128 resultados para Dopamine Transporter
em Université de Lausanne, Switzerland
Resumo:
The dopamine (DA) transporter (DAT) is a plasma membrane glycoprotein expressed in dopaminergic (DA-) cells that takes back DA into presynaptic neurons after its release. DAT dysfunction has been involved in different neuro-psychiatric disorders including Parkinson's disease (PD). On the other hand, numerous studies support that the glial cell line-derived neurotrophic factor (GDNF) has a protective effect on DA-cells. However, studies in rodents show that prolonged GDNF over-expression may cause a tyrosine hydroxylase (TH, the limiting enzyme in DA synthesis) decline. The evidence of TH down-regulation suggests that another player in DA handling, DAT, may also be regulated by prolonged GDNF over-expression, and the possibility that this effect is induced at GDNF expression levels lower than those inducing TH down-regulation. This issue was investigated here using intrastriatal injections of a tetracycline-inducible adeno-associated viral vector expressing human GDNF cDNA (AAV-tetON-GDNF) in rats, and doxycycline (DOX; 0.01, 0.03, 0.5 and 3mg/ml) in the drinking water during 5weeks. We found that 3mg/ml DOX promotes an increase in striatal GDNF expression of 12× basal GDNF levels and both DA uptake decrease and TH down-regulation in its native and Ser40 phosphorylated forms. However, 0.5mg/ml DOX promotes a GDNF expression increase of 3× basal GDNF levels with DA uptake decrease but not TH down-regulation. The use of western-blot under non-reducing conditions, co-immunoprecipitation and in situ proximity ligation assay revealed that the DA uptake decrease is associated with the formation of DAT dimers and an increase in DAT-α-synuclein interactions, without changes in total DAT levels or its compartmental distribution. In conclusion, at appropriate GDNF transduction levels, DA uptake is regulated through DAT protein-protein interactions without interfering with DA synthesis.
Resumo:
Tonically active cholinergic interneurons (TANs) from the nucleus accumbens (NAc) are centrally involved in reward behavior. TANs express a vesicular glutamate transporter referred to as VGLUT3 and thus use both acetylcholine and glutamate as neurotransmitters. The respective roles of each transmitter in the regulation of reward and addiction are still unknown. In this study, we showed that disruption of the gene that encodes VGLUT3 (Slc17a8) markedly increased cocaine self-administration in mice. Concomitantly, the amount of dopamine (DA) release was strongly augmented in the NAc of VGLUT3(-/-) mice because of a lack of signaling by metabotropic glutamate receptors. Furthermore, dendritic spines and glutamatergic synaptic transmission on medium spiny neurons were increased in the NAc of VGLUT3(-/-) mice. Increased DA and glutamate signaling in the NAc are hallmarks of addiction. Our study shows that TANs use glutamate to reduce DA release and decrease reinforcing properties of cocaine in mice. Interestingly, we also observed an increased frequency of rare variations in SLC17A8 in a cohort of severe drug abusers compared with controls. Our findings identify VGLUT3 as an unexpected regulator of drug abuse.
