Monoamine Oxidase Inhibitor Pargyline Research Articles

Simultaneous mass spectrometric quantification of trace amines, their precursors and metabolites

ObjectivesTrace amines are powerful neuromodulators influencing the release and reuptake of catecholamines. These low concentrated endogenous amines impact mood, cognition, and hormone regulation. Dysregulation of trace amines have been associated with a variety of diseases, such as schizophrenia, Parkinson’s disease, migraine, depression and more. Succesfull simultaneous quantification of trace amines, their precursors and metabolites would benefit both research and patient care. Since these compounds have various functional groups and are present in biological matrices with large concentration difference, their simultaneous quantification is an analytical challenge. Our goal was to develop a highly sensitive LC-MS/MS assay to simultaneously quantify trace amines, their precursors and metabolites in plasma. MethodsOur method is based on a simple two-step in-matrix derivatization protocol: propionic anhydride (PA) and 3-Ethyl-1-[3-(dimethylamino)propyl]carbodiimide (EDC) in combination with 2,2,2-trifluoroethylamine (TFEA) followed by online solid phase extraction combined with LC-MS/MS. Fifteen metabolites can be measured simultaneously, three precursors, eight trace amines and four metabolites. Validation of this method was performed according to international validation guidelines. The pre-analytical stability of trace amines was assessed. ResultsThis novel method was successful in quantifying trace amines, their precursors, and metabolites in plasma. Using just 50 µl human plasma, we were able to accomplish limit of quantification for 2-phenylethylamine and N-methyl-phenylethylamine of 0.2 nmol/L and 0.1 nmol/L for tyramine and n-methyltyramine. Inter-and intra-assay imprecision was < 15 % for all analytes. Stability assessment showed susceptibility of certain trace amines e.g. 2-phenylethylamine and N-methyl-phenylethylamine to enzymatic degradation in plasma. The addition of the monoamine oxidase inhibitor pargyline to plasma prevented this enzymatic degradation. ConclusionsWe developed a novel LC-MS/MS method that1) uses a new double derivatization technique, 2) is automated with online SPE, 3) uses far less sample volume then previous methods and 4) detects more components in the same sample (eight trace amines, three precursors, and four metabolites) with high specificity and selectivity. Furthermore, addition of MAO A/B inhibitor prevents degradation and guarantees more accurate quantification of trace amines.

Uptake and Metabolization of Serotonin by Granulosa Cells Form a Functional Barrier in the Mouse Ovary

Serotonin (5-HT) plays an essential role in regulating female reproductive function in many animals. 5-HT accumulates in the mammalian ovary with the involvement of membrane serotonin transporter SERT and is functionally active in the oocytes of growing follicles, but shows almost no activity in follicular cells. In this study, we clarified the interplay between 5-HT membrane transport and its degradation by monoamine oxidase (MAO) in the mammalian ovary. Using pharmacologic agents and immunohistochemical staining of the cryosections of ovaries after serotonin administration in vitro, we demonstrated the activity of transport and degradation systems in ovarian follicles. The MAO inhibitor pargyline increased serotonin accumulation in the granulosa cells of growing follicles, indicating the activity of both serotonin uptake and degradation by MAO in these cells. The activity of MAO and the specificity of the membrane transport of serotonin was confirmed in primary granulosa cell culture treated with pargyline and fluoxetine. Moreover, the accumulation of serotonin is more effective in the denuded oocytes and occurs at lower concentrations than in the oocytes within the follicles. This confirms that the activity of SERT and MAO in the granulosa cells surrounding the oocytes impedes the accumulation of serotonin in the oocytes and forms a functional barrier to serotonin.

Monoamine Oxidase-Dependent Pro-Survival Signaling in Diabetic Hearts Is Mediated by miRNAs.

