Monoamine Neurons Research Articles

The Neuronal Monoamine Transporter VMAT2 Is Regulated by the Trimeric GTPase Go2

Monoamines such as noradrenaline and serotonin are stored in secretory vesicles and released by exocytosis. Two related monoamine transporters, VMAT1 and VMAT2, mediate vesicular transmitter uptake. Previously we have reported that in the rat pheochromocytoma cell line PC 12 VMAT1, localized to peptide-containing secretory granules, is controlled by the heterotrimeric G-protein Go(2). We now show that in BON cells, a human serotonergic neuroendocrine cell line derived from a pancreatic tumor expressing both transporters on large, dense-core vesicles, VMAT2 is even more sensitive to G-protein regulation than VMAT1. The activity of both transporters is only downregulated by Galphao(2), whereas comparable concentrations of Galphao(1) are without effect. In serotonergic raphe neurons in primary culture VMAT2 is also downregulated by pertussis toxin-sensitive Go(2). By electron microscopic analysis from prefrontal cortex we show that VMAT2 and Galphao(2) associate preferentially to locally recycling small synaptic vesicles in serotonergic terminals. In addition, Go(2)-dependent modulation of VMAT2 also works when using a crude synaptic vesicle preparation from this brain area. We conclude that regulation of monoamine uptake by the heterotrimeric G proteins is a general feature of monoaminergic neurons that controls the content of both large, dense-core and small synaptic vesicles.

The monoamine neurons of the rat brain preferentially express a splice variant of alpha1B subunit of the N-type calcium channel.

The N-type voltage-dependent calcium channels play a significant role in neurotransmitter release. The alpha1B subunit of the N-type calcium channel functions as the primary subunit that forms the pore and contains the structural motifs that mediate the pharmacological and gating properties of the channel. We report on an isoform of the alpha1B subunit that is preferentially expressed by the monoaminergic neurons of the rat brain. This isoform contains a 21-amino acid cassette in the synprint site present in the cytoplasmic loop between domains IIS6 and IIIS1. RT-PCR of micropunched tissue was used to show preferential expression of this isoform in regions of the brain containing monoaminergic neurons and to a lesser extent in the cerebellum. Double-label in situ hybridization was used to show expression of this isoform mRNA in dopaminergic neurons of the ventral mesencephalon. The expression of two distinct N-type calcium channels containing these alpha1B subunit isoforms by the monoaminergic neurons may provide for synapse-specific regulation of neurotransmitter release.

Neurotoxic effects of (±)fenfluramine and phentermine, alone and in combination, on monoamine neurons in the mouse brain

Neurotoxic effects of (±)fenfluramine and phentermine, alone and in combination, on monoamine neurons in the mouse brain

Neurotoxic effects of +/-fenfluramine and phenteramine, alone and in combination, on monoamine neurons in the mouse brain.

Until recently, (+/-)fenfluramine (FEN) was widely prescribed as an appetite suppressant. In animals, FEN is a potent and selective brain serotonin neurotoxin. The present studies assessed the effects of phentermine (PHEN), an appetite suppressant frequently used clinically in combination with FEN, on FEN-induced serotonin neurotoxicity. Groups (n = 6/group) of mice were treated with FEN (10 mg/kg), PHEN (20 mg/kg or 40 mg/kg), FEN (10 mg/kg) plus PHEN (20 mg/kg or 40 mg/kg), or vehicle twice daily for four days. Food intake and body weight were measured during and after drug treatment. Brains were evaluated for regional brain serotonin and dopamine axonal markers two weeks after drug treatment. PHEN enhanced the anorectic and weight-reducing effects of FEN. PHEN also significantly enhanced FEN's long-term toxic effects on 5-HT axons. This effect was evident in some (hypothalamus, striatum) but not all (hippocampus, cortex) brain regions examined. PHEN alone produced no long-term effects on 5-HT axonal markers. However, whether given alone or in combination with FEN, PHEN produced significant, dose-related decreases in striatal DA axonal markers. These results, coupled with those from previous studies, suggest that PHEN has the potential to exacerbate FEN-induced serotonin neurotoxicity, if utilized in certain doses. Further, the present results indicate that PHEN possesses dopamine (DA) neurotoxic potential. The relevance of these data to humans previously treated with FEN/PHEN is discussed.

Molecular identification of the corticosterone-sensitive extraneuronal catecholamine transporter.

