 1993).

Even though its role in mental disorders has been investi-gated less intensively than other NTs, the noradrenergic sys-tem is implicated in the development of schizophrenia (Raoand Mller 1994). Elevation of plasma and cerebrospinalfluid (CSF) norepinephrine (NE) levels has been reported inseveral studies (Van Kammen et al. 1990; Pickar et al. 1990;Van Kammen and Kelly 1991), and in addition elevated lev-els of NE in limbic areas of post mortem schizophrenic braintissues were found (Farley et al. 1978). However, conflictingstudies also exist (Rao and Mller 1994; Bird et al. 1979).Despite this, noradrenergic activity has been correlated with

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Received January 23, 1998.

T.G. Borda, G. Cremaschi, and L. Sterin-Borda.1 Centrode Estudios Farmacolgicos y Botnicos (CEFYBO), ConsejoNacional de Investigaciones Cientficas y Tcnicas de laRepblica Argentina (CONICET) y Universidad de BuenosAires, U.B.A., Serrano 669, 1414 Buenos Aires, Argentina.

1Author for correspondence.

both positive and negative schizophrenic symptoms (Raoand Mller 1994; Van Kammen et al. 1990; Pickar et al.1990; Van Kammen 1991).

The implication of a noradrenergic system in schizophre-nia is also demonstrated by the fact that neuroleptic drugsmay produce some therapeutic effects via adrenergic recep-tors (Van Kammen 1991). The typical neuroleptic halo-peridol exerts its potent antipsychotic effect partly by itsability to antagonize dopamine D2 receptors, but possible actionon other central NT receptors has been suggested (Rao andMller 1994). It was determined that haloperidol also blocksthe serotonin 5HT2 receptor and, to a lesser extent,1- and2-adrenoceptor activity (Leysen et al. 1988). In fact,long-term haloperidol treatment was shown to decrease thefiring rate of noradrenergic neurons in the rat (Dinan andAstron-Jones 1985). However, it was demonstrated thathaloperidol acts peripherally as an1-adrenoceptor agonist,enhancing the sialagogue response to adrenergic drugs in therat submandibular gland (Pazo et al. 1985).

In a previous study (Borda and Cremaschi 1997), wefound that haloperidol was able to potentiate the effect ofhigh doses of a muscarinic cholinergic agonist on phos-phoinositide (PI) turnover stimulation in cerebral frontal cor-tex preparations, and that this effect could be blunted by the1 antagonist prazosin. To further characterize haloperidolaction on central1 adrenergic receptors, here we studied itseffect in comparison with NE action on1 adrenergic medi-ated stimulation of PI hydrolysis on rat frontal cerebral cor-tex preparations.

Therefore, in this study we show that haloperidol interactswith 1-adrenoceptors activating them, similarly to, althoughto a lesser extent than, NE, behaving as a partial agonist.Also, its effects were not indirectly due to NE release butwere mediated through the direct activation of postsynapticreceptors. The possibility that sustained activation of1-adrenoceptor upon chronic haloperidol treatment leadingto receptor desensitization is also discussed.

AnimalsFemale Wistar rats (obtained from the Pharmacology Depart-

ment, School of Dentistry, University of Buenos Aires) werehoused in our colony in small groups and kept in automaticallycontrolled lighting (lights on 08:0019:00) and uniform tempera-ture (25C) conditions. All animals were used at 34 months ofage. Central noradrenergic denervation was accomplished by theintracerebro-ventricular injection of 6-hydroxydopamine (6-OHDA)(250 g in 10 L of 0.9% saline containing 0.1% ascorbate)(Kendall et al. 1985). Injections were repeated after 24 h, andsham-lesioned animals received the vehicle alone. Animals weresacrificed after 14 days. The animals were cared for in accordancewith the principles and guidelines of the Canadian Council onAnimal Care.

