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ACCELERATED COMMUNICATION
3,4-Methylenedioxymethamphetamine (MDMA, “Ecstasy”)
Induces Fenfluramine-Like Proliferative Actions on Human
Cardiac Valvular Interstitial Cells in Vitro
VINCENT SETOLA, SANDRA J. HUFEISEN, K. JANE GRANDE-ALLEN, IVAN VESELY, RICHARD A. GLENNON,
BRUCE BLOUGH, RICHARD B. ROTHMAN, and BRYAN L. ROTH
Departments of Biochemistry (V.S., S.J.H., B.L.R.) and the National Institutes of Mental Health Psychoactive Drug Screening Program (S.J.H.,
B.L.R.), Case Western Reserve University School of Medicine, Cleveland, Ohio; Department of Biomedical Engineering, Lerner Research
Institute, Cleveland Clinic Foundation, Cleveland, Ohio, (K.J.G.-A., I.V.); Department of Medicinal Chemistry, Virginia Commonwealth University,
Richmond, Virginia (R.A.G.); Chemistry and Life Sciences Group, Research Triangle Institute International, Research Triangle Park, North
Carolina (B.B.); and Clinical Psychopharmacology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes
of Health, Baltimore, Maryland (R.B.R.)
Received February 10, 2003; accepted March 18, 2003 This article is available online at http://molpharm.aspetjournals.org
ABSTRACT
Recent findings have implicated the 5-hydroxytryptamine 2B (5-
HT
2B
) serotonin receptor in mediating the heart valve fibroplasia
[valvular heart disease (VHD)] and primary pulmonary hyperten-
sion observed in patients taking the now-banned appetite sup-
pressant fenfluramine (Pondimin, Redux). Via large-scale, random
screening of a portion of the receptorome, we have discovered
that the amphetamine derivative 3,4-methylenedioxymetham-
phetamine (MDMA, “Ecstasy”) and its N-demethylated metabolite
3,4-methylenedioxyamphetamine (MDA) each preferentially bind
to and activate human recombinant 5-HT
2B
receptors. We also
demonstrate that MDMA and MDA, like fenfluramine and its N-de-
ethylated metabolite norfenfluramine, elicit prolonged mitogenic
responses in human valvular interstitial cells via activation of
5-HT
2B
receptors. We also report that pergolide and dihydroer-
gotamine, two drugs recently demonstrated to induce VHD in
humans, potently activate 5-HT
2B
receptors, thus validating this
assay system for its ability to predict medications that might
induce VHD. Our discovery that MDMA and a major metabolite,
MDA, induce prolonged mitogenic responses in vitro similar to
those induced by fenfluramine and norfenfluramine in vivo (i.e.,
valvular interstitial cell fibroplasia) predict that long-term MDMA
use could lead to the development of fenfluramine-like VHD. Be-
cause of the widespread abuse of MDMA, these findings have
major public health implications. These findings also underscore
the necessity of screening current and future drugs at h5-HT
2B
receptors for agonist actions before their use in humans.
In September of 1997, the highly effective appetite sup-
pressant fenfluramine (Pondimin), a component of the
drug combination “Fen-Phen”, and the optically pure (⫹)-
isomer dexfenfluramine (Redux) were voluntarily removed
the from the marketplace at the urging of the United
States Food and Drug Administration because of their
association with heart valve fibroplasia and dysfunction, a
condition known as valvular heart disease (VHD). Since
then, several independent echocardiographic studies of pa-
tients who received long-term fenfluramine therapy re-
vealed an increased prevalence of valvular heart disease
(Connolly et al., 1997; Jick et al., 1998; Weissman et al.,
1998; Weissman, 2001). Histopathological examination of
resected valves has revealed proliferative foci containing
interstitial cells and increased levels of extracellular ma-
trix (Steffee et al., 1999). Identical pathology has been seen
in resected valves harvested from persons undergoing
This work was supported in part by National Institutes of Mental Health
(NIMH) grants R01-MH57635, K02-MH01366, and the NIMH Psychoactive
Drug Screening Program (N01-MH80002) (to B.L.R.). V.S. was supported in
part by an American Heart Association Pre-Doctoral Award.
