515065
article2014
JOP28410.1177/0269881113515065Journal of PsychopharmacologyMarazziti et al.
Original Paper
Clozapine effects on adenylyl cyclase activity and serotonin type 1A receptors in human brain post-mortem Donatella Marazziti1, Stefano Baroni1, Lionella Palego1, Laura Betti2, Gino Giannaccini2, Maura Castagna3, Antonio G Naccarato3, Antonio Luccachini2, Mario Catena-Dell’Osso4 and Liliana Dell’Osso1
Journal of Psychopharmacology 2014, Vol. 28(4) 320–328 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0269881113515065 jop.sagepub.com
Abstract Although the pharmacological profile of the atypical antipsychotic clozapine has been extensively studied in animal models, little information is available on its effects in the human brain. In particular, much interest is focused on the understanding of clozapine activity on serotonin (5-HT) neurotransmission, particularly on 5-HT receptor of type 1A (5-HT1A) that seems to play a pivotal role in the control of the 5-HT system. The present work, therefore, aimed at evaluating the effects of clozapine and its major metabolite, norclozapine, on the modulation of adenylyl cyclase (AC) velocity via 5-HT1A receptors in human post-mortem brain regions, in particular the prefrontal cortex, hippocampus and raphe nuclei. Concomitantly, the ability of the two compounds to displace the specific binding of the 5-HT1A receptor agonist [3H]-8-hydroxy-(2-di-N-propylamino) tetralin ([3H]-8-OH-DPAT) was evaluated in the same brain areas. The results showed that both clozapine and norclozapine, although with a 20fold lower affinity, displaced [3H]8-OH-DPAT binding in all of the brain regions analysed, suggesting their interaction with 5-HT1A receptors. At the same time, clozapine and, to a lesser extent, norclozapine were found to inhibit the forskolin (FK)-stimulated AC system, while decreasing cyclic adenosine monophosphate (cAMP) concentrations in the hippocampus only. The receptor characterisation of the clozapine effect on AC observed in the hippocampus by the use of antagonists showed a mixed profile, involving not only the 5-HT1A receptor but also a muscarinic (M) receptor subtype, most likely the M4 one. These findings, while considering all the limitations due to the use of post-mortem tissues, are strongly suggestive of a regiondependent pharmacological action of clozapine in the human brain that may explain its peculiar clinical effects and open up research towards novel targets for future antipsychotic drugs.
Keywords Clozapine, psychosis, adenylyl cyclase, human brain, serotonin type 1A receptors, post-mortem tissues
Introduction The understanding of the neurochemical substrates underlying schizophrenia still represents a main goal for psychiatrists and pharmacologists. The dopamine (DA) hypothesis postulated that positive symptoms would be the result of the hyperactive DA mesolimbic pathway, while the negative ones would be due to decreased DA activity in mesocortical projections to the prefrontal cortex (Abi-Dargham et al., 2000; Carlsson, 1978; Iversen, 1977; Laruelle et al., 1996). The observation that typical antipsychotics, which are DA receptors of type 2 (D2) blockers, improve positive symptoms but can provoke a worsening of negative symptoms, has strongly supported this theory (Meltzer and Sumiyoshi, 2008; Schooler, 1994). On the contrary, atypical antipsychotics show a broader pharmacological profile than the typical ones, since they act through mechanisms involving other neurotransmitters, in particular serotonin (5-HT) (Remington, 2003; Roth et al., 2004; Stahl, 2008). As a consequence, on the basis of pharmacological data, the original DA hypothesis has been modified to include abnormalities of different neurotransmitters and neuroreceptors (Hirvonen and Hietala, 2011; Kuroki et al., 2008; Stahl, 2008). Interestingly, preclinical and clinical evidence has increasingly drawn attention towards the possible role of serotonin type
1A (5-HT1A) receptors in the pathophysiology of schizophrenia. In animal models, 5-HT1A receptor activation was found to reverse extrapyramidal symptoms of typical antipsychotics (Prinssen et al., 1999), and to improve anxiety and depression (Blier and Ward, 2003). In humans, post-mortem and positron emission tomography (PET) studies highlighted that 5-HT1A receptors were up-regulated in the frontal cortex of schizophrenic patients (Burnet et al., 1997; Tauscher et al., 2002). Further, adjunctive treatment with tandospirone, a 5-HT1A partial agonist, 1Department
of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy 2Department of Pharmacy, University of Pisa, Pisa, Italy 3Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy 4Department of Clinical and Experimental Medicine, Polytechnic University of ‘Marche’, Ancona, Italy Corresponding author: Donatella Marazziti, Department of Clinical and Experimental Medicine, Psychiatry Unit, University of Pisa, Via Roma 67, 56100 Pisa, 56100, Italy. Email: [email protected]
Downloaded from jop.sagepub.com at TEXAS SOUTHERN UNIVERSITY on December 12, 2014
321
Marazziti et al. was shown to reduce memory dysfunctions in some schizophrenic patients (Sumiyoshi et al., 2001). The activation of 5-HT1A receptors would enhance the release of DA in the prefrontal cortex (Arborelius et al., 1993; Diaz-Mataix et al., 2005), while decreasing the functional impairment of mesocortical DA transmission which, as already mentioned, is believed to underlie negative symptoms of schizophrenia (Millan, 2000; Weinberger and Berman, 1996). Accordingly, atypical antipsychotics, which have been shown to act as partial agonists at recombinant human and native rat 5-HT1A receptors, can increase the fronto-cortical release of DA and improve negative symptoms (Ichikawa et al., 2002; Li et al., 1998; Newman-Tancredi et al., 1998; Rollema et al., 2000; Sharif et al., 2004). Clozapine, the prototype of an atypical antipsychotic first introduced in the clinical practice, is a tricyclic dibenzazepinic compound which shows a lower affinity for D2 and greater affinity for D4 receptors than typical antipsychotics (Coward, 1992). Clozapine displays high affinity and antagonistic properties for 5-HT receptors in particular the 5-HT2A, 5-HT2C, 5-HT3, 5-HT6 and 5-HT7 subtypes (Agid et al., 2010; Heiser et al., 2004; Millan, 2000). Beside these activities, clozapine seems to interact, as an agonist, with 5-HT1A receptors which have been correlated either to the regulation of mood-anxiety levels or to cognitive processes (Drevets et al., 2007). In fact, in transfected cell lines expressing 5-HT1A receptors, clozapine was found to inhibit the adenylyl cyclase (AC) activity stimulated by forskolin (FK), an effect which was sensitive to 5-HT1A antagonists (Assie et al., 1997; Newman-Tancredi et al., 1996; Sharif et al., 2004). A similar inhibition of AC activity was reported in the rat brain, albeit mediated by muscarinic M4 receptors (Olianas et al., 1997). Further, the partial agonistic properties of clozapine on 5-HT1A receptors was confirmed by the use of guanosine-5’-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding assays in membranes of transfected cells and of rat hippocampus (Millan et al., 1998; Newman-Tancredi et al., 1998; Odagaki and Toyoshima, 2007). Although the pharmacological profile of clozapine has been extensively studied in several models, there is a gap in the literature concerning the human brain. In a binding study performed by using the high-affinity agonist [3H]-8-hydroxy-(2-di-Npropylamino) tetralin ([3H]-8-OH-DPAT) (Larsson et al., 1990) to evaluate the affinities of a range of antipsychotics to human hippocampal 5-HT1A receptors, clozapine showed the highest affinity (Mason and Reynolds, 1992). Moreover, there is no data regarding the pharmacological effects of the major metabolite of clozapine, norclozapine, on native human brain 5-HT1A receptors. In the present work, we aimed, therefore, at evaluating the effects of clozapine and norclozapine on FK-stimulated AC activity in three human brain regions obtained post-mortem, considered at moderate and high 5-HT1A receptor density: the prefrontal cortex, hippocampus and raphe nuclei. In order to verify if these compounds could bind to 5-HT1A receptors, we also carried out competition binding experiments by means of the 5-HT1A agonist [3H]8-OH-DPAT in the same brain areas.
Materials and methods Chemicals [3H]-8-OH-DPAT (specific activity: 135 Ci/mmol), [32P]-α-ATP (specific activity: 30 Ci/mmol) and [3H]- cyclyc adensine
monophosphate (cAMP) (specific activity: 27 Ci/mmol) were purchased from Perkin-Elmer Life Science (Milan, Italy). FK, adenosine triphosphate (ATP); cAMP, isobutyl-methyl-xantine (IBMX), guanosine triphosphate (GTP), 8-OH-DPAT, yohimbine, pindobind 5-HT1A, buspirone, tropicamide, clozapine and norclozapine were purchased from Sigma–Aldrich Corporation (Milan, Italy). Reagents for total protein determination were purchased from Bio-Rad (Bio-Rad Laboratories, CA, USA). All the other reagents used were of the best analytical grade.
