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0022-3565/06/3191-237–246 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 319, No. 1 U.S. Government work not protected by U.S. copyright 103622/3139867 JPET 319:237–246, 2006 Printed in U.S.A.

Interaction of and Related Compounds at the Vesicular

John S. Partilla, Allison G. Dempsey, Ameet S. Nagpal, Bruce E. Blough, Michael H. Baumann, and Richard B. Rothman Clinical Psychopharmacology Section, Intramural Research Program, National Institute on Abuse, National Institutes of Health, Department of Health and 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 February 26, 2006; accepted July 7, 2006 Downloaded from

ABSTRACT -type agents interact with the vesicular mono- findings derive from this comprehensive study. First, our work amine transporter type 2 (VMAT2), promoting the release of indicates that most agents are VMAT2 substrates. Second, our intravesicular and an increase in cytoplasmic data strongly suggest that amphetamine-type agents deplete neurotransmitter. Some compounds, such as , “re- vesicular neurotransmitter via a carrier-mediated exchange jpet.aspetjournals.org lease” neurotransmitter by inhibiting the ability of VMAT2 to mechanism rather than via a weak effect, although this accumulate neurotransmitter in the vesicle, whereas other conclusion needs to be confirmed via direct measurement of types of compounds can release neurotransmitter via a carrier- vesicular pH. Third, our data fail to reveal differential VMAT2 mediated exchange mechanism. The purpose of this study was interactions among agents that do and do not produce long- to determine, for 42 mostly amphetamine-related compounds, term 5-hydroxytryptamine depletion. Fourth, the data reported 3 their mode of interaction with the VMAT2. We used a crude revealed the presence of two pools of [ H]amine within the vesicular fraction prepared from rat caudate to assay VMAT2 vesicle, one pool that is free and one pool that is tightly asso- activity. Test compounds were assessed in several assays, ciated with the ATP/ complex that helps store amine. at ASPET Journals on January 21, 2020 3 including 1) inhibition of [ H] binding, 2) Finally, the VMAT2 assays we have developed should prove 3 inhibition of vesicular [ H] uptake, and 3) release of useful for guiding the synthesis and evaluation of novel VMAT2 preloaded [3H]dopamine and [3H]. Several important agents as possible treatment agents for addictive disorders.

The vesicular monamine transporter type 2 (VMAT2) transporters, as uptake inhibitors or as sub- pumps its substrates dopamine, , , strates, also termed releasers (Rothman et al., 2001). Re- epinephrine, and into vesicular storage vesicles uptake inhibitors bind to transporter , but they are against a gradient. This process is powered by the vesicular not themselves transported. These elevate extracellu- ϩ H -ATPase and the exchange of two intravesicular protons lar transmitter concentrations by blocking transporter-medi- for one substrate molecule (Schuldiner et al., 1998). Once in ated recapture of transmitter molecules from the . the vesicle, substrates form a complex with ATP proteins, Substrate-type releasers bind to transporter proteins, and which may account for the very high concentrations of sub- these drugs are subsequently transported into the cytoplasm strates in the granule (Cooper et al., 2003). Although much is of nerve terminals. Releasers elevate extracellular transmit- known about the bioenergetics of VMAT function, less is 2 ter concentrations by a two-pronged mechanism: 1) they pro- known about the interactions of a wide array of amphet- mote efflux of transmitter by a process of transporter-medi- amine-related compounds at VMAT (Schuldiner et al., 1995; 2 ated exchange, and 2) they increase cytoplasmic levels of Perera et al., 2003). In contrast, much more is known about transmitter by disrupting storage of transmitters in vesicles how a variety of different agents interact with the plasma (Rudnick and Clark, 1993; Rudnick, 1997). This latter action increases the pool of neurotransmitter available for release This study was supported by the Intramural Research Program of the National Institutes of Health, National Institute on Drug Abuse, and National by transporter-mediated exchange. Because substrate-type Institute on Drug Abuse Grant R01 DA12970 (to B.E.B.) releasing agents must be transported into nerve terminals to Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. promote transmitter release, inhibitors can block doi:10.1124/jpet.106.103622. the effects of releasers. With appropriate assay methods that

