RESUMEN
Concurrent cocaine and alcohol use is among the most frequent drug combination, and among the most dangerous in terms of deleterious outcomes. Cocaine increases extracellular monoamines by blocking dopamine (DA), norepinephrine (NE) and serotonin (5-HT) transporters (DAT, NET and SERT, respectively). Likewise, ethanol also increases extracellular monoamines, however evidence suggests that ethanol does so independently of DAT, NET and SERT. Organic cation transporter 3 (OCT3) is an emergent key player in the regulation of monoamine signaling. Using a battery of in vitro, in vivo electrochemical, and behavioral approaches, as well as wild-type and constitutive OCT3 knockout mice, we show that ethanol's actions to inhibit monoamine uptake are dependent on OCT3. These findings provide a novel mechanistic basis whereby ethanol enhances the neurochemical and behavioral effects of cocaine and encourage further research into OCT3 as a target for therapeutic intervention in the treatment of ethanol and ethanol/cocaine use disorders.
Asunto(s)
Trastornos Relacionados con Cocaína , Cocaína , Ratones , Animales , Dopamina , Etanol/farmacología , Proteínas Portadoras , Cocaína/farmacología , Serotonina , Ratones Noqueados , Cationes , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Proteínas de Transporte de Serotonina en la Membrana PlasmáticaRESUMEN
Human organic cation transporter 2 (hOCT2) is involved in the transport of endogenous and exogenous organic cations mainly in cells of the kidney and the brain. Here, we focus on the regulation of hOCT2 by direct protein-protein interaction. Screening within a mating-based split-ubiquitin-yeast-two-hybrid system (mBSUS) revealed the lysosomal-associated protein transmembrane 4 alpha (LAPTM4A) as a potential interacting protein. Interaction of LAPTM4A and hOCT2 was confirmed by pulldown assays, FRET microscopy analysis and immunofluorescence microscopy. Functionally, overexpression of LAPTM4A significantly decreased ASP(+) uptake in HEK293 cells stably transfected with hOCT2, suggesting a negative regulation of hOCT2-mediated transport. Furthermore, overexpression of LAPTM4A leads to a significantly decreased hOCT2 plasma membrane expression in surface biotinylation experiments. In addition, significant expression of LAPTM4A in human kidney was demonstrated by immunoblotting and immunofluorescence.In this work, LAPTM4A has been identified as interaction partner of hOCT2. LAPTM4A regulates the function of hOCT2 by influencing its trafficking to/from the cell membrane and processing it via the intracellular sorting machinery.
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Endocitosis/fisiología , Proteínas de Transporte de Membrana/fisiología , Proteínas de Transporte de Catión Orgánico/metabolismo , Transporte Biológico , Endosomas/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Túbulos Renales Proximales/metabolismo , Lisosomas/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/fisiología , Transportador 2 de Cátion Orgánico , Mapeo de Interacción de Proteínas , ARN Mensajero/metabolismoRESUMEN
The emergence of new synthetic cathinones continues to be a matter of public health concern. In fact, they are quickly replaced by new structurally related alternatives. The main goal of the present study was to characterize the pharmacological profile, the psychostimulant and rewarding properties of novel cathinones (pentedrone, N-ethyl-pentedrone, α-PVP, N,N-diethyl-pentedrone and α-PpVP) which only differs in their amino terminal substitution. Rat synaptosomes were used for [3H]dopamine uptake experiments. HEK293 transfected cells (hDAT, hSERT, hOCT; human dopamine, serotonin and organic cation transporter) were also used for [3H]monoamine uptake and transporter binding assays. Molecular docking was used to investigate the effect of the amino substitutions on the biological activity. Hyperlocomotion and conditioned place preference paradigm were used in order to study the psychostimulant and rewarding effects in mice. All compounds tested are potent inhibitors of DAT with very low affinity for SERT, hOCT-2 and -3, and their potency for inhibiting DAT increased when the amino-substituent expanded from a methyl to either an ethyl-, a pyrrolidine- or a piperidine-ring. Regarding the in vivo results, all the compounds induced an increase in locomotor activity and possess rewarding properties. Results also showed a significant correlation between predicted binding affinities by molecular docking and affinity constants (Ki) for hDAT as well as the cLogP of their amino-substituent with their hDAT/hSERT ratios. Our study demonstrates the role of the amino-substituent in the pharmacological profile of novel synthetic cathinones as well as their potency inhibiting DA uptake and ability to induce psychostimulant and rewarding effects in mice.
