RESUMO
The present study addressed the role of N-linked glycosylation of the human dopamine transporter (DAT) in its function with the help of mutants, in which canonical N-glycosylation sites have been removed (N181Q, N181Q,N188Q, and N181Q,N188Q,N205Q), expressed in human embryonic kidney-293 cells. Removal of canonical sites produced lower molecular weight species as did enzymatic deglycosylation or blockade of glycosylation, and all three canonical sites were found to carry sugars. Prevention of N-glycosylation reduced both surface and intracellular DAT. Although partially or non-glycosylated DAT was somewhat less represented at the surface, no evidence was found for preferential exclusion of such material from the plasma membrane, indicating that glycosylation is not essential for DAT expression. Non-glycosylated DAT was less stable at the surface as revealed by apparently enhanced endocytosis, consonant with weaker DAT immunofluorescence at the cell surface and stronger presence in cytosol in confocal analysis of the double and triple mutant. Non-glycosylated DAT did not transport dopamine as efficiently as wild-type DAT as judged from the sharp reduction in uptake V(max), and prevention of N-glycosylation enhanced the potency of cocaine-like drugs in inhibiting dopamine uptake into intact cells without changing their affinity for DAT when measured in membrane preparations prepared from these cells. Thus, non-glycosylated DAT at the cell surface displays appreciably reduced catalytic activity and altered inhibitor sensitivity compared with wild type.
Assuntos
Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Substituição de Aminoácidos , Biotinilação , Linhagem Celular , DNA Complementar/genética , Antagonistas de Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina , Glicosilação , Humanos , Moduladores de Transporte de Membrana , Proteínas de Membrana Transportadoras/antagonistas & inibidores , Proteínas de Membrana Transportadoras/química , Microscopia Confocal , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tunicamicina/farmacologiaRESUMO
Dopamine transporter (DAT) trafficking was assessed by functional measurements of dopamine uptake and by biotinylation of surface proteins followed by gel electrophoresis and Western blotting. In human embryonic kidney (HEK)-293 cells expressing human DAT (HEK-hDAT), pretreatment with dopamine (0.1-100 microM) followed by washout caused reductions in subsequent dopamine uptake (reflected in Vmax) with effective dopamine concentrations in the 10 to 100 microM range and pretreatment times of 10 to 60 min. Reductions assessed after 60-min pretreatment with 100 microM dopamine corresponded with decreases measured in surface DAT by the noncleavable biotin method, which were caused, at least in part, by enhanced endocytosis as monitored with cleavable biotin. Pretreatment of rat striatal synaptosomes with dopamine (10 and 100 microM) also caused reductions in DAT uptake activity (Vmax), and again the underlying mechanism seemed to be a diminished presence of DAT at the surface of synaptosomes as measured by the noncleavable biotin method. The copresence of cocaine during pretreatment with dopamine prevented the down-regulation of surface DAT. The present results show that DAT surface residency can be regulated by substrate acting on it, not only in cells heterologously expressing DAT but also in situ in rat brain tissue.
Assuntos
Corpo Estriado/citologia , Dopamina/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras/biossíntese , Proteínas do Tecido Nervoso , Sinaptossomos/metabolismo , Animais , Transporte Biológico , Biotinilação , Linhagem Celular , Proteínas da Membrana Plasmática de Transporte de Dopamina , Humanos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
An industrial chemical used in synthetic rubber production, 1,3-butadiene, is toxic to reproduction in rats and mice. Bioactivation of butadiene to reactive intermediates, i.e. diepoxybutane and other metabolites, is responsible for this toxicity. The present study examines the biochemical and developmental mechanisms of diepoxybutane at the feto-maternal placental axis during gestation. Female Sprague-Dawley rats were administered four daily intraperitoneal doses of diepoxybutane in groups (0.25, 0.30, 0.35, or 0.40 mmol in sesame oil per kg body weight, n = 6/group) during postimplantation (gestation days 5-8) and euthanized on gestation day 9 or 12 for retrieval of uterine and ovarian tissues, and serum for assays. The results demonstrate that this timely diepoxybutane treatment significantly decreased placental levels of pituitary adenylate cyclase-activating polypeptide mRNA expression that was measured by reverse transcription-polymerase chain reaction and of matrix metalloproteinase-9 activity that was determined by gelatin zymography, and serum progesterone levels on gestation days 9 and 12. From a developmental standpoint, fetal growth and viability were reduced in correlation with treatment-related effects of diepoxybutane on implantation losses and fetal resorptions on gestation day 9. Additionally, fetal mortality was maximally increased due to significantly pronounced, dose-independent effects on these parameters on gestation day 12. This trend towards more severe embryolethal treatment effects from gestation day 9 to 12 suggests that fetal metabolism in the gravid uteri of rats may be more sensitive to diepoxybutane exposure as pregnancy progresses. The inhibitory actions of diepoxybutane on placental pituitary adenylate cyclase-activating polypeptide expression and matrix metalloproteinase activity may contribute towards altering placental molecular support for fetal development and viability. Moreover, the reproductive toxicity of diepoxybutane in rats appears to be linked to progesterone action.
Assuntos
Desenvolvimento Embrionário , Compostos de Epóxi/farmacologia , Ovário/efeitos dos fármacos , Útero/efeitos dos fármacos , Animais , Sequência de Bases , Primers do DNA , Relação Dose-Resposta a Droga , Feminino , Metaloproteinase 9 da Matriz/metabolismo , Neuropeptídeos/genética , Ovário/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Útero/enzimologia , Útero/metabolismoRESUMO
The results of the study demonstrate that the weak estrogenic action of bisphenol A (a daily subcutaneous dose of 200 mg/kg on 4 consecutive days, administered before or after decidual induction that occurs on day 4 of pseudopregnancy) on deciduoma growth in pseudopregnant Sprague-Dawley rats, was functionally associated with the hormonal status of the uterus. Whereas bisphenol A displayed uterotrophic action during the pre-decidual, estrogen related period, it inhibited decidual growth and progesterone secretion during the post-decidual, progesterone-dominated period. The estrogenic action of bisphenol A on uterine decidual growth was not correlated with the reduced levels of estrogen receptor binding sites and mRNA expression, nor the unchanged serum estradiol concentrations. BPA action appeared to be antagonized by progesterone.