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Vol. 280, Issue 2, 941-947, 1997
Departments of Psychiatry (T.T., K.T.F.), Pharmacology and Toxicology (K.T.F.) and Neuroscience (K.T.F.), University of Utah School of Medicine and the Veterans Administration Medical Center, Salt Lake City, Utah
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Abstract |
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 Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The role of nitric oxide (NO) in the long-term, amine-depleting effects of methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA) was investigated in the rodent central nervous system. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) antagonized the dopamine- and serotonin-depleting effects of both METH and MDMA. The protective actions of L-NAME in METH-treated mice were reversed by prior administration of the NO generator isosorbide dinitrate. However, pretreatment with NG-monomethyl-L-arginine or NG-nitro-L-arginine, two other NO synthase inhibitors, failed to block the neurotoxic effects of METH or MDMA. L-NAME was also the only NO synthase inhibitor that antagonized the hyperthermic effects of METH, reducing colonic temperatures in mice by a mean of 3°C, in comparison with control. Moreover, if the hypothermic effects of L-NAME in METH-treated mice were prevented by raising the ambient room temperature, the dopamine-depleting actions of the stimulant were fully restored. The latter findings suggest that it is the hypothermic actions of L-NAME, rather than its NO inhibitory properties, that are responsible for the prevention of neurotoxicity. Together with the results of the NG-monomethyl-L-arginine and NG-nitro-L-arginine experiments, the data suggest that NO plays little or no role in the toxic mechanism of action of METH or MDMA.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Amphetamine and several of its
analogs (METH and MDMA) produce persistent decreases in the CNS
concentrations of DA and serotonin (5-HT) in rodents and monkeys (for
reviews, see Seiden and Ricaurte, 1987
; Finnegan and Schuster, 1989;
Gibb et al., 1990). The decline in neurotransmitter levels
is accompanied by equally persistent decreases in the number of DA and
5-HT reuptake sites and in the synthetic capacities of the enzymes
tyrosine and tryptophan hydroxylase. Coupled with morphological data
showing the presence of degenerating axons and terminals in these same
brain areas (Ellison et al., 1978; Ricaurte et
al., 1982; Molliver et al., 1990), the data have been
interpreted as indicating that the amphetamines damage DA and 5-HT
neurons in experimental animals. Although previous studies have
implicated changes in both dopaminergic and glutamatergic neurotransmission in the mechanism of the toxicity (Schmidt et al., 1985; Stone et al., 1988; Sonsalla et
al., 1989; Finnegan et al., 1990), the neurochemical
events responsible for the neuronal damage remain uncertain.
NO is a versatile substance implicated in the regulation of macrophage
killing, vascular tone and neurotransmission (for reviews, see Moncada
et al., 1991; Nathan, 1992; Iadecola et al.,
1994). NO is a known mediator of tissue injury in the periphery; based on this observation, its involvement in the pathophysiology of cell
death in the CNS has received considerable recent attention. As
reviewed previously (Choi, 1988; Coyle and Puttfarcken, 1993), glutamate release and the overstimulation of glutamate receptors appear
to underlie the neuron-damaging effects of several acute CNS insults,
including ischemia/hypoxemia, hypoglycemia and traumatic injury.
Glutamate rapidly stimulates NO production by a mechanism that involves
NMDA receptor stimulation and the translocation of calcium (Garthwaite
et al., 1988, 1989; Bredt and Snyder, 1989, 1990; Knowles
et al., 1989). The resultant increase in intracellular calcium is thought to trigger a calmodulin-mediated phosphorylation event that in turn activates the enzyme responsible for the synthesis of NO (NOS). In vitro, the inhibition of NOS prevents the
neuron-damaging effects of NMDA and glutamate in cortical cell culture
(Dawson et al., 1991). In vivo, CNS NO
concentrations become elevated shortly after the onset of cerebral
ischemia (Kader et al., 1993; Malinski et al.,
1993; Sato et al., 1993), whereas the inhibition of NO
production is reported to decrease cellular damage, as indicated by
reductions in infarction volume (Nowicki et al., 1991;
Buisson et al., 1992; Nagafuji et al., 1992;
Ashwal et al., 1993). Results such as these have lead to the
proposal that the neuron-damaging effects of glutamate are mediated by
NO. Reductions in cell damage (Garthwaite and Garthwaite, 1994) or
infarction volume (Yamamoto et al., 1992; Kuluz et
al., 1993) have not always been observed, however, suggesting that
any role for NO in glutamate-induced neurotoxicity is complex.