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Alcaloides/química , Alcaloides/farmacología , Simulación del Acoplamiento Molecular/métodos , Psicotrópicos/química , Psicotrópicos/farmacología , Recompensa , Animales , Estimulantes del Sistema Nervioso Central/química , Estimulantes del Sistema Nervioso Central/farmacología , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Locomoción/efectos de los fármacos , Locomoción/fisiología , Masculino , Ratones , RatasRESUMEN
Glutamate transporter associated protein 3-18 (GTRAP3-18) is an endoplasmic reticulum (ER)-localized protein belonging to the prenylated rab-acceptor-family interacting with small Rab GTPases, which regulate intracellular trafficking events. Its impact on secretory trafficking has not been investigated. We report here that GTRAP3-18 has an inhibitory effect on Rab1, which is involved in ER-to-Golg trafficking. The effects on the early secretory pathway in HEK293 cells were: reduction of the rate of ER-to-Golgi transport of the vesicular stomatitis virus glycoprotein (VSVG), slowed accumulation of a Golgi marker plasmid in pre-Golgi structures after Brefeldin A treatment and inhibition of cargo concentration of the neuronal glutamate transporter excitatory amino-acid carrier 1 (EAAC1) into transpor complexes in HEK293 cells, an effect that could be completely reversed in the presence of an excess of Rab1. In accordance with the known role of Rab1 in neurite formation, overexpression of GTRAP3-18 significantly inhibited the length of outgrowing neurites in differentiated CAD cells. The inhibitory effect of GTRAP3-18 on neurite growth was rescued by co-expression with Rab1, supporting the conclusion that GTRAP 3-18 acted by inhibiting Rab1 action. Finally, we hypothesized that expression of GTRAP3-18 in the brain shoul be lower at stages of active synaptogenesis compared to early developmental stages. This was the case as expression of GTRAP3-18 declined from E17 to P0 and adult rat brains. Thus, we propose a model where protein trafficking and neuronal differentiation are directly linked by the interaction of Rab1 and its regulator GTRAP3-18.
Asunto(s)
Proteínas Portadoras/metabolismo , Diferenciación Celular/fisiología , Neuronas/fisiología , Transporte de Proteínas/fisiología , Proteínas de Unión al GTP rab1/metabolismo , Animales , Biomarcadores/metabolismo , Brefeldino A/metabolismo , Proteínas Portadoras/genética , Línea Celular , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Humanos , Neuronas/citología , Inhibidores de la Síntesis de la Proteína/metabolismo , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Proteínas de Unión al GTP rab1/genéticaRESUMEN
Aberrant serotonergic neurotransmission in the brain is considered at the core of the pathophysiological mechanisms involved in neuropsychiatric disorders. Gene by environment interactions contribute to the development of depression and involve modulation of the availability and functional activity of the serotonin transporter (SERT). Using behavioral and in vivo electrophysiological approaches together with biochemical, molecular-biological and molecular imaging tools we establish Flotillin-1 (Flot1) as a novel protein interacting with SERT and demonstrate its involvement in the response to chronic corticosterone (CORT) treatment. We show that genetic Flot1 depletion augments chronic CORT-induced behavioral despair and describe concomitant alterations in the expression of SERT, activity of serotonergic neurons and alterations of the glucocorticoid receptor transport machinery. Hence, we propose a role for Flot1 as modulatory factor for the depressogenic consequences of chronic CORT exposure and suggest Flotillin-1-dependent regulation of SERT expression and activity of serotonergic neurotransmission at the core of the molecular mechanisms involved.
