Download Hypothalamic-Pituitary-Thyroid Axis and Sympathetic Nervous

0022-3565/03/3051-159 –166$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics
JPET 305:159–166, 2003
Vol. 305, No. 1
44982/1051103
Printed in U.S.A.
Hypothalamic-Pituitary-Thyroid Axis and Sympathetic Nervous
System Involvement in Hyperthermia Induced by 3,4Methylenedioxymethamphetamine (Ecstasy)
JON E. SPRAGUE, MATTHEW L. BANKS, VALERIE J. COOK, and EDWARD M. MILLS
The Department of Pharmaceutical and Biomedical Sciences, The Raabe College of Pharmacy, Ohio Northern University, Ada, Ohio (J.E.S.,
M.L.B., V.J.C.); and National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland (E.M.M.)
Received September 30, 2002; accepted December 18, 2002
The substituted amphetamine 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) is commonly associated with
an increase in body temperature in both humans (Dar and
McBrien, 1996; Mallick and Bodenham, 1997) and rodents
(Gordon et al., 1991). Because of its association with weekends and rave parties, emergency room personnel often refer
to severe forms of this hyperthermia as “Saturday Night
Fever” (Williams et al., 1998), which can be associated with
rhabdomyolysis, multiorgan failure, and death (Walubo and
Seger, 1999). Although deaths from overdose remain rare,
the prevalence and especially hospitalizations resulting from
MDMA exposure have dramatically increased from 250 hospitalizations in 1994 to over 2850 in 1999 (Drug Abuse Warning Network, 2000). Much evidence from rodent and nonhuman primate studies suggests that MDMA also induces longterm serotonergic neurotoxicity that seems to be ostensibly
This research was sponsored by an Undergraduate Research Initiative
Grant and funded by the Ohio Northern University College of Pharmacy.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.102.044982.
thermic response seen after MDMA. Prazosin, an ␣1-antagonist
(0.2 mg/kg i.p.), administered 30 min before MDMA significantly
attenuated the MDMA-induced increase in rectal temperature,
but had no effect on skeletal muscle temperature. Cyanopindolol, a ␤3-antagonist (4 mg/kg s.c.), administered 30 min before MDMA (40 mg/kg s.c.) significantly attenuated the increase
in skeletal muscle temperature, but had no effect on the rise in
rectal temperature. The combination of prazosin and cyanopindolol resulted in an abolishment of MDMA-induced hyperthermia. The mechanisms of thermogenesis induced by MDMA
seem to result from an interaction between the hypothalamicpituitary-thyroid axis and the sympathetic nervous system,
wherein mechanisms leading to core and skeletal muscle hyperthermia after MDMA exposure seem to be differentially regulated by ␣1- and ␤3-adrenergic receptors.
linked to hyperthermia (Broening et al., 1995; Farfel and
Seiden, 1995; Malberg et al., 1996). Although the importance
of their elucidation cannot be overstated, the fundamental
biological mechanisms involved in heat production and progression to hyperthermia after MDMA exposure are unknown. Furthermore, we do not understand clearly the associations between hyperthermia and many of the pathological
changes induced by MDMA.
Gordon et al. (1991) proposed that MDMA induces a dysfunction in central nervous system thermoregulatory mechanisms that are influenced by ambient temperature. Several
laboratories have shown that when MDMA is given to rats in
a 24°C or greater environment, hyperthermia results
(Schmidt et al., 1990; Gordon et al., 1991). If the ambient
temperature is lowered to 10°C, however, a hypothermic
response occurs (Gordon et al., 1991). The set point for either
a hyperthermic or hypothermic response seems to be above or
below 20 –22°C (Gordon et al., 1991; Malberg and Seiden,
1998) with most neurotoxicity studies being conducted at
23–24°C.
In addition to evidence suggesting that MDMA acutely
ABBREVIATIONS: MDMA, 3,4-methylenedioxymethamphetamine; SNS, sympathetic nervous system; UCP, uncoupling proteins; HPT, hypothalamic-pituitary-thyroid; HYPO, hypophysectomized; TX, thyroparathyroidectomized; 5-HT, 5-hydroxytryptamine, serotonin; T4, thyroxine.
159
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 6, 2017
ABSTRACT
An acute and potentially life-threatening complication associated with the recreational use of the 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) is hyperthermia. In the present
study, Sprague-Dawley rats treated with MDMA (40 mg/kg s.c.)
responded with a significant increase (maximal at 1 h) in rectal
and skeletal muscle temperatures that lasted for at least 3 h
post-treatment. Hypophysectomized (HYPO) and thyroparathyroidectomized (TX) animals treated with MDMA (40 mg/kg
s.c.) did not become hyperthermic and in fact displayed a
significant hypothermia. The HYPO and TX animals were also
resistant to the serotonergic neurotoxic effects of MDMA assessed by serotonin measurements 4 to 7 days later in the
striatum and hippocampus. MDMA (40 mg/kg s.c.) induced a
significant increase in thyroxine levels 1 h post-treatment. Thyroid hormone replacement in TX animals returned the hyper-
160
Sprague et al.
Materials and Methods
The present study was carried out in accordance with protocols
approved by the Ohio Northern University Animal Care and Use
Committee.
