In vitro inhibition of mitochondrial respiratory rate by antidepressants

Abstract

Mitochondria represent a possible drug target with unexplored therapeutic and toxicological potential. The possibility was suggested that antidepressants, mood stabilizers and other drugs may show some therapeutic and/or toxic effects through their action on mitochondrial functions. There are no sufficient data about the effect of these drugs on mitochondrial respiration in the brain. We investigated the in vitro effects of amitriptyline, fluoxetine, tianeptine, ketamine, lithium, valproate, olanzapine, chlorpromazine and propranolol on mitochondrial respiration in crude mitochondrial fractions of pig brains. Respiration was energized using substrates of complex I or complex II and dose dependent drug-induced changes in mitochondrial respiratory rate were measured by high-resolution respirometry. Antidepressants, but not mood stabilizers, ketamine and propranolol were found to inhibit mitochondrial respiratory rate. The effective dose of antidepressants reaching half the maximal respiratory rate was in the range of 0.07–0.46 mmol/L. Partial inhibition was found for all inhibitors. Differences between individual drugs with similar physicochemical properties indicate selectivity of drug-induced changes in mitochondrial respiratory rate. Our findings suggest that mood stabilizers do not interfere with brain mitochondrial respiration, whereas direct mitochondrial targeting is involved in mechanisms of action of pharmacologically different antidepressants.

Introduction

Impaired function of mitochondria leads to impaired bioenergetics, decreased ATP production, impaired calcium homeostasis, increased production of free radicals and oxidative stress and to initiation of apoptotic processes (Fišar and Hroudová, 2010, Hroudová and Fišar, 2011). There is mounting evidence for the role of mitochondrial dysfunction in the pathophysiology and treatment of neurodegenerative diseases, including mood disorders (Kato and Kato, 2000, Stork and Renshaw, 2005). Thus, mitochondria are now the target for therapeutic interventions and enhancement of mitochondrial function may represent a critical component for the optimal treatment of neurodegenerative and stress-related diseases (Quiroz et al., 2008, Frantz and Wipf, 2010, Schapira, 2012). In contrast, drug-induced disruption of mitochondrial oxidative phosphorylation is a mechanism of toxicity, which disturbs mitochondrial respiration and thereby alters energy metabolism.

At the mitochondrial level, there are several potential drug targets that can lead to toxicity. Thus, it is important to test for mitochondrial toxicity in the early phase of new drug development, as impairment of mitochondrial function can induce various pathological conditions or can increase progression of existing diseases (Chan et al., 2005, Boelsterli and Lim, 2007, Fišar et al., 2010, Fišar et al., 2011, Scatena, 2012). E.g., valproic acid and nefazodone as central nervous system drugs showed both mitochondrial liability and potential hepatic and cardiovascular toxicity (Dykens and Will, 2007, Boelsterli and Lim, 2007).

Although a wide range of pharmacologically different antidepressants and mood stabilizers is available, molecular mechanisms of their therapeutic or side effects have not yet been sufficiently clarified. There is little information about the association between the therapeutic and/or adverse effects of these drugs and mitochondrial respiration. Assessment of mitochondrial dysfunctions and drug cytotoxicity may be important in drug development as well as in the study of drug interactions (Dykens et al., 2008). The hypothesis that antipsychotic-induced extrapyramidal side effects may be due to inhibition of the mitochondrial respiratory chain was reported previously (Maurer and Möller, 1997).

1040 drugs and other bioactive compounds were tested as potential inhibitors of mitochondrial permeability transition in rat liver mitochondria (Stavrovskaya et al., 2004). It was found that heterocyclics, including antipsychotics, antidepressants, antihistaminics and others mediate dose-dependent protection that is not related to therapeutic class. Recently, the effects of selected antidepressants (clomipramine, desipramine, norfluoxetine, tianeptine) on mitochondrial functions such as caspase-3 activity, membrane potential, mitochondrial electron transport chain complex activities and mitochondrial oxygen consumption were investigated in a model cell system or in isolated mitochondria (Abdel-Razaq et al., 2011). The data suggest that complex I may be more sensitive to antidepressant inhibition than other complexes of the electron transport chain (ETC).

More than 60 different types of compounds, including antidepressants and mood stabilizers (Hroudova and Fisar, 2010), are well-known inhibitors of complex I. Due to its complexity, complex I is vulnerable to lipophilic molecules. Complex II has been less studied in mitochondrial pharmacotoxicology. For complex III, there are a large number of inhibitors with no known clinical value. There are a number of well-known entities that inhibit complex IV. Moreover, some polycationic molecules can interact with mobile electron carriers.