ABBREVIATIONS: VMAT2, vesicular monoamine transporter type 2; MDMA, 3,4-methylenedioxymethamphetamine; DHTBZ, dihydrotetrabena- zine; mCPP, 1-(m-chlorophenyl); TFMPP, 1-(m-trifluoromethylphenyl)piperazine. 237 238 Partilla et al.

assess both the uptake and releasing properties of test was restored to osmolality in a volume of 10 ml by adding concen- agents, new insights are possible. For example, whereas (ϩ)- trated solutions to create the uptake buffer: 25 mM HEPES, 100 mM pseudophenmetrazine is a substrate, potassium tartrate, 1.7 mM L-ascorbic acid, 0.05 EGTA, 0.1 mM (Ϫ)-pseudophenmetrazine is a dopamine transporter inhibi- EDTA, 2 mM Mg-ATP, 1 ␮M , and 0.1 mM , pH 7.4. Buffered S3 was incubated at 25°C for 15 min before use. VMAT tor (Rothman et al., 2002). 2 uptake assays were performed in 96-well plates. Each well was Our laboratory previously characterized the interaction of preloaded with 50 ␮l of uptake buffer or test drug at the appropriate a wide range of amphetamine-like agents at the biogenic concentration and 200 ␮lof60nM[3H]dopamine in uptake buffer. amine transporters (Rothman et al., 2001, 2002). In these The reaction was initiated by addition of 250 ␮l of preparation studies, we developed methods that determined whether the (20 ␮g of protein) and stopped after 5 min by rapid vacuum filtration test compound is a substrate or inhibitor of the transporter. over GF/B filters presoaked in 2% polyethyleneimine, using a model The major purpose of this study was to determine the mode MWR-96T-4 cell harvester (Brandel Inc.). Filters were washed twice

of interaction (VMAT2 substrate or inhibitor) for a wide with 2 ml of ice-cold uptake buffer without indatraline and pargyline range of test compounds. Toward this end, we developed and with 2 mM MgSO4 instead of Mg-ATP. Radioactivity retained on methods that allow the relatively rapid determination of filters was quantified using a Trilux (PerkinElmer Life and Analyt- substrate versus inhibitor activities of test compounds using ical Sciences, Boston, MA) liquid scintillation counter at 40% effi- ciency. Dopamine (100 ␮M) was used to determine nonspecific activ- three major endpoints: 1) inhibition of [3H]dihydrotetrabena- ity. Control experiments showed that the uptake of [3H]dopamine zine (DHTBZ) binding, 2) inhibition of [3H]dopamine and 3 3 was saturable with respect to time, it was proportional to protein [ H]tyramine uptake, and 3) release of preloaded [ H]dopam- concentration, and it was entirely dependent on the presence of ATP Downloaded from 3 ine and [ H]tyramine. By systematically evaluating the abil- (data not shown).

ity of various amphetamine-like agents to alter VMAT2-me- For VMAT2 release assays, buffered S3 preparations were incu- diated binding, uptake, and release, our data show that most bated for 15 min at 4°C before use. VMAT2 release assays were amphetamine-like agents tested are substrates for the 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 VMAT2, with EC50 values in the range of 5 to 50 M, and, transferring 500 ␮l to 96-well plates containing the appropriate

unexpectedly, that can exist in two dis- jpet.aspetjournals.org ␮ tinct compartments in the vesicle. concentration of test drug in 50 l 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 Materials and Methods uptake assay. Nonspecific activity was determined in the presence of 100 ␮M dopamine. This value was subtracted from the other values Preparation of a Crude Vesicular Fraction. Rat caudate was to yield a “specific” activity. To determine the rate of dopamine- dissected from frozen rat purchased from Pel-Freez (Rogers, induced efflux of [3H]amine, vesicles were prepared as described 3