ABBREVIATIONS: VHD, valvular heart disease; 5-HT. 5-hydroxytryptamine; h, human; PPH, primary pulmonary hypertension; MDMA, 3,4-
methylenedioxymethamphetamine; HEK, human embryonic kidney; VIC, interstitial valvular cells; DMEM, Dulbecco’s modified Eagle’s medium;
MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; DA, dopamine; NE, norepinephrine; GBR12935, 1-[2-
(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine; IP, inositol phosphate; RTI-229, 3

-(4-iodophenyl)tropane-2

-pyrrolidine carboxamide;
MDA, 3,4-methylenedioxyamphetamine; SB206553, 5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole.
0026-895X/03/6306-1223–1229
MOLECULAR PHARMACOLOGY Vol. 63, No. 6
U.S. Government work not protected by U.S. copyright 2458/1069686
Mol Pharmacol 63:1223–1229, 2003 Printed in U.S.A.
1223
long-term administration of certain ergot derivatives (e.g.,
ergotamine and methysergide) and from those suffering
from carcinoid syndrome (Steffee et al., 1999).
Recently, we proposed that drugs (and/or their metabo-
lites) associated with VHD should preferentially bind with
high affinity to a single, proximal molecular target (receptor,
channel, or transporter), whereas similar medications (e.g.,
fluoxetine, phentermine) not associated with VHD would not
(Rothman et al., 2000). Via screening of VHD-associated and
non–VHD-associated drugs at a limited number of recombi-
nant receptors, transporters, and ion channels, we discovered
that VHD-associated drugs shared high affinity for only the
human 5-HT
2B
(h5-HT
2B
) receptor (Rothman et al., 2000). In
functional assays, we demonstrated that VHD-associated
drugs were all h5-HT
2B
receptor agonists (Rothman et al.,
2000). Fitzgerald et al. (2000) also reported that fenflura-
mine and a major metabolite, norfenfluramine, were agonists
at recombinant h5-HT
2B
receptors and independently sug-
gested that the 5-HT
2B
receptor was responsible for fenflu-
ramine-induced VHD. Launay et al. (2002) subsequently dis-
covered that activation of the 5-HT
2B
receptor is also
responsible for fenfluramine-induced primary pulmonary hy-
pertension (PPH).
Recently, we pioneered the use of large-scale screening of
psychoactive drugs at a huge panel of recombinant receptors
(i.e., “receptorome”) to identify the
-opioid receptor as the
site of action of the novel hallucinogen Salvinorin A (Roth et
al., 2002; Sheffler and Roth, 2003). We now report the results
of a receptorome screen of the club drug 3,4-methyl-
enedioxymethamphetamine (MDMA, “Ecstasy”) that we per-
formed to identify novel molecular targets responsible for the
actions of MDMA in humans. These studies reveal that
MDMA, like fenfluramine and other drugs known to be as-
sociated with heart valve fibroplasia, preferentially binds to
and activates h5-HT
2B
receptors. Additionally, we demon-
strate that MDMA induces prolonged mitogenic responses in
primary cultures of human cardiac interstitial valve cells.
Because drugs that activate h5-HT
2B
receptors induce VHD
and PPH in humans, these findings have major public health
implications.
Materials and Methods
HEK293 and COS-7 Cell Culture and Transfection. Stably
and transiently transfected cells were maintained as detailed previ-
ously (Roth et al., 2002). For transfections with the h5-HT
2B
recep-
tor, HEK293 or COS-7 cells were seeded in 100-mm dishes and
transfected using 6
g of plasmid and 36
l of Fugene (Roche,
Indianapolis, IN) according to the manufacturer’s protocol as de-
scribed previously (Rothman et al., 2000).
Radioligand Binding Assays and Phosphatidylinositol Hy-
drolysis Assays. Radioligand binding assays were performed as
described previously using the resources of the National Institute of
Mental Health Psychoactive Drug Screening Program (Rothman et
al., 2000). Phosphatidylinositol hydrolysis assays were performed
using HEK293 cells transiently expressing human 5-HT
2B
receptors
as reported previously (Rothman et al., 2000).
VIC Isolation and Culture. Human heart valves were obtained
from donor hearts deemed unsuitable for transplantation, or from
hearts that were removed from transplant recipients at the Cleve-
land Clinic Foundation. All Cleveland Clinic patients who have tis-
sue surgically removed have authorized its subsequent use for re-
search purposes (protocols approved by the CCF IRB 2378). To
remove the cells from the tissue, the specimens were placed into
sterile containers, immersed in a solution of collagenase-II (2 mg/ml;
Worthington Biochemicals, Freehold, NJ) in serum-free medium,
then agitated in an incubated shaker (140 rpm, 20 min, 37°C).
