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Increased platelet vesicular monoamine transporter density in adult schizophrenia patients
European Neuropsychopharmacology 12 (2002) 343–347 www.elsevier.com / locate / euroneuro
Increased platelet vesicular monoamine transporter density in adult schizophrenia patients a b a,b a, Michal Zucker , Avi Valevski , Abraham Weizman , Moshe Rehavi * a
b
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Geha Psychiatric Hospital and Felsenstein Medical Research Center, Beilinson Campus, Petah Tikva and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Received 20 September 2001; accepted 30 April 2002
Abstract Vesicular monoamine transporter (VMAT2) plays a major role in the synaptic accumulation and release of monoamines. In the present study, we assessed high affinity [ 3 H]dihydrotetrabenazine binding to platelet VMAT2 in a group of treated (n59) and untreated (n54) patients with schizophrenia and age- and sex-matched controls. Significantly elevated platelet VMAT2 density (Bmax ) (53%, P,0.0001) was observed in the mixed population of schizophrenia patients. The affinity of the ligand (Kd ) to platelet VMAT2 was similar in both groups. The increased platelet VMAT2 density may indicate a schizophrenia-related hyperactivity of the monoaminergic system or an adaptive response to chronic drug treatment. 2002 Elsevier Science B.V. All rights reserved. Keywords: Schizophrenia; Platelet; Vesicular monoamine transporter; Dihydrotetrabenazine; Monoamines
1. Introduction Schizophrenia is a chronic and disabling neuropsychiatric disorder of unknown etiology. On the basis of the strong correlation between the therapeutic potency of antipsychotic drugs in schizophrenia and their inhibitory activity at the D2 receptors, some researchers have hypothesized that a dysregulation in dopaminergic neurotransmission may play a role in the pathophysiology of the disorder (Seeman et al., 1976). However, many of the atypical antipsychotic drugs have a higher affinity for 5-HT2 receptors than for D2 receptors. Using platelet plasma membrane serotonin transporter as a peripheral model for plasma membrane serotonin transporter in the brain, studies have revealed alterations in the expression of serotonin transporter in patients with major depression (Paul et al., 1981), obsessive–compulsive disorder (Weizman et al., 1986; Sallee et al., 1996), and *Corresponding author. Tel.: 1972-3-640-8759; fax: 1972-3-6409113. E-mail address: [email protected] (M. Rehavi).
post-traumatic stress disorder (Fichtner et al., 1994), and in women treated with hormonal preparations (Weizman et al., 1987b). Findings in schizophrenia patients, however, were equivocal. Some investigators reported a decrease in imipramine binding (Arora et al., 1986) whereas others did not (Weizman et al., 1987a). Brain vesicular monoamine transporter (VMAT2) transports intracellular monoamines into the synaptic vesicles and is expressed in all monoaminergic neurons (Edwards, 1992). VMAT2 expression seems to be almost insensitive to treatment with drugs known to regulate monoaminergic neurotransmission, such as antipsychotics, monoamine oxidase inhibitors, and monoamine reuptake blockers (Naudon et al., 1994; Vander Borght et al., 1995; Wilson and Kish, 1996; Zucker et al., 2001a). Recently, positronemission tomography (PET) studies in treated patients with schizophrenia have shown an increase in VMAT2 expression in the ventral brainstem, but not in the thalamus (Zubieta et al., 2001) or striatum (Taylor et al., 2000). VMAT2 expression was higher in the thalamus and ventral brainstem of treated patients with bipolar disorder (Zubieta et al., 2001).
0924-977X / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0924-977X( 02 )00041-X
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M. Zucker et al. / European Neuropsychopharmacology 12 (2002) 343 – 347
VMAT2 is also expressed in the platelets (Cesura et al., 1990; Zucker et al., 2001b), and a line of evidence suggests that brain and platelet vesicular monoamine transporters have identical coding sequences and similar pharmacological properties (Lesch et al., 1993, 1994). Therefore, like for serotonin transporters, platelet VMAT2 may serve as a possible peripheral model for brain VMAT2 (Zucker et al., 2001b). Recently, Carlsson et al. (2001) suggested that schizophrenia might involve a dysregulation of several neurotransmitters. Therefore, we hypothesized that the disorder may be associated with an alteration in the expression of VMAT2, which is essential to the transmission of dopamine, serotonin and norepinephrine. Specifically, a VMAT2-induced increase in capacity to accumulate and release monoamines could lead to higher monoaminergic activity. This could explain the efficacy of agents with antagonistic activity at the monoaminergic receptors in the treatment of schizophrenia. In the present study, we assessed the pharmacodynamic characteristics of platelet VMAT2, using [ 3 H]dihydrotetrabenazine ([ 3 H]TBZOH) as a ligand, in untreated and treated schizophrenia patients.