Sample collection The sample collection from the human brain regions under investigation, in particular, the prefrontal cortex, hippocampus and raphe nuclei, was carried out during autopsy sessions by qualified anatomists from the Department of Translational Research and Nez Technologies in Medicine and Surgery of the Dipartimento di Chirurgia of the University of Pisa. Brain samples were taken as parts of tissues kept for diagnostic scopes, always following the procedure approved by the Ethics Committee of the University of Pisa. The brain specimens were obtained from a total of 15 subjects (ten men and five women, mean age±standard error of the mean (SEM): 69±2.9 years), who had died from causes not involving primarily or secondarily the central nervous system (CNS) (myocardial infarction and cardiovascular diseases: n= 9; kidney failure: n=3; pulmonary diseases: n=3), and were not suffering from chronic metabolic diseases, dementia, neurological or major psychiatric disorders, as shown by their medical charts. The post-mortem delay (PMD, the time between demise and tissue dissection/freezing) ranged between 10–45 h (mean±SEM: 26.5 ± 3.1 h). After collection by anatomists, brain samples were kept on ice and immediately processed for membrane preparation.
AC assay Brain samples were homogenised by hand in 50 vols (w/v) of icecold 10 mM HEPES–NaOH buffer, pH=7.4, containing 0.32 M sucrose, using a glass Potter-teflon homogeniser (20–30 strokes). Homogenates were then centrifuged at 1000 g for 10 min at +4°C. Supernatants were collected and centrifuged at 13,500 g for 20 min at +4°C. The resulting pellet was re-suspended in 50 vols (w/v) of ice-cold 10 mM HEPES–NaOH buffer, pH 7.4. After incubating membranes on ice for 10 min to displace endogenous compounds, the homogenate was centrifuged at 35,000 g for 20 min at +4°C. The resulting pellet was frozen in liquid nitrogen until assay, which was carried out within one week. On the day of assay, frozen membranes were suspended in 2 mL of 10 mM HEPES NaOH buffer, pH 7.4, and centrifuged at 35,000 g for 20 min at +4°C. The resulting pellet was then diluted in the same buffer, yielding a final protein concentration of about 0.6 mg/mL. Protein evaluation was performed by means of the Bradford Bio-Rad kit, using γ-globulin as the standard. This suspension was pre-incubated at 24°C for 10–15 min before assay and diluted as above. An exogenous substrate enzyme method was chosen herein for determining AC activity and cAMP production in human postmortem cerebral tissues, following a procedure modified from Salomon (1979) and a double column chromatographic separation procedure accordingly to Johnson et al. (1994). Briefly, the
Downloaded from jop.sagepub.com at TEXAS SOUTHERN UNIVERSITY on December 12, 2014
322
Journal of Psychopharmacology 28(4)
Table 1. Basal adenylyl cyclase (AC) and forskolin (FK)-stimulated AC activity in prefrontal cortex, hippocampus and raphe nuclei. The percentage of AC stimulation with FK for basal values of 100% is also reported.
Prefrontal cortex Hippocampus Raphe nuclei
Basal AC velocity (a)
FK-stimulated AC velocity (a)
% stimulation
135±56c 87±49 84±41
346±184b,c 191±120b 160±84b
253±60 223±75 203±70
Data are presented as mean ± standard deviation (SD) (n=15). aMeasured as pmol cyclic adenosine monophosphate (cAMP)/min mg protein. bSignificantly higher FK-stimulated vs. basal AC velocity in the three areas: ANOVA F(1,42)=78.85, p 4.5, p10 μM in subjects’ samples where no effect was found and, as such, not detectable in our assay, as the maximal agonist concentration used herein was 10 μM. The inhibitory effect of clozapine on AC activity was characterised by the use of 5-HT1A receptor antagonists and the muscarinic M4 receptor antagonist tropicamide. At the tested doses, pindobind 5-HT1A, provoked a strong rightward shift of the dose-response curve, without changing the drug efficacy. This suggests that pindobind 5-HT1A competitively antagonises clozapine-mediated AC inhibition via 5-HT1A receptors. Buspirone also behaved as a competitive antagonist, while being, however, much less potent than pindobind 5-HT1A, as it slightly shifted towards right the clozapine dose-response curve, without substantial changes in efficacy. An
intermediate antagonism effect, between pindobind 5-HT1A and buspirone, was observed for higher doses of tropicamide. Taken together, these findings indicate that the clozapine inhibition of AC activity in the hippocampus is not a pure effect, as it results from the interaction with 5-HT1A and M4 receptor subtypes, both co-localised in the hippocampus and coupled to Gi proteins and AC inhibition. Although present results are preliminary, they might be due to cross-talk between 5-HT and muscarinic receptor subtypes in the human hippocampus, as observed in other systems and model (Jordan and Devi, 1999). The mixed 5-HT1A/ M4 inhibitory response observed herein also provides further support for the existence of functional relationships between acetylcholine and serotonergic neurotransmission in the hippocampus (Vizi and Kiss, 1998), together with new insights on the regulation of the cerebral cholinergic tonus in cognitive processes (Tzavara et al., 2003). Some doubt may arise concerning the identification of the muscarinic receptor subtype involved in the clozapine-mediated AC inhibitory response: some authors, in fact, have reported a partial selectivity of tropicamide for M4 receptors (Lazareno and Birdsall, 1993). Moreover, M4 receptors have been mainly localised in the striatum, while being
Downloaded from jop.sagepub.com at TEXAS SOUTHERN UNIVERSITY on December 12, 2014
326
Journal of Psychopharmacology 28(4)
co-localised with M2 receptors in the rodent hippocampus, where each subtype displays a distinct role in the regulation of the cognitive function (Tzavara et al., 2003). The issue can be clarified through immunohistochemistry, co-localisation and coimmunoprecipitation studies of muscarinic and serotonergic receptor subtypes in human tissues. The use of human cerebral tissues post-mortem would permit obtaining valuable information on the molecular mechanisms of clozapine’s action under native conditions. Possible limitations can derive from ethical issues, the small sample size of subjects investigated and postmortem degradation. In the present study, age or PMD did not modify clozapine-mediated inhibition of AC velocity within the sample of autopsy subjects examined. At the same time, subjects evaluated in our work represent an old population, mean age of 70 years, not allowing us to comprehensively estimate the effect of age on these parameters. The possible effect of gender was not determined herein since there was a prevalence of men compared to women. However, our results overall confirm previous evidence of clozapine interaction with 5-HT1A receptors coming from studies performed in transfected cells and rat brain, underlining the validity of the nmodel, obviously, the comparison between native tissues and cell lines should be always carefully evaluated, given the variability of plasmatic membrane components (Millan et al., 1998; Newman-Tancredi et al., 1998; Odagaki and Toyoshima, 2007). Third, in the present work, the competition binding data were more homogenous than AC experiments, while demonstrating that clozapine and norclozapine were both able to interact with 5-HT1A receptors in all brain areas under investigation, with higher affinities in the hippocampus and cortex respectively. Moreover, by binding experiments, clozapine was shown to displace the [3H]8-OH-DPAT specific binding to the prefrontal cortex, hippocampus and raphe nuclei displaying monophasic curves; norclozapine was found to similarly displace the [3H]8OH-DPAT binding to all the three brain areas, although with a lower affinity (15-40 fold) than the mother compound. In summary, our results indicate that clozapine and its major metabolite norclozapine interact with 5-HT1A receptors in the three brain regions investigated. However, clozapine and, to a poor extent, even norclozapine, can inhibit the AC system in the hippocampus only, while decreasing the intracellular cAMP concentrations. These findings are strongly suggestive of a regiondependent pharmacological action of clozapine. It was reported that the effectiveness of atypical antipsychotics on negative (cognitive and affective) symptoms of schizophrenia was linked to DA release in the prefrontal cortex, possibly through the activation of 5-HT1A receptors (Bortolozzi et al., 2010; Chou et al., 2003). Conversely, our data support at least an indirect or partial role of 5-HT1A receptors in DA release from brain cortices, since we observed no effect of the drug on AC in the human prefrontal cortex. At the same time, data in the literature show that 5-HT1A receptors are increased in the cortex of schizophrenic patients by binding experiments using either agonists (Hashimoto et al., 1991; Sumiyoshi et al., 1996) or antagonists (Tauscher et al., 2002), encouraging the further exploration of the role of the receptor coupling state in psychotic symptoms. Apart from such a question, the finding that clozapine has a region-dependent pharmacological effect in the human brain seems rather in favour of the possibility that atypical
