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Haloperidol regulates the binding of guanine nucleotides to synaptic membranes
through the NMDA receptor
N. Natsvlishvili, E. Juravliova, D. Dzneladze and D. Mikeladze*
Department of Neurochemistry, I. Beritashvili Institute of Physiology, Georgian Academy of Sciences,14 Gotua St., 380060 Tbilisi, Georgia The NMDA receptor is believed to be important in a wide range of nervous system functionsincluding neuronal migration, synapse formation, learning and memory. In addition, it isinvolved in excitotoxic neuronal cell death that occurs in a variety of acute and chronicneurological disorders. Besides agonist/coagonist sites, other modulator sites, includingthe butyrophenone site, may regulate the NMDA receptor. It has been shown that manyneuronal modulator mechanisms may be co-coordinated by a group of binding proteinsthat both clusters NMDA receptors and links them to signalling pathways within the cell.
We have found that 5'-guanylylimidodiphosphate (Gpp(NH)p) inhibited the binding of[3H]haloperidol to both the cortical and hippocampal synaptic membranes with high affinityand, reciprocally, haloperidol reduced the binding of [3H]Gpp(NH)p to the membranes.
Both effects are abolished by addition of anti-p21Ras. Affinity-purified preparations of theNMDA receptor, which were immunoprecipitated by anti-p21Ras contained only the ε2(NR2A/NR2B) subunits of NMDA receptors and nNOS. These data suggest that the possibleproteins participating in the formation of the macromolecular signalling complexes inpostsynaptic density may be nNOS and p21Ras.
Keywords: haloperidol, NMDA receptor, nNOS, p21Ras
1. INTRODUCTION
found that a p21Ras-activating protein, SynGAP (GTPaseactivating protein), is exclusively localized at synapses and The NMDA receptor is believed to be important in a associated with the PSD95 and NMDA receptors [7, 13].
wide range of nervous system functions including neuronal It has also been shown that NMDA receptor stimulation migration, synapse formation and learning and memory.
leads to activation of p21Ras through generation of nitric In addition, it is involved in the excitotoxic neuronal cell oxide (NO) in a cGMP-dependent pathway via nNOS [24].
death that occurs in a variety of acute and chronic These data suggest that p21Ras may also participate in the neurological disorders (see [21] for a review).
formation of supramolecular complexes with NMDA It has been shown that many neuronal modulator mechanisms may be co-coordinated by a group of binding Besides its agonist/coagonist sites, the NMDA receptor proteins that both clusters NMDA receptors and links them is regulated by other modulator action, including voltage- to signalling pathways within the cell. The NR2A and dependent blocking of the channel by Mg2+, voltage- NR2B subunits of the receptor bind to the SH2 domains independent actions of Zn2+, the redox state of the receptor, of phospholipase C in a tyrosine phosphorylation- and sites for arachidonic acid, ethanol, neurosteroids, pH dependent manner [11]. Members of the PSD-95/SAP90 and polyamines [23]. It has been found that haloperidol, a family will also cluster NMDA receptors when co- therapeutically useful antipsychotic drug, inhibits neuronal expressed in transfected cells and are associated with the NMDA responses and has neuroprotective effects against NMDA receptor in neuronal membranes [14,18]. PSD-95 NMDA-induced neurotoxicity [12,19]. Results from also binds to non-inducible nitric oxide synthase (nNOS) Whittemore et al. [22] suggest that a noncompetitive by interaction of its PDZ domain with a PDZ domain in allosteric modulator site expressed by isoforms of the nNOS [2] thus suggesting that PDZ domains may receptor containing the NR1/NR2B subunit mediates participate in the assembly of macromolecular signalling haloperidol’s action on the NMDA receptor. The ligand complexes involving NMDA receptors and one or more binding experiments of [8], as well as point mutation signalling molecules at the postsynaptic density.
studies [3] showed that haloperidol interacts with One of the possible signalling proteins participating in polyamine sensitive sites of the NR2B subunits.
the formation of the macromolecular signalling complexes Haloperidol induces the expression of immediate early in postsynaptic density may be p21Ras [20]. It has been
genes [15] and genes associated with synaptic plasticity [9].
NMDA receptor antagonists diminished the c-fos mRNAincrease produced by haloperidol, indicating that theNMDA receptor is involved in this process [17, 25].