Diabetes leads to cardiomyopathy and heart failure, the leading cause of death for diabetic patients. Monoamine oxidase (MAO) inhibition in diabetic cardiomyopathy prevents oxidative stress, mitochondrial and endoplasmic reticulum stress and the development of diastolic dysfunction. However, it is unclear whether, in addition to the direct effects exerted on the mitochondria, MAO activity is able to post-transcriptionally regulate cardiomyocyte function and survival in diabetes. To this aim, we performed gene and miRNA expression profiling in cardiac tissue from streptozotocin-treated mice (model of type 1 diabetes (T1D)), administered with either vehicle or MAOs inhibitor pargyline for 12 weeks. We found that inhibition of MAO activity in T1D hearts leads to profound transcriptomic changes, affecting autophagy and pro-survival pathways activation. MAO activity in T1D hearts increased miR-133a-3p, -193a-3p and -27a-3p expression. These miRNAs target insulin-like growth factor receptor 1 (Igf1r), growth factor receptor bound protein 10 and inositol polyphosphate 4 phosphatase type 1A, respectively, all components of the IGF1R/PI3K/AKT signaling pathway. Indeed, AKT activation was significantly downregulated in T1D hearts, whereas MAO inhibition restored the activation of this pro-survival pathway. The present study provides an important link between MAO activity, transcriptomic changes and activation of pro-survival signaling and autophagy in diabetic cardiomyopathy.

Monoamine oxidase-dependent histamine catabolism accounts for post-ischemic cardiac redox imbalance and injury

Monoamine oxidase (MAO), a mitochondrial enzyme that oxidizes biogenic amines generating hydrogen peroxide, is a major source of oxidative stress in cardiac injury. However, the molecular mechanisms underlying its overactivation in pathological conditions are still poorly characterized.Here, we investigated whether the enhanced MAO-dependent hydrogen peroxide production can be due to increased substrate availability using a metabolomic profiling method. We identified N1-methylhistamine -the main catabolite of histamine- as an important substrate fueling MAO in Langendorff mouse hearts, directly perfused with a buffer containing hydrogen peroxide or subjected to ischemia/reperfusion protocol. Indeed, when these hearts were pretreated with the MAO inhibitor pargyline we observed N1-methylhistamine accumulation along with reduced oxidative stress. Next, we showed that synaptic terminals are the major source of N1-methylhistamine. Indeed, in vivo sympathectomy caused a decrease of N1-methylhistamine levels, which was associated with a marked protection in post-ischemic reperfused hearts. As far as the mechanism is concerned, we demonstrate that exogenous histamine is transported into isolated cardiomyocytes and triggers a rise in the levels of reactive oxygen species (ROS). Once again, pargyline pretreatment induced intracellular accumulation of N1-methylhistamine along with decrease in ROS levels. These findings uncover a receptor-independent mechanism for histamine in cardiomyocytes.In summary, our study reveals a novel and important pathophysiological causative link between MAO activation and histamine availability during pathophysiological conditions such as oxidative stress/cardiac injury.

Monoamine oxidase-dependent endoplasmic reticulum-mitochondria dysfunction and mast cell degranulation lead to adverse cardiac remodeling in diabetes

Monoamine oxidase (MAO) inhibitors ameliorate contractile function in diabetic animals, but the mechanisms remain unknown. Equally elusive is the interplay between the cardiomyocyte alterations induced by hyperglycemia and the accompanying inflammation. Here we show that exposure of primary cardiomyocytes to high glucose and pro-inflammatory stimuli leads to MAO-dependent increase in reactive oxygen species that causes permeability transition pore opening and mitochondrial dysfunction. These events occur upstream of endoplasmic reticulum (ER) stress and are abolished by the MAO inhibitor pargyline, highlighting the role of these flavoenzymes in the ER/mitochondria cross-talk. In vivo, streptozotocin administration to mice induced oxidative changes and ER stress in the heart, events that were abolished by pargyline. Moreover, MAO inhibition prevented both mast cell degranulation and altered collagen deposition, thereby normalizing diastolic function. Taken together, these results elucidate the mechanisms underlying MAO-induced damage in diabetic cardiomyopathy and provide novel evidence for the role of MAOs in inflammation and inter-organelle communication. MAO inhibitors may be considered as a therapeutic option for diabetic complications as well as for other disorders in which mast cell degranulation is a dominant phenomenon.