Animals. Sham, hypophysectomized (HYPO), and thyroparathyroidectomized (TX) adult male Sprague-Dawley rats (weighing 175–
200 g) were obtained from Harlan (Indianapolis, IN). All animals
were housed in groups of three and given ad libitum access to food
and drinking water. Housing conditions were maintained at a constant temperature of 23°C and a 12:12-h light/dark cycle. For hypophysectomy, anesthetized animals were placed in a ventral recumbency in a Hoffman-Reiter hypophysectomy instrument. A 19-gauge
needle was inserted through the hollow right ear bar. The needle was
pushed through the bone with the bevel side down until the needle
stopper made contact with the ear bar. The needle was rotated in a
semicircle two or three times. The needle was then rotated so the
bevel pointed downward and the pituitary was slowly aspirated into
the water-filled syringe. The pituitary was then examined to ensure
complete removal. Once confirmed, HYPO animals were given 5%
sucrose solution as a drinking source for 5 days postsurgery. For
thyroparathyroidectomy, anesthetized animals had the ventral cervical area shaved and swabbed with surgical scrub. A 1- to 1.5-cm
midventral incision was made from just caudal to the pharynx to the
cranial edge of the pectoral muscle cutting through the underlying
fat until the sternohyoid muscle was exposed. The trachea was
exposed and the thyroid gland located. The isthmus was then held
and the thyroid gland was separated from the trachea. TX animals
were given 2 to 4% calcium lactate solution as a drinking source for
the duration of the experiment. HYPO animals were administered
MDMA (40 mg/kg s.c.) or saline 72 h postsurgery. TX and their
corresponding shams were treated with MDMA (40 mg/kg s.c.) or
saline 1-week postsurgery.
Drugs and Chemicals Cyanopindolol, a ␤3-receptor antagonist,
was purchased from Tocris Cookson, Inc. (Ellisville, MO). MDMA
was generously donated by Dr. David E. Nichols (Purdue University,
West Lafayette, IN). Prazosin, an ␣1-receptor antagonist, and all
other reagents were purchased from Sigma-Aldrich (St. Louis, MO)
or VWR Scientific Products (Columbus, OH).
MDMA Effects on Thermogenesis. Basal skeletal muscle
and/or rectal temperatures were taken in all animals before administering MDMA or saline. Skeletal muscle temperatures were taken
in the biceps femoris using a thermocouple with the electrode inserted into an 18-gauge needle. Rectal temperatures were taken with
a rectal probe (Physitemp Instruments, Clifton, NJ) attached to the
thermocouple and white petrolatum was applied to the probe before
insertion. Skeletal muscle and rectal temperatures were taken at 1,
2, and 3 h post-treatment. Skeletal muscle temperatures were not
monitored in TX animals due to the stress of the surgeries in general.
Assessment of MDMA-Induced Serotonergic Neurotoxicity.
To assess neurotoxicity, 5-HT levels were measured from each hemisphere of the striatum and hippocampus 7 days after treatment. TX
animals were assessed 4 days post-treatment. Samples were collected by brain dissection of the specific brain regions over ice. Tissue
samples were then frozen in liquid nitrogen and stored at ⫺80°C
until analysis could be performed. These samples were subsequently
sonicated, using a sonic dismembrator (Fisher Scientific Co., Pittsburgh, PA), for 15 s at a setting of approximately 4 while suspended
in 100 ␮l of mobile phase.
High-performance liquid chromatography EC (Waters) was conducted to determine 5-HT levels. The mobile phase (consisting of
0.05 M sodium phosphate, 0.03 M citric acid buffer with a pH range
of 2.1–3.9, 0.1 mM EDTA, 0 – 0.05% sodium octyl sulfate, and 0 –30%
methanol) was pumped through a Nova-Pak C18 (5 ␮m 3.9 ⫻ 300
mm) reverse-phase column at a flow rate of 0.7 ml/min with the
potential set at 1 nA and an E of ⫹750 mV. Millennium software was
used to integrate and analyze the raw data for the determination of
5-HT levels compared with internal standard curves (R2 ⫽ 0.95).
Detection limits were 5 pg/␮l.
MDMA Effects on Thyroxine (T4) Levels. Twelve SpragueDawley rats were assigned to receive saline or MDMA (40 mg/kg s.c.)
to assess the effects of MDMA on T4 levels 1 h post-MDMA administration. The 1-h time point was selected based on previous results
that showed MDMA-induced hyperthermia to be most robust at the
1-h time point. Animals were anesthetized using chloroform and
blood was subsequently drawn from the left ventricle. Measurement
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 6, 2017
disturbs central nervous system thermoregulatory functions,
MDMA-induced activation of the sympathetic nervous system (SNS) and subsequent alterations in vascular hemodynamics may also play an important role in heat production
and redistribution. Peripheral heat production in organs
such as brown and white fat and skeletal muscle is regulated
in part by norepinephrine (Bianco et al., 1988; Rubio et al.,
1995a,b). Recently, Pedersen and Blessing (2001) showed
that MDMA induces cutaneous vasoconstriction and that
this cutaneous restriction in blood flow contributes to the
increase in core body temperature seen after treatment with
MDMA. These authors also showed that sympathectomy only
partially attenuated the hyperthermic response seen after
MDMA. Recently, Fernandez et al. (2002) showed that ganglionic blockade only partially attenuated the hyperthermic
effect induced by MDMA. Taken together, these studies suggest that the hyperthermic response to MDMA involves more
than merely the activation of the SNS and changes in regional blood flow.
