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 Previous Article
The Journal of Neuroscience, February 15, 2001, 21(4):1413-1419
Methamphetamine-Induced Rapid and Reversible Changes in Dopamine
Transporter Function: An In Vitro Model
Verónica
Sandoval,
Evan L.
Riddle,
Yvette V.
Ugarte,
Glen
R.
Hanson, and
Annette E.
Fleckenstein
Department of Pharmacology and Toxicology, University of Utah, Salt
Lake City, Utah 84112
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ABSTRACT |
This laboratory has demonstrated that a single methamphetamine
(METH) injection rapidly and reversibly decreases the activity of the
dopamine transporter (DAT), as assessed ex vivo in
synaptosomes prepared from treated rats. This decrease does not occur
because of residual drug introduced by the original injection or nor is it associated with a change in binding of the DAT ligand WIN35428. The
purpose of this study was to elucidate the mechanism or mechanisms of
this METH effect by determining whether direct application of this
stimulant to synaptosomes causes changes in DAT similar to those
observed ex vivo. Similar to the ex vivo
effect, incubation of striatal synaptosomes with METH decreased DAT
activity, but not WIN35428 binding: the effect on activity was not
eliminated by repeated washing of synaptosomes. Also, as observed
ex vivo, incubation with
3,4-methylenedioxymethamphetamine, but not cocaine or methylphenidate,
caused a METH-like reduction in DAT function. The rapid and reversible
METH-induced diminution in DAT activity did not occur because of a
change in membrane potential, as assessed in vitro and
ex vivo by
[3H]tetraphenylphosphonium accumulation. However,
the METH-related decline in DAT function may be attributed to
phosphorylation because NPC15437, a protein kinase C inhibitor,
attenuated the METH-induced decline in DAT function. Similarities
between previously reported effects ex vivo of a single
METH injection on serotonin and norepinephrine transporter function and
effects of direct METH application in vitro were also
found. Together, these data demonstrate that the in
vitro incubation model mimics the rapid and reversible effects observed after a single METH injection.
Key words:
in vitro; rapid and reversible; serotonin; norepinephrine; transport; phosphorylation
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INTRODUCTION |
It has been established that the
monoamine transporters [i.e., dopamine (DA), serotonin (5-HT), and
norepinephrine (NE)] are the biological targets for stimulants such as
amphetamine, a metabolite of methamphetamine (METH) (Azzaro et al.,
1973 ). These transporters are affected acutely by promoting the release
of neurotransmitters via a carrier-mediated efflux (Fischer and Cho,
1979; Liang and Rutledge, 1982), as well as through the reversal of the
transporter (Sulzer et al., 1995). In addition to these acute changes
in transporter activity, multiple high-dose administrations of METH
cause a persistent loss of transporter protein (Wagner et al., 1980;
Ricaurte et al., 1982), as well as depress tyrosine hydroxylase
activity (Kogan et al., 1976; Hotchkiss and Gibb, 1980).
Investigations from this laboratory have demonstrated that the acute
effects caused by a single and multiple administrations of METH are
distinct from long-term transporter deficits presumably associated with
nerve terminal degeneration (Fleckenstein et al., 1997; Kokoshka et
al., 1998b). For instance, dopamine transporter (DAT) function rapidly
and reversibly decreases after a single injection of METH (Fleckenstein
et al., 1997), whereas multiple administrations of METH cause a rapid
decrease in DAT activity that is partially reversed (Fleckenstein et
al., 1997; Kokoshka et al., 1998b). Similar to DAT activity, serotonin
transporter (SERT) function is also rapidly and reversibly affected by
single and multiple METH administrations (Kokoshka et al., 1998a;
Haughey et al., 2000b). These acute transporter changes reflect a
Vmax reduction and do not occur
because of residual METH introduced by the original injection, as
suggested by findings that the effects observed are still present after
successive washing of METH-treated synaptosomes (Fleckenstein et al.,
1997; Kokoshka et al., 1998a,b). Like the activities of DAT and SERT,
norepinephrine transporter (NET) activity decreases after both single
and multiple METH administrations (Fleckenstein et al., 1997; Kokoshka
et al., 1998a; Haughey et al., 2000a). However, the changes in NET
activity occur because of a direct competitive interaction of METH with
the transporter; as suggested by findings that the effect is associated
with an increase in Km and is
eliminated by successive synaptosomal washing (Haughey et al.,
2000a).