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Corticosterona/metabolismo , Depresión/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Animales , Femenino , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Neuronas Serotoninérgicas/metabolismoRESUMEN
Traditionally, substrate translocation by neurotransmitter transporters has been described by the alternate access model. Recent structural data obtained with three distantly related transporters have also been interpreted as supportive of this model, because conformational correlates were visualized (inward-facing conformation, occluded state). However, the experimental evidence is overwhelmingly in favour of a more complex mode of operation: Transporters also exist in conformations that do not seal the permeation pathway. These conformations support a channel-like activity, including random permeation of substrate and co-substrate ions in a single-file mode. It is likely that the channel-like activity is modified by the interaction of the transporters with accessory proteins and regulatory kinases. Finally, channel-like activity is instrumental to understand the mechanism of action of amphetamines.
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Neurotransmisores/metabolismo , Proteínas de Transporte de Neurotransmisores/metabolismo , Sinapsis/fisiología , Anfetaminas/farmacología , Animales , Transporte Biológico , Humanos , Activación del Canal Iónico , Canales Iónicos/química , Canales Iónicos/metabolismo , Potenciales de la Membrana , Modelos Neurológicos , Proteínas de Transporte de Neurotransmisores/química , Proteínas de Transporte de Neurotransmisores/efectos de los fármacos , Conformación ProteicaRESUMEN
Cellular localization of neurotransmitter transporters is important for the precise control of synaptic transmission. By removing the neurotransmitters from the synaptic cleft, these transporters terminate signalling and affect duration and intensity of neurotransmission. Thus, a lot of work has been invested in the determination of the cellular compartment to which neurotransmitter transporters localize. In particular, the polarized distribution has received substantial attention. However, trafficking of transporters in the early secretory pathway has been largely ignored. Oligomer formation is a prerequisite for newly formed transporters to pass the stringent quality control mechanisms of the endoplasmic reticulum (ER), and this quaternary structure is also the preferred state which transporters reside in at the plasma membrane. Only properly assembled transporters are able to recruit the coatomer coat proteins that are needed for ER-to-Golgi trafficking. In this review, we will start with a brief description on transporter oligomerization that underlies ER-to-Golgi trafficking, followed by an introduction to ER-to-Golgi trafficking of neurotransmitter transporters. Finally, we will discuss the importance of oligomer formation for the pharmacological action of the illicitly used amphetamines and its derivatives.
Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Transporte de Neurotransmisores/metabolismo , Procesamiento Proteico-Postraduccional , Anfetaminas/farmacología , Animales , Proteínas Bacterianas/química , Estimulantes del Sistema Nervioso Central/farmacología , Cristalización , Aparato de Golgi/metabolismo , Humanos , Modelos Biológicos , Proteínas de Transporte de Neurotransmisores/química , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Transmisión SinápticaRESUMEN
BACKGROUND AND PURPOSE: 4-Methyl-N-methylcathinone (mephedrone) is a synthetic stimulant that acts as a substrate-type releaser at transporters for dopamine (DAT), noradrenaline (NET) and 5-HT (SERT). Upon systemic administration, mephedrone is metabolized to several phase I compounds: the N-demethylated metabolite, 4-methylcathinone (nor-mephedrone); the ring-hydroxylated metabolite, 4-hydroxytolylmephedrone (4-OH-mephedrone); and the reduced keto-metabolite, dihydromephedrone. EXPERIMENTAL APPROACH: We used in vitro assays to compare the effects of mephedrone and synthetically prepared metabolites on transporter-mediated uptake and release in HEK293 cells expressing human monoamine transporters and in rat brain synaptosomes. In vivo microdialysis was employed to examine the effects of i.v. metabolite injection (1 and 3 mg·kg(-1) ) on extracellular dopamine and 5-HT levels in rat nucleus accumbens. KEY RESULTS: In cells expressing transporters, mephedrone and its metabolites inhibited uptake, although dihydromephedrone was weak overall. In cells and synaptosomes, nor-mephedrone and 4-OH-mephedrone served as transportable substrates, inducing release via monoamine transporters. When administered to rats, mephedrone and nor-mephedrone produced elevations in extracellular dopamine and 5-HT, whereas 4-OH-mephedrone did not. Mephedrone and nor-mephedrone, but not 4-OH-mephedrone, induced locomotor activity. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that phase I metabolites of mephedrone are transporter substrates (i.e. releasers) at DAT, NET and SERT, but dihydromephedrone is weak in this regard. When administered in vivo, nor-mephedrone increases extracellular dopamine and 5-HT in the brain whereas 4-OH-mephedrone does not, suggesting the latter metabolite does not penetrate the blood-brain barrier. Future studies should examine the pharmacokinetics of nor-mephedrone to determine its possible contribution to the in vivo effects produced by mephedrone.