Thyroid hormone is the primary endocrine regulator of
metabolism and thermogenesis. Surprisingly, there seem to
be no studies that have directly examined the role of thyroid
hormone in the hyperthermic response to MDMA. Three
lines of evidence suggest that thyroid hormone may be linked
to the hyperthermic response to MDMA. 1) Fekete et al.
(2000) observed that amphetamines induce the cocaine- and
amphetamine-regulated transcript in the hypothalamic
paraventricular nucleus, resulting in an increased biosynthesis of thyrotropin-releasing hormone. This study predicts
that MDMA may also increase thyroid hormone levels. 2) At
the cellular level, thyroid hormone seems to play both a
permissive and synergistic role in norepinephrine- or SNSmediated thermogenesis (Bianco et al., 1988; Rubio et al.,
1995a,b). According to recent evidence, these effects may be
mediated by a family of mitochondrial uncoupling proteins
(UCP), members of which mediate nonshivering thermogenesis (Argyropoulos and Harper, 2002). 3) The synergism between thyroid hormone and norepinephrine-dependent
mechanisms of thermogenesis, including the activation and
transcriptional regulation of UCP (Gong et al., 1997), seems
to be mediated specifically by the thyroid hormone receptor
and ␣1- and ␤3-adrenergic receptors (Silva, 1995).
Because the role, if any, that thyroid hormone plays in
MDMA-induced hyperthermia is not known, we examined
the role of and the interactions between the SNS and the
hypothalamic-pituitary-thyroid (HPT) axis in the development of the hyperthermia induced by MDMA. We hypothesized that MDMA administration would induce an increase
in thyroid hormone levels, ultimately facilitating norepinephrine-mediated thermogenesis. Furthermore, we predicted, as evidence would suggest, that acute hyperthermia
plays a prominent role in MDMA-induced chronic neurotoxicity.
MDMA-Mediated Hyperthermia
of T4 levels was conducted with a Snap T4 Test. Snap T4 test is an
enzyme linked immunosorbent assay for the quantitative measurement of total T4 in serum. The Snap T4 test uses a competitive
enzyme immunoassay format.
In the test procedure, the serum sample is first incubated with an
anti-T4 antibody-enzyme conjugate. During incubation, T4 present in
the serum sample will bind to the conjugate. The T4 concentration is
then calculated from the ratio of T4 test stripe color to reference
stripe color developed from standard curves (R2 ⫽ 0.95) with detection limits of 0.4 ␮g/dl.
Statistical Analysis. Data were analyzed by analysis of variance
with a Student-Newman-Keuls post hoc test or a t test. Significance
was set at p ⱕ 0.05. All biochemical measurements were based on
tissue wet weight and are represented as percentage of saline control
for ease of presentation. Control groups in all studies were treated
with saline only. All figure legends contain the control values and
sample size.
Results
Fig. 1. Effects of MDMA (40 mg/kg s.c.) on rat rectal (A) and skeletal
muscle (B) temperature. Each value is the mean ⫾ S.E.M. (n ⫽ 4). ⴱ,
significantly different from the corresponding control group (p ⬍ 0.01).
resulted in a significant (p ⬍ 0.001) decrease in rectal temperature at the 2- and 3-h time points. Similar results were
seen in the skeletal muscle temperature measurements (Fig.
2). MDMA yielded a significant (p ⬍ 0.001) increase in skeletal muscle temperatures at the 1-, 2-, and 3-h time points. In
contrast, the HYPO animals treated with MDMA demonstrated a significant (p ⬍ 0.001) decrease in skeletal muscle
temperatures at the 1-, 2-, and 3-h time points.
Effects of Hypophysectomy on MDMA-Induced Serotonergic Neurotoxicity. Striatal 5-HT concentrations were
significantly (p ⬍ 0.03) decreased in the MDMA treatment
group compared with sham only (Fig. 3A). Hippocampal
5-HT concentrations were also significantly (p ⬍ 0.01) decreased in the MDMA treatment group compared with all
other treatment groups (Fig. 3B). Hypophysectomy attenuated this decrease in 5-HT levels seen in both regions.
Effects of MDMA on T4 Levels. MDMA induced a significant (p ⬍ 0.007) increase in T4 levels 1 h post-treatment.
Theses results are shown in Fig. 4.
Effects of Thyroparathyroidectomy on MDMA-Induced Hyperthermia. As was seen in the HYPO animals,
thyroparathyroidectomy resulted in a hypothermic response
(p ⬍ 0.01) after treatment with MDMA. MDMA alone induced a significant (p ⬍ 0.01) elevation in rectal temperature. The thyroparathyroidectomized treatment group began
the study with a baseline temperature that was significantly
(p ⬍ 0.05) less than the sham control group (Fig. 5A).
Fig. 2. Effects of MDMA (40 mg/kg s.c.) on rectal (A) and skeletal muscle
(B) temperature in HYPO rats. Each value is the mean ⫾ S.E.M. for
rectal temperatures (n ⫽ 12) and for skeletal muscle (n ⫽ 6) temperatures. ⴱ, significantly different from all treatment groups (p ⬍ 0.001).
Sham and HYPO treatment groups received saline (s.c.). MDMA only
treatment groups also received sham surgeries.
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 6, 2017
Effects of MDMA on Rat Rectal and Skeletal Muscle
Temperature. MDMA induced a statistically significant increase (p ⬍ 0.01) in rat rectal (Fig. 1A) and skeletal muscle
(Fig. 1B) temperature at the 1-, 2-, and 3-h time points.