The mechanism or mechanisms responsible for these METH-induced acute
effects on DAT and SERT was not apparent from reports of these rapid
changes. However, recent in vitro studies by Kim et al.
(2000) may have important relevance to these rapid in vivo outcomes of METH administration. This group reported that incubation of
striatal synaptosomes in the presence of 10 µM
METH caused a reduction in DAT activity that was not likely to be
because of METH acting as a competitive inhibitor of dopamine uptake. The characteristics of the in vitro synaptosomal effects of
METH reported by these investigators resemble some of the features associated with the rapid and reversible responses of DAT to in vivo administration of METH, as described above. The present study delineates this association and suggests that the in vitro
effect of METH and the acute and reversible consequence of in
vivo treatment with this stimulant on monoamine transporters are
likely the same phenomenon. In addition, it was demonstrated in
vitro as well as in vivo that the METH-induced
reduction in DAT activity does not occur because of changes in
synaptosomal membrane potential, as assessed by
[3H]tetraphenylphosphonium
([3H]TPP+)
accumulation. Instead, using the in vitro synaptosomal
system, it was established that some protein kinase C (PKC) inhibitors, including the highly selective PKC inhibitor NPC15437, block the METH-induced decline in DA uptake. This suggests that phosphorylation may mediate this effect and that the synaptosomal in vitro
system is an appropriate model to help elucidate the mechanism or
mechanisms responsible for the decrease in DA uptake caused by METH treatment.
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MATERIALS AND METHODS |
Animals. Male Sprague Dawley rats (140-330 gm;
Simonsen Laboratories, Gilroy, CA) were group-housed in wire hanging
cages, maintained at 22 ± 1°C on a 14/10 hr light/dark cycle,
and provided with food and water ad libitum. On the day of
the experiment, six to eight rats were housed in nontransparent
Plexiglas cages lined with screened sawdust shavings. Rats were killed
by decapitation. All procedures were conducted in accordance with
approved National Institutes of Health guidelines.
Drugs and chemicals. (±)-METH hydrochloride,
3,4-methylenedioxymethamphetamine hydrochloride (MDMA)
("ecstasy"), and cocaine hydrochloride were generously supplied by
the National Institute on Drug Abuse (Rockville, MD). Methylphenidate
hydrochloride (MPD) was obtained from Ciba Geigy (Summit, NJ). Drugs
were administered as indicated in figure legends; doses were calculated
as the respective free base. Pargyline hydrochloride, valinomycin,
HEPES, EDTA, chelerythrine chloride, and NPC15437 were
purchased from Sigma (St. Louis, MO). Desipramine hydrochloride was
purchased from Research Biochemicals International (Natick, MA).
Citalopram hydrochloride was obtained from H. Lundbeck A/S (Copenhagen,
Denmark). [7,8-3H]Dopamine (49 Ci/mmol)
and
[3H]TPP+
(32 Ci/mmol) were purchased from Amersham Life Sciences (Arlington Heights, IL).
[ring-2,5,6-3H]levo-Norepinephrine (51.8 Ci/mmol) and
[N-methyl-3H]WIN35428 (84.5 Ci/mmol) were purchased from New England Nuclear (Boston, MA).
[3H]Neurotransmitter uptake and
binding assays. Uptake of
[3H]neurotransmitter was determined as
described previously by Kokoshka et al. (1998b). Briefly, fresh
striatal tissue was homogenized in ice-cold 0.32 M sucrose and centrifuged (800 × g for 12 min; 4°C), the supernatant (S1) fractions were
carefully removed and centrifuged (22,000 × g for 15 min; 4°C) to obtain the synaptosomal-containing pellet (P2).
The resulting pellet (P2) was resuspended in an ice-cold modified
Krebs' buffer (in mM): 126 NaCl, 4.8 KCl, 1.3 CaCl2, 16 sodium phosphate, 1.4 MgSO4, 11 dextrose, 1 ascorbic acid, pH 7.4. In
METH preincubation experiments, samples were preincubated with 10 µM METH for 30 min at 37°C, unless otherwise
specified in the figure legends (i.e., in the legend to Fig. 6).
