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NEUROPHARMACOLOGY
Clinical Psychopharmacology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland. (J.S.P., A.G.D., A.S.N., M.H.B., R.B.R.); and Chemistry and Life Sciences Group, Research Triangle Institute International, Research Triangle Park, North Carolina (B.E.B.)
Received for publication
February 26, 2006
Accepted
July 7, 2006.
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Abstract |
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). Once in the vesicle, substrates form a complex with ATP proteins, which may account for the very high concentrations of substrates in the granule (Cooper et al., 2003). Although much is known about the bioenergetics of VMAT2 function, less is known about the interactions of a wide array of amphetamine-related compounds at VMAT2 (Schuldiner et al., 1995; Perera et al., 2003). In contrast, much more is known about how a variety of different agents interact with the plasma biogenic amine transporters, as uptake inhibitors or as substrates, also termed releasers (Rothman et al., 2001). Re-uptake inhibitors bind to transporter proteins, but they are not themselves transported. These drugs elevate extracellular transmitter concentrations by blocking transporter-mediated recapture of transmitter molecules from the synapse. Substrate-type releasers bind to transporter proteins, and these drugs are subsequently transported into the cytoplasm of nerve terminals. Releasers elevate extracellular transmitter concentrations by a two-pronged mechanism: 1) they promote efflux of transmitter by a process of transporter-mediated exchange, and 2) they increase cytoplasmic levels of transmitter by disrupting storage of transmitters in vesicles (Rudnick and Clark, 1993; Rudnick, 1997). This latter action increases the pool of neurotransmitter available for release by transporter-mediated exchange. Because substrate-type releasing agents must be transported into nerve terminals to promote transmitter release, reuptake inhibitors can block the effects of releasers. With appropriate assay methods that assess both the uptake and releasing properties of test agents, new insights are possible. For example, whereas (+)-pseudophenmetrazine is a dopamine transporter substrate, (-)-pseudophenmetrazine is a dopamine transporter inhibitor (Rothman et al., 2002).
Our laboratory previously characterized the interaction of a wide range of amphetamine-like agents at the biogenic amine transporters (Rothman et al., 2001, 2002). In these studies, we developed methods that determined whether the test compound is a substrate or inhibitor of the transporter. The major purpose of this study was to determine the mode of interaction (VMAT2 substrate or inhibitor) for a wide range of test compounds. Toward this end, we developed methods that allow the relatively rapid determination of substrate versus inhibitor activities of test compounds using three major endpoints: 1) inhibition of [3H]dihydrotetrabenazine (DHTBZ) binding, 2) inhibition of [3H]dopamine and [3H]tyramine uptake, and 3) release of preloaded [3H]dopamine and [3H]tyramine. By systematically evaluating the ability of various amphetamine-like agents to alter VMAT2-mediated binding, uptake, and release, our data show that most amphetamine-like agents tested are substrates for the VMAT2, with EC50 values in the range of 5 to 50 µM, and, unexpectedly, that neurotransmitters can exist in two distinct compartments in the vesicle.
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Materials and Methods |
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). Freshly excised caudates were homogenized for 30 s in 0.32 M sucrose using a Polytron (Brinkmann Instruments, Westbury, NY) and spun at 800g for 12 min at 4°C. The pellet was discarded and synaptosomal fragments in the supernatant were pelleted by centrifugation at 22,000g for 15 min at 4°C (P2). The pellet was diluted to 8 ml with distilled water and homogenized with 6 strokes of a Potter-Elvehjem tissue grinder. Reagents were added to yield the following final concentrations: 25 mM HEPES, 100 mM potassium tartrate, 5 mM MgCl2, 10 mM NaCl, 1.7 mM L-ascorbic acid, 0.05 mM EGTA, 0.1 mM EDTA, and 100 µM pargyline, pH 7.4 (binding buffer) in a final volume of 10 ml.
