In vitro neuronal and vascular responses to 5-hydroxytryptamine: Modulation by 4-methylthioamphetamine, 4-methylthiomethamphetamine and 3,4-methylenedioxymethamphetamine

Article abstract of DOI:10.1016/S0014-2999(02)01586-8

4-Methylthioamphetamine and 4-methylthiomethamphetamine are thioarylethylamines structurally related to 3,4-methylenedioxymethamphetamine (MDMA, 'Ecstasy'). This study compared effects of these agents and MDMA on 5-hydroxytryptamine (5-HT) signalling systems in the brain and vasculature in vitro. Both 4-methylthioamphetamine and 4-methylthiomethamphetamine (100 μM) reduced the rate of specific high affinity [³H]5-HT reuptake in isolated rat brain synaptosomes to 14% and 10% of control, respectively. The concentration required for half-maximal inhibition (IC₅₀) of [³H]5-HT reuptake by 4-methylthioamphetamine (0.27 μM) was significantly lower (P<0.005) than that for inhibition by MDMA (1.28 μM) and that for inhibition by 4-methylthiomethamphetamine (0.89 μM). Both 4-MTA and 4-MTMA caused a significant release of preloaded [³H]5-HT from synaptosomes, but were significantly less effective than MDMA at the concentrations tested (1-100 μM). In isolated rat aorta, a 15-min preincubation with 4-methylthioamphetamine or 4-methylthiomethamphetamine significantly reduced the maximal contraction (E_max) induced by 5-HT to 71% or 91% of control (P<0.05 in each case), respectively. In addition, 4-methylthiomethamphetamine (100 μM), but not 4-methylthioamphetamine, significantly increased the concentration of 5-HT required for half-maximal contraction (EC₅₀) from 4.13 to 20.08 μM (P<0.0001). In contrast, MDMA did not significantly alter the E_max or the EC₅₀ of 5-HT-induced aortic contraction. It is concluded that both 4-methylthioamphetamine and 4-methylthiomethamphetamine are potent inhibitors of [³H]5-HT reuptake in the brain. Furthermore, unlike MDMA, they both directly inhibit 5-HT-mediated vascular contraction. These results suggest that these compounds may be potentially more harmful than MDMA in the context of human misuse.

Full text of DOI:10.1016/S0014-2999(02)01586-8

European Journal of Pharmacology 444 (2002) 61–67
www.elsevier.com/locate/ejphar
In vitro neuronal and vascular responses to 5-hydroxytryptamine:
modulation by 4-methylthioamphetamine, 4-methylthiomethamphetamine
and 3,4-methylenedioxymethamphetamine
James E.J. Murphy a,b, James J. Flynn a, Dara M. Cannon a,b, Patrick J. Guiry c,
Peter McCormack c, Alan W. Baird d, Gethin J. McBean a, Alan K. Keenan b,
*
aDepartment of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland
bDepartment of Pharmacology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland
cCentre for Synthesis and Chemical Biology, Department of Chemistry, Conway Institute of Biomolecular and Biomedical Research,
University College Dublin, Dublin 4, Ireland
dDepartment of Veterinary Physiology and Biochemistry, Conway Institute of Biomolecular and Biomedical Research,
University College Dublin, Dublin 4, Ireland
Received 27 March 2002; accepted 5 April 2002
Abstract
4-Methylthioamphetamine and 4-methylthiomethamphetamine are thioarylethylamines structurally related to 3,4-methylenedioxyme-
thamphetamine (MDMA, ‘Ecstasy’). This study compared effects of these agents and MDMA on 5-hydroxytryptamine (5-HT) signalling
systems in the brain and vasculature in vitro. Both 4-methylthioamphetamine and 4-methylthiomethamphetamine (100 AM) reduced the rate
of specific high affinity [3H]5-HT reuptake in isolated rat brain synaptosomes to 14% and 10% of control, respectively. The concentration
required for half-maximal inhibition (IC50) of [3H]5-HT reuptake by 4-methylthioamphetamine (0.27 AM) was significantly lower ( P<0.005)
than that for inhibition by MDMA (1.28 AM) and that for inhibition by 4-methylthiomethamphetamine (0.89 AM). Both 4-MTA and 4-
MTMA caused a significant release of preloaded [3H]5-HT from synaptosomes, but were significantly less effective than MDMA at the
concentrations tested (1–100 AM). In isolated rat aorta, a 15-min preincubation with 4-methylthioamphetamine or 4-methylthiomethamphet-
amine significantly reduced the maximal contraction (Emax) induced by 5-HT to 71% or 91% of control ( P<0.05 in each case), respectively.