After return to the sterile flow hood, all surfaces were rubbed with
a sterile cotton swab to remove the endothelial cells. The valve
specimens were then finely minced and then digested with colla-
genase-III (1 mg/ml) in an incubated shaker (4 h, 140 rpm, 37°C).
Each resulting cell suspension was filtered (70
m) to remove
debris, and the cell pellet was resuspended in DMEM/Ham’s F12
medium (1:1, containing low glucose with HEPES) supplemented
with 10% fetal bovine serum and 1% antibiotic-antimycotic solu-
tion (Invitrogen, Carlsbad, CA). The culture was incubated in a
humidified atmosphere of 95% air/5% CO
2
at 37°C with changes of
medium every 48 h.
[
3
H]Thymidine Deoxyribose Incorporation Assay. Subcon-
fluent VIC seeded in 24-well clusters were incubated overnight in
serum-free DMEM (Invitrogen). Cells were then treated over the
course of 3 days with various concentrations of test agents. Twelve
hours before the end of the treatment period, cells were pulsed with
2to5
Ci/ml [
3
H]thymidine deoxyribose (PerkinElmer Life Sciences,
Boston, MA). After treatment, the medium was removed and the
cells were washed thoroughly with ice-cold phosphate-buffered sa-
line, pH 7.4. Ice-cold 10% trichloroacetic acid was then added and the
cells were incubated for 30 min at 4°C. The cells were again washed
thoroughly with ice-cold phosphate-buffered saline, pH 7.4, and then
lysed in 0.5 N NaOH. After neutralization with glacial acetic acid,
samples were assayed for [
3
H]thymidine deoxyribose incorporation
by liquid scintillation counting. Values are reported as the mean ⫾
S.E.M. of triplicate determinations and are representative of three
independent experiments.
Immunoblot Analysis of Erk 1/2
MAPK
Phosphorylation.
VIC seeded in 24-well clusters were incubated overnight first in
DMEM containing 5% dialyzed fetal bovine serum and then in
serum-free DMEM. Cells were treated over the course of 15 min
with 10
M fenfluramine, norfenfluramine, MDMA, or MDA. Af-
ter treatment, the medium in each well was replaced with 200
l
of 1⫻Laemmli sample buffer and collected. Samples were re-
solved on 10% SDS-polyacrylamide gels and electroblotted onto
nitrocellulose membranes. The membranes were probed for phos-
pho-Erk 1/2
MAPK
immunoreactivity using a 1:1000 dilution of
polyclonal primary antibody (Cell Signaling Inc., Beverly, MA)
and a 1:1000 dilution of horseradish peroxidase-conjugated goat
anti-rabbit IgG (Vector Laboratories, Burlingame, CA) according
to the manufacturer’s recommendations. Immunoreactivity was
revealed using LumiLight horseradish peroxidase substrate
(Roche) and imaged on a Kodak Digital Science Image Station
440CF (Eastman Kodak, Rochester, NY). Densitometric analysis
was performed using Scion Image software (Scion Corporation,
Frederick, MD). Samples were similarly analyzed for total Erk
1/2
MAPK
immunoreactivity, and the resulting values were used to
correct phospho-Erk 1/2
MAPK
measurements for slight differences
in sample protein content. Values are reported as the mean ⫾
S.E.M. of duplicate determinations and are representative of
three independent experiments.
[
3
H]DA, [
3
H]NE, and [
3
H]5-HT Release Assays. Following
published procedures (Rothman et al., 2001), rat caudate (for
[
3
H]DA release) or whole brain minus cerebellum and caudate (for
[
3
H]NE and [
3
H]5-HT release) was homogenized in ice-cold 10%
sucrose containing 1
M reserpine. Nomifensine (100 nM) and
GBR12935 (100 nM) were also added to the sucrose solution for
[
3
H]5-HT release experiments to block any potential [
3
H]5-HT
reuptake into NE and DA nerve terminals. After 12 strokes with
a Potter-Elvehjem homogenizer, homogenates were centrifuged at
1000gfor 10 min at 0 to 4°C and the supernatants were retained
on ice (synaptosomal preparation). Each rat brain (approximately
1200 mg) produced enough tissue for 250 test tubes for the [
3
H]DA
and [
3
H]5-HT release assays and for 125 test tubes for the [
3
H]NE
release assay.