Resumo:
Background: T reatment o f chronic hepatitis C i s evolving, a nd direct acting antivirals ( DAAs) are now a dded to p egylated interferon-α ( Peg- INF-α) and ribavirin (RBV) for the treatment o f hepatitis C v irus ( HCV) genotype 1 infection. DAAs c ause d ifferent side effects and can even worsen RBV induced hemolytic anemia. T herefore, identifying host genetic d eterminants of R BV bioavailability and therapeutic e fficacy will remain crucial for individualized treatment. Recent d ata showed associations between R BV induced h emolytic anemia and genetic polymorphisms o f concentrative nucleoside transporters s uch as C NT3 (SLC28A3) and i nosine t riphosphatase (ITPA). T o analyze t he association of genetic variants of SLC28 transporters and ITPA with RBV induced hemolytic anemia and treatment o utcome. Methods: I n our study, 173 patients f rom t he S wiss Hepatitis C C ohort Study and 2 2 patients from Swiss Association for the Study of the Liver study 24 (61% HCV g enotype 1, 3 9% genotypes 2 o r 3) were analyzed for SLC28A2 single nucleotide p olymorphism (SNP) rs11854484, SLC28A3 rs56350726 and SLC28A3 rs10868138 as well as ITPA SNPs rs1127354 and rs7270101. RBV serum levels during treatment were measured in 49 patients. Results: SLC28A2 r s11854484 genotype TT was associated with significantly higher dosage- and body weight-adjusted RBV levels as compared to genotypes TC and CC (p=0.04 and p=0.02 at weeks 4 and 8, respectively). ITPA SNPs rs1127354 and rs7270101 were associated with h emolytic a nemia both in genotype as w ell as i n allelic a nalyses. SLC28A3 rs56350726 genotype TT (vs. AT/AA, RR=2.1; 95% CI 1.1-4.1) as well as the T allele (vs. A; RR=1.8, 95% CI 1.1-3.2) were associated with increased SVR rates. The combined analysis of overall ITPA activity and SLC28 v ariants together revealed n o significant a dditive effects on either treatment-related anemia or SVR. Conclusions: T he newly identified association between RBV serum levels a nd SLC28A2 rs11854484 genotype as well as the replicated association of ITPA and SLC28A3 g enetic p olymorphisms w ith RBV induced hemolytic anemia and treatment r esponse underpin the need for further studies on host genetic d eterminants of R BV bioavailability and therapeutic e fficacy f or individualized treatment of chronic hepatitis C.
Resumo:
It was found recently that locomotor and rewarding effects of psychostimulants and opiates were dramatically decreased or suppressed in mice lacking alpha1b-adrenergic receptors [alpha1b-adrenergic receptor knock-outs (alpha1bAR-KOs)] (Drouin et al., 2002). Here we show that blunted locomotor responses induced by 3 and 6 mg/kg d-amphetamine in alpha1bAR-KO mice [-84 and -74%, respectively, when compared with wild-type (WT) mice] are correlated with an absence of d-amphetamine-induced increase in extracellular dopamine (DA) levels in the nucleus accumbens of alpha1bAR-KO mice. Moreover, basal extracellular DA levels in the nucleus accumbens are lower in alpha1bAR-KO than in WT littermates (-28%; p < 0.001). In rats however, prazosin, an alpha1-adrenergic antagonist, decreases d-amphetamine-induced locomotor hyperactivity without affecting extracellular DA levels in the nucleus accumbens, a finding related to the presence of an important nonfunctional release of DA (Darracq et al., 1998). We show here that local d-amphetamine releases nonfunctional DA with the same affinity but a more than threefold lower amplitude in C57BL6/J mice than in Sprague Dawley rats. Altogether, this suggests that a trans-synaptic mechanism amplifies functional DA into nonfunctional DA release. Our data confirm the presence of a powerful coupling between noradrenergic and dopaminergic neurons through the stimulation of alpha1b-adrenergic receptors and indicate that nonfunctional DA release is critical in the interpretation of changes in extracellular DA levels. These results suggest that alpha1b-adrenergic receptors may be important therapeutic pharmacological targets not only in addiction but also in psychosis because most neuroleptics possess anti-alpha1-adrenergic properties.
Resumo:
Brain-derived neurotrophic factor (BDNF) promotes synaptic plasticity via an enhancement in expression of specific synaptic proteins. Recent results suggest that the neuronal monocarboxylate transporter MCT2 is a postsynaptic protein critically involved in synaptic plasticity and long-term memory. To investigate in vivo whether BDNF can modulate the expression of MCT2 as well as other proteins involved in synaptic plasticity, acute injection of BDNF was performed in mouse dorsal hippocampal CA1 area. Using immunohistochemistry, it was found that MCT2 expression was enhanced in part of the CA1 area and in the dentate gyrus 6 h after a single intrahippocampal injection of BDNF. Similarly, expression of the immediate early genes Arc and Zif268 was enhanced in the same hippocampal areas, in accordance with their role in synaptic plasticity. Immunoblot analysis confirmed the significant enhancement in MCT2 protein expression. In contrast, no changes were observed for the glial monocarboxylate transporters MCT1 and MCT4. When other synaptic proteins were investigated, it was found that postsynaptic density 95 (PSD95) and glutamate receptor 2 (GluR2) protein levels were significantly enhanced while no effect could be detected for synaptophysin, synaptosomal-associated protein 25 (SNAP25), αCaMKII and GluR1. These results demonstrate that MCT2 expression can be upregulated together with other key postsynaptic proteins in vivo under conditions related to synaptic plasticity, further suggesting the importance of energetics for memory formation.