AR). A crude vesicular fraction was prepared from rat caudate pu- above for the release assays, and the amount of retained [ H]amine at ASPET Journals on January 21, 2020 tamen with minor modifications of published procedures (Teng et al., was measured at several time points after the addition of 1 ␮M 1998). Freshly excised caudates were homogenized for 30 s in 0.32 M dopamine. sucrose using a Polytron (Brinkmann Instruments, Westbury, NY) Experimental Design, Data Analysis, and Statistics. Inhibi- and spun at 800g for 12 min at 4°C. The pellet was discarded and tion/release curves were generated using eight drug concentrations synaptosomal fragments in the supernatant were pelleted by centrif- per curve. For [3H]dopamine uptake inhibition assays and ugation at 22,000g for 15 min at 4°C (P2). The pellet was diluted to [3H]DHTBZ binding, the data of three independent experiments 8 ml with distilled water and homogenized with 6 strokes of a were pooled and fit to the two-parameter logistic (eq. 1) using Potter-Elvehjem tissue grinder. Reagents were added to yield the MLAB-PC (Civilized Software, Bethesda, MD), as described previ-

following final concentrations: 25 mM HEPES, 100 mM potassium ously (Rothman et al., 2001), for the best-fit estimates of the IC50 and

tartrate, 5 mM MgCl2, 10 mM NaCl, 1.7 mM L-ascorbic acid, 0.05 slope factor. For release assays, the data were calculated as a per- mM EGTA, 0.1 mM EDTA, and 100 ␮M pargyline, pH 7.4 (binding centage of inhibition and then fit to eq. 2 for the best-fit estimates of

buffer) in a final volume of 10 ml. the EC50 and plateau level (EMAX), using either MLAB-PC or Kalei- [3H]Dihydrotetrabenazine Binding. [3H]DHTBZ was used to daGraph 3.6 software (Synergy Software, Reading, PA). Graphs were label synaptosomal vesicles from rat caudate putamen with minor generated with KaleidaGraph 3.6 software. The equations used are modifications of published procedures (Teng et al., 1998). Crude as follows: vesicles, prepared as described above, were added to 12- ϫ 75-mm ϭ ͑ ϩ ͑ ͒N͒ polystyrene test tubes prefilled with 300 ␮l of binding buffer con- Y 100/ 1 X/IC50 (1) taining test drugs and 2 nM [3H]DHTBZ (20 Ci/mmol). Assays were

terminated after4hat25°C by rapid vacuum filtration over GF/B Y ϭ EMAX ϫ ͓Drug͔/͓͑Drug͔ ϩ EC50͒. (2) filters presoaked in ice-cold 2% polyethyleneimine followed by two 3 3 rinse cycles with ice-cold binding buffer without pargyline, using a To determine the KM and VMAX of [ H]dopamine and [ H]tyramine model M-48 cell harvester (Brandel Inc., Gaithersburg, MD). Radio- uptake, each was displaced with either dopamine or activity retained on filters was quantified by a Taurus (Micromedic, tyramine, and the pooled data of three experiments were fit to the

Huntsville, AL) liquid scintillation counter at 40% efficiency. Non- Michaelis-Menten equation for the best-fit estimate of the VMAX and ␮ 3 specific binding was determined in the presence of 20 M tetraben- KM. Dopamine-induced efflux of [ H]amine data were fit to a mono- azine. Control experiments showed that binding was saturable with exponential decay equation for the best-fit estimates of the Koff respect to time and was proportional to protein concentration (data (Rothman et al., 1991). not shown). Chemicals and Reagents. [3H]Dopamine (3,4[7-3H]dihydroxy-

