Journal of Neurochemistry, 2004, 89, 685–694 doi:10.1111/j.1471-4159.2004.02397.

Dopamine transporter-mediated cytotoxicity of b-carbolinium

derivatives related to Parkinson’s disease: relationship to
transporter-dependent uptake

Alexander Storch,* Yu-I Hwang,* Debra A. Gearhart, J. Warren Beach,à Edward J. Neafsey,§
Michael A. Collins§ and Johannes Schwarz¶
*Department of Neurology, University of Ulm, Ulm, Germany
Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia, USA
àPharmacological and Biomedical Sciences, University of Georgia, Athens, Georgia, USA
§Department of Cell Biology, Neurobiology and Anatomy, Loyola University Stritch School of Medicine, Maywood, Illinois, USA
¶Department of Neurology, University of Leipzig, Leipzig, Germany

Abstract
Endogenous or exogenous b-carboline (bC) 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 test-
ing the differential cytotoxicity and cellular uptake of 12 bCs in
human embryonic kidney HEK-293 cells ectopically expres-
sing 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 fluores-
cence-based uptake assay. All bCs 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 ¼ 2[N]-methyl-
norharmanium > 2[N]-methyl-harmanium > 2[N],9[N]-dimeth-
yl-norharminium. Consistently, only 2[N]-methylated bCs 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 bCs at the DAT (Km),
but not with Vmax. Our data suggest that DAT-mediated cel-
lular uptake of 2[N]-methylated bCs represents a potential
mechanism for selective toxicity towards dopaminergic neu-
rons and may be relevant for the pathogenesis of Parkinson’s
disease.
Keywords: 1-methyl-4-pyridinium (MPP+), b-carboline deriv-
atives, dopamine transporter, neurotoxin, Parkinson’s dis-
ease.
J. Neurochem. (2004) 89, 685–694.

b-Carboline (bC) derivatives (pyrido-indoles) are hetero-

cyclic molecules occurring both exogenously and endogen-
ously, which are structurally related to the parkinsonian
neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) and its active metabolite 1-methyl-4-phenylpyridi-
nium (MPP+ for structures refer to Fig. 1). bC indole
alkaloids are widespread in our environment and in most
diets, such as grilled or broiled food, as well as smoke
including tobacco smoke (Sugimura and Nagao 1979;
Wakabayashi et al. 1997; Collins and Neafsey 2000;
Received 31 October, 2003; revised manuscript received 16 January,
2004; accepted 19 January, 2004.
Address correspondence and reprint requests to Alexander Storch,
University of Ulm, Department of Neurology, Oberer Eselsberg 45,
89081 Ulm, Germany. E-mail: alexander.storch@medizin.uni-ulm.de
Abbreviations used: CSF, cerebrospinal fluid; DAT, dopamine trans-
porter; HEK-293, human embryonic kidney 293 cells; HEK-hDAT,
dopamine transporter transfected HEK-293 cells; IQ, isoquinoline;
MPP+, 1-methyl-4-phenylpyridinium ion; MPTP, 1-methyl-4-phenyl-
2,3,6-tetrahydropyridine; MTT, [4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide; THbC, 1,2,3,4-tetrahydro-b-carboline; wt, wild type.

2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 685–694 685
686 A. Storch et al.
Herraiz 2000). bCs are synthesized from indolamines in
mammals and further metabolized by sequential N-methy-
lation forming 2[N]-methylated b-carbolinium ions and
subsequently 2[N],9[N]-dimethylated b-carbolinium cations
(Collins et al. 1992; Matsubara et al. 1992; Gearhart et al.
1997; Gearhart et al. 2000; Gearhart et al. 2002). The latter
methylating activity on the indole nitrogen as well as its
products (2[N],9[N]-dimethylated b-carbolinium cations)
seem to be elevated in parkinsonian brain (Matsubara et al.
1995; Gearhart et al. 2000). These chemical alterations
metabolically bioactivate the molecules, greatly increasing
both the mitochondrial inhibitory activity and the dop-
aminergic toxicity in vitro and in vivo (Hoppel et al. 1987;
Albores et al. 1990; Fields et al. 1992; Krueger et al.
1993). Consistently, some of these bCs are reported to
cause nigrostriatal neurodegeneration and behavioral
impairment in several animals, including non-human pri-
mates (Collins and Neafsey 1985; Collins et al. 1986;
Matsubara et al. 1998a), most likely by the described
sequential biotransfomation via N-methylations to b-carbo-
linium ions.
In vitro studies demonstrated cytotoxicity of N-methylated
b-carbolinium ions, in particular 2[N]-methyl-harmalinium;
2[N],9[N]-dimethyl-norharmanium; 2[N],9[N]-dimethyl-har-
manium, on PC12 cells as well as primary mesencephalic
cultures from rat with partial selectivity for dopaminergic
neurons (Cobuzzi et al. 1994; Collins et al. 1995; Matsubara
et al. 1998a). As for MPP+, cellular uptake of bC or rather
b-carbolinium ions by the dopamine transporter (DAT) is
considered to be involved in the selectivity of dopaminergic
toxicity of these compounds. Indeed, Drucker et al. (1990)
showed that 2[N]-methylated b-carbolinium ions are able to
inhibit [3H]dopamine uptake into rat striatal synaptosomes,
but IC50 values are much lower compared with MPP+
(12 lM to approx. 150 lM for b-carbolinium ions and
0.5 lM for MPP+, respectively; (Drucker et al. 1990).
Furthermore, the partially competitive nature of inhibition
by two effective compounds, 2[N]-methyl-harminium and
harmine, indicate that uptake of bCs is mediated in part by
synaptosomal DAT (Drucker et al. 1990). Accumulation of