2. Experimental procedures
2.1. Subjects Thirteen hospitalised patients with schizophrenia (seven males, six females) were recruited from Geha Psychiatric Hospital to participate in the study. All underwent a psychiatric interview according to the guidelines of the Structured Clinical Interview for DMS-IV Axis I Disorders [SCID-P] (First et al., 1996), and a diagnosis of schizophrenia was established according to the DSM-IV-TR criteria (American Psychiatric Association, 2000). Nine patients were being treated with antipsychotics only, two had been drug-free for at least 3 months (due to non compliance) and two were never treated (due to refusal to get psychiatric help). Symptoms were assessed with the Positive and Negative Syndrome Scale [PANSS] (Kay et al., 1987). Fifteen age- and sex-matched healthy subjects from the community served as controls. The study was approved by the Geha psychiatric hospital Review Board, and informed consent was obtained from all participants after the nature of the study was fully explained to them.
2.2. Platelet membrane preparation Blood samples (25 ml) were collected between 8:00 and 10:00 h into tubes containing an anticoagulant solution of 16 mM citrate buffer and 1 mM EDTA. Platelet-rich plasma was separated from blood cells by low-speed centrifugation (3503g for 10 min), resuspended in 20 ml
Hepes buffer 50 mM pH 8.0, and centrifuged at 17003g for 20 min. The pellet was disrupted with Brinkman polytron in 20 ml Hepes buffer 50 mM pH 8.0 containing 300 mM sucrose (buffer A) and centrifuged twice at 27 0003g for 20 min. It was then resuspended in 4.5 ml buffer A to yield a protein concentration of about 1 mg / ml.
2.3. [ 3 H] TBZOH binding For [ 3 H]TBZOH binding, 100 ml membranes were incubated at 25 8C with 50 ml of [ 3 H]TBZOH (eight concentrations, 0.5–8.0 nM) (specific activity: 20 Ci / mmol; American Radiolabeled Chemicals Inc., St. Louis MO) and 50 ml buffer A. After 30 min of incubation, the mixture was filtered with vacuum on glass fiber filters (GF / C). The filters were washed four times with ice-cold buffer A, and the radioactivity was counted in scintillation liquid in a b-counter (Packard 1600 TR). Nonspecific binding was measured in parallel samples in the presence of 1 mM tetrabenazine (Fluka, Buch, Switzerland). This ligand was shown to inhibit [ 3 H]TBZOH binding to human platelet VMAT2 in a competitive manner (IC 50 510 nM) (Zucker et al., 2001b). The nonspecific binding at Kd value (3.2 nM) did not exceed 20%. Protein concentration was determined according to the method of Bradford (1976). The ligand affinity (Kd ) to VMAT2 and [ 3 H]TBZOH binding capacity (Bmax ) were assessed by Scatchard analysis.
2.4. Statistical analysis Two-tailed Student’s t-test was used for between-group comparisons. Pearson correlation test was used as appropriate. All results are expressed as mean6S.D.