VMAT2 Uptake and Release Assays. Crude synaptic vesicles phenylethylamine; specific activity 20.5 Ci/mmol) was purchased were prepared as described above. P2 preparations were diluted to 8 from PerkinElmer Life and Analytical Sciences. [3H]Tyramine (50 ml with ice-cold distilled water, they were homogenized with 6 Ci/mmol), [2-3H]dihydrotetrabenazine (20 Ci/mmol), and tetrabena- strokes of a Potter-Elvehjem tissue grinder, and then they were zine were purchased from American Radiolabeled Chemicals (St. incubated on ice for 30 min followed by centrifugation at 22,000g for Louis, MO). , reserpine, dopamine, norepinephrine, tyra- 15 min at 4°C. The pellet was discarded and the supernatant (S3) mine, parachloroamphetamine, metachlorophenylpiperazine, and Amphetamines and VMAT2 239 trifluoromethylphenylpiperazine were purchased from Sigma/RBI 1.7 ␮M (norepinephrine). This pattern of activity is similar to (Natick, MA). Histamine and 5-hydroxytryptamine were purchased that observed for the plasma membrane biogenic amine from Sigma-Aldrich (St. Louis, MO). (ϩ)-, (Ϫ)-nor- transporters, where uptake blockers inhibit both biogenic Ϯ , and ( )-norfenfluramine were a gift from SRI Inter- amine transporter binding and function, and substrates in- national (Menlo Park, CA). All other drugs in the study were pro- hibit function much more potently than they inhibit trans- vided by the Research Center Pharmacy (National porter binding (Rothman et al., 1999). The remaining test Institute on Drug Abuse, National Institutes of Health, Baltimore, MD). The sources of other reagents are published (Rothman et al., agents behaved as substrates, having very low affinity for 3 2001). [ H]DHTBZ binding and much higher potency at inhibiting 3 vesicular [ H]dopamine uptake. The IC50 values for these agents for inhibiting [3H]dopamine uptake ranged from 0.5 Results ␮M for 1-napthyl-2-aminopropane to 92 ␮M for (ϩ)-pseudo- [3H]Dihydrotetrabenazine Binding and [3H]Dopam- . It is noteworthy that these agents are all ine Vesicular Uptake. The first series of experiments (Ta- much more potent at the plasma membrane biogenic amine ble 1) determined the IC50 values of test agents for inhibiting transporters than at the VMAT2 (Rothman et al., 2001, [3H]DHTBZ binding and for blocking vesicular uptake of 2002). Interestingly, histamine, although generally thought 3 [ H]dopamine. As expected, known vesicular uptake inhibi- to be a substrate for VMAT2, was very weak in both assays. tors, such as , ketanserin, and reserpine were [3H]Dopamine and [3H]Tyramine Release Experi- 3 3 potent inhibitors of both [ H]DHTBZ binding and [ H]do- ments. We next assessed the ability of known VMAT2 sub- Downloaded from pamine uptake. Known VMAT2 substrates (dopamine, nor- strates (dopamine and norepinephrine) and uptake inhibi- epinephrine, and serotonin) were inactive at inhibiting tors (tetrabenazine and ketanserin) to release preloaded 3 3 3 [ H]DHTBZ binding but had IC50 values for inhibition of [ H]dopamine and [ H]tyramine. The results (Fig. 1) showed [3H]dopamine uptake ranging from 0.68 ␮M (serotonin) to that substrates fully released [3H]tyramine and [3H]dopa-

TABLE 1 jpet.aspetjournals.org ͓3 ͔ ͓3 ͔ Interaction of test agents with VMAT2: H DHTBZ binding versus H dopamine uptake inhibition ͓3H͔DHTBZ (2 nM) binding and ͓3H͔dopamine (60 nM) uptake inhibition assays were conducted as described under Materials and Methods. The data of three experiments ϭ were combined (n 24 points) and fit to the two-parameter logistic equation for the best-fit estimates of the IC50 and slope factor (N). The N values are not reported since Ϯ they were all about 1.0. Each value is IC50 S.D. Drug Known/Suspected Activitya ͓3H͔DHTBZ Binding ͓3H͔Dopamine Uptake

