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The Journal of Biological Chemistry Dec 2007D(3) dopamine receptors are expressed by dopamine neurons and are implicated in the modulation of presynaptic dopamine neurotransmission. The mechanisms underlying this...
D(3) dopamine receptors are expressed by dopamine neurons and are implicated in the modulation of presynaptic dopamine neurotransmission. The mechanisms underlying this modulation remain ill defined. The dopamine transporter, which terminates dopamine transmission via reuptake of released neurotransmitter, is regulated by receptor- and second messenger-linked signaling pathways. Whether D3 receptors regulate dopamine transporter function is unknown. We addressed this issue using a fluorescent imaging technique that permits real time quantification of dopamine transporter function in living single cells. Accumulation of the fluorescent dopamine transporter substrate trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium (ASP(+)) in human embryonic kidney cells expressing human dopamine transporter was saturable and temperature-dependent. In cells co-expressing dopamine transporter and D3 receptors, the D2/D3 agonist quinpirole produced a rapid, concentration-dependent, and pertussis toxin-sensitive increase of ASP(+) uptake. Similar agonist effects were observed in Neuro2A cells and replicated in human embryonic kidney cells using a radioligand uptake assay in which binding to and activation of D3 receptors by [(3)H]dopamine was prevented. D3 receptor stimulation activated phosphoinositide 3-kinase and MAPK. Inhibition of either kinase prevented the quinpirole-induced increase in uptake. D3 receptor activation differentially affected dopamine transporter function and subcellular distribution depending on the duration of agonist exposure. Biotinylation experiments revealed that the rapid increase of uptake was associated with increased cell surface and decreased intracellular expression and increased dopamine transporter exocytosis. In contrast, prolonged agonist exposure reduced uptake and transporter cell surface expression. These results demonstrate that D3 receptors regulate dopamine transporter function and identify a novel mechanism by which D3 receptors regulate extracellular dopamine concentrations.
Topics: Cell Line; Dopamine; Dopamine Agonists; Dopamine Plasma Membrane Transport Proteins; Dose-Response Relationship, Drug; Exocytosis; Gene Expression Regulation; Humans; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase Kinases; Neurons; Neurotransmitter Agents; Phosphatidylinositol 3-Kinases; Quinpirole; Receptors, Dopamine D3; Second Messenger Systems
PubMed: 17923483
DOI: 10.1074/jbc.M611758200 -
Journal of Neurochemistry Oct 2021Methcathinone (MCAT) is a psychostimulant of abuse that can cause both persistent striatal dopaminergic and serotonergic, as well as hippocampal serotonergic, deficits....
Methcathinone (MCAT) is a psychostimulant of abuse that can cause both persistent striatal dopaminergic and serotonergic, as well as hippocampal serotonergic, deficits. Evidence suggests that the rapid effects of stimulants that are structurally and mechanistically similar to MCAT on monoamine transporter function may contribute to the abuse liability and/or persistent monoaminergic deficits caused by these agents. Thus, effects of MCAT on 1) striatal dopamine (DA) transporter (DAT); and 2) striatal and hippocampal serotonin transporter (SERT) function, as determined in tissues from adult male rats, were assessed. As reported previously, a single administration of MCAT rapidly (within 1 hr) decreases striatal [ H]DA uptake. Similarly, incubation of rat synaptosomes with MCAT at 37℃ (but not 4˚C) decreased striatal [ H]DA uptake. Incubation with MCAT likewise decreased [ H]5HT but not vesicular [ H]DA uptake. MCAT incubation in vitro was without effect on [ H]DA uptake in striatal synaptosomes prepared from MCAT-treated rats. The decrease in [ H]DA uptake caused by MCAT incubation: (a) reflected a decrease in V , with minimal change in K , and (b) was attenuated by co-incubation with the cell-permeable calcium chelator, N,N'-[1,2-ethanediylbis(oxy-2,1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]-1,1'-bis[(acetyloxy)methyl] ester-glycine (BAPTA-AM), as well as the non-selective protein kinase-C (PKC) inhibitors bisindolylmaleimide-1 (BIM-1) and 2-[1-3(Aminopropyl)indol-3-yl]-3(1-methyl-1H-indol-3-yl)maleimide (or Bisindolylmaleimide VIII; Ro-31-7549). Taken together, these results suggest that in vitro MCAT incubation may model important aspects of MCAT administration in vivo, and that calcium and PKC contribute to the in vitro effects of MCAT on DAT.