After 30 min, resuspended P2 fractions were "washed" by
centrifugation (22,000 × g for 15 min; 4°C). The
resulting pellet (P3) was then resuspended in ice-cold Krebs' buffer
and once again centrifuged (22,000 × g for 15 min;
4°C) to obtain a P4 pellet that was subsequently resuspended and
assayed. Assays were conducted in Krebs' buffer. Each assay tube
contained synaptosomal tissue (i.e., resuspended P4 obtained from 1.5, 7.5, and 10 mg original wet weight from striatal and hippocampal
tissue, for dopamine, serotonin and norepinephrine, respectively) and 1 µM pargyline. Nonspecific values for DA, 5-HT, and NE transport were determined in the presence of 1 mM cocaine, 10 µM
citalopram, and 1 µM desipramine, respectively.
After preincubation of assay tubes for 10 min at 37°C, assays were
initiated by addition of
[3H]neurotransmitter [final
concentrations were (in nM): 0.5 for DA and 5 for
5-HT and NE]. Samples were incubated at 37°C for 3 (DA and 5-HT) or
5 (NE) min. [3H]WIN35428 binding (0.5 nM final concentration) was conducted in
phosphate-buffered 0.32 M sucrose, pH 7.4, with
synaptosomes obtained from 2 mg (original wet weight) of striatal
tissue per reaction tube, and samples were incubated on ice for 2 hr.
Samples were then filtered through Whatman GF/B filters
(Brandel, Gaithersburg, MD) soaked previously in 0.05%
polyethylenimine. Filters were washed rapidly three times with 3 ml of
ice-cold 0.32 M sucrose using a filtering
manifold (Brandel). Radioactivity trapped in filters was counted
using a liquid scintillation counter. The remaining synaptosomal tissue
(i.e., resuspended P4 not used for the uptake assay) was retained, and
protein was determined according to the method of Lowry et al.
(1951).
[3H]TPP+
accumulation assay.
[3H]TPP+
accumulation was measured using a modification of the method of Pauwels
and Laduron (1986). Briefly, fresh tissue was homogenized in ice-cold
0.32 M sucrose containing 3.0 mM HEPES and 1.0 mM EDTA,
pH 7.4. The homogenate was centrifuged (800 × g for 12 min; 4°C), and the supernatant (S1) fractions were carefully removed
and centrifuged (22,000 × g for 15 min; 4°C) to
obtain the synaptosomal-containing pellet (P2). The resulting pellet
(P2) was resuspended in ice-cold sucrose-HEPES. In METH incubation
experiments, samples were incubated with 10 µM
METH for 30 min at 37°C. After 30 min preincubation periods, resuspended P2 fractions were washed by centrifugation (22,000 × g for 15 min; 4°C). The resulting pellet (P3) was then
resuspended in ice-cold sucrose-HEPES and once again centrifuged
(22,000 × g for 15 min; 4°C) to obtain a P4 pellet
that was subsequently resuspended and assayed. Assays were conducted in
5 and 75 mM KCl incubation buffers containing the
following (in mM): 135 NaCl, 50 HEPES, 1.8 CaCl2, 0.8 MgSO4, and 5.5 glucose, pH 7.4. Each assay tube contained synaptosomal tissue (i.e.,
resuspended P4 obtained from 16 mg of original wet weight from striatal
tissue). Nonspecific values for
[3H]TPP+
accumulation were determined in the presence of 75 mM KCl incubation buffer. Valinomycin (final
concentration: 0.5 µM and 5% ethanol) was
added to all assay tubes to eliminate the accumulation of [3H]TPP+ by
intrasynaptosomal mitochondria (Scott and Nicholls, 1980). After
preincubation of assay tubes for 10 min at 30°C, assays were
initiated by addition of
[3H]TPP+
(final concentration, 5 nM). Samples were
incubated at 30°C for 10 min. Samples were then filtered through
Whatman GF/B filters soaked previously in 5 mM KCl buffer. Filters were washed rapidly three times with 3 ml of ice-cold 0.32 M sucrose
using a Brandel filtering manifold. Radioactivity trapped in filters
was counted using a liquid scintillation counter. The remaining
synaptosomal tissue (i.e., resuspended P4 not used for the uptake
assay) was retained, and protein was determined according to the method
of Lowry et al. (1951). Membrane potential calculations were made by
applying the Nernst equation and using 3.6 µl/mg as the
intrasynaptosomal volume (Ramos et al., 1979).