Mechanisms by which NO could produce cell damage include the inhibition
of iron-containing enzymes such as complexes I and II of the
mitochondrial respiratory chain (Stadler et al., 1991), thiol inactivation and protein ribosylation (Dimmeler et
al., 1992; Zhang et al., 1994), alterations in DNA
synthesis (Wink et al., 1991) or the reaction of NO with
superoxide to generate the potent oxidant peroxynitrite and other
destructive oxygen radicals (Radi et al., 1991). Because
several of the above mechanisms may also play a role in
amphetamine-induced neuronal damage, it is possible that the modes of
action of NO and the amphetamines may overlap. Indeed, the idea that NO
might be directly involved in the toxic mechanism of action of the
amphetamines is especially attractive because glutamate has been
suggested to play a central role in both. METH is reported to increase
synaptic concentrations of glutamate (Nash and Yamamoto, 1992, 1993;
Abekawa et al., 1994), and the neurotoxic effects of the
stimulants, like glutamate, are blocked by NMDA antagonists (Sonsalla
et al., 1989, 1991; Finnegan et al., 1990, 1991).
Although open to debate (Bowyer et al., 1993, 1994; Farfel
and Seiden, 1995), one interpretation of these data is that the
amphetamines damage neurons by stimulating glutamate release. If
increases in NO mediate the injurious effects of excess glutamate, it
might be predicted that NOS inhibitors would also block the toxic
effects of the amphetamines. The aim of the present studies was to test
this hypothesis by evaluating the abilities of several NOS inhibitors
to block the long-term, amine-depleting effects of METH in mice and
MDMA in rats.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Animals and drugs. Male CF-1 mice (30 g) or male Sprague-Dawley rats (200 g) were used (Sasco, Omaha, NE). Animals were housed in an American Association for the Accreditation of Laboratory Animal Care-approved facility, under conditions of constant room temperature (23°C) and humidity (45%). The rodents were provided with 12 hr of light per day; food and water were given ad libitum. All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care Committee.
The NOS inhibitors L-NAME, NG-monomethyl-L-arginine and NG-nitro-L-arginine were purchased from the Sigma Chemical Co. (St. Louis, MO). Isosorbide dinitrate, a compound that spontaneously generates NO in solution, was also obtained from Sigma. METH and MDMA were generously donated by the National Institute on Drug Abuse (Washington, DC). Drug doses are expressed as the free base, and all drugs were dissolved in 0.9% saline immediately before injection. METH or MDMA was administered s.c. to rats four times, with injections separated by intervals of 2 hr. In general, multiple doses of the NOS inhibitors were given concurrently with the administration of the stimulants. Doses of the NOS inhibitors used were based on published literature values and on pilot studies. The exact dose, route of administration and schedule for each drug used in the experiments are detailed in the figure legends.Assay of catecholamines and indoleamines.
One week after
drug administration, mice were sacrificed by cervical dislocation,
whereas rats were sacrificed by decapitation. The striatum of both
species was rapidly dissected free, and the tissue samples were frozen
in liquid nitrogen (Finnegan et al., 1989). Samples of the
hippocampus and frontal cortex were also obtained from rats. Levels of
amines in the striatum (DA, homovanillic acid, dihydroxyphenylacetic
acid, 5-HT and 5-hydroxyindoleacetic acid) and in the hippocampus and
frontal cortex (5-HT and 5-hydroxyindoleacetic acid) were assayed using
high-pressure liquid chromatography, coupled with electrochemical
detection, as described previously (Finnegan et al., 1989).
The concentrations of the various amines (in nanograms per milligram of
tissue) were determined by comparison with standard curves.
Measurement of colonic temperature. Colonic temperatures of mice were obtained using an electronic thermometer attached to a rectal temperature probe (TRI-R instruments, Jamaica, NY). The probe was lubricated with surgical lubricant (Surgilube; Surgical Supply, Inc., Melville, NY) and inserted exactly 2.5 cm into the rectum for 30 sec. Colonic temperature was recorded immediately before drug administration (base line) and at 30-min intervals thereafter.
Statistics. A one-factor analysis of variance was used when the data involved measurements of catecholamines and indoleamines (Sigmastat; Jandel Scientific). When the overall analysis was statistically significant, differences between individual groups were compared post hoc by Student t test (corrected for multiple comparisons by the Bonferroni method). Colonic temperature data were analyzed using a two-factor analysis of variance (treatment × time) with repeated measures across time. Differences between individual groups were compared post hoc using the Student-Newman-Kuels method. The significance level was set at P < .05.
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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As shown in figure 1, METH significantly decreased
the concentrations of DA in the striatum of mice sacrificed 1 week
after completion of the multiple-dose regimen (38% of control).