Myocardial interstitial levels of serotonin and its major metabolite 5-hydroxyindole acetic acid during ischemia-reperfusion.

By monitoring myocardial interstitial levels of 5-HT and its metabolite, 5-hydroxyindole acetic acid, we investigated 5-HT kinetics during myocardial ischemia-reperfusion. 5-HT accumulates but 5-HT degradation is suppressed during ischemia. After reperfusion, 5-HT degradation is enhanced and this degradation is dependent on monoamine oxidase activity but not fluoxetine-sensitive uptake transporter.

The modulatory action of harmane on serotonergic neurotransmission in rat brain

The naturally occurring β-carboline, harmane, has been implicated in various physiological and psychological conditions. Some of these effects are attributed to its interaction with monoaminergic systems. Previous literature indicates that certain β-carbolines including harmane modulate central monoamine levels partly through monoamine oxidase (MAO) inhibition. However, this is not always the case and thus additional mechanisms may be involved. This study set to assess the potential modulatory role of harmane on the basal or K+ stimulated release of preloaded radiolabelled noradrenaline (NA), dopamine (DA) and serotonin (5-HT) in rat brain cortex in vitro in the presence of the MAO inhibitor pargyline. Harmane displayed an overt elevation in K+ -evoked [3H]5-HT release; whilst little and no effect was reported with [3H]DA and [3H]NA respectively. The effect of harmane on [3H]5-HT efflux was partially compensated in K+-free medium. Further analyses demonstrated that removal of Ca2+ ions and addition of 1.2mM EGTA did not alter the action of harmane on [3H]5-HT release from rat brain cortex. The precise mechanism of action however remains unclear but is unlikely to reflect an involvement of MAO inhibition. The current finding aids our understanding on the modulatory action of harmane on monoamine levels and could potentially be of therapeutic use in psychiatric conditions such as depression and anxiety.

Analgesic Effects of β-Phenylethylamine and Various Methylated Derivatives in Mice

Administration of β-phenylethylamine (PEA), the simplest endogenous neuroamine, and various methylated PEA derivatives including α-methyl PEA (amphetamine, AMP) elicits analgesia in mice. Five or 20 min after intraperitoneal PEA injection of as little as 6 mg/kg resulted in an increased latency response time (from 2.4 ± 0.4 to 8.5 ± 2.3 or 7.0 ± 3.0 s, respectively) to the thermal stimulus (hot-plate test), which reached statistical significance at the 15 mg/kg (20 min; 13.1 ± 0.4 s) or 25 mg/kg dose (5 min; 15.3 ± 4.1 s). This PEA effect, was dose-dependent (albeit non-linear: 6, 12, 15, 25, 50 and 100 mg/kg), reached the cut-off time of 45 s at the upper PEA dose (5 min), and it was consistently enhanced by pretreatment with the monoamine oxidase inhibitor pargyline (P). Methylated PEA derivatives (15 and 100 mg/kg dose) produced various degrees of analgesia (in decreasing order p-Me PEA > PEA > N,N-diMe PEA > N-Me PEA) which, likewise to PEA itself, were consistently increased by P and declined over time (mice tested 5, 20 and 60 min after amine injection); small but statistically significant o- and β-Me PEA antinociceptive effects (5 min) were observed only at the higher dose (in the presence of P for β-Me PEA). A small analgesic effect was observed after the administration of AMP (5 or 10 mg/kg) which failed, even after P, to reach statistically significance. Independent of the amine and concentration tested, individual compound's antinociceptive properties were reliably increased by P (exception of AMP), decreased by reserpine (R) or haloperidol (H), and remained essentially unchanged after naloxone (N) administration suggesting the involvement of catecholamines, but not opioid peptides, in their observed analgesic effects. Injection of P + N produced results similar to those seen after P alone. Under the experimental conditions described neither P, R, H or N had any effects by themselves. These findings suggest additional understanding of the mechanism of action responsible for the analgesic effects of these amines would be of interest, leading further to controlled studies on their alleged usefulness as weight reducing agents and sport performance enhancers.