Asunto(s)
Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Metanfetamina/análogos & derivados , Animales , Células Cultivadas , Células HEK293 , Humanos , Masculino , Metanfetamina/química , Metanfetamina/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Whether amphetamine enhances noradrenergic activity by uptake blockade or a releasing action is still a matter of debate. In order to gain insight into the interaction of amphetamine with the noradrenaline transporter its cDNA was transfected into COS-7 cells (NAT-cells) or cotransfected with the cDNA of the vesicular monoamine transporter (NAT/VMAT-cells); cells were loaded with [3H]noradrenaline, superfused and the efflux analysed for total tritium and [3H]noradrenaline. In NAT-cells amphetamine stimulated [3H]noradrenaline efflux concentration-dependently when added to the superfusion buffer at 0.01, 0.1 and 1 microM. By contrast, 10 or 100 microM amphetamine stimulated efflux to a smaller extent or not at all; however, on switching back to amphetamine-free buffer a prompt increase of efflux was observed. Cocaine did not increase efflux per se and blocked the amphetamine-induced efflux. In NAT/VMAT-cells amphetamine stimulated efflux in a concentration-dependent manner. The effect showed saturation at 1 microM and was not suppressed at higher concentrations. Cocaine also elicited efflux from NAT/VMAT-cells concentration-dependently; the maximum was reached at approximately 1 microM and amounted to only about half of the amphetamine-induced efflux. It is concluded that amphetamine can induce noradrenaline transporter mediated release only at high nanomolar to low micromolar concentrations. At higher concentrations it blocks the noradrenaline transporter; in this case, the releasing action of amphetamine, like that of cocaine, is dependent on a vesicular pool of noradrenaline.
Asunto(s)
Anfetamina/farmacología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas de Transporte de Membrana , Neuropéptidos , Norepinefrina/metabolismo , Simportadores , Animales , Transporte Biológico , Células COS , Humanos , Glicoproteínas de Membrana/metabolismo , Neurotransmisores/metabolismo , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática , Perfusión , Transfección , Proteínas de Transporte Vesicular de Aminas Biógenas , Proteínas de Transporte Vesicular de MonoaminasRESUMEN
Human embryonic kidney 293 (HEK-293) cells stably transfected with the human serotonin (5-HT) or dopamine transporter (hSERT, hDAT), or the rat GABA transporter GAT-1 were incubated with saturating concentrations of transporter substrates (hSERT: [(3)H]5-HT, [(3)H]N-methyl-phenyl-pyridinium (MPP+); hDAT: [(3)H]dopamine, [(3)H]MPP(+); rGAT: [(3)H]GABA). Uptake velocities decreased significantly over time for [(3)H]5-HT and [(3)H]dopamine (already visible at 1 min), but not for [(3)H]MPP(+) or [(3)H]GABA. In efflux experiments cells were preloaded and substrate diffusion into the medium was studied following the addition of appropriate uptake inhibitors. Fractional effluxes were (% min(-1)) 1.27, 0.72, 0.27 and 0.08 for [(3)H]5-HT, [(3)H]dopamine, [(3)H]MPP(+) and [(3)H]GABA, respectively. The results suggest that in uptake experiments the more lipophilic substrates [(3)H]5-HT and [(3)H]dopamine leave the cells by diffusion already after a short time (1 min) of accumulation.