1224 Setola et al.
Synaptosomal preparations were incubated to steady state with 5
nM [
3
H]DA (30 min), 7 nM [
3
H]NE (60 min), or 5 nM [
3
H]5-HT (60
min) in uptake buffer without bovine serum albumin, plus 1
M
reserpine, in a polypropylene beaker with stirring at 25°C. Nomi-
fensine (100 nM) and GBR12935 (100 nM) were added to the buffer
for [
3
H]5-HT release experiments, whereas RTI-229 (5 nM) was
added to the buffer for [
3
H]NE release experiments. After incubation
to steady state, 850
l of synaptosomes preloaded with [
3
H]neuro-
transmitter were added to 12 ⫻75-mm polystyrene test tubes that
contained 150
l of test drug in uptake buffer. After 5 min ([
3
H]DA
and [
3
H]5-HT) or 30 min ([
3
H]NE), the release reaction was termi-
nated by dilution with 4 ml of wash buffer (10 mM Tris-HCl, pH 7.4,
containing 0.9% NaCl at 25°C) followed by rapid vacuum filtration
over Whatman GF/B filters using a Brandel Harvester (Brandel Inc.,
Gaithersburg, MD). The filters were rinsed twice with 4 ml of wash
buffer using the Brandel Harvester, and the retained tritium was
counted by a Taurus liquid scintillation counter at 40% efficiency
after an overnight extraction in 3 ml of Cytoscint (ICN Biomedicals
Inc., Costa Mesa, CA).
Results
Screening the Receptorome Reveals the h5-HT
2B
Re-
ceptor As a Primary Molecular Target for MDMA and
MDA. To discover novel molecular targets for the effects of
psychoactive compounds (see Roth et al., 2002, for a recent
example), we screened the club drugs MDMA and MDA at a
Fig. 1. Large-scale screening of the receptorome reveals that MDMA preferentially interacts with the human 5-HT
2B
serotonin receptor. Top, K
i
values
for various drugs screened at a large number of mainly human recombinant receptors, ion channels, or transporters using the resources of the National
Institute of Mental Health Psychoactive Drug Screening Program. For these studies, test compounds were initially screened at 10
M. When greater
than 50% inhibition of radioligand specific binding was obtained, K
i
values were determined in quadruplicate. A three-dimensional mesh plot of the
data was made in which the K
i
values were color-coded. The red arrow indicates that MDMA has preferentially high affinity for h5-HT2B receptors.
Bottom left, representative isotherms showing radioligand displacement from h5-HT
2B
receptors expressed in COS-7 cells, the nonlinear regression
of which was used to determine IC
50
values. K
i
values were calculated using the Cheng-Prusoff approximation. Bottom right, K
i
values for MDMA in
bar chart format; the arrow shows the K
i
value for the h5-HT
2B
receptor; K
i
values ⬎10,000 nM are set to zero for clarity. Red arrow, K
i
value for
MDMA.
MDMA Activates Valvulopathogenic 5-HT
2B
Receptors 1225
large number of recombinant (mostly human) neurotrans-
mitter and hormone receptors, ion channels, and transport-
ers. To our surprise, MDMA exhibited preferentially high
affinity for the h5-HT
2B
receptor (Fig. 1A), a receptor previ-
ously implicated in fenfluramine-induced VHD (Fitzgerald et
al., 2000; Rothman et al., 2000) and PPH (Launay et al.,
2002). As shown in Fig. 1A and Table 1, other valvulopathic
drugs are also characterized by preferentially high affinities
for h5-HT
2B
receptors (Fig. 1A; Table 1). We also discovered
that two additional commonly prescribed medications bind to
and activate 5-HT
2B
receptors: 1) pergolide, a drug used in
treating Parkinson’s disease that was recently associated
with VHD of the fenfluramine-type (Pritchett et al., 2002)
and 2) dihydroergotamine, a drug used in treating migraine
headaches, which was reported several years ago to induce
VHD (Creutzig, 1992). As is shown in Table 1, both pergolide
and dihydroergotamine have high affinities for h5-HT
2B
re-
ceptors.
We subsequently examined the abilities of MDMA and its
N-demethylated metabolite (MDA) to activate human, re-
combinant 5-HT
2B
receptors. These studies identified MDA
as a more potent and efficacious agonist than MDMA (Fig.