Effects of the monoamine oxidase inhibitors pargyline and tranylcypromine on cellular proliferation in human prostate cancer cells

Chemotherapy is one of the therapeutic strategies that has been used for the inhibition of cancer cell proliferation in several types of cancer, including prostate cancer. Although monoamine oxidase (MAO) inhibitors, phytoestrogen and antioxidants used in chemotherapy have been systematically studied, their effects on cancer cell growth remain to be fully understood. The purpose of this study was to investigate the effects of the MAO inhibitors, pargyline and tranylcypromine on cell survival in human prostate carcinoma (LNCaP-LN3) cells. After treating LNCaP-LN3 cells with pargyline or tranylcypromine, we examined cell proliferation, cell cycle pattern, apoptosis and the expression levels of apoptosis-related genes. The proliferation of cells exposed to pargyline decreased in a dose- and time-dependent manner, while tranylcypromine-treated cells showed the opposite results. Treatment with pargyline significantly induced cell cycle arrest at the G1 phase compared to the control and tranylcypromine-treated cells. In addition, pargyline induced an increase in the cell death rate by promoting apoptosis; however, tranylcypromine had no effect on LNCaP-LN3 cells. Based on our results, we suggest that pargyline is more powerful than tranylcypromine for the treatment of human prostate cancer.

Dopamine oxidation facilitates rotenone-dependent potentiation of N-methyl-d-aspartate currents in rat substantia nigra dopamine neurons

Rotenone is a mitochondrial poison that causes dopamine cell death and is used as a model of Parkinson's disease in rodents. Recently, we showed that rotenone augments currents evoked by N-methyl-D-aspartate (NMDA) by relieving voltage-dependent Mg(2+) block in rat substantia nigra compacta (SNC) dopamine neurons. Because rotenone is well known to generate reactive oxygen species (ROS), we conducted the present experiments to evaluate the role of ROS in mediating the effect of rotenone on NMDA current augmentation. Using patch pipettes to record whole-cell currents from SNC neurons in slices of rat brain, we found that the ability of rotenone (100 nM) to increase NMDA (3-30 μM) current was antagonized by the antioxidant agent n-acetylcysteine (1 mM). In contrast, mercaptosuccinate (1 mM), which blocks glutathione peroxidase and raises tissue levels of H(2)O(2), mimicked rotenone by augmenting inward currents evoked by NMDA. Because oxidation of dopamine can also generate ROS, we explored the role of dopamine on this action of rotenone. We prepared dopamine-depleted midbrain slices from rats that had been pretreated with reserpine (5 mg/kg ip) and alpha-methyl-para-tyrosine (AMPT, 250 mg/kg ip). Dopamine depletion blocked the ability of rotenone (100 nM) to increase inward current evoked by NMDA (30 μM). Rotenone-dependent augmentation of NMDA current was also blocked by the monoamine oxidase inhibitor pargyline (100 μM) in slices prepared from normal rats. In contrast, the dopamine precursor levodopa potentiated the action of rotenone on NMDA current. These results suggest that ROS and/or dopamine oxidation products mediate the ability of rotenone to potentiate NMDA currents. Because excessive NMDA receptor stimulation can produce excitotoxicity, our results suggest that oxidative metabolism of dopamine might facilitate the neurotoxicity of rotenone.