Asunto(s)
Proteínas Portadoras/biosíntesis , Línea Celular/metabolismo , Glicoproteínas de Membrana/biosíntesis , Proteínas de la Membrana/biosíntesis , Proteínas de Transporte de Membrana/biosíntesis , Proteínas del Tejido Nervioso , Inhibidores de la Captación de Neurotransmisores/metabolismo , Transportadores de Anión Orgánico , 1-Metil-4-fenilpiridinio/metabolismo , Animales , Difusión , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Proteínas Transportadoras de GABA en la Membrana Plasmática , Humanos , Riñón , Ratas , Serotonina/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Ácido gamma-Aminobutírico/metabolismoRESUMEN
BACKGROUND: Patients with Parkinson disease (PD) may be akinetic/rigid, be tremor dominant, or have comparable severity of these motor symptoms (classic). The pathophysiologic basis of different PD phenotypes is unknown. This study assessed pallidal and striatal dopamine level patterns in different motor subgroups of PD and normal control brains. METHODS: Globus pallidus and striatum dopamine (DA) levels were measured with high performance liquid chromatography in eight autopsy confirmed PD and five control frozen brains. RESULTS: DA levels in the external globus pallidus (GPe) of normal brains were nearly six times greater than in the internal pallidum (GPi). In PD, the mean loss of DA was marked (-82%) in GPe and moderate (-51%) in GPi. DA loss of variable degree was seen in different subdivisions of GPe and GPi in PD; however, DA levels were near normal in the ventral (rostral and caudal) GPi of PD cases with prominent tremor. There was marked loss of DA (-89%) in the caudate and severe loss (-98.4%) in the putamen in PD. The pattern of pallidal DA loss did not match the putaminal DA loss. CONCLUSION: There is sufficient loss of dopamine (DA) in external globus pallidus and the internal globus pallidum (GPi) as may contribute to the motor manifestations of Parkinson disease (PD). The possible functional disequilibrium between GABAergic and DAergic influences in favor of DA in the caudoventral parts of the GPi may contribute to resting tremor in tremor dominant and classic PD cases.
Asunto(s)
Dopamina/metabolismo , Globo Pálido/química , Globo Pálido/metabolismo , Trastornos de la Destreza Motora/metabolismo , Enfermedad de Parkinson/metabolismo , Anciano , Anciano de 80 o más Años , Química Encefálica/fisiología , Femenino , Globo Pálido/patología , Humanos , Masculino , Persona de Mediana Edad , Trastornos de la Destreza Motora/clasificación , Trastornos de la Destreza Motora/patología , Enfermedad de Parkinson/clasificación , Enfermedad de Parkinson/patologíaRESUMEN
The formation of oligomeric structures has been proposed for a large number of membrane proteins, including G-protein-coupled receptors and ion channels. Biochemical studies employing gel filtration, cross-linking or co-immunoprecipitation techniques showed that the serotonin [5-hydroxytryptamine (5-HT)] transporter is also capable of forming oligomers. We investigated whether the human serotonin transporter (hSERT) can be visualized as an oligomer in the plasma membrane of intact cells. To test this working hypothesis, we generated fusion proteins of hSERT and spectral variants of green fluorescent protein [cyan and yellow fluorescent proteins (CFP and YFP, respectively)]. When expressed in HeLa or HEK-293 cells, the resulting fusion proteins (CFP-hSERT and YFP-hSERT) were inserted into the plasma membrane and were indistinguishable from wild-type hSERT on functional testing (5-HT uptake assays, inhibition of 5-HT uptake by blockers such as imipramine). Oligomers were visualized by fluorescence resonance energy transfer (FRET) microscopy in living cells using complementary methods. Interestingly, oligomerization was not confined to hSERT; FRET was also observed between CFP-and YFP-labelled rat gamma-aminobutyric acid transporter. Gel filtration experiments showed that most of the protein was recovered as higher molecular weight complexes; almost no monomeric form was detected. This indicates that the homo-oligomeric form is the favoured state of hSERT in living cells. The formation of oligomers was not significantly affected by co-incubation with transporter substrates or blockers. Based on our observations, oligomer formation might not be essential for the physiological function of the transporter protein, the re-uptake of substrates. Furthermore, we conclude that constitutive oligomer formation might be a general property of Na(+)/Cl(-)-dependent neurotransmitter transporters.