[3H]Dihydrotetrabenazine Binding. [3H]DHTBZ was used to label synaptosomal vesicles from rat caudate putamen with minor modifications of published procedures (Teng et al., 1998). Crude vesicles, prepared as described above, were added to 12- x 75-mm polystyrene test tubes prefilled with 300 µl of binding buffer containing test drugs and 2 nM [3H]DHTBZ (20 Ci/mmol). Assays were terminated after 4 h at 25°C by rapid vacuum filtration over GF/B filters presoaked in ice-cold 2% polyethyleneimine followed by two rinse cycles with ice-cold binding buffer without pargyline, using a model M-48 cell harvester (Brandel Inc., Gaithersburg, MD). Radioactivity retained on filters was quantified by a Taurus (Micromedic, Huntsville, AL) liquid scintillation counter at 40% efficiency. Nonspecific binding was determined in the presence of 20 µM tetrabenazine. Control experiments showed that binding was saturable with respect to time and was proportional to protein concentration (data not shown).
VMAT2 Uptake and Release Assays. Crude synaptic vesicles were prepared as described above. P2 preparations were diluted to 8 ml with ice-cold distilled water, they were homogenized with 6 strokes of a Potter-Elvehjem tissue grinder, and then they were incubated on ice for 30 min followed by centrifugation at 22,000g for 15 min at 4°C. The pellet was discarded and the supernatant (S3) was restored to osmolality in a volume of 10 ml by adding concentrated solutions to create the uptake buffer: 25 mM HEPES, 100 mM potassium tartrate, 1.7 mM L-ascorbic acid, 0.05 EGTA, 0.1 mM EDTA, 2 mM Mg-ATP, 1 µM indatraline, and 0.1 mM pargyline, pH 7.4. Buffered S3 was incubated at 25°C for 15 min before use. VMAT2 uptake assays were performed in 96-well plates. Each well was preloaded with 50 µl of uptake buffer or test drug at the appropriate concentration and 200 µl of 60 nM [3H]dopamine in uptake buffer. The reaction was initiated by addition of 250 µl of tissue preparation (20 µg of protein) and stopped after 5 min by rapid vacuum filtration over GF/B filters presoaked in 2% polyethyleneimine, using a model MWR-96T-4 cell harvester (Brandel Inc.). Filters were washed twice with 2 ml of ice-cold uptake buffer without indatraline and pargyline and with 2 mM MgSO4 instead of Mg-ATP. Radioactivity retained on filters was quantified using a Trilux (PerkinElmer Life and Analytical Sciences, Boston, MA) liquid scintillation counter at 40% efficiency. Dopamine (100 µM) was used to determine nonspecific activity. Control experiments showed that the uptake of [3H]dopamine was saturable with respect to time, it was proportional to protein concentration, and it was entirely dependent on the presence of ATP (data not shown).
For VMAT2 release assays, buffered S3 preparations were incubated for 15 min at 4°C before use. VMAT2 release assays were initiated by preloading vesicles in the uptake buffer with 60 nM [3H]dopamine or 60 nM [3H]tyramine for 20 min at 25°C and by transferring 500 µl to 96-well plates containing the appropriate concentration of test drug in 50 µl of uptake buffer. The release reaction was terminated after 10 min ([3H]dopamine) or 2 min ([3H]tyramine), and samples were processed as described for the uptake assay. Nonspecific activity was determined in the presence of 100 µM dopamine. This value was subtracted from the other values to yield a "specific" activity. To determine the rate of dopamine-induced efflux of [3H]amine, vesicles were prepared as described above for the release assays, and the amount of retained [3H]amine was measured at several time points after the addition of 1 µM dopamine.
Experimental Design, Data Analysis, and Statistics. Inhibition/release curves were generated using eight drug concentrations per curve. For [3H]dopamine uptake inhibition assays and [3H]DHTBZ binding, the data of three independent experiments were pooled and fit to the two-parameter logistic (eq. 1) using MLAB-PC (Civilized Software, Bethesda, MD), as described previously (Rothman et al., 2001), for the best-fit estimates of the IC50 and slope factor. For release assays, the data were calculated as a percentage of inhibition and then fit to eq. 2 for the best-fit estimates of the EC50 and plateau level (EMAX), using either MLAB-PC or KaleidaGraph 3.6 software (Synergy Software, Reading, PA). Graphs were generated with KaleidaGraph 3.6 software. The equations used are as follows:
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To determine the KM and VMAX of [3H]dopamine and [3H]tyramine uptake, each radioligand was displaced with either dopamine or tyramine, and the pooled data of three experiments were fit to the Michaelis-Menten equation for the best-fit estimate of the VMAX and KM. Dopamine-induced efflux of [3H]amine data were fit to a monoexponential decay equation for the best-fit estimates of the Koff (Rothman et al., 1991).
