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THE SEROTONIN RECEPTOR ANTAGONISTS NAN-190, METHIOTHEPIN,
METHYSERGIDE, MIANSERIN OR ONDANSETRON PROTECT AGAINST
METHAMPHETAMINE-INDUCED HYPERTHERMIA IN MICE
Omer T. Ginawi, Aymen K. Al-Suwailem, Abdulhakeem A. Al-Majed
Department of Pharmacology, College of Pharmacy,King Saud University, P.O. Box 2457,
Riyadh 11451, Saudi Arabia
THE SEROTONIN RECEPTOR ANTAGONISTS NAN-190, METHIOTHEPIN,
METHYSERGIDE, MIANSERIN OR ONDANSETRON PROTECT AGAINST
METHAMPHETAMINE-INDUCED HYPERTHERMIA IN MICE
Omer T. Ginawi‫٭‬, Aymen K. Al-Suwailem, Abdulhakeem A. Al-Majed
The effect of some serotonin receptor antagonists was investigated on the body
temperature of male mice. These antagonists produced significant reductions of body
temperature in methamphetamine-induced hyperthermic male mice. Thus, the hyperthermia
induced by methamphetamine (11 µmol/kg,i.p.) was antagonized by the previous i.p.
administration of 1.06 µmol/kg NAN-190 (5-HT1A receptor antagonist), 22.1 µmol/kg
methiothepin (5-HT1B/1D receptor antagonist), 2.13 µmol/kg methysergide (5-HT 2A/2B receptor
antagonist), 3.3 µmol/kg mianserin (5-HT2C and α2 - adrenoceptor antagonist) or 0.003
µmol/kg ondansetron (5-HT3 receptor antagonist). The present results indicate that central
serotonin receptors may be involved in the hyperthermic effect of methamphetamine.
Ondansetron, being effective at a relatively small dose (0.003 µmol/kg) as compared to other
antagonists used in this study, may be a potential antidote for methamphetamine-induced
hyperthermia.
Keywords: methamphetamine, serotonin (5-HT), hyperthermia, Nan-190, methiothepin,
methysergide, mianserin, ondansetron.
__________________________________________________________________________
Department of Pharmacology, College of Pharmacy,King Saud University, P.O. Box 2457,
Riyadh 11451, Saudi Arabia
‫٭‬Author for correspondence
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Introduction
Some aspects of locomotor activity and the stereotyped behavior induced by
methamphetamine, a known drug of abuse worldwide, are probably a consequence of the
release of dopamine from dopaminergic nerve terminals, particularly in the neostriatum. With
higher doses of methamphetamine, disturbances of perception and overt psychotic behavior
occur. These effects may be due to release of 5-hydroxytryptamine (5-HT, serotonin) from
tryptaminergic neurons and of dopamine in the mesolimbic system (1). Other suggested
mechanisms for the actions of methamphetamine include reuptake inhibition of biogenic
amines and inhibition of monoamine oxidase. Both mechanisms may result in increased
concentrations of the biogenic amines centrally and peripherally (1). Neurochemical studies
indicate that methamphetamine increased central serotonin (5-HT) levels more markedly than
other psychomotor stimulants such as amphetamine or cocaine. However, several studies
indicate that 5-HT agonists and uptake inhibitors did not produce any methamphetamine-like
stimulus effects when administrated alone, but if taken together with methamphetamine; the
methamphetamine effects were significantly potentiated (2).
In addition to these mechanisms, methamphetamine may exert direct effects on central
receptors for biogenic amines. Previously it was difficult to investigate such drug-receptor
interactions, especially with serotonergic systems. However with the newly proposed
serotonin receptor subtypes (3) and with the innovation of new selective agonist and
antagonist drugs at these receptors, it became possible to try to investigate the receptor
subtypes involved in the actions of methamphetamine.
Methamphetamine can induce hyperthermia (4) especially if it was used in overdose.