However, the signalling pathway whereby haloperidol Tel: +99 532 37 47 24; Fax: +99 532 94 10 45; induces the expression of genes through the NMDA receptor is unknown. In this study we have tried Journal of Biological Physics and Chemistry 1 (2001) 24–28
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Effects of haloperidol on NMDA-receptor N. Natzvlishvili, E. Juravliova, D. Dzneladze and D. Mikeladze 25 to demonstrate that the action of haloperidol may be synaptic membranes. It was found that Gpp(NH)p inhibits mediated by p21Ras, which forms a macromolecular the binding of 3H-haloperidol to the both cortical (IC = 1012.8 nM, data not shown) and hippocampal (IC = 900.6 nM) synaptic membranes with high affinity (fig.1). On the contrary haloperidol reduces the binding 2. MATERIALS AND METHODS
Membrane preparations from rat cortex or hippo- campus were obtained after tissue homogenization in 20volumes of ice-cold 0.32 M sucrose, containing 0.1 M phenylmethylsulphonyl fluoride (PMSF), 3 mM EDTA,5 units/ml aprotinin and 5 µg/ml pepstatin A. The homogenate was centrifuged at 1000 g for 10 min, the pelletwas removed, and the supernatant was centrifuged at 20000 g for 20 min. The pellet was resuspended in 20 mM Tris- HCl, pH 7.4 buffer, containing 0.1 mM PMSF, 2 mM EDTA, 5 µg/ml pepstatin and 5 units/ml aprotinin (buffer A) to yield a suspension of 5 mg protein/ml and Solubilization was carried out using 1% sodium deoxy- cholate at a detergent/protein ratio = 4/1 (mg/mg) in buffer A during 1 hour at 4 °C followed by centrifugation at 100000 g for 1 hour. The final supernatant was dialyzedagainst 20 mM Tris-HCl (pH 7.4) containing 2 mM EDTA Figure 1. Inhibition of 5 nM [3H]haloperidol binding by GppNHp and 0.1 mM PMSF (buffer B) and was applied on either in rat hippocampal synaptic membranes in the presence (•) and dextrorphan-Sepharose or trihexyphenidyl (THP)-Sepha- absence (!) of anti-Ras. 100 % binding corresponds to the specific rose columns (1 x 10 cm) pre-equilibrated in buffer A.
binding of the control in the absence of GppNHp. Binding wascarried out as described in the experimental procedures. The curve The columns were washed with 40 ml buffer A and is representative of three independent experiments. Specific matrix-binding proteins were eluted by 100 mM dex- binding in the absence of GppNHp (dpm ± S.E.M.) from trorphan or by 100 mM phencyclidine in buffer A. The eluates were dialyzed against buffer B and their bindingactivity was determined.
[3H]haloperidol binding to synaptic membranes, and the solubilized or affinity-purified preparations, weredetermined in buffer A, containing 200–300 µg/ml protein and 5 nM [3H]haloperidol. Nonspecific binding wascalculated after addition 0.5 mM of haloperidol to the medium. The incubation was carried out during 1 hour at 4 oC and the mixture was filtered through Whatman GF/B filters pretreated with 0.05 polyethylenimine.
[3H]Gpp(NH)p binding assays were carried out in the presence of 5 nM [3H]Gpp(NH)p as described for[3H]haloperidol binding. 1 mM Gpp(NH)p was used to define the nonspecific binding. Radioactivity retained on the filter was determined by liquid scintillation The binding of THP and dextrorphan to Sepharose 4B was carried out as Egly et al. [10] suggested for triflufenazine immobilization [6]. Protein concentration was determined using a dye-binding method (Bio-Rad).
Figure 2. Inhibition of 5 nM [3H]GppNHp binding byhaloperidol in rat hippocampal synaptic membranes in thepresence (•) and absence (!) of anti-Ras. 100 % binding 3.RESULTS
corresponds to the specific binding of the control in the absenceof haloperidol. Binding was carried out as described in the 3.1 Binding experiments
experimental procedures. The curve is representative of threeindependent experiments. Specific binding in the absence of The guanine nucleotide analogue, 5'-guanylyli- haloperidol (dpm ± S.E.M.) from [3H]GppNHp was 2773 ± 248.
midodiphosphate (Gpp(NH)p), was examined for its effecton 3H-haloperidol binding to rat cortical and hippocampal ______________________________________________________________________________
26 N. Natzvlishvili, E. Juravliova, D. Dzneladze and D. Mikeladze Effects of haloperidol on NMDA-receptor of [3H] Gpp(NH)p to the membranes (fig. 2). Both effects solubilization by 1% sodium deoxycholate (see Materials are abolished by addition of anti-p21Ras.