Catecholaminergic signaling regulates various physiological functions, such as blood pressure1 and is implicated in drug dependence, affective disorders and male aggressive behavior2,3. The actions of released catecholamines are terminated by sodium-driven, high-affinity transporters in the plasma membrane of the releasing neurons4,5 and by a corticosterone-sensitive, low-affinity, high-capacity extraneuronal transport system6, originally named uptake2, found in sympathetically innervated tissues7 and in central nervous system glia8. Here we report the molecular identification and pharmacological characterization of the extraneuronal catecholamine transporter, which is unrelated to the family of sodium-driven neuronal monoamine transporters5.

Biochemical aspects of Parkinson's disease.

The importance of the striatal dopamine (DA) deficiency and the DA substituting property of levodopa for the pathophysiology and therapy of Parkinson's disease (PD) is reiterated. In addition, it is shown that in PD, significantly reduced DA levels are also found in the nucleus accumbens, external and internal segments of the globus pallidus, the substantia nigra reticulata, and the subthalamic nucleus. It is proposed that, in addition to the critical role played by the striatal DA loss, the DA changes in the extrastriatal nuclei of the basal ganglia are importantly involved in the pathophysiologic mechanisms resulting in the parkinsonian movement disorder, and that the therapeutic and/or side effects of DA substitution therapy may, in part, be mediated through these brain regions which, like the striatum, suffer DAergic deafferentation in PD. From observations in brain of patients with secondary parkinsonism, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine parkinsonism in the rhesus monkey, as well as the regional DA transporter distribution in the primate substantia nigra, it is concluded that PD may be caused by any exogenous and/or endogenous toxin using the transporter system for DA and to some degree the other brain monoamines (noradrenaline, serotonin), to enter, and damage, the respective monoamine neurons. Based on converging evidence, the view is advanced that endogenous, genetically based (excessive) formation, or accumulation, of toxic DA transporter substrates, such as isoquinoline or beta-carboline derivatives, may in fact represent the primary cause of substantia nigra cell degeneration in patients with PD.

A Leucine-based Motif Mediates the Endocytosis of Vesicular Monoamine and Acetylcholine Transporters

Specific transport proteins mediate the packaging of neurotransmitters into secretory vesicles and consequently require targeting to the appropriate intracellular compartment. To identify residues in the neuron-specific vesicular monoamine transporter (VMAT2) responsible for endocytosis, we examined the effect of amino (NH2-) and carboxyl (COOH-)-terminal mutations on steady state distribution and internalization. Deletion of a critical COOH-terminal domain sequence (AKEEKMAIL) results in accumulation of VMAT2 at the plasma membrane and a 50% reduction in endocytosis. Site-directed mutagenesis shows that replacement of the isoleucine-leucine pair within this sequence by alanine-alanine alone reduces endocytosis by 50% relative to wild type VMAT2. Furthermore, the KEEKMAIL sequence functions as an internalization signal when transferred to the plasma membrane protein Tac, and the mutation of the isoleucine-leucine pair also abolishes internalization of this protein. The closely related vesicular acetylcholine transporter (VAChT) contains a similar di-leucine sequence within the cytoplasmic COOH-terminal domain that when mutated results in accumulation of VAChT at the plasma membrane. The VAChT di-leucine sequence also confers internalization when appended to two other proteins and in one of these chimeras, conversion of the di-leucine sequence to di-alanine reduces the internalization rate by 50%. Both VMAT2 and VAChT thus use leucine-based signals for efficient endocytosis and as such are the first synaptic vesicle proteins known to use this motif for trafficking.

Prenatal Cocaine Exposure Alters Signal Transduction in the Brain D1 Dopamine Receptor Systema.

Cocaine use during pregnancy may result in persistent behavioral abnormalities in the newborn. Animal studies show behavioral and neurochemical alterations in offspring that were exposed to cocaine prenatally. The monoamine neurons, including those containing dopamine, appear and become operational prenatally and mature during early postnatal life. It is therefore conceivable that exposure to cocaine during gestation may critically affect normal development and subsequently cause protracted postnatal neurochemical and behavioral changes. The data we obtained demonstrate that prenatal exposure to cocaine in the rabbit impairs signal transduction via the D1 but not the D2 dopamine receptor system. This is reflected in impaired dopamine-stimulated [35S]GTPγS binding to Gαs without affecting binding of the nucleotide to Gαi in both cortex and striatum of rabbit offspring. This selective reduction in D1 dopamine receptor-mediated activation of Gs protein increased in severity as the dose of cocaine administered to the pregnant dams was increased. Maximal impairment was observed after treatment with two daily injections of 3 mg/kg of cocaine HC1. The reduction in dopamine-stimulated GTP binding to Gαs did not result from a decrease in concentration of membrane Gαs protein or D1 dopamine receptors. The data also indicate that in utero cocaine exposure causes persistent uncoupling of the D1 dopamine receptors from their associated Gs protein which appears as early as gestational day 22 and persists to postnatal day 100. The reduction in D1 dopamine receptor-mediated signal transduction may be mediated by posttranslational modifications of the D1 dopamine receptor or of Gsα such as phosphorylation, which result in altered coupling between these membrane components. The resultant attenuated D1 dopamine receptor-mediated signaling may ultimately underlie both long-lasting behavioral dysfunction and morphologic changes which are associated with prenatal cocaine exposure in the rabbit.