Data analysis. The data presented represent means ± SEM. Statistical analyses between two groups were conducted using the two-tailed, unpaired Student's t test. Analyses among
multigroup data were conducted using ANOVA, followed by a Fisher
least significant difference test. Differences among groups were
considered significant if the probability of error was <5%.
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RESULTS |
Results presented in Figure 1
indicate that preincubation of striatal synaptosomes at 37°C with
various METH concentrations (i.e., 1-100 µM) reduced
[3H]DA uptake by ~50%. Based on the
amphetamine brain levels (i.e., 8-15 µM) reported by
Clausing et al. (1995) after multiple administrations of 5 mg/kg drug
and based on the data presented in Figure 1, a 10 µM METH concentration was used in subsequent experiments.
This METH-induced reduction in [3H]DA
uptake occurred rapidly (i.e., within 5 min) and increased in magnitude
with increasing time of preincubation (Fig.
2A). The largest
decrease was achieved at 30 min; consequently, this time point was used
in the subsequent experiments because viability of synaptosomes was
compromised with longer periods of preincubation. These reductions in
DAT function caused by METH preincubation were not associated with a
significant change in binding of the DAT ligand,
[3H]WIN35428 (Fig.
2B). The decline in DAT activity after the 30 min
preincubation in the presence of 10 µM METH was
associated with a diminution in transporter
Vmax (in femtomoles per milligram of
protein per 3 min: 2162 and 1324 for control and METH-treated synaptosomes, respectively) with no change in
Km (in nM: 98 and 96 for control and METH-treated synaptosomes, respectively). In contrast, no drug effect was observed after preincubation of striatal synaptosomes at 4°C, suggesting that the reduction in
[3H]DA uptake did not occur because of
residual drug (i.e., the same METH concentration was used at 37°C and
4°C, and yet no effect was observed at 4°C).
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Figure 1.
METH preincubation at various concentrations
decreased [3H]DA uptake. Striatal synaptosomes
were preincubated with various concentrations of METH (in
µM: 1-100) or assay buffer for 30 min at 37°C and
later assayed at 37°C for the influx of [3H]DA.
Before assaying DA influx, synaptosomal preparations were washed two
extra times, as described in Materials and Methods. Values represent
means, and vertical lines are 1 SEM of determinations
from three independent experiments. Asterisks indicate
values for METH-treated synaptosomes that differ significantly from
controls (p  0.05).
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Figure 2.
Preincubation with METH decreased
[3H]DA uptake without changing
[3H]WIN35428 binding in striatal synaptosomes.
Synaptosomes were preincubated with 10 µM METH or assay
buffer for various time points (5-30 min) at 37°C. Before assaying
[3H]DA uptake and
[3H]WIN35428 binding, synaptosomal preparations
were washed two extra times, as described in Materials and Methods.
Values represent means, and vertical lines are 1 SEM of
determinations from three independent experiments.
Asterisks indicate values for METH-treated synaptosomes
that differ significantly from controls (p 0.05).
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The results presented in Figure 3
indicate that the METH-induced decrease was not restricted to DAT,
because a reduction in [3H]5-HT uptake
was observed when synaptosomes were preincubated with 10 µM METH at 37°C. Similar to DAT activity, SERT function was not altered by METH when synaptosomes were preincubated at 4°C.
The data in Figure 4 demonstrate that
[3H]NE uptake (as assessed in washed
hippocampal synaptosomes because the striatum contains few NE
terminals), unlike [3H]DA uptake, was
not decreased by METH preincubation.
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Figure 3.
Effect of METH preincubation on
[3H]5-HT uptake. Striatal synaptosomes were
preincubated with 10 µM METH or assay buffer for 30 min
at 37 or 4°C. Before assaying [3H]5-HT influx,
synaptosomal preparations were washed two extra times, as described in
Materials and Methods. Columns represent means, and
vertical lines are 1 SEM of determinations from three
independent experiments. The asterisk indicates a value
for METH-treated synaptosomes that differ significantly from the
respective control group (p 0.05).
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Figure 4.