3. Results The demographic data of the patients and controls are given in Table 1. All participants were physically healthy and did not abuse drugs or alcohol. As there was no difference between the treated (n59) and untreated (n54) patients in Bmax [10946252 vs. 8536224 fmol / mg protein (222% vs. control), t51.64, df511, P50.13] or Kd (3.3660.93 vs. 3.4761.01 nm, t50.18, df511, P50.86), the findings for all the schizophrenia patients (n513) were pooled. VMAT2 density was found to be significantly higher in the schizophrenia patients (n513) than the controls (10206261 vs. 6686187 fmol / mg protein, t5 4.15, df526; P,0.0006) (Fig. 1), but there was no difference between the groups in Kd (3.4060.93 vs. 2.7361.19 nm, t51.64, df526; P50.11, NS). Sub-analysis of the data for the untreated patients alone (N54) as compared to the controls, revealed higher (128%)
M. Zucker et al. / European Neuropsychopharmacology 12 (2002) 343 – 347
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Table 1 Clinical and epidemiological data of the study population Schizophrenia patients (n513)
Healthy controls (n515)
Age range (years) Male / Female Age of onset (years) Duration of illness (years)
23–52 7/6 27.4666.50 11.6167.41
18–58 9/7 – –
Medications (dose range)a Atypical antipsychotics Clozapine (n53) mg / day Risperidone (n52) mg / day
150–325 2–6
– –
7.5–30 32 100 mg / 3 days 12.5–500 40 25 mg / 3 weeks 5.762.8
– – – – – – –
70.3618.3 19.3865.74 19.1568.35 31.7768.07
– – – –
Typical antipsychotics Haloperidol (n52) mg / day Perphenazine (n51) mg / day Zuclopenthixol (n51) Levomepromazine (n52) mg / day Penfluridol (n51) mg / week Fluphenazine decanoate (n51) Number of hospitalisations Symptom rating (mean6S.D.)b Total Positive Negative General a b
Some patients were treated with more than one antipsychotic. Positive and negative syndrome scale.
VMAT2 density, however, the results did not reach a significant level (t51.7, df517, P50.11, NS). Platelet VMAT2 density did not correlate with the severity of schizophrenia as assessed by the PANSS total score (r50.23, n513; P50.45, NS), and positive (r50.05, n513; P50.88, NS) and negative (r50.24, n513; P5 0.43, NS) and general (r50.03, n513; P50.92, NS) scores. To evaluate the possible in vitro interaction between [ 3 H]TBZOH binding and the antipsychotic drugs adminis-
Fig. 1. The density of [ 3 H]TBZOH binding sites (Bmax ) in platelet membranes of the study population. Values are mean6S.D. For details of the binding assay (see Experimental procedures).
tered to the patients, we assessed the inhibitory potency of the drugs on [ 3 H]TBZOH binding to platelet membranes. Only risperidone and haloperidol exhibited a modest (in the micromolar range) inhibitory effect (IC 50 54.5310 27 and 1.5310 26 M, respectively).
4. Discussion The major finding of this study was the high platelet VMAT2 density in schizophrenia patients compared to normal healthy controls. The VMAT2 Bmax values did not correlate significantly with the severity of the psychosis; however, most of the patients (9 / 13) were being maintained on antipsychotic drugs. Although the untreated schizophrenia patients (n54) exhibited lower Bmax values (222%) than the treated patients, the difference was not significant. This suggests that antipsychotic treatment per se is not responsible for the elevation in platelet VMAT2 density. This assumption is further supported by animal studies showing that brain VMAT2 is insensitive to treatment with agents active at the nigrostriatal dopaminergic pathway, such as haloperidol (Naudon et al., 1994; Vander Borght et al., 1995), the D2-agonist bromocriptine, and the dopamine reuptake inhibitor cocaine (Wilson and Kish, 1996). In our study, only two antipsychotics, haloperidol and risperidone, showed a tendency to inhibit, and not to augment, [ 3 H]TBZOH binding
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to human platelet membranes. Moreover, during the binding assay the platelet membranes were extensively washed, thus it seems unlikely that the presence of the drugs affect the binding values. Thus, the excessive VMAT2 binding sites in schizophrenia patients are apparently associated with the disorder, and not the exposure to antipsychotics. However, the Bmax values of the untreated patient (N54), although being higher (128% vs. controls), did not differ significantly from that of the controls. Furthermore, considering the high percentage of patients being treated with antipsychotics, we cannot conclusively rule out the role of neuroleptics or duration of disease and treatment in the increase in [ 3 H]TBZOH binding. It also remains unclear whether the elevated VMAT2 binding in platelets represents a parallel change in the brain. Interestingly, in vivo PET studies of brain VMAT2 levels in treated schizophrenia patients using [ 11 C]TBZOH as a ligand demonstrated increased VMAT2 density in the ventral brain stem (Zubieta et al., 2001), but not in the striatum (Taylor et al., 2000). As overexpression of VMAT2 in monoaminergic neurons is associated with increased monoamine storage and quantal release of monoamines from synaptic terminals (Fon et al., 1997; Reimer et al., 1998), its putative occurrence in brain regions relevant to schizophrenia could lead to functional overactivity of brain dopaminergic, serotonergic and noradrenergic pathways. It is of note that the VMAT2 blocker reserpine has been shown to be effective in the treatment of some drug-resistant schizophrenia patients, although its use is limited by its side effects (Jalenques, 1996). In conclusion, we observed a significant increase in platelet VMAT2 binding capacity in a mixed population of treated and untreated schizophrenia patients as compared to normal controls. This increase may indicate a hyperfunction of monoaminergic pathways in schizophrenia or an adaptive response to chronic antipsychotic treatment. Further controlled large-scale studies including treated and untreated patients with schizophrenia and other mental disorders are needed to confirm our finding and to demonstrate its specificity.