Chemicals and Reagents. [3H]Dopamine (3,4[7-3H]dihydroxyphenylethylamine; specific activity 20.5 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences. [3H]Tyramine (50 Ci/mmol), [2-3H]dihydrotetrabenazine (20 Ci/mmol), and tetrabenazine were purchased from American Radiolabeled Chemicals (St. Louis, MO). Ketanserin, reserpine, dopamine, norepinephrine, tyramine, parachloroamphetamine, metachlorophenylpiperazine, and trifluoromethylphenylpiperazine were purchased from Sigma/RBI (Natick, MA). Histamine and 5-hydroxytryptamine were purchased from Sigma-Aldrich (St. Louis, MO). (+)-Norfenfluramine, (-)-norfenfluramine, and (±)-norfenfluramine were a gift from SRI International (Menlo Park, CA). All other drugs in the study were provided by the Addiction Research Center Pharmacy (National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD). The sources of other reagents are published (Rothman et al., 2001).
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). The remaining test agents behaved as substrates, having very low affinity for [3H]DHTBZ binding and much higher potency at inhibiting vesicular [3H]dopamine uptake. The IC50 values for these agents for inhibiting [3H]dopamine uptake ranged from 0.5 µM for 1-napthyl-2-aminopropane to 92 µM for (+)-pseudophenmetrazine. It is noteworthy that these agents are all much more potent at the plasma membrane biogenic amine transporters than at the VMAT2 (Rothman et al., 2001, 2002). Interestingly, histamine, although generally thought to be a substrate for VMAT2, was very weak in both assays.
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[3H]Dopamine and [3H]Tyramine Release Experiments. We next assessed the ability of known VMAT2 substrates (dopamine and norepinephrine) and uptake inhibitors (tetrabenazine and ketanserin) to release preloaded [3H]dopamine and [3H]tyramine. The results (Fig. 1) showed that substrates fully released [3H]tyramine and [3H]dopamine and that uptake inhibitors were apparent partial releasers, probably due to leakage of [3H]amine from the vesicle. The EC50 and EMAX values for release are reported in Table 2. The EC50 values of the four test drugs for [3H]tyramine and [3H]dopamine release were similar. These data suggest that the [3H]tyramine and [3H]dopamine release assays distinguish between substrates and inhibitors of VMAT2: substrates have EMAX values close to 100% and uptake inhibitors have EMAX values much less than 100%.
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We next examined the ability of (+)-fenfluramine and (+)-3,4-methylenedioxymethamphetamine [MDMA], 1-(m-chlorophenyl)piperazine (mCPP), and 1-(m-trifluoromethylphenyl)piperazine (TFMPP) to release preloaded [3H]dopamine and [3H]tyramine. As reported in Fig. 2 and Table 2, the results showed that all four agents fully released [3H]tyramine, indicating that all are substrates for VMAT2. However, only mCPP was a full releaser of [3H]dopamine (EMAX = 100%). For example, the EMAX values of (+)-fenfluramine and (+)-MDMA for [3H]dopamine release were 68.1 ± 2.0 and 65.3 ± 3.0, respectively. TFMPP extrapolated to be a full releaser.
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To explain the different EMAX values observed for [3H]dopamine and [3H]tyramine release, we hypothesized the existence of two pools for [3H]dopamine: "free" dopamine and dopamine tightly associated with the ATP and protein present in the vesicles, metaphorically "crystallized" out. We further hypothesized that [3H]tyramine would mostly be free or loosely associated with the ATP/protein complex, but tyramine would still be able to displace [3H]dopamine from the ATP/protein complex (Cooper et al., 2003). According to this hypothesis, substrates fully release [3H]tyramine, because it is mostly free, and differ in their ability to release [3H]dopamine depending on the access of the agent to the crystallized pool. Stated somewhat differently, the EMAX value of an agent at [3H]dopamine release reflects the degree to which it can displace or exchange for [3H]dopamine in the crystallized pool. High EMAX values mean a higher degree of access, and lower EMAX values indicate a lower degree of access to the crystallized pool.