Effects of Hypophysectomy on MDMA-Induced Hyperthermia. MDMA produced a significant (p ⬍ 0.001) increase in rectal temperature at the 1-, 2-, and 3-h time points.
Treatment of HYPO animals with the same dose of MDMA
161
162
Sprague et al.
Fig. 3. Effects of MDMA (40 mg/kg s.c.) on 5-HT levels in the striatum (A)
and hippocampus (B) of hypophysectomized rats 7 days after treatment.
Each value is the mean ⫾ S.E.M. (n ⫽ 6). ⴱ, significantly different from
sham only (p ⬍ 0.03). ⴱⴱ, significantly different from all treatment groups
(p ⬍ 0.01). Sham striatal 5-HT levels were 280.6 ⫾ 8.2 pg/mg tissue
weight and sham hippocampal 5-HT levels were 100.6 ⫾ 11.7 pg/mg
tissue weight. Sham and HYPO treatment groups received saline (s.c.).
MDMA only treatment groups also received sham surgeries.
Effects of Levothyroxine Supplementation on
MDMA-Induced Thermogenic Response in TX Animals. Replacing thyroid hormones with levothyroxine (100
␮g/kg s.c. ⫻ 5 days) resulted in a significant hyperthermic
response compared with control (p ⬍ 0.01) albeit still significantly less than MDMA alone (p ⬍ 0.01). TX animals treated
with MDMA only responded with a significant (p ⬍ 0.001)
hypothermic response (Fig. 5B).
Effects of Thyroparathyroidectomy on MDMA-Induced Serotonergic Neurotoxicity. Striatal 5-HT concentrations were significantly (p ⬍ 0.01) decreased in the MDMA
treatment group compared with sham only (Fig. 6A). Hippocampal 5-HT concentrations were also significantly (p ⬍
0.01) decreased in the MDMA treatment group compared
with all other treatment groups (Fig. 6B). Thyroparathyroidectomy attenuated this decrease in 5-HT levels seen in both
regions.
Effects of Prazosin on MDMA-Induced Hyperthermia. The ␣1-receptor antagonist prazosin significantly (p ⬍
0.01) attenuated this rise in rectal temperature but did not
Fig. 5. A, effects of MDMA (40 mg/kg s.c.) on rat rectal temperature in TX
animals. Each value is the mean ⫾ S.E.M. (n ⫽ 6 –7) for rectal temperatures. ⴱ, significantly different from sham only (p ⬍ 0.05). ⴱⴱ, significantly different from all other groups (p ⬍ 0.01). ⌿, significantly different
from sham and MDMA (p ⬍ 0.01). Sham and TX treatment groups
received saline (s.c.). MDMA only treatment groups also received sham
surgeries. B, effects of levothyroxine (100 ␮g/kg s.c. ⫻ 5 days) supplementation in TX animals on MDMA (40 mg/kg s.c.) induced hyperthermia. Each value is the mean ⫾ S.E.M. (n ⫽ 5– 6) for rectal temperatures.
ⴱ, significantly different from all other groups (p ⬍ 0.01).
completely eliminate the hyperthermic response (Fig. 7). In
the prazosin only treatment group, rectal temperatures remained constant throughout the monitoring period (data not
shown). MDMA resulted in a significant (p ⬍ 0.001) hypothermia in the TX animals that was significantly (p ⬍ 0.05)
potentiated by prazosin at the 3-h time point.
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 6, 2017
Fig. 4. Effects of MDMA (40 mg/kg s.c.) on T4 levels 1 h postadministration. ⴱ, significantly different from control (p ⬍ 0.007). Each column
represents a mean ⫾ S.E.M. (n ⫽ 6). Saline T4 levels were 8.6 ⫾ 0.6 ␮g/dl.
MDMA-Mediated Hyperthermia
163
Fig. 8. Effects of MDMA (40 mg/kg s.c.) and prazosin (0.2 mg/kg i.p. 30
min before MDMA) on rat skeletal muscle temperature. Each value is the
mean ⫾ S.E.M. (n ⫽ 6) for skeletal muscle temperatures. ⴱ, significantly
different from control and prazosin (p ⬍ 0.001). ⴱⴱ, significantly different
from all other groups (p ⬍ 0.01).
Fig. 6. Effects of MDMA (40 mg/kg s.c.) on 5-HT levels in the rat striatum
(A) and hippocampus (B) in TX animals 4 days after treatment. Each
value is the mean percentage of control ⫾ S.E.M. (n ⫽ 6 –7). ⴱ, significantly different from all other groups (p ⬍ 0.05). Sham striatal 5-HT
levels were 307.35 pg/mg and sham hippocampal 5-HT levels were 164.45
pg/mg. Sham and TX treatment groups received saline (s.c.). MDMA only
treatment groups also received Sham surgeries.
Fig. 7. Effects of MDMA (40 mg/kg s.c.) on rat rectal temperature in TXand prazosin (0.2 mg/kg i.p. 30 min before MDMA)-treated animals. Each
value is the mean ⫾ S.E.M. (n ⫽ 6 –7) for rectal temperatures. ⴱ, significantly different from all other groups (p ⬍ 0.001). ⴱⴱ, significantly
different from all other groups except TX ⫹ MDMA ⫹ prazosin (p ⬍
0.001). ⌿, significantly different from all other groups except TX ⫹
MDMA (p ⬍ 0.001).
