![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
NEUROPHARMACOLOGY
Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah
Received November 6, 2003; accepted January 23, 2004.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). This long-term monoamine depletion is reflected by damage to dopamine nerve terminals (Lorez, 1981; Ricaurte et al., 1982), decreased tyrosine hydroxylase activity (Kogan et al., 1976; Hotchkiss and Gibb, 1980), and decreases in dopamine uptake sites (Wagner et al., 1980). Similar deficits have been observed in nonhuman primates (Seiden et al., 1976; Woolverton et al., 1989; Melega et al., 1997) and humans (Wilson et al., 1996).
Several factors contribute to the persistent dopamine deficits caused by methamphetamine. For example, studies from Bowyer et al. (1994) and Albers and Sonsalla (1995) suggest that hyperthermia contributes to the long-term neurotoxic effects of methamphetamine on striatal dopamine neurons since prevention of elevated core body temperatures attenuates this deficit. A second contributor may be alterations in the distribution of methamphetamine in the brain. Studies by Schmidt et al. (1985) and Gygi et al. (1996) suggest that pretreatment regimens can alter the distribution of methamphetamine between brain and plasma 2 h after the methamphetamine challenge administration and possibly contribute to the protection against the neurotoxic effects of methamphetamine. Furthermore, recent studies suggested that aberrant monoamine transporter function contributes to the neurotoxic effects of methamphetamine. For example, multiple high-dose administration of methamphetamine causes a rapid and partially reversible decrease in plasmalemmal dopamine transporter (DAT) uptake activity (Fleckenstein et al., 1997), and post-treatment with DAT reuptake inhibitors such as amfonelic acid can block the neurotoxic effects of methamphetamine (Marek et al., 1990).
Similarly, multiple high-dose administrations of methamphetamine cause a rapid decrease in vesicular dopamine uptake through the vesicular monoamine transporter-2 (VMAT-2; Brown et al., 2000, 2002; Hogan et al., 2000). Drugs such as methlyphenidate reversed this methamphetamine-induced decrease in activity and attenuated the persistent dopamine deficits caused by methamphetamine (Sandoval et al., 2003).
In addition to factors noted above, several studies have demonstrated that the dopamine deficits caused by methamphetamine can be attenuated by pretreatment with methamphetamine. For example, pretreatment with multiple low-dose injections of methamphetamine (Stephans and Yamamoto, 1996) or with multiple escalating doses of methamphetamine prior to a high-dose methamphetamine challenge administration can lead to the development of tolerance to the acute neurotoxic effects on dopamine and serotonin systems (Schmidt et al., 1985; Gygi et al., 1996), as assessed 18 h after the last challenge injection, and the long-term deficits to monoamine content, as assessed 4 and 7 days after the challenge administration (Stephans and Yamamoto, 1996; Johnson-Davis et al., 2003). Mechanisms underlying the protection afforded by methamphetamine pretreatment are unknown. Hence, the purpose of this study was to explore the ability of a tolerance-producing methamphetamine regimen to prevent acute alterations in factors linked to the long-term dopamine deficits caused by methamphetamine. Tolerance was defined as a diminished response to methamphetamine after our dosing regimen.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Animals. Male Sprague-Dawley rats (Charles River Canada, Montreal, Canada) weighing 290 to 310 g at the time of experiment were housed four per cage in hanging wire cages in a light- and temperature-controlled room. Rats had free access to food and water. The Institutional Animal Care and Use Committee of the University of Utah approved the animal care and experimental procedures. These procedures were also in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
Pharmacological Procedures. Our laboratory did not use our previously published protocol for these experiments (Johnson-Davis et al., 2003) because we were unable to purchase rats from the Simonsen distributor at the time of the experiments. Due to a change in vendor, to Charles Rivers, the methamphetamine pretreatment and challenge doses were reduced, and the frequency of administration was altered for the present study. The evening before the experiment, rats were weighed and then rehoused in plastic cages. The tolerance-dosing regimen consisted of saline or methamphetamine pretreatments on days 1, 3, and 5. On day 1, a total of four injections of either saline or 2.0 mg/kg of methamphetamine were administered (s.c.) with a 4-h interval between each dose. Rats were allowed a 40-h drug-free period between each pretreatment dosing regimen. The doses for methamphetamine were increased to 4.0 mg/kg on day 3 and 6 mg/kg on day 5. Sixty-six hours after the last pretreatment regimen on day 5, rats were either challenged with four injections of saline or 8 mg/kg methamphetamine (s.c.) with a 4-h interval between each injection. Where indicated, core body (rectal) temperatures were assessed every h during the pretreatment and challenge dosing regimens while the rats were in the cages. Rectal temperatures were measured using a BAT-12 model thermometer and a thermocoupled rectal probe (model RET-2) (Physiotemp Instruments, Clifton, NJ).
