Inhibition of nicotinic acetylcholine receptors and calcium channels by clozapine in bovine adrenal chromaffin cells

doi:10.1016/S0006-2952(01)00577-9

Biochemical Pharmacology

Volume 61, Issue 8, 15 April 2001, Pages 1011-1019

https://doi.org/10.1016/S0006-2952(01)00577-9 Get rights and content

Abstract

The effects of clozapine on the activities of nicotinic acetylcholine receptors (nAChRs) and voltage-sensitive calcium channels (VSCCs) were investigated and compared with those of chlorpromazine (CPZ) in bovine adrenal chromaffin cells. [³H]Norepinephrine ([³H]NE) secretion induced by activation of nAChRs was inhibited by clozapine and CPZ with half-maximal inhibitory concentrations (ic₅₀) of 10.4 ± 1.1 and 3.9 ± 0.2 μM, respectively. Both cytosolic calcium increase and inward current in the absence of extracellular calcium induced by nicotinic stimulation were also inhibited by clozapine and CPZ, but the greater inhibition was achieved by CPZ. In addition, [³H]nicotine binding to chromaffin cells was inhibited by clozapine and CPZ with ic₅₀ values of approximately 19 and 2 μM, respectively. On the other hand, [³H]NE secretion induced by high K⁺ was inhibited by clozapine and CPZ with similar ic₅₀ values of 15.5 ± 3.8 and 17.1 ± 3.9 μM, respectively. Our results suggest that clozapine, as well as CPZ, inhibits nAChRs and VSCCs, thereby causing inhibition of catecholamine secretion, and that clozapine is much less potent than CPZ in inhibiting nAChRs.

Introduction

The dopamine hypothesis of schizophrenia proposes that schizophrenia results from too much dopaminergic activity and that the antipsychotic drugs used act by blocking dopamine receptors in the nervous system. The strongest evidence of the dopamine hypothesis is that classical antipsychotic drugs such as CPZ and haloperidol bind and block dopamine D₂ receptors in a way that is directly related to their clinical antipsychotic potencies [1]. The report that the number of D₂ or D₂-like receptors is elevated in post-mortem schizophrenia brain tissue [2] supports the dopamine hypothesis. However, the dopamine hypothesis of schizophrenia is profoundly challenged by the atypical antipsychotic drug clozapine.

Clozapine is a neuroleptic agent that is structurally different from CPZ, a phenothiazine with two benzene rings linked by a sulfur and a nitrogen atom. Clozapine consists of a dibenzodiazepine derivative with a piperazinyl side chain. It has been classified as an atypical neuroleptic drug due to its unique neuropharmacological profile (for review see [3]). Clozapine has been useful in treatment-resistant patients and in treating the negative symptoms of schizophrenia, while it has a high affinity for D₄ receptors rather than D₂ receptors [4]. In addition, in some patients treated with typical antipsychotics, the therapeutic response was still poor despite D₂ receptor blockage. On the other hand, all patients treated with clozapine showed significant clinical improvement with a lower level of D₂ receptor blockage [5].

In addition to dopamine receptors, many membrane proteins, such as voltage-sensitive Ca²⁺, Na⁺, and K⁺ channels, have been reported to be inhibited by rather high concentrations of many classical antipsychotics [6], [7], [8], [9], [10]. We recently reported that CPZ inhibited nAChRs and L-type VSCCs, thereby causing inhibition of catecholamine secretion in rat pheochromocytoma cells [11]. In contrast to the extensive studies of classical antipsychotics with regard to their inhibitory effects on neurotransmitter release and the activity of ion channels and nAChRs, little attention has been given to the action of clozapine. Indeed, comparative studies of classical and atypical antipsychotics might provide a clue toward an understanding of the clinical efficacy of clozapine and the side effects caused by classical antipsychotics. Furthermore, investigation of the effect of clozapine on catecholamine secretion and ion fluxes and comparison with the inhibitory effects induced by CPZ are necessary to understand the clinical efficacy and physiological effects of clozapine. Adrenal chromaffin cells have been widely used as a model system by which to investigate the mechanism of neurosecretion [12], [13], [14]. Both nicotinic cholinergic agonists and high K⁺ have been extensively used to investigate the mechanisms of calcium increase and catecholamine secretion induced by activation of nAChRs and VSCCs [14], [15]. By using the well-established model system, we investigated the effect of clozapine on catecholamine secretion and ion fluxes induced by the activation of nAChRs and VSCCs. Our results suggest that clozapine inhibits nAChRs and VSCCs, leading to the subsequent inhibition of calcium increase and catecholamine secretion. Furthermore, clozapine was much less potent than CPZ in the inhibition of nAChRs.