One prediction of this hypothesis is that the capacity of the vesicle for [3H]dopamine uptake should exceed that of [3H]tyramine, because [3H]dopamine is being sequestered in the crystallized pool. To test this hypothesis, we determined the VMAX values of the vesicles for [3H]tyramine and [3H]dopamine uptake. The Km and VMAX values for [3H]tyramine and [3H]dopamine are reported in Table 3. The results indicate that the VMAX for [3H]tyramine uptake is 44% lower than that of [3H]dopamine uptake. A second prediction of this hypothesis is that whereas dopamine will completely release [3H]dopamine and [3H]tyramine, tyramine will completely release [3H]tyramine but only partially release [3H]dopamine. Consistent with this prediction, the EMAX of tyramine versus [3H]tyramine was 100%, but 87% versus [3H]dopamine (Table 2). A third prediction of this hypothesis is that preloaded [3H]dopamine will efflux more slowly than [3H]tyramine, because the net efflux rate of [3H]dopamine will be strongly influenced by its presumably slower "dissociation" from the crystallized pool. As reported in Fig. 3, dopamine (1 µM)-stimulated [3H]tyramine efflux was approximately 10-fold faster than that observed for [3H]dopamine efflux.
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Via what is termed the "weak base" effect, amphetamine-type agents can deplete vesicular biogenic amine content by degrading the pH gradient that powers the transporter (Sulzer and Rayport, 1990). To determine whether this effect occurred under our assay conditions, we compared the effects of (+)-amphetamine and NH4Cl on vesicular [3H]dopamine release. As reported in Fig. 4, (+)-amphetamine, at micromolar concentrations, reduced retained [3H]dopamine in a dose-dependent manner, reaching a plateau at approximately 40% of control. In contrast, NH4Cl, at millimolar concentrations, reduced retained [3H]dopamine below the "nonspecific" level determined with 100 µM dopamine. (+)-Amphetamine (100 µM) and NH4Cl (100 µM and 5 mM) did not alter the pH of the buffer.
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; Cooper et al., 2003; Sulzer et al., 2005). Amphetamine-type drugs interact with the VMAT2 (Schuldiner et al., 1993) in a complex manner (for review, see Fleckenstein and Hanson, 2003). Amphetamines can inhibit VMAT2 function via competitive blockade (Gonzalez et al., 1994) and also deplete vesicular biogenic amine content by degrading the pH gradient that powers the transporter (Sulzer and Rayport, 1990). In addition, amphetamine alters the distribution of vesicles between the cytoplasm and plasma membrane (Fleckenstein and Hanson, 2003). Some evidence suggests that the ability of MDMA to release neuronal serotonin (Mlinar and Corradetti, 2003) and amphetamine to release neuronal dopamine (Jones et al., 1998) is dependent on release of vesicular neurotransmitter.
We previously characterized the interaction of a wide range of amphetamine-like agents at the biogenic amine transporters (Rothman et al., 2001, 2002), using methods that determined whether the test compound is a substrate or inhibitor of the transporter. In the present study, we sought to apply this approach to the VMAT2. We developed methods that allow the relatively rapid determination of substrate versus inhibitor activities of test compounds and examined the interaction of a wide range of compounds on VMAT2 function, using three major endpoints: 1) inhibition of [3H]DHTBZ binding, 2) inhibition of [3H]dopamine uptake, and 3) release of preloaded [3H]dopamine and [3H]tyramine. In undertaking these experiments, we also were interested to see whether differential interactions at the VMAT2 might distinguish between serotonin transporter substrates that produce long-term serotonin depletion (fenfluramine and MDMA) and serotonin transporter substrates that do not (mCPP and TFMPP). Our results show that most amphetamine-like agents tested are substrates for the VMAT2, with EC50 values in the range of 5 to 50 µM; that VMAT2 interactions do not predict the ability of a serotonin transporter substrate to produce long-term serotonin depletion; and that most agents are much more potent at the plasma membrane biogenic amine transporters than at the VMAT2.