Effect of METH preincubation on
[3H]NE uptake. Hippocampal and striatal
synaptosomes were preincubated with 10 µM METH or assay
buffer for 30 min at 37°C and assayed at 37°C for the influx of
[3H]NE and [3H]DA,
respectively. Before assaying [3H]NE and
[3H]DA influx, synaptosomal preparations were
washed two extra times, as described in Materials and Methods.
Columns represent means, and vertical lines
are 1 SEM of determinations from four independent experiments. The
asterisk indicates a value for METH-treated synaptosomes
that differs significantly from the control
(p 0.05).
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To determine whether the in vitro phenomenon was
related to that of an in vivo single or multiple
administration of METH, rats were treated in vivo with a
single injection (15 mg/kg, s.c.) of METH or saline. The synaptosomes
prepared from drug- or saline-treated rats (in vivo) were
then preincubated in the presence of 10 µM METH
(in vitro), as described above, and DAT activity was
assessed by [3H]DA uptake. As shown in
Figure 5, METH preincubation in
vitro did not further diminish DAT activity in synaptosomal
preparations from drug-treated rats compared with the two 10 µM METH groups, suggesting that the in
vitro and ex vivo models represent the same
phenomenon.
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Figure 5.
Effects of a single administration and
preincubation with METH on [3H]DA uptake. Rats
received METH (15 mg/kg, s.c.) or saline (1 ml/kg, s.c.) 1 hr before
decapitation. Striatal synaptosomes were preincubated with 10 µM METH or assay buffer for 30 min at 37°C, as
described in Materials and Methods, and assayed for the influx of
[3H]DA. Before assaying DA influx, synaptosomal
preparations were washed two extra times, as described in Materials and
Methods. Columns represent means, and vertical
lines are 1 SEM of determinations from four independent
experiments. Asterisks indicate values for METH-treated
synaptosomes that differ significantly from the saline/control group
(p 0.05).
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To determine the selectivity of the transient METH-related reduction in
DAT function observed in vitro, synaptosomes were preincubated with (in µM): 10 MDMA, 10 cocaine,
or 10 MPD. MDMA, like METH, decreased
[3H]DA uptake (i.e., to 47 and 51% of
control for MDMA and METH, respectively) (Fig.
6). In contrast, preincubation with
cocaine and MPD was without effect in
[3H]DA uptake (Fig. 6).
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Figure 6.
Effect of psychostimulant preincubation on
[3H]DA uptake. Striatal synaptosomes were
preincubated with (in µM): 10 METH, 10 MDMA, 10 cocaine,
and 10 MPD or assay buffer for 30 min at 37°C. Before assaying
[3H]DA influx, synaptosomal preparations were
washed two extra times, as described in Materials and Methods.
Columns represent means, and vertical lines
are 1 SEM of determinations from three independent experiments.
Asterisks indicate values for drug-treated synaptosomes
that differ significantly from their respective controls
(p 0.05).
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The in vitro model was used next to determine whether
synaptosomal membrane potential changes contributed to the METH-induced decline in DAT activity. To achieve this objective,
[3H]TPP+
accumulation assays were conducted in vitro. As presented in Table 1, in vitro incubation
of striatal synaptosomes with 10 µM METH did
not alter synaptosomal membrane potential. Furthermore, [3H]TPP+
accumulation assays were also conducted ex vivo after a
single injection of METH; these assays, like those conducted in
vitro, showed no alterations in synaptosomal membrane
potential.
Finally, the in vitro model was used to examine the role of
phosphorylation in the METH-induced DAT changes. Synaptosomes were
pretreated with 15 µM NPC15437 for 5 min before
METH exposure. The results presented in Figure
7 demonstrate that pretreatment with
NPC15437 significantly attenuated the in vitro METH-induced decrease in synaptosomal [3H]DA uptake.
In contrast, pretreatment with a second PKC inhibitor, chelerythrine,
did not attenuate the METH-induced reduction in synaptosomal
[3H]DA uptake (Table
2).
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Figure 7.
Effect of NPC15437 pretreatment on the decrease in
[3H]DA uptake in striatal synaptosomes induced by
METH preincubation. Striatal synaptosomes were pretreated with 15 µM NPC15437 for 5 min and subsequently preincubated with
10 µM METH or assay buffer for 30 min at 37°C. Before
assaying [3H]DA influx, synaptosomal preparations
were washed two extra times, as described in Materials and Methods.