Monoamine Tissue Content. Rats were decapitated, and the striatum was dissected from the brains 7 days after the last methamphetamine challenge injection. Tissues were quickly frozen on dry ice and then stored at -80°C. The striatum and hippocampus was sonicated (Branson Sonifier 250; Branson Ultrasonics Corporation, Danbury, CT) in 1 ml of tissue buffer [0.05 M sodium phosphate/0.03 M citric acid with 15% (v/v) methanol, pH 2.5], then centrifuged at 18,800g for 15 min at 4°C to separate the supernatant from the protein. The supernatant was centrifuged at 18,800g for 10 min at 4°C, then 20 µl was injected onto a high-performance liquid chromatography system (Dynamax AI-200 Autosampler and SD-200 pump; Varian, Walnut, CA) coupled to an electrochemical detector (Eox = +0.70 V; Varian Star 9080) to quantitate the dopamine and serotonin concentrations, as described by Chapin et al. (1986). A Whatman PartiSphere C-18 column (250 x 4.6 mm, 5 µm; Whatman, Clifton, NJ) was used to separate the monoamines. The mobile phase consisted of 23% (v/v) MeOH, 0.03% (w/v) sodium octyl sulfate, 0.1 mM EDTA, 0.05 M sodium phosphate dibasic, and 0.03 M citric acid. The pH of the mobile phase was 2.87, and the flow rate was 1 ml/min. Protein content was determined as described by Lowry et al. (1951).
Vesicular [3H]Dopamine Uptake. Synaptic vesicles were prepared from rat striatum as described previously (Brown et al., 2000), 2 h after the last methamphetamine challenge injection. The 2-h time point was selected to be consistent with the 2-h time point evaluated for concentrations of methamphetamine in the brain. Vesicular [3H]dopamine uptake was determined as described by Teng et al. (1997) with the following modifications: synaptic vesicles were isolated in two ultracentrifugation steps (20,000g, 20 min; and 100,000g, 45 min); vesicles were incubated at 30°C for 3 min in the presence of [3H]dopamine (final concentration, 30 nM); and nonspecific uptake was determined by incubating synaptic vesicles at 4°Cin the absence of ATP. Protein concentrations were determined using the Bradford protein assay (Bradford, 1976).
VMAT-2 Immunoreactivity. Striatal synpatosomes were prepared as described previously (Fleckenstein et al., 1997). Fresh striatal tissue was homogenized in ice-cold 0.32 M sucrose and centrifuged (800g, 12 min, 4°C). The resulting supernatant was then centrifuged (22,000g, 15 min, 4°C) and the whole synaptosomal fraction pellet (P2) was resuspended in cold distilled deionized water at a concentration of 50 mg/ml (original wet weight of tissue), and a portion was saved for Western blot analysis. The remainder of the synaptosomal samples was centrifuged (22,000g, 20 min, 4°C) to pellet the membrane-associated fraction (P3), which was resuspended at 50 mg/ml (original wet weight of tissue) and saved for Western blot analysis. Prior to resuspension of P3, the resulting supernatant [nonmembrane-associated fraction (S3)] was removed and saved for Western blot analysis.
Western Blot Analysis. Western blot analysis was performed as described previously by Riddle et al. (2002). The binding of VMAT-2 antibody was performed using 50 µg of protein from P2, P3, and S3. The bound primary VMAT-2 antibody (1:1000 dilution) was visualized with anti-rabbit immunoglobulin antibody (1:2000 dilution). The antigen-antibody complexes were visualized by chemiluminescence. Bands on blots were quantified by densitometry measuring net intensity using Kodak 1D image analysis software (Eastman Kodak, Rochester, NY). Western blots were run on a 15-well gel. Therefore, to run a blot with an n = 6 per group, only two groups were compared on each blot. The same samples from each group were used to run the four Western blots needed to make all of the two group comparisons. Each blot was developed on a separate film, and the values from the Western blot data were measured by densitometric analysis.
[3H]Dopamine Uptake via Plasmalemmal Transporters. Uptake of [3H]dopamine was determined in striatal synaptosomes prepared according to the method described by Fleckenstein el al. (1997). One hour after the last methamphetamine challenge injection, rats were decapitated, and striatal tissues were dissected and homogenized in cold 0.32 M sucrose and centrifuged (800g, 12 min, 4°C). The supernatant was then centrifuged (22,000g, 15 min, 4°C), and the resulting pellet (P2) was resuspended in ice-cold modified Krebs' buffer (126 nM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 16 mM sodium phosphate, 1.4 mM MgSO4, 11 mM dextrose, 1 mM ascorbic acid, pH 7.4) Assays were conducted in Krebs' buffer. Each assay tube contained synaptosomal tissue (the resuspended P2 from 1.5 mg of original wet weight striatal tissue) and 1 µM pargyline. Nonspecific values were determined in the presence of 10 µM cocaine. Assay tubes were pre-incubated for 10 min at 37°C, and then [3H]dopamine (0.5 nM final concentration) was added to initiate the reaction. Assay tubes were incubated for 3 min at 37°C, then samples were filtered through Whatman GF/B filters (Brandel Inc., Gaithersburg, MD) soaked previously in 0.05% polyethylenimine. Filters were washed rapidly three times with 3 ml of ice-cold 0.32 M sucrose by using a Brandel filtering manifold. The radioactivity trapped in filters was counted using a liquid scintillation counter. Remaining resuspended P2 samples were assayed for protein concentrations according to the method of Lowry et al. (1951).