and Methods) and subsequent centrifugation was applied In the next series of experiments the effects of other either on THP-Sepharose or dextrorphan-sepharose NMDA receptor and sigma antagonists on the binding of columns. Proteins binding THP-Sepharose or dextrorphan- [3H] Gpp(NH)p to the hippocampal synaptic membranes Sepharose from columns were eluted by 10 µM phen- were examined. We found that out of the series of NMDA cyclidine or by 10 µM dextrorphan respectively.
antagonists tested, haloperidol is significantly more potent SDS-PAG electrophoresis has shown that eluates from at decreasing [3H]Gpp(NH)p binding. Phencyclidine THP-sepharose (preparation 1) contain 5 major protein (PCP),MK-801((+)-5-methyl-10,11-dihydro-5H- fractions with molecular weights of 23000, 48000, 56000, dibenzo[a,d]cyclohepten-5,10-imine) and dextrorphan 11500 and 125000, and eluates from dextrorphan- were less potent than haloperidol (Table 1). These data sepharose (preparation 2) contain 6 protein fractions with demonstrate that the action of haloperidol on the binding molecular weights of 23000, 32000, 48000, 56000, 11500,125000 (data not shown). As the dextrorphan has higheraffinity to a sigma receptor, is possible that the additional Table 1. The inhibition of [3H]GppNHp bindinga to synaptic protein in the preparation 2 is one of the sigma binding membranes, and solubilized and affinity-purified preparations.
Pharmacological specificities of solubilized and both affinity-purified preparations were studied by competitionbinding experiments. MK-801 was found to have the highest affinity for the proteins of solubilized preparations, while pentazocine, a sigma opiate agonist, the lowest (data not shown). This shows that sodium deoxycholate solubilizes NMDA-glutamate receptors highly effectively,but sigma-binding proteins relatively ineffectively. The pharmacological profile of the affinity-purified prepa-rations does not significantly differ by specificity from the solubilized preparations, indicating that the pharmaco- logical specificity of NMDA receptors does not changeduring affinity chromatography.
It was also revealed that both preparation 1 and preparation 2 bound [3H] GppNp and did not lose sensitivity to haloperidol. The data for haloperidol are presented in Table 1; similar results are obtained for theother drugs. In spite of the fact that the IC is in this case a100 % binding corresponds to the specific binding of 5nM lower against the membranous preparation, the specificity [3H]GppNHp in the absence of drugs. Binding was measured as of interaction with the drugs is the same (data not shown).
described in the experimental procedures. Specific binding in Thus it is possible to conclude that the supramolecular the absence of drugs (dpm ± S.E.M.) from [3H]GppNHp was complex of NMDA- receptor after affinity chromatography 2773 ± 248 for membranes, 946±85 for the solubilized contains proteins binding in the same way as haloperidol preparation and 234± 37 for the affinity-purified preparation.
b Mean ± SEM calculated from 3 independent experiments.
3.3 Immunoprecipitation of the NMDA receptor
of guanine nucleotide with synaptic membranes is specificand can be directed at a system of p21Ras.
For identification of proteins in the supramolecular complex of NMDA receptor both affinity-purified 3.2 Purification and characterization of haloperidol
preparations were immunoprecipitated by anti-Ras and binding proteins
analyzed by Western-blot. It was found that only the ε2 (NR2A/NR2B) subunits of the NMDA receptors were It has been shown previously that many neuronal present in both immunoprecipitated preparations. In modulator mechanisms may be co-coordinated by a group addition it was revealed that both preparations contained of binding proteins that both clusters NMDA receptors nNOS and did not contain the ε1 subunit of the NMDA and links them to signalling pathways within the cell [21].
The possible signalling proteins participating in the Thus, it is possible to conclude that the supramolecular formation of the macromolecular signalling complexes in complex obtained after dextrorphan-Sepharose and THP- postsynaptic density may be nNOS and p21Ras [20]. For Sepharose chromatography contains the NR2A/NR2B identification of the target protein for haloperidol in the subunits of the NMDA receptor, p21Ras and nNOS.
supramolecular complex of NMDA receptors the Furthermore this macromolecular complex is sensitive to solubilization and affinity chromatography was performed.
haloperidol and binds guanine nucleotides.
For this purpose, the final supernatant followed ______________________________________________________________________________
Effects of haloperidol on NMDA-receptor N. Natzvlishvili, E. Juravliova, D. Dzneladze and D. Mikeladze 27 mediated by the haloperidol/ifenprodil binding sites of theNMDA receptor. Accordingly, it is possible to concludethat regulation of the Ras-signalling pathway via theNMDA receptor may be carried out by polyamine(ifenprodil) sites.