antipsychotics act through different area-dependent mechanisms in the CNS. There are several possible explanations of these brain regional mechanisms, most of them associated with the mixed pharmacological profile of this atypical antipsychotic compound. The absence of a clozapine-mediated functional effect on FK-stimulated AC velocity in the human cortex and raphe could imply that the drug behaves as an antagonist upon 5-HT1A FK-stimulated AC inhibition in these areas. On the other hand, present findings might be ascribed to the existence of a 5-HT1A receptor variance within the brain, such as different 5-HT1A mRNA expression, presence of 5-HT1A receptor isoforms, or diverse coupling/regulatory states. This has been suggested by immuno-histochemistry studies which have revealed differences in the 5-HT1A receptor labelling patterns between the cortex, raphe nuclei and hippocampus (Anthony and Azmitia, 1997), suggesting that structural changes can underlie the different functionality of the receptor within the CNS. Other authors have also underscored the existence of differential gene expression patterns after 5-HT1A activation in the hippocampus and cortex of the rat brain (Tilakaratne and Friedman, 1996). As aforementioned, additional heterogeneity of human brain 5-HT1A receptors could derive from the receptor coupling state and/or G protein isoforms reserve, as well as from different receptor trafficking and protein kinase regulation in the various brain areas which, in turn, would orientate the stimulation of other signal transduction pathways, including ion channel opening or phospholipase C modulation (Banerjee et al., 2007; Borsini et al., 1999). Again, an increased positive cAMP response through the release of Gi/o protein β/γ subunits after 5-HT1A receptor activation has been proposed (Uezono et al., 2004). Therefore, clozapine could provoke functional effects also in the cortex and raphe nuclei through other G protein subunits/isoforms. This would explain the observed clozapine ability to release DA from both rat prefrontal cortex and hippocampus involving, partially, 5-HT1A receptors (Chung et al., 2004). These authors also report that clozapine provokes acetylcholine (ACh) release from the same regions, presumably via M4 receptors. The use of different experimental models (rat brain and human brain autopsy samples) or the presence of species differences in 5-HT1A receptor sensitisation states could explain all these findings. Concomitantly, clozapine central effects seem to reflect multiple and complex receptor-mediated molecular mechanisms in discrete brain regions, apparently redundant or opposite but permitting the adaptation of a physiological response to different stimuli and stressors. Compounds acting as partial agonists on a G protein-coupled receptor subtype could be inactive and behave as apparent antagonists depending on their coupling state, interaction with other receptor subtypes and effector availability in the brain region (Marazziti et al, 2002; Monferini et al., 1993). Therefore, taking into consideration all the limitations due to the use of post-mortem tissues, clozapine’s regional effects on AC activity might derive from the different reserve of target neurotransmitter receptors in different brain areas. In fact, this compound interacts not only with 5-HT1A receptors, but also, as an antagonist or inverse agonist, with other 5-HT receptor subtypes, such as the 5-HT7 ones (Rauly-Lestienne et al., 2007; Sprouse et al., 2004): this indicates that, beside 5-HT1A receptor regulatory pathways, clozapine-mediated AC activity decrease can be potentiated by the drug inhibitory effects on other excitatory
Downloaded from jop.sagepub.com at TEXAS SOUTHERN UNIVERSITY on December 12, 2014
327
Marazziti et al. 5-HT receptor subtypes. At the same time, since the observed clozapine-mediated inhibition of FK-stimulated AC has been found to be a mixed event, our results would be additionally dependent on the relative 5-HT/M receptor subtype reserve/ availability in the different brain regions. In fact, this atypical antipsychotic drug has been found to act as a partial agonist also at the level of M1, M2 and M3 receptors (Olianas et al., 1999). In addition, the distribution/efficiency of AC isoforms within the human brain could have also contributed to the observed regional differences in clozapine responses on AC activity.
Conclusions In conclusion, the observations that the inhibition of FK-activated AC activity by clozapine may be mediated by 5-HT1A and M4 receptors in the human brain with a specific regional pattern provides new insights on the therapeutic clinical properties of this compound, as well as suggesting novel targets for future antipsychotic drugs.
Acknowledgements The authors would like to thank Dr. Maria F Cioffi for her valuable assistance during the carrying out of this work.
Conflict of interest The authors declare that there are no conflict of interest.
Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
References Abi-Dargham A, Rodenhiser J, Printz D, et al. (2000) Increased baseline occupancy of D2 receptors by dopamine in schizophrenia. Proc Natl Acad Sci U S A 97: 8104–8109. Agid O, Foussias G, Singh S, et al. (2010) Where to position clozapine: Re-examining the evidence. Can J Psychiatry 55: 677–684. Anthony TE and Azmitia EC (1997) Molecular characterization of antipeptide antibodies against the 5-HT1A receptor: Evidence for state-dependent antibody binding. Brain Res Mol Brain Res 50: 277–284. Arborelius L, Nomikos GG, Hacksell U, et al. (1993) (R)-8-OH-DPAT preferentially increases dopamine release in rat medial prefrontal cortex. Acta Physiol Scand 148: 465–466. Assie MB, Cosi C and Koek W (1997) 5-HT1A receptor agonist properties of the antipsychotic, nemonapride: Comparison with bromerguride and clozapine. Eur J Pharmacol 334: 141–147. Banerjee P, Mehta M and Kanjilal B (2007) The 5-HT1A receptor: A signaling hub linked to emotional balance. Chapter 7, In: Chattopadhyay A (ed) Serotonin Receptors in Neurobiology. Frontiers in Neuroscience. Boca Raton: CRC Press. Blier P and Ward NM (2003) Is there a role for 5-HT1A agonists in the treatment of depression? Biol Psychiatry 53: 193–203. Borsini F, Brambilla A, Grippa N, et al. (1999) Behavioral effects of flibanserin (BIMT 17). Pharmacol Biochem Behav 64: 137–146. Bortolozzi A, Masana M, Diaz-Mataix L, et al. (2010) Dopamine release induced by atypical antipsychotics in prefrontal cortex requires 5-HT(1A) receptors but not 5-HT(2A) receptors. Int J Neuropsychopharmacol 13: 1299–1314. Burnet PW, Eastwood SL and Harrison PJ (1997) [3H]WAY-100635 for 5-HT1A receptor autoradiography in human brain: A comparison
with [3H]8-OH-DPAT and demonstration of increased binding in the frontal cortex in schizophrenia. Neurochem Int 30: 565–574. Carlsson A (1978) Antipsychotic drugs, neurotransmitters, and schizophrenia. Am J Psychiatry 135: 165–173. Cheng Y and Prusoff WH (1973) Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 22: 3099–3108. Chou YH, Halldin C and Farde L (2003) Occupancy of 5-HT1A receptors by clozapine in the primate brain: A PET study. Psychopharmacology (Berl) 166: 234–240. Chung YC, Li Z, Dai J, et al. (2004) Clozapine increases both acetylcholine and dopamine release in rat ventral hippocampus: Role of 5-HT1A receptor agonism. Brain Res 1023: 54–63. Coward DM (1992) General pharmacology of clozapine. Br J Psychiatry Suppl: 5–11. Cowburn RF, Marcusson JO, Eriksson A, et al. (1994) Adenylyl cyclase activity and G-protein subunit levels in postmortem frontal cortex of suicide victims. Brain Res 633: 297–304. Diaz-Mataix L, Scorza MC, Bortolozzi A, et al. (2005) Involvement of 5-HT1A receptors in prefrontal cortex in the modulation of dopaminergic activity: Role in atypical antipsychotic action. J Neurosci 25: 10831–10843. Drevets WC, Thase ME, Moses-Kolko EL, et al. (2007) Serotonin-1A receptor imaging in recurrent depression: Replication and literature review. Nucl Med Biol 34: 865–877. Hashimoto T, Nishino N, Nakai H, et al. (1991) Increase in serotonin 5-HT1A receptors in prefrontal and temporal cortices of brains from patients with chronic schizophrenia. Life Sci 48: 355–363. Heiser P, Schulte E, Hausmann C, et al. (2004) Effects of clozapine and its metabolites on the 5-HT2 receptor system in cortical and hippocampal cells in vitro. Prog Neuropsychopharmacol Biol Psychiatry 28: 297–302. Hirvonen J and Hietala J (2011) Dysfunctional brain networks and genetic risk for schizophrenia: Specific neurotransmitter systems. CNS Neurosci Ther 17: 89–96. Ichikawa J, Li Z, Dai J, et al. (2002) Atypical antipsychotic drugs, quetiapine, iloperidone, and melperone, preferentially increase dopamine and acetylcholine release in rat medial prefrontal cortex: Role of 5-HT1A receptor agonism. Brain Res 956: 349–357. Iversen LL (1977) Dopaminergic mechanisms in schizophrenia. Proc R Soc Med 70: 1–4. Johnson RA, Alvarez R and Salomon Y (1994) Assay of AC catalytic activity. In: Iyengar R (ed) Methods in Enzymology. San Diego: Academic Press, pp. 31–56. Jordan BA and Devi LA (1999) G protein coupled receptor heterodimerization modulates receptor function. Nature 399: 697–700. Kuroki T, Nagao N and Nakahara T (2008) Neuropharmacology of second-generation antipsychotic drugs: A validity of the serotonindopamine hypothesis. Prog Brain Res 172: 199–212. Larsson LG, Renyi L, Ross SB, et al. (1990) Different effects on the responses of functional pre- and postsynaptic 5-HT1A receptors by repeated treatment of rats with the 5-HT1A receptor agonist 8-OHDPAT. Neuropharmacology 29: 85–91. Laruelle M, Abi-Dargham A, van Dyck CH, et al. (1996) Single photon emission computerized tomography imaging of amphetamineinduced dopamine release in drug-free schizophrenic subjects. Proc Natl Acad Sci U S A 93: 9235–9240. Lazareno S and Birdsall NJ (1993) Pharmacological characterization of acetylcholine-stimulated [35S]-GTP gamma S binding mediated by human muscarinic m1-m4 receptors: Antagonist studies. Br J Pharmacol 109: 1120–1127. Li XM, Perry KW, Wong DT, et al. (1998) Olanzapine increases in vivo dopamine and norepinephrine release in rat prefrontal cortex, nucleus accumbens and striatum. Psychopharmacology (Berl) 136: 153–161.