Methamphetamine and Amphetamine Concentrations. The striatum was dissected from the brain of the saline/methamphetamine and methamphetamine/methamphetamine groups 66 h after the last 6 mg/kg pretreatment injection (t = 0, corresponding to the time for the first injection of the methamphetamine challenge administration), then 30 min, 1 h, and 2 h after the last methamphetamine challenge injection (8 mg/kg) and quickly frozen on dry ice, then stored at -80°C. On the day of extraction, brain tissues were thawed on ice. Brain samples were weighed and sonicated in 1 ml of doubled deionized water using a Branson Sonifier 250. When samples were equilibrated to room temperature, 500 µl of each sample was transferred into 16- x 100-mm silanized glass test tubes with screw caps. Two hundred fifty nanograms of deuterated methamphetamine and deuterated amphetamine were added as an internal standard to 500 µl of homogenate brain samples. Samples were vortexed for 5 s, and then the pH was made alkaline (pH > 12) with 100 µl of concentrated ammonium hydroxide. Five hundred-microliter aliquots of brain homogenates were extracted with 5 ml of n-butyl chloride: acetonitrile [4:1 (v/v)] at room temperature for 30 min with gentle rocking. Samples were centrifuged for 20 min at 1,200g, and then the organic layer containing methamphetamine and amphetamine was transferred to silanized glass test tubes (13 x 100 mm) with screw caps. Samples were evaporated halfway to dryness at 20°C, then 50 µl of 1% HCl in methanol was added to stabilize the analytes before samples were completely dried down. When samples were completely evaporated to dryness, samples were reconstituted in 100 µl of 95% formic acid/5% acetonitrile solution and transferred into vials prior to analysis by liquid chromatography/mass spectrometry/mass spectrometry. Methamphetamine and amphetamine concentrations in brain were determined with a ThermoFinnigan TSQ7000 tandem mass spectrometer operating in atmospheric pressure chemical ionization mode. Chromatographic separation was achieved on a Metasil Basic 3µ, 100- x 3.0-mm column (MetaChem Technologies Inc., Torrance, CA) with a mobile phase consisting of 95% formic acid (0.1%) and 5% acetonitrile. Selected reaction monitoring of ions was performed, and transitions were monitored as follows: m/z 150.1 
m/z 91 (methamphetamine), m/z 158 m/z 92 (methamphetamine-d8), m/z 136 m/z 91 (amphetamine), and m/z 141.1 m/z 92 (amphetamine-d5). Accuracy was within 20% of the fortified nondeuterated methamphetamine and amphetamine target samples from brain homogenate quality control samples. The limit of quantitation for this assay was 5 ng/ml.
Statistical Analysis. Dopamine content, VMAT-2 uptake, and DAT uptake were analyzed with one-way ANOVA followed by least significant difference post hoc comparison (version 11.5; SPSS, Chicago, IL). Methamphetamine and amphetamine concentrations and VMAT-2 immunoreactivity were analyzed by using Student's t test. Analysis of data for core rectal temperatures was first attempted using ANOVA with repeated measures; however, the underlying statistical assumptions for this test (compound symmetry and homogeneity of variance) were not met. Therefore, a one-way ANOVA followed by post hoc testing with Student's t test was performed at each individual time point when the assumption of homogeneity of variance was met. The fixed factor was "treatment"; the dependent variable was temperature. When the assumption of homogeneity of variance was not met for a particular time point, data were analyzed using Kruskal-Wallis Analysis of Variance by Ranks, followed by post hoc testing with Mann-Whitney U. Statistical significance for all comparisons was set at p < 0.05. Data represent means ± S.E.M.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
On each day of the pretreatment regimen, rats that received methamphetamine had greater core body temperatures than those that received saline. Figure 2 demonstrates that likewise, on the day of the challenge, the high-dose methamphetamine administration elevated core body temperatures in both the saline/methamphetamine and methamphetamine/methamphetamine groups, which differed from the saline/saline-treated rats at each time point (p < 0.05). Importantly, there was a general trend that the methamphetamine/methamphetamine group had lower body temperatures than the saline/methamphetamine group throughout the experiment, but it was only significant at three time points.
|
To determine whether alterations in brain methamphetamine concentrations contributed to the development of tolerance, striatal methamphetamine and amphetamine concentrations were measured. Neither methamphetamine nor amphetamine were detected 66 h after the last methamphetamine pretreatment injection (i.e., t = 0 in Fig. 3, corresponding to the time immediately prior to the first methamphetamine challenge injection). Results presented in Fig. 3 demonstrate that methamphetamine and amphetamine levels were elevated similarly at 0.5, 1, and 2 h after the methamphetamine challenge injection, with a statistical significant difference (i.e., greater methamphetamine levels in the methamphetamine/methamphetamine versus saline/methamphetamine) only observed at 0.5 h.