2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 685–694
2-[14C]methyl-harmanium into synaptosomes is partially
blocked by the potent DAT inhibitor nomifensine (Drucker
et al. 1990). Matsubara and coworkers measured the uptake
of the neutral bC norharmane and quaternary b-carboliniums
2[N]-methyl-norhamanium and 2[N],9[N]-dimethyl-norhar-
manium, respectively, using a HPLC-based method (Mat-
subara et al. 1998b) and found that norharmane is an
unfavorable substrate for the DAT, while the 2[N]-methyla-
ted compounds showed DAT-mediated influx into striatal
synaptosomes with 1–4 times greater Km values and  10
times smaller maximal uptake velocity (Vmax). Thus,
quaternization of bCs strikingly increases the affinity for
the DAT. These data suggest that uptake of b-carbolinium
cations, in particular 2[N]-methylated bCs, by the DAT
molecule may be an important factor in the partial selectivity
of these compounds towards dopaminergic neurons.
Although uptake through the DAT has been characterized
for several bC derivatives, its relevance for selective
dopaminergic toxicity of these compounds remains unclear.
In particular, there is no systematic study investigating
whether bC uptake kinetics are sufficient to increase
accumulation of bC derivatives to concentrations that disrupt
vital cellular structures, most likely inhibition of mitochon-
drial respiratory function and subsequent cell death (Albores
et al. 1990; Fields et al. 1992; Krueger et al. 1993). We used
human embryonic kidney HEK-293 cells stably transfected
with the human DAT to compare DAT-mediated cytotoxicity
and cellular uptake of 12 neutral and quaternary compounds
from two classes of bC derivatives [10 bCs, two 3,4-dihydro-
b-carbolines (DHbC)] listed in Fig. 1.
Materials and methods
Materials
MPP+ iodide (a) was from Research Biochemical International
(RBI; Natick, USA), norharmane (b), 2[N]-propylnorharmanium
iodide (d), harmane (f), harmine (i) and harmaline (l) were purchased
from Sigma (St. Louis, MO, USA). 1-{2-[Bis(4-fluorophenyl)
methoxy]ethyl}-4-[3-phenylpropyl]-piperazine dihydrochloride
Fig. 1 Structures of 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP), 1-me-
thyl-4-phenylpyridinium (MPP+) as well as
b-carboline and 3,4-dihydro-b-carboline
derivatives.

(GBR 12909) was purchased from RBI. Geniticin (G418) was from
Gibco (Eggenstein, Germany). All other chemicals were of
analytical grade and from Sigma.
Synthesis of methylated b-carbolinium derivatives
All 2[N ]-methylated and 2[N],9[N]-dimethylated bC (c,e,g,h,j,k)
and DHbC derivatives (m) were prepared as iodide or chloride salts
as previously reported (Albores et al. 1990; Drucker et al. 1990;
Gearhart 1997). In brief, 2[N]-methylated compounds were syn-
thesized in the laboratories of M. A. Collins or J. W. Beach by
treatment of the appropriate non-methylated bC or DHbC with
excess of methyl iodide in methanol, ethanol, or acetone. Cooling to
ice-bath temperature generally precipitated the 2[N]-methylated
compound, which was then isolated by filtration; occasionally, silver
chloride was used to convert the iodide salt of a bC to the
corresponding chloride salt. The 2[N],9[N]-dimethylated bCs were
synthesized by sequential reaction of respective non-methylated bCs
with methyl iodide. Briefly, non-methylated bCs were first 9[N]-
methylated by methyl iodide in the presence of sodium hydride
(Gearhart 1997); after decomposing the sodium hydride, the 9[N]-
methylated bC was 2[N]-methylated using methyl iodide as
described above. The structures of recrystallized products, a single
peak by HPLC, was confirmed by NMR and melting point
comparison (Ho et al. 1973).
Cell culture
Human embryonic kidney HEK-293 cells were purchased from
American Tissue Type Culture Collection (ATCC, reference no.
CRL 1573; Rockville, USA). HEK-293 cells were grown in
minimum essential medium with Earl’s salts and glutamine (Gibco,
Rockville, MD, USA) supplemented with 10% heat inactivated fetal
bovine serum (PAA, Cölbe, Germany), at 37C in a humidified
atmosphere of 5% CO2 in air. HEK-293 cells stably transfected with
the human dopamine transporter gene (HEK-hDAT cells) were
prepared and characterized as previously described (Storch et al.
1999; Storch et al. 2002), and cultured at the conditions above
except for adding 400 lg/mL geneticin (G418) to the medium.
Toxicity assays
Cell viability was assessed by the colorimetric MTT method
(Denizot and Lang 1986), which offers precise quantification of cell
viability in mammalian cell cultures (Da Costa et al. 1999; Storch
et al. 1999; Storch et al. 2002). Cells of interest were seeded in
96-well plates at a density of 10 000–12 500 cells/well (in 200 lL
medium). The cultures were grown for 48 h, then various
concentrations of the substance of interest in 50 lL medium were
added. After incubation at 37C for 72 h, 30 lL of MTT reagent
(0.5 mg/mL MTT in PBS containing 10 mM HEPES) was added to
each well and incubated in the CO2 incubator for 1 h. After drying
the culture plate at 37C for 1 h, the resulting formazan dye was
extracted with acid-isopropanol (100 lL of 0.04 M HCl in isopro-
panol) and the absorbance was measured spectrophotometrically
with a computer-driven spectrophotometer immunoreader (Molecu-
lar Dynamics, Krefeld, Germany) at a wavelength of 570 nm with
reference at 630 nm. Wells without cells were used as blanks and
were subtracted as background from each sample. MTT reduction
was expressed as percentage of the untreated control. The data
obtained with the MTT method corresponded very closely to data