Resumo:
Land plants have developed a cuticle preventing uncontrolled water loss. Here we report that an ATP-binding cassette (ABC) subfamily G (ABCG) full transporter is required for leaf water conservation in both wild barley and rice. A spontaneous mutation, eibi1.b, in wild barley has a low capacity to retain leaf water, a phenotype associated with reduced cutin deposition and a thin cuticle. Map-based cloning revealed that Eibi1 encodes an HvABCG31 full transporter. The gene was highly expressed in the elongation zone of a growing leaf (the site of cutin synthesis), and its gene product also was localized in developing, but not in mature tissue. A de novo wild barley mutant named "eibi1.c," along with two transposon insertion lines of rice mutated in the ortholog of HvABCG31 also were unable to restrict water loss from detached leaves. HvABCG31 is hypothesized to function as a transporter involved in cutin formation. Homologs of HvABCG31 were found in green algae, moss, and lycopods, indicating that this full transporter is highly conserved in the evolution of land plants.
Resumo:
Based on homology with GLUT1-5, we have isolated a cDNA for a novel glucose transporter, GLUTX1. This cDNA encodes a protein of 478 amino acids that shows between 29 and 32% identity with rat GLUT1-5 and 32-36% identity with plant and bacterial hexose transporters. Unlike GLUT1-5, GLUTX1 has a short extracellular loop between transmembrane domain (TM) 1 and TM2 and a long extracellular loop between TM9 and TM10 that contains the only N-glycosylation site. When expressed in Xenopus oocytes, GLUTX1 showed strong transport activity only after suppression of a dileucine internalization motif present in the amino-terminal region. Transport activity was inhibited by cytochalasin B and partly competed by D-fructose and D-galactose. The Michaelis-Menten constant for glucose was approximately 2 mM. When translated in reticulocytes lysates, GLUTX1 migrates as a 35-kDa protein that becomes glycosylated in the presence of microsomal membranes. Western blot analysis of GLUTX1 transiently expressed in HEK293T cells revealed a diffuse band with a molecular mass of 37-50 kDa that could be converted to a approximately 35-kDa polypeptide following enzymatic deglycosylation. Immunofluorescence microscopy detection of GLUTX1 transfected into HEK293T cells showed an intracellular staining. Mutation of the dileucine internalization motif induced expression of GLUTX1 at the cell surface. GLUTX1 mRNA was detected in testis, hypothalamus, cerebellum, brainstem, hippocampus, and adrenal gland. We hypothesize that, in a similar fashion to GLUT4, in vivo cell surface expression of GLUTX1 may be inducible by a hormonal or other stimulus.
Resumo:
A recent study with 69 Japanese liver transplants treated with tacrolimus found that the MDR13435 C >T polymorphism, but not the MDR12677 G >T polymorphism, was associated with differences in the intestinal expression level of CYP3A4 mRNA. In the present study, over 6 h, we measured the kinetics of a 75 microg oral dose of midazolam, a CYP3A substrate, in 21 healthy subjects genotyped for the MDR13435 C >T and 2677 G >T polymorphism. No statistically significant differences were found in the calculated pharmacokinetic parameters between the three 3435 C >T genotypes (TT, CT and CC group, respectively: Cmax (mean +/- SD: 0.30 +/- 0.08 ng/ml, 0.31 +/- 0.09 ng/ml and 0.31 +/- 0.11 ng/ml; Apparent clearance: 122 +/- 29 l/h, 156 +/- 92 l/h and 111 +/- 35 l/h; t1/2: 1.9 +/- 1.1 h, 1.6 +/- 0.90 h and 1.7 +/- 0.7 h). In addition, the 30-min 1'OH midazolam to midazolam ratio, a marker of CYP3A activity, determined in 74 HIV-positive patients before the introduction of antiretroviral treatment, was not significantly different between the three 3435 C >T genotypes (mean ratio +/- SD: 3.65 +/- 2.24, 4.22 +/- 3.49 and 4.24 +/- 2.03, in the TT, CT and CC groups, respectively). Similarly, no association was found between the MDR12677 G >T polymorphism and CYP3A activity in the healthy subjects or in the HIV-positive patients. The existence of a strong association between the activity of CYP3A and MDR13435 C >T and 2677 G >T polymorphisms appears unlikely, at least in Caucasian populations and/or in the absence of specific environmental factors.