References American Psychiatric Association, 2000. Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, DSM-IV-TR. American Psychiatric Association, Washington DC. Arora, R., Locascio, J., Meltzer, H., 1986. 3 H-imipramine binding in blood platelets of schizophrenic patients. Psychiatry Res. 19, 215–224. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72, 248–254. Carlsson, A., Waters, N., Holm-Waters, S., Tedroff, J., Nilsson, M., Carlsson, M.L., 2001. Interactions between monoamines, glutamate and GABA in schizophrenia: new evidence. Annu. Rev. Pharmacol. Toxicol. 41, 237–260. Cesura, A.M., Bertocci, B., Da Prada, M., 1990. Binding of [ 3 H]dihydrotetrabenazine and [ 125 I]azidoiodoketanserin photoaffinity
labeling of the monoamine transporter of platelet 5-HT organelles. Eur. J. Pharmacol. 186, 95–104. Edwards, R.H., 1992. The transport of neurotransmitters into synaptic vesicles. Curr. Opin. Neurobiol. 2, 568–594. Fichtner, C.G., Arora, R.C., O’Connor, F.L., Crayton, J.W., 1994. Platelet paroxetine binding and fluoxetine pharmacotherapy in posttraumatic stress disorder: preliminary observations on a possible predictor of clinical treatment response. Life Sci. 54, 39–44. First, M.B., Spitzer, R.L., Gibbon, M., Williams, J., 1996. Structured Clinical Interview for DMS-IV Axis I Disorders (SCID), clinical version: User’s guide. American Psychiatric Press, Washington DC. Fon, E.A., Pothos, E.N., Sun, B.-C., Killeen, N., Sulzer, D., Edwards, R.H., 1997. Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action. Neuron 19, 1271–1283. Jalenques, I., 1996. Drug-resistant schizophrenia—treatment options. CNS Drugs 5, 8–23. Kay, S.R., Fitzbein, A., Opler, L.A., 1987. The Positive and Negative Syndrome Scale (PANSS) for schizophrenia. Schizophr. Bull. 13, 261–275. Lesch, K.P., Gross, J., Wolozin, B.L., Murphy, D.L., Riederer, P., 1993. Extensive sequence divergence between the human and rat brain vesicular monoamine transporter: possible molecular basis for species differences in the susceptibility to MPP 1 . J. Neural Transm. 93, 75–82. Lesch, K.P., Gross, J., Wolozin, B.L., Franzek, E., Bengel, D., Riederer, P., Murphy, D.L., 1994. Direct sequencing of the reserpine-sensitive vesicular monoamine transporter complementary DNA in unipolar depression and manic depressive illness. Psychiatr. Genet. 4, 153–160. Naudon, L., Leroux-Nicollet, I., Costentin, J., 1994. Short-term treatments with haloperidol or bromocriptine do not alter the density of the monoamine vesicular transporter in the substantia nigra. Neurosci. Lett. 173, 1–4. Paul, S.M., Rehavi, M., Skolnick, P., Ballenger, J.C., Goodwin, F.K., 1981. Depressed patients have decreased binding of tritiated imipramine to platelet serotonin transporter. Arch. Gen. Psychiatry 38, 1315–1317. Reimer, R.J., Fon, E.A., Edwards, R.H., 1998. Vesicular neurotransmitter transport and the presynaptic regulation of quantal size. Curr. Opin. Neurobiol. 