Antagonist Functional Selectivity: 5-HT2A Serotonin Receptor Antagonists Differentially Regulate 5-HT2A Receptor Protein Level In Vivo

Department of Pharmacology, University of North Carolina, Chapel Hill Medical School, Chapel Hill, North Carolina 27599, USA.
Journal of Pharmacology and Experimental Therapeutics (Impact Factor: 3.97). 07/2011; 339(1):99-105. DOI: 10.1124/jpet.111.183780
Source: PubMed


Dysregulation of the 5-HT(2A) receptor is implicated in both the etiology and treatment of schizophrenia. Although the essential role of 5-HT(2A) receptors in atypical antipsychotic drug actions is widely accepted, the contribution of 5-HT(2A) down-regulation to their efficacy is not known. We hypothesized that down-regulation of cortical 5-HT(2A) receptors contributes to the therapeutic action of atypical antipsychotic drugs. To test this hypothesis, we assessed the effect of chronically administered antipsychotics (clozapine, olanzapine, and haloperidol) and several 5-HT(2A) antagonists [ketanserin, altanserin, α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol (M100907), α-phenyl-1-(2-phenylethyl)-4-piperidinemethano (M11939), 4-[(2Z)-3-{[2-(dimethylamino)ethoxy]amino}-3-(2-fluorophenyl)prop-2-en-1-ylidene]cyclohexa-2,5-dien-1-one (SR46349B), and pimavanserin], on the phencyclidine (PCP)-induced hyperlocomotor response and cortical 5-HT(2A) receptor levels in C57BL/6J mice. Clozapine and olanzapine, but not haloperidol, induced receptor down-regulation and attenuated PCP-induced locomotor responses. Of the selective 5-HT(2A) antagonists tested, only ketanserin caused significant receptor protein down-regulation, whereas SR46349B up-regulated 5-HT(2A) receptors and potentiated PCP-hyperlocomotion; the other 5-HT(2A) receptor antagonists were without effect. The significance of these findings with respect to atypical antipsychotic drug action is discussed.