There are several interconnections and feedbacks within the system of oxidative phosphorylation (OXPHOS). Thus, inhibition of the activation of one component (e.g. complex I or complex IV) does not provide any information about the final effect of a drug on cellular respiration and energetics. The bioenergetics function of mitochondria can be investigated by measuring the rate of ATP formation and the efficiency of the process (the P/O ratio, i.e. the ratio of ATP formed over oxygen consumed) (Merlo-Pich et al., 2004). Measurement of oxygen consumption and its sensitivity to substrates, uncouplers and inhibitors can be a good indicator of mitochondrial phosphorylative capacity. To provide a routine approach to the study of oxygen kinetics, multiple substrate-uncoupler-inhibitor titration protocols for high-resolution respirometry were developed for the accurate measurement using small amounts of tissue, cells and isolated mitochondria (Pesta and Gnaiger, 2012).

Respiratory rate is a parameter characterizing functioning of the OXPHOS system as a whole. Drug effect on respiratory rate was measured in the work presented here using pharmacologically different psychotropic drugs known to affect specific mitochondrial functions: 1. amitriptyline, fluoxetine and tianeptine (antidepressants); 2. ketamine (glutamate N-methyl-d-aspartate receptor antagonist; anesthetic and potential antidepressant) (Rezin et al., 2009, de Oliveira et al., 2009); 3. lithium and valproate (mood stabilizers) (Haas et al., 1981, Ponchaut et al., 1992, Silva et al., 1997, Bachmann et al., 2009, Maurer et al., 2009); 4. olanzapine (atypical antipsychotic and mood stabilizer) (Lauterbach et al., 2010); 5. chlorpromazine (typical antipsychotic); and 6. propranolol (non-selective beta blocker) (Katyare and Rajan, 1991, Robinson et al., 2009, Kaasik et al., 2010, Wills et al., 2012). Our study was aimed at discovering the effects of antidepressants and mood stabilizers on mitochondrial respiratory rate. The effect of chlorpromazine was measured for comparison, as its mitochondrial effects are well-known. Propranolol, a drug which can induce depressive symptoms, was tested to compare the effect of a molecule with similar physicochemical properties as antidepressants, however with a diametrically different pharmacological mode of action.

Amitriptyline is a tricyclic antidepressant, whose primary biochemical action related to its therapeutic effects is serotonin and norepinephrine reuptake inhibition. Its adverse effects are due to the antagonism of α₁-adrenergic, histamine and muscarinic acetylcholine receptors. Recently, amitriptyline was found to be an inhibitor of mitochondrial functions, e.g. mitochondrial membrane permeability transition (Stavrovskaya et al., 2004). Acute in vivo administration of amitriptyline caused a general pattern of decrease in brain oxidative metabolism (González-Pardo et al., 2008). Amitriptyline treatment induced oxidative stress in several tissues, including brain, in a dose-dependent manner. However, coenzyme Q was increased in the brain after long-term treatment, probably in order to counteract oxidative damage (Bautista-Ferrufino et al., 2011).

Fluoxetine together with its active metabolite norfluoxetine is an antidepressant of the selective serotonin reuptake inhibitor (SSRI) class. Its adverse side effects are less pronounced compared to amitriptyline and include sexual dysfunctions, nausea, insomnia, somnolence, anorexia, anxiety, nervousness, asthenia and tremor. Fluoxetine and norfluoxetine inhibit many isoenzymes of the cytochrome P450 system that make drug metabolism possible. Furthermore, they inhibit the activity of P-glycoprotein that plays an important role in drug transport and metabolism. The simultaneous use of fluoxetine with serotonergic agents can result in a serotonin syndrome. Fluoxetine has multiple effects on the energy metabolism of rat liver mitochondria, being potentially toxic in high doses (Souza et al., 1994). It affects apoptosis through an increase of the voltage sensitivity of the mitochondrial voltage-dependent anion channel (Nahon et al., 2005). Fluoxetine induces inhibition of OXPHOS and decreases the activity of ATP synthase in rat brain mitochondria (Curti et al., 1999). Apoptotic effects, reduction of membrane potential, inhibition of the activity of mitochondrial complexes and decrease of state 3 respiration were observed after exposure to norfluoxetine (Abdel-Razaq et al., 2011).

Tianeptine is an antidepressant drug, which was first classified as a selective serotonin reuptake enhancer. However, the more recent view is that the therapeutic mechanism of action of tianeptine can be attributed to its effect on the glutamatergic system and the reversal of impaired neuroplasticity associated with stress (McEwen et al., 2010). Its side effects are less pronounced compared to amitriptyline and include dry mouth, constipation, dizziness, drowsiness, postural hypotension, insomnia and vivid dreams, headaches and hypomania induction. Tianeptine inhibits complex I activity; other mitochondrial complexes, apoptotic effects and reduction of membrane potentials were not affected following exposure of model cell systems or isolated mitochondria to tianeptine (Abdel-Razaq et al., 2011).