Prenatal Exposure to the Pesticide Dieldrin or the GABAA Receptor Antagonist Bicuculline Differentially Alters Expression of GABAA Receptor Subunit mRNAs in Fetal Rat Brainstem

We have previously shown that GABA acts as a trophic signal for monoamine neurons in embryonic day 14 (E14) rat brainstem cultures [Liu et al., J Neurosci 1997a;17:2420–2428]. The organochlorine pesticide dieldrin and the classical GABA_A receptor antagonist bicuculline interfere with the trophic actions of GABA and alter expression of several GABA_A receptor subunit mRNA transcripts in these cultures [Liu et al., J Neurosci Res 1997b;49:645–653]. In the present study, we investigated whether prenatal exposure to dieldrin or bicuculline from E12–17 would alter mRNA expression of α1, β3, γ1, γ2S and γ2L GABA_A receptor subunits in fetal (E17) rat brainstem using competitive RT-PCR to absolutely quantify these transcripts. The effects of dieldrin and bicuculline on expression of GABA_A receptor subunit transcripts were similar across subunits. Dieldrin and bicuculline decreased expression of α1, β3 and γ1 transcripts compared to vehicle-injected controls, but did not significantly alter expression of γ2S and γ2L transcripts. Taken together, these studies indicate that in utero exposure to organochlorine pesticides acting as GABA_A receptor antagonists may alter the expression and subunit composition of developing GABA_A receptors. If these changes persist, they could have long-lasting effects on developing GABAergic neural circuitry, GABA_A receptor function and GABA-mediated behaviors.

Prenatal Exposure to Neurotoxicants Dieldrin or Lindane Alters tert-ButylbicyclophosphorothionateBinding to GABA A Receptors in Fetal Rat Brainstem

GABA acts as a trophic signal for cultured embryonic rat monoamine neurons by activating GABA_A receptors. These effects are blocked by the organochlorine insecticide dieldrin and the classic GABA_A antagonist bicuculline. Both dieldrin and another organochlorine insecticide, lindane, block the effects of GABA on the GABA_A receptor by binding directly to the Cl^– channel. Therefore, prenatal exposure to these chemicals could lead to disturbances in the trophic actions of GABA on monoamine neurotransmitter systems in the embryonic brain and produce alterations in GABA_A receptor expression and function. Effects of daily prenatal exposure to organochlorine insecticide (dieldrin or lindane) or bicuculline from embryonic day (E)12–17 were determined in brains of E17 fetal rats using t-[³⁵S]butyl-bicyclophosphorothionate ([³⁵S]TBPS) binding. This radioligand was chosen because, like organochlorine insecticides, it binds directly to GABA_A receptor/Cl^– channels. [³⁵S]TBPS binding was analyzed in extensively washed membranes from E17 brainstem and whole brain with the brainstem removed (‘rest of brain’) at a TBPS concentration that approximated the K_D determined in [³⁵S]TBPS saturation binding experiments performed on normal E17 rat brainstem. In utero exposure to dieldrin, lindane, or bicuculline from E12-E17 caused a significant reduction in the amount of [³⁵S]TBPS binding in E17 brainstem compared to vehicle-injected controls, but had no significant effect on ‘rest of brain’. These data suggest that in utero exposure to organochlorine insecticides that act as GABA_A antagonists negatively regulate expression of GABA_A receptors in fetal brainstem. If these effects persist, they could lead to disturbances in postnatal functions of the ascending GABAergic system, possibly with behavioral consequences.

Increase in monoamine levels caused by emotional stress in mice brain regions is attenuated by Saiko-ka-ryukotsu-borei-to.

