Dopamine transporter-mediated cytotoxicity of beta-carbolinium derivatives related to Parkinson's disease: relationship to transporter-dependent uptake.
Endogenous or exogenous beta-carboline (betaC) 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 betaCs 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 betaCs and MPP(+) showed general cytotoxicity in parental HEK-293 cells after 72 h with half-maximal toxic concentrations (TC(50) 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 TC(50) 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 betaCs were transported into the cell through the DAT with up to five times greater K(m) and 12-220 times smaller V(max) values compared with dopamine and MPP(+). There was a weak relation of DAT-mediated selectivity with the affinity of betaCs at the DAT (K(m)), but not with V(max). Our data suggest that DAT-mediated cellular uptake of 2[N]-methylated betaCs represents a potential mechanism for selective toxicity towards dopaminergic neurons and may be relevant for the pathogenesis of Parkinson's disease.
Storch
Alexander
A
Department of Neurology, University of Ulm, Ulm, Germany. alexander.storch@medizin.uni-ulm.de
Hwang
Yu-I
YI
Gearhart
Debra A
DA
Beach
J Warren
JW
Neafsey
Edward J
EJ
Collins
Michael A
MA
Schwarz
Johannes
J
D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine / Q000494:pharmacology; D015655:1-Methyl-4-phenylpyridinium / Q000633:toxicity; D002243:Carbolines / Q000493:pharmacokinetics* / Q000633:toxicity*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006246:Harmaline / Q000031:analogs & derivatives / Q000493:pharmacokinetics / Q000633:toxicity; D006247:Harmine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000633:toxicity; D006801:Humans; D020128:Inhibitory Concentration 50; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008562:Membrane Glycoproteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008745:Methylation; D009419:Nerve Tissue Proteins; D010302:Parkinson Disease, Secondary / Q000139:chemically induced / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology
D002243:Carbolines; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D008562:Membrane Glycoproteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010879:Piperazines; C493214:SLC6A3 protein, human; D006247:Harmine; C043425:vanoxerine; C010262:norharman; D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; D006246:Harmaline; D015655:1-Methyl-4-phenylpyridinium