Section snippets

Materials

Clozapine, CPZ, and DMPP were purchased from Sigma. Fura-2/acetoxymethylester, SBFI/acetoxymethylester, and Pluronic F-127 were obtained from Molecular Probes, Inc. [³H]NE and [³H]nicotine were purchased from NEN Life Science Products.

Chromaffin cell preparation

Chromaffin cells were isolated from bovine adrenal medulla by two-step collagenase digestion as previously described [12]. For measurement of [³H]NE secretion and the [³H]nicotine binding assay, cells were plated in 24-well plates at a density of 5 × 10⁵ cells per

Inhibitory effects of clozapine and CPZ on DMPP-induced [³H]NE secretion

In order to study the effect of clozapine on catecholamine secretion, [³H]NE-loaded chromaffin cells were treated with clozapine. Clozapine (up to 100 μM) by itself did not induce significant [³H]NE secretion (data not shown). DMPP, a well-known activator of nAChRs [14], [15], [20], [21], [22], induced [³H]NE secretion by 17.2 ± 2.4% of the total endogenous content. Clozapine decreased DMPP-induced [³H]NE secretion in a concentration-dependent manner, with a half-maximal inhibitory

Discussion

Our results clearly show that, like CPZ, clozapine inhibits both nAChRs and VSCCs. The inhibitory effects lead to the subsequent inhibition of calcium increase and catecholamine secretion. We previously reported that CPZ inhibited the nAChR- and VSCC-mediated catecholamine secretion of rat pheochromocytoma PC12 cells by blocking nAChRs and VSCCs [10]. Therefore, clozapine is similar to CPZ in that both antipsychotics inhibit catecholamine secretion induced by the activation of nAChRs and VSCCs.

Acknowledgements

We thank the MyoungSin Company Inc. (Pohang, Korea) and especially Mr. Hwan Seok, chief director, and Mr. Byung-Soon Kang for providing the bovine adrenal glands. We are grateful to Ms. Eun-Mi Hur for her technical assistance in patch clamp recordings. We also thank Ms. G. Hoschek for editing the manuscript. This work was supported by grants from the Brain Science and Engineering Research Program, the KOSEF, and the National Research Laboratory Program sponsored by the Korean Ministry of