2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 685–694
Dopamine transporter and b-carbolines 687
obtained with direct cell counting using trypan blue staining (for
technical details, see Storch et al. 1999; Storch et al. 2002).
Uptake assay
To measure the cellular uptake of bC and DHbC derivatives through
the DAT, we established an uptake method based on the strong
autofluorescence of pyrido-indoles at excitation wavelength of 280
or 310 nm, and an emission wavelength of 450 nm (Spenser 1956).
HEK-293 or HEK-hDAT cells were distributed into 48-well plates at
a density of 2.5–5 · 105 cells/well 48 h before the uptake
experiment was performed. At the beginning of the uptake
experiment, each well was washed with 0.5 mL of ice-cold uptake
buffer (in mM: 1 MgCl2, 1 CaCl2 in PBS, pH 7.4) and further
incubated with 0.5 mL of uptake buffer for 10–15 min. Then
100 lL of buffer containing the bC derivative of interest
(0–500 lM) were added to each well and the wells were incubated
for 10 min at 37C. For initial time-course experiments, cells were
incubated at 37C with norharmane derivatives (b,d,e) (100 lM) for
different time periods (0 up to 60 min) before the uptake was
stopped. Uptake was stopped by aspirating the substance solution
and adding 0.5 mL of ice-cold uptake buffer to each well and
immediately aspirating the buffer and washing twice with 0.5 mL of
ice-cold buffer. Non-specific transport was determined in parallel
assays conducted in the presence of 3 lM GBR12909 (Storch et al.
1999; Storch et al. 2002). The intracellular bC was extracted by
solubilizing the cells with 100 lL of lysis buffer (0.1 M HCl
containing 2 mM EDTA) for 1 h on a plate shaker followed by three
freeze-thaw cycles. The transported compounds were quantified by
measuring their fluorescence using a microplate fluorometer
(Molecular Dynamics) at a wavelength of 280 and 452 nm for
excitation and emission, respectively (bandwidth of filters: 20 nm).
Wells filled with lysis buffer were used as blanks and subtracted as
background from each sample. Calculation of absolute concentra-
tions of each bC was obtained by comparison with external
standards of known concentrations of the respective bC in lysis
buffer.
Data analysis and statistics
We used the MTT assay to evaluate toxicity of each compound at
eight to 10 different concentrations (0.01–1000 lM). Each experi-
ment was replicated at least three times. To compare the toxic
effects of bC derivatives on different cell lines, we calculated the
half-maximal toxic concentration (TC50 value) for all compounds
on wild type cell lines (TC50wt) and cell lines expressing the DAT
(TC50DAT) using non-linear curve fitting (Origin, Version 7.0;
OriginLab) according to the mathematical model Y ¼ A1 + (A2 –
A1)/[1 + 10(log B – x)nH], where A1 is the limit when the
concentration approaches 0, A2 is the limit when the concentration
approaches the maximum, B is the concentration that yields a 50%
reduction of cell viability (TC50), and nH the Hill slope. The ratio
TC50wt/TC50DAT was defined as IDAT. The unpaired t-test was used
to compare the TC50wt and TC50DAT values of a given compound.
We evaluated six to eight different concentrations (0–125 or
500 lM, depending on the availability of the compound) for each
bC derivative, to calculate the kinetic parameters for cellular
uptake of each compound into HEK-hDAT cells. Each experiment
was repeated at least three times. Vmax, Km and nH values were
calculated using non-linear regression analysis similar to the
688 A. Storch et al.

analysis of toxicity curves. Results were expressed as means ±
SEM and compared using Student’s unpaired or paired t-test.
Results
b-Carboline derivatives are toxic towards HEK-293 (wt)
cells
To study the importance of the DAT for selective
dopaminergic toxicity of bC derivatives, we used non-
neuronal HEK-293 cells stably transfected with the DAT
gene (HEK-hDAT cells) (Storch et al. 1999; Storch et al.
2002). These cells show extremely high sensitivity and
selectivity for MPP+-toxicity (TC50DAT ¼ 0.14 lM after
72 h; IDAT ¼ 4107) and display selectivity for 2[N]-
methylated isoquinolines related to PD and structurally
similar to MPP+ (Storch et al. 1999; Storch et al. 2002).
First, we investigated the toxicity of all bC derivatives
towards the parental cell line HEK-293 (wt) using the
MTT assay. Using bC concentrations up to 1000 lM, all
compounds showed concentration-dependent cytotoxicity
on untransfected HEK-293 (wt) cells with TC50WT values
in the high micromolar range. The rank order of potency
was: 2[N],9[N]-dimethyl-harminium (k) > harmine (i) >
harmaline (l) > harmane (f) ¼ 2[N]-methyl-harmalinium
(m) > 2[N],9[N]-dimethyl-harmanium (h) > 2[N],9[N]-di-
methyl-norharmanium (e) > 2[N]-methyl-harminium (j) >
norharmane (b) ¼ 2[N]-methyl-harmanium (g) > 2[N]-Pro-
pyl-norharmanium (d) > 2[N]-methyl-norharmanium (c).
Table 1 summarizes the calculated TC50wt values obtained
by non-linear regression analysis of the toxicity curves.
DAT enhances toxicity of 2[N]-methylated and
2[N],9[N]-dimethylated bCs
As shown in Table 1, only 2[N]-methylated bC derivatives
exhibited significantly enhanced cytotoxicity (lower TC50
values relative to wt cells) in HEK-293 cells functionally
expressing the human DAT; however, potency and selectivity
were less than those of MPP+. The rank order of selectivity
(IDAT) was: MPP+ (a) >> 2[N],9[N]-dimethyl-harminium
(k) > 2[N]-methyl-harminium (j) > 2[N],9[N]-dimethyl-har-
manium (h) ¼ 2[N]-methyl-norharmanium (c) > 2[N]-
methyl-harmanium (g) > 2[N],9[N]-dimethyl-norharmanium
(e). As an example for toxicity curves, Fig. 2 shows the
effects of norharmane (b), 2[N]-methyl-norharmanium (c)
and 2[N],9[N]-dimethyl-norharmanium (e), respectively, on
cell viability of untransfected HEK-293 and HEK-hDAT
cells. Table 1 summarizes the calculated TC50DAT and IDAT
values obtained by non-linear regression analysis of the
toxicity curves. The DAT inhibitor GBR12909 (1 lM)
protected bC-treated HEK-hDAT cells against DAT-depend-
ent toxicity; the extent of protection afforded by GBR12909
approached the level of cell survival seen in untransfected
HEK-293 (wt) cells after treatment with the same concen-
tration of each toxin (Fig. 3a–e).
DAT transports 2[N]-methylated and
2[N],9[N]-dimethylated bCs
To measure cellular uptake of bC derivatives in HEK-293
(wt) and HEK-hDAT cells, we established an uptake assay
based on the strong autofluorescence of pyrido-indoles
(Spenser 1956). Initially, we investigated the time-course of

Table 1 Toxicity of b-carboline derivatives

Compound TC50wt (lM) TC50DAT (lM) IDAT
in HEK-293 (wt) and HEK-hDAT cells
Pyridine derivatives
(a) MPP+ 575.0 ± 27.9 0.14 ± 0.02** 4107
b-Carboline derivatives
(b) Norharmane 129.9 ± 8.2 176.7 ± 17.4 0.7
(c) 2[N]-Methyl-norharmanium 326.8 ± 19.3 147.0 ± 11.2** 2.2
(d) 2[N]-Propyl-norharmanium 259.9 ± 12.5 236.3 ± 15.7 1.1
(e) 2[N],9[N]-Dimethyl-norharmanium 82.8 ± 5.7 62.1 ± 5.1* 1.3
(f) Harmane 63.1 ± 7.9 72.9 ± 9.3 0.9
(g) 2[N]-Methyl-harmanium 204.7 ± 6.2 126.4 ± 4.2** 1.6
(h) 2[N],9[N]-Dimethyl-harmanium 72.7 ± 6.3 29.4 ± 0.6** 2.5
(i) Harmine 36.3 ± 8.9 37.4 ± 9.2 1.0
(j) 2[N]-Methyl-harminium 103.1 ± 21.2 34.3 ± 3.7* 3.0
(k) 2[N],9[N]-Dimethyl-harminium 21.3 ± 2.8 4.7 ± 0.4** 4.5
3,4-Dihydro-b–carboline (DHbC) derivatives
(l) Harmaline 52.9 ± 4.7 76.4 ± 10.6 0.7
(m) 2[N]-Methyl-harmalinium 62.4 ± 3.0 46.8 ± 5.1 1.3