Monoamine oxidase activity promotes oxidative stress and cell death in cardiomyocytes exposed to high glucose

Mitochondrial respiratory chain and NADPH oxidase are described as major sources of reactive oxygen species (ROS) in diabetic cardiomyopathy. Oddly, the role of monoamine oxidase (MAO) has been overlooked so far. These mitochondrial flavoenzymes are responsible for oxidative stress and cell death in failing and ischemic/reperfused hearts. Here we assessed whether MAO contribute to high glucose (HG) induced cell damage. Neonatal rat cardiomyocytes were cultured in high glucose (HG, 30 mM) for 24 or 48 hours in the absence or presence of MAO inhibitor pargyline and compared to those cultured in normal (NG, 5 mM) or high mannitol (HM, 30mM) containing media. Mitochondrial ROS production, detected by Mitotracker Red, was significantly increased in HG myocytes (+40%, p&lt;0.001) as compared to other cells. Pargyline completely prevented this increase. Apoptosis and necrosis, assessed by bisbenzimide and propidium iodide staining respectively, were 1.8 and 2‐fold higher in HG (p&lt;0.05). MAO inhibition significantly reduced cell death both after 24 and 48 hours of culture in HG. In conclusion, here we demonstrate that MAO play a major role in the oxidative stress and cell death in cardiomyocytes exposed to HG. MAO may amplify the effects of existing ROS triggers or HG may directly enhance MAO activity. Both phenomena are likely to be relevant for the onset and progression of diabetic cardiomyopathy.

The effects of pargyline and 2-phenylethylamine on D1-like dopamine receptor binding

2-Phenylethylamine (PE) potentiates neuronal responses to dopamine by an unknown post-synaptic mechanism. Here, whether PE modifies D1-like receptor binding was examined. An unexpected effect of the monoamine oxidase inhibitor pargyline was observed, which did not involve competition for ligand binding. PE did not affect ligand binding in the presence or absence of pargyline. It is concluded that the effect of pargyline does not involve elevation of endogenous PE, and PE effects on dopaminergic neurotransmission are not due to altered D1-like receptor binding.

Effects of monoamine oxidase inhibitor and cytochrome P450 2D6 status on 5-methoxy- N, N-dimethyltryptamine metabolism and pharmacokinetics

5-Methoxy- N, N-dimethyltryptamine (5-MeO-DMT) is a natural psychoactive indolealkylamine drug that has been used for recreational purpose. Our previous study revealed that polymorphic cytochrome P450 2D6 (CYP2D6) catalyzed 5-MeO-DMT O-demethylation to produce active metabolite bufotenine, while 5-MeO-DMT is mainly inactivated through deamination pathway mediated by monoamine oxidase (MAO). This study, therefore, aimed to investigate the impact of CYP2D6 genotype/phenotype status and MAO inhibitor (MAOI) on 5-MeO-DMT metabolism and pharmacokinetics. Enzyme kinetic studies using recombinant CYP2D6 allelic isozymes showed that CYP2D6.2 and CYP2D6.10 exhibited 2.6- and 40-fold lower catalytic efficiency ( V max/ K m), respectively, in producing bufotenine from 5-MeO-DMT, compared with wild-type CYP2D6.1. When co-incubated with MAOI pargyline, 5-MeO-DMT O-demethylation in 10 human liver microsomes showed significantly strong correlation with bufuralol 1′-hydroxylase activities ( R 2 = 0.98; P < 0.0001) and CYP2D6 contents ( R 2 = 0.77; P = 0.0007), whereas no appreciable correlations with enzymatic activities of other P450 enzymes. Furthermore, concurrent MAOI harmaline sharply reduced 5-MeO-DMT depletion and increased bufotenine formation in human CYP2D6 extensive metabolizer hepatocytes. In vivo studies in wild-type and CYP2D6-humanized (Tg- CYP2D6) mouse models showed that Tg- CYP2D6 mice receiving the same dose of 5-MeO-DMT (20 mg/kg, i.p.) had 60% higher systemic exposure to metabolite bufotenine. In addition, pretreatment of harmaline (5 mg/kg, i.p.) led to 3.6- and 4.4-fold higher systemic exposure to 5-MeO-DMT (2 mg/kg, i.p.), and 9.9- and 6.1-fold higher systemic exposure to bufotenine in Tg- CYP2D6 and wild-type mice, respectively. These findings indicate that MAOI largely affects 5-MeO-DMT metabolism and pharmacokinetics, as well as bufotenine formation that is mediated by CYP2D6.