Similar declines in the levels of striatal dihydroxyphenylacetic acid and homovanillic acid were also noted (data not shown). Pretreatment with the NOS inhibitor L-NAME blocked the DA-depleting
effects of METH in a dose-related fashion. The dose-dependent nature of the antagonism was most apparent at the two lowest doses of
L-NAME tested (37 and 75 mg/kg/injection) and appeared to
plateau at higher doses (150 and 300 mg/kg/injection). The
administration of L-NAME alone (150 mg/kg/injection) did
not significantly affect the concentration of striatal DA. These
observations are consistent with the notion that NO is involved in the
toxic mode of action of METH.
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The protection by NOS inhibitors against glutamate- or NMDA-induced
neuronal cell damage is reversed by the administration of compounds
that spontaneously liberate NO (Dawson et al., 1991). We
therefore investigated whether the NO generator isosorbide dinitrate
would restore the toxic effects of METH in the presence of
L-NAME. Consistent with the findings in the previous
experiment, METH alone profoundly lowered striatal DA concentrations to
23% of control (fig. 2), whereas pretreatment with
L-NAME provided significant protection against the toxicity
(79% of control). Administration of isosorbide dinitrate, however,
restored the DA-depleting effects of the stimulant in mice concurrently
given L-NAME and METH; that is, isosorbide dinitrate
appeared to reverse the protective effects of L-NAME (fig.
2). The restoration of METH-induced toxicity was most dramatic in the
mice receiving the larger dose of isosorbide dinitrate (500 mg/kg/injection); the mean DA depletion in this group was the same as
that observed in mice given METH alone (23% of control). The
administration of isosorbide dinitrate by itself or in combination with
METH produced no significant effect on striatal DA levels, in
comparison with the appropriate control. These data appear to provide
additional support for the proposal that threshold concentrations of NO
are required for the expression of METH-induced neurotoxicity.
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Two other NOS inhibitors were investigated, to rule out the possibility
that the neuroprotective effects of L-NAME might have resulted from some nonspecific action. In one set of experiments, mice
were injected with 50 mg/kg
NG-nitro-L-arginine twice each day
for 4 days and then given METH on day 5. This dosing regimen for
NG-nitro-L-arginine was reported to
inhibit NOS activity in the mouse CNS by >95% (Dwyer et
al., 1991). Surprisingly, no protection was observed (fig.
3). Higher multiple doses of
NG-nitro-L-arginine (75 mg/kg/injection) likewise provided no protection. Similar results were
found when a third NOS inhibitor,
NG-monomethyl-L-arginine, was
studied; that is, pretreatment of mice with multiple doses of
NG-monomethyl-L-arginine did not
alter the DA-depleting effects of METH (fig. 3).
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The role of NO in the neurotoxic effects of MDMA was also investigated.
MDMA is structurally related to METH but preferentially targets CNS
5-HT, rather than DA, neurons in rats (Stone et al., 1987).
As shown in figure 4, pretreatment with
L-NAME significantly attenuated the long-term,
5-HT-depleting effects of MDMA in both the hippocampus and frontal
cortex of rats. As was found for METH, however, the neuroprotective
effects of L-NAME were not shared by a second inhibitor of
NOS, in this case
NG-nitro-L-arginine. The finding
that L-NAME blocks the amine-depleting effects of METH and
MDMA but other NOS inhibitors do not suggests that the protective
actions of the drug arise as a consequence of some action other than
its inhibition of NOS.
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Recent studies have shown that temperature plays an important role in
the neuron-damaging effects of METH. Placing the animals in a cold
environment (e.g., a walk-in refrigerator) during the period
of METH administration, for example, or administering drugs that lower
body temperature blocks the toxicity (Bowyer et al., 1993,
1994). Based on these findings, we examined the effects of
L-NAME (fig. 5, top) or
NG-nitro-L-arginine (fig. 5, bottom)
on colonic temperature in METH-treated mice. As illustrated in figure
5, top, L-NAME (administered at doses that provide
significant protection against the toxic effects of METH) induced a
profound hypothermic response when given concurrently with the
stimulant. Compared with saline- or METH-alone-treated mice, animals
given the combination of L-NAME and METH displayed a mean
decrease in colonic temperature of almost 3°C. In contrast, colonic
temperatures in mice treated concurrently with
NG-nitro-L-arginine and METH (fig.
5, bottom) were not significantly different from those observed in
saline- or METH-alone-treated mice; that is,
NG-nitro-L-arginine did not produce
hypothermia when given with METH.