MDMA-induced a significant rise (p ⬍ 0.001) in skeletal
muscle temperature that was not altered by prazosin pretreatment at the 1- and 2-h time points (Fig. 8). At the 3-h
time point, prazosin attenuated (p ⬍ 0.01) this rise in skeletal muscle temperature. Basal skeletal muscle temperature
Fig. 9. Effects of MDMA (40 mg/kg s.c.) and cyanopindolol (4 mg/kg s.c. 30
min before MDMA) administration on rat rectal (A) and skeletal muscle
(B) temperature. Each value is the mean ⫾ S.E.M. (n ⫽ 6) for rectal
temperatures. ⴱ, significantly different from control and cyanopindolol
(p ⬍ 0.0001). ⴱⴱ, significantly different from all other treatment groups
(p ⬍ 0.001). ⌿, significantly different from MDMA only (p ⬍ 0.05).
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 6, 2017
was significantly (p ⬍ 0.001) reduced after saline and prazosin treatment and remained constant throughout the remaining duration of the monitoring period (Fig. 8).
Effects of Cyanopindolol on MDMA-Induced Hyperthermia. The ␤3-receptor antagonist cyanopindolol had no
effect on the MDMA-mediated increase in rectal temperature
but significantly (p ⬍ 0.001) attenuated the increase in skeletal muscle temperature (Fig. 9B). Combining cyanopindolol
and prazosin eliminated the hyperthermic response in both
the rectum and skeletal muscle (Fig. 10).
164
Sprague et al.
Discussion
Here, we show that when administered to rats, MDMA
acutely increases plasma levels of thyroid hormone T4 and
induces a similarly acute and robust elevation in core and
skeletal muscle temperatures. Surgical removal of either the
pituitary or thyroid glands abolished the hyperthermic response and produced a significant hypothermia, in addition
to blocking subsequent serotonergic neurotoxicity, consistent
with previous evidence suggesting that the neurotoxic effects
of MDMA are greatly influenced by the hyperthermic response (Broening et al., 1995; Farfel and Seiden, 1995; Malberg and Seiden, 1998). Hyperthermia was specifically associated with elevations in thyroid hormone and TX animals
showed a hyperthermic response to MDMA when thyroid
hormone was replaced. We confirmed previous data (Pedersen and Blessing, 2001; Fernandez et al., 2002) suggesting
that MDMA-induced activation of the SNS contributes to the
hyperthermic response using antagonists of ␣1- and ␤3-adrenergic receptors prazosin and cyanopindolol, respectively.
To date, MDMA research has focused exclusively on druginduced changes in core temperatures using rectal probes
exclusively, and these techniques may inadvertently overlook the possibility that hyperthermia may arise from heat
generation in different tissue sites. By using thermocouple
devices to record rectal and skeletal muscle temperature, we
observed that ␣1- and ␤3-adrenergic antagonists differentially attenuated hyperthermia according to the tissue examined, supporting the notion that the hyperthermic response
to MDMA is heterogeneous and may be the result of heat
generation and differential perfusion within multiple tissues.
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 6, 2017
Fig. 10. Effects of MDMA (40 mg/kg s.c.) and combination treatment with
prazosin (0.2 mg/kg i.p.) and cyanopindolol (4 mg/kg s.c.) 30 min before
MDMA on rat rectal (A) and skeletal muscle (B) temperature. Each value
is the mean ⫾ S.E.M. (n ⫽ 6). ⴱ, significantly different from all other
groups (p ⬍ 0.05).
Alone, each drug only partially blocked the hyperthermic
response in skeletal muscle (cyanopindolol) or core (prazosin). Combination pretreatment with the antagonists, however, completely blocked MDMA-induced hyperthermia in
both locations, strongly suggesting that combination sympatholytic drug therapy may be a superior acute treatment
option for clinical cases of MDMA-mediated hyperthermia.
Based on the findings of Pedersen and Blessing (2001)
suggesting that cutaneous vasoconstriction contributes to the
increase in core temperature seen after treatment with
MDMA, and those of McDaid and Docherty (2001) showing
that the vascular effects of MDMA seem to involve predominately ␣1-adrenergic receptors, we tested the effects of prazosin on MDMA-mediated thermogenesis. Our results showing only a partial attenuation of the core hyperthermia by
prazosin parallel those of Pedersen and Blessing (2001), who
saw only a partial antagonism with surgical sympathectomy.
The ganglionic blocker chlorisondamine has also been shown
to reduce the amplitude of the hyperthermia induced by
MDMA (Fernandez et al., 2002). Based upon these studies,
we hypothesized that MDMA-induced core hyperthermia
would be attenuated with prazosin pretreatment. Our results
with prazosin’s effects on MDMA-mediated core temperature
changes confirmed these previous observations. Skeletal
muscle hyperthermia was, however, unaffected by ␣1-blockade.
Skeletal muscle thermogenesis occurs by three primary
mechanisms: 1) contraction, or shivering, 2) thermogenic calcium cycling mediated by the dantrolene-sensitive ryanodine
receptor (Paul-Pletzer et al., 2002), and 3) activation of mitochondrial proton leak by UCP-3, which is highly expressed
in skeletal muscle and has recently been associated with
skeletal muscle thermogenesis in transgenic mice overexpressing UCP-3 (Curtin et al., 2002) and yeast (Hinz et al.,
1999). Dantrolene is the primary pharmacological line of
defense in hospitalizations for MDMA-induced hyperthermia
(Dar and McBrien, 1996). Despite its widespread use, dantrolene fails to adequately control the hyperthermic response
seen after MDMA ingestion (Dar and McBrien, 1996). In
animals, the skeletal muscle relaxant xylazine also fails to
reduce the hyperthermic response (our unpublished observations). These data suggest that other mechanisms may contribute more readily to hyperthermia.