Unlike other studies that use purified synaptic vesicles (Teng et al., 1998), we used a crude preparation of synaptic vesicles that contained 1 µM indatraline to block any residual plasma membrane biogenic amine transporters. Under these conditions, we obtained data similar to those reported by others. For example, similar to our data, two groups reported that (+)-amphetamine inhibited [3H]DHTBZ binding with an IC50 value greater than 10 µM (Rostene et al., 1992; Zucker et al., 2001). Moreover, Teng et al. (1998) reported that (+)-amphetamine released [3H]dopamine from purified vesicles with an EC50 = 2.2 µM, a value almost identical to what we observed (2.5 µM; Table 2).
As with the biogenic amine transporters, VMAT2 can be assessed both by binding and functional assays. Binding assays label the transporter with a compound that inhibits the transporter ([3H]DHTBZ), and functional assays measure the ability of the transporter to translocate a substrate ([3H]dopamine) across the cell membrane. Our previous work with the biogenic amine transporters indicated that transporter inhibitors had similar potencies in both types of assays, whereas substrates were much more potent in the functional assay than the binding assay. Indeed, this seemed to be the case for the VMAT2 as well (Table 1). Compounds known to be VMAT2 substrates (dopamine, norepinephrine, and serotonin), inhibited [3H]dopamine uptake, with IC50 values between 590 and 1700 nM, whereas they were inactive in the [3H]DHTBZ assay. In contrast, known VMAT2 uptake inhibitors (tetrabenazine, ketanserin, and reserpine) had similar potencies in both assays. Using this approach to define transporter activity, the data indicated that all of the active test agents examined (Table 1) were putative substrates. Among the VMAT2 substrates, 1-napthyl-2-aminopropane was the most potent at inhibiting [3H]dopamine uptake (IC50 = 500 nM).
A number of test drugs displayed no activity in the [3H]dopamine uptake inhibition assay (Table 1). For example, (+)-phenmetrazine and (-)-phenmetrazine, the major metabolites of phendimetrazine (Rothman et al., 2002), were essentially inactive. Interestingly, (-)-pseudophenmetrazine and (+)-pseudophenmetrazine, which were inhibitor and substrate, respectively, at the dopamine transporter, also differed in their interaction at the VMAT2, because only (-)-pseudophenmetrazine was active in the [3H]dopamine release assay. (+)-Pseudophenmetrazine had an IC50 value of 92 µM in the [3H]dopamine uptake inhibition assay, and it was inactive in the [3H]dopamine release assay, suggesting that (+)-pseudophenmetrazine may be a VMAT2 inhibitor. However, the very low potency of (+)-pseudophenmetrazine in these assays rules out the possibility of a definitive experiment to establish this point.
We next developed release assays similar to those used for the plasma membrane biogenic amine transporters (Rothman et al., 2001). In this procedure, [3H]substrate is incubated to steady state, and test drugs are then added. Samples are filtered a short time later, and the drug-induced "release" was calculated based on the amount of [3H]substrate retained on the filter. We examined the ability of test agents to release both [3H]dopamine and [3H]tyramine. Our initial experiments indicated that the substrates dopamine and norepinephrine fully released both [3H]substrates (Fig. 1), whereas tetrabenazine and ketanserin, known VMAT2 inhibitors, were partial releasers. These data suggested that the degree of release (EMAX) could be used to distinguish between substrates and inhibitors. However, the data obtained with the first set of test agents were not clear-cut. When a broader array of compounds were examined, it became clear that, with few exceptions, most agents fully released [3H]tyramine but partially released [3H]dopamine.