Figure 3. Co-immunoprecipitation of NR2A/NR2B subunits REFERENCES
of NMDA-receptor and nNOS by anti-Ras. Preparations 1 and 2were precipitated using the Ras antibody, separated on 1. Brenman, J.E., Chao, D.S., Gee, S.H., et al. Interaction SDS-PAGE, and analyzed using immunoblots probed with of nitric oxid synthase with the post synaptic density nNOS, NR2A/NR2B subunits and SAP antibodies as desribed protein PSD-95 and alpa-1-syntrophin mediated by in “Materials and Methods”. Lanes 1 and 2 – nNOS in prepara- PDZ domains. Cell 84 (1996) 757–767.
tions 1 and 2 respectively; lanes 3 and 4 – NR2A/NR2B in prepa- 2. Brenman, J.E., Christopherson, K.S., Graven, S., et al.
rations 1 and 2 respectively; lane 5 – SAP(PSD-95). The blot isrepresentative of three independent experiments.
Cloning and characterization of post synaptic density
93, a nitric oxide synthase interacting protein.
J.Neurosci. 16 (1996) 7407–7415.
3. Brimecombe, J.C., Gallagher, M.J., Lynch, D.R., 4. DISCUSSION
Aizenman, E. An NR2B point mutation affectinghaloperidol and CP101, 606 sensitivity of single Several PDZ-containing proteins have been shown to recombinant N-methyl-D-aspartate receptors.
interact closely with ion-channel linked receptors (see for J. Pharmacol. Exp. Therapeutics 286 (1998) 627–34.
review [5]). PSD-95 and PSD-93 were reported to interact 4. Boegman, R.J., Vincent, S.R. Involvement of adenosine directly with the C-terminal domains of two different and glutamate receptors in the induction of c-fos in the subunits (NR2A and NR2B) of the NMDA receptor [1,14].
striatum by haloperidol. Synapse 22 (1996) 70–77.
The multivalent PDZ-containing protein CIPP (channel 5. Carner, C.C., Nash, J., Huganir, R.L. PDZ domains in interacting PDZ-domain protein) was shown to bind to all synapse assembly and signaling. Trends Cell. Biol. 10
four NR2 subunits [16]. On the other hand nNOS and synaptic Ras-GTPase activating protein (synGap),which 6. Charbonneau, H., Porath, J. Purification of plant have been reported [7,13] to interact with PDZ-containing calmodulin by flufenazin-sepharose affinity chromato- proteins, could further contribute to a complex protein graphy. Biochem. Biophys. Res. Comm. 90 (1979)
scaffold. Our data has shown that the affinity-purified NMDA receptor preparation was immunoprecipitated by 7. Chen, H.J., Rojas-Soto, M., Oquni, A., Kennedy, M.B.
anti-Ras and contained nNOS. The protein complex does A synaptic Ras-GTPase activating protein (p135 not contain SAP, however, and this fact indicates that the SynGAP) inhibited by CaM kinase II. Neuron 20
interaction of Ras and nNOS with the NR2B subunit is possibly carried out by other proteins, such as PSD-93 8. Coughenour, L.L., Cordon, J.J. Characterization of haloperidol and trifluperidol as subtype-selective N- Ras signalling at the postsynaptic membrane may be methyl-D-aspartate receptor antagonists using [3H]TCP involved in the modulation of excitatory synaptic and [3H]Ifenprodil binding in rat brain membranes.
transmission [20]. On the basis of our results it is possible J. Pharmacol. Exp. Therapeutics 280 (1997) 584–592.
to conclude that the macromolecular complex containing 9. Eastwood, S.I., Heffernan, J., Harrison, P.J. Chronic NR2B and Ras may be directly regulated by haloperidol.
haloperidol treatment affects the expression of synaptic As shown in fig. 1, haloperidol decreases the binding of and neuronal plasticity-associated genes. Mol. 3H-GppNp with synaptic membranes in a dose-dependent Psychiatry 2 (1997) 322–329.
manner. Neither PCP, MK-801, dextrorphan nor penta- 10. Egly, J.-M., Porath, J. Change transfer and water zocine change the binding of 3H-GppNp with synaptic mediated chromatography. II. Adsorption of nucleo- membranes, which suggests that haloperidol binds tides and related compounds in acriflavin-Sephadex.
elsewhere than at the PCP-site of the NMDA receptor. Ilyin J. Chromatogr. 168 (1979) 35–47.