Downloaded from jop.sagepub.com at TEXAS SOUTHERN UNIVERSITY on December 12, 2014
328
Journal of Psychopharmacology 28(4)
Marazziti D, Marracci S, Palego L, et al. (1994) Localization and gene expression of serotonin 1A (5HT1A) receptors in human brain postmortem. Brain Res 658: 55–59. Marazziti D, Palego L, Giromella A, et al. (2002) Region-dependent effects of flibanserin and buspirone on adenylyl cyclase activity in the human brain. Int J Neuropsychopharmacol 5: 131–140. Mason SL and Reynolds GP (1992) Clozapine has sub-micromolar affinity for 5-HT1A receptors in human brain tissue. Eur J Pharmacol 221: 397–398. Meltzer HY and Sumiyoshi T (2008) Does stimulation of 5-HT(1A) receptors improve cognition in schizophrenia? Behav Brain Res 195: 98–102. Millan MJ (2000) Improving the treatment of schizophrenia: Focus on serotonin (5-HT)(1A) receptors. J Pharmacol Exp Ther 295: 853–861. Millan MJ, Gobert A, Newman-Tancredi A, et al. (1998) S 16924 ((R)2-[1-[2-(2,3-dihydro-benzo[1,4] dioxin-5-Yloxy)-ethyl]-pyrrolidin3yl]-1-(4-fluoro-phenyl)-ethanone), a novel, potential antipsychotic with marked serotonin (5-HT)1A agonist properties: I. Receptorial and neurochemical profile in comparison with clozapine and haloperidol. J Pharmacol Exp Ther 286: 1341–1355. Monferini E, Gaetani P, Rodriguez y Baena R, et al. (1993) Pharmacological characterization of the 5-hydroxytryptamine receptor coupled to adenylyl cyclase stimulation in human brain. Life Sci 52: 61–65. Newman-Tancredi A, Chaput C, Verriele L, et al. (1996) Clozapine is a partial agonist at cloned, human serotonin 5-HT1A receptors. Neuropharmacology 35: 119–121. Newman-Tancredi A, Gavaudan S, Conte C, et al. (1998) Agonist and antagonist actions of antipsychotic agents at 5-HT1A receptors: A [35S]GTPgammaS binding study. Eur J Pharmacol 355: 245–256. Odagaki Y and Toyoshima R (2007) 5-HT1A receptor agonist properties of antipsychotics determined by [35S]GTPgammaS binding in rat hippocampal membranes. Clin Exp Pharmacol Physiol 34: 462–466. Ohm TG, Bohl J and Lemmer B (1991) Reduced basal and stimulated (isoprenaline, Gpp(NH)p, forskolin) adenylate cyclase activity in Alzheimer’s disease correlated with histopathological changes. Brain Res 540: 229–236. Olianas MC, Maullu C and Onali P (1997) Effects of clozapine on rat striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity and on the human cloned m4 receptor. Br J Pharmacol 122: 401–408. Olianas MC, Maullu C and Onali P (1999) Mixed agonist-antagonist properties of clozapine at different human cloned muscarinic receptor subtypes expressed in Chinese hamster ovary cells. Neuropsychopharmacology 20: 263–270. Palego L, Giromella A, Marazziti D, et al. (1999) Effects of postmortem delay on serotonin and (+)8-OH-DPAT-mediated inhibition of adenylyl cyclase activity in rat and human brain tissues. Brain Res 816: 165–174. Prinssen EP, Kleven MS and Koek W (1999) Interactions between neuroleptics and 5-HT(1A) ligands in preclinical behavioral models for antipsychotic and extrapyramidal effects. Psychopharmacology (Berl) 144: 20–29. Rauly-Lestienne I, Boutet-Robinet E, Ailhaud MC, et al. (2007) Differential profile of typical atypical and third generation antipsychotics at human 5-HT7a receptors coupled to adenylyl cyclase: Detection of agonist and inverse agonist properties. Naunyn Schmiedebergs Arch Pharmacol 376: 93–105.
Remington G (2003) Understanding antipsychotic ‘atypicality’: A clinical and pharmacological moving target. J Psychiatry Neurosci 28: 275–284. Rollema H, Lu Y, Schmidt AW, et al. (2000) 5-HT(1A) receptor activation contributes to ziprasidone-induced dopamine release in the rat prefrontal cortex. Biol Psychiatry 48: 229–237. Roth BL, Hanizavareh SM and Blum AE (2004) Serotonin receptors represent highly favorable molecular targets for cognitive enhancement in schizophrenia and other disorders. Psychopharmacology (Berl) 174: 17–24. Salomon Y (1979) Adenylate cyclase assay. Adv Cyclic Nucleotide Res 10: 35–55. Schooler NR (1994) Deficit symptoms in schizophrenia: Negative symptoms versus neuroleptic-induced deficits. Acta Psychiatr Scand Suppl 380: 21–26. Sharif NA, Drace CD, Williams GW, et al. (2004) Cloned human 5-HT1A receptor pharmacology determined using agonist binding and measurement of cAMP accumulation. J Pharm Pharmacol 56: 1267–1274. Sprouse J, Reynolds L, Li X, et al. (2004) 8-OH-DPAT as a 5-HT7 agonist: Phase shifts of the circadian biological clock through increases in cAMP production. Neuropharmacology 46: 52–62. Stahl MS (2008) Stahl’s essential psychopharmacology. Neuroscientific basis and practical applications, New York: Cambridge University Press. Sumiyoshi T, Matsui M, Yamashita I, et al. (2001) The effect of tandospirone, a serotonin(1A) agonist, on memory function in schizophrenia. Biol Psychiatry 49: 861–868. Sumiyoshi T, Stockmeier CA, Overholser JC, et al. (1996) Serotonin1A receptors are increased in postmortem prefrontal cortex in schizophrenia. Brain Res 708: 209–214. Tauscher J, Kapur S, Verhoeff NP, et al. (2002) Brain serotonin 5-HT(1A) receptor binding in schizophrenia measured by positron emission tomography and [11C]WAY-100635. Arch Gen Psychiatry 59: 514–520. Tilakaratne N and Friedman E (1996) Genomic responses to 5-HT1A or 5-HT2A/2C receptor activation is differentially regulated in four regions of rat brain. Eur J Pharmacol 307: 211–217. Tong J, Ross BM, Sherwin AL, et al. (2001) Dopamine D1-stimulated adenylyl cyclase activity in cerebral cortex of autopsied human brain. Neurochem Int 39: 117–125. Tzavara ET, Bymaster FP, Felder CC, et al. (2003) Dysregulated hippocampal acetylcholine neurotransmission and impaired cognition in M2, M4 and M2/M4 muscarinic receptor knockout mice. Mol Psychiatry 8: 673–679. Uezono Y, Kaibara M, Murasaki O, et al. (2004) Involvement of G protein betagamma-subunits in diverse signaling induced by G(i/o)-coupled receptors: Study using the Xenopus oocyte expression system. Am J Physiol Cell Physiol 287: C885–C894. Vizi ES and Kiss JP (1998) Neurochemistry and pharmacology of the major hippocampal transmitter systems: Synaptic and nonsynaptic interactions. Hippocampus 8: 566–607. Weinberger DR and Berman KF (1996) Prefrontal function in schizophrenia: Confounds and controversies. Philos Trans R Soc Lond B Biol Sci 351: 1495–1503. Yamamoto K, Yamamoto Y, Matsumoto H, et al. (1997) Unusual postmortem autolytic change in the liver: Wavy transformation of hepatocytes. Med Sci Law 37: 256–259.
Downloaded from jop.sagepub.com at TEXAS SOUTHERN UNIVERSITY on December 12, 2014
article2014
JOP28410.1177/0269881113515065Journal of PsychopharmacologyMarazziti et al.