Measurement of total labelled inositol phosphates (IPs)Rat strips from the frontal cerebral cortex were incubated for

120 min in 0.5 mL of KrebsRinger bicarbonate buffer (KRB)gassed with 5% CO2 95% O2 with 1 Ci (1 Ci = 37 GBq)[myo-3H]inositol ([3H]MI) (specific activity 15 Ci/mmol) fromDuPont - New England Nuclear (Mississauga, Ont.). LiCl (10 mM)was added for inositol monophosphate accumulation according to

the technique of Berridge et al. (1982). Haloperidol was added30 min before the end of the incubation period, and the blockerswere added 30 min before the addition of either haloperidol or NE.Water-soluble IPs were extracted after 120 min incubation, follow-ing the method of Berridge et al. (1982). Cerebral cortical stripswere quickly washed with KRB and homogenized in 0.3 mL ofKRB with 10 mM LiCl and 2 mL chloroformmethanol (1:2, v/v)to stop the reaction. Then, chloroform (0.62 mL) and water (1 mL)were added. Samples were centrifuged at 3000 g for 10 min, andthe aqueous phase of the supernatants (12 mL) was applied to a0.7-mL column of Bio-Rad AG (Mississauga, Ont.) (formate form)1 8, anion-exchange resin (100200 mesh), suspended in 0.1 Mformic acid, which had been previously washed with 10 mMTris-formic, pH 7.4. The resin was then washed with 20 volumesof 5 mM myo-inositol followed by 6 volumes of water, and IPswere eluted with 1 M ammonium formate in 0.1 M formic acid.One-millilitre fractions were recovered, and radioactivity was de-termined by scintillation counting. Peak areas were determinedby triangulation. Results corresponding to the second peak wereexpressed as absolute values of area units under the curve permilligram of wet weight tissue (area/mg) following the criteria ofSimpsons equation (Camusso et al. 1995). To confirm the absenceof [3H]MI in the eluted peaks of IPs, chromatography in silica gel60 F254 sheets (Merck, Darmstadt, Germany) was performed usingpropan-2-ol 6 M NH4OH (14:5, v/v) as the developing solvent(Hokin-Neaverson and Sadeghian 1976).

DrugsNorepinephrine (NE), cocaine, prazosin, 6-hydroxydopamine

(6-OHDA), haloperidol, 2-nitro-4-carboxyphenyl-N,N-diphenyl-carbamate(NCDC), pertussis toxin (PTX) (fromBordetella pertus-sis), and1-[6-([(17)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino)hexyl]-1H--pyrrole-2,5-dione) (U-73122) were purchased from SigmaChemica l Corp . , S t . Lou is , Mo. Both1-[1-[4,4-bis(4-fluorophenyl)butyl]-4-piperidinyl]-1,3-dihydro-2H-benzimidazol-2one (pimozide) and3-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]methyl-1H-indole(L-741,626) were from Tocris Cookson Inc. (Ballwin, Mo.). Stocksolutions were freshly prepared in the corresponding buffers. Thedrugs were diluted in the bath to achieve the final concentrationsstated in the text.

Statistical analysisStudentst-test for unpaired values was used to determine the

levels of significance. When multiple comparisons were neces-sary, after analysis of variance, the StudentNewmanKeuls testwas applied. Differences between means were considered signifi-cant if p 0.05.

Haloperidol stimulation of PI turnover on rat frontalcerebral cortex preparations

Many receptors including1 adrenergic, muscarinic ace-tylcholine (mACh), H1 histaminergic, and 5HT2 receptorsare coupled to PI turnover, which could be elevated throughinositol phosphate (IP) accumulation. As shown in Fig. 1,haloperidol was able to increase IP formation on rat cerebralfrontal strips. Because of its multiple receptor interactions,several pharmacologic blockers were studied to assess whichis (are) the receptor(s) involved in haloperidol-mediated ac-tion on PI turnover.

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As also shown in Fig. 1, only the1 adrenergic antagonistprazosin was able to inhibit haloperidol action, with themAChR antagonist atropine, H1 histaminergic antagonistpirilamine, and2 adrenoceptor blocker yohimbine being in-effective. To further assess whether PI turnover by halo-peridol was blocked by other dopamine receptor antagonistsand whether these antagonists were also able to stimulate PIturnover, experiments were conducted using the D2 antago-nist pimozide and the high-affinity D2 antagonist L-741,626.The two drugs were ineffective both in stimulating PI turn-over and in blocking haloperidol-mediated stimulation of IPformation (Table 1).

As the extent of stimulation of [3H]inositol metabolism byNE in brain slices is a good measure of1 adrenergic recep-tor responsiveness in this tissue (Minneman and Johnson

1984), the effect of haloperidol on PI turnover was com-pared with that induced by NE. As shown in Fig. 2,haloperidol action was similar to that obtained with the en-dogenous agonist NE.