Asunto(s)
Proteínas Portadoras/química , Glicoproteínas de Membrana/química , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Espectrometría de Fluorescencia/métodos , Antidepresivos/farmacología , Línea Celular , Transferencia de Energía , Humanos , Unión Proteica , Proteínas de Transporte de Serotonina en la Membrana PlasmáticaRESUMEN
The diagnostic hallmarks of amyotrophic lateral sclerosis (ALS) are degeneration of upper and lower motor neurons and of corticospinal tracts. Here, we demonstrate the suitability of the gliosis marker [3H]PK11195 for quantitative evaluation of tract degeneration in ALS in vitro. Binding of [3H]PK11195 was increased in lateral and ventral white matter of ALS spinal cords but not in the anterior horn, in spite of a dramatic loss in muscarinic binding sites and a high level of oxidatively modified protein. Labeling of activated microglia with [11C]PK11195 may also allow tract degeneration in ALS to be visualized in vivo.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Gliosis/patología , Degeneración Nerviosa/patología , Tractos Piramidales/patología , Adulto , Anciano , Esclerosis Amiotrófica Lateral/fisiopatología , Antineoplásicos , Autorradiografía , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Biomarcadores/análisis , Gliosis/fisiopatología , Antígenos HLA-DR/metabolismo , Humanos , Isoquinolinas , Microglía/efectos de los fármacos , Microglía/metabolismo , Persona de Mediana Edad , Degeneración Nerviosa/fisiopatología , Estrés Oxidativo/fisiología , Tractos Piramidales/fisiopatología , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/metabolismo , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/metabolismo , TritioRESUMEN
Quantitative aspects of inward and outward transport of substrates by the human plasmalemmal serotonin transporter (hSERT) were investigated. Uptake and superfusion experiments were performed on human embryonic kidney 293 cells permanently expressing the hSERT using [(3)H]serotonin (5-HT) and [(3)H]1-methyl-4-phenylpyridinium (MPP(+)) as substrates. Saturation analyses rendered K(m) values of 0.60 and 17.0 microM for the uptake of [(3)H]5-HT and [(3)H]MPP(+), respectively. Kinetic analysis of outward transport was performed by prelabeling the cells with increasing concentrations of the two substrates and exposing them to a saturating concentration of p-chloroamphetamine (PCA; 10 microM). Apparent K(m) values for PCA induced transport were 564 microM and about 7 mM intracellular [(3)H]5-HT and [(3)H]MPP(+), respectively. Lowering the extracellular Na(+) concentrations in uptake and superfusion experiments revealed differential effects on substrate transport: at 10 mM Na(+) the K(m) value for [(3)H]5-HT uptake increased approximately 5-fold and the V(max) value remained unchanged. The K(m) value for [(3)H]MPP(+) uptake also increased, but the V(max) value was reduced by 50%. When efflux was studied at saturating prelabeling conditions of both substrates, PCA as well as unlabeled 5-HT and MPP(+) (all substances at saturating concentrations) induced the same efflux at 10 mM and 120 mM Na(+). Thus, notwithstanding a 50% reduction in the V(max) value of transport into the cell, MPP(+) was still able to induce maximal outward transport of either substrate. Thus, hSERT-mediated inward and outward transport seems to be independently modulated and may indicate inconsistencies with the classical model of facilitated exchange diffusion.