In addition, 4-methylthiomethamphetamine (100 AM), but not 4-methylthioamphetamine, significantly increased the concentration of 5-HT
required for half-maximal contraction (EC50) from 4.13 to 20.08 AM ( P<0.0001). In contrast, MDMA did not significantly alter the Emax or
the EC50 of 5-HT-induced aortic contraction. It is concluded that both 4-methylthioamphetamine and 4-methylthiomethamphetamine are
potent inhibitors of [3H]5-HT reuptake in the brain. Furthermore, unlike MDMA, they both directly inhibit 5-HT-mediated vascular
contraction. These results suggest that these compounds may be potentially more harmful than MDMA in the context of human misuse.
D 2002 Elsevier Science B.V. All rights reserved.
Keywords: 4-MTA (4-methylthioamphetamine); 4-MTMA (4-methylthiomethamphetamine); MDMA (3,4-methylenedioxymethamphetamine); 5-HT (5-
hydroxyhyptamine, serotonin); Reuptake; Synaptosome; Aortic contraction
1. Introduction
amines, alcohol and methadone) were detected in 4-MTA-
containing samples (EMCDDA, 1999; Elliott, 2000). 4-
MTA has also been identified as a constituent of a so-called
herbal stimulant (De Boer et al., 1999).
4-Methylthiomethamphetamine (4-MTA) has recently
emerged as a 3,4-methylenedioxymethamphetamine
(MDMA)-like drug of abuse, and was classified as a
Schedule 1 controlled substance in 1999 (Groombridge,
1998; Poortman and Lock, 1999). Five deaths involving
4-MTA were recorded in the European Union in that year,
although in all but one case, other agents (MDMA, amphet-
The major acute neurochemical effects of 4-MTA in the
rat are an increase in 5-hydroxytryptamine (5-HT) release
and an inhibition of 5-HT reuptake, as well as inhibition of
monoamine oxidase A (Scorza et al., 1999; Huang et al.,
1992), which are all well-documented effects of MDMA
(see review by Green et al., 1995). Unlike MDMA, how-
ever, 4-MTA reportedly lacks toxicity to serotonergic neuro-
nes, on the basis of a study showing that no long-term
*
Corresponding author. Tel.: +353-1-7161561; fax: +353-1-2692749.
E-mail address: alan.keenan@ucd.ie (A.K. Keenan).
0014-2999/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S0014-2999(02)01586-8

62
J.E.J. Murphy et al. / European Journal of Pharmacology 444 (2002) 61–67
nitrostyrene derivative obtained from the nitroaldol/dehy-
dration reaction of 4-methylthiobenzaldehyde and nitro-
ethane (Butterick and Unrau, 1974). 4-MTMA was synthes-
ised by a variation of the method of Butterick and Unrau
(1974). MDMA, 4-MTA and 4-MTMA were prepared as
their (F)-HCl salts. Their structures and purity ( > 99%)
were confirmed by NMR, microanalysis and mass spectro-
scopy. All concentrations of MDMA, 4-MTA and 4-MTMA
used refer to the (F)-HCl salt.
Fig. 1. The chemical structures of 4-methylthioamphetamine (R=H) and 4-
methylthiomethamphetamine (R=CH3).
2.2. Synaptosomal preparation
depletion of 5-HT occurred following acute administration
to rats (Huang et al., 1992). 4-Methylthiomethamphetamine
(4-MTMA) has not been identified to date on the illicit
market and there have been no reports of its effects on
serotonergic neurones. However, due to its structural sim-
ilarity to 4-MTA and because it is N-methylated, as is
MDMA (Fig. 1), it is probable that 4-MTMA also targets
5-HT-releasing neurones.