Resumo:
Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of dopaminergic (DA) neurons of the substantia nigra pars compacta with unknown aetiology. 6-Hydroxydopamine (6-OHDA) treatment of neuronal cells is an established in vivo model for mimicking the effect of oxidative stress found in PD brains. We examined the effects of 6-OHDA treatment on human neuroblastoma cells (SH-SY5Y) and primary mesencephalic cultures. Using a reverse arbitrarily primed polymerase chain reaction (RAP-PCR) approach we generated reproducible genetic fingerprints of differential expression levels in cell cultures treated with 6-OHDA. Of the resulting sequences, 23 showed considerable homology to known human coding sequences. The results of the RAP-PCR were validated by reverse transcription PCR, real-time PCR and, for selected genes, by Western blot analysis and immunofluorescence. In four cases, [tomoregulin-1 (TMEFF-1), collapsin response mediator protein 1 (CRMP-1), neurexin-1, and phosphoribosylaminoimidazole synthetase (GART)], a down-regulation of mRNA and protein levels was detected. Further studies will be necessary on the physiological role of the identified proteins and their impact on pathways leading to neurodegeneration in PD.
Resumo:
BACKGROUND:HIV-1-infected patients vary considerably by their response to antiretroviral treatment, drug concentrations in plasma, toxic events, and rate of immune recovery. This variability could have a genetic basis. We did a pharmacogenetics study to analyse the association between response to antiretroviral treatment and allelic variants of several genes. METHODS:In 123 patients, we did PCR analyses of the gene for the multidrug-resistance transporter (MDR1), which codes for P-glycoprotein, of genes coding for isoenzymes of cytochrome P450, CYP3A4, CYP3A5, CYP2D6, and CYP2C19, and of the gene for the chemokine receptor CCR5. We measured concentrations in plasma of the antiretroviral agents efavirenz and nelfinavir by high-performance liquid-chromatography, and measured levels of P-glycoprotein expression, CD4-cell count, and HIV-1 viraemia. FINDINGS: Median drug concentrations in patients with the MDR1 3435 TT, CT, and CC genotypes were at the 30th, 50th, and 75th percentiles, respectively (p=0.0001). In patients with CYP2D6 extensive-metaboliser or poor-metaboliser alleles, median drug concentrations were at percentiles 45 and 62.5, respectively (p=0.04). Patients with the MDR1 TT genotype 6 months after starting treatment had a greater rise in CD4-cell count (257 cells/microL) than patients with the CT (165 cells/microL) and CC (121 cells/microL) genotype (p=0.0048), and the best recovery of naïve CD4-cells. INTERPRETATION:The polymorphism MDR1 3435 C/T predicts immune recovery after initiation of antiretroviral treatment. This finding suggests that P-glycoprotein has an important role in admittance of antiretroviral drugs to restricted compartments in vivo.