Columns represent means, and vertical lines
are 1 SEM of determinations from three independent experiments.
Asterisks indicate values for METH-treated synaptosomes
that differ significantly from controls; #, a value for
NPC15437-treated synaptosomes that differs significantly from
METH-treated synaptosomes (p 0.05).
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DISCUSSION |
The intent of this study was to elucidate the mechanism whereby
METH administration rapidly and reversibly reduces DAT activity in
striatal synaptosomes. Recently, the findings by Kim et al. (2000)
suggested that in vitro application of METH to the
synaptosomes may cause an effect similar to that observed rapidly after
in vivo administrations of METH. For instance, this group
reported that the in vitro METH-induced reduction in
[3H]DA uptake was not associated with a
change in [3H]WIN35428 binding, as shown
previously in vivo by Fleckenstein et al. (1997) and
Kokoshka et al. (1998b). Like the in vivo findings of
Fleckenstein et al. (1997) and Kokoshka et al. (1998b), this group also
reported that the in vitro changes in transporter activity occurred because of a decline in Vmax.
It was also determined that, as observed ex vivo in
synaptosomes prepared from METH-treated rats (Kokoshka et al., 1998a),
glutamate uptake was unaltered after in vitro preincubation
with 10 µM METH. Moreover, this in vitro system also parallels previous in vivo reports
that showed no alteration in [3H]DA
uptake in the presence of cocaine and MPD (Fleckenstein et al., 1999).
Thus, the observations made by this group resemble those effects of
METH previously reported in vivo, which further suggested
that the in vitro synaptosomal system is an appropriate model for the rapid and reversible changes caused by in vivo
administrations of METH.
To confirm the possibility that this in vitro model
resembles those effects observed in vivo, we further
characterized the in vitro effect and found substantial
parallels. For example, the proposed in vitro model not only
resembles the METH-induced ex vivo changes in DAT function,
but is also similar to the METH-induced effects in other monoamine
transporter activities, such as SERT and NET. Kokoshka et al. (1998a)
and Fleckenstein et al. (1999) showed that SERT function, like DAT
activity, is reduced after a single administration of METH. This
decrease was not a result of residual METH introduced by the original
injection of the drug (Kokoshka et al., 1998a). Similarly, results in
Figure 3 are consistent with these in vivo findings of
Kokoshka et al. (1998a) and Fleckenstein et al. (1999), in that SERT
function decreased by 22% after synaptosomes were exposed in
vitro to 10 µM METH at 37°C. These data
further establish that this reduction in
[3H]5-HT uptake did not occur because of
a direct interaction of residual METH with the transporter protein,
because synaptosomal exposure to 10 µM METH did
not alter [3H]5-HT uptake at 4°C,
whereas transporter activity was reduced at 37°C after successive
washing of the synaptosomal preparation (Fig. 3). Unlike DAT and SERT,
NET activity was not reduced after hippocampal synaptosomes were
exposed to 10 µM METH (Fig. 4). These results
are concordant with the recent observations of Haughey et al. (2000a)
that demonstrated that unlike DAT and SERT,
[3H]NE uptake returned to control levels
after successive washing of drug-treated synaptosomes (Fig. 4).
Interestingly, the results presented in Figure 5 further support the
possibility that the in vitro model represents acute changes
in DAT function after in vivo METH treatment. These data demonstrate that after in vitro preincubation with 10 µM METH, the DAT activity is reduced comparably
in striatal synaptosomes prepared from saline- and METH-treated
animals. The fact that the in vivo treatment with METH did
not further add to the decline in DAT activity caused by in
vitro preincubation with this drug suggests that these two
treatments cause the same phenomenon.
These comparisons are significant for the following reasons: first, the
reversible changes after in vivo treatment with METH are
likely the same as those resulting from in vitro
preincubation with this stimulant. The similarities between the
in vitro and the rapid in vivo models are
confirmed by findings indicating that, similar to the in
vivo model (Fleckenstein et al., 1999), cocaine and MPD were
without effect on DAT activity after the in vitro
preincubation (Fig. 6). Second, the rapid transporter phenomenon does
not necessarily reflect a neurotoxic process because it (1) occurs
quickly and is reversible and (2) is comparably caused by MDMA, a drug
not particularly toxic to DA systems (Stone et al., 1987; O'Hearn et
al., 1988; Ricaurte et al., 1993) except at high doses in rats (Commins
et al., 1987) and mice (Logan et al., 1988). Still, the decrease in DAT
function may, under certain conditions, promote the development of
neurotoxicity; this possibility remains to be explored.