|
Since the pretreatment regimen altered methamphetamine-induced hyperthermia during the challenge administration (Fig. 2), it was important to determine whether this decrease in core body temperatures afforded neuroprotection by attenuating the acute methamphetamine-induced decrease in VMAT-2 function. Hence, vesicular dopamine uptake was measured in rats that were treated with methamphetamine at room temperature and/or in a 6°C environment to allow or prevent methamphetamine-induced hyperthermia, respectively. Results presented in Fig. 4A demonstrate that multiple injections of methamphetamine (4 x 8 mg/kg; 2-h interval; s.c.) elevated core body temperatures and that methamphetamine-induced hyperthermia was blocked in rats that were treated with methamphetamine at 6°C. Figure 4B shows that at room temperature (24°C), methamphetamine decreased vesicular dopamine uptake 72% when compared with the value for the saline-treated rats (24°C) (p < 0.05). The prevention of methamphetamine-induced hyperthermia did not attenuate this decrease in vesicular dopamine uptake. At 6°C, methamphetamine decreased vesicular dopamine uptake 75% when compared with the value for the saline-treated rats (p < 0.05). Vesicular dopamine uptake was not altered in rats that were treated with saline at 6°C.
|
Figure 5 demonstrates that the high-dose methamphetamine administration decreased plasmalemmal DAT uptake 72% when compared with the value for the saline/saline group (p < 0.05), 1 h after the last methamphetamine injection on the day of the challenge administration. In rats that were pretreated with methamphetamine prior to the high-dose challenge administration, dopamine transporter uptake was decreased 58% when compared with the value for the saline/saline group (p < 0.05). Dopamine transporter uptake was not statistically different between the methamphetamine/methamphetamine and the saline/methamphetamine groups. The pretreatment regimen per se did not significantly alter DAT uptake when compared with the saline/saline control.
|
Figure 6 demonstrates that the high-dose methamphetamine administration decreased vesicular dopamine uptake 57% when compared with the value for the saline/saline control (p < 0.05), 2 h after the last methamphetamine injection on the day of the challenge administration. In rats that were pretreated with methamphetamine prior to the high-dose challenge administration, the decrease in vesicular dopamine uptake was attenuated when compared with the value for the saline/methamphetamine group (p < 0.05). The pretreatment regimen alone did not significantly alter vesicular dopamine uptake when compared with the saline/saline control. Vesicular dopamine uptake was significantly different between the saline/methamphetamine and methamphetamine/methamphetamine groups. Vesicular uptake in the saline/methamphetamine group was decreased 39% when compared with the value for the methamphetamine/methamphetamine group (p < 0.05).
|
To determine whether the attenuation of the methamphetamine-induced decrease in VMAT-2 function was due to alterations in VMAT-2 protein levels, Western blot analysis was performed on the three tissue fractions: S3 (vesicularenriched fraction), P3 (membrane-bound vesicles), and P2 2 h after the last injection of the methamphetamine challenge administration. In S3, results shown in Fig. 7A illustrate that the pretreatment regimen alone (methamphetamine/saline group) does not alter VMAT-2 immunoreactivity when compared with the saline/saline control (p > 0.05). Figure 7B demonstrates that the challenge methamphetamine administration alone decreased VMAT-2 immunoreactivity when compared with saline/saline control. In Fig. 7C, there was no difference in VMAT-2 immunoreactivity between the methamphetamine/saline and methamphetamine/methamphetamine groups. In Fig. 7D, there was a significant difference in VMAT-2 immunoreactivity between the saline/methamphetamine and methamphetamine/methamphetamine groups (p < 0.05).
|
Results from P2 resembled the data shown in S3. Only the saline/methamphetamine group was significantly less than the other groups (p < 0.05); however, none of the remaining groups differed from one another (data not shown). Furthermore, there was no difference in VMAT-2 immunoreactivity among the P3 fractions (data not shown).
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Gygi et al., 1996; Stephans and Yamamoto, 1996; Johnson-Davis et al., 2003). Although using a different dosing paradigm, our data confirm previous findings by illustrating that methamphetamine pretreatment can attenuate the long-term depletion of striatal dopamine content 7 days after the challenge administration.