Wistar rats (200–250 g) were killed by cervical disloca-
tion and the brains quickly removed and placed in homog-
enisation medium at 4 jC containing 0.32 M sucrose, 1.0
mM EDTA and 0.25 mM dithiothreitol. Whole brain (minus
the cerebellum) was homogenised and a synaptosomal
fraction isolated by Percoll density gradient centrifugation,
according to the method of Dunkley et al. (1988). The
synaptosomal fraction was removed and washed twice in
ice-cold Krebs’ bicarbonate medium of the following com-
position (mM): NaCl 109.6, KCl 4.72, KH2PO4 1.2, MgSO4
1.2, NaHCO3 25, D-(+)-glucose 11 and CaCl2 2.5, pH 7.4,
gassed with 95% O2/5% CO2, followed by centrifugation
for 15 min at 16,000g. The final pellet, containing purified
synaptosomes, was resuspended in Krebs’ medium and
maintained on ice until required.
The only reported cardiovascular effects of 4-MTA are a
decrease in blood pressure and heart rate in rats treated with 5
mg/kg 4-MTA (Li et al., 1996). There are no reports to date of
4-MTMA actions in the cardiovascular system. MDMA, on
the other hand, is known to induce cardiac arrhythmias
(Dowling et al., 1987; Milroy et al., 1996), sudden cardiac
death (Suarez and Riemersma, 1988), ventricular tachycar-
dia, hypertension (Hayner and McKinney, 1986; Vollen-
weider et al., 1998; Mas et al., 1999) and impaired
parasympathetic activity (Brody et al., 1998) in humans. In
rats, MDMA has been reported to increase heart rate (Gordon
et al., 1991). Interestingly, we have recently demonstrated
impairment of a peripheral 5-HT-mediated response in rats
chronically treated with MDMA (Cannon et al., 2001): twice
daily administration of 20 mg/kg MDMA for up to 4 days
resulted in reduced 5-HT-mediated contraction of aorta iso-
lated 1 or 7 days later.
2.3. Synaptosomal transport procedure
5-HT transport was initiated by the addition of 25 Al of
the synaptosomal fraction to 975 Al of Krebs’ bicarbonate
medium, maintained at 37 jC, containing [3H]5-HT (4.0–
6.0Â105 Bq) diluted with unlabelled 5-HT to give a final
concentration of 25 nM–1 AM (final specific activity 80–
2400 Bq/pmol). Transport was arrested after 4 min by the
addition of 200 Al of ice-cold 4 mM 5-HT and the tubes
were immediately placed on ice prior to centrifugation for
10 min at 13,000g at 4 jC. The pellet was washed with
homogenisation buffer and centrifuged as before. The final
pellet was solubilised overnight in 2% sodium dodecyl
sulphate and radiolabel incorporation was measured by
liquid scintillation spectroscopy. Rates of transport are
expressed as specific uptake of [3H]5-HT (pmol/min/mg
protein). This was determined by subtracting nonspecific
transport (defined in the presence of 100 AM alaproclate)
from total transport. 4-MTA, 4-MTMA or MDMA were
added at time zero of the transport assay and measurements
were performed on each synaptosomal fraction in triplicate.
Protein was determined by the method of Markwell et al.
(1978).
The aim of the present study was to increase knowledge
of the structural specificity of the action of amphetamine-
like compounds on 5-HT signalling systems in rat brain and
vasculature. This was addressed by comparing the effects of
4-MTMA, 4-MTA and MDMA on the reuptake of 5-HT and
release of preloaded [3H]5-HT from rat brain synaptosomes,
as well as on the responsiveness of isolated rat aorta to 5-
HT.
2. Materials and methods
2.1. Synthesis of MDMA, 4-MTA and 4-MTMA
MDMA was synthesised by the method of Braun et al.
(1980). In brief, isosafrole was oxidised to 3,4-methylene-
dioxyphenylacetone, which was then reacted with methyl-
amine. The product of this reaction was treated with sodium
cyanoborohydride to yield the corresponding MDMA deriv-
ative. 4-MTA was synthesised via LiAlH4 reduction of the
2.4. [3H]5-HT release from synaptosomes
Release of preloaded [3H]5-HT from crude synapto-
somes was measured using the method of Rothman et al.

J.E.J. Murphy et al. / European Journal of Pharmacology 444 (2002) 61–67
63
Table 1
Inhibition of [3H]5-HT uptake by MDMA, 4-MTA and 4-MTMA
2.6. Materials
[3H]-5-hydroxytryptamine (specific activity=944 GBq
(25.5 Ci)/mmol) was purchased from NEN, Amsterdam.