UCP-3 is both induced and activated by increased intracellular cAMP downstream of ␤3-adrenergic receptors and
thyroid hormone (Gong et al., 1997). ␤3-Adrenergic agonists
and thyroid hormone are thought to produce a synergistic
activation of thermogenesis in animals (Silva, 1995). In the
present study, the ␤3-antagonist, cyanopindolol, attenuated
the rise in skeletal muscle temperature after MDMA treatment but had no effect on core temperature. Together, the
data suggests that mitochondrial UCP-3 may contribute to
increased skeletal muscle temperatures after MDMA.
Three other lines of evidence suggest that UCPs may be
involved in the thermogenic response to MDMA. First, clinical presentations of severe hyperthermia induced by MDMA
can include rhabdomyolysis, wherein skeletal muscle cells
lose viability, lyse, and release myoglobin, which can lead to
renal failure (Walubo and Seger, 1999). We recently demonstrated that overexpression of UCP-2, a homolog of UCP-3,
induces ATP depletion and oncosis in transiently transfected
and retrovirally infected cultured cells (Mills et al., 2002).
MDMA-Mediated Hyperthermia
oxidase-B (Falk et al., 2002) or the dopamine transporter
(Kanthasamy et al., 2002) also attenuate the serotonergic
neurotoxicity induced by MDMA without altering the hyperthermic response.
Lowering body temperature protects against brain damage
induced by a variety of insults, most likely by simply slowing
neurochemical processes. Bowyer et al. (1993) showed that if
animals were placed in a cold environment, both dopamine
release and neurotoxicity induced by methamphetamine
treatment were decreased. Thus, agents that reduce body
temperature may decrease the neurochemical effects of
MDMA and provide protection against its acute peripheral
effects and chronic neurotoxicity. Our results using surgically modified animals are consistent with previous suggestions that blocking hyperthermia protects against subsequent neurotoxicity. As our data also suggest, however,
hypothermia may be required for the magnitude of neurologic protection observed in our studies.
The results of the present study and supporting evidence
that is extant in the literature would argue strongly for an
interactive role between the SNS and the HPT axis in the
hyperthermic response to MDMA. We propose that MDMA
acutely activates the HPT axis and the SNS, and stimulates
thyroid-, ␣1-adrenergic-, and ␤3-adrenergic-dependent core
and skeletal muscle hyperthermia by the activation of uncoupling proteins. Furthermore, we propose that clinical intervention consistent with this mechanism of MDMA-induced
hyperthermia may prove superior in protecting individuals
from some of the acute peripheral and delayed neurological
toxicities that may be seen after MDMA misuse.
Acknowledgments
We are grateful for technical assistance provided by Dr. David
Houx and Michele Smith in the assessment of T4 levels. We are also
appreciative of the generous gift of MDMA by Dr. David E. Nichols
and of the constructive comments provided by Dr. Toren Finkel
during the preparation of this manuscript.
References
Argyropoulos G and Harper ME (2002) Uncoupling proteins and thermoregulation.
J Appl Physiol 92:2187–2198.
Burrows KB, Gudelsky G, and Yamamato BK (2000) Rapid and transient inhibition
of mitochondrial function following methamphetamine or 3,4-methylenedioxymethamphetamine administration. Eur J Pharmacol 398:11–18.
Bianco AC, Sheng X, and Silva JE (1988) Triiodothyronine amplifies norepinephrine
stimulation of uncoupling protein gene transcription by a mechanism not requiring protein synthesis. J Biol Chem 263:18168 –18175.
Bowyer JF, Gough B, Slikker W Jr, Lipe GW, Newport GD, and Holson RR (1993)
Effects of a cold environment or age on methamphetamine-induced dopamine
release in the caudate putamen of female rats. Pharmacol Biochem Behav 44:87–
98.
Broening HW, Bowyer JF, and Slikker W (1995) Age-dependent sensitivity of rats to
the long-term effects of the serotonergic neurotoxicant (⫹)-3,4-methylenedioxymethamphetamine (MDMA) correlates with the magnitude of MDMAinduced hyperthermia. J Pharmacol Exp Ther 275:325–333.
Coulombe P and Dussault JH (1977) Catecholamine metabolism in thyroid disease.
II. Norepinephrine secretion rate in hyperthyroidism and hypothyroidism. J Clin
Endocrinol Metab 44:1185–1189.
Curtin NA, Clapham JC, and Barclay CJ (2002) Excess recovery heat production by
isolated muscles from mice overexpressing uncoupling protein-3. J Physiol (Lond)
542:231–235.
Dar KJ and McBrien ME (1996) MDMA induced hyperthermia: report of a fatality
and review of current therapy. Intensive Care Med 22:995–996.
Dicker A, Raasmaja A, Cannon B, and Nedergaard J (1992) Increased ␣1adrenoceptor density in brown adipose tissue indicates recruitment drive in hypothyroid rats. Am J Physiol 263:E654 –E662.
Drug Abuse Warning Network (2000) The DAWN report: club drugs, 2000 December;
pp 1–10, Office of Applied Studies, Substance Abuse, and Mental Health Services
Administration, Washington, DC.