Several laboratories have shown that hyperthermia plays an important role in mediating the long-term neurotoxic effects of methamphetamine on striatal dopamine neurons (Bowyer et al., 1994; Albers and Sonsalla, 1995). For example, lowering the environmental temperature to 4°C while administering a neurotoxic regimen of methamphetamine, as well as administering pharmacological drugs that produce hypothermia in combination with a neurotoxic regimen of methamphetamine, attenuates the methamphetamine-induced depletion of striatal dopamine (Bowyer et al., 1992; Ali et al., 1996). Collectively, these experiments demonstrate a link between hyperthermia and methamphetamine-induced neurotoxicity, suggesting that the attenuation of methamphetamine-induced hyperthermia could play a role in tolerance to the neurotoxic effects of the stimulant. Thus, it was important to determine whether the development of tolerance to the neurotoxic effects of methamphetamine was due to the attenuation of methamphetamine-induced hyperthermia during the challenge administration. Results from this present experiment demonstrate that methamphetamine pretreatment can attenuate methamphetamine-induced hyperthermia at a few time points during the challenge administration when using this 4-h tolerance-dosing regimen. This suggests that hyperthermia plays a small role in the tolerance phenomenon. However, other factors clearly contribute to the protection from the neurotoxic effects of methamphetamine as well. Findings that other mechanisms contribute to the tolerance are consistent with our previous study in which we reported that, when dosing with a 2-h interval, both the pretreated and nontreated rats had similar core body temperatures on the day of the challenge administration (Johnson-Davis et al., 2003).
Since the pretreatment regimen altered methamphetamine-induced hyperthermia during the challenge administration, it was important to determine whether this decrease in core body temperatures afforded neuroprotection by attenuating the acute methamphetamine-induced decrease in VMAT-2 function. This was particularly important given the report by Ugarte et al. (2003) that hyperthermia contributed to the decrease in VMAT-2 function in mice caused by methamphetamine-induced toxicity. Hence, vesicular dopamine uptake was measured in rats that were treated with methamphetamine at room temperature and/or in a 6°C environment to allow or prevent methamphetamine-induced hyperthermia, respectively. Our results are consistent with previous findings from Brown et al. (2002) that the decrease in VMAT-2 function, after a single methamphetamine injection to rats, is not temperature dependent. Despite the blockade of methamphetamine-induced hyperthermia, exposure to methamphetamine decreased VMAT-2 uptake similarly at both 24°C and 6°C. Therefore, these data suggest that the attenuation in methamphetamine-induced hyperthermia during the methamphetamine challenge administration did not afford any protection against the methamphetamine-induced decrease in VMAT-2 function.
In addition to the role of hyperthermia, some studies suggest that alterations in methamphetamine redistribution could contribute to the tolerance of the neurotoxic effects of methamphetamine. Schmidt et al. (1985) and Gygi et al. (1996) reported, using different dosing paradigms, that methamphetamine pretreatment altered the distribution of methamphetamine between the brain and plasma following a challenge methamphetamine administration. Specifically, methamphetamine-pretreated rats had one-half the amount of methamphetamine in the brain and twice the amount of methamphetamine in plasma when compared with nonpretreated rats. These studies suggested that this reduction in brain methamphetamine concentration could lead to a decrease in the amount of neurotoxicity to dopamine neurons and, therefore, induce tolerance. In contrast to these findings, the present data indicate that methamphetamine pretreated rats have similar methamphetamine concentrations in the brain than the nonpretreated rats with our current dosing paradigm. Methamphetamine concentrations in the brains of the pretreated rats were significantly higher at the 30-min time point but were similar to the nonpretreated rats at the 1- and 2-h time points. Thus, using our tolerance-inducing dosing regimen, this phenomenon cannot be attributed to alterations in brain methamphetamine concentrations between the pretreated and nonpretreated rats.
Lastly, recent studies suggest that aberrant monoamine transporter function contributes to the neurotoxic effects of methamphetamine. For example, a study by Fumagalli et al. (1999) showed that impairment of the function of VMAT-2 (i.e., as demonstrated in VMAT-2 knockout mice) potentiates methamphetamine-induced neurotoxicity to dopaminergic neurons in vivo. Likewise, Fumagalli et al. (1998) illustrated that DAT (-/-) knockout mice were protected against neurotoxicity to dopamine neurons when exposed to methamphetamine. Clearly, the role of monoamine transporter function is complex. The current theory for methamphetamine-induced neurotoxicity suggests that the decrease in VMAT-2 function, by methamphetamine, results in the disruption of vesicular storage of dopamine. This disruption can lead to the accumulation of interneuronal dopamine, which oxidizes into reactive oxygen species (Graham et al., 1978; Hastings, 1995), and damage the nerve terminals of dopamine neurons (Cubells et al., 1994; Liu and Edwards, 1997). Hence, for this study, it was important to explore the roles of the VMAT-2 and DAT in the induction of tolerance to the long-term persistent dopaminergic deficits caused by methamphetamine.