Acid amyloglucosidase and carbohydrate regulation. 3. The induction of sulphonylurea-stimulated insulin release and its dependence on intracellular monoamines.

The induction of sulphonylurea-stimulated insulin release and its dependence on intracellular monoamine levels in the β-cells was studied in mice. Model experiments in vitro on the interaction between monoamines and purified fungal acid amyloglucosidase were performed.1) Glibenclamide-induced hypoglycemia in mice was prolonged by pretreatment with a small dose of purified fungal acid amyloglucosidase, which itself did not influence the blood glucose level.2) Two minutes following the intravenous injection of glibenclamide, the "free" activity of acid amyloglucosidase in islet homogenates prepared in osmotically protected media was increased. This increase was found to be dose dependent.3) Glibenclamide-induced insulin release in adrenalectomized animals was significantly inhibited by pretreatment with the lysosomal stabilizer dexamethasone and significantly enhanced by pretreatment with the lysosomal labilizer progesterone.4) Glibenclamide-induced insulin release in animals pretreated with a small dose of purified fungal acid amyloglucosidase was considerably enhanced. This increase was significantly inhibited following administration of the monoamine precursors L-5-hydroxytryptophan (L-5-HTP) and L-3,4-dihydroxyphenylalanine (L-DOPA) and also after pretreatment with the non-hydrazine monoamine oxidase inhibitor pargyline. L-DOPA was found to be a more potent inhibitor than L-5-HTP. Combined treatment with pargyline and L-DOPA further decreased the response.5) Model experiments in vitro on the interaction between amines and acid amyloglucosidase activity (purified fungal acid amyloglucosidase) showed a pH-dependent inhibitory action of the monoamines dopamine (DA) and 5-hydroxytryptamine (5-HT) on amyloglucosidase activity. DA was more potent than 5-HT.6) From the data presented it is assumed that the induction of sulphonylurea-stimulated insulin release is elicited through the following events in the β-cell: Lysosomal activation - Increase of the "free" activity of the lysosomal acid amyloglucosidase - Hydrolytic glycogenosis through acid amyloglucosidase (reaction product: glucose) - Insulin release. From a functional point of view it is proposed that lysosomes taking part in insulin secreting processes should be designated crinosomes.

Effects of co-administration of antidepressants and monoamine oxidase inhibitors on 5-HT-related behavior in rats

5-hydroxytryptamine (5-HT) syndrome is a dangerous condition of 5-HT excess that can occur during co-administration of a monoamine oxidase (MAO) inhibitor and an antidepressant. We investigated the effects of acute administration of MAO inhibitors and subchronic administration of tricyclic and heterocyclic antidepressants, and a serotonin-noradrenaline reuptake inhibitor (SNRI) on 5-HT syndrome in rats treated with 5-hydroxytryptophan (5-HTP). The irreversible and non-selective MAO inhibitor pargyline, and the reversible and selective MAO-A inhibitor clorgyline, produced increases in 5-HT syndrome in the 5-HTP-treated rats, while subchronic co-administration of imipramine partly intensified and partly attenuated the syndrome, whereas milnacipran only attenuated the syndrome. Co-administration of mianserin partly intensified and partly attenuated the syndrome but the attenuating effect was dominant. Administration of the irreversible and selective MAO-B inhibitor selegiline did not produce any increase in 5-HT syndrome in the 5-HTP-treated rats, compared with the saline control. These data suggest that non-selective MAO and selective MAO-A inhibitors can induce 5-HT syndrome in humans when co-administered with antidepressants. Furthermore, the risk of 5-HT syndrome may be lower with the selective MAO-B inhibitor selegiline.