Topics: Animals; Central Nervous System Stimulants; Corpus Striatum; Dopamine Plasma Membrane Transport Proteins; Male; Propiophenones; Protein Kinase C; Rats; Rats, Sprague-Dawley; Synaptosomes
PubMed: 34320222
DOI: 10.1111/jnc.15483 -
Toxicology in Vitro : An International... Oct 2021Dopamine (DA) metabolism and cell trafficking are critical for the proper functioning of DA neurons. Disruption of these DA processes can yield toxic products and is...
Dopamine (DA) metabolism and cell trafficking are critical for the proper functioning of DA neurons. Disruption of these DA processes can yield toxic products and is implicated in neurological conditions including Parkinson's disease (PD). To investigate pathogenic mechanisms involving DA neurons, in vitro models that recapitulate DA metabolism and trafficking in vivo are crucial. N27 cells are a widely used model for PD; however, these cells exhibit little expression of the DA transporter (DAT) confounding studies of DA uptake and metabolism. This lack of adequate DAT expression calls into question the use of this cell line as a model to study DA cell trafficking and metabolism. To overcome this problem, we stably expressed the human DAT (hDAT) in N27 cells to develop cells that we named N27-BCD. This approach allows for characterization of toxicants that may alter DA metabolism, trafficking, and/or interactions with DAT. N27-BCD cells are more sensitive to the neurotoxins 1-methyl-4-phenylpyridinium (MPTP/MPP+) and 6-hydroxydopamine (6-OHDA). N27-BCD cells allowed for clear observation of DA metabolism, whereas N27 cells did not. Here, we propose that stable expression of hDAT in N27 cells yields a useful model of DA neurons to study the impact of altered DA cell trafficking and metabolism.
Topics: 1-Methyl-4-phenylpyridinium; Animals; Cell Line; Dopamine; Dopamine Plasma Membrane Transport Proteins; Humans; Models, Biological; Oxidopamine; Rats
PubMed: 34252731
DOI: 10.1016/j.tiv.2021.105210 -
Neurochemistry International Jul 2014Alterations in dopamine (DA) signaling underlie the most widely held theories of molecular and circuit level perturbations that lead to risk for attention-deficit... (Review)
Review
Alterations in dopamine (DA) signaling underlie the most widely held theories of molecular and circuit level perturbations that lead to risk for attention-deficit hyperactivity disorder (ADHD). The DA transporter (DAT), a presynaptic reuptake protein whose activity provides critical support for DA signaling by limiting DA action at pre- and postsynaptic receptors, has been consistently associated with ADHD through pharmacological, behavioral, brain imaging and genetic studies. Currently, the animal models of ADHD exhibit significant limitations, stemming in large part from their lack of construct validity. To remedy this situation, we have pursued the creation of a mouse model derived from a functional nonsynonymous variant in the DAT gene (SLC6A3) of ADHD probands. We trace our path from the identification of these variants to in vitro biochemical and physiological studies to the production of the DAT Val559 mouse model. We discuss our initial findings with these animals and their promise in the context of existing rodent models of ADHD.
Topics: Amphetamine; Animals; Attention Deficit Disorder with Hyperactivity; Central Nervous System Stimulants; Disease Models, Animal; Dopamine Plasma Membrane Transport Proteins; Gene Knock-In Techniques; Humans; Methylphenidate; Mice; Mice, Neurologic Mutants; Mutation; Sensation
PubMed: 24332984
DOI: 10.1016/j.neuint.2013.11.009 -
Rhode Island Medical Journal (2013) Sep 2023The Dopamine Transporter Scan (DaT) is a radionuclear imaging technique which was approved by the FDA to differentiate essential tremor (ET) from Parkinson's disease... (Review)
Review
The Dopamine Transporter Scan (DaT) is a radionuclear imaging technique which was approved by the FDA to differentiate essential tremor (ET) from Parkinson's disease (PD). The scan is a crude indicator of the number of dopamine-secreting cells and is abnormal in presynaptic parkinsonian syndromes. In this article we review this and other possible clinical situations in which a DaT scan may be useful.
Topics: Humans; Dopamine Plasma Membrane Transport Proteins; Parkinsonian Disorders; Parkinson Disease; Radionuclide Imaging
PubMed: 37643339
DOI: No ID Found -
Molecular Psychiatry Apr 2016Attention-deficit/hyperactivity disorder (ADHD) is a common, highly heritable neuropsychiatric disorder with hyperactivity as one of the hallmarks. Aberrant dopamine...