Each compound was tested in both HEK-293 (wt) and HEK-hDAT cell lines. TC50wt and TC50DAT
represent the effective concentration (in lM) inducing a 50% decrease of cell viability of HEK-293
(wt) and HEK-hDAT cells, respectively, measured with the MTT assay after 72 h of exposure. IDAT,
defined as the ratio TC50wt/TC50DAT, represents an index of DAT-dependent toxicity. *TC50DAT
significantly different from corresponding TC50wt at p < 0.05, **p < 0.01.

2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 685–694

 Dopamine transporter and b-carbolines 689

Fig. 2 Cytotoxicity of norharmane (a), 2[N]-methyl-norharmanium (b)

and 2[N],9[N]-dimethyl-norharmanium (c), on HEK-293 cells perma-
nently expressing the human DAT. Wild-type cells (open symbols) and
cells permanently transfected with the DAT gene (filled symbols) were
treated with various concentrations of the compound of interest or
vehicle for 72 h and cell viability was assessed using the MTT assay.
Fig. 3 Effects of the DAT inhibitor GBR12909 on cytotoxicity of
For half-maximal toxic concentrations (TC50 values) see Table 1. Data
2[N]-methyl-norharmanium (a), 2[N]-methyl-harminium (b), 2[N],9[N]-
points represent the mean ± SEM of at least three independent
dimethyl-harmanium (c), 2[N]-methyl-harminium (d) and 2[N],9[N]-
experiments (running in triplicate). * indicates p < 0.05 when com-
dimethyl-harminium (e) on HEK-293 cells permanently expressing
pared with untransfected HEK-293 cells.
the human DAT. HEK-hDAT cells were treated with the b–carboline
derivative of interest for 72 h in the absence (open bars) or presence
uptake of the three norharmane derivatives (b,c,e) to
(filled bars) of GBR12909 (1 lM). For comparison with cells not
demonstrate time-dependency and saturation of uptake. As
expressing the DAT, cell survival of HEK-293 (wt) cells in the pres-
shown in Fig. 4(a), GBR 12909-sensitive cellular uptake of ence of the respective b-carboline is displayed (gray bars). Cell
norharmane (b), 2[N]-methyl-norharmanium (c) and viability was assessed using the MTT assay. Data points represent
2[N],9[N]-dimethyl-norharmanium (e) into HEK-hDAT cells the mean ± SEM of at least three independent experiments (running
was linear for up to 10 min with subsequent saturation of in triplicate). * indicates p < 0.05 when compared with control (no
cellular uptake. To measure the kinetic parameters for uptake, GBR12909).

2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 685–694

690 A. Storch et al.

all 2[N]-methylated and 2[N],9[N]-dimethylated bCs into

Fig. 4 Uptake of norharmane, 2[N]-methyl-norharmanium or
2[N],9[N]-dimethyl-norharmanium in HEK-293 cells stably transfected
with the human DAT gene. (a) Time-course of specific DAT-mediated
uptake of norharmane derivatives into HEK-hDAT cells. The cells were
incubated with 100 lM of the respective norharmane derivative for up
to 60 min. Then the uptake was stopped and the accumulated com-
pound was measured using its fluorescence (see text for details).
(b–d) Kinetics of the transport of norharmane (b), 2[N]-methyl-nor-
harmanium (c) or 2[N],9[N]-dimethyl-norharmanium (d) into wild-type
HEK-293 and HEK-hDAT cells, respectively. The cells were incubated
with increasing concentrations of the norharmane derivative for
10 min. For calculated Vmax and Km values see Table 2. Data points
represent the mean ± SEM of three independent experiments.
the bC of interest was incubated with HEK-hDAT cells at
concentrations of 0–125 or 500 lM for 10 min. Thus, we
were able to detect significant GBR12909-sensitive uptake of
HEK-hDAT cells. Figure 4(b–d) show the uptake curves for
norharmane (b), 2[N]-methyl-norharmanium (c) and
2[N],9[N]-dimethyl-norharmanium (e) in HEK-293 (wt)
and HEK-hDAT cells. Table 2 summarizes the calculated
Km, Vmax and nH values obtained by non-linear regression
analysis of the uptake curves. Except for 2[N],9[N]-dimethyl-
harmanium (h), the affinities of all transported compounds
were less than the affinity of dopamine or MPP+ (a) with Km
values up to five times greater than that of dopamine/MPP+
(Table 2). Furthermore, the uptake velocities (Vmax) of all bC
derivatives were 12–220 times smaller than the Vmax values
for dopamine or MPP+ (Table 2). In contrast, bCs lacking a
2[N]-methyl group (b,f,i,l) did not show significant
GBR12909-sensitive uptake into HEK-hDAT cells (for
example, see Fig. 4b). The parental cell line HEK-293 (wt)
not expressing the DAT molecule showed no significant
GBR12909-sensitive accumulation of all bC derivatives (for
examples, see Fig. 4b–d).
DAT-mediated toxicity correlates with cellular uptake
through the DAT
All bC derivatives that exhibited significant DAT-mediated
toxicity were specifically transported into the cell via the
DAT, and there was no compound showing DAT-dependent
toxicity but no cellular uptake through the DAT (compare
Table 1 with Table 2). On the other hand, only 2[N]-methyl-
harmalinium (m) showed specific cellular uptake through the
DAT, but no DAT-mediated toxicity. To correlate DAT-
mediated toxicity with uptake kinetics of bC derivatives, we
plotted the Km and Vmax values vs. IDAT (an index of DAT-
mediated toxicity) for a given compound. As shown in
Fig. 5, there was no relation between Vmax and IDAT values
(adjusted R2 value of 0.05, p ¼ 0.2; Pearson’s correlation