Alaproclate HCl and nomifensine were purchased from
RBI, UK. All other chemicals and compounds were pur-
chased from Sigma, Dorset, UK and were of the highest
grade commercially available.
5-HT (nM)
Rate of transport (pmol/mg protein/min)
50
100
500
No drug
4.54F0.84
0.23F0.04*
0.23F0.14*
0.35F0.05*
10.38F1.76
0.55F0.19**
1.02F0.26**
1.22F0.39*
18.34F1.96
4-MTA (100 AM)
4-MTMA (100 AM)
MDMA (100 AM)
2.61F0.35***
1.99F1.36***
2.44F0.59**
Specific (alaproclate-sensitive) transport of 5-HT was determined at 50, 100
and 500 nM [3H]5-HT in the absence or presence of 100 AM 4-MTA, 4-
MTMA or MDMA for 4 min at 37 jC. The results are the meansFS.E.M.
of at least three independent determinations measured in triplicate.
* P<0.05 compared to uptake of the corresponding concentration of
[3H]5-HT in the absence of drug.
2.7. Data analysis
Data were analysed using the software package Kaleida-
Graph under nonlinear regression analysis and fitting to
either a one site rectangular hyperbola (Michaelis Menten
curve) or sigmoidal concentration–response curve as appro-
priate. Determination of statistical significance was per-
formed by a two-tailed unpaired Student’s t-test, and a P
value <0.05 was considered significant. Data are presented
as the meansFS.E.M. for n determinations as indicated.
** P<0.01 compared to uptake of the corresponding concentration of
[3H]5-HT in the absence of drug.
*** P<0.001 compared to uptake of the corresponding concentration of
[3H]5-HT in the absence of drug.
(2000). Briefly, rat whole brain was homogenised in ice-
cold 10% sucrose containing 1 AM reserpine, 100 nM
nomifensine and 100 nM GBR12935 to block any potential
[3H]5-HT reuptake into noradrenergic and dopaminergic
nerve terminals. The homogenate was centrifuged at
1000g for 10 min at 4 jC, and the supernatant (‘synapto-
somal preparation’) was incubated with 5 nM [3H]5-HT
(specific activity 9.65Â105 Bq (26 ACi)/nmol) in Krebs’
phosphate buffer pH 7.4, containing 154.4 mM NaCl, 2.9
mM KCl, 4.9 mM KH2PO4, 1.1 mM CaCl2, 0.83 mM
MgCl2, 5 mM glucose, 1 mg/ml ascorbic acid, 50 AM
pargyline, 1 AM reserpine, 100 nM nomifensine and 100
nM GBR12935. After preincubation, synaptosomes (900 Al)
were incubated for 5 min at 25 jC in the presence of test
drug. The releasing action of each drug was terminated by
dilution with 4 ml wash buffer (10 mM Tris–HCl, pH 7.4,
containing 0.9% NaCl at 25 jC), followed by rapid vacuum
filtration over GF/B filters. The quantity of radioactivity
retained by the filters was determined by liquid scintillation
counting following overnight solubilisation in 5 ml Ecoscint
(National Diagnostics, UK). Nonspecific binding of [3H]5-
HT was measured by incubations in the presence of 100 AM
tyramine. The results are expressed as the fraction (%) of
radioactivity retained by the tissue in the absence of drug.