Methamphetamine-induced hyperthermia may have serious consqeuences on the user such
as rhabdomyolysis, culminating in fatal acute renal failure (5).
There is no known specific antidote for methamphetamine-induced hyperthermia.
Haloperidol, the dopamine antagonist, has been used to reduce many of the behavioural
actions
induced
by
methamphetamine.
However,
haloperidol
precipitates
serious
extrapyramidal side effects in the user. Therefore, search for a suitable treatment of the
serious outcomes of poisoning with methamphetamine, such as rhabdomyolysis, is
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encouraged. Serotonin plays an important role in thermoregulation. Methamphetamine
releases serotonin especially when used at high poisoning doses and causes hyperthermia.
Therefore, the present study was designed to identify some of the serotonin receptors
involved in the hyperthermic action of methamphetamine, by using some of the available
selective 5-HT antagonists.
Materials and Methods
Drugs
Drugs used in this study were: methamphetamine hydrochloride (Merck AG. Darmstadt,
Germany), NAN–190 hydrobromide (Sigma Chemical Co., St. Louis, USA), methiothepin
(Winlab, Leicestershire, UK), methysergide (Research Biochemicals Inc., Wayland, USA),
mianserin hydrochloride (Sigma Chemical Co., St. Louis, USA) and ondansetron
hydrochloride dihydrate (Glaxo Laboratories Ltd, Greenford, UK). All drugs were dissolved in
0.9%NaCl solution. Doses are expressed as µmol/kg of the salt. Doses were administered in
a volume of 10 ml/kg, intraperitoneally.
Animals
Male Swiss albino mice (obtained from the Animal Care Center, College of Pharmacy,
King Saud University) weighing 25-30 grams were used. The animals were housed, 10 mice
per cage, under conditions of constant room temperature (22 ± 1oC), humidity and light cycle
(7 a.m. to 7 p.m.). They were given access to food (standard lab chow, Grain Silos and Flour
Mills Organization, Riyadh) and water ad libitum. Each mouse was used only once.
Treatment protocol
In each of the experimental procedures planned, two groups (each group consisting of 10
mice) were designated for each antagonist study. The first group was for antagonist + saline
study, and the second group was for antagonist + methamphetamine study. In addition, two
groups (each group consisting of 10 mice) were designated for cotrol purposes. These were
saline + saline group and saline + methamphetamine group.
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The first drug (i.e. saline or antagonist) was given 30 min before the second drug (saline or
methamphetamine) .
Measurement of body temperature
Temperature measurements were made at room temperature (22±1°C). The range of
body temperature for all animals used in this experiment was (37 - 37.8o C). Measurement
of body temperature was performed before any drug administration and at 30 and 90
minutes after injection of the second drug. Rectal temperature was measured by gently
inserting a lubricated temperature probe (model YSI 400, France) 2 cm into the rectum of
the mouse for 30 seconds. The probe was connected to a digital thermometer (Apelex,
Pb0331, Panlab, France). Increases or decreases in body temperature were calculated
by subtraction of the post-injection from the pre-injection rectal temperature readings.
Statistics
Statistical analysis of the results was performed by using one way ANOVA. For
significant results, a post-hoc comparison between the means was done by Tukey-krammer
test. Significance was accepted at P = 0.05.
Results
Appropriate dosages for methamphetamine and for each of the 5-HT antagonists were
determined from pilot experiments, conforming with doses of these drugs commonly
employed in previous studies cited in the literature.
Methamphetamine at a dose of 11 µmol/kg, i.p. (2 mg/kg), significantly increased the
normal body temperature of male mice during an observation period of 90 min. This dose of
methamphetamine was selected for challenge with 5-HT antagonists in all experiments of this
study.
The effect of NAN-190 on methamphetamine-induced hyperthermia in male mice:
Escalating
doses
of
NAN-190
were
tested
against
methamphetamine-induced
hyperthermia in male mice. Practically, the least possible dose of NAN-190, which
significantly (P< 0.001) abolished the hyperthermia-induced by methamphetamine during the
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90 min observation period, was 1.06 µmol/kg, i.p. ( 0.5 mg/kg), as shown in Fig 1. This dose
of NAN-190 did not alter the normal body temperature of mice when used alone.