Asunto(s)
1-Metil-4-fenilpiridinio/metabolismo , Proteínas Portadoras/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Serotonina/metabolismo , Sodio/farmacología , Transporte Biológico/efectos de los fármacos , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/genética , Células Cultivadas , Humanos , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Transfección , TritioRESUMEN
HEK 293 cells stably expressing the human serotonin transporter (hSERT) were grown on coverslips, preincubated with [(3)H]5-hydroxytryptamine (5-HT), and superfused. Substrates of the hSERT [e.g., p-chloroamphetamine (PCA)], increased the basal efflux of [(3)H]5-HT in a concentration-dependent manner. 5-HT reuptake blockers (e.g., imipramine, paroxetine) also raised [(3)H]5-HT efflux, reaching approximately one-third of the maximal effect of the hSERT substrates. In uptake experiments, both groups of substances inhibited [(3)H]5-HT uptake. Using the low-affinity substrate [(3)H]N-methyl-4-phenylpyridinium (MPP(+)) to label the cells in superfusion experiments, reuptake inhibitors failed to enhance efflux. Similar results were obtained using human placental choriocarcinoma (JAR) cells that constitutively express the hSERT at a low level. By contrast, PCA raised [(3)H]MPP(+) efflux in both types of cells, and its effect was inhibited by paroxetine. The addition of the Na(+),K(+)-ATPase inhibitor ouabain (100 microM) to the superfusion buffer enhanced basal efflux of [(3)H]5-HT-loaded hSERT cells by approximately 2-fold; the effect of PCA (10 microM) was strongly augmented by ouabain, whereas the effect of imipramine was not. The Na(+)/H(+) ionophore monensin (10 microM) also augmented the effect of PCA on efflux of [(3)H]5-HT as well as on efflux of [(3)H]MPP(+). In [(3)H]5-HT-labeled cells, the combination of imipramine and monensin raised [(3)H]5-HT efflux to a greater extent than either of the two substances alone. In [(3)H]MPP(+)-labeled cells, imipramine had no effect on its own and fully reversed the effect of monensin. The results suggest that the [(3)H]5-HT efflux caused by uptake inhibitors is entirely due to interrupted high-affinity reuptake, which is ongoing even under superfusion conditions.
Asunto(s)
Proteínas Portadoras/fisiología , Glicoproteínas de Membrana/fisiología , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Serotonina/metabolismo , 1-Metil-4-fenilpiridinio/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Fenfluramina/farmacología , Humanos , Riñón/metabolismo , Monensina/farmacología , Inhibidores de la Monoaminooxidasa/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Transfección , p-Cloroanfetamina/farmacologíaRESUMEN
Human embryonic kidney 293 cells stably transfected with the rat plasmalemmal serotonin transporter (rSERT) were incubated with 5-[3H]hydroxytryptamine ([3H]5-HT) and superfused. Substrates of the rSERT, such as p-chloroamphetamine (PCA) or methylenedioxymethamphetamine, concentration-dependently increased basal efflux of [3H]5-HT. 5-HT reuptake blockers (e.g., imipramine, citalopram) also caused an enhancement of [3H]5-HT efflux, reaching about half the maximal effect of the rSERT substrates. In uptake experiments, both groups of substances concentration-dependently inhibited 5-HT uptake. EC50 values obtained in superfusion experiments significantly correlated with IC50 values from uptake studies (r2 = 0.92). Addition of the Na+,K(+)-ATPase inhibitor ouabain (100 microM) to or the omission of K+ from the superfusion buffer accelerated basal efflux. The effect of PCA (10 microM) was markedly enhanced by both measures, whereas the effect of uptake inhibitors remained unchanged. When [3H]MPP+, a substrate with low affinity for the rSERT, was used instead of [3H]5-HT for labeling the cells, uptake inhibitors failed to augment efflux. By contrast, PCA accelerated [3H]MPP+ efflux, and its effect was strongly enhanced in the presence of ouabain. The results suggest that the [3H]5-HT efflux caused by substrates of rSERT is carrier-mediated, whereas efflux induced by uptake inhibitors is a consequence of interrupted high-affinity reuptake that is ongoing even under superfusion conditions.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , 1-Metil-4-fenilpiridinio/farmacocinética , Animales , Línea Celular , Inhibidores Enzimáticos/farmacología , Humanos , Ouabaína/farmacología , Perfusión , Ratas , Serotonina/farmacocinética , Serotoninérgicos/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Transfección , p-Cloroanfetamina/farmacologíaRESUMEN
Amphetamine and related substances induce dopamine release. According to a traditional explanation, this dopamine release occurs in exchange for amphetamine by means of the dopamine transporter (DAT). We tested this hypothesis in human embryonic kidney 293 cells stably transfected with the human DAT by measuring the uptake of dopamine, tyramine, and D- and L-amphetamine as well as substrate-induced release of preloaded N-methyl-4-[3H]phenylpyridinium ([3H]MPP+). The uptake of substrates was sodium-dependent and was inhibited by ouabain and cocaine, which also prevented substrate-induced release of MPP+. Patch-clamp recordings revealed that all four substrates elicited voltage-dependent inward currents (on top of constitutive leak currents) that were prevented by cocaine. Whereas individual substrates had similar affinities in release, uptake, and patch-clamp experiments, maximal effects displayed remarkable differences. Hence, maximal effects in release and current induction were approximately 25% higher for D-amphetamine as compared with the other substrates. By contrast, dopamine was the most efficacious substrate in uptake experiments, with its maximal initial uptake rate exceeding those of amphetamine and tyramine by factors of 20 and 4, respectively. Our experiments indicate a poor correlation between substrate-induced release and the transport of substrates, whereas the ability of substrates to induce currents correlates well with their releasing action.