Chlorpromazine is a typical antipsychotic. It is a very effective antagonist of D2-type dopamine receptors, but also produces anticholinergic, antihistaminic and weak antiadrenergic effects. It is known as an inhibitor of mitochondrial functions, e.g. complex I (Modica-Napolitano et al., 2003), mitochondrial nitric oxide synthase (Lores-Arnaiz et al., 2004), mitochondrial phospholipase A₂ and mitochondrial membrane permeability transition (Furuno et al., 2001, Stavrovskaya et al., 2004).

Common physicochemical properties of drugs may be associated with their similar effect on certain cellular functions. Cationic amphiphilic drugs include antidepressants, antipsychotics, calcium channel blockers, beta receptor blockers, antihistaminics, and antifungals. The technically incorrect term “cationic amphiphilic drug” comprises drug compounds that have a hydrophobic part consisting of a nonpolar ring part and a hydrophilic group with one or more nitrogen containing groups, which can bear a net positive charge at physiological pH. Cationic amphiphilic drugs may induce phospholipidosis (Xia et al., 2000, Reasor and Kacew, 2001). However, the relationship between drug-induced phospholipidosis and adverse drug effects remains unexplained (Anderson and Borlak, 2006).

Drug lipophilicity, charge, polar surface area and membrane potential influence mitochondrial drug delivery, with the uptake of positively charged, lipophilic molecules being the most efficient (Durazo et al., 2011). From a physicochemical point of view, amitriptyline, fluoxetine, tianeptine, ketamine, olanzapine, chlorpromazine and propranolol are all cationic amphiphilic drugs, which can accumulate in mitochondria.

The mitochondrial hypothesis states that impaired energy metabolism of brain cells is involved in the pathophysiology of mood disorders and in the effects of antidepressants and mood stabilizers. We presume that the therapeutic or side effects of drugs administered in the treatment of depression may involve the targeted regulation of mitochondrial functions and the subsequent effect on neuroplasticity, disease related inflammatory responses, calcium homeostasis, production of reactive oxygen and nitrogen species and other processes related to the complex response to stress, neurotoxicity or impaired neurotransmission. On the basis of this hypothesis, we studied the effects of antidepressants and mood stabilizers on specific enzymes and complexes of the respiratory chain (Fišar et al., 2010, Hroudova and Fisar, 2010). In the study presented here, we measured the effects of these drugs on the respiratory rate of intact mitochondria, i.e. we measured drug induced changes on mitochondrial function as a whole. We considered functional parameters such as respiratory rate, mitochondrial permeability transition or inner membrane potential (i.e. measurements that reflect activity of the intact respiratory system) to be more suitable for evaluating drug effects on cellular energetics than the measurement of specific mitochondrial enzymes. Our study has the potential to contribute to a better understanding of the role of mitochondria in the mechanism of action of antidepressants and to evaluate positive and negative effects of the tested drugs on brain bioenergetics.

There is a need both for predictive and retrospective in vitro assays of drug-induced mitochondrial toxicity. A large number of drugs that have been withdrawn from the market or stopped during development due to hepatotoxicity, nephrotoxicity or cardiotoxicity have been reported to disturb mitochondrial functions. The aim of the present study was to investigate the effects of antidepressants and mood stabilizers of different chemical structures on mitochondrial respiratory rate in intact brain mitochondria. In vitro effects of pharmacologically different antidepressants (amitriptyline, fluoxetine, tianeptine), mood stabilizers (lithium, valproate, olanzapine) and other drugs (ketamine, chlorpromazine, propranolol) on the mitochondrial respiratory rate were measured in crude mitochondrial fractions isolated from pig brain using high-resolution respirometry. Pig mitochondria are relatively often used in studies of mitochondrial functions and enzymes. Moreover, the pig model is commonly used in immunological studies due to the similarity of the human and pig immune system, and more precise measurement in the brain structures is possible in pig brains compared to rat or mice brains. Respiration was characterized by respiratory state 3 (Chance and Williams, 1955, Gnaiger, 2009) using substrates for electron supply either through complex I (malate- and pyruvate-energized mitochondria) or through complex II (succinate-energized mitochondria) of ETC. Extended-range titration of mitochondrial suspensions by drugs enables the determination of half maximal inhibitory concentrations and other inhibitory parameters applicable for evaluating the potential mitochondrial toxicity of individual drugs.