Table 2 Apparent uptake kinetics for dop-

Compound Km (lM) Vmax (pmol/min/well) nH
amine transporter mediated uptake
Dopamine 23.0 ± 3.7* 244 ± 27* 1.05 ± 0.13*
(a) MPP+ 25.3 ± 5.9* 167 ± 35* 1.25 ± 0.21*
b-Carboline derivatives
(b) Norharmane N.A. N.A. N.A.
(c) 2[N]-Methyl-norharmanium 58.0 ± 8.7 21.3 ± 1.3 1.40 ± 0.20
(e) 2[N],9[N]-Dimethyl-norharmanium  114 1.2 ± 0.3 1.10 ± 0.30
(f) Harmane N.A. N.A. N.A.
(g) 2[N]-Methyl-harmanium 48.3 ± 9.2 12.8 ± 0.9 1.07 ± 0.16
(h) 2[N],9[N]-Dimethyl-harmanium 15.0 ± 2.1 8.0 ± 0.4 1.77 ± 0.44
(i) Harmine N.A. N.A. N.A.
(j) 2[N]-Methyl-harminium 74.1 ± 9.6 17.3 ± 1.3 1.64 ± 0.21
(k) 2[N],9[N]-Dimethyl-harminium 32.7 ± 5.0 2.7 ± 0.2 1.98 ± 0.58
3,4-Dihydro-b-carboline (DHbC) derivatives
(l) Harmaline N.A. N.A. N.A.
(m) 2[N]-Methyl-harmalinium 31.7 ± 2.6 1.1 ± 0.1 2.87 ± 0.58

*Data from Storch et al. (1999, 2002) obtained with [3H]dopamine and [3H]MPP+. N.A., not
accumulated through the dopamine transporter.

2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 685–694


Fig. 5 Correlation of DAT-mediated toxicity and uptake kinetics. The
index of DAT-mediated toxicity (IDAT) is plotted versus Km values (a)
and Vmax values (b) for all bC derivatives with significant GBR12909-
sensitive uptake into HEK-hDAT cells (see Table 1 for IDAT values and
Table 2 for uptake parameters).
coefficient: 0.05), but a weak relation between Km and IDAT
values (adjusted R2 value of 0.36, p ¼ 0.09; Pearson’s
correlation coefficient: – 0.31).
Discussion
A growing body of evidence from morphological (Waters
et al. 1988; Pakkenberg et al. 1991; Cerruti et al. 1993;
Hersch et al. 1997), molecular biological and genetic studies
(Le Couteur et al. 1997; Morino et al. 2000; Nishimura et al.
2002) indicates that the DAT may be responsible for the
selectivity of dopaminergic cell death in PD (for review, see
Storch and Schwarz 2000). Furthermore, impairment of
mitochondrial energy production caused by a reduction of
mitochondrial enzyme activities is an important factor
leading to cell death in PD (for review, see (Lang and
Lozano 1998a,b). Exogenous or endogenous toxins trans-
ported into the cell by the DAT and subsequently affecting
mitochondrial respiration, may play a role in the pathogen-
esis of PD. bC derivatives, structurally related to MPTP/
MPP+, are considered as reasonable candidates with eviden-
ces for dopaminergic toxicity (Cobuzzi et al. 1994; Collins
et al. 1995; Matsubara et al. 1998a), inhibition of mitoch-
ondrial respiration (Hoppel et al. 1987; Albores et al. 1990;
Fields et al. 1992; Krueger et al. 1993), and transmembrane
transportation via the DAT (Drucker et al. 1990; Matsubara
et al. 1998b). Furthermore, increased brain levels of bCs and
their synthesizing enzymes were detected in patients with
PD: 2[N],9[N]-dimethylnormarmanium (e) was found in the
cerebrospinal fluid (CSF) of half of the tested PD patients but
not in controls, and the total content of methylated bCs was
significantly higher in PD patients than in controls (Matsub-
ara et al. 1995). Moreover, enzymatic bC N-methyltransf-
erase activities are present in mammalian and human brain
(Gearhart et al. 1997; Gearhart et al. 2000; Gearhart et al.
2002), with four-fold increased activity of bC 9[N]-methyl-
transferase activity in the supernatant fraction of postmortem
frontal cortex samples of PD patients compared with control
(Gearhart et al. 2000). Those studies are consistent with the