To explain the different EMAX values observed with the [3H]dopamine and [3H]tyramine release assays, we hypothesized, based on the fact that dopamine tightly associates with the ATP and protein present in the vesicles (Cooper et al., 2003), that [3H]dopamine existed in two pools within the vesicle: a free and a crystallized out pool. The latter pool presumably represents [3H]dopamine tightly associated with the ATP/protein complex. We further hypothesized that [3H]tyramine would mostly be free or loosely associated with the ATP/protein complex but that tyramine would still be able to displace [3H]dopamine from the ATP/protein complex. According to this hypothesis, substrates fully release [3H]tyramine, because it is mostly free, and they differ in their ability to release [3H]dopamine depending on the access of the agent to the crystallized pool.
Three experiments support this hypothesis. First, the VMAX of vesicular [3 H]tyramine uptake is substantially lower than that for [3H]dopamine uptake (Table 3), consistent with [3H]dopamine being sequestered into a location to which [3H]tyramine has limited access. Second, whereas dopamine completely released [3H]tyramine, tyramine partially released [3H]dopamine (EMAX = 87%). Three, preloaded [3H]dopamine effluxed more slowly than [3H]tyramine, because the net efflux rate of [3H]dopamine was reduced by its presumably slower dissociation from the crystallized pool (Fig. 3). These experiments support the hypothesis that VMAT2 substrates have EMAX values of approximately 100% with the [3H]tyramine release assay and that EMAX values in the [3H]dopamine release assay reflect the degree of access to the crystallized pool.
Dopamine (pKa = 8.9) and tyramine (pKa = 9.74) are primary amines, and like the other primary (amphetamine, pKa = 9.8) and secondary amines, these agents would be positively charged at the acidic pH present in the vesicles. Thus, the finding that [3H]dopamine is apparently sequestered into a different compartment than [3H]tyramine cannot arise from differences in net charge. Moreover, it is unlikely that the slower release of [3H]dopamine, compared with [3H]tyramine, results from some of the [3H]dopamine undergoing oxidation, because oxidation is limited both by the presence of ascorbic acid and the monoamine oxidase inhibitor pargyline in the assay buffer.
An intriguing observation to emerge from this study is that agents differ in their ability to be sequestered in the vesicle via a tight association with the ATP/protein complex. As noted above, our data suggest that the EMAX value of a substrate for releasing [3H]dopamine reflects its access to this compartment. Compounds such as the endogenous VMAT2 substrates, which have EMAX values of approximately 100%, tightly associate with the ATP/protein complex. Agents such as (+)-methamphetamine, with much lower EMAX values (65%), presumably are sequestered to lesser extent. If this hypothesis is correct, then agents with EMAX values near 100%, such as mCPP, TFMPP, and 1-napthyl-2-aminopropane, might be sequestered in the vesicle and act as false neurotransmitters.
A number of contrasting results emerged between the [3H]tyramine and [3H]dopamine release assays. For example, (+)-norfenfluramine is approximately 3-fold more potent in the [3H]dopamine assay, but (-)-norfenfluramine has similar potencies in both assays. (+)-Amphetamine is approximately 7-fold more potent in the [3H]dopamine assay, but (-)-amphetamine was approximately 4.4-fold more potent in the [3H]tyramine assay. The reasons for these differences are not clear. One possibility is that the association and dissociation constants of the free [3H]dopamine with the ATP/protein complex contribute to the measured EC50 value of a test agent. Unfortunately, our experimental approach is not sensitive enough to measure the kinetics of this system.
Our work also provides some additional insight into the mechanisms of amphetamine-induced neurotransmitter release. According to the weak base hypothesis (Sulzer and Rayport, 1990; Schuldiner et al., 1993), amphetamine depletes vesicular biogenic amine content by degrading the pH gradient that powers the transporter. However, our data (Fig. 4) indicate that raising intravesicular pH with NH4Cl in the presence of 100 µM dopamine reduces vesicular [3H]dopamine only at millimolar concentrations. It is noteworthy that neither amphetamine nor any other test agent besides NH4Cl, released [3H]dopamine to a level below that produced by 100 µM dopamine, indicating that even extraordinarily high concentrations of amphetamine-type drugs do not deplete vesicular amine via the free-base effect. For these agents to do so would probably require millimolar concentrations, which are far beyond the range they might achieve in vivo.
