HEK-293 Cells Expressing the Human Dopamine Transporter are Susceptible to Low Concentrations of 1-Methyl-4-Phenylpyridinium (MPP⁺) via Impairment of Energy Metabolism

Abstract

The dopamine transporter may play a crucial role in the selective vulnerability of dopaminergic neurons in respect to l-Methyl-4-phenylpyridinium ion (MPP⁺) toxicity. We stably expressed the human dopamine transporter in human embryonic kidney cells (HEK-293 cells) to investigate MPP⁺-induced cytotoxicity as well as changes of cellular energy metabolism by measuring the intracellular adenine and pyridine nucleotide content using HPLC. The permanent ectopic expression of the human dopamine transporter in HEK-293 cells conferred time and dose-dependent cytotoxicity low concentrations of MPP⁺ with an IC₅₀ value of 740 nM after 48 h. MPP⁺ initially induced a fast increase of cellular NADH content within the first 6 hours, followed by a slow reduction of intracellular ATP content (IC₅₀ value of 690 nM after 48 h) as well as reduction of intracellular ATP/ADP ratio. These changes of cellular energy metabolism consistently preceded the reduction of cell viability. The toxic effects of MPP⁺ were blocked by dopamine transporter inhibition. Forcing glycolysis by addition of D-glucose to the culture medium had significant protective effects against MPP⁺ toxicity, whereas antioxidants did not show any effects. This study provides evidence that cellular damage induced by low concentrations of MPP⁺ (i) is mediated via transmembrane uptake of MPP⁺ by the dopamine transporter and (ii) is primarily due to cellular energy depletion, probably caused by inhibition of mitochondrial complex I activity.