Results from this experiment demonstrates that acutely (i.e., 1 h after the last methamphetamine challenge injection) methamphetamine impaired DAT uptake similarly in both the pretreated and nonpretreated rats. This suggests that tolerance was not caused by an acute prevention of the methamphetamine-induced decrease in plasmalemmal dopamine uptake. In contrast, 2 h after the last methamphetamine challenge injection, there was an attenuation in the methamphetamine-induced decrease in VMAT-2 function in the methamphetamine/methamphetamine rats. The decrease in vesicular uptake was not due to residual methamphetamine because isolation of the vesicles removes any residual methamphetamine from the preparation to levels of <1 nM (Fleckenstein et al., 1997), a concentration that is below the necessary concentration to alter dopamine uptake in our preparations. Concurrent with this decrease in vesicular dopamine uptake, Western blot analysis demonstrated that exposure to methamphetamine caused a significant decrease in VMAT-2 immunoreactivity in S3. This finding is consistent with data from Riddle et al. (2002) that showed that exposure to a high-dose methamphetamine administration redistributes VMAT-2 protein from the nerve terminal and could possibly explain the mechanism underlying the methamphetamine-induced decrease in VMAT-2 uptake as shown by Hogan et al. (2000) and Brown et al. (2000, 2002). In addition, data from our experiment showed the rats that were pretreated with methamphetamine prior to the challenge administration had greater VMAT-2 immunoreactivity in S3 when compared with the saline/methamphetamine rats. This finding could explain the attenuation in the methamphetamine-induced decrease in VMAT-2 function and suggests that the pretreatment regimen attenuates the ability of the high-dose methamphetamine challenge administration to traffic vesicles out of the nerve terminal, therefore providing neuroprotection to striatal dopamine function.
One possible mechanism for the apparent redistribution of VMAT-2 and, presumably, associated synaptic vesicles, is that the methamphetamine pretreatment regimen causes dopamine to preferentially act on the D2 dopamine autoreceptor since previous data suggest that D2 agonists are more potent at D2 autoreceptors than postsynaptic D2 receptors (Drukarch and Stoof, 1990). The methamphetamine pretreatment regimen could possibly alter the function of proteins involved in synaptic vesicle trafficking. For example, D2 receptors are coupled to the inhibitory G-protein to decrease adenylyl cyclase activity and, thus, decrease cAMP formation (Stoof and Kebabian, 1981; Vallar and Meldolesi, 1989). Therefore, activation of the D2 autoreceptor would lead to the decrease in protein kinase A activity and could possibly prevent the phosphorylation of synapsin. Synapsins are proteins that are associated with synaptic vesicle membranes in the nerve terminal (De Camilli et al., 1983a,b; Huttner et al., 1983) and functions to cluster synaptic vesicles in the cytoplasm (Bahler and Greengard, 1987) and regulate neurotransmitter release from the nerve terminal when phosphorylated (Llinas et al., 1985; Bahler and Greengard, 1987). Consequently, activation of D2 receptors and associated decrease in protein kinase A activity during the methamphetamine pretreatment regimen could prevent phosphorylation of synapsin and potentially inhibit synaptic vesicles from trafficking, thus allowing more vesicles to cluster in the nonmembrane-associated fraction. This mechanism may explain why the methamphetamine-pretreated rats had more VMAT-2 immunoreactivity in S3 than the nonpretreated rats and provided neuroprotection against the high-dose methamphetamine challenge administration by allowing more VMAT-2 to sequester dopamine. Of course, results from our present study do not preclude the involvement of other possible mechanisms through which tolerance could occur, such as a potential up-regulation of antioxidant activity.
In summary, pretreatment with methamphetamine prior to a high-dose methamphetamine challenge administration attenuated the acute methamphetamine-induced decrease in VMAT-2 uptake and redistribution of VMAT-2 immunoreactivity within the nerve terminal. This attenuation in VMAT-2 uptake was not due to blocking methamphetamine-induced hyperthermia nor was the protection due to alterations in the concentration of methamphetamine in the brain because the methamphetamine- and saline-pretreated rats had similar amounts of methamphetamine and amphetamine at 30 min to 2 h after the last methamphetamine challenge injection. These data are the first to demonstrate an association between the prevention of acute alterations in vesicular dopamine uptake and the development of tolerance to the neurotoxic effects of methamphetamine.
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS. DAT, dopamine transporter; VMAT-2, vesicular monoamine transporter-2; P2, whole synaptosomal fraction pellet; P3, membrane-associated fraction; S3, nonmembrane-associated fraction; ANOVA, analysis of variance; METH, methamphetamine.
Address correspondence to: Dr. Diana G. Wilkins, Codirector, Center for Human Toxicology, Research Associate Professor, Department of Pharmacology and Toxicology, University of Utah, 20 South 2030 East, Room 490, Salt Lake City, UT 84112-9457. Email: dwilkins{at}alanine.pharm.utah.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Albers DS and Sonsalla PK (1995) Methamphetamine-induced hyperthermia and dopaminergic neurotoxicity in mice: pharmacological profile of protective and nonprotective agents. J Pharmacol Exp Ther 275: 1104-1114.