Original Paper
Clozapine effects on adenylyl cyclase activity and serotonin type 1A receptors in human brain post-mortem Donatella Marazziti1, Stefano Baroni1, Lionella Palego1, Laura Betti2, Gino Giannaccini2, Maura Castagna3, Antonio G Naccarato3, Antonio Luccachini2, Mario Catena-Dell’Osso4 and Liliana Dell’Osso1
Journal of Psychopharmacology 2014, Vol. 28(4) 320–328 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0269881113515065 jop.sagepub.com
Abstract Although the pharmacological profile of the atypical antipsychotic clozapine has been extensively studied in animal models, little information is available on its effects in the human brain. In particular, much interest is focused on the understanding of clozapine activity on serotonin (5-HT) neurotransmission, particularly on 5-HT receptor of type 1A (5-HT1A) that seems to play a pivotal role in the control of the 5-HT system. The present work, therefore, aimed at evaluating the effects of clozapine and its major metabolite, norclozapine, on the modulation of adenylyl cyclase (AC) velocity via 5-HT1A receptors in human post-mortem brain regions, in particular the prefrontal cortex, hippocampus and raphe nuclei. Concomitantly, the ability of the two compounds to displace the specific binding of the 5-HT1A receptor agonist [3H]-8-hydroxy-(2-di-N-propylamino) tetralin ([3H]-8-OH-DPAT) was evaluated in the same brain areas. The results showed that both clozapine and norclozapine, although with a 20fold lower affinity, displaced [3H]8-OH-DPAT binding in all of the brain regions analysed, suggesting their interaction with 5-HT1A receptors. At the same time, clozapine and, to a lesser extent, norclozapine were found to inhibit the forskolin (FK)-stimulated AC system, while decreasing cyclic adenosine monophosphate (cAMP) concentrations in the hippocampus only. The receptor characterisation of the clozapine effect on AC observed in the hippocampus by the use of antagonists showed a mixed profile, involving not only the 5-HT1A receptor but also a muscarinic (M) receptor subtype, most likely the M4 one. These findings, while considering all the limitations due to the use of post-mortem tissues, are strongly suggestive of a regiondependent pharmacological action of clozapine in the human brain that may explain its peculiar clinical effects and open up research towards novel targets for future antipsychotic drugs.
Keywords Clozapine, psychosis, adenylyl cyclase, human brain, serotonin type 1A receptors, post-mortem tissues
Introduction The understanding of the neurochemical substrates underlying schizophrenia still represents a main goal for psychiatrists and pharmacologists. The dopamine (DA) hypothesis postulated that positive symptoms would be the result of the hyperactive DA mesolimbic pathway, while the negative ones would be due to decreased DA activity in mesocortical projections to the prefrontal cortex (Abi-Dargham et al., 2000; Carlsson, 1978; Iversen, 1977; Laruelle et al., 1996). The observation that typical antipsychotics, which are DA receptors of type 2 (D2) blockers, improve positive symptoms but can provoke a worsening of negative symptoms, has strongly supported this theory (Meltzer and Sumiyoshi, 2008; Schooler, 1994). On the contrary, atypical antipsychotics show a broader pharmacological profile than the typical ones, since they act through mechanisms involving other neurotransmitters, in particular serotonin (5-HT) (Remington, 2003; Roth et al., 2004; Stahl, 2008). As a consequence, on the basis of pharmacological data, the original DA hypothesis has been modified to include abnormalities of different neurotransmitters and neuroreceptors (Hirvonen and Hietala, 2011; Kuroki et al., 2008; Stahl, 2008). Interestingly, preclinical and clinical evidence has increasingly drawn attention towards the possible role of serotonin type
1A (5-HT1A) receptors in the pathophysiology of schizophrenia. In animal models, 5-HT1A receptor activation was found to reverse extrapyramidal symptoms of typical antipsychotics (Prinssen et al., 1999), and to improve anxiety and depression (Blier and Ward, 2003). In humans, post-mortem and positron emission tomography (PET) studies highlighted that 5-HT1A receptors were up-regulated in the frontal cortex of schizophrenic patients (Burnet et al., 1997; Tauscher et al., 2002). Further, adjunctive treatment with tandospirone, a 5-HT1A partial agonist, 1Department
of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy 2Department of Pharmacy, University of Pisa, Pisa, Italy 3Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy 4Department of Clinical and Experimental Medicine, Polytechnic University of ‘Marche’, Ancona, Italy Corresponding author: Donatella Marazziti, Department of Clinical and Experimental Medicine, Psychiatry Unit, University of Pisa, Via Roma 67, 56100 Pisa, 56100, Italy. Email: [email protected]
Downloaded from jop.sagepub.com at TEXAS SOUTHERN UNIVERSITY on December 12, 2014
321
Marazziti et al. was shown to reduce memory dysfunctions in some schizophrenic patients (Sumiyoshi et al., 2001). The activation of 5-HT1A receptors would enhance the release of DA in the prefrontal cortex (Arborelius et al., 1993; Diaz-Mataix et al., 2005), while decreasing the functional impairment of mesocortical DA transmission which, as already mentioned, is believed to underlie negative symptoms of schizophrenia (Millan, 2000; Weinberger and Berman, 1996). Accordingly, atypical antipsychotics, which have been shown to act as partial agonists at recombinant human and native rat 5-HT1A receptors, can increase the fronto-cortical release of DA and improve negative symptoms (Ichikawa et al., 2002; Li et al., 1998; Newman-Tancredi et al., 1998; Rollema et al., 2000; Sharif et al., 2004). Clozapine, the prototype of an atypical antipsychotic first introduced in the clinical practice, is a tricyclic dibenzazepinic compound which shows a lower affinity for D2 and greater affinity for D4 receptors than typical antipsychotics (Coward, 1992). Clozapine displays high affinity and antagonistic properties for 5-HT receptors in particular the 5-HT2A, 5-HT2C, 5-HT3, 5-HT6 and 5-HT7 subtypes (Agid et al., 2010; Heiser et al., 2004; Millan, 2000). Beside these activities, clozapine seems to interact, as an agonist, with 5-HT1A receptors which have been correlated either to the regulation of mood-anxiety levels or to cognitive processes (Drevets et al., 2007). In fact, in transfected cell lines expressing 5-HT1A receptors, clozapine was found to inhibit the adenylyl cyclase (AC) activity stimulated by forskolin (FK), an effect which was sensitive to 5-HT1A antagonists (Assie et al., 1997; Newman-Tancredi et al., 1996; Sharif et al., 2004). A similar inhibition of AC activity was reported in the rat brain, albeit mediated by muscarinic M4 receptors (Olianas et al., 1997). Further, the partial agonistic properties of clozapine on 5-HT1A receptors was confirmed by the use of guanosine-5’-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding assays in membranes of transfected cells and of rat hippocampus (Millan et al., 1998; Newman-Tancredi et al., 1998; Odagaki and Toyoshima, 2007). Although the pharmacological profile of clozapine has been extensively studied in several models, there is a gap in the literature concerning the human brain. In a binding study performed by using the high-affinity agonist [3H]-8-hydroxy-(2-di-Npropylamino) tetralin ([3H]-8-OH-DPAT) (Larsson et al., 1990) to evaluate the affinities of a range of antipsychotics to human hippocampal 5-HT1A receptors, clozapine showed the highest affinity (Mason and Reynolds, 1992). Moreover, there is no data regarding the pharmacological effects of the major metabolite of clozapine, norclozapine, on native human brain 5-HT1A receptors. In the present work, we aimed, therefore, at evaluating the effects of clozapine and norclozapine on FK-stimulated AC activity in three human brain regions obtained post-mortem, considered at moderate and high 5-HT1A receptor density: the prefrontal cortex, hippocampus and raphe nuclei. In order to verify if these compounds could bind to 5-HT1A receptors, we also carried out competition binding experiments by means of the 5-HT1A agonist [3H]8-OH-DPAT in the same brain areas.
Materials and methods Chemicals [3H]-8-OH-DPAT (specific activity: 135 Ci/mmol), [32P]-α-ATP (specific activity: 30 Ci/mmol) and [3H]- cyclyc adensine
monophosphate (cAMP) (specific activity: 27 Ci/mmol) were purchased from Perkin-Elmer Life Science (Milan, Italy). FK, adenosine triphosphate (ATP); cAMP, isobutyl-methyl-xantine (IBMX), guanosine triphosphate (GTP), 8-OH-DPAT, yohimbine, pindobind 5-HT1A, buspirone, tropicamide, clozapine and norclozapine were purchased from Sigma–Aldrich Corporation (Milan, Italy). Reagents for total protein determination were purchased from Bio-Rad (Bio-Rad Laboratories, CA, USA). All the other reagents used were of the best analytical grade.