2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 685–694
Dopamine transporter and b-carbolines 691
concept that bC derivatives as well as biological methylation
may play a role in the pathogenesis of at least a subset of PD
cases (Waring et al. 1989; Charlton and Mack 1994;
Charlton and Crowell 1995).
The importance of DAT-mediated cellular uptake of bCs,
in particular 2[N]-methylated derivatives, with respect to
their dopaminergic toxicity remains unclear; there has been
no systematic evaluation of bCs to determine the potency,
selectivity and structural requirements for both DAT-depend-
ent cytotoxicity and cellular uptake. Therefore, we used
HEK-293 cells stably transfected with the DAT gene (HEK-
hDAT cells) to examine the cytotoxic effects and transmem-
brane uptake in a series of bCs that are structurally related to
MPTP/MPP+. HEK-hDAT cells show high sensitivity and
selectivity for MPP+-mediated toxicity (Storch et al. 1999;
Storch et al. 2002), and these cells have dopamine uptake
kinetics that are similar to those reported for other ectopic
expression systems (Pifl et al. 1993; Buck and Amara 1994;
Pifl et al. 1996; Kitayama et al. 1998), but different from
studies using rat synaptosomes, with 5–10 times higher Km
values (Pifl et al. 1993; Matsubara et al. 1998b). After
entering the cell via the DAT, there might be relevant
differences in the intracellular toxic pathways between
dopaminergic neurons and our non-neuronal HEK-293 cell
system expressing the DAT. As shown for MPP+, not only
the inhibition of mitochondrial complex I activity is respon-
sible for its toxicity towards dopaminergic cells, but also the
cytosolic clearance of the neurotoxin by the vesicular
monoamine transporter 2 (VMAT2) and interactions with
dopamine or the enzyme tyrosine hydoxylase (TH) (Przed-
borski 2000). Most of these factors are not expressed in
HEK-293 cells. However, HEK-hDAT cells represent a
valuable tool for studying the involvement of the DAT
molecule in mediating the selective dopaminergic toxicity of
various neurotoxins that may have relevance in PD.
We report that all bCs tested displayed a concentration-
dependent toxicity (TC50 values were in the high micromolar
range) towards the parental HEK-293 (wt) cell line, which
does not express the DAT protein (Table 1). There is no clear
structure-activity relationship between bC derivatives and
their cytotoxic effects in HEK-293 (wt) cells. Indeed, the
rank order of toxicity of bCs in HEK-293 (wt) cells does not
correlate with bC lipophilicity (Biagi et al. 1989), nor with
the potency of bCs to inhibit mitochondrial respiration
(Hoppel et al. 1987; Albores et al. 1990; Fields et al. 1992;
Krueger et al. 1993). In contrast, only three 2[N]-methylated
compounds [2[N]-methyl-norharmanium (c), 2[N]-methyl-
harmanium (g) and 2[N]-methyl-harminium (j)), and all
2[N],9[N]-dimethylated compounds (2[N],9[N]-dimethyl-
norharmanium (e), 2[N],9[N]-dimethyl-harmanium (h) and
2[N],9[N]-dimethyl-harminium (k)] displayed significantly
enhanced toxicity against HEK-hDAT cells relative to the
parental cell line. Significantly, N-methylated bC-mediated
selective toxicity was completely blocked by the DAT
692 A. Storch et al.
inhibitor GBR12909, suggesting that DAT expression con-
fers a susceptibility to the toxic effects of certain bCs. These
results in HEK-hDAT cells exposed to 2[N]-methylated and
2[N],9[N]-dimethylated bCs parallel the toxicity of the same
compounds towards dopaminergic neurons in fetal rat
mesencephalic cultures (Cobuzzi et al. 1994; Collins et al.
1995; Collins et al. 1996; Matsubara et al. 1998a). Further-
more, in both cell systems the toxicity of N-methylated bCs
is potentiated by addition of a methoxy group on position 7
of the aromatic ring [substances 2[N]-methyl-harminium (j)
and 2[N],9[N]-dimethyl-harminium (k)].
In order to compare DAT-mediated toxicity of bC
derivatives with their cellular uptake through the DAT
molecule, we also measured DAT-dependent transmembrane
uptake of bCs in HEK-hDAT cells. Previously published
methods for bC uptake only measured DAT binding or
dopamine uptake inhibitory capacities (Drucker et al. 1990).
Our uptake assay utilizing the fluorescent properties of bCs
has the advantages of measuring cellular accumulation of
bCs including estimates for uptake kinetic parameters (Km
and Vmax). We demonstrated that all 2[N]-methylated bCs
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+. In line with our results,
Matsubara et al. (1998b) used an HPLC-based uptake assay
to show that 2[N]-methyl-norharmanium (c) and 2[N],9[N]-
dimethyl-norharmanium (e), but not norharmane (b) were
accumulated into rat striatal synaptosomes through the DAT
(Matsubara et al. 1998b).
All bC derivatives with significant DAT-mediated toxicity
were specifically transported into the cell via the DAT and
there is no compound showing DAT-dependent toxicity but
no cellular uptake through the DAT. There is a weak relation
of DAT-mediated selectivity (IDAT) with the affinity of bCs at
the DAT (Km), but not with uptake velocity through the DAT
(Vmax). Thus, we speculate that in particular the lower
affinities (greater Km values) of bCs to the DAT compared
with that of MPP+ might explain the moderate selectivity of
these compounds towards DAT-expressing cells, e.g. dop-
aminergic neurons. Interestingly, only one bC with specific
cellular uptake through the DAT, namely 2[N]-methyl-
harmalinium (m), displayed no significant DAT-mediated
toxicity. However, this compound had high Km value, very
low maximal uptake velocity (Vmax), and low mitochondrial
inhibitory capacity (Krueger et al. 1993), which could
explain the lack of toxicity. In line with these results, the
dopaminergic toxicity of 2[N]-methyl-harmalinium (m) was
not prevented by DAT inhibitors (Collins et al. 1995),
suggesting a dissimilar DAT-independent mechanism of
toxicity compared with other 2[N]-methylated bCs.
The group of b-carboline derivatives used in the present
study for analyzing DAT-mediated toxicity and uptake does
not allow to measure quantitative structure-activity (selec-
tivity) relationships, but some qualitative trends are apparent.

2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 685–694
The intermolecular distance between the nitrogen atom and
the centrinoid of the benzene or catechol ring is one of
the most important factors for the affinity of compounds at
the dopamine transporter molecule. The conformation of the
dopamine molecule at the uptake site of the dopamine
transporter is the extended or trans form (Horn 1974;
Meiergerd and Schenk 1994). The distance between the
nitrogen atom and the centrinoid in MPP+ and b-carboline
derivatives is very close to that of the extended dopamine
conformation. The charge of the nitrogen atom seems to be
another factor not only for the affinity of the compounds to
the transporter molecule, but also for the transmembrane
transport of the compounds by the DAT (Matsubara et al.
1998b). Indeed, DAT-mediated toxicity and uptake were
restricted to quaternary 2[N]-methylated bCs with a net
charge comparable to dopamine, whereas structurally iden-
tical neutral compounds lack selective toxicity towards
DAT-expressing cell lines (see Fig. 1). Interestingly, addi-
tion of a methoxy group on position 7 of the aromatic ring
increases the DAT-mediated toxicity of N-methylated bCs,
but not their affinity or uptake velocity at the DAT
molecule.
Taken together, our data demonstrate that DAT-dependent
cellular uptake of 2[N]-methylated bCs confers DAT-
mediated cytotoxicity of bC derivatives. Thus, the DAT
molecule mediates the partial selectivity of cytotoxicity
induced by 2[N]-methylated bCs towards dopaminergic
neurons in vitro and in vivo (Matsubara et al. 1998a). The
low affinities of bCs at the DAT protein are most likely the
rate-limiting factor for selective dopaminergic toxicity.
Conclusively, high concentrations of and/or prolonged
exposure to bC derivatives might be necessary to produce
significant dopaminergic toxicity. This does not rule out the
possible involvement of bC derivatives in the pathophys-
iology of PD since the slow progression of this disease
suggests a mild and prolonged degenerative process.
However, the present results together with morphological
and genetic evidences for the involvement of the DAT in
the etiopathogenesis of PD (for review, see Storch and
Schwarz 2000) further support the hypothesis that selective
toxic insults towards dopaminergic neurons via DAT-
mediated cellular uptake of exogenous and/or endogenous
2[N]-methylated bC derivatives play a pivotal role in
dopaminergic neurodegeneration in PD.
Acknowledgements
The authors would like to thank Sinje Jankowski, Andrea Weinhold
and Thomas Lenk for excellent technical assistance. This study was
supported by the University of Ulm Medical School Research
Foundation (to AS) and the Ministerium für Wissenschaft, Fors-
chung und Kunst Baden-Württemberg (Förderprogramm Entwick-
lung von Ersatzmethoden zur Vermeidung von Tierversuchen) (to
AS and JS).