2A; Tables 1 and 2). In this regard, we reported previously
that the N-dealkylated metabolites of drugs known to induce
either VHD or PPH (e.g., norfenfluramine and methylergono-
vine) are also more potent and efficacious 5-HT
2B
receptor
agonists than their respective parent compounds (Table 2;
Rothman et al., 2000). Importantly, the EC
50
values for ac-
tivating phosphoinositide hydrolysis at h5-HT
2B
receptors
for MDMA (2000 nM) and MDA (190 nM) are nearly identical
to the plasma concentrations found in humans after a single
recreational dose (150 mg) of MDMA in humans. For in-
stance, after a single 150-mg dose of MDMA, de la Torre et al.
(2000) reported a C
max
for MDMA of 2000 nM and a C
max
for
MDA of 150 nM. Table 2 also demonstrates that both pergol-
ide and dihydroergotamine, drugs recently demonstrated to
induce VHD of the fenfluramine-type in humans (Pritchett et
al., 2002), are also potent h5-HT
2B
agonists.
Because the (⫹)-stereoisomer of fenfluramine, dexfenflura-
mine, also used as an anorexigenic (Redux), was associated
with VHD and PPH, we evaluated optically pure prepara-
tions of MDMA and MDA for potency and efficacy at human
5-HT
2B
receptors. We detected no significant difference in
efficacy between the R- and S-stereoisomers of either MDMA
or MDA; with respect to potency, the S-stereoisomer of
MDMA was slightly more potent than the R-stereoisomer,
whereas the R- and S-stereoisomers of MDA exhibited no
statistically significant difference in potency (Fig. 2B and
Table 2).
MDMA and MDA are widely appreciated to release the
biogenic amine neurotransmitters from nerve terminals via a
carrier-mediated exchange mechanism see (Baumann et al.,
2000; Rothman and Baumann, 2002). We thus determined
the EC
50
values of the compounds under consideration for
releasing [
3
H]5-HT, [
3
H]NE, and [
3
H]DA from rat brain syn-
aptosomes. Norfenfluramine is more potent than fenflura-
mine at releasing [
3
H]NE and [
3
H]DA. MDMA is most potent
at releasing [
3
H]5-HT, but still potently releases [
3
H]NE and
[
3
H]DA (Table 2). MDA differs from MDMA in that its most
potent action is in releasing [
3
H]NE (Table 2). There is a
pronounced enantioselectivity in the actions of MDMA and
MDA as indicated by the more potent effects of (S)-MDA and
(S)-MDMA compared with (R)-MDA and (R)-MDMA, respec-
tively (Table 2). Perhaps the key feature to emerge from this
analysis is that the potency of (R)-MDMA, MDA and its
stereoisomers, in the biogenic amine release assays is similar
to their potency at 5-HT
2B
receptors, indicating that MDMA
will activate 5-HT
2B
receptors at typical pharmacological
doses.
Valvulopathic Drugs Induce Prolonged Mitogenic
Responses in Human Heart Valve Interstitial Cells.
Because much of the evidence implicating 5-HT
2B
receptor
activation in drug-induced VHD is inferential, we set out to
directly test the mitogenic activity of valvulopathic drugs
using primary cultures of human heart valve interstitial cells
(hVICs). In preliminary studies, we established that hVICs
express functional 5-HT
2B
receptors coupled to phosphoino-
sitide hydrolysis (data not shown). We next evaluated the
abilities of selected VHD-associated drugs to elicit mitogenic
responses from hVICs. For these studies, we incubated se-
rum-starved hVICs for 48 h with fenfluramine, norfenflura-
mine, MDMA, MDA, SB206553 (a 5-HT
2B/2C
antagonist), or
5-HT and measured [
3
H]thymidine incorporation into newly-
synthesized DNA.
The VHD-associated drugs fenfluramine and norfenflura-
mine each induced statistically significant mitogenic re-
sponses in hVICs (Fig. 3A). MDMA, MDA, and 5-HT, but not
TABLE 1
MDMA, MDA, and other valvulopathic drugs bind to human
recombinant 5-HT
2B
receptors
Data represent mean ⫾S.E.M. for at least three separate determinations in which
K
i
values were measured by radioligand displacement assays (see Materials and
Methods). Fenfluramine is known to induce VHD in humans.