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The data raise the possibility that the hypothermia produced by
L-NAME may be responsible for its neuroprotective effects. If this is true, preventing the hypothermia might restore the toxicity
of METH. This was accomplished by periodically moving the animals from
the laboratory (23°C) to an incubator (33°C) for 10 min whenever
their colonic temperatures were observed to fall below 37°C. In this
fashion, mice given L-NAME and METH concurrently and mice
given METH alone were made similar with respect to body temperature. As
shown in figure 6, striatal DA levels in
L-NAME/METH-treated mice whose colonic temperatures were
maintained at or above 37°C were similar to those of animals given
METH alone; that is, prevention of hypothermia appeared to restore the
toxicity of the stimulant in the presence of L-NAME.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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It is a commonly held maxim that drugs exert multiple
pharmacological effects and that any given pharmacological action can be produced by many different drugs. The findings here highlight the
fact that several commonly used inhibitors of NOS are no exception to
this rule; they also emphasize the value of using multiple drugs in an
effort to circumvent the problem of nonspecificity. We observed that
the NOS inhibitor L-NAME protected against the long-term,
DA-depleting effects of METH, a finding that tends to support the
notion that this diffusible gas plays a key role in the neurotoxic
mechanism of action of the stimulant. In accordance with this idea,
pretreatment with the NO donor isosorbide dinitrate restored the
DA-depleting effects of METH in L-NAME-treated mice. L-NAME also partially prevented the long-term,
5-HT-depleting effects of the related neurotoxin MDMA. These findings
are reminiscent of those provided by other investigators, showing that
inhibitors of NOS prevented the neuron-damaging effects of glutamate
and its analogs in cell culture (Dawson et al., 1991) and
in vivo (Nowicki et al., 1991; Buisson et
al., 1992; Nagafuji et al., 1992; Ashwal et
al., 1993), a result that was often reversed by the addition of NO
donors or the NOS substrate L-arginine. The conclusion that
NO is involved in METH-induced toxicity is also attractive because much
of the work on NO and neuronal cell death has centered on the action of
glutamate, an excitatory amino acid that has also been implicated in
the toxic mechanism of action of both METH and MDMA (Sonsalla et
al., 1989; Finnegan et al., 1990; Nash and Yamamoto,
1992).
Our subsequent studies, however, seem to dispute this initial
conclusion because
NG-monomethyl-L-arginine and
NG-nitro-L-arginine, two other
NG-substituted L-arginine analogs
commonly used to inhibit NOS activity (for reviews, see Moncada
et al., 1991; Nathan, 1992; Iadecola et al.,
1994), provided no protection against the amine-depleting effects of
METH or MDMA. Although the negative findings might be explained by
inadequate dosing or perhaps by reductions in the delivery of the
inhibitors to the CNS, we view these explanations as unlikely. Reduced
delivery might occur because the inhibition of NOS is known to produce
substantial effects on cerebral vascular tone, blood flow and arterial
blood pressure (Iadecola et al., 1994). However, METH- or
MDMA-induced amine depletions in animals pretreated with
NG-monomethyl-L-arginine or
NG-nitro-L-arginine were very
similar to those observed in animals given the neurotoxins alone,
indicating that such hemodynamic effects did not affect the delivery of
the neurotoxins, and by extension the NO inhibitors, to the brain. That
L-NAME was neuroprotective, of course, is also consistent
with this conclusion. Inadequate dosing of the two NOS inhibitors is
another possibility. However, Dwyer et al. (1991) reported
that the systemic administration of 50 mg/kg
NG-nitro-L-arginine twice daily for
4 days inhibited NOS activity in the CNS by 95%. We evaluated the
effects of 50 and 75 mg/kg/injection NG-nitro-L-arginine using the same
schedule of administration and found no protection. These doses may
actually be in excess of those required, because other investigators
have shown that considerably lower doses (e.g., 5-10 mg/kg
NG-nitro-L-arginine) are capable of
reducing infarction volume and neurological deficits in a rodent model
of stroke (Nowicki et al., 1991; Nagafuji et al.,
1992). The systemic administration of a single 30 mg/kg dose of
NG-monomethyl-L-arginine profoundly
reduces blood flow in the cerebral cortex and various deep structures
of brain (Tanaka et al., 1991; Adachi et al.,
1993), although this finding probably results more from the inhibition
of endothelial NOS. Nonetheless, because animals in the present
experiments were treated multiple times with equivalent or higher doses
of the two inhibitors, adequate inhibition of the brain isozyme seems
likely to have been achieved. Because only one of the three NOS
inhibitors used blocked the amine-depleting effects of METH or MDMA, we
conclude that NO is unlikely to be involved in the toxic mechanism of
action of the stimulants. Other factors, evidently, must account for
the neuroprotective effects of L-NAME.
