HEK-293 cells expressing the human dopamine transporter are susceptible to low concentrations of 1-methyl-4-phenylpyridine (MPP+) via impairment of energy metabolism.
Selective dopaminergic neurotoxicity induced by 1-methyl-4-phenylpyridine (MPP+) is believed to be due to the transmembrane uptake by the dopamine transporter and subsequent inhibition of mitochondrial complex I and/or production of free radicals. However, little is known about the molecular sequence of intracellular events leading to cell death induced by low concentrations of MPP+. Here we stably express the human dopamine transporter (hDAT) in human embryonic kidney HEK-293 cells to correlate cytotoxicity and indices of cellular energy metabolism after exposure to low concentrations of MPP+. The permanent ektopic expression of hDAT in HEK-293 cells confers time and dose-dependent cytotoxicity at nanomolar concentrations of MPP+ with an IC50 value of 740 nM after 48 h. MPP+ initially induces a fast increase of cellular NADH content within the first 6 h, followed by a slow reduction of intracellular ATP (IC50 value of 690 nM after 48 h) as well as reduction of intracellular ATP/ADP ratio. These changes of cellular energy metabolism precede reduction of cell viability. The toxic effects of MPP+ are blocked by the hDAT inhibitor GBR12909 with EC50 values of 110 and 60 nM for cytotoxicity and ATP depletion, respectively. Antioxidants such as D-alpha-tocopherol and ascorbic acid do not have significant protective effects against MPP+ toxicity. This study shows that HEK-293 cells expressing the hDAT gene are highly sensitive to MPP+ due to (i) transmembrane uptake of MPP+ by the dopamine transporter, (ii) cellular energy depletion, probably caused by inhibition of mitochondrial complex I activity and (iii) that the toxicity is independent from the presence of antioxidants. This cell system may serve as a screening system for endogenous and exogenous compounds with similar effects compared to MPP+ as well as protective agents.
Storch
A
A
Department of Neurology, University of Ulm Medical School, Germany. alexander.storch@medizin.uni-ulm.de
Ludolph
A C
AC
Schwarz
J
J
D015655:1-Methyl-4-phenylpyridinium / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D050483:Dopamine Plasma Membrane Transport Proteins; D004734:Energy Metabolism; D006801:Humans; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D014162:Transfection
D000975:Antioxidants; D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D015655:1-Methyl-4-phenylpyridinium