Ali SF, Newport GD, and Slikker W Jr (1996) Methamphetamine-induced dopaminergic toxicity in mice: role of environmental temperature and pharmacological agents. Ann N Y Acad Sci 801: 187-198.[Medline]
Bahler M and Greengard P (1987) Synapsin I bundles F-actin in a phosphorylation-dependent manner. Nature 326: 704-707.[CrossRef][Medline]
Bowyer JF, Davies DL, Schmued L, Broening HW, Newport GD, Slikker W Jr, and Holson RR (1994) Further studies of the role of hyperthermia in methamphetamine neurotoxicity. J Pharmcol Exp Ther 268: 1571-1580.
Bowyer JF, Tank AW, Newport GD, Slikker W Jr, and Holson RR (1992) The influence of environmental temperature on the transient effects of methamphetamine on dopamine levels and dopamine release in rat striatum. J Pharmacol Exp Ther 260: 817-824.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.[CrossRef][Medline]
Brown JM, Hanson GR, and Fleckenstein AE (2000) Methamphetamine rapidly decreases vesicular dopamine uptake. J Neurochem 74: 2221-2223.[CrossRef][Medline]
Brown JM, Riddle EL, Sandoval V, Weston RK, Hanson JE, Crosby MJ, Ugarte YV, Gibb JW, Hanson GR, and Fleckenstein AE (2002) A single methamphetamine administration rapidly decreases vesicular dopamine uptake. J Pharmacol Exp Ther 302: 497-501.
Chapin DS, Lookingland KJ, and Moore KE (1986) Effects of LC mobile phase composition on retention times for biogenic amines and their precursors and metabolites. Curr Sep 7: 68-71.
Cubells JF, Rayport S, Rajindron G, and Sulzer D (1994) Methamphetamine neurotoxicity involves vacuolation of endocytic organelles and dopamine-dependent intracellular stress. J Neurosci 14: 2260-2771.[Abstract]
De Camilli P, Cameron R, and Greengard P (1983a) Synapsin I (protein I), a nerve terminal-specific phosphoprotein: I. Its general distribution in synapses of the central and peripheral nervous system demonstrated by immunofluorescence in frozen and plastic sections. J Cell Biol 96: 1337-1354.
De Camilli P, Harris SM Jr, Huttner WB, and Greengard P (1983b) Synapsin I (Protein I), a nerve terminal-specific phosphoprotein: II. Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agaroseembedded synaptosomes. J Cell Biol 96: 1355-1373.
Drukarch B and Stoof JC (1990) D-2 dopamine autoreceptor selective drugs: do they really exist? Life Sci 47: 361-376.[CrossRef][Medline]
Fleckenstein AE, Metzger RR, Wilkins DG, Gibb JW, and Hanson GR (1997) Rapid and reversible effects of methamphetamine on dopamine transporters. J Pharmacol Exp Ther 282: 834-838.
Fumagalli F, Gainetdinov RR, Valenzano KJ, and Caron MG (1998) Role of dopamine transporter in methamphetamine-induced neurotoxicity: evidence from mice lacking the transporter. J Neurosci 18: 4861-4869.
Fumagalli F, Gainetdinov RR, Wang YM, Valenzano KJ, Miller GW, and Caron MG (1999) Increased methamphetamine neurotoxicity in heterozygous vesicular monoamine transporter 2 knock-out mice. J Neurosci 19: 2424-2431.
Gibb JW, Hanson GR, and Johnson M (1994) Neurochemical mechanisms of toxicity, in Amphetamine and Its Analogs (Cho AK and Segal DS eds) pp 269-295, Academic Press, San Diego, CA
Graham DG, Tiffany SM, Bell WR Jr, and Gutknecht WF (1978) Autooxidation versus covalent binding of quinines as the mechanism of toxicity of dopamine, 6-hydroxydopamine and related compounds toward C1300 neuroblastoma cells in vitro. Mol Pharmacol 14: 644-653.
Gygi MP, Gygi SP, Johnson M, Wilkins DG, Gibb JW, and Hanson GR (1996) Mechanisms for tolerance to methamphetamine effects. Neuropharmacololgy 35: 751-757.
Hastings TG (1995) Enzymatic oxidation of dopamine: role of prostaglandin H synthase. J Neurochem 64: 919-924.[Medline]
Hogan KA, Staal RGW, and Sonsalla PK (2000) Analysis of VMAT2 binding after methamphetamine or MPTP treatment: disparity between homogenates and vesicle preparations. J Neurochem 74: 2217-2220.[CrossRef][Medline]
Hotchkiss AJ and Gibb JW (1980) Long-term effects of multiple doses of methamphetamine on tryptophan hydroxylase and tyrosine hydroxylase activity in rat brain. J Pharmacol Exp Ther 214: 257-262.
Huttner WB, Schiebler W, Greengard P, and De Camilli P (1983) Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation. J Cell Biol 96: 1374-1388.