et al. [12] indicated that haloperidol selectively inhibits 11. Gurd, J.W., Bisson, N. The N-methyl-d-aspartate NMDA receptors composed of NR1 and NR2B subunits receptor subunits NR2A and NR2B bind to the by acting at a noncompetitive allosteric site distinct from SH2-domains of phospholipase C. J. Neurochem. 69
the glutamate-, glycine- and PCP-binding sites, but related to the ifenprodil binding sites. Brimecombe et al. [3] found 12. Ilyin, V.I., Whittemore, E.R., Guastella, J., et al.
an NR2B point mutation affecting the haloperidol and Subtype-specific inhibition of NMDA receptors by CP101 (ifenprodil analogue) sensitivity of a single haloperidol. Mol. Pharmacol, 50 (1996) 1541–1550.
recombinant NMDA receptor. Together, these observations 13. Kim, J.H., Liao, D., Lau, L.F., Huganir, R.L. SynGAP: suggest that the unique effect of haloperidol on 3H-GppHp a synaptic RasGAP that associates with the PSD-95/ binding (and possibly on the activity of p21Ras) may be SAP90 protein family. Neuron 20 (1998) 683–691.
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28 N. Natzvlishvili, E. Juravliova, D. Dzneladze and D. Mikeladze Effects of haloperidol on NMDA-receptor 14. Kornau, H.-C., Schenker, L.T., Kennedy, M.B., 22. Whittemore, E.R., Ilyin, V.I., Woodward, R.M.
Seeburg, P.H. Domain interaction between NMDA Antagonism of N-methyl-D-aspartate receptors by σ- receptor subunits and the postsynaptic density protein site ligands: potency, subtype selectivity and PSD-95. Science 269 (1995) 1737–1740.
mechanisms of inhibition. J. Pharmacol. Exp. 15. Kouradi, C., Heckers, S. Haloperidol-induced Fos Therapeutics 282 (1997) 326–338.
expression in striatum is dependent upon trancription 23. Yamakura, T., Shimoji, K. Subunit- and site-specific factor cyclic AMP response element binding protein.
pharmacology of the NMDA receptor channel.
Neuroscience 65 (1995) 1051–1061.
Prog. Neurobiology 59 (1999) 279–298.
16. Kurschner, C., Mermelstein, P.G., Holden, W.T., 24. Yun, H-Y., Gonzalez-Zulueta, M., Dawson, V.L., Surmeier, D.J. CIPP, a novel multivalent PDZ domain Dawson, T.M. Nitric oxide mediates N-methyl-D- protein, selectively interacts with kir4.0 family aspartate receptor-induced activation of p21ras.
members, NMDA receptor subunits, neurexins, and Proc. Natl Acad. Sci. 95 (1998) 5773–5778.
neurogulins. Mol. Cell Neurosci. 11 (1998) 161–172.
25. Ziolkowska, B., Hollt, V. The NMDA receptor 17. Morinobu, S., Strausbaugh, H., Terwilliger, R., antagonist MK-801 markedly reduces the induction Duman, R.S. Regulation of c-fos and NGF-A by of c-fos gene by haloperidol in the mouse striatum.
antidepressant treatment. Synapse 25 (1997) 313–
Neurosci. Lett. 156 (1993) 39–42.
18. Neithammer, M., Kim, E., Sheng, M. Interaction between the C terminus of NMDA receptor subunits APPENDIX
and multiple members of the PSD-95 family ofmembrane-associated guanylate kinases. J. Neurosci. Alphabetical list of abbreviations:
16 (1996) 2157–2163.
19. Nishikawa, H., Hashino, A., Kume, R., et al.
Gpp(NH)p: 5’guanylylimidodiphosphate;
Involvement of direct inhibition of NMDA receptors EDTA: ethylenediaminetetraacetic acid;
in the effects of -receptor ligands on glutamate neuroto- MK-80: (+)-5-methyl-10,11-dihydro-5H-dibenzo-
xicity in vitro. Eur. J. Pharmacol. 404 (2000) 41–48.
20. Orban, P.C., Chapman, P.F., Brambilla, R. Is the NMDA: N-methyl-D-aspartate;
Ras-MAPkinase signalling pathway necessary for nNOS: neural nitric oxide synthase;
long-term memory formation? Trends Neurosci. 22
PMSF: phenylmethylsulphonylfluoride;
(1999) 38–44.
PSD-95: postsynaptic density protein;
21. Ozawa, S., Kamiya, H., Tsuzuki, K. Glutamate SAP-90: synapse associated protein;
receptors in the mammalian central nervous system.
THP: trihexyphenidylhydrochloride.
Prog. Neurobiol. 54 (1998) 581–618.

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