The effect of Saiko-ka-ryukotsu-borei-to (SRBT) on the stress-induced increase of monoamines in brain regions was investigated in three mouse emotional stress models. Dopamine (DA) and 3,4-dihydroxy-phenyl acetic acid (DOPAC) contents were elevated significantly by electric shock stress, psychological stress and conditioned fear stress in thalamus, hypothalamus and amygdala. The DA and DOPAC levels were decreased by preadministration of SRBT (600 mg/kg, p.o.) in the last two models, but were not altered in electric shock stress. Therefore, this compound seems to be effective in stress involving emotional factors. These results indicate that SRBT affects the brain monoamine neurons leading to psychological change in mice.

Nigrostriatal dopamine neurons express low levels of GTP cyclohydrolase I protein.

A previous study using the in situ hybridization technique showed that serotonin neurons contain substantially more GTP cyclohydrolase I mRNA than do either dopamine or norepinephrine neurons. The objective of the current study was to determine whether these differences in mRNA abundance are predictive of the amount of GTP cyclohydrolase I protein available for tetrahydrobiopterin biosynthesis. The double-label immunofluorescence technique was used to localize GTP cyclohydrolase I protein to the tyrosine hydroxylase-positive A9 dopamine neurons of the substantia nigra and the A6 norepinephrine neurons of the locus ceruleus or the tryptophan hydroxylase-positive B6/B7 serotonin neurons of the dorsal raphe nucleus. Although GTP cyclohydrolase I immunofluorescence within serotonin and norepinephrine neurons was relatively intense, the fluorescence signal within dopamine neurons was faint to nondetectable. An immunoautoradiographic technique was developed to quantify these apparent differences in GTP cyclohydrolase I protein expression at the cellular level. Significant differences between all three neurochemical subdivisions were found and comparisons showed that on average serotonin neurons contain between 2.3- and 7.3-fold more GTP cyclohydrolase I protein than do either norepinephrine or dopamine neurons, respectively. Nigrostriatal dopamine neurons thus appear to synthesize and maintain tetrahydrobiopterin at low levels. Because dopamine and norepinephrine neurons express essentially equal amounts of GTP cyclohydrolase I mRNA, posttranscriptional events may serve to maintain low levels of GTP cyclohydrolase I protein within dopamine neurons. Phenotypic differences in GTP cyclohydrolase I protein expression across populations of monoamine neurons may be an important control point in neurotransmitter biosynthesis.

Cytokine regulation of embryonic rat dopamine and serotonin neuronal survival in vitro

Interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) are cytokines with pleiotropic effects in the central nervous system (CNS), including an emerging role in neurodevelopment. This study measured the effects of cytokines on the survival of tyrosine hydroxylase (TH) immunoreactive dopamine neurons from the substantia nigra (SN), and 5-hydroxytryptamine (5-HT) immunoreactive serotonin neurons from the rostral raphe (RR), using cultures from embryonic day 14 (E14) rat brain. IL-1β, IL-6, and TNF-α were added to cell cultures at 1, 10 and 100 U/ml. After 3 days in vitro, TH and 5-HT neurons were counted. The survival of 5-HT neurons was significantly reduced by 20–30% at 10 U/ml of IL-6. IL-1β and TNF-α at doses of 1 and 10 U/ml appeared to have a similar effect on the survival of these neurons, but this effect was not statistically significant. Comparable non-significant reductions of survival also occurred for TH neurons at the lower doses of IL-6 and TNF-α. In separate experiments, SN and RR cultures were exposed to the cytokines at a higher dose (1000 U/ml), causing a significant 30–40% decrease in the survival of TH neurons, but little or no change in 5-HT neuronal survival. Taken together, these results show that IL-1β, IL-6, and TNF-α can affect developing monoamine neurons at physiologically relevant concentrations, and that high doses differentially inhibit the survival of TH and 5-HT neurons after short exposures.

Parkinson disease: a new link between monoamine oxidase and mitochondrial electron flow.