Falk EM, Cook VJ, Nichols DE, and Sprague JE (2002) An antisense oligonucleotide
targeted at MAO-B attenuates rat striatal serotonergic neurotoxicity induced by
MDMA. Pharmacol Biochem Behav 72:617– 622.
Farfel GM and Seiden LS (1995) Role of hypothermia in the mechanism of protection
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 6, 2017
Second, we recently reported that MDMA regulates the levels of UCP-3 mRNA in rat skeletal muscle (Sprague et al.,
2002). Third, MDMA has also been shown to increase proton
leak in rat striatum, a specific functional correlate of UCP
activity (Burrows et al., 2000).
MDMA-mediated dopamine release and subsequent activation of hypothalamic D1 receptors has been shown to play
an essential role in this hyperthermic response (Mechan et
al., 2002). Activation of the hypothalamic axis following
MDMA treatment is also confirmed by increased c-fos expression in the supraoptic and median preoptic nucleus of the
hypothalamus following MDMA treatment (Stephenson et
al., 1999). Curiously, our hypophysectomized and thyroparathyroidectomized animals showed a hypothermic response to
MDMA. Hypothyroidism has been shown to stimulate the
SNS by increasing the amounts of norepinephrine in the
plasma (Coulombe and Dussault, 1977). Despite increased
amounts of norepinephrine, the normal thermogenic response to norepinephrine and cold is blunted in hypothyroid
animals (Triandafillou et al., 1982; Sundin et al., 1984), supporting the notion that thyroid hormone plays a permissive
role in SNS-mediated thermogenesis. According to Bianco et
al. (1988), this blunted response is probably due to a reduced
synergistic action between T3 and norepinephrine at the gene
level. In the absence of T3, as would presumably be the case
with our thyroparathyroectomized animals, the norepinephrine response would be attenuated (Bianco et al., 1988). This
defect in the norepinephrine signaling pathway may alter the
stimulatory action of cAMP on UCP-3 activity and/or expression (Gripois and Valens, 1982; Young et al., 1982). Hypothyroidism has also been associated with an up-regulation of ␣1(Dicker et al., 1992) and ␤3-receptors (Rubio et al., 1995b)
and a down-regulation of ␤1- and ␤2-receptors (Rubio et al.,
1995a). We cannot rule out the possibility that changes in
sympathetic receptor levels in our TX animals may well play
a role in the hypothermic response seen in this study. Our
data showing that levothyroxine supplementation restores
the hyperthermic response to MDMA further supports our
hypothesis that thyroid hormone is required for MDMAmediated thermogenesis.
Much controversy surrounds the role of hyperthermia in
the neurotoxic effects of MDMA in experimental animals.
The 5-HT2A/2C receptor antagonists ketanserin (Nash, 1990)
and MDL 11,939 (Schmidt, 1987) not only prevent the neurotoxicity but also block the hyperthermia induced by a low
dose (10 mg/kg) of MDMA. In both of these reports, higher
doses of MDMA still produced hyperthermia, but serotonergic neurotoxicity was nevertheless blocked. Malberg et al.
(1996) showed that the ability of ketanserin to block the
neurotoxicity of MDMA is lost by increasing the body temperature of the animal. Those authors further reported that
pretreatment of the animals with ␣-methyl-para-tyrosine, a
tyrosine hydroxylase inhibitor, induced hypothermia and
prevented the neurotoxicity of subsequently administered
MDMA, but that warming the animals negated these protective effects. Although these data clearly suggest a link between hyperthermia and subsequent neurotoxicity, not all
agents that prevent MDMA-induced neurotoxicity necessarily do so by blocking the hyperthermic response. Fluoxetine
fails to prevent MDMA-induced hyperthermia, yet still affords protection against the neurotoxic process (Malberg et
al., 1996). Antisense oligonucleotides targeting monoamine
165
166
Sprague et al.
Williams PG, and Parness J (2002) Identification of the dantrolene binding sequence on the skeletal muscle ryanodine receptor. J Biol Chem 277:34918 –34923.
Pedersen NP and Blessing WW (2001) Cutaneous vasoconstriction contributes to
hyperthermia induced by 3,4-methylenedioxymethamphetamine (Ecstasy) in conscious rabbits. J Neurosci 21:8648 – 8654.
Rubio A, Raasmaja A, Maia AL, Kim K, and Silva JE (1995a) Effects of thyroid
hormone on norepinephrine signaling in brown adipose tissue. I: ␤1- and ␤2Adrenergic receptors and cyclic adenosine monophosphate generation. Endocrinology 136:3267–3276.
Rubio A, Raasmaja A, and Silva JE (1995b) Effects of thyroid hormone on norepinephrine signaling in brown adipose tissue. II: Differential effects of thyroid
hormone on ␤3-adrenergic receptors in brown and white adipose tissue. Endocrinology 136:3277–3284.
Schmidt CJ (1987) Neurotoxicity of the psychedelic amphetamine, methylenedioxymethamphetamine. J Pharmacol Exp Ther 240:1–7.
Schmidt CJ, Black CK, and Taylor VL (1990) Antagonism of the neurotoxicity due to
a single administration of 3,4-methylenedixymethamphetamine. Eur J Pharmacol
181:59 –70.
Silva JE (1995) Thyroid hormone control of thermogenesis and energy balance.
Thyroid 5:481– 492.
