Chagkutip Jaturaporn, Vaughan Roxanne A, Govitrapong Piyarat, Ebadi Manuchair
Department of Pharmacology, University of North Dakota School of Medicine and Health Sciences, Grand Forks 58202, USA.
Biochem Biophys Res Commun. 2003 Nov 7;311(1):49-54. doi: 10.1016/j.bbrc.2003.09.155.
The mechanisms whereby 1-methyl-4-phenylpyridinium (MPP(+)) mediates cell death and Parkinsonism are still unclear. We have shown that dopamine transporter (DAT) is required for MPP(+)-mediated cytotoxicity in HEK-293 cells stably transfected with human DAT. Furthermore, MPP(+) produced a concentration- and time-dependent reduction in the uptake of [3H]dopamine. We observed a significant decrease in [3H]WIN 35428 binding in the intact cells with MPP(+). The saturation analysis of the [3H]WIN 35428 binding obtained from total membrane fractions revealed a decrease in the transporter density (B(max)) with an increase in the dissociation equilibrium constant (K(d)) after MPP(+) treatment. Furthermore, biotinylation assays confirmed that MPP(+) reduced both plasma membrane and intracellular DAT immunoreactivity. Taken together, these findings suggest that the reduction in cell surface DAT protein expression in response to MPP(+) may be a contributory factor in the down-regulation of DAT function while enhanced lysosomal degradation of DAT may signal events leading to cellular toxicity.
1-甲基-4-苯基吡啶离子（MPP(+)）介导细胞死亡和帕金森症的机制仍不清楚。我们已经表明，在稳定转染人多巴胺转运体（DAT）的HEK-293细胞中，MPP(+)介导的细胞毒性作用需要多巴胺转运体（DAT）。此外，MPP(+)使[3H]多巴胺摄取呈浓度和时间依赖性降低。我们观察到，MPP(+)处理的完整细胞中[3H]WIN 35428结合显著减少。从总膜组分获得的[3H]WIN 35428结合的饱和分析显示，MPP(+)处理后转运体密度（B(max)）降低，解离平衡常数（K(d)）增加。此外，生物素化分析证实，MPP(+)降低了质膜和细胞内DAT免疫反应性。综上所述，这些发现表明，响应MPP(+)时细胞表面DAT蛋白表达的降低可能是DAT功能下调的一个促成因素，而DAT溶酶体降解增强可能预示导致细胞毒性的事件。