␮M Tetrabenazine Inhibitor 0.016 Ϯ 0.0014 0.12 Ϯ 0.01 Ketanserin Inhibitor 0.12 Ϯ 0.013 0.32 Ϯ 0.03 at ASPET Journals on January 21, 2020 Reserpine Inhibitor 0.71 Ϯ 0.04 0.22 Ϯ 0.02 Histamine Substrate Ͼ100 Ͼ45 Dopamine Substrate Ͼ100 0.92 Ϯ 0.05 Norepinephrine Substrate Ͼ100 1.72 Ϯ 0.17 Serotonin Substrate Ͼ100 0.68 Ϯ 0.05 Tyramine Substrate Ͼ100 0.59 Ϯ 0.05 Ͼ100 2.44 Ϯ 0.18 (Ϯ)-MDMA Substrate Ͼ100 19.5 Ϯ 1.2 (ϩ)-MDMA Substrate Ͼ100 22.8 Ϯ 1.9 (Ϫ)-MDMA Substrate Ͼ100 31.1 Ϯ 1.9 (Ϯ)- Substrate Ͼ250 14.9 Ϯ 1.3 (ϩ)-Methamphetamine Substrate Ͼ100 9.10 Ϯ 1.02 (Ϫ)-Methamphetamine Substrate Ͼ100 19.3 Ϯ 2.7 (Ϯ)-Fenfluramine Substrate 134 Ϯ 7.6 11.5 Ϯ 0.7 (ϩ)-Fenfluramine Substrate 139 Ϯ 8.7 11.6 Ϯ 0.8 (Ϫ)-Fenfluramine Substrate 124 Ϯ 6.9 4.37 Ϯ 0.48 (Ϯ)-Norfenfluramine Substrate 171 Ϯ 6.0 6.11 Ϯ 0.40 (ϩ)-Norfenfluramine Substrate 87.2 Ϯ 8.1 4.34 Ϯ 0.38 (Ϫ)-Norfenfluramine Substrate 220 Ϯ 21 23.1 Ϯ 3.4 (Ϯ)-Amphetamine Substrate Ͼ100 3.29 Ϯ 0.20 (ϩ)-Amphetamine Substrate 185 Ϯ 15 3.27 Ϯ 0.27 (Ϫ)-Amphetamine Substrate Ͼ100 29.0 Ϯ 2.3 Parachloroamphetamine Substrate Ͼ100 4.66 Ϯ 0.34 mCPP Substrate 179 Ϯ 15 13.6 Ϯ 0.7 TFMPP Substrate 277 Ϯ 24 14.0 Ϯ 1.2 (Ϫ)- Inactive Ͼ100 Ͼ100 1-Naphthyl-2-aminopropane Substrate Ͼ100 0.50 Ϯ 0.02 Substrate Ͼ100 4.12 Ϯ 0.19 Inactive Ͼ100 Ͼ100 (ϩ)-Phenmetrazine Inactive Ͼ100 Ͼ100 (Ϫ)-Phenmetrazine Inactive Ͼ100 90.0 Ϯ 7.5 (ϩ)-Pseudophenmetrazine Inactive Ͼ100 92.0 Ϯ 10.0 (Ϫ)-Pseudophenmetrazine Substrate Ͼ100 22.7 Ϯ 1.8 1- Inactive Ͼ100 Ͼ100 Diethylpropion Inactive Ͼ100 Ͼ100 (ϩ)-Diethylnorephedrine Inactive Ͼ100 59.8 Ϯ 4.9 (Ϫ)-Diethylnorephedrine Inactive Ͼ100 Ͼ100 Inactive Ͼ100 Ͼ100 a Italics indicate suspected activity. 240 Partilla et al. mine and that uptake inhibitors were apparent partial re- and [3H]tyramine. As reported in Fig. 2 and Table 2, the leasers, probably due to leakage of [3H]amine from the vesi- results showed that all four agents fully released [3H]tyra- cle. The EC50 and EMAX values for release are reported in mine, indicating that all are substrates for VMAT2. However, 3 3 ϭ Table 2. The EC50 values of the four test drugs for [ H]tyra- only mCPP was a full releaser of [ H]dopamine (EMAX 3 ϩ mine and [ H]dopamine release were similar. These data 100%). For example, the EMAX values of ( )-fenfluramine suggest that the [3H]tyramine and [3H]dopamine release as- and (ϩ)-MDMA for [3H]dopamine release were 68.1 Ϯ 2.0 says distinguish between substrates and inhibitors of and 65.3 Ϯ 3.0, respectively. TFMPP extrapolated to be a full