Since it has been demonstrated that monoamine oxidase isable to deaminate a significant proportion of NE under theconditions used in the PI breakdown assays (Fowler et al.1986), NE action in the presence of pargyline was also ex-amined. It can be seen in Fig. 2 that intrinsic activity ofhaloperidol was less than 1 ( = 0.72), indicating that it be-haves as a partial agonist on these cerebral preparations. It isworth noting that pargyline had no effect on the haloperidoldoseresponse curve, giving the same maximum effect and apEC50 = 8.0, both in the presence and absence of themonoamine oxidase inhibitor. However, maximum NE effectwas significantly higher in the presence of pargyline, whileits potency was only slightly augmented (pEC50 NE alone =7.7, pEC50 NE + pargyline = 8.0).

Haloperidol is a partial agonist of 1 adrenergicreceptor on rat cerebral frontal cortex

To firmly assess that haloperidol is a partial agonist of1-adrenoceptors on rat frontal cerebral cortex, doseresponsecurves of the full agonist NE in the presence of haloperidolon PI hydrolysis stimulation and vice versa were performedon frontal cortical strips. As can be seen in Fig. 3A, the

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Fig. 1. Haloperidol-mediated stimulation of phosphoinositidehydrolysis in rat frontal cerebral cortex preparations. Effects ofneurotransmitter receptor antagonists. Rat frontal cerebral cortexpreparations, prelabelled with [3H]MI as indicated in Methodsand materials were incubated for 60 min in the absence (Basal)or presence of haloperidol (105 M) alone (A) or with thefollowing blockers (105 M): atropine (B), pirilamine (C),prazosin (D), or yohimbine (E). Results shown are means SEM of n = 7 experiments. It is worth noting that none of thedrugs has an effect per se on basal IP formation. *Differssignificantly from basal values,p 0.01.

Additionsa IP formationb (fold of stimulation)

Haloperidol 3.10.2Haloperidol + pimozide 3.20.1Haloperidol + L-741,626 2.90.2Pimozide 0.80.1L-741,626 1.00.1

aDrugs (105 M) were added 30 min before the end of the incubationperiod or 30 min before the addition of haloperidol.bInositol phosphate(IP) formation in cerebral frontal cortex was measured as indicated inMethods and materials, and results were referred as fold of stimulationabove basal values (26.0 4.5 area/mg) of at least three experiments.

Table 1. Effect of D2 receptor antagonists on PI turnover byhaloperidol.

Fig. 2. Doseresponse curves of PI turnover to NE orhaloperidol. Inositol phosphate formation in frontal cerebralcortex was measured as indicated above in the presence ofincreasing concentrations of haloperidol (d) or NE (j).Doseresponse curves of NE in the presence of 1M pargylinewere also performed (u). Results shown are means SEM ofn = 5 experiments.

doseresponse curve of NE in the presence of 108 Mhaloperidol was shifted to the right, reaching the sameEmaxvalues as the curve for the agonist alone. Furthermore, asshown in Fig. 3B, the doseresponse curve of haloperidol inthe presence of 106 M NE showed an inhibitory effect thatreached anEmax similar to that obtained with the partial ago-nist alone.

Haloperidol exerts its action through direct activationof 1-adrenoceptors

To analyze whether haloperidol action on PI turnover wasa direct effect or whether it was mediated through the re-lease of NE from the presynaptic nerve terminal, we studiedhaloperidol action in the presence of cocaine, a NE neuronaluptake blocker (Trendelemburg 1963). As can be seen inFig. 4, cocaine was able to potentiate NE activation of PIturnover but had no effect on haloperidol stimulation of IPaccumulation.

Moreover, the action of chemical denervation was alsostudied. Figure 5 shows that haloperidol was able to stimu-late PI hydrolysis, in a similar fashion, on both normal and6-hydroxydopamine (6-OHDA) denervated cerebral cortexpreparations. The full agonist NE was also active on bothtissues, but a potentiation was evident on 6-OHDA prepara-tions.

Haloperidol interaction with 1-adrenoceptors leads tophospholipase C action

To further confirm that haloperidol-mediated PI turnoveractivation involves the direct activation of1-adrenoceptorsleading to phospholipase C (PLC) activation, we analyzedthe effects of two PLC blockers, NCDC and U-73122. Asshown in Fig. 6, PLC blockers were able to abrogate bothhaloperidol- and NE-mediated IP activation. The inhibitionof their action by the1-adrenoceptor antagonist prazosin isalso shown. It is worth noting that both haloperidol and NEstimulation of IP formation was unaffected by preincubationof brain slices with PTX (310g/mL) (data not shown).

