Dopamine transporter-mediated cytotoxicity of β-carbolinium derivatives related to Parkinson's disease: Relationship to transporter-dependent uptake

Abstract

Endogenous or exogenous β-carboline (βC) derivatives structurally related to the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP+) may contribute to dopaminergic neurodegeneration in Parkinson's disease (PD). We addressed the importance of the dopamine transporter (DAT) for selective dopaminergic toxicity by testing the differential cytotoxicity and cellular uptake of 12 βCs in human embryonic kidney HEK-293 cells ectopically expressing the DAT gene. Cell death was measured using [4,5-Dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion assays, and uptake by a fluorescence-based uptake assay. All βCs and MPP+ showed general cytotoxicity in parental HEK-293 cells after 72 h with half-maximal toxic concentrations (TC50 values) in the upper micromolar range. Besides MPP +, only 2[N]-methylated compounds showed enhanced cytotoxicity in DAT expressing HEK-293 cells with 1.3- to 4.5-fold reduction of TC50 values compared with parental cell line. The rank order of selectivity was: MPP+ 2[N],9[N]-dimethyl-harminium > 2[N]-methyl-harminium > 2[N],9[N]-dimethyl-harmanium = 2[N]-methyl-norharmanium > 2[N]-methyl-harmanium > 2[N],9[N]-dimethyl-norharminium. Consistently, only 2[N]-methylated βCs were transported into the cell through the DAT with up to five times greater Km and 12-220 times smaller Vmax values compared with dopamine and MPP+. There was a weak relation of DAT-mediated selectivity with the affinity of βCs at the DAT (Km), but not with Vmax. Our data suggest that DAT-mediated cellular uptake of 2[N]-methylated βCs represents a potential mechanism for selective toxicity towards dopaminergic neurons and may be relevant for the pathogenesis of Parkinson's disease.