Johnson-Davis KL, Fleckenstein AE, and Wilkins DG (2003) The role of hyperthermia and metabolism as mechanisms of tolerance to methamphetamine neurotoxicity. Eur J Pharmacol 482: 151-154.[CrossRef][Medline]
Kogan FJ, Nichols WK, and Gibb JW (1976) Influence of methamphetamine on nigral and striatal tyrosine hydroxylase activity and on striatal dopamine levels. Eur J Pharmacol 36: 363-371.[CrossRef][Medline]
Liu Y and Edwards RH (1997) The role of vesicular transport proteins in synaptic transmission and neural degeneration. Annu Res Neurosci 20: 125-156.
Llinas R, McGuinness TL, Leonard CS, Sugimori M, and Greengard P (1985) Intraterminal injection of synapsin I or calcium/calmodulin-dependent protein kinase II alters neurotransmitter release at the squid giant synapse. Proc Natl Acad Sci USA 82: 3035-3039.
Lorez H (1981) Fluorescence histochemistry indicates damage of striatal dopamine nerve terminals in rats after multiple doses of methamphetamine. Life Sci 28: 911-916.[CrossRef][Medline]
Lowry OH, Rosebrough N, Farr A, and Randall R (1951) Protein measurements with folin phenol reagent. J Biol Chem 193: 265-275.
Marek GJ, Vosmer G, and Seiden LS (1990) Dopamine uptake inhibitors block long-term neurotoxic effects of methamphetamine upon dopaminergic neurons. Brain Res 513: 274-279.[CrossRef][Medline]
Melega WP, Raleigh MJ, Stout DB, Lacan G, Huang SC, and Phelps ME (1997) Recovery of striatal dopamine function after acute amphetamine- and methamphetamine-induced neurotoxicity in the vervet monkey. Brain Res 766: 113-120.[CrossRef][Medline]
Ricaurte GA, Guillery RW, Seiden LS, Schuster CR, and Moore RY (1982) Dopamine nerve terminal degeneration produced by high doses of methylamphetamine in the rat brain. Brain Res 235: 93-103.[CrossRef][Medline]
Riddle EL, Topham MK, Haycock JW, Hanson GR, and Fleckenstein AE (2002) Differential trafficking of the vesicular monoamine transporter-2 by methamphetamine and cocaine. Eur J Pharmacol 449: 71-74.[CrossRef][Medline]
Sandoval V, Riddle EL, Hanson GR, and Fleckenstein AE (2003) Methylphenidate alters vesicular monoamine transport and prevents methamphetamine-induced dopaminergic deficits. J Pharmacol Exp Ther 304: 1181-1187.
Schmidt CJ, Gehlert DR, Peat MA, Sonsalla PK, Hanson GR, Wamsley JK, and Gibb JW (1985) Studies on the mechanism of tolerance to methamphetamine. Brain Res 343: 305-313.[CrossRef][Medline]
Seiden LS, Fischman MW, and Schuster CR (1976) Long-term methamphetamine induced changes in brain catecholamines in tolerant rhesus monkeys. Drug Alcohol Depend 1: 215-219.[CrossRef][Medline]
Stephans S and Yamamoto B (1996) Methamphetamine pretreatment and the vulnerability of the striatum to methamphetamine neurotoxicity. Neuroscience 72: 593-600.[CrossRef][Medline]
Stoof JC and Kebabian JW (1981) Opposing roles for D-1 and D-2 dopamine receptors in efflux of cyclic AMP from rat neostriatum. Nature 294: 366-368.[CrossRef][Medline]
Teng L, Crooks PA, Sonsalla PK, and Dwoskin LP (1997) Lobeline and nicotine evoked [3H]overflow from rat striatal slices preloaded with [3H]dopamine: differential inhibition of synaptosomal and vesicular [3H]dopamine uptake. J Pharmacol Exp Ther 280: 1432-1444.
Ugarte YV, Rau KS, Riddle EL, Hanson GR, and Fleckenstein AE (2003) Methamphetamine rapidly decreases mouse vesicular dopamine uptake: role of hyperthermia and dopamine D2 receptors. Eur J Pharmacol 472: 165-171.[CrossRef][Medline]
Vallar L and Meldolesi J (1989) Mechanisms of signal transduction at the dopamine D2 receptor. Trends Pharmacol Sci 10: 74-77.[CrossRef][Medline]
Wagner GC, Ricaute G, Seiden LS, Schuster CR, Miller RJ, and Westley J (1980) Long-lasting depletions of striatal dopamine and loss of dopamine uptake sites following repeated administration of methamphetamine. Brain Res 181: 151-160.[CrossRef][Medline]
Wilson JM, Kalasinsky KS, Levey AI, Bergeron C, Reiber G, Anthony RM, Schmunk GA, Shannak K, Haycock JW, and Kish SJ (1996) Striatal dopamine nerve terminal markers in human, chronic methamphetamine users. Nat Med 2: 699-703.[CrossRef][Medline]
Woolverton WL, Ricaurte GA, Forno LS, and Seiden LS (1989) Long-term effects of chronic methamphetamine administration in rhesus monkeys. Brain Res 486: 73-78.[CrossRef][Medline]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