References
Albores R., Neafsey E. J., Drucker G., Fields J. Z. and Collins M. A.
(1990) Mitochondrial respiratory inhibition by N-methylated beta-
carboline derivatives structurally resembling N-methyl-4-phenyl-
pyridine. Proc. Natl Acad. Sci. USA 87, 9368–9372.
Biagi G. L., Pietrogrande M. C., Barbaro A. M., Guerra M. C., Borea P.
A. and Forti G. C. (1989) Study of the lipophilic character of a
series of beta-carbolines. J. Chromatogr. 469, 121–126.
Buck K. J. and Amara S. G. (1994) Chimeric dopamine-norepinephrine
transporters delineate structural domains influencing selectivity for
catecholamines and 1-methyl-4-phenylpyridinium. Proc. Natl
Acad. Sci. USA 91, 12584–12588.
Cerruti C., Walther D. M., Kuhar M. J. and Uhl G. R. (1993) Dopamine
transporter mRNA expression is intense in rat midbrain neurons
and modest outside midbrain. Brain Res. Mol Brain Res. 18, 181–
186.
Charlton C. G. and Mack J. (1994) Substantia nigra degeneration and
tyrosine hydroxylase depletion caused by excess S-adenosyl-
methionine in the rat brain: support for an excess methylation
hypothesis for parkinsonism. Mol. Neurobiol. 9, 149–161.
Charlton C. G. and Crowell B. Jr (1995) Striatal dopamine depletion,
tremors, and hypokinesia following the intracranial injection of
S-adenosylmethionine: a possible role of hypermethylation in
parkinsonism. Mol. Chem. Neuropathol. 26, 269–284.
Cobuzzi R. J. Jr, Neafsey E. J. and Collins M. A. (1994) Differential
cytotoxicities of N-methyl-beta-carbolinium analogues of MPP+ in
PC12 cells: insights into potential neurotoxicants in Parkinson’s
disease. J. Neurochem. 62, 1503–1510.
Collins M. A. and Neafsey E. J. (1985) Beta-carboline analogues of
N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP): endog-
enous factors underlying idiopathic parkinsonism? Neurosci. Lett.
55, 179–184.
Collins M. A. and Neafsey E. J. (2000) b-Carboline analogs of MPP+ as
environmental neurotoxins, In: Neurotoxic Factors in Parkinson’s
Disease and Related Disorders (Storch, A. and Collins, M. A.,
eds), pp. 115–130. Kluwer Academic, Plenum Press, New York.
Collins M. A., Neafsey E. J., Cheng B. Y., Hurley-Gius K., Ung-Chhun
N. A., Pronger D. A., Christiansen M. A. and Hurley-Gius D.
(1986) Endogenous analogs of MPTP: indolamine-derived tetra-
hydro-b-carbolines as potential causative factors in Parkinson’s
diseases. Adv. Neurol. 45, 179–182.
Collins M. A., Neafsey E. J., Matsubara K., Cobuzzi R. J. Jr and
Rollema H. (1992) Indole-N-methylated beta-carbolinium ions as
potential brain-bioactivated neurotoxins. Brain Res. 570, 154–
160.
Collins M. A., Slobodnik L. and Neafsey E. J. (1995) Inhibitors of NO
synthase and poly(ADP-ribose) synthase (PARS) do not block
toxic actions of b-carbolinium cations or MPP+ in mesencephalic
cultures. Abstr. Soc. Neurosci. 21, 1259.
Collins M. A., Neafsey E. J. and Matsubara K. (1996) b-Carbolines:
metabolism and neurotoxicity. Biogen. Amines 12, 171–180.
Da Costa A. O., de Assis M. C., Marques E. A. and Plotkowski M. C.
(1999) Comparative analysis of three methods to assess viability of
mammalian cells in culture. Biocell 23, 65–72.
Denizot F. and Lang R. (1986) Rapid colorimetric assay for cell growth and
survival: modifications to the tetrazolium dye procedure giving
improved sensitivity and reliability. J. Immunol. Methods 89, 271–
277.
Drucker G., Raikoff K., Neafsey E. J. and Collins M. A. (1990)
Dopamine uptake inhibitory capacities of beta-carboline and
3,4-dihydro-beta-carboline analogs of N-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP) oxidation products. Brain Res.
509, 125–133.