The pharmacological profile of haloperidol mainly in-cludes the blockade of the dopamine D2 receptor, and it isalso classically considered to antagonize the activity of5HT2 receptors and1- and2-adrenoreceptors, although toa lesser extent (Rao and Mller 1994).

Haloperidol interaction with1-adrenoceptors on rat fron-tal cerebral cortex has been well characterized by bindingassays performed in vitro (Leysen et al. 1992) and by exvivo autoradiography (Schotte et al. 1993). In fact,long-term haloperidol treatment decreased the firing rate ofnoradrenergic neurons in the rat (Dinan and Astron-Jones1985). However, previous data from our laboratory (Bordaand Cremaschi 1997) showed that haloperidol was able topotentiate mAChR coupled with inositol phospholipid

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Fig. 3. Haloperidol as a partial agonist of1 adrenergic receptor on rat frontal cerebral cortex. (A) Doseresponse curves of NE in thepresence of haloperidol: formation of IPs on frontal cerebral cortex preparations was measured as doseresponse curves of the fullagonist NE in the absence (u) or presence (d) of 108 M haloperidol. (B) Doseresponse curves of haloperidol in the absence (d) orpresence (u) of 106 M NE. In both cases 1M of pargyline was also used. Results shown are means SEM ofn = 6 experiments.

breakdown in rat cerebral cortex, and that this effect wasblunted by the1 antagonist prazosin. As binding data aremostly related to receptor occupancy while functional studiesbetter reflect transductional pathways triggered by a drug, theaim of this paper was to better investigate the agonistichaloperidol action on PI turnover from cerebral frontal cortexpreparations. We found a concentration-dependent increase inIP accumulation that was only blunted by the1 antagonistprazosin. Interestingly, blockers of other cerebral receptors,known to be coupled with PI turnover, were ineffective. Fur-thermore, PI turnover by haloperidol was not affected bypreincubation of cortical slices with other selective D2 dopa-mine receptor antagonists, namely pimozide and L-741,626.Also, these two antagonists were unable to stimulate PI turn-over.

The effect of haloperidol was similar to and even higherthan that obtained with the full agonist NE, which was usedto compare their actions. This difference could be due to ag-onist degradation; in fact when we used pargyline to preventNE degradation by monoaminooxidase, the NE doseresponsecurve was significantly higher than that obtained withhaloperidol in the same conditions, showing the highest effi-cacy for NE. However, both drugs seemed to be similar inpotencies.

Moreover, doseresponse curves of both the full agonistand haloperidol demonstrated that the typical neurolepticbehaved as a partial agonist on frontal cerebral cortex1-adrenoceptors. As it is clear that in many systems the re-sponse is not directly proportional to receptor occupancyand that full agonists can produce maximal effects evenwhen a small fraction of the receptors are occupied(Jenkinson et al. 1995), it can be assumed that in this tissuethere is no receptor reserve for haloperidol actions; it is pos-

sible that there would be spare receptors for NE but not forhaloperidol in frontal cortical slices, thus acting as a partialagonist. In fact, doseresponse curves of NE in the presenceof EC50 concentration of haloperidol started at a value thatwas higher than the basal and was shifted to the right.Candura et al. (1990) observed a similar inhibition of the NEdoseresponse curve by haloperidol, and obtained the sameEmax values with NE alone and NE plus haloperidol, buthaloperidol alone had no effect per se. Furthermore, whenperforming the doseresponse curve of haloperidol in thepresence of 106 M NE, we found that the effect due to NEalone was inhibited by increasing haloperidol concentra-tions, reaching theEmax values of haloperidol alone.

Fowler and Thorell (1987) also found that haloperidol de-creased the maximum NE effect in a dose-dependent man-ner. However, they were not able to show any effect ofhaloperidol on the PI basal values. This could be explained bydifferences in the methods that had been used. Thus, they per-formed tissue incubations in the presence of highconcentrations (20 mM) of potassium. Candy et al. (1985)postulated that raised extracellular potassium enhanced, in adifferent magnitude, the PI response of some agonists, in-cluding NE, probably depending on the intracellular path-ways activated by each agonist. The high extracellularpotassium that led to higher basal values would probablymake it difficult to appreciate the partial agonistic effect ofhaloperidol. Nevertheless, the possibility of discrepancy be-cause of the different cortical preparation regions, as well as1-adrenoceptor heterogeneity (Hieble et al. 1986), couldnot be discarded.