Drug K
i
(pK
i
)
nM
Fenfluramine 4134 (5.9 ⫾0.2)
Norfenfluramine 15 (7.83 ⫾0.06)
Dihydroergotamine 15.2 (7.82 ⫾0.06)
Pergolide 14 (7.8 ⫾0.1)
(R,S)-MDMA 500 (6.30 ⫾0.08)
(R,S)-MDA 100 (6.8 ⫾0.1)
Fig. 2. MDMA and MDA potently activate h5-HT
2B
-serotonin receptors
in vitro. Concentration-dependent stimulation of phosphatidylinositide
hydrolysis via activation of h5-HT
2B
receptors expressed in HEK293 cells
by fenfluramine, norfenfluramine, (R,S)-MDMA, (R)-MDMA, (S)-MDMA,
(R,S)-MDA, (R)-MDA, or (S)-MDA was assayed as described under Ma-
terials and Methods. Data represent mean ⫾S.E.M. for n⫽3 separate
experiments of percentage stimulation of [
3
H]IP accumulation relative to
the full agonist 5-HT.
1226 Setola et al.
the 5-HT
2B/2C
receptor antagonist SB206553, each caused
similar responses (Fig. 3A). The mitogenic response elicited
by each drug was abrogated by coincubation with the
5-HT
2B/2C
receptor antagonist SB206553, demonstrating
that the mitogenic response was caused by 5-HT
2B
receptor
activation (Fig. 3B), because heart valve cells do not express
5-HT
2C
receptors (Roy et al., 2000).
Immunoblot analysis of vehicle- and drug-treated hVIC ly-
sates revealed that short-term (10-min) treatment of serum-
starved cells with either norfenfluamine, MDMA, MDA, or
5-HT induced an increase (statistically significant for all drugs
but MDMA) in Erk 1/2
MAPK
phosphorylation, an early mitro-
genic marker, compared with vehicle-treated cells (Fig. 4). In-
terestingly, the 5-HT
2B/2C
receptor antagonist SB206553 caused
a statistically significant decrease in [
3
H]thymidine deoxyribose
incorporation and no increase in Erk 1/2
MAPK
phosphorylation
compared with vehicle-treated cells, suggesting that 5-HT
2B
receptors regulate basal mitogensis in hVICs. In fact, we have
observed that basal Erk 1/2
MAPK
phosphorylation, which is
quite high in serum-starved VICs compared with serum-
starved HEK cells (data not shown), hinders the detection of a
statistically significant mitogenic response to drug treatment.
Discussion
The major finding of the present study is that MDMA and
MDA, in a manner identical to drugs demonstrated to induce
VHD and PPH in humans, bind to and activate human re-
combinant 5-HT
2B
receptors and induce mitogenesis in hu-
man heart valve interstitial cells in vitro. Importantly,
MDMA and MDA activate h5-HT
2B
receptors within the
same concentration ranges at which they 1) occur in plasma
TABLE 2
MDMA and MDA, similar to other valvulopathic drugs, activates h5-HT2B serotonin receptors in vitro
Data represent mean ⫾S.E.M. For estimates of efficacy (relative to 5-HT) and potency (EC
50
) for activation of PI hydrolysis (n⫽3 separate experiments) or estimates of
potency (EC
50
) for in vitro neurotransmitter release, assays used rat brain synaptosomes preloaded with 关
3
H兴5-HT, 关
3
H兴DA, or 关
3
H兴NE (see Materials and Methods for details).
Data for methysergide and methylergonovine are from ref. 10. The chemical structures are shown below the table, and the arrows indicate the dealkylated nitrogen. Drugs
in bold are those known to induce VHD in humans.
Release EC
50
Drug pEC
50
for 5-HT
2B
-Mediated PI
Hydrolysis
Relative Efficacy for 5-HT
2B
-Mediated PI
Hydrolysis 5-HT DA NE
nM nM
5-HT 1 (9.0 ⫾0.1) 1.00 ⫾0.06 44 ⫾3⬎10,000 ⬎10,000
Fenfluramine 400 (6.4 ⫾0.2) 0.13 ⫾0.02 108 ⫾4⬎10,000 740 ⫾30
Norfenfluramine 60 (7.2 ⫾0.1) 0.96 ⫾0.03 104 ⫾3 1900 ⫾200 170 ⫾10
Dihydroergotamine 30 (7.52 ⫾0.09) 0.73 ⫾0.02 N.D. N.D. N.D.
Pergolide 53 (7.27 ⫾0.09) 1.12 ⫾0.04 N.D. N.D. N.D.