8, 405–412. Sallee, F.R., Richman, H., Beach, K., Sethuraman, G., Nesbitt, L., 1996. Platelet serotonin transporter in children and adolescents with obsessive-compulsive disorder or Tourette’s syndrome. J. Am. Acad. Child Adolesc. Psychiatry 35, 1647–1656. Seeman, P., Lee, T., Chau-Wong, M., Wong, K., 1976. Antipsychotic drug doses and neuroleptic / dopamine receptors. Nature 261, 717–719. Taylor, S.F., Koeppe, R.A., Tandon, R., Zubieta, J.-K., Frey, K.A., 2000. In vivo measurement of the vesicular monamine transporter in schizophrenia. Neuropsychopharmacology 23, 667–675. Vander Borght, T., Kilbourn, M., Desmond, T., Kuhl, D., Frey, K., 1995. The vesicular monoamine transporter is not regulated by dopaminergic drugs treatment. Eur. J. Pharmacol. 294, 577–583. Weizman, A., Carmi, M., Hermesh, H., Shahar, A., Apter, A., Tyano, S., Rehavi, M., 1986. High affinity imipramine binding and serotonin uptake in platelets of eight adolescent and ten adult obsessive-compulsive patients. Am. J. Psychiatry 143, 335–339. Weizman, A., Gonen, N., Tyano, S., Szekely, G.A., Rehavi, M., 1987a. Platelet [ 3 H]imipramine binding in autism and schizophrenia. Psychopharmacology 91, 101–103. Weizman, A., Morgenstern, H., Kaplan, B., Amiri, Z., Tyano, S., Ovadia, Y., Rehavi, M., 1987b. Up-regulatory effect of triphasic oral contraceptive on platelet 3 H-imipramine binding sites. Psychiatry Res. 23, 23–29. Wilson, M., Kish, S.J., 1996. The vesicular monoamine transporter, in contrast to the dopamine transporter, is not altered by chronic cocaine self-administration in the rat. J. Neurosci. 16, 3507–3510. Zubieta, J.K., Taylor, S.F., Huguelet, P., Koeppe, R.A., Kilbourn, M.R.,
M. Zucker et al. / European Neuropsychopharmacology 12 (2002) 343 – 347 Frey, K.A., 2001. Vesicular monoamine transporter concentrations in bipolar disorder type I, schizophrenia and healthy subjects. Biol. Psychiatry 49, 110–116. Zucker, M., Weizman, A., Harel, D., Rehavi, M., 2001a. Changes in vesicular monoamine transporter (VMAT2) and synaptophysin in rat
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substantia nigra and prefrontal cortex induced by psychotropic drugs. Neuropsychobiology 44, 187–191. Zucker, M., Weizman, A., Rehavi, M., 2001b. Characterization of highaffinity [ 3 H]TBZOH binding to the human platelet vesicular monoamine transporter. Life Sci. 69, 2311–2317.