In addition, chronic treatment with haloperidol wasfound to decrease NE-sensitive IP accumulation in frontalcortex and striatum from rat brains (Li et al. 1993). Inview of our results, it is possible that sustained activationof 1-adrenoceptors upon chronic treatment with halo-peridol, leading toreceptor desensitization, would explain

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Fig. 5. Effects of chemical denervation on haloperidol and NEaction on frontal cerebral cortices IP formation. IP formation onfrontal cerebral cortices of normal rats or rats treated with6-hydroxydopamine (6-OHDA) incubated alone (Basal) or with106 M haloperidol (A) or 106 M NE (B) are shown. Data aremeans SEM ofn = 4 experiments. Values differ significantlyfrom basal values: *p 0.01, p 0.005.

Fig. 4. Effect of cocaine on haloperidol and NE effects onfrontal cerebral cortex PI turnover. Cerebral preparations offrontal cortex were incubated as was previously described alone(Basal) or in the presence 106 M of haloperidol or NE (A) asindicated in the figure or in the presence of cocaine 106 M (B).The effect of cocaine alone is also shown (C). Data shown aremeans SEM ofn = 5 experiments. Values differ significantlyfrom basal values:* p 0.05, p 0.01, p 0.005.

the decreased response to NE in these experimental condi-tions.

Direct interaction of haloperidol with cerebral1-adrenoceptorwas also demonstrated by the fact that cocaine had no effecton haloperidol-induced stimulation of IP accumulation andalso because chemical denervation did not affect haloperidolaction. Further, haloperidol-induced PI turnover stimulationvia its interaction with1-adrenoceptors involved the cou-pling of G protein PLC activation, as indicated by theblockade of this effect, as well asNE-mediated stimulation ofPI turnover, with NCDC and with the aminosteroid PLC-specificantagonist U-73122. Because it was previously demonstratedthat the major coupling system of the1 adrenergic receptoris the activation of PLC through a PTX-insensitive G protein(Cotecchia et al. 1990.), and that PTX-insensitive Gq pro-teins were shown to stimulate the activity of PLC frombovine brain (Smrcka et al. 1991), the effect of PTX on bothhaloperidol and NE actions was studied. PTX, at concentrationsthat were shown to be effective in in vitro blocking Gi inhi-bition of adenylate cyclase (Goin et al. 1994), did not abro-gate both effects, so it seems probable that aPTX-insensitive G protein is involved in the phenomenon.

It is well known that neuroleptic drug effects in negativeschizophrenia are still limited (Rao and Mller 1994). In ad-dition to their antipsychotic actions, neuroleptic drugs exertside effects such as extrapyramidal motor disturbances (Raoand Mller 1994) because of their multireceptor interactions.Thus, the agonistic1-adrenoceptor activity described herein cerebral frontal cortex for haloperidol would be involvedin these effects. In fact, it has been described that negativesymptoms were related to noradrenergic activity (Van

Kammen 1991) and to hypofrontality in chronic schizophre-nia.

Although future research in this field is necessary to con-firm the role of haloperidol-mediated activation of cerebral1-adrenoceptors in schizophrenic symptomatology, it wouldpresent a sound basis for the involvement of neurotransmit-ter interactions in neuropsychiatric disorders.

The authors thank Mrs. Elvita Vannucchi for excellenttechnical assistance. Financial support was provided throughgrants from the Consejo Nacional de InvestigacionesCientficas y Tcnicas de la Repblica Argentina (CONICET)and the Ciencia y Tecnica Universidad de Buenos Aires(UBACyT).

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Fig. 6. Action of the phospholipase C inhibitors on bothhaloperidol and NE effects on PI turnover. Frontal cerebralcortices prelabelled with [3H]MI were incubated alone (Basal) orin the presence of 106 M haloperidol or NE as indicated in thefigure (A). The effect upon both haloperidol and NE actions ofphospholipase C inhibitors, 5 106 M NCDC (B) and1 106 M U-73122 (C) in comparison with the1 antagonistprazosin blockade (D). Data shown are the means SEM ofn = 6 experiments. Values differ significantly from basal values:*p 0.01, p 0.005

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