Sample collection The sample collection from the human brain regions under investigation, in particular, the prefrontal cortex, hippocampus and raphe nuclei, was carried out during autopsy sessions by qualified anatomists from the Department of Translational Research and Nez Technologies in Medicine and Surgery of the Dipartimento di Chirurgia of the University of Pisa. Brain samples were taken as parts of tissues kept for diagnostic scopes, always following the procedure approved by the Ethics Committee of the University of Pisa. The brain specimens were obtained from a total of 15 subjects (ten men and five women, mean age±standard error of the mean (SEM): 69±2.9 years), who had died from causes not involving primarily or secondarily the central nervous system (CNS) (myocardial infarction and cardiovascular diseases: n= 9; kidney failure: n=3; pulmonary diseases: n=3), and were not suffering from chronic metabolic diseases, dementia, neurological or major psychiatric disorders, as shown by their medical charts. The post-mortem delay (PMD, the time between demise and tissue dissection/freezing) ranged between 10–45 h (mean±SEM: 26.5 ± 3.1 h). After collection by anatomists, brain samples were kept on ice and immediately processed for membrane preparation.
AC assay Brain samples were homogenised by hand in 50 vols (w/v) of icecold 10 mM HEPES–NaOH buffer, pH=7.4, containing 0.32 M sucrose, using a glass Potter-teflon homogeniser (20–30 strokes). Homogenates were then centrifuged at 1000 g for 10 min at +4°C. Supernatants were collected and centrifuged at 13,500 g for 20 min at +4°C. The resulting pellet was re-suspended in 50 vols (w/v) of ice-cold 10 mM HEPES–NaOH buffer, pH 7.4. After incubating membranes on ice for 10 min to displace endogenous compounds, the homogenate was centrifuged at 35,000 g for 20 min at +4°C. The resulting pellet was frozen in liquid nitrogen until assay, which was carried out within one week. On the day of assay, frozen membranes were suspended in 2 mL of 10 mM HEPES NaOH buffer, pH 7.4, and centrifuged at 35,000 g for 20 min at +4°C. The resulting pellet was then diluted in the same buffer, yielding a final protein concentration of about 0.6 mg/mL. Protein evaluation was performed by means of the Bradford Bio-Rad kit, using γ-globulin as the standard. This suspension was pre-incubated at 24°C for 10–15 min before assay and diluted as above. An exogenous substrate enzyme method was chosen herein for determining AC activity and cAMP production in human postmortem cerebral tissues, following a procedure modified from Salomon (1979) and a double column chromatographic separation procedure accordingly to Johnson et al. (1994). Briefly, the
Downloaded from jop.sagepub.com at TEXAS SOUTHERN UNIVERSITY on December 12, 2014
322
Journal of Psychopharmacology 28(4)
Table 1. Basal adenylyl cyclase (AC) and forskolin (FK)-stimulated AC activity in prefrontal cortex, hippocampus and raphe nuclei. The percentage of AC stimulation with FK for basal values of 100% is also reported.
Prefrontal cortex Hippocampus Raphe nuclei
Basal AC velocity (a)
FK-stimulated AC velocity (a)
% stimulation
135±56c 87±49 84±41
346±184b,c 191±120b 160±84b
253±60 223±75 203±70
Data are presented as mean ± standard deviation (SD) (n=15). aMeasured as pmol cyclic adenosine monophosphate (cAMP)/min mg protein. bSignificantly higher FK-stimulated vs. basal AC velocity in the three areas: ANOVA F(1,42)=78.85, p 4.5, p10 μM in subjects’ samples where no effect was found and, as such, not detectable in our assay, as the maximal agonist concentration used herein was 10 μM. The inhibitory effect of clozapine on AC activity was characterised by the use of 5-HT1A receptor antagonists and the muscarinic M4 receptor antagonist tropicamide. At the tested doses, pindobind 5-HT1A, provoked a strong rightward shift of the dose-response curve, without changing the drug efficacy. This suggests that pindobind 5-HT1A competitively antagonises clozapine-mediated AC inhibition via 5-HT1A receptors. Buspirone also behaved as a competitive antagonist, while being, however, much less potent than pindobind 5-HT1A, as it slightly shifted towards right the clozapine dose-response curve, without substantial changes in efficacy. An
intermediate antagonism effect, between pindobind 5-HT1A and buspirone, was observed for higher doses of tropicamide. Taken together, these findings indicate that the clozapine inhibition of AC activity in the hippocampus is not a pure effect, as it results from the interaction with 5-HT1A and M4 receptor subtypes, both co-localised in the hippocampus and coupled to Gi proteins and AC inhibition. Although present results are preliminary, they might be due to cross-talk between 5-HT and muscarinic receptor subtypes in the human hippocampus, as observed in other systems and model (Jordan and Devi, 1999). The mixed 5-HT1A/ M4 inhibitory response observed herein also provides further support for the existence of functional relationships between acetylcholine and serotonergic neurotransmission in the hippocampus (Vizi and Kiss, 1998), together with new insights on the regulation of the cerebral cholinergic tonus in cognitive processes (Tzavara et al., 2003). Some doubt may arise concerning the identification of the muscarinic receptor subtype involved in the clozapine-mediated AC inhibitory response: some authors, in fact, have reported a partial selectivity of tropicamide for M4 receptors (Lazareno and Birdsall, 1993). Moreover, M4 receptors have been mainly localised in the striatum, while being
Downloaded from jop.sagepub.com at TEXAS SOUTHERN UNIVERSITY on December 12, 2014
326
Journal of Psychopharmacology 28(4)
co-localised with M2 receptors in the rodent hippocampus, where each subtype displays a distinct role in the regulation of the cognitive function (Tzavara et al., 2003). The issue can be clarified through immunohistochemistry, co-localisation and coimmunoprecipitation studies of muscarinic and serotonergic receptor subtypes in human tissues. The use of human cerebral tissues post-mortem would permit obtaining valuable information on the molecular mechanisms of clozapine’s action under native conditions. Possible limitations can derive from ethical issues, the small sample size of subjects investigated and postmortem degradation. In the present study, age or PMD did not modify clozapine-mediated inhibition of AC velocity within the sample of autopsy subjects examined. At the same time, subjects evaluated in our work represent an old population, mean age of 70 years, not allowing us to comprehensively estimate the effect of age on these parameters. The possible effect of gender was not determined herein since there was a prevalence of men compared to women. However, our results overall confirm previous evidence of clozapine interaction with 5-HT1A receptors coming from studies performed in transfected cells and rat brain, underlining the validity of the nmodel, obviously, the comparison between native tissues and cell lines should be always carefully evaluated, given the variability of plasmatic membrane components (Millan et al., 1998; Newman-Tancredi et al., 1998; Odagaki and Toyoshima, 2007). Third, in the present work, the competition binding data were more homogenous than AC experiments, while demonstrating that clozapine and norclozapine were both able to interact with 5-HT1A receptors in all brain areas under investigation, with higher affinities in the hippocampus and cortex respectively. Moreover, by binding experiments, clozapine was shown to displace the [3H]8-OH-DPAT specific binding to the prefrontal cortex, hippocampus and raphe nuclei displaying monophasic curves; norclozapine was found to similarly displace the [3H]8OH-DPAT binding to all the three brain areas, although with a lower affinity (15-40 fold) than the mother compound. In summary, our results indicate that clozapine and its major metabolite norclozapine interact with 5-HT1A receptors in the three brain regions investigated. However, clozapine and, to a poor extent, even norclozapine, can inhibit the AC system in the hippocampus only, while decreasing the intracellular cAMP concentrations. These findings are strongly suggestive of a regiondependent pharmacological action of clozapine. It was reported that the effectiveness of atypical antipsychotics on negative (cognitive and affective) symptoms of schizophrenia was linked to DA release in the prefrontal cortex, possibly through the activation of 5-HT1A receptors (Bortolozzi et al., 2010; Chou et al., 2003). Conversely, our data support at least an indirect or partial role of 5-HT1A receptors in DA release from brain cortices, since we observed no effect of the drug on AC in the human prefrontal cortex. At the same time, data in the literature show that 5-HT1A receptors are increased in the cortex of schizophrenic patients by binding experiments using either agonists (Hashimoto et al., 1991; Sumiyoshi et al., 1996) or antagonists (Tauscher et al., 2002), encouraging the further exploration of the role of the receptor coupling state in psychotic symptoms. Apart from such a question, the finding that clozapine has a region-dependent pharmacological effect in the human brain seems rather in favour of the possibility that atypical