Increased platelet vesicular monoamine transporter density in adult schizophrenia patients a b a,b a, Michal Zucker , Avi Valevski , Abraham Weizman , Moshe Rehavi * a
b
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Geha Psychiatric Hospital and Felsenstein Medical Research Center, Beilinson Campus, Petah Tikva and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Received 20 September 2001; accepted 30 April 2002
Abstract Vesicular monoamine transporter (VMAT2) plays a major role in the synaptic accumulation and release of monoamines. In the present study, we assessed high affinity [ 3 H]dihydrotetrabenazine binding to platelet VMAT2 in a group of treated (n59) and untreated (n54) patients with schizophrenia and age- and sex-matched controls. Significantly elevated platelet VMAT2 density (Bmax ) (53%, P,0.0001) was observed in the mixed population of schizophrenia patients. The affinity of the ligand (Kd ) to platelet VMAT2 was similar in both groups. The increased platelet VMAT2 density may indicate a schizophrenia-related hyperactivity of the monoaminergic system or an adaptive response to chronic drug treatment. 2002 Elsevier Science B.V. All rights reserved. Keywords: Schizophrenia; Platelet; Vesicular monoamine transporter; Dihydrotetrabenazine; Monoamines
1. Introduction Schizophrenia is a chronic and disabling neuropsychiatric disorder of unknown etiology. On the basis of the strong correlation between the therapeutic potency of antipsychotic drugs in schizophrenia and their inhibitory activity at the D2 receptors, some researchers have hypothesized that a dysregulation in dopaminergic neurotransmission may play a role in the pathophysiology of the disorder (Seeman et al., 1976). However, many of the atypical antipsychotic drugs have a higher affinity for 5-HT2 receptors than for D2 receptors. Using platelet plasma membrane serotonin transporter as a peripheral model for plasma membrane serotonin transporter in the brain, studies have revealed alterations in the expression of serotonin transporter in patients with major depression (Paul et al., 1981), obsessive–compulsive disorder (Weizman et al., 1986; Sallee et al., 1996), and *Corresponding author. Tel.: 1972-3-640-8759; fax: 1972-3-6409113. E-mail address: [email protected] (M. Rehavi).
post-traumatic stress disorder (Fichtner et al., 1994), and in women treated with hormonal preparations (Weizman et al., 1987b). Findings in schizophrenia patients, however, were equivocal. Some investigators reported a decrease in imipramine binding (Arora et al., 1986) whereas others did not (Weizman et al., 1987a). Brain vesicular monoamine transporter (VMAT2) transports intracellular monoamines into the synaptic vesicles and is expressed in all monoaminergic neurons (Edwards, 1992). VMAT2 expression seems to be almost insensitive to treatment with drugs known to regulate monoaminergic neurotransmission, such as antipsychotics, monoamine oxidase inhibitors, and monoamine reuptake blockers (Naudon et al., 1994; Vander Borght et al., 1995; Wilson and Kish, 1996; Zucker et al., 2001a). Recently, positronemission tomography (PET) studies in treated patients with schizophrenia have shown an increase in VMAT2 expression in the ventral brainstem, but not in the thalamus (Zubieta et al., 2001) or striatum (Taylor et al., 2000). VMAT2 expression was higher in the thalamus and ventral brainstem of treated patients with bipolar disorder (Zubieta et al., 2001).
0924-977X / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0924-977X( 02 )00041-X
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M. Zucker et al. / European Neuropsychopharmacology 12 (2002) 343 – 347
VMAT2 is also expressed in the platelets (Cesura et al., 1990; Zucker et al., 2001b), and a line of evidence suggests that brain and platelet vesicular monoamine transporters have identical coding sequences and similar pharmacological properties (Lesch et al., 1993, 1994). Therefore, like for serotonin transporters, platelet VMAT2 may serve as a possible peripheral model for brain VMAT2 (Zucker et al., 2001b). Recently, Carlsson et al. (2001) suggested that schizophrenia might involve a dysregulation of several neurotransmitters. Therefore, we hypothesized that the disorder may be associated with an alteration in the expression of VMAT2, which is essential to the transmission of dopamine, serotonin and norepinephrine. Specifically, a VMAT2-induced increase in capacity to accumulate and release monoamines could lead to higher monoaminergic activity. This could explain the efficacy of agents with antagonistic activity at the monoaminergic receptors in the treatment of schizophrenia. In the present study, we assessed the pharmacodynamic characteristics of platelet VMAT2, using [ 3 H]dihydrotetrabenazine ([ 3 H]TBZOH) as a ligand, in untreated and treated schizophrenia patients.
2. Experimental procedures
2.1. Subjects Thirteen hospitalised patients with schizophrenia (seven males, six females) were recruited from Geha Psychiatric Hospital to participate in the study. All underwent a psychiatric interview according to the guidelines of the Structured Clinical Interview for DMS-IV Axis I Disorders [SCID-P] (First et al., 1996), and a diagnosis of schizophrenia was established according to the DSM-IV-TR criteria (American Psychiatric Association, 2000). Nine patients were being treated with antipsychotics only, two had been drug-free for at least 3 months (due to non compliance) and two were never treated (due to refusal to get psychiatric help). Symptoms were assessed with the Positive and Negative Syndrome Scale [PANSS] (Kay et al., 1987). Fifteen age- and sex-matched healthy subjects from the community served as controls. The study was approved by the Geha psychiatric hospital Review Board, and informed consent was obtained from all participants after the nature of the study was fully explained to them.