2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 685–694
Dopamine transporter and b-carbolines 693
Fields J. Z., Albores R. R., Neafsey E. J. and Collins M. A. (1992)
Inhibition of mitochondrial succinate oxidation – similarities and
differences between N-methylated beta-carbolines and MPP+.
Arch. Biochem. Biophys. 294, 539–543.
Gearhart D. A. (1997) N-Methylation of b-carbolines as a potential
bioactivation route in Parkinson’s disease. Department of Neuro-
science, Loyola University, Chicago, IL, USA. p. 269.
Gearhart D. A., Neafsey E. J. and Collins M. A. (1997) Characterization
of brain beta-carboline-2-N-methyltransferase, an enzyme that may
play a role in idiopathic Parkinson’s disease. Neurochem. Res. 22,
113–121.
Gearhart D. A., Collins M. A., Lee J. M. and Neafsey E. J. (2000)
Increased beta-carboline 9N-methyltransferase activity in the
frontal cortex in Parkinson’s disease. Neurobiol. Dis. 7,
201–211.
Gearhart D. A., Neafsey E. J. and Collins M. A. (2002) Phenyletha-
nolamine N-methyltransferase has beta-carboline 2N-methyl-
transferase activity: hypothetical relevance to Parkinson’s disease.
Neurochem. Int. 40, 611–620.
Herraiz T. (2000) Analysis of the bioactive alkaloids tetrahydro-beta-
carboline and beta-carboline in food. J. Chromatogr. A 881, 483–
499.
Hersch S. M., Yi H., Heilman C. J., Edwards R. H. and Levey A. I.
(1997) Subcellular localization and molecular topology of the
dopamine transporter in the striatum and substantia nigra. J. Comp.
Neurol. 388, 211–227.
Ho B. T., Gardner P. M. and Walker K. E. (1973) Inhibition of MAO by
b-carbolinium halides. J. Pharm. Sci. 62, 36–39.
Hoppel C. L., Grinblatt D., Kwok H. C., Arora P. K., Singh M. P., Sayre
L. M. and Greenblatt D. (1987) Inhibition of mitochondrial res-
piration by analogs of 4-phenylpyridine and 1-methyl-4-phenyl-
pyridinium cation (MPP+), the neurotoxic metabolite of MPTP.
Biochem. Biophys. Res. Commun. 148, 684–693.
Horn A. S. (1974) The conformation of dopamine at its uptake site;
further studies with rigid analogues. J. Pharm. Pharmacol. 26,
735–737.
Kitayama S., Mitsuhata C., Davis S., Wang J. B., Sato T., Morita K., Uhl
G. R. and Dohi T. (1998) MPP+ toxicity and plasma membrane
dopamine transporter: study using cell lines expressing the wild-
type and mutant rat dopamine transporters. Biochim. Biophys. Acta
1404, 305–313.
Krueger M. J., Tan A. K., Ackrell B. A. and Singer T. P. (1993) Is
complex II involved in the inhibition of mitochondrial respiration
by N-methyl-4-phenylpyridinium cation (MMP+) and N-methyl-
beta-carbolines? Biochem. J. 291, 673–676.
Lang A. E. and Lozano A. M. (1998a) Parkinson’s disease: second of
two parts. N. Engl. J. Med. 339, 1130–1143.
Lang A. E. and Lozano A. M. (1998b) Parkinson’s disease: first of two
parts. N. Engl. J. Med. 339, 1044–1053.
Le Couteur D. G., Leighton P. W., McCann S. J. and Pond S. (1997)
Association of a polymorphism in the dopamine-transporter gene
with Parkinson’s disease. Mov. Disord. 12, 760–763.
Matsubara K., Neafsey E. J. and Collins M. A. (1992) Novel S-adeno-
sylmethionine-dependent indole-N-methylation of beta-carbolines
in brain particulate fractions. J. Neurochem. 59, 511–518.
Matsubara K., Kobayashi S., Kobayashi Y., Yamashita K., Koide H.,
Hatta M., Iwamoto K., Tanaka O. and Kimura K. (1995) beta-
Carbolinium cations, endogenous MPP+ analogs, in the lumbar
cerebrospinal fluid of patients with Parkinson’s disease. Neurology
45, 2240–2245.
Matsubara K., Gonda T., Sawada H., Uezono T., Kobayashi Y.,
Kawamura T., Ohtaki K., Kimura K. and Akaike A. (1998a)
Endogenously occurring beta-carboline induces parkinsonism in
694 A. Storch et al.
nonprimate animals: a possible causative protoxin in idiopathic
Parkinson’s disease. J. Neurochem. 70, 727–735.
Matsubara K., Senda T., Uezono T. et al. (1998b) Structural significance
of azaheterocyclic amines related to Parkinson’s disease for dop-
amine transporter. Eur. J. Pharmacol. 348, 77–84.
Meiergerd S. M. and Schenk J. O. (1994) Striatal transporter for dop-
amine: catechol structure-activity studies and susceptibility to
chemical modification. J. Neurochem. 62, 998–1008.
Morino H., Kawarai T., Izumi Y., Kazuta T., Oda M., Komure O., Udaka
F., Kameyama M., Nakamura S. and Kawakami H. (2000) A single
nucleotide polymorphism of dopamine transporter gene is associ-
ated with Parkinson’s disease. Ann. Neurol. 47, 528–531.
Nishimura M., Kaji R., Ohta M., Mizuta I. and Kuno S. (2002)
Association between dopamine transporter gene polymorphism and
susceptibility to Parkinson’s disease in Japan. Mov. Disord. 17,
831–832.
Pakkenberg B., Moller A., Gundersen H. J., Mouritzen Dam A. and
Pakkenberg H. (1991) The absolute number of nerve cells in
substantia nigra in normal subjects and in patients with Parkinson’s
disease estimated with an unbiased stereological method. J. Neurol.
Neurosurg. Psychiatry 54, 30–33.
Pifl C., Giros B. and Caron M. G. (1993) Dopamine transporter
expression confers cytotoxicity to low doses of the parkinsonism-
inducing neurotoxin 1-methyl-4-phenylpyridinium. J. Neurosci.
13, 4246–4253.
Pifl C., Hornykiewicz O., Giros B. and Caron M. G. (1996) Catechol-
amine transporters and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-
dine neurotoxicity: studies comparing the cloned human
noradrenaline and human dopamine transporter. J. Pharmacol.
Exp. Ther. 277, 1437–1443.
Przedborski S. (2000) Advances in our knowledge of MPTP action and
mechanism. In: Neurotoxic Factors in Parkinson’s Disease and

2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 685–694
Related Disorders (Storch, A. and Collins, M. A., eds), pp. 41–54.
Kluwer Academic, Plenum Publishers, New York, USA.
Spenser I. D. (1956) The structure of N-alkyl-b-carboline anhydro-bases.
J. Chem. Soc. 3659–3663.
Storch A. and Schwarz J. (2000) The dopamine transporter: Involvement
in selective dopaminergic degeneration, In: Neurotoxic Factors in
Parkinson’s Disease and Related Disorders (Storch, A. and Col-
lins, M. A., eds), pp. 17–40. Kluwer Academic, Plenum Press,
New York, USA.
Storch A., Ludolph A. C. and Schwarz J. (1999) HEK-293 cells
expressing the human dopamine transporter are susceptible to low
concentrations of 1-methyl-4-phenylpyridine (MPP+) via impair-
ment of energy metabolism. Neurochem. Int. 35, 393–403.
Storch A., Ott S., Hwang Y. I., Ortmann R., Hein A., Frenzel S., Mat-
subara K., Ohta S., Wolf H. U. and Schwarz J. (2002) Selective
dopaminergic neurotoxicity of isoquinoline derivatives related to
Parkinson’s disease: studies using heterologous expression systems
of the dopamine transporter. Biochem. Pharmacol. 63, 909–920.
Sugimura T. and Nagao M. (1979) Mutagenic factors in cooked foods.
CRC Crit Rev. Toxicol. 6, 189–209.
Wakabayashi K., Totsuka Y., Fukutome K., Oguri A., Ushiyama H. and
Sugimura T. (1997) Human exposure to mutagenic/carcinogenic
heterocyclic amines and comutagenic beta-carbolines. Mutat. Res.
376, 253–259.
Waring R. H., Steventon G. B., Sturman S. G., Heafield M. T., Smith M.
C. and Williams A. C. (1989) S-Methylation in motorneuron dis-
ease and Parkinson’s disease. Lancet 2, 356–357.
Waters C. M., Peck R., Rossor M., Reynolds G. P. and Hunt S. P. (1988)
Immunocytochemical studies on the basal ganglia and substantia
nigra in Parkinson’s disease and Huntington’s chorea. Neuro-
science 25, 419–438.