3. Results
3.1. The effects of 4-MTA, 4-MTMA and MDMA on
synaptosomal [3H]5-HT transport
The maximum rate of [3H]5-HT reuptake into isolated rat
synaptosomes (Vmax) was 20.16F2.77 pmol/min/mg protein
with a Km (substrate concentration required for half-max-
imal rate of uptake) of 395F99 nM. Addition of either 4-
MTA, 4-MTMA or MDMA (100 AM) almost completely
abolished [3H]5-HT uptake over the entire concentration
range of [3H]5-HT (1–500 nM; Table 1). For example, at
500 nM [3H]5-HT, 4-MTA, 4-MTMA, or MDMA reduced
the rate of reuptake to 14%, 10%, or 13% of control,
respectively. Kinetic analysis of [3H]5-HT uptake in the
presence of each of the drugs showed a significant reduction
in the Vmax, without any significant change in the Km, as
Table 2
Kinetic analysis of synaptosomal [3H]5-HT uptake by MDMA, 4-MTA and
4-MTMA
Drug
Km (nM)
Vmax (pmol/mg protein/min)
2.5. Measurement of aortic contraction
None
395F99
426F106
296F120
681F155
20.16F2.77
4.95F1.69*
3.66F2.35*
6.11F1.00**
4-MTA
4-MTMA
MDMA
Descending thoracic aortae were isolated, mounted in a
tissue bath, attached to a force transducer and maintained at
37 jC in Krebs’–Hensleit bicarbonate buffer of the follow-
ing composition (mM): NaCl 112.6, KCl 4.7, KH2PO4 1.2,
MgSO4 –7H2O 1.2, NaHCO3 25, (D)-(+)-glucose 12,
CaCl2–2H2O 1.9, gassed with 95% O2/5% CO2. After
equilibration of rings for 45 min at a basal tension of 1.3
g, changes in isometric tension were measured after 15 min
preincubation with drug, using the PowerLab data acquis-
ition package.
Specific (alaproclate-sensitive) uptake was determined over a concentration
range of 25–500 nM [3H]5-HT for 4 min at 37 jC. Drugs (100 AM) were
added at time zero of the assay, and the rate of uptake was determined at
each concentration of substrate in triplicate in at least four independent
experiments. The Km and Vmax were determined by nonlinear regression
analysis by applying the Michaelis Menten equation to the data and are
expressed as the meansFS.E.M.
* P<0.01 compared to data obtained in the absence of drug.
** P<0.05 compared to data obtained in the absence of drug.

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J.E.J. Murphy et al. / European Journal of Pharmacology 444 (2002) 61–67
3.2. The effects of 4-MTA, 4-MTMA and MDMA on [3H]5-
HT release from synaptosomes
4-MTA, 4-MTMA and MDMA (1–100 AM) all caused
release of preloaded [3H]5-HT from synaptosomes, but the
quantity of [3H]5-HT released by MDMA was significantly
greater than for either 4-MTA or 4-MTMA. For example, the
quantity of radioactivity retained by the synaptosomes after 5
min incubation with 10 AM MDMA fell to 42.7F3.5% of
control, whereas this figure was 60.0F2.7% of control for 10
AM 4-MTA and 58.3F4.3% of control for 10 AM 4-MTMA
(Fig. 3). With 100 AM drug, the quantity of [3H]5-HT
released was 52.0F2.5% (MDMA), 70.3F2.3% (4-MTA)
and 66.5F4.6% of control (4-MTMA). There was no sig-
nificant difference in the amount of [3H]5-HT released by 4-
MTA and 4-MTMA at any of the concentrations tested.
3.3. The effects of 4-MTA, 4-MTMA and MDMA on 5-HT-
induced rat aortic ring contraction
Preincubation of aortic rings with 100 AM 4-MTA for 15
min significantly reduced ( P<0.05) the maximal contraction
(Emax) to 5-HT to 71% (0.84F0.03 g) that of control rings
(1.20F0.06 g), as shown in Fig. 4A. Similar treatment with
4-MTMA significantly reduced ( P<0.05) the Emax to 91%
(1.07F0.05 g) that of control rings (1.17F0.07 g; Fig. 4B)
while control Emax values (0.92F0.02 g) were not signifi-
cantly altered (0.82F0.02 g) in rings treated with 100 AM
MDMA (Fig. 4C). The concentrations of 5-HT required for
half-maximal contraction (EC50) in control, 4-MTA-treated
and 4-MTMA-treated tissues were 4.13, 8.75 and 20.08 AM,
Fig. 2. Inhibition of synaptosomal 5-HT uptake by 4-MTA, 4-MTMA and
MDMA. Synaptosomes were incubated for 4 min at 37 jC in the presence
of [3H]5-HT as specified in Section 2. (A) IC50 plot showing the inhibition
of uptake of 500 nM [3H]5-HT by 1 nM–1 mM 4-MTA (o), 4-MTMA (5)
or MDMA ( w ). (B) IC50 plot showing the inhibition of uptake of 50 nM
(
) and 500 nM ( w ) [3H]5-HT by MDMA. Data are presented as the
y
meansFS.E.M. of four individual experiments, each performed in triplicate.