(R,S)-MDMA 2000 (5.8 ⫾0.1) 0.32 ⫾0.02 72 ⫾3 278 ⫾7 110 ⫾10
(R)-MDMA 900 (6.0 ⫾0.2)* 0.27 ⫾0.02 340 ⫾20 3700 ⫾100 560 ⫾40
(S)-MDMA 6000 (5.2 ⫾0.2)*
†
0.38 ⫾0.03 74 ⫾3 142 ⫾4 136 ⫾9
(R,S)-MDA 190 (6.73 ⫾0.05) 0.80 ⫾0.02 160 ⫾7 190 ⫾6 108 ⫾7
(R)-MDA 150 (6.83 ⫾0.05) 0.76 ⫾0.02 310 ⫾10 900 ⫾30 290 ⫾10
(S)-MDA 100 (6.9 ⫾0.1) 0.81 ⫾0.04 100 ⫾498⫾450⫾5
Methysergide 150 (6.8 ⫾0.1) 0.18 ⫾0.02 N.D. N.D. N.D.
Methylergonovine 0.8(9.2 ⫾0.1) 0.40 ⫾0.02 N.D. N.D. N.D.
N.D., not determined
* Significantly different (P⬍0.05) from the other enantiomer by two-tailed ttest.
†Significantly different from racemate (P⬍0.05) by two-tailed ttest.
MDMA Activates Valvulopathogenic 5-HT
2B
Receptors 1227
after a single recreational dose and 2) release biogenic
amines, an activity widely accepted to be a major pharmaco-
logical effect of these agents. We also report that two com-
monly prescribed medications reported to induce VHD in
humans, pergolide and dihydroergotamine (Creutzig, 1992;
Pritchett et al., 2002), also activate h5-HT
2B
receptors in
vitro. Previous studies suggested that VHD-associated drugs
cause heart valve dysfunction via activation of heart valve
interstitial cell 5-HT
2B
receptors. Our current report brings
to five the number of medications known to activate 5-HT
2B
receptors (e.g., fenfluramine, ergotamine, dihydroergota-
mine, pergolide, and methysergide), each of which induces
VHD of the fenfluramine-type in humans. With the exception
of fenfluramine, all of the drugs currently reported to produce
VHD in humans are ergot derivatives. Because of the wide-
spread use of ergot derivatives for treating diseases such as
migraine headaches and Parkinson’s disease, these findings
are likely to have negative implications for drug discovery
efforts that use ergolines or ergopeptines as lead candidates.
Our finding that amphetamine derivatives (e.g., fenflura-
mine, MDMA, MDA, and norfenfluramine) also activate h5-
HT
2B
receptors demonstrates that drugs of other classes also
need to be screened for potential valvulopathogenic actions.
In this regard, we are in the process of a large-scale screen of
various drugs currently approved for use in humans to iden-
tify potential valvulopathogenic drugs by virtue of their abil-
ity to bind to and activate recombinant h5-HT
2B
receptors
(V. S. Setola, S. J. Hufeisen, K. J. Grande-Allen, I. Vesely,
R. A. Glennon, B. Blough, R. B. Rothman, B. L. Roth, manu-
script in preparation).
Because there is no suitable animal model for predicting
the valvulopathogenic actions of drugs, we evaluated the
mitogenic effect of various drugs on hVICs, a novel in vitro
model system. Because hVICs are the cells affected in drug-
induced VHD, hVICs represent the most physiologically and
pharmacologically relevant model system for VHD predic-
tion. We report here that several drugs known to induce VHD
in humans, as well as MDMA and MDA, elicit prolonged
mitogenic responses in hVICs. Our results strongly suggest,
therefore, that MDMA and MDA are valvulopathogenic; ret-
rospective echocardiographic studies in human MDMA users
are currently in progress to test this notion.