Two factors that contribute to the progression of Parkinson disease are a brain defect in mitochondrial respiration and the generation of hydrogen peroxide (H2O2) by monoamine oxidase (MAO). Here we show that the two are linked. Metabolism of the neurotransmitter dopamine, or other monoamines (benzylamine, tyramine), by intact rat brain mitochondria suppresses pyruvate- and succinate-dependent electron transport. MAO inhibitors prevent this action. Mitochondrial damage is also reversed during electron flow. A probable explanation is that MAO-generated H2O2 oxidizes glutathione to glutathione disulfide (GSSG), which undergoes thiol-disulfide interchange to form protein mixed disulfides, thereby interfering reversibly with thiol-dependent enzymatic function. In agreement with this premise, direct addition of GSSG to mitochondria resulted in similar reversible inhibition of electron transport. In addition, the monoamines induced an elevation in protein mixed disulfides within mitochondria. These observations imply that (i) heightened activity and metabolism of neurotransmitter by monoamine neurons may affect neuronal function, and (ii) apparent defects in mitochondrial respiration associated with Parkinson disease may reflect, in part, an established increase in dopamine turnover. The experimental results also target mitochondrial repair mechanisms for further investigation and may, in time, lead to newer forms of therapy.

GABAAReceptors Mediate Trophic Effects of GABA on Embryonic Brainstem Monoamine NeuronsIn Vitro

The inhibitory neurotransmitter GABA may act as a trophic signal for developing monoamine neurons in embryonic rat brain, because GABA neurons and their receptors appear in brainstem during generation of monoamine neurons. To test this hypothesis, we used dissociated cell cultures from embryonic day 14 rat brainstem, which contains developing serotonin (5-HT), noradrenaline (tyrosine hydroxylase; TH), and GABA neurons. Immunocytochemistry and reverse transcription-PCR (RT-PCR) revealed the presence of multiple alpha, beta, gamma, and delta subunits in these cultures. Competitive RT-PCR demonstrated high levels of beta3 subunit transcripts. Expression of functional GABAA receptors was demonstrated using 36Cl- flux assays. To investigate GABAergic regulation of neuronal survival and growth, cultures were treated for 1-3 d in vitro with 10 microM GABA and/or GABAA antagonist (bicuculline or the pesticide dieldrin). The effects of treatments were quantified by analysis of immunoreactive 5-HT, TH, and GABA neurons. GABAA receptor ligands differentially regulated neuronal survival and growth depending on neurotransmitter phenotype. GABA exerted positive effects on monoamine neurons, which were countered by bicuculline (and dieldrin, 5-HT neurons only). By itself, bicuculline produced inhibitory effects on both 5-HT and TH neurons, whereas dieldrin potently inhibited 5-HT neurons only. GABA neurons responded positively to both antagonists, but more strongly to bicuculline. Taken together, these results demonstrate that the activation/inhibition of GABAA receptors produces opposite effects on the development of embryonic monoamine and GABA neurons. This suggests that these neurotransmitter phenotypes may express GABAA receptors that differ in fundamental ways, and these differences determine the developmental responses of these cells to GABAergic stimuli.

An affinity-modulating site on neuronal monoamine transport proteins.

The dissociation rates of [3H]nisoxetine, [3H]GBR 12935 and [3H]citalopram from, respectively, the rat brain noradrenaline, dopamine and 5-HT transporters were found to be markedly affected by several drugs. Sertraline strongly attenuated the rate of dissociation of [3H]nisoxetine from the noradrenaline transporter, while citalopram strongly attenuated that of [3H]citalopram from the 5-HT transporter. The effect of both drugs were stereospecific. Less potent affinity-modulating drugs were identified with regards to [3H]GBR 12935 dissociation from the dopamine transporter. All three neuronal monoamine transporters may thus have specific affinity-modulating sites which change the function of the transporters with possible implication for the reuptake of monoamines released during synaptic activity.

Catecholamines, Opioids, and Vagal Afferents in the Nucleus of the Solitary Tract

There is prominent nonsynaptic vesicular release of catecholamines and/or opioid peptides in the nucleus of the solitary tract (NTS), and this occurs mainly from large dense-core vesicles (LDCVs). Catecholamines and/or opioids in the NTS can modulate both cardiorespiratory and nociceptive reflexes. In addition, both noradrenergic and adrenergic neurons in this region contain endogenous opioid peptides and receive direct synaptic input from vagal afferents. The functional sites for storage and release of catecholamines and for the physiological effects of catecholamines and/or opioids in the NTS, however, are largely unknown. To identify these sites, previously characterized antipeptide antisera is used to examine the immunocytochemical localization of the vesicular monoamine transporter-2 (VMAT2), the transporter responsible for the reserpine-sensitive vesicular uptake of monoamines in neurons; the β2-adrenergic receptor (βAR), a receptor subtype implicated in the presynaptic regulation of noradrenaline release; and the μopioid receptor (μOR), the major opioid-receptor subtype involved in modulation of vagal reflexes. The results suggest that occupancy of βAR in the NTS can modulate not only the presynaptic release and postsynaptic responses of catecholaminergic neurons, but also the activity of neighboring glia. These findings, as well as the presence of βAR on glia and μOR at extrasynaptic sites on dendrites and axons, suggest that catecholamines and opioids are involved in parasynaptic transmission.