antipsychotics act through different area-dependent mechanisms in the CNS. There are several possible explanations of these brain regional mechanisms, most of them associated with the mixed pharmacological profile of this atypical antipsychotic compound. The absence of a clozapine-mediated functional effect on FK-stimulated AC velocity in the human cortex and raphe could imply that the drug behaves as an antagonist upon 5-HT1A FK-stimulated AC inhibition in these areas. On the other hand, present findings might be ascribed to the existence of a 5-HT1A receptor variance within the brain, such as different 5-HT1A mRNA expression, presence of 5-HT1A receptor isoforms, or diverse coupling/regulatory states. This has been suggested by immuno-histochemistry studies which have revealed differences in the 5-HT1A receptor labelling patterns between the cortex, raphe nuclei and hippocampus (Anthony and Azmitia, 1997), suggesting that structural changes can underlie the different functionality of the receptor within the CNS. Other authors have also underscored the existence of differential gene expression patterns after 5-HT1A activation in the hippocampus and cortex of the rat brain (Tilakaratne and Friedman, 1996). As aforementioned, additional heterogeneity of human brain 5-HT1A receptors could derive from the receptor coupling state and/or G protein isoforms reserve, as well as from different receptor trafficking and protein kinase regulation in the various brain areas which, in turn, would orientate the stimulation of other signal transduction pathways, including ion channel opening or phospholipase C modulation (Banerjee et al., 2007; Borsini et al., 1999). Again, an increased positive cAMP response through the release of Gi/o protein β/γ subunits after 5-HT1A receptor activation has been proposed (Uezono et al., 2004). Therefore, clozapine could provoke functional effects also in the cortex and raphe nuclei through other G protein subunits/isoforms. This would explain the observed clozapine ability to release DA from both rat prefrontal cortex and hippocampus involving, partially, 5-HT1A receptors (Chung et al., 2004). These authors also report that clozapine provokes acetylcholine (ACh) release from the same regions, presumably via M4 receptors. The use of different experimental models (rat brain and human brain autopsy samples) or the presence of species differences in 5-HT1A receptor sensitisation states could explain all these findings. Concomitantly, clozapine central effects seem to reflect multiple and complex receptor-mediated molecular mechanisms in discrete brain regions, apparently redundant or opposite but permitting the adaptation of a physiological response to different stimuli and stressors. Compounds acting as partial agonists on a G protein-coupled receptor subtype could be inactive and behave as apparent antagonists depending on their coupling state, interaction with other receptor subtypes and effector availability in the brain region (Marazziti et al, 2002; Monferini et al., 1993). Therefore, taking into consideration all the limitations due to the use of post-mortem tissues, clozapine’s regional effects on AC activity might derive from the different reserve of target neurotransmitter receptors in different brain areas. In fact, this compound interacts not only with 5-HT1A receptors, but also, as an antagonist or inverse agonist, with other 5-HT receptor subtypes, such as the 5-HT7 ones (Rauly-Lestienne et al., 2007; Sprouse et al., 2004): this indicates that, beside 5-HT1A receptor regulatory pathways, clozapine-mediated AC activity decrease can be potentiated by the drug inhibitory effects on other excitatory
Downloaded from jop.sagepub.com at TEXAS SOUTHERN UNIVERSITY on December 12, 2014
327
Marazziti et al. 5-HT receptor subtypes. At the same time, since the observed clozapine-mediated inhibition of FK-stimulated AC has been found to be a mixed event, our results would be additionally dependent on the relative 5-HT/M receptor subtype reserve/ availability in the different brain regions. In fact, this atypical antipsychotic drug has been found to act as a partial agonist also at the level of M1, M2 and M3 receptors (Olianas et al., 1999). In addition, the distribution/efficiency of AC isoforms within the human brain could have also contributed to the observed regional differences in clozapine responses on AC activity.
Conclusions In conclusion, the observations that the inhibition of FK-activated AC activity by clozapine may be mediated by 5-HT1A and M4 receptors in the human brain with a specific regional pattern provides new insights on the therapeutic clinical properties of this compound, as well as suggesting novel targets for future antipsychotic drugs.
Acknowledgements The authors would like to thank Dr. Maria F Cioffi for her valuable assistance during the carrying out of this work.
Conflict of interest The authors declare that there are no conflict of interest.
Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
References Abi-Dargham A, Rodenhiser J, Printz D, et al. (2000) Increased baseline occupancy of D2 receptors by dopamine in schizophrenia. Proc Natl Acad Sci U S A 97: 8104–8109. Agid O, Foussias G, Singh S, et al. (2010) Where to position clozapine: Re-examining the evidence. Can J Psychiatry 55: 677–684. Anthony TE and Azmitia EC (1997) Molecular characterization of antipeptide antibodies against the 5-HT1A receptor: Evidence for state-dependent antibody binding. Brain Res Mol Brain Res 50: 277–284. Arborelius L, Nomikos GG, Hacksell U, et al. (1993) (R)-8-OH-DPAT preferentially increases dopamine release in rat medial prefrontal cortex. Acta Physiol Scand 148: 465–466. Assie MB, Cosi C and Koek W (1997) 5-HT1A receptor agonist properties of the antipsychotic, nemonapride: Comparison with bromerguride and clozapine. Eur J Pharmacol 334: 141–147. Banerjee P, Mehta M and Kanjilal B (2007) The 5-HT1A receptor: A signaling hub linked to emotional balance. Chapter 7, In: Chattopadhyay A (ed) Serotonin Receptors in Neurobiology. Frontiers in Neuroscience. Boca Raton: CRC Press. Blier P and Ward NM (2003) Is there a role for 5-HT1A agonists in the treatment of depression? Biol Psychiatry 53: 193–203. Borsini F, Brambilla A, Grippa N, et al. (1999) Behavioral effects of flibanserin (BIMT 17). Pharmacol Biochem Behav 64: 137–146. Bortolozzi A, Masana M, Diaz-Mataix L, et al. (2010) Dopamine release induced by atypical antipsychotics in prefrontal cortex requires 5-HT(1A) receptors but not 5-HT(2A) receptors. Int J Neuropsychopharmacol 13: 1299–1314. Burnet PW, Eastwood SL and Harrison PJ (1997) [3H]WAY-100635 for 5-HT1A receptor autoradiography in human brain: A comparison
with [3H]8-OH-DPAT and demonstration of increased binding in the frontal cortex in schizophrenia. Neurochem Int 30: 565–574. Carlsson A (1978) Antipsychotic drugs, neurotransmitters, and schizophrenia. Am J Psychiatry 135: 165–173. Cheng Y and Prusoff WH (1973) Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 22: 3099–3108. Chou YH, Halldin C and Farde L (2003) Occupancy of 5-HT1A receptors by clozapine in the primate brain: A PET study. Psychopharmacology (Berl) 166: 234–240. Chung YC, Li Z, Dai J, et al. (2004) Clozapine increases both acetylcholine and dopamine release in rat ventral hippocampus: Role of 5-HT1A receptor agonism. Brain Res 1023: 54–63. Coward DM (1992) General pharmacology of clozapine. Br J Psychiatry Suppl: 5–11. Cowburn RF, Marcusson JO, Eriksson A, et al. (1994) Adenylyl cyclase activity and G-protein subunit levels in postmortem frontal cortex of suicide victims. Brain Res 633: 297–304. Diaz-Mataix L, Scorza MC, Bortolozzi A, et al. (2005) Involvement of 5-HT1A receptors in prefrontal cortex in the modulation of dopaminergic activity: Role in atypical antipsychotic action. J Neurosci 25: 10831–10843. Drevets WC, Thase ME, Moses-Kolko EL, et al. (2007) Serotonin-1A receptor imaging in recurrent depression: Replication and literature review. Nucl Med Biol 34: 865–877. Hashimoto T, Nishino N, Nakai H, et al. (1991) Increase in serotonin 5-HT1A receptors in prefrontal and temporal cortices of brains from patients with chronic schizophrenia. Life Sci 48: 355–363. Heiser P, Schulte E, Hausmann C, et al. (2004) Effects of clozapine and its metabolites on the 5-HT2 receptor system in cortical and hippocampal cells in vitro. Prog Neuropsychopharmacol Biol Psychiatry 28: 297–302. Hirvonen J and Hietala J (2011) Dysfunctional brain networks and genetic risk for schizophrenia: Specific neurotransmitter systems. CNS Neurosci Ther 17: 89–96. Ichikawa J, Li Z, Dai J, et al. (2002) Atypical antipsychotic drugs, quetiapine, iloperidone, and melperone, preferentially increase dopamine and acetylcholine release in rat medial prefrontal cortex: Role of 5-HT1A receptor agonism. Brain Res 956: 349–357. Iversen LL (1977) Dopaminergic mechanisms in schizophrenia. Proc R Soc Med 70: 1–4. Johnson RA, Alvarez R and Salomon Y (1994) Assay of AC catalytic activity. In: Iyengar R (ed) Methods in Enzymology. San Diego: Academic Press, pp. 31–56. Jordan BA and Devi LA (1999) G protein coupled receptor heterodimerization modulates receptor function. Nature 399: 697–700. Kuroki T, Nagao N and Nakahara T (2008) Neuropharmacology of second-generation antipsychotic drugs: A validity of the serotonindopamine hypothesis. Prog Brain Res 172: 199–212. Larsson LG, Renyi L, Ross SB, et al. (1990) Different effects on the responses of functional pre- and postsynaptic 5-HT1A receptors by repeated treatment of rats with the 5-HT1A receptor agonist 8-OHDPAT. Neuropharmacology 29: 85–91. Laruelle M, Abi-Dargham A, van Dyck CH, et al. (1996) Single photon emission computerized tomography imaging of amphetamineinduced dopamine release in drug-free schizophrenic subjects. Proc Natl Acad Sci U S A 93: 9235–9240. Lazareno S and Birdsall NJ (1993) Pharmacological characterization of acetylcholine-stimulated [35S]-GTP gamma S binding mediated by human muscarinic m1-m4 receptors: Antagonist studies. Br J Pharmacol 109: 1120–1127. Li XM, Perry KW, Wong DT, et al. (1998) Olanzapine increases in vivo dopamine and norepinephrine release in rat prefrontal cortex, nucleus accumbens and striatum. Psychopharmacology (Berl) 136: 153–161.