The data were analysed by fitting to a sigmoidal concentration–response
curve using the KaleidaGraph application from SynergyR.
indicated in Table 2. To determine the IC50 for inhibition of
[3H]5-HT reuptake, a range of inhibitor concentrations (10
nM–100 AM) was tested at a substrate concentration of 500
nM 5-HT and the concentration–response curves for 4-
MTA, 4-MTMA and MDMA are shown in Fig. 2A. The
corresponding IC50 values (midpoints of each curve) were:
0.27 AM for 4-MTA ( P<0.005 w.r.t. 4-MTMA or MDMA);
0.89 AM for 4-MTMA and 1.28 AM for MDMA. The IC50
for inhibition of [3H]5-HT uptake by MDMA was not
altered when the substrate concentration was lowered to
50 nM 5-HT (1.35 AM; Fig. 2B).
Fig. 3. Stimulation of efflux of [3H]5-HT from synaptosomes by 4-MTA, 4-
MTMA and MDMA. Release of preloaded [3H]5-HT was measured for 5
min at 25 jC in the presence of 1, 10 and 100 AM 4-MTA, 4-MTMA or
MDMA, as described in Section 2. The results are expressed as the fraction
(%) of the radioactivity retained by the tissue in the absence of drug, and are
presented as the meansFS.E.M. of four separate experiments, each
measured in triplicate. 5 No drug, 4-MTA, 4-MTMA, l MDMA.

J.E.J. Murphy et al. / European Journal of Pharmacology 444 (2002) 61–67
65
4-MTA was not significantly different from control. Neither
4-MTA, 4-MTMA, nor MDMA themselves induced con-
traction in the aorta in the absence of 5-HT (data not shown).
4. Discussion
This study shows that 4-MTA and 4-MTMA, like
MDMA target 5-HT signalling systems in brain and vascu-
lature. In the brain, MDMA, 4-MTA and 4-MTMA are all
effective inhibitors of synaptosomal [3H]5-HT transport
(Fig. 1). This is manifest as micromolar IC50 values for
[3H]5-HT reuptake into cerebral synaptosomes. Steele et al.
(1987) also showed that synaptosomal uptake of 5-HT is
directly blocked by MDMA, with an IC50 that compares
well with the data from our experiments. In the case of 4-
MTA, our data confirm the earlier work of Huang et al.
(1992) and show that 4-MTA is more potent than MDMA.
In a separate study reported by Johnson et al. (1991),
another para-substituted amphetamine, p-chloroamphet-
amine was also more potent than MDMA. N-methylation,
however, appears to result in a reduction of inhibitory
potency, since 4-MTMA and MDMA were both less effec-
tive inhibitors of [3H]5-HT reuptake than the nonmethylated
analogue, 4-MTA. Stone et al. (1987) compared the neuro-
toxic potential of 3,4-methylenedioxyamphetamine (MDA)
with that of its N-methylated (MDMA) and N-ethylated
derivatives: in that case, both MDA and MDMA dramati-
cally decreased brain concentrations of 5-HT, whereas the
N-ethylated compound was much less potent. The present
findings strengthen the conclusions of Stone et al. (1987)
that increased N-alkyl substitution leads to reduced potency.
Experiments have shown that both 4-MTA and 4-MTMA
cause release of preloaded [3H]5-HT from synaptosomes,
but that at the concentrations tested, they are less effective in
this regard than MDMA. Huang et al. (1992) also demon-
strated that 4-MTA stimulated [3H]5-HT release from rat
cortical slices over a similar concentration range and with a
potency comparable to that of p-chloroamphetamine. The
greater releasing action of MDMA shown in our experi-
ments suggests that replacement of the methylenedioxy
group in MDMA with the methylthio moiety in 4-MTA
and 4-MTMA has a greater impact on the ability of these
compounds to cause release of 5-HT than does methylation
of the side chain, as in MDMA and 4-MTMA. Thus, the
structural specificity for stimulation of 5-HT release differs
from that of either inhibition of 5-HT transport or 5-HT-
mediated contraction of the aorta, which is discussed below.