Evaluation of marble burying behavior: induced alteration of monoamine metabolism in mouse brain

Evaluation of marble burying behavior (MBB) housing-induced alteration of monoamine metabolism in mouse brain was performed by measuring metabolite levels with HPLC-ECD. Isolated housing of mice, each in a cage (22 x 32 x 14 cm; sawdust, 1-mm diameter; 5 cm in thickness) with 15 evenly spaced glass marbles on the floor (2.5-cm diameter; control, without marbles) for 24-168 hr, 5-HIAA contents were decreased in three regions: the midbrain, thalamus and hypothalamus. 5-HT turnover was not inhibited except for in the hypothalamus due to the decreases of 5-HT in the other two regions. On the other hand, DOPAC content and DA turnover were decreased in four regions: striatum, midbrain, thalamus, hypothalamus. The decrease in hypothalamic monoamine neurons was observed notably after 72 hr of MBB housing. No alterations were observed in feeding, water-drinking, spontaneous locomotor activity, number of buried marbles, serum corticosterone and serum glucose concentrations during MBB keeping compared with the control mice. These results suggested that the isolated mouse housed in a cage with evenly spaced glass marbles for a long period may be a model animal for alteration of monoamine metabolism in brain regions without physical infringement.

GABA A Receptors Mediate Trophic Effects of GABA on Embryonic Brainstem Monoamine Neurons In Vitro

GABA A Receptors Mediate Trophic Effects of GABA on Embryonic Brainstem Monoamine Neurons In Vitro

Amezinium and debrisoquine are substrates of uptake1 and potent inhibitors of monoamine oxidase in perfused lungs of rats.

Previous studies have resulted in the classification of amezinium as a selective inhibitor of neuronal monoamine oxidase (MAO), because it is a much more potent MAO inhibitor in intact tissues, in which it is accumulated in noradrenergic neurones by uptake1, than in tissue homogenates. In the present study, the effects of amezinium on the deamination of noradrenaline were investigated in intact lungs of rats, since the pulmonary endothelial cells are a site where the catecholamine transporter is non-neuronal uptake1. In addition, another drug that is both a substrate of uptake1 and a MAO inhibitor, debrisoquine, was investigated in the study. The first aim of the study was to show whether amezinium and debrisoquine are substrates of uptake1 in rat lungs. After loading of isolated perfused lungs with 3H-noradrenaline (MAO and catechol-O-methyltransferase (COMT) inhibited), the efflux of 3H-noradrenaline was measured for 30 min. When 1 mumol/l amezinium or 15 mumol/l debrisoquine was added for the last 15 min of efflux, there was a rapid and marked increase in the fractional rate of loss of 3H-noradrenaline, which was reduced by about 70% when 1 mumol/l desipramine was present throughout the efflux period. These results showed that both drugs were substrates for uptake1 in rat lungs. In lungs perfused with 1 nmol/l 3H-noradrenaline (COMT inhibited), 10, 30 and 300 nmol/l amezinium caused 58%, 76% and 74% inhibition of noradrenaline deamination, respectively, and 30, 300 and 3000 nmol/l debrisoquine caused 56%, 89% and 96% inhibition of noradrenaline deamination, respectively. When MAO-B was also inhibited, 10 nmol/l amezinium caused 84% inhibition of the deamination of noradrenaline by MAO-A in the lungs. In contrast, in hearts perfused with 10 nmol/l 3H-noradrenaline under conditions where the amine was accumulated by uptake2 (COMT, uptake1 and vesicular transport inhibited), 10 nmol/l amezinium had no effect and 300 nmol/l amezinium caused only 36% inhibition of deamination of noradrenaline. The results when considered with previous reports in the literature show that amezinium is about 1000 times more potent and debrisoquine is about 20 times more potent for MAO inhibition in rat lungs than in tissue homogenates, and the reason for their high potencies in the intact lungs is transport and accumulation of the drugs in the pulmonary endothelial cells by uptake1. Amezinium is much less potent as a MAO inhibitor in cells with the uptake2 transporter, such as the myocardial cells of the heart. The results also confirmed previous reports that amezinium is highly selective for MAO-A.