Resumo:
Pachydermoperiostosis, or primary hypertrophic osteoarthropathy (PHO), is an inherited multisystem disorder, whose features closely mimic the reactive osteoarthropathy that commonly accompanies neoplastic and inflammatory pathologies. We previously described deficiency of the prostaglandin-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (HPGD) as a cause of this condition, implicating elevated circulating prostaglandin E(2) (PGE(2) ) as causative of PHO, and perhaps also as the principal mediator of secondary HO. However, PHO is genetically heterogeneous. Here, we use whole-exome sequencing to identify recessive mutations of the prostaglandin transporter SLCO2A1, in individuals lacking HPGD mutations. We performed exome sequencing of four probands with severe PHO, followed by conventional mutation analysis of SLCO2A1 in nine others. Biallelic SLCO2A1 mutations were identified in 12 of the 13 families. Affected individuals had elevated urinary PGE(2) , but unlike HPGD-deficient patients, also excreted considerable quantities of the PGE(2) metabolite, PGE-M. Clinical differences between the two groups were also identified, notably that SLCO2A1-deficient individuals have a high frequency of severe anemia due to myelofibrosis. These findings reinforce the key role of systemic or local prostaglandin excess as the stimulus to HO. They also suggest that the induction or maintenance of hematopoietic stem cells by prostaglandin may depend upon transporter activity. Hum Mutat 33:1175-1181, 2012. © 2012 Wiley Periodicals, Inc.
Resumo:
Addictive properties of drugs of misuse are generally considered to be mediated by an increased release of dopamine (DA) in the ventral striatum. However, recent experiments indicated an implication of alpha1b-adrenergic receptors in behavioural responses to psychostimulants and opiates. We show now that DA release induced in the ventral striatum by morphine (20 mg/kg) is completely blocked by prazosin (1 mg/kg), an alpha1-adrenergic antagonist. However, morphine-induced increases in DA release in the ventral striatum were found to be similar in mice deleted for the alpha1b-adrenergic receptor (alpha1b-AR KO) and in wild-type (WT) mice, suggesting the presence of a compensatory mechanism. This acute morphine-evoked DA release was completely blocked in alpha1b-AR KO mice by SR46349B (1 mg/kg), a 5-HT2A antagonist. SR46349B also completely blocked, in alpha1b-AR KO mice, the locomotor response and the development of behavioural sensitization to morphine (20 mg/kg) and D-amphetamine (2 mg/kg). Accordingly, the concomitant blockade of 5-HT2A and alpha1b-adrenergic receptors in WT mice entirely blocked acute locomotor responses but also the development of behavioural sensitization to morphine, D-amphetamine or cocaine (10 mg/kg). We observed, nevertheless, that inhibitory effects of each antagonist on locomotor responses to morphine or D-amphetamine were more than additive (160%) in naïve WT mice but not in those sensitized to either drug. Because of these latter data and the possible compensation by 5-HT2A receptors for the genetic deletion of alpha1b-adrenergic receptors, we postulate the existence of a functional link between these receptors, which vanishes during the development of behavioural sensitization.
Resumo:
AIMS/HYPOTHESIS: Excess glucose transport to embryos during diabetic pregnancy causes congenital malformations. The early postimplantation embryo expresses the gene encoding the high-Km GLUT2 (also known as SLC2A2) glucose transporter. The hypothesis tested here is that high-Km glucose transport by GLUT2 causes malformations resulting from maternal hyperglycaemia during diabetic pregnancy. MATERIALS AND METHODS: Glut2 mRNA was assayed by RT-PCR. The Km of embryo glucose transport was determined by measuring 0.5-20 mmol/l 2-deoxy[3H]glucose transport. To test whether the GLUT2 transporter is required for neural tube defects resulting from maternal hyperglycaemia, Glut2+/- mice were crossed and transient hyperglycaemia was induced by glucose injection on day 7.5 of pregnancy. Embryos were recovered on day 10.5, and the incidence of neural tube defects in wild-type, Glut2+/- and Glut2-/- embryos was scored. RESULTS: Early postimplantation embryos expressed Glut2, and expression was unaffected by maternal diabetes. Moreover, glucose transport by these embryos showed Michaelis-Menten kinetics of 16.19 mmol/l, consistent with transport mediated by GLUT2. In pregnancies made hyperglycaemic on day 7.5, neural tube defects were significantly increased in wild-type embryos, but Glut2+/- embryos were partially protected from neural tube defects, and Glut2-/- embryos were completely protected from these defects. The frequency of occurrence of wild-type, Glut2+/- and Glut2-/- embryos suggests that the presence of Glut2 alleles confers a survival advantage in embryos before day 10.5. CONCLUSIONS/INTERPRETATIONS: High-Km glucose transport by the GLUT2 glucose transporter during organogenesis is responsible for the embryopathic effects of maternal diabetes.