Downloaded from jop.sagepub.com at TEXAS SOUTHERN UNIVERSITY on December 12, 2014
328
Journal of Psychopharmacology 28(4)
Marazziti D, Marracci S, Palego L, et al. (1994) Localization and gene expression of serotonin 1A (5HT1A) receptors in human brain postmortem. Brain Res 658: 55–59. Marazziti D, Palego L, Giromella A, et al. (2002) Region-dependent effects of flibanserin and buspirone on adenylyl cyclase activity in the human brain. Int J Neuropsychopharmacol 5: 131–140. Mason SL and Reynolds GP (1992) Clozapine has sub-micromolar affinity for 5-HT1A receptors in human brain tissue. Eur J Pharmacol 221: 397–398. Meltzer HY and Sumiyoshi T (2008) Does stimulation of 5-HT(1A) receptors improve cognition in schizophrenia? Behav Brain Res 195: 98–102. Millan MJ (2000) Improving the treatment of schizophrenia: Focus on serotonin (5-HT)(1A) receptors. J Pharmacol Exp Ther 295: 853–861. Millan MJ, Gobert A, Newman-Tancredi A, et al. (1998) S 16924 ((R)2-[1-[2-(2,3-dihydro-benzo[1,4] dioxin-5-Yloxy)-ethyl]-pyrrolidin3yl]-1-(4-fluoro-phenyl)-ethanone), a novel, potential antipsychotic with marked serotonin (5-HT)1A agonist properties: I. Receptorial and neurochemical profile in comparison with clozapine and haloperidol. J Pharmacol Exp Ther 286: 1341–1355. Monferini E, Gaetani P, Rodriguez y Baena R, et al. (1993) Pharmacological characterization of the 5-hydroxytryptamine receptor coupled to adenylyl cyclase stimulation in human brain. Life Sci 52: 61–65. Newman-Tancredi A, Chaput C, Verriele L, et al. (1996) Clozapine is a partial agonist at cloned, human serotonin 5-HT1A receptors. Neuropharmacology 35: 119–121. Newman-Tancredi A, Gavaudan S, Conte C, et al. (1998) Agonist and antagonist actions of antipsychotic agents at 5-HT1A receptors: A [35S]GTPgammaS binding study. Eur J Pharmacol 355: 245–256. Odagaki Y and Toyoshima R (2007) 5-HT1A receptor agonist properties of antipsychotics determined by [35S]GTPgammaS binding in rat hippocampal membranes. Clin Exp Pharmacol Physiol 34: 462–466. Ohm TG, Bohl J and Lemmer B (1991) Reduced basal and stimulated (isoprenaline, Gpp(NH)p, forskolin) adenylate cyclase activity in Alzheimer’s disease correlated with histopathological changes. Brain Res 540: 229–236. Olianas MC, Maullu C and Onali P (1997) Effects of clozapine on rat striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity and on the human cloned m4 receptor. Br J Pharmacol 122: 401–408. Olianas MC, Maullu C and Onali P (1999) Mixed agonist-antagonist properties of clozapine at different human cloned muscarinic receptor subtypes expressed in Chinese hamster ovary cells. Neuropsychopharmacology 20: 263–270. Palego L, Giromella A, Marazziti D, et al. (1999) Effects of postmortem delay on serotonin and (+)8-OH-DPAT-mediated inhibition of adenylyl cyclase activity in rat and human brain tissues. Brain Res 816: 165–174. Prinssen EP, Kleven MS and Koek W (1999) Interactions between neuroleptics and 5-HT(1A) ligands in preclinical behavioral models for antipsychotic and extrapyramidal effects. Psychopharmacology (Berl) 144: 20–29. Rauly-Lestienne I, Boutet-Robinet E, Ailhaud MC, et al. (2007) Differential profile of typical atypical and third generation antipsychotics at human 5-HT7a receptors coupled to adenylyl cyclase: Detection of agonist and inverse agonist properties. Naunyn Schmiedebergs Arch Pharmacol 376: 93–105.
Remington G (2003) Understanding antipsychotic ‘atypicality’: A clinical and pharmacological moving target. J Psychiatry Neurosci 28: 275–284. Rollema H, Lu Y, Schmidt AW, et al. (2000) 5-HT(1A) receptor activation contributes to ziprasidone-induced dopamine release in the rat prefrontal cortex. Biol Psychiatry 48: 229–237. Roth BL, Hanizavareh SM and Blum AE (2004) Serotonin receptors represent highly favorable molecular targets for cognitive enhancement in schizophrenia and other disorders. Psychopharmacology (Berl) 174: 17–24. Salomon Y (1979) Adenylate cyclase assay. Adv Cyclic Nucleotide Res 10: 35–55. Schooler NR (1994) Deficit symptoms in schizophrenia: Negative symptoms versus neuroleptic-induced deficits. Acta Psychiatr Scand Suppl 380: 21–26. Sharif NA, Drace CD, Williams GW, et al. (2004) Cloned human 5-HT1A receptor pharmacology determined using agonist binding and measurement of cAMP accumulation. J Pharm Pharmacol 56: 1267–1274. Sprouse J, Reynolds L, Li X, et al. (2004) 8-OH-DPAT as a 5-HT7 agonist: Phase shifts of the circadian biological clock through increases in cAMP production. Neuropharmacology 46: 52–62. Stahl MS (2008) Stahl’s essential psychopharmacology. Neuroscientific basis and practical applications, New York: Cambridge University Press. Sumiyoshi T, Matsui M, Yamashita I, et al. (2001) The effect of tandospirone, a serotonin(1A) agonist, on memory function in schizophrenia. Biol Psychiatry 49: 861–868. Sumiyoshi T, Stockmeier CA, Overholser JC, et al. (1996) Serotonin1A receptors are increased in postmortem prefrontal cortex in schizophrenia. Brain Res 708: 209–214. Tauscher J, Kapur S, Verhoeff NP, et al. (2002) Brain serotonin 5-HT(1A) receptor binding in schizophrenia measured by positron emission tomography and [11C]WAY-100635. Arch Gen Psychiatry 59: 514–520. Tilakaratne N and Friedman E (1996) Genomic responses to 5-HT1A or 5-HT2A/2C receptor activation is differentially regulated in four regions of rat brain. Eur J Pharmacol 307: 211–217. Tong J, Ross BM, Sherwin AL, et al. (2001) Dopamine D1-stimulated adenylyl cyclase activity in cerebral cortex of autopsied human brain. Neurochem Int 39: 117–125. Tzavara ET, Bymaster FP, Felder CC, et al. (2003) Dysregulated hippocampal acetylcholine neurotransmission and impaired cognition in M2, M4 and M2/M4 muscarinic receptor knockout mice. Mol Psychiatry 8: 673–679. Uezono Y, Kaibara M, Murasaki O, et al. (2004) Involvement of G protein betagamma-subunits in diverse signaling induced by G(i/o)-coupled receptors: Study using the Xenopus oocyte expression system. Am J Physiol Cell Physiol 287: C885–C894. Vizi ES and Kiss JP (1998) Neurochemistry and pharmacology of the major hippocampal transmitter systems: Synaptic and nonsynaptic interactions. Hippocampus 8: 566–607. Weinberger DR and Berman KF (1996) Prefrontal function in schizophrenia: Confounds and controversies. Philos Trans R Soc Lond B Biol Sci 351: 1495–1503. Yamamoto K, Yamamoto Y, Matsumoto H, et al. (1997) Unusual postmortem autolytic change in the liver: Wavy transformation of hepatocytes. Med Sci Law 37: 256–259.
Downloaded from jop.sagepub.com at TEXAS SOUTHERN UNIVERSITY on December 12, 2014