If these acute reversible changes in DAT function do not occur because
of damage to the monoamine terminals, the mechanism or mechanisms by
which METH causes the transient reduction in DAT activity is unclear.
Using the in vitro model to address this issue, and knowing
that monoamine transporters belong to the
Na+/Cl -dependent
family (Uhl and Hartig, 1992; Borowsky and Hoffman, 1995), changes in
ion fluxes were investigated as a possible mechanism causing the acute
decline in DAT activity. The results suggest that ion fluxes did not
contribute to the transient diminution of DAT action, as suggested by
the findings that the synaptosomal membrane potential was not altered
in vitro in the presence of 10 µM
METH, nor was it altered in vivo after a single injection of
METH, as assessed by
[3H]TPP+
accumulation (Table 1).
Another mechanism whereby METH might cause rapid and reversible
reductions in DAT activity is phosphorylation. It has been determined
that DAT is extensively glycosylated and that its sequence contains
phosphorylation consensus sites for protein kinase A, PKC, and
Ca2+-calmodulin kinase (Giros et al.,
1991; Kilty et al., 1991; Shimada et al., 1991; Vandenbergh et al.,
1992), suggesting that these transporters may be subject to regulation
by phosphorylation. This is supported by studies using DAT-expressing
COS cells that have shown that activation of PKC by phorbol
esters leads to a decrease in the transport of DA into the transfected
cell line (Kitamaya et al., 1994). This observation is also in
accordance with other reports that state that PKC-induced regulation
decreases DAT activity in cell culture systems (Huff et al., 1997;
Zhang et al., 1997) as well as in mouse and rat synaptosomes (Copeland et al., 1996; Vaughan et al., 1997). The role of phosphorylation in the METH-related changes in DAT function was examined using the PKC
inhibitors NPC15437 and chelerythrine. As demonstrated by Kim et al.
(2000), chelerythrine application did not prevent the
METH-induced decrease in DAT function. However, NPC15437 treatment attenuated the METH-induced decrease in DA uptake. Interestingly, Kim
et al. (2000) reported that another PKC inhibitor,
bisindolylmaleimide (BIS), attenuated the METH-induced decrease in DA
uptake; however, BIS per se decreased DA uptake and, hence, the authors
concluded that a role for PKC in an in vitro model remained
to be established. Our data accomplish this by demonstrating that a PKC
inhibitor can substantially attenuate the METH-induced decrease in DAT
function, without eliciting an effect per se.
A possible consequence of phosphorylation is internal trafficking of
the monoamine transporters. METH-induced internalization is suggested
by the findings of Saunders et al. (2000), who demonstrated that
treatment with amphetamine internalized the human DAT expressed on
human embryonic kidney cells, thereby decreasing its function. These
observations are consistent with reports by Pristupa et al. (1998) and
Melikian and Buckley (1999) that showed that activation of PKC results
in decreased transporter activity that is attributable to intracellular
transporter sequestration.
In summary, these observations demonstrate that METH in
vitro alters DAT activity in a manner that parallels what occurs
in vivo after a single administration of this stimulant.
Furthermore, we speculate that this METH-induced decrease in DA uptake
may be mediated by internalization of DAT via a PKC-mediated mechanism. Other related drugs such as MDMA, but not cocaine or MPD, appear to cause a similar decrease in DA uptake. This effect appears to be
reversible and perhaps relates to an important means of physiologically
regulating the activity of DA neurons.
| |
FOOTNOTES |
Received Aug. 21, 2000; revised Dec. 5, 2000; accepted Dec. 6, 2000.
This research was supported by United States Public Health Service
Grants DA00869, DA04222, DA11389, and DA00378.
Correspondence should be addressed to Dr. Annette E. Fleckenstein,
Department of Pharmacology and Toxicology, 30 South 2000 East, Room
201, University of Utah, Salt Lake City, UT 84112. E-mail:
fleckenstein{at}hsc.utah.edu.
| |
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INTRODUCTION