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Clozapine is the first atypical antipsychotic, and improves positive and negative symptoms of many patients with schizophrenia resistant to treatment with other antipsychotic agents. Clozapine induces minimal extrapyramidal side effects, but is more often associated with seizures. A large number of studies have been conducted to elucidate pharmacological profiles of clozapine and its major active metabolite, N-desmethylclozapine (NDMC). However, there are only a limited number of electrophysiological studies examining their effects on synaptic transmission. In this study, we examined effects of clozapine and NDMC on synaptic transmission by measuring inhibitory and excitatory postsynaptic currents in rat cultured hippocampal neurons. We found that clozapine and NDMC have qualitatively similar actions. They depressed the inhibitory transmission at 1–30 μM, and the excitatory transmission at 30 μM, the former being much more sensitive. The depression of IPSCs by 30 μM of these drugs was associated with an increase in the paired-pulse ratio. The GABA-induced currents were suppressed by these drugs, but less sensitive than IPSCs. The AMPA-induced currents were slightly potentiated by these drugs at 30 μM. At 30 μM, clozapine and NDMC slightly suppressed Ca²⁺ and Na⁺ channels. These results strongly suggest that clozapine and NMDC depress the inhibitory synaptic transmission mainly by antagonizing postsynaptic GABA_A receptors, but at higher concentrations additionally by acting on presynaptic site, possibly in part through inhibition of presynaptic Ca²⁺ and Na⁺ channels. Preferential depression of inhibitory synaptic transmission by clozapine and NDMC might contribute to therapeutic actions and/or side-effects of clozapine.
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Low voltage-activated T-type calcium channels are involved in the regulation of the neuronal excitability, and could be subject to many antipsychotic drugs. The effects of clozapine, an atypical antipsychotic drug, on recombinant Ca_v3.1 T-type calcium channels heterologously expressed in human embryonic kidney 293 cells were examined using whole-cell patch-clamp recordings. At a standard holding potential of − 100 mV, clozapine inhibited Ca_v3.1 currents with an IC₅₀ value of 23.7 ± 1.3 μM in a use-dependent manner. However, 10 μM clozapine inhibited more than 50% of the Ca_v3.1 currents in recordings at a more physiologically relevant holding potential of −75 mV. Clozapine caused a significant hyperpolarizing shift in the steady-state inactivation curve of the Ca_v3.1 channels, which is presumably the main mechanism accounting for the inhibition of the Ca_v3.1 currents. In addition, clozapine slowed Ca_v3.1 deactivation and inactivation kinetics but not activation kinetics. Clozapine-induced changes in deactivation and inactivation rates of the Ca_v3.1 channel gating would likely facilitate calcium influx via Ca_v3.1 T-type calcium channels. Thus, clozapine may exert its therapeutic and/or side effects by altering cell's excitability and firing properties through actions on T-type calcium channels.
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However, few reports have addressed antipsychotic interactions with nicotinic cholinergic receptors. Clozapine and chlorpromazine are known to displace [3H]-nicotine in bovine chromaffin cells (Park et al., 2001) as does chlorpromazine in rat pheochromocytoma cells (Lee et al., 1999). The typical antipsychotic chlorpromazine is known to interact with functional NNRs as a non-competitive inhibitor (Heidmann et al., 1983; Heidmann and Changeux, 1986).
It has been suggested that the interaction of antipsychotic medications with neuronal nicotinic receptors may increase the cognitive dysfunction associated with schizophrenia and may explain why current therapies only partially address this core feature of the illness. In the present studies we compared the effects of the atypical antipsychotics quetiapine, clozapine and N-desmethylclozapine to those of the typical antipsychotics haloperidol and chlorpromazine on the α4β2 and α7 nicotinic receptor subtypes. The binding of [³H]-nicotine to rat cortical α4β2 receptors and [³H]-methyllycaconitine to rat hippocampal α7 receptors was not affected by any of the compounds tested. However, Rb⁺ efflux evoked either by nicotine or the selective α4β2 agonist TC-1827 from α4β2 receptors expressed in SH-EP1 cells and nicotine-evoked [³H]-dopamine release from rat striatal synaptosomes were non-competitively inhibited by all of the antipsychotics. Similarly, α-bungarotoxin-sensitive epibatidine-evoked [³H]-norepinephrine release from rat hippocampal slices and acetylcholine-activated currents of α7 nicotinic receptors expressed in oocytes were inhibited by haloperidol, chlorpromazine, clozapine and N-desmethylclozapine. The inhibitory effects on nicotinic receptor function produced by the antipsychotics tested occurred at concentrations similar to plasma levels achieved in schizophrenia patients, suggesting that they may lead to clinically relevant effects on cognition.
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Clozapine (CZP), a dibenzodiazepine derivative with a piperazinyl side chain, is in clinical use as an antipsychotic drug. This study investigated the effect of CZP on the modulation of the PI3K/Akt/GSK-3β pathway in PTEN-negative U-87MG glioblastoma cells. Treatment with CZP rapidly inhibited the basal and EGF-induced phosphorylation of Akt. The inhibition of Akt resulted in the dephosphorylation of GSK-3β and increased GSK-3β kinase activity. A voltage-sensitive Ca²⁺ channel blocker and calmodulin (CaM) antagonists inhibited Akt phosphorylation, whereas elevation of the intracellular Ca²⁺ concentration prevented CZP-induced dephosphorylation of Akt and GSK-3β, suggesting that Ca²⁺/CaM participates in the inhibition of Akt by CZP in U-87MG cells. In addition, similar to LY294002, CZP arrested cell cycle progression at G0/G1 phase, which was accompanied by decreased expression of cyclin D1. The reduction in the cyclin D1 level induced by CZP was abrogated by the inhibition of GSK-3β, the inhibition of proteasome-dependent proteolysis, or an increase in the intracellular Ca²⁺ concentration. These results suggest that the antipsychotic drug CZP modulates the PI3K/Akt/GSK-3β pathway by counteracting Ca²⁺/CaM in PTEN-negative U-87MG glioblastoma cells.
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We reported previously that the protopanaxatriol saponins in Panax ginseng greatly reduce the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh). However, protopanaxadiol saponins showed only slight inhibitory effects. Recent studies have demonstrated that oligosaccharides connected to the hydroxyl groups of the aglycone in ginseng saponins (ginsenosides) are in turn hydrolyzed in the digestive tract and absorbed into the circulation following oral administration of ginseng. Therefore, the present study was performed to investigate the effects of the major ginsenoside metabolites (M1, M2, M3, M4, M5, M11, and M12) on catecholamine secretion. All of these metabolites were shown to be potent inhibitors of ACh-evoked secretion, and M4 was the most effective. M4 blocked not only the ACh-induced Na⁺ influx into the chromaffin cells but also the ACh-induced inward current into Xenopus oocytes expressing human α3β4 neuronal nicotinic ACh receptors. M4 reduced the secretion induced by high K⁺, an activator of voltage-sensitive Ca²⁺ channels, to a much lesser extent than that evoked by ACh. M1, M2, M3, M5, and M12 are protopanaxadiol saponin-derived metabolites. Therefore, these results imply that the protopanaxadiol saponins are prodrugs, and they show more potent inhibitory activity following metabolism in the digestive tract. The results further suggest that the metabolites act on nicotinic ACh receptors, blocking Na⁺ influx through the receptors, and consequently reduce the catecholamine secretion from bovine adrenal chromaffin cells. The inhibitory effect of ginsenoside metabolites is probably one of the mechanisms of action responsible for the pharmacological effects of ginseng.