The effect of methiothepin on methamphetamine-induced hyperthermia:
The least possible dose of methiothepin, which significantly (P< 0.001) abolished the
hyperthermia-induced by methamphetamine during the 90 min observation period was 22.1
µmol/kg, i.p.( 0.1 mg/kg), as shown in Fig 2. However, this dose of methiothepin caused a
significant reduction in normal body temperature of male mice when used alone (P< 0.05).
The effect of methysergide on methamphetamine-induced hyperthermia:
Various doses of methysergide were tested against methamphetamine-induced
hyperthermia in male mice. The least possible dose of methysergide, which significantly
(P<0.001) abolished the hyperthermia-induced by methamphetamine during the 90 min
observation period (Fig 3.) was 2.13 µmol/kg, i.p. (1 mg/kg). This dose of methysergide did
not affect the normal body temperature of mice when used alone.
The effect of mianserin on methamphetamine-induced hyperthermia:
The least possible dose of mianserin, which significantly (P< 0.001) abolished the
hyperthermia-induced by methamphetamine during the 90 min observation period was 3.3
µmol/kg, i.p. (1mg/kg), as shown in Fig 4. This dose of mianserin did not affect the normal
body temperature of mice when used alone.
The effect of ondansetron on methamphetamine-induced hyperthermia:
An extremely small dose of ondansetron (0.003 µmol/kg, i.p. = 0.001 mg/kg ) significantly
(P< 0.001) abolished the hyperthermia-induced by methamphetamine during the 90 min
observation period (Fig 5.). This dose of ondansetron had no effect on the normal body
temperature of mice when used alone.
Discussion
In this study, methamphetamine produced a significant increase in body temperature. The
hyperthermia-induced by methamphetamine have previously been explained to be due to
methamphetamine’s increases of oxidative stress, which may mediate the toxic effects of this
drug. Systemic methamphetamine increases hydroxyl radical formation and leads to protein
modification in the striatum (6-8). In addition, energy use is increased after systemic
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administration of methamphetamine, as evidenced by an immediate and sustained increase
in the extracellular concentrations of lactate in the striatum (9). Furthermore, adenosine
triphosphate (ATP) concentrations are rapidly lost in brain slices from the mouse striatum
after methamphetamine pretreatment (10).
Methamphetamine-induced hyperthermia is also related to the toxicity of
methamphetamine on both serotonin (5-HT) and dopamine (DA) neurons. Following an initial
massive release of these neurotransmitters, methamphetamine causes long lasting
decreases in tissue concentrations of 5-HT and DA (11-13), as well as decreases in the
activities of their respective rate-limiting enzymes, tryptophan hydroxylase and tyrosine
hydroxylase, respectively(14).
Serotonin is a biogenic amine that is distributed widely in various body tissues and cell
types and possesses a diversity of pharmacological effects at both central and peripheral
sites. Serotonin is involved in the control of temperature regulation, cardiovascular function,
muscle contraction and endocrine regulation. Seven major families of 5-HT receptors have
been identified, all of which have distinct pharmacological properties, regional distributions
and physiological functions. The physiological effects of 5-HT are mediated by at least four
groups of 5-HT receptors, which have been distinguished pharmacologically according to the
second messenger systems to which they are coupled (15).
In the present study, and with the exception of methiothepin, all 5-HT antagonists used
(i.e. NAN-190, methysergide, mianserin and ondansetron) did not alter the body temperature
when used alone. However, all the 5-HT antagonists tested in this study, including
methiothepin abolished methamphetamine-induced hyperthermia. These findings may be
related to the ability of these antagonists to act directly or indirectly to block the actions of
released 5-HT on the central neuronal pathways involved in the temperature regulation and
therefore prevent any temperature elevation (16-18).