Absence of tissue-bound semicarbazide-sensitive amine oxidase activity in carp tissues

We have previously reported that carp ( Cyprinus carpio) tissue mitochondria contain a novel form of monoamine oxidase (MAO), which belongs neither to MAO-A nor to MAO-B of the mammalian enzyme. This conclusion results from the findings that the carp MAO was equally sensitive to a selective MAO-A inhibitor clorgyline and to the MAO-B selective inhibitor l-deprenyl, when tyramine, a substrate for both forms, serotonin or β-phenylethylamine, a substrate for either A or B-form of mammalian MAO, was used. In the present study, we tried to detect another amine oxidase, termed tissue-bound semicarbazide-sensitive amine oxidase (SSAO), activity in carp tissues. As definition of SSAO was used, such as insensitivity to inhibition of the kynuramine oxidizing activity by an MAO inhibitor pargyline and high sensitivity to the SSAO inhibitor semicarbazide. The results indicated that the oxidizing activity was selectively and almost completely inhibited by 0.1 mM pargyline alone or a combination of 0.1 mM pargyline plus 0.1 mM semicarbazide, but not by 0.1 mM semicarbazide alone. We also tried to detect any SSAO activity by changing experimental conditions, such as lower incubation temperature, higher enzyme protein concentration, a lower substrate concentration and different pH's in the reaction, as the enzyme source. However, still no SSAO activity could be detected in the tissues. These results conclusively indicate that carp tissues so far examined do not contain SSAO activity.

Reversal of learned helplessness by selective serotonin reuptake inhibitors in rats is not dependent on 5-HT availability

Serotonin (5-HT) and 5-HT 1A receptors have been suggested to play a pivotal role in the mechanism of action of antidepressant drugs, particularly in the case of selective serotonin reuptake inhibitors (SSRIs). In the rat learned helplessness (LH) paradigm, a valid animal model of human depression, repeated treatment with the 5-HT 1A receptor agonist 8-OH-DPAT (0.125 and 0.5 mg/kg) and several classes of antidepressants such as the tricyclic agent desipramine (30 and 60 mg/kg), the monoamine oxidase inhibitor (MAOI) pargyline (60 mg/kg) and the SSRIs fluoxetine (15 and 30 mg/kg), paroxetine (15 and 30 mg/kg) and sertraline (30 mg/kg) improved behavioural deficit in helpless rats. The involvement of serotonergic mechanisms in the antidepressant-like effect of these agents was investigated using the selective 5-HT 1A receptor antagonist WAY 100,635 and the 5-HT synthesis inhibitor p-chlorophenylalanine (PCPA). Pretreatment with WAY 100,635 blocked the 8-OH-DPAT-induced reduction in escape failures, but did not counteract the antidepressant effect of fluoxetine and paroxetine. PCPA given alone did not modify helpless behaviour nor did it affect the behavioural effect of 8-OH-DPAT, fluoxetine and paroxetine. Adaptive changes in 5-HT 1A receptor function were studied by measuring 8-OH-DPAT-mediated hypothermia and lower lip retraction (LLR) in the animals 24 h after LH test session. Fluoxetine and paroxetine treatments caused a marked reduction in agonist-induced responses, an effect completely prevented by WAY 100,635 and PCPA. In conclusion, whereas direct agonist activity at postsynaptic 5-HT 1A receptors attenuated helpless behaviour, the antidepressant-like effect of SSRIs was found to be independent of their actions on either 5-HT 1A receptor function or extracellular 5-HT.