Some evidence suggests that the ability of MDMA to release neuronal serotonin (Mlinar and Corradetti, 2003) and amphetamine to release neuronal dopamine (Jones et al., 1998) is dependent on release of vesicular amine. Consistent with these in vivo data, our results (Table 2) indicate that (±)-amphetamine and (±)-MDMA release vesicular dopamine at the pharmacologically relevant EC50 values of 4.3 and 27 µM, respectively. In contrast, other amphetamine-type agents, such as phentermine, phenmetrazine, and 1-benzylpiperazine, are potent releasers of neuronal dopamine (Baumann et al., 2000, 2005; Rothman et al., 2002), but they are inactive at VMAT2. Agents such as these may prove to be valuable control compounds for determining the importance of vesicular release for the in vivo actions of amphetamine-type agents.
Certain serotonin transporter substrates, such as fenfluramine (McCann et al., 1997) and MDMA (Green et al., 2003), are described as "neurotoxic" based on their ability to produce, when administered at high doses, persistent decreases in markers of the presynaptic serotonin nerve terminal., although recent data strongly suggest that these agents may not cause axotomy in the rat (Rothman et al., 2003; Wang et al., 2004). As reviewed previously, being a serotonin transporter substrate is necessary, but not sufficient, for a drug to produce long-term serotonin depletion (Rothman and Baumann, 2002a). For example, 1-naphthyl-2-aminopropane, mCPP, and TFMPP are serotonin transporter substrates (Baumann et al., 2004) that do not deplete brain serotonin (Baumann et al., 2001; Rothman et al., 2005). Thus, in the present study we explored the hypothesis that neurotoxic serotonin transporter substrates (MDMA and fenfluramine) differ from non-neurotoxic serotonin transporter substrates in their VMAT2 interactions. As described below, the data suggest that this is not the case.
The non-neurotoxic serotonin transporter substrates (1-napthyl-2-aminopropane, TFMPP, and mCPP) differ from the neurotoxic serotonin transporter substrates [(+)-MDMA and (+)-fenfluramine] in their EMAX values in the [3H]dopamine release assay. The former substrates have EMAX values of approximately 80 to 100%, whereas the latter substrates have EMAX values of approximately 65%. However, other compounds with known serotonin-depleting activity do not fit into this framework. For example, the EMAX value of (+)-norfenfluramine (67.6%), a neurotoxic compound (Johnson and Nichols, 1990), and parachloroamphetamine (74.4%) are similar to that of TFMPP (82.5%). Moreover, it is not clear whether some of these agents would achieve high enough concentrations in the nerve terminal to actually interact at VMAT2. For example, the high concentrations of the piperazine serotonin transporter substrates needed to affect VMAT2 function (
20 µM) compared with their potency as serotonin transporter substrates (30-60 nM) (Rothman and Baumann, 2002b; Baumann et al., 2005) suggests that this mechanism might not occur in vivo.
In summary, our comprehensive study of the interaction of 42 mostly amphetamine-related agents with VMAT2 has led to several important findings. First, our work indicates that most agents are VMAT2 substrates. Second, our data strongly suggest that amphetamine-type agents deplete vesicular neurotransmitter via a carrier-mediated exchange mechanism rather than via the weak base effect, although this conclusion needs to be confirmed via direct measurement of vesicular pH. Third, our data fail to reveal differential VMAT2 interactions among agents that do and do not produce long-term 5-HT depletion. Fourth, the data revealed the presence of two pools of [3H]amine within the vesicle, one pool that is free and one pool that is tightly associated with the ATP/protein complex that helps store amine. Finally, the VMAT2 assays we have developed should prove useful for guiding the synthesis and evaluation of novel VMAT2 agents as possible treatment agents for addictive disorders (Miller et al., 2004).
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: VMAT2, vesicular monoamine transporter type 2; MDMA, 3,4-methylenedioxymethamphetamine; DHTBZ, dihydrotetrabenazine; mCPP, 1-(m-chlorophenyl)piperazine; TFMPP, 1-(m-trifluoromethylphenyl)piperazine.
Address correspondence to: Dr. Richard B. Rothman, Clinical Psychopharmacology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan Shock Dr., Baltimore, MD 21224. E-mail: rrothman{at}mail.nih.gov
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