NAN-190 is not a pure selective antagonist at 5-HT1A receptors. Recently, NAN-190 has
been reported to be a partial agonist of presynaptic and an antagonist of postsynaptic 5-HT1A
receptors and alpha1-adrenoceptors (19). Therefore, the results of NAN-190 in the present
study should be cautiously interpreted. The same investigators, however (19), studied the
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effects of MM77 and MP245 (antagonists of postsynaptic 5-HT1A receptors), as well as 8-OHDPAT (a 5-HT1A agonist) and prazosin (an alpha1-adrenoceptor antagonist) on the body
temperature of mice. All of these agents induced a dose-dependent hypothermia in mice,
indicating the involvement of their respective receptors in thermoregulation.
The results of the present study indicate the possible involvement of 5-HT1,2,3 receptors (or
some of their subtypes) in methamphetamine-induced hyperthermia. Thus, 5-HT1A receptors
(which are blocked by NAN-190), 5-HT1B/1D receptors (which are blocked by methiothepin), 5HT 2A/2B (which are blocked by methysergide), 5-HT2C (which are blocked by mianserin) and
5-HT3( which are blocked by ondansetron ) are involved in methamphetamine-induced
hyperthermia. A relatively small dose of onadansetron,as compared to the other 5-HT
antagoists used in this study, has successfully reversed methamphetamine-induced
hyperthermia. This observation suggests that 5-HT3 receptor is a possible site of action in
methamphetamine-induced hyperthermia. However, this is not to exclude 5-HT4-7 receptors,
which were not investigated in this study.
Little is known about the role of 5-HT in skeletal muscle cells, the site for rhabdomyolysis.
However, 5-HT exerts direct effects at 5-HT receptors in skeletal muscles. It has been shown
that administration of 5-HT produced an increase in the intracellular concentration of cyclic
3',5'-AMP in skeletal muscle preparations via specific 5-HT receptors on skeletal muscle ( 20).
The 5-HT3 receptor is a ligand-gated ion channel and activation of this receptor leads to
increased adenylyl cyclase activity and hence muscle contraction. Therefore, it is not
unusual that blockers of this receptor may antagonize muscle contraction and may protect
against hyperthermia and rhabdomyolysis.
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Legends
- Fig 1. Effect of NAN-190 alone (0.5 mg/kg, i.p) and in combination with
methamphetamine (met 2 mg/kg, i.p) on the normal body temperature of
male mice.
***: P< 0.001, as compared to saline control.
###: P< 0.001, as compared to saline + methamphetamine group.
- Fig 2. Effect of methiothepin (mtio) alone (0.1 mg/kg, i.p) and in
combination with methamphetamine (2 mg/kg, i.p) on the normal body
temperature of male mice.
*: P< 0.05, as compared to saline control.
***: P< 0.001, as compared to saline control.
###: P< 0.001, as compared to saline + methamphetamine group.
- Fig 3. Effect of methysergide (methys) alone (1 mg/kg, i.p) and in
combination with methamphetamine (2 mg/kg, i.p) on the normal body
temperature of male mice.
***: P< 0.001, as compared to saline control.
###: P< 0.001, as compared to saline + methamphetamine group.
- Fig 4. Effect of mianserin (mian) alone (1 mg/kg, i.p) and in combination with
methamphetamine (2 mg/kg, i.p) on the normal body temperature of male
mice.
***: P< 0.001, as compared to saline group.
###: P< 0.001, as compared to saline + methamphetamine group.
- Fig 5. Effect of ondansetron (ond) alone (0.001 mg/kg, i.p) and in
combination with methamphetamine (2 mg/kg, i.p) on the normal body
temperature of male mice.
***: P< 0.001, as compared to saline control.
###: P< 0.001, as compared to saline + methamphetamine group.
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Fig 1.
13
Fig 2.
14
Fig 3.
15
Fig 4.
16
Fig 5.
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