These studies also showed that h5-HT
2B
receptor activa-
tion plays a critical role in the transduction of a mitogenic
signal by VHD-associated drugs, strongly supporting the hy-
pothesis that h5-HT
2B
receptor agonists are likely to cause
VHD. In this regard, we demonstrated that mitogenesis was
abrogated by coincubation with a 5-HT
2B/2C
-selective antag-
onist (SB206553; see http://kidb.bioc.cwru.edu/pdsp.php for
comprehensive pharmacological profile of SB206553). Be-
cause human cardiac valves express large quantities of
5-HT
2B
receptors and do not express appreciable amounts of
5-HT
2C
receptors (Fitzgerald et al., 2000), it is likely that the
inhibition by SB206553 is principally caused by 5-HT
2B
re-
ceptor blockade. It is conceivable that the residual stimula-
tion of [
3
H]thymidine incorporation not blocked by SB206553
might be caused by activation of mitogenic 5-HT
2A
receptors,
because human heart valves express 5-HT
2A
mRNA [al-
though sheep VICs apparently predominantly express
5-HT
2A
receptors (Xu et al., 2002)], and the drugs studied
herein are low-affinity, low-efficacy 5-HT
2A
agonists (Nash et
al., 1994; Rothman et al., 2000; Roy et al., 2000). Arguing
against such a role for 5-HT
2A
receptors in the mitogenic
response of hVICs is the observation that the genetic ablation
of 5-HT
2B
receptors, but not of 5-HT
2A
receptors (J. Gingrich,
personal communication), interferes with myocardioblast
proliferation during embryonic development, suggesting that
the activation of mitogenic pathways by 5-HT
2A
receptors in
heart valves is not essential for cardiac development (Nebigil
et al., 2000a,b). Taken together, these results imply that
Fig. 3. MDMA and MDA induce mitogenesis in human heart valve
interstitial cells in vitro. A, stimulation of [
3
H]thymidine deoxyribose
incorporation in VICs treated for 48 h with either vehicle (V), 5-HT, the
5-HT
2B/2C
receptor antagonist SB206553 (SB), fenfluramine (F), norfen-
fluramine (NF), MDMA (X), or MDA (M) reveals a prolonged mitogenic
response that is blocked by pretreatment with SB (B). All drugs used at
10
M except SB206553, which was used at 1
M. *, P⬍0.05; **, P⬍
0.01; ***, P⬍0.001, significant difference from vehicle-treated cells by
two-tailed ttest. C, immunoblot analysis of Erk 1/2 phosphorylation in
VICs treated for 10 min with norfenfluramine, MDMA, MDA, SB206553,
or 5-HT reveals a short-term mitogenic response (i.e., increase in percent
of total cellular Erk 1/2 phosphorylated; see Materials and Methods for
details) after exposure to 5-HT
2B
receptor agonists. *, P⬍0.05; **, P⬍
0.01, significant difference from vehicle-treated cells by two-tailed ttest.
1228 Setola et al.
activation of mitogenic pathways by 5-HT
2A
receptors is in-
essential for cardiac development and that the 5-HT
2B
recep-
tor is most likely responsible for the mitogenic responses
induced by valvulopathogenic drugs. Other findings implicat-
ing the h5-HT
2B
receptor as the proximal molecular target
responsible for fenfluramine-like VHD are the observations
that h5-HT
2B
receptors 1) are enriched in human heart
valves; 2) are essential for normal cardiac development; and
3) induce, upon activation, prolonged mitogenic responses in
heterologous expression systems (Fitzgerald et al., 2000;
Nebigil et al., 2000b).
Our discovery that pergolide and dihydroergotamine, two
drugs reported to induce VHD in humans (Creutzig, 1992;
Pritchett et al., 2002), also activate h5-HT
2B
receptors in
vitro validates the use of recombinant h5-HT
2B
receptors to
screen for valvulopathogenic potential. Of equal importance,
recent data have implicated the 5-HT
2B
receptor in the
pathogenesis of primary pulmonary hypertension, a severe
and frequently fatal illness (Launay et al., 2002). Impor-
tantly, in this regard, fenfluramine use increases the risk of
developing primary pulmonary hypertension (Abenhaim et
al., 1996). Thus, these data further highlight the necessity of
screening current and potential pharmacotherapies for ago-
nist potencies and efficacies at human 5-HT
2B
receptors and
validate the use of 5-HT
2B
receptor-expressing cell lines as
models to do so. The data presented herein are thus of major
public health importance because they suggest that MDMA
abuse, which is at an all-time high, puts an expanding pop-
ulation at increased risk for developing VHD and primary
pulmonary hypertension.
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Address correspondence to: Bryan L. Roth MD, PhD, Department of
Biochemistry; RM W438, School of Medicine, Case Western Reserve Uni-
versity, 2109 Adelbert Road, Cleveland, OH 44106-4935. E-mail: roth@
biocserver.cwru.edu
MDMA Activates Valvulopathogenic 5-HT
2B
Receptors 1229