Sprague JE, Banks ML, and Mills EM (2002) UCP3 involvement in the peripheral
hyperthermia induced by 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy). Soc Neurosci Abstr 469.5.
Sundin U, Mills I, and Fain JN (1984) Thyroid-catecholamine interactions in isolated
brown adipocytes. Metabolism 33:1028 –1033.
Stephenson CP, Hunt GE, Topple AN, and McGregor IS (1999) The distribution of
3,4-methylenedioxymethamphetamine ‘Ecstasy’-induced c-fos expression in rat
brain, Neuroscience 92:1011–1023.
Triandafillou J, Gwilliam C, and Himms-Hagen J (1982) Role of thyroid hormone in
cold-induced changes in rat brown adipose tissue mitochondria. Can J Biochem
60:530 –537.
Walubo A and Seger D (1999) Fatal multi-organ failure after suicidal overdose with
MDMA, ‘Ecstasy’: case report and review of the literature. Human Exp Toxicol
18:119 –125.
Williams H, Dratcu L, Taylor R, Roberts M, and Oyefeso A (1998) “Saturday night
fever”: ecstasy related problems in a London accident and emergency department.
J Accid Emerg Med 15:322–326.
Young JB, Saville E, and Landsberg L (1982) Effect of thyroid state on norepinephrine (NE) turnover in rat brown adipose tissue (BAT): potential importance of the
pituitary. Clin Res 30:407A.
Address correspondence to: Dr. Jon E. Sprague, Associate Professor of
Pharmacology, Department of Pharmaceutical and Biomedical Sciences, The
Raabe College of Pharmacy, Ohio Northern University, Ada, OH 45810. Email: [email protected]
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 6, 2017
against serotonergic toxicity. I. Experiments using 3,4-methylenedioxymethamphetamine, discipline, CGS 19755 and NBQX. J Pharmacol Exp Ther 272:860 –
867.
Fekete C, Mihaly E, Lu-Guang L, Kelly J, Clausen J, Mao Q, Rand WM, Moss LG,
Kuhar M, Emerson CH, et al. (2000) Association of cocaine- and amphetamineregulated transcript-immunoreactive elements with thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and its
role in the regulation of the hypothalamic-pituitary-thyroid axis during fasting.
J Neurosci 20:9228 –9234.
Fernandez F, Aguerre S, Mormede P, and Chaouloff F (2002) Influences of the
corticotropic axis and sympathetic activity on neurochemical consequences of
3,4-methylenedioxymethamphetamine (MDMA) administration in Fischer 344
rats. Eur J Neurosci 16:607– 618.
Gong D, He Y, Karas M, and Reitman M (1997) Uncoupling protein-3 is a mediator
of thermogenesis regulated by thyroid hormone, ␤3-adrenergic agonists and leptin.
J Biol Chem 272:24129 –24132.
Gordon CJ, Watkinson WP, O’Callaghan JP, and Miller DB (1991) Effects of 3,4methylenedioxymethamphetamine on autonomic thermoregulatory responses of
the rat. Pharmacol Biochem Behav 14:644 – 653.
Gripois D and Valens M (1982) Uptake and turnover rate of norepinephrine in
interscapular brown adipose tissue of the young rat. Influence of hypothyroidism.
Biol Neonate 42:113–119.
Hinz W, Faller B, Gruninge S, Gazzotti P, and Chiesi M (1999) Recombinant human
uncoupling protein-3 increases thermogenesis in yeast cells. FEBS Lett 448:57–
61.
Kanthasamy A, Sprague JE, Shotwell JR, and Nichols DE (2002) Unilateral infusion
of a dopamine transporter antisense into the substantia nigra protects against
MDMA-induced serotonergic deficits in the ipsilateral striatum. Neuroscience 114:
917–924.
Malberg JE, Sabo KE, and Seiden L (1996) Co-administration of MDMA with drugs
that protect against MDMA neurotoxicity produces different effects on body temperature in the rat. J Pharmacol Exp Ther 278:258 –267.
Malberg JE and Seiden L (1998) Small changes in ambient temperature cause large
changes in 3,4-methylenedioxymethamphetamine (MDMA)-induced serotonin
neurotoxicity and core body temperature in the rat. J Neurosci 18:5086 –5094.
Mallick A and Bodenham AR (1997) MDMA induced hyperthermia: a survivor with
an initial body temperature of 42.9 degrees C. J Accid Emerg Med 14:336 –338.
McDaid J and Docherty JR (2001) Vascular actions of MDMA involve ␣1 and
␣2-adrenoceptors in the anaesthetized rat. Br J Pharmacol 133:429 – 439.
Mechan AO, Esteban B, O’Shea E, Elliott JM, Colado MI, and Green AR (2002) The
pharmacology of the acute hyperthermic response that follows the administration
of 3,4-methylenedioxymethamphetamine (MDMA, ‘ecstasy’) to rats. Br J Pharmacol 135:170 –180.
Mills EM, Xu D, Fegusson MM, Combs CA, Xu Y, and Finkel T (2002) Regulation of
cellular oncosis by uncoupling protein 2. J Biol Chem 277:27385–27392.
Nash JF (1990) Ketanserin pretreatment attenuates MDMA-induced DA release in
the striatum as measured by in-vivo microdialysis. Life Sci 47:2401–2408.
Paul-Pletzer K, Yamamoto T, Bhat MB, Ma J, Ikemoto N, Jimenez LS, Morimoto H,