One possibility in this regard is the effect of L-NAME on
core body temperature. Recent studies have shown that reducing body temperature by placing rats in a cold environment prevents METH-induced neurotoxicity, whereas increasing body temperature potentiates the
toxicity (Bowyer et al., 1993, 1994). Similar findings have been reported for MDMA (Schmidt et al., 1990). These data
indicate that, whatever the mechanism of the toxicity, it is evidently strongly regulated by body temperature. When colonic temperatures in
mice treated with L-NAME and METH were investigated, we
found that the NO inhibitor induced profound hypothermia, reducing the mean core body temperature by almost 3°C, in comparison with saline- or METH-alone-treated mice. This reduction in colonic temperature is
twice that previously noted to be effective in blocking the neuron-damaging actions of METH (Bowyer et al., 1993),
indicating that the hypothermic actions of L-NAME could
very well account for its neuroprotective effects. In contrast,
NG-nitro-L-arginine, one of the NO
inhibitors that did not protect against METH- or MDMA-induced toxicity,
produced no significant effect on body temperature in METH-treated
mice. Moreover, periodic exposure to warmer ambient temperatures fully
restored the DA-depleting effects of METH in L-NAME-treated
mice. All of these data support the argument that it is the hypothermic
actions of L-NAME, and not its NO inhibitory properties,
that are responsible for its ability to antagonize the long-term,
amine-depleting effects of METH and MDMA. The ability of isosorbide
dinitrate to restore the toxicity in L-NAME/METH-treated
mice is puzzling in this regard but perhaps reflects an antagonism of
the hypothermia produced by L-NAME. This was not
investigated, however, and remains a topic for future research.
Our conclusions concerning L-NAME are consistent with a
number of recent studies demonstrating that the neuroprotective effects of a variety of agents appear to result from their common ability to
block the hyperthermic actions of the amphetamines and to lower body
temperature. Glutamatergic antagonists, such as MK-801 and CGS19755,
reduce METH-induced toxicity to a degree predicted by their inhibition
of the hyperthermia, whereas increasing the ambient temperature
abolishes the neuroprotection (Bowyer et al., 1994; Albers
and Sonsalla, 1995). NMDA antagonists are also known to block the
5-HT-depleting effects of MDMA (Finnegan et al., 1990), and
in a analogous series of experiments Farfel and Seiden (1995) showed
that MK-801 and CGS19755 do so by inducing hypothermia in MDMA-treated
animals. Similarly, Albers and Sonsalla (1995) have reported that the
mechanism by which several dopaminergic drugs (e.g., the
catecholamine synthesis inhibitor 
-methyl-p-tyrosine or
the DA receptor antagonists haloperidol, sulpiride and SCH 23390) block
METH-induced toxicity involves reductions in body temperature. In
addition to dopaminergic and glutamatergic antagonists, hypothermia
appears to explain the protective actions of a variety of other drugs
(ethanol, pentobarbital, diethyldithiocarbamate, aminooxyacetic acid,
propranolol and dilantin) (Albers and Sonsalla, 1995; Miller and
O'Callaghan, 1995). All of these data suggest that hypothermia is a
common factor in the protective effects of many different drugs, and
they support the contention that reductions in body temperature may
similarly underlie the ability of L-NAME to block the toxic
effects of METH and MDMA. Given the structural diversity of these
compounds, it is likely that multiple mechanisms are involved in their
capacity to induce hypothermia and that, therefore, it is the
hypothermia itself that is key. Although this is undoubtedly an
important clue, the significance of the hypothermia with respect to the
mechanism of toxicity remains to be determined.
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Acknowledgments |
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The authors gratefully acknowledge the technical assistance of Jennifer Clikeman in the execution of these studies.
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Footnotes |
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Accepted for publication October 21, 1996.
Received for publication May 10, 1996.
1 This work was supported in part by grants from the Office of Veterans Affairs and the National Institute on Drug Abuse (DA07239).
Send reprint requests to: Kevin T. Finnegan, M.D., Ph.D., Psychiatry Service (116A), Veterans Administration Medical Center, 500 Foothill Blvd., Salt Lake City, UT 84148.
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Abbreviations |
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CNS, central nervous system; DA, dopamine; 5-HT, 5-hydroxytryptamine; MDMA, 3,4-methylenedioxymethamphetamine; METH, methamphetamine; l-NAME, NG-nitro-L-arginine methyl ester; NMDA, N-methyl-D-aspartate; NO, nitric oxide; NOS, nitric oxide synthase.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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