Attention-deficit/hyperactivity disorder (ADHD) is a common, highly heritable neuropsychiatric disorder with hyperactivity as one of the hallmarks. Aberrant dopamine signaling is thought to be a major theme in ADHD, but how this relates to the vast majority of ADHD candidate genes is illusive. Here we report a Drosophila dopamine-related locomotor endophenotype that is shared by pan-neuronal knockdown of orthologs of the ADHD-associated genes Dopamine transporter (DAT1) and Latrophilin (LPHN3), and of a gene causing a monogenic disorder with frequent ADHD comorbidity: Neurofibromin (NF1). The locomotor signature was not found in control models and could be ameliorated by methylphenidate, validating its relevance to symptoms of the disorder. The Drosophila ADHD endophenotype can be further exploited in high throughput to characterize the growing number of candidate genes. It represents an equally useful outcome measure for testing chemical compounds to define novel treatment options.
Topics: Animals; Attention Deficit Disorder with Hyperactivity; Disease Models, Animal; Dopamine; Dopamine Plasma Membrane Transport Proteins; Drosophila; Male; Methylphenidate; Neurofibromin 1; Receptors, Peptide; Signal Transduction
PubMed: 25962619
DOI: 10.1038/mp.2015.55 -
The European Journal of Neuroscience Jan 2017The objective of this review is to evaluate the evidence that recreational methamphetamine exposure might damage dopamine neurones in human brain, as predicted by... (Review)
Review
The objective of this review is to evaluate the evidence that recreational methamphetamine exposure might damage dopamine neurones in human brain, as predicted by experimental animal findings. Brain dopamine marker data in methamphetamine users can now be compared with those in Parkinson's disease, for which the Oleh Hornykiewicz discovery in Vienna of a brain dopamine deficiency is established. Whereas all examined striatal (caudate and putamen) dopamine neuronal markers are decreased in Parkinson's disease, levels of only some (dopamine, dopamine transporter) but not others (dopamine metabolites, synthetic enzymes, vesicular monoamine transporter 2) are below normal in methamphetamine users. This suggests that loss of dopamine neurones might not be characteristic of methamphetamine exposure in at least some human drug users. In methamphetamine users, dopamine loss was more marked in caudate than in putamen, whereas in Parkinson's disease, the putamen is distinctly more affected. Substantia nigra loss of dopamine-containing cell bodies is characteristic of Parkinson's disease, but similar neuropathological studies have yet to be conducted in methamphetamine users. Similarly, it is uncertain whether brain gliosis, a common feature of brain damage, occurs after methamphetamine exposure in humans. Preliminary epidemiological findings suggest that methamphetamine use might increase risk of subsequent development of Parkinson's disease. We conclude that the available literature is insufficient to indicate that recreational methamphetamine exposure likely causes loss of dopamine neurones in humans but does suggest presence of a striatal dopamine deficiency that, in principle, could be corrected by dopamine substitution medication if safety and subject selection considerations can be resolved.
Topics: Animals; Central Nervous System Stimulants; Dopamine; Dopamine Agents; Dopamine Plasma Membrane Transport Proteins; Humans; Methamphetamine; Parkinson Disease
PubMed: 27519465
DOI: 10.1111/ejn.13363 -
Neuroscience Sep 2009The behavioral effects of cocaine are affected by gene knockout (KO) of the dopamine transporter (DAT), the serotonin transporter (SERT) and the norepinephrine...
The behavioral effects of cocaine are affected by gene knockout (KO) of the dopamine transporter (DAT), the serotonin transporter (SERT) and the norepinephrine transporter (NET). The relative involvement of each of these transporters varies depending on the particular behavioral response to cocaine considered, as well as on other factors such as genetic background of the subjects. Interestingly, the effects of these gene knockouts on cocaine-induced locomotion are quite different from those on reward assessed in the conditioned place preference paradigm. To further explore the role of these genes in the rewarding effects of cocaine, the ability of five daily injections of cocaine to induce conditioned locomotion was assessed in DAT, SERT and NET KO mice. Cocaine increased locomotor activity acutely during the initial conditioning session in SERT KO and NET KO, but not DAT KO, mice. Surprisingly, locomotor responses in the cocaine-paired subjects diminished over the five conditioning sessions in SERT KO mice, while locomotor responses increased in DAT KO mice, despite the fact that they did not demonstrate any initial locomotor responses to cocaine. Cocaine-induced locomotion was unchanged over the course of conditioning in NET KO mice. In the post-conditioning assessment, conditioned locomotion was not observed in DAT KO mice, and was reduced in SERT KO and NET KO mice. These data reaffirm the central role of dopamine and DAT in the behavioral effects of cocaine. Furthermore, they emphasize the polygenic basis of cocaine-mediated behavior and the non-unitary nature of drug reward mechanisms, particularly in the context of previous studies that have shown normal cocaine-conditioned place preference in DAT KO mice.