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    • "As seen in A (middle row), a profuse laminar distribution of 5-HT 2A R immunoreactivity was detected in the rat mPFC using the Immunostar 5-HT 2A R antibody from Neuromics (see Table 1). The strongest 5-HT 2A R immunoreactivity was seen in layer V. Though quite profuse, the 5-HT 2A R immunostaining produced by the antibody is typical for this cortical region (Weber and Andrade, 2010; Yadav et al., 2011b). Under higher magnification , a punctate 5-HT 2A R expression could be seen on the soma and initial segment of cells in the region (see sample cell in B, center and left photo). "
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    ABSTRACT: Neural function within the medial prefrontal cortex (mPFC) regulates normal cognition, attention and impulse control, implicating neuroregulatory abnormalities within this region in mental dysfunction related to schizophrenia, depression and drug abuse. Both serotonin -2A (5-HT2A) and -2C (5-HT2C) receptors are known to be important in neuropsychiatric drug action and are distributed throughout the mPFC. However, their interactive role in serotonergic cortical regulation is poorly understood. While the main signal transduction mechanism for both receptors is stimulation of phosphoinositide production, they can have opposite effects downstream. 5-HT2A versus 5-HT2C receptor activation oppositely regulates behavior and can oppositely affect neurochemical release within the mPFC. These distinct receptor effects could be caused by their differential cellular distribution within the cortex and/or other areas. It is known that both receptors are located on GABAergic and pyramidal cells within the mPFC, but it is not clear whether they are expressed on the same or different cells. The present work employed immunofluorescence with confocal microscopy to examine this in layers V-VI of the prelimbic mPFC. The majority of GABA cells in the deep prelimbic mPFC expressed 5-HT2C receptor immunoreactivity. Furthermore, most cells expressing 5-HT2C receptor immunoreactivity notably co-expressed 5-HT2A receptors. However, 27% of 5-HT2C receptor immunoreactive cells were not GABAergic, indicating that a population of prelimbic pyramidal projection cells could express the 5-HT2C receptor. Indeed, some cells with 5-HT2C and 5-HT2A receptor co-labeling had a pyramidal shape and were expressed in the typical layered fashion of pyramidal cells. This indirectly demonstrates that 5-HT2C and 5-HT2A receptors may be commonly co-expressed on GABAergic cells within the deep layers of the prelimbic mPFC and perhaps co-localized on a small population of local pyramidal projection cells. Thus a complex interplay of cortical 5-HT2A and 5-HT2C receptor mechanisms exists, which if altered, could modulate efferent brain systems implicated in mental illness. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Mar 2015 · Neuroscience
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    • "Clozapine is typically considered a potent inverse agonist at the 5-HT2AR (Weiner et al, 2001); however, this classification is complicated by the findings that chronic administration causes downregulation of this receptor in brain (Matsubara and Meltzer, 1989; Moreno et al, 2013; Peroutka and Snyder, 1980; Yadav et al, 2011) and that it can internalize the 5-HT2AR both in vitro and in vivo (Bhatnagar et al, 2001; Raote et al, 2013; Willins et al, 1998, 1999). In addition to causing 5-HT2AR internalization and downregulation , clozapine has also been shown to promote protein kinase B (Akt) phosphorylation in cultured neuronal cells and the rat prefrontal cortex (Dwyer and Donohoe, 2003; Lu et al, 2004; Sutton and Rushlow, 2011; Xi et al, 2011), thereby demonstrating that clozapine can act like an agonist. "
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    ABSTRACT: The G protein-coupled serotonin 2A receptor (5-HT2AR) is a prominent target for atypical antipsychotic drugs, such as clozapine. Although clozapine is known to inhibit 5-HT2AR signaling through G protein-dependent mechanisms, it differs from classic GPCR antagonists, in that it also induces 5-HT2AR internalization and activates Akt signaling via a 5-HT2AR-mediated event. In this regard, clozapine may also be considered a functionally selective agonist. The cognate neurotransmitter at the 5-HT2AR, serotonin, also induces 5-HT2AR internalization and Akt phosphorylation. Serotonin promotes interactions with the scaffolding and regulatory protein, βarrestin2, which results in the recruitment and activation of Akt. These interactions prove to be critical for serotonin-induced, 5-HT2AR-mediated behavioral responses in mice. Herein, we sought to determine whether clozapine also utilizes βarrestin2-mediated mechanisms to induce 5-HT2AR signaling, and whether this interaction contributes to its behavioral effects in mice. We demonstrate that unlike serotonin, clozapine-mediated 5-HT2AR internalization and Akt phosphorylation is independent of receptor interactions with βarrestin2. Moreover, clozapine-mediated suppression of MK-801 and PCP-induced hyperlocomotion is βarrestin2 independent, although it is dependent upon Akt. These results demonstrate that pharmacologically oppositional ligands, serotonin and clozapine, utilize differential mechanisms to achieve the same 5-HT2AR-meadiated downstream events: Akt phosphorylation and receptor internalization. While βarrestin2 has no effect on clozapine's actions in vivo, Akt phosphorylation is required for clozapine's efficacy in blocking MK-801- and PCP-induced models of schizophrenic behaviors in mice.Neuropsychopharmacology accepted article preview online, 17 February 2014; doi:10.1038/npp.2014.38.
    Preview · Article · Feb 2014 · Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology
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    • "To investigate the 5-HT 2A receptor mechanism further we used ketanserin. Although widely used as a selective 5-HT 2A antagonist (Chen et al., 2003; De Paula et al., 2012; Yadav et al., 2011), ketanserin also exhibits a weaker affinity for the 5-HT 2C receptor (Alex and Pehek, 2007), which has been demonstrated as having an inhibitory effect on DA efflux in the NAc shell in vivo (Di Matteo et al., 2000). Unlike the phasic excitatory effect of 5-HT 2A receptors (Navailles and De Deurwaerdere, 2011), the 5-HT 2C subtype receptors exert both a tonic and phasic inhibitory control over DA efflux in the NAc shell with high constitutive activity (Navailles et al., 2006). "
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    ABSTRACT: Harmine is a β-carboline alkaloid and major component of ayahuasca, a traditional South American psychoactive tea with anecdotal efficacy for treatment of cocaine dependence. Harmine is an inhibitor of monoamine oxidase A (MAO-A) and interacts in vitro with several pharmacological targets which modulate dopamine (DA) neurotransmission. In vivo studies have demonstrated dopaminergic effects of harmine, attributed to monoamine oxidase inhibitor (MAOI) activity, however none have directly demonstrated a pharmacological mechanism. This study investigated the acute effects, and pharmacological mechanism(s), of harmine on electrically evoked DA efflux parameters in the nucleus accumbens both in the absence and presence of cocaine. Fast cyclic voltammetry in rat brain slices was used to measure electrically evoked DA efflux in accumbens core and shell. Harmine (300 nM) significantly augmented DA efflux (148±8% of baseline) in the accumbens shell. Cocaine augmented efflux in shell additive to harmine (260±35%). Harmine had no effect on efflux in the accumbens core or on reuptake in either sub-region. The effect of harmine in the shell was attenuated by the 5-HT(2A/2C) antagonist ketanserin. The MAOI moclobemide (10 µM) had no effect on DA efflux. These data suggest that harmine augments DA efflux via a novel, shell-specific, presynaptic 5-HT(2A) receptor-dependent mechanism, independent of MAOI activity. A DA-releasing 'agonist therapy' mechanism may thus contribute to the putative therapeutic efficacy of ayahuasca for cocaine dependence.
    Full-text · Article · Oct 2012 · Journal of Psychopharmacology
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