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¹: Abbreviations: CPZ, chlorpromazine; nAChR, nicotinic acetylcholine receptor; VSCC, voltage-sensitive calcium channel; DMPP, 1,1-dimethyl-4-phenylpiperazinium iodide; SBFI, sodium-binding benzofuran isophthalate; [³H]NE, [³H]norepinephrine; DMEM/F-12, Dulbecco’s modified Eagle’s medium/F-12; [Ca²⁺]_i, cytosolic free Ca²⁺ concentration; [Na⁺]_i, cytosolic free Na⁺ concentration; and EPS, extrapyramidal side effects.

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Inhibition of nicotinic acetylcholine receptors and calcium channels by clozapine in bovine adrenal chromaffin cells1

Abstract

Introduction

Section snippets

Materials

Chromaffin cell preparation

Inhibitory effects of clozapine and CPZ on DMPP-induced [3H]NE secretion

Discussion

Acknowledgements

Trends Pharmacol Sci

Lancet

Brain Res

Biochem Pharmacol

J Biol Chem

Eur J Pharmacol

Mol Cells

FEBS Lett

Addict Behav

Schizophr Res

Biol Psychiatry

Neuropsychopharmacology

Biol Psychiatry

Compr Psychiatry

Brain receptors for antipsychotic drugs and dopaminedirect binding assays

Proc Natl Acad Sci USA

Bimodal distribution of dopamine receptor densities in brains of schizophrenics

Science

Adverse effects of antipsychotic agents. Do newer agents offer advantages?

Drugs

Calmodulin antagonists depress calcium and potassium currents in ventricular and vascular myocytes

Am J Physiol

Differential block of sodium and calcium channels by chlorpromazine in mouse neuroblastoma cells

J Physiol

Mechanisms of potassium channel blocker in rat alveolar epithelial cells

J Pharmacol Exp Ther

Characterization of inhibition by haloperidol and chlorpromazine of a voltage-activated K+ current in rat phaeochromocytoma cells

Br J Pharmacol

Inhibition of nicotinic acetylcholine receptors and calcium channels by clozapine in bovine adrenal chromaffin cells¹

Inhibitory effects of clozapine and CPZ on DMPP-induced [³H]NE secretion

Characterization of inhibition by haloperidol and chlorpromazine of a voltage-activated K⁺ current in rat phaeochromocytoma cells