There is no direct evidence demonstrating accumulation
of MDMA, 4-MTA or 4-MTMA into synaptosomes, never-
theless, the ability of these compounds to stimulate 5-HT
release is consistent with their being taken up by the 5-HT
transporter, resulting in drug/5-HT exchange, which would
not be expected for a nontransported inhibitor. This view is
strengthened by the observation that the 5-HT releasing
action of MDMA can be blocked by 5-HT uptake inhibitors,
Fig. 4. The effect of 4-MTA, 4-MTMA and MDMA on 5-HT induced
contraction in the rat thoracic aortic ring. Isolated rings were mounted in a
tissue bath, and either 100 AM 4-MTA (o) (A), 4-MTMA (5) (B) or
MDMA ( w ) (C) were added 15 min prior to addition of 5-HT. Control
responses to 5-HT are also shown (Â). Data are presented as the
meansFS.E.M. of five (4-MTA, 4-MTMA) or nine (MDMA) individual
experiments, each performed in duplicate. The data were analysed by fitting
to a sigmoidal concentration–response curve to determine the EC50 and
Emax for aortic contraction.
respectively (EC50 for 4-MTMA significantly different from
that for control, and 4-MTA, P<0.0001 in each case). In
separate experiments, EC50 values for control and MDMA-
treated tissues were not significantly different (2.99 and 5.39
AM, respectively). Additionally, the EC50 in the presence of

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J.E.J. Murphy et al. / European Journal of Pharmacology 444 (2002) 61–67
such as fluoxetine (Hekmatpanah and Peroutka, 1990), and
the same is true for p-chloroamphetamine-stimulated 5-HT
release (Rudnick and Wall, 1992). It is likely that 4-MTA
and 4-MTMA also act by a similar mechanism, although our
data do not rule out the possibility that these compounds, by
binding to the transporter, initiate uncoupled efflux of 5-HT.
The altered order of potency of inhibition of transport
between 4-MTA, 4-MTMA and MDMA, as opposed to
stimulation of 5-HT efflux, may be an indication that
structural modification of MDMA alters the balance
between behaviour as a substrate or as an inhibitor of the
5-HT transporter: 4-MTA and 4-MTMA may not be as well
taken up as MDMA, although they are better inhibitors of 5-
HT transport.
which is followed later by a central action; this might also
explain the reportedly slower onset of its central effects
compared to MDMA (EMCDDA, 1999).
The evidence presented here for peripheral vascular
effects of 4-methylthio substituted amphetamines would
be consistent with targeting by these agents of a wide range
of peripheral organs, following oral administration. Indeed,
significant levels of MDMA are known to be concentrated
in organs such as the liver, bladder and intestine following
oral administration (Cho et al., 1990; Sakai et al., 1983), and
it is likely that the 4-methylthio compounds, being lip-
ophilic, would also accumulate at these sites.
In conclusion, this study demonstrates that whereas 4-
MTA is a more potent inhibitor than MDMA and 4-MTMA
of [3H]5-HT reuptake in the brain, MDMA is most potent at
causing [3H]5-HT release. Both 4-MTA and 4-MTMA,
unlike MDMA, are potent inhibitors of 5-HT-mediated
responses in the vasculature. Whether MDMA and its 4-
methylthio analogues specifically target 5-HT systems, or
also modulate noradrenergic neurotransmission in the brain
and periphery, is currently the subject of a comprehensive
evaluation of the central and peripheral vascular toxicity of
these compounds.
Both 4-MTA and 4-MTMA were effective inhibitors of
5-HT-mediated contraction of the rat aorta in vitro. This
result is particularly interesting, in view of the fact that
MDMA had no significant effect in this respect and raises
the question of how 4-MTA and 4-MTMA might differ in
their mechanism of action from MDMA. In contrast to the
brain, MDMA does not inhibit transport of 5-HT by non-
neuronal transporters in the aorta (Cannon et al., 2001). It is
not known whether 4-MTA or 4-MTMA affect the function
of such transporters in the peripheral vasculature, but an
inhibition of 5-HT reuptake into nonneuronal tissue would
not be expected to lead to the reduced 5-HT responsiveness
observed in our experiments The fact that neither 4-MTA
nor 4-MTMA altered contractility directly (data not shown),
coupled with the observed shifts in the concentration–
response curves (Fig. 4A and B) suggests that these agents
may be acting as antagonists, though the nature of this
antagonism is unclear at this stage.
Acknowledgements
This work was funded by the Conway Institute.
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