2.2. Platelet membrane preparation Blood samples (25 ml) were collected between 8:00 and 10:00 h into tubes containing an anticoagulant solution of 16 mM citrate buffer and 1 mM EDTA. Platelet-rich plasma was separated from blood cells by low-speed centrifugation (3503g for 10 min), resuspended in 20 ml
Hepes buffer 50 mM pH 8.0, and centrifuged at 17003g for 20 min. The pellet was disrupted with Brinkman polytron in 20 ml Hepes buffer 50 mM pH 8.0 containing 300 mM sucrose (buffer A) and centrifuged twice at 27 0003g for 20 min. It was then resuspended in 4.5 ml buffer A to yield a protein concentration of about 1 mg / ml.
2.3. [ 3 H] TBZOH binding For [ 3 H]TBZOH binding, 100 ml membranes were incubated at 25 8C with 50 ml of [ 3 H]TBZOH (eight concentrations, 0.5–8.0 nM) (specific activity: 20 Ci / mmol; American Radiolabeled Chemicals Inc., St. Louis MO) and 50 ml buffer A. After 30 min of incubation, the mixture was filtered with vacuum on glass fiber filters (GF / C). The filters were washed four times with ice-cold buffer A, and the radioactivity was counted in scintillation liquid in a b-counter (Packard 1600 TR). Nonspecific binding was measured in parallel samples in the presence of 1 mM tetrabenazine (Fluka, Buch, Switzerland). This ligand was shown to inhibit [ 3 H]TBZOH binding to human platelet VMAT2 in a competitive manner (IC 50 510 nM) (Zucker et al., 2001b). The nonspecific binding at Kd value (3.2 nM) did not exceed 20%. Protein concentration was determined according to the method of Bradford (1976). The ligand affinity (Kd ) to VMAT2 and [ 3 H]TBZOH binding capacity (Bmax ) were assessed by Scatchard analysis.
2.4. Statistical analysis Two-tailed Student’s t-test was used for between-group comparisons. Pearson correlation test was used as appropriate. All results are expressed as mean6S.D.
3. Results The demographic data of the patients and controls are given in Table 1. All participants were physically healthy and did not abuse drugs or alcohol. As there was no difference between the treated (n59) and untreated (n54) patients in Bmax [10946252 vs. 8536224 fmol / mg protein (222% vs. control), t51.64, df511, P50.13] or Kd (3.3660.93 vs. 3.4761.01 nm, t50.18, df511, P50.86), the findings for all the schizophrenia patients (n513) were pooled. VMAT2 density was found to be significantly higher in the schizophrenia patients (n513) than the controls (10206261 vs. 6686187 fmol / mg protein, t5 4.15, df526; P,0.0006) (Fig. 1), but there was no difference between the groups in Kd (3.4060.93 vs. 2.7361.19 nm, t51.64, df526; P50.11, NS). Sub-analysis of the data for the untreated patients alone (N54) as compared to the controls, revealed higher (128%)
M. Zucker et al. / European Neuropsychopharmacology 12 (2002) 343 – 347
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Table 1 Clinical and epidemiological data of the study population Schizophrenia patients (n513)
Healthy controls (n515)
Age range (years) Male / Female Age of onset (years) Duration of illness (years)
23–52 7/6 27.4666.50 11.6167.41
18–58 9/7 – –
Medications (dose range)a Atypical antipsychotics Clozapine (n53) mg / day Risperidone (n52) mg / day
150–325 2–6
– –
7.5–30 32 100 mg / 3 days 12.5–500 40 25 mg / 3 weeks 5.762.8
– – – – – – –
70.3618.3 19.3865.74 19.1568.35 31.7768.07
– – – –
Typical antipsychotics Haloperidol (n52) mg / day Perphenazine (n51) mg / day Zuclopenthixol (n51) Levomepromazine (n52) mg / day Penfluridol (n51) mg / week Fluphenazine decanoate (n51) Number of hospitalisations Symptom rating (mean6S.D.)b Total Positive Negative General a b
Some patients were treated with more than one antipsychotic. Positive and negative syndrome scale.