Topics: Animals; Cocaine; Conditioning, Psychological; Dopamine Plasma Membrane Transport Proteins; Mice; Mice, Knockout; Motor Activity; Norepinephrine Plasma Membrane Transport Proteins; Serotonin Plasma Membrane Transport Proteins
PubMed: 19482066
DOI: 10.1016/j.neuroscience.2009.05.058 -
Molecular Psychiatry Dec 2017The dopamine transporter (DAT) is an important regulator of brain dopamine (DA) homeostasis, controlling the intensity and duration of DA signaling. DAT is the target...
The dopamine transporter (DAT) is an important regulator of brain dopamine (DA) homeostasis, controlling the intensity and duration of DA signaling. DAT is the target for psychostimulants-like cocaine and amphetamine-and plays an important role in neuropsychiatric disorders, including attention-deficit hyperactivity disorder and drug addiction. Thus, a thorough understanding of the mechanisms that regulate DAT function is necessary for the development of clinical interventions to treat DA-related brain disorders. Previous studies have revealed a plethora of protein-protein interactions influencing DAT cellular localization and activity, suggesting that the fine-tuning of DA homeostasis involves multiple mechanisms. We recently reported that G-protein beta-gamma (Gβγ) subunits bind directly to DAT and decrease DA clearance. Here we show that Gβγ induces the release of DA through DAT. Specifically, a Gβγ-binding/activating peptide, mSIRK, increases DA efflux through DAT in heterologous cells and primary dopaminergic neurons in culture. Addition of the Gβγ inhibitor gallein or DAT inhibitors prevents this effect. Residues 582 to 596 in the DAT carboxy terminus were identified as the primary binding site of Gβγ. A TAT peptide containing the Gβγ-interacting domain of DAT blocked the ability of mSIRK to induce DA efflux, consistent with a direct interaction of Gβγ with the transporter. Finally, activation of a G-protein-coupled receptor, the muscarinic M5R, results in DAT-mediated DA efflux through a Gβγ-dependent mechanism. Collectively, our data show that Gβγ interacts with DAT to promote DA efflux. This novel mechanism may have important implications in the regulation of brain DA homeostasis.
Topics: Animals; Binding, Competitive; Brain; Cells, Cultured; Cricetulus; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dopaminergic Neurons; Female; GTP-Binding Protein beta Subunits; GTP-Binding Protein gamma Subunits; Humans; Membrane Potentials; Rats, Sprague-Dawley; Receptor, Muscarinic M5
PubMed: 28894302
DOI: 10.1038/mp.2017.176 -
Neuroscience Oct 2010We investigated the functional relationship between the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein syntaxin 1A (syn 1A) and the...
We investigated the functional relationship between the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein syntaxin 1A (syn 1A) and the dopamine transporter (DAT) by treating rat striatal tissue with Botulinum Neurotoxin C (BoNT/C) and co-transfecting syn 1A with DAT in non-neuronal cells, followed by analysis of DAT activity, phosphorylation, and regulation. Treatment of striatal slices with BoNT/C resulted in elevated dopamine (DA) transport Vmax and reduced DAT phosphorylation, while heterologous co-expression of syn 1A led to reduction in DAT surface expression and transport Vmax. Syn 1A was present in DAT immunoprecipitation complexes, supporting a direct or indirect interaction between the proteins. Phorbol ester regulation of DA transport activity was retained in BoNT/C-treated synaptosomes and syn 1A transfected cells, demonstrating that protein kinase C (PKC) and syn 1A effects occur through independent processes. These findings reveal a novel mechanism for regulation of DAT activity and phosphorylation, and suggest the potential for syn 1A to impact DA neurotransmission through effects on reuptake.
Topics: Animals; Botulinum Toxins; Corpus Striatum; Dopamine Plasma Membrane Transport Proteins; LLC-PK1 Cells; Male; Phorbol Esters; Phosphorylation; Protein Transport; Rats; Rats, Sprague-Dawley; Receptor Cross-Talk; Swine; Synaptosomes; Syntaxin 1; Transfection
PubMed: 20643191
DOI: 10.1016/j.neuroscience.2010.07.025