RESUMO
The dopamine transporter (DAT) plays a major role in modulating dopamine (DA) neurotransmission by controlling the levels of this neurotransmitter in the extracellular space. We have isolated 8.3 kb of the 5'-flanking regulatory region of the human DAT (hDAT) gene and identified numerous potential elements involved in transcriptional control of the DAT. A series of hDAT-luciferase reporter constructs encompassing increasing amounts of 5'-flanking sequence was utilized in transient transfection assays assessing basal activity and response to selected stimuli. Our results suggest that the proximal hDAT 5'-flanking region displays a strong, nonselective promoter activity that is silenced through regulatory elements present in the distal portion of the 5'-flanking sequence. Although potential cyclic AMP responsive elements (CRE) were identified on the sequence, hDAT constructs were unresponsive to cyclic AMP induction. The transcription factor nurr1 increases the transcriptional activity of several larger hDAT constructs, consistent with the presence of several putative NGFI-B response elements (NBRE). The cloning and functional analysis of an extensive portion of the 5'-flanking regulatory region of the hDAT gene provides further insights into the factors involved in the regulation of this gene.
Assuntos
Proteínas de Transporte/genética , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/metabolismo , DNA/genética , DNA/isolamento & purificação , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Humanos , Luciferases/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares , Células PC12 , Regiões Promotoras Genéticas/genética , Ratos , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta/genética , Deleção de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/farmacologia , Células Tumorais CultivadasRESUMO
Neurokinin-3 receptor expression within rat midbrain dopamine neurons was demonstrated using a combination of in situ hybridization and receptor autoradiographic techniques. Continuous intranigral infusion of the neurokinin-3 receptor agonist senktide selectively increased striatal dopamine metabolism over a period of several days, followed by apparent development of tolerance. In contrast, in animals with moderate unilateral 6-hydroxydopamine-induced lesions of nigrostriatal dopamine cells, intranigral senktide infusion increased dopamine turnover in the surviving dopamine neurons and reduced functional dopamine asymmetry (reflected by spontaneous rotations) over the 2-week period tested. Thus nigral neurokinin receptors can modulate normal dopamine cell activity and may provide a therapeutic target in the treatment of Parkinson's disease.