Asunto(s)
Anfetamina/farmacología , Proteínas Portadoras/fisiología , Dopamina/farmacología , Glicoproteínas de Membrana , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Tiramina/farmacología , Anfetamina/farmacocinética , Transporte Biológico/fisiología , Proteínas Portadoras/genética , Línea Celular Transformada , Dopamina/farmacocinética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Electrofisiología , Humanos , Técnicas de Placa-Clamp , Transfección , Tiramina/farmacocinéticaRESUMEN
Recent biochemical studies indicate that the serotonin transporter can form oligomers. We investigated whether the human serotonin transporter (hSERT) can be visualized as an oligomer in the plasma membrane of intact cells. For this purpose, we generated fusion proteins of hSERT and spectral variants of the green fluorescent protein (cyan and yellow fluorescent proteins, CFP and YFP, respectively). When expressed in human embryonic kidney 293 cells, the resulting fusion proteins (CFP-hSERT and YFP-hSERT) were efficiently inserted into the plasma membrane and were functionally indistinguishable from wild-type hSERT. Oligomers were visualized by fluorescence resonance energy transfer microscopy in living cells using two complementary methods, i.e. ratio imaging and donor photobleaching. Interestingly, oligomerization was not confined to hSERT; fluorescence resonance energy transfer was also observed between CFP- and YFP-labeled rat gamma-aminobutyric acid transporter. The bulk of serotonin transporters was recovered as high molecular weight complexes upon gel filtration in detergent solution. In contrast, the monomers of CFP-hSERT and YFP-hSERT were essentially undetectable. This indicates that the homo-oligomeric form is the favored state of hSERT in living cells, which is not significantly affected by coincubation with transporter substrates or blockers. Based on our observations, we conclude that constitutive oligomer formation might be a general property of Na(+)/Cl(-)-dependent neurotransmitter transporters.
Asunto(s)
Proteínas Portadoras/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Transportadores de Anión Orgánico , Animales , Biopolímeros , Línea Celular , Transferencia de Energía , Proteínas Transportadoras de GABA en la Membrana Plasmática , Humanos , Ratas , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Espectrometría de FluorescenciaRESUMEN
Secretoneurin (SN) is a neuropeptide derived from secretogranin II that is found in brain and endocrine tissues. The aim of the present study was to determine the influence of this novel peptide on dopamine (DA) release from rat striatum using the microdialysis technique. Rat SN (1-30 mumol/L added to the dialysis buffer) enhanced DA outflow of awake rats in a concentration-dependent way without marked effects on the outflow of 3,4-dihydroxyphenylacetic acid or homovanillic acid. The increase in extracellular DA content caused by the peptide was observed throughout the entire period of administration (up to 4 h). Human SN and its 15-amino-acid C-terminal sequence also increased DA outflow, but the effects were smaller than those of rat SN. Two other peptides derived from secretogranin II were without effect on DA efflux. These results establish that SN has a pronounced effect on DA release under in vivo conditions.