Inhibition of vesicular monoamine transporter enhances vulnerability of dopaminergic cells: relevance to Parkinson's disease

Parkinson's disease is a neurodegenerative disorder associated with progressive loss of dopaminergic cells in the substantia nigra. Oxidative stress has been implicated in the pathogenesis of the disease, and dopamine has been suggested as a contributing factor that generates reactive oxygen species due to its unstable catechol moiety. We have previously shown that tetrahydrobiopterin (BH4), an obligatory cofactor for dopamine synthesis, also contributes to the vulnerability of dopamine-producing cells by generating oxidative stress. This study shows that the presence of dopamine in the cytosol enhances the cell's vulnerability to BH4. Upon exposure to ketanserin, a vesicular monoamine transporter inhibitor, BH4-induced dopaminergic cell death is exacerbated, accompanied by increased lipid peroxidation and protein bound quinone. While intracellular amount of DOPAC is elevated by ketanserin, the monoamine oxidase inhibitor pargyline showed no significant protection. Instead, the thiol agent N-acetylcysteine and quinone reductase inducer dimethyl fumarate abolish BH4/ketanserin-induced cell death, suggesting that quinone production plays an important role. Therefore, it can be concluded that the presence of dopamine in the cytosol seems to contribute to the cells’ vulnerability to BH4 and that vesicular monoamine transporter plays a protective role in dopaminergic cells by sequestering dopamine not only from monoamine oxidase but also from BH4-induced oxidative stress.

EGb761 pretreatment reduces monoamine oxidase activity in mouse corpus striatum during 1-methyl-4-phenylpyridinium neurotoxicity.

EGb761 produces reversible inhibition of both monoamine oxidase (MAO) isoforms in the central nervous system. 1-Methyl-4-phenylpyridinium (MPP+) neurotoxicity is prevented by treatment with the MAO inhibitor pargyline. We investigated EGb761's effect on striatal MAO activity during MPP+ neurotoxicity. C-57 black mice were pretreated with EGb761 (10 mg/kg) daily for 17 days followed by administration of MPP+ (0.72 mg/kg). MPP+ enhanced striatal MAO (30%) activity at 6 h, and EGb761 prevented this effect. MAO-B activity in striatum was enhanced (70%) 6 h after MPP+ administration and was reduced to almost normal levels in EGb761 + MPP+ group compared to MPP+ group. Pretreatment with EGb761 partially prevented (32%) the striatal dopamine-depleting effect of MPP+ and prevented the reduction in striatal tyrosine hydroxylase activity (100%). Results suggest that EGb761 supplements may be effective in reducing MAO activity as well as enhancement in dopamine metabolism, thereby preventing MPP+-neurotoxicity.

Norepinephrine-deficient mice lack responses to antidepressant drugs, including selective serotonin reuptake inhibitors.

Mice unable to synthesize norepinephrine (NE) and epinephrine due to targeted disruption of the dopamine beta-hydroxylase gene, Dbh, were used to critically test roles for NE in mediating acute behavioral changes elicited by different classes of antidepressants. To this end, we used the tail suspension test, one of the most widely used paradigms for assessing antidepressant activity and depression-related behaviors in normal and genetically modified mice. Dbh(-/-) mice failed to respond to the behavioral effects of various antidepressants, including the NE reuptake inhibitors desipramine and reboxetine, the monoamine oxidase inhibitor pargyline, and the atypical antidepressant bupropion, even though they did not differ in baseline immobility from Dbh(+/-) mice, which have normal levels of NE. Surprisingly, the effects of the selective serotonin reuptake inhibitors (SSRIs) fluoxetine, sertraline, and paroxetine were also absent or severely attenuated in the Dbh(-/-) mice. In contrast, citalopram (the most selective SSRI) was equally effective at reducing immobility in mice with and without NE. Restoration of NE by using L-threo-3,4-dihydroxyphenylserine reinstated the behavioral effects of both desipramine and paroxetine in Dbh(-/-) mice, thus demonstrating that the reduced sensitivity to antidepressants is related to NE function, as opposed to developmental abnormalities resulting from chronic NE deficiency. Microdialysis studies demonstrated that the ability of fluoxetine to increase hippocampal serotonin was blocked in Dbh(-/-) mice, whereas citalopram's effect was only partially attenuated. These data show that NE plays an important role in mediating acute behavioral and neurochemical actions of many antidepressants, including most SSRIs.