VMAT2 density, however, the results did not reach a significant level (t51.7, df517, P50.11, NS). Platelet VMAT2 density did not correlate with the severity of schizophrenia as assessed by the PANSS total score (r50.23, n513; P50.45, NS), and positive (r50.05, n513; P50.88, NS) and negative (r50.24, n513; P5 0.43, NS) and general (r50.03, n513; P50.92, NS) scores. To evaluate the possible in vitro interaction between [ 3 H]TBZOH binding and the antipsychotic drugs adminis-
Fig. 1. The density of [ 3 H]TBZOH binding sites (Bmax ) in platelet membranes of the study population. Values are mean6S.D. For details of the binding assay (see Experimental procedures).
tered to the patients, we assessed the inhibitory potency of the drugs on [ 3 H]TBZOH binding to platelet membranes. Only risperidone and haloperidol exhibited a modest (in the micromolar range) inhibitory effect (IC 50 54.5310 27 and 1.5310 26 M, respectively).
4. Discussion The major finding of this study was the high platelet VMAT2 density in schizophrenia patients compared to normal healthy controls. The VMAT2 Bmax values did not correlate significantly with the severity of the psychosis; however, most of the patients (9 / 13) were being maintained on antipsychotic drugs. Although the untreated schizophrenia patients (n54) exhibited lower Bmax values (222%) than the treated patients, the difference was not significant. This suggests that antipsychotic treatment per se is not responsible for the elevation in platelet VMAT2 density. This assumption is further supported by animal studies showing that brain VMAT2 is insensitive to treatment with agents active at the nigrostriatal dopaminergic pathway, such as haloperidol (Naudon et al., 1994; Vander Borght et al., 1995), the D2-agonist bromocriptine, and the dopamine reuptake inhibitor cocaine (Wilson and Kish, 1996). In our study, only two antipsychotics, haloperidol and risperidone, showed a tendency to inhibit, and not to augment, [ 3 H]TBZOH binding
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to human platelet membranes. Moreover, during the binding assay the platelet membranes were extensively washed, thus it seems unlikely that the presence of the drugs affect the binding values. Thus, the excessive VMAT2 binding sites in schizophrenia patients are apparently associated with the disorder, and not the exposure to antipsychotics. However, the Bmax values of the untreated patient (N54), although being higher (128% vs. controls), did not differ significantly from that of the controls. Furthermore, considering the high percentage of patients being treated with antipsychotics, we cannot conclusively rule out the role of neuroleptics or duration of disease and treatment in the increase in [ 3 H]TBZOH binding. It also remains unclear whether the elevated VMAT2 binding in platelets represents a parallel change in the brain. Interestingly, in vivo PET studies of brain VMAT2 levels in treated schizophrenia patients using [ 11 C]TBZOH as a ligand demonstrated increased VMAT2 density in the ventral brain stem (Zubieta et al., 2001), but not in the striatum (Taylor et al., 2000). As overexpression of VMAT2 in monoaminergic neurons is associated with increased monoamine storage and quantal release of monoamines from synaptic terminals (Fon et al., 1997; Reimer et al., 1998), its putative occurrence in brain regions relevant to schizophrenia could lead to functional overactivity of brain dopaminergic, serotonergic and noradrenergic pathways. It is of note that the VMAT2 blocker reserpine has been shown to be effective in the treatment of some drug-resistant schizophrenia patients, although its use is limited by its side effects (Jalenques, 1996). In conclusion, we observed a significant increase in platelet VMAT2 binding capacity in a mixed population of treated and untreated schizophrenia patients as compared to normal controls. This increase may indicate a hyperfunction of monoaminergic pathways in schizophrenia or an adaptive response to chronic antipsychotic treatment. Further controlled large-scale studies including treated and untreated patients with schizophrenia and other mental disorders are needed to confirm our finding and to demonstrate its specificity.
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