VMAT2: substrates have EMAX values close to 100% and releaser. uptake inhibitors have EMAX values much less than 100%. To gain additional insight into the relationship between We next examined the ability of (ϩ)-fenfluramine and (ϩ)- [3H]dopamine and [3H]tyramine release, we determined the 3,4-methylenedioxymethamphetamine [MDMA], 1-(m-chlo- releasing effect of a wider array of test agents. As reported in rophenyl)piperazine (mCPP), and 1-(m-trifluoromethylphe- Table 2, most agents fully or almost completely released nyl)piperazine (TFMPP) to release preloaded [3H]dopamine [3H]tyramine and partially released [3H]dopamine. (Ϫ)-Am- Downloaded from jpet.aspetjournals.org at ASPET Journals on January 21, 2020

Fig. 1. Release of preloaded vesicular [3H]amine by test agents. Release assays were conducted as described under Materials and Methods. 3 3 Endogenous VMAT2 substrates dopamine (A) and norepinephrine (B) completely release both [ H]dopamine and [ H]tyramine. VMAT2 inhibitors 3 3 tetrabenazine (C) and ketanserin (D) partially release both [ H]dopamine and [ H]tyramine. The EC50 and EMAX values are reported in Table 3. Each value is the mean Ϯ S.D. (n ϭ 3). Amphetamines and VMAT2 241

TABLE 2

Release EC50 and EMAX values for selected compounds ͓3H͔Amine release assays were conducted as described under Materials and Methods. The data of three experiments were combined (n ϭ 24 points), and the best-fit estimates Ϯ of the EC50 and EMAX were determined using MLAB-PC or KaleidaGraph 3.6 software. Each value is S.D. a ͓3 ͔ ͓3 ͔ ͓3 ͔ ͓3 ͔ Drug Known/Suspected Activity H Tyramine EC50 H Tyramine EMAX H Dopamine EC50 H Dopamine EMAX ␮M%␮M% Tetrabenazine Inhibitor 0.06 Ϯ 0.01 54.7 Ϯ 3.1 0.18 Ϯ 0.06 45.3 Ϯ 2.9 Ketanserin Inhibitor 0.21 Ϯ 0.04 65.8 Ϯ 3.0 0.41 Ϯ 0.06 46.3 Ϯ 1.3 Reserpine Inhibitor 0.13 Ϯ 0.03 57.1 Ϯ 3.3 0.23 Ϯ 0.06 50 Ϯ 3 Histamine Substrate Ͼ200 1700 Ϯ 300 116 Ϯ 9 Dopamine Substrate 0.69 Ϯ 0.09 104 Ϯ 4 0.41 Ϯ 0.03 101 Ϯ 1 Norepinephrine Substrate 2.66 Ϯ 0.63 117 Ϯ 10 1.4 Ϯ 0.2 118 Ϯ 5 Serotonin Substrate 0.51 Ϯ 0.07 107 Ϯ 4 0.29 Ϯ 0.02 102 Ϯ 2 Tyramine Substrate 0.47 Ϯ 0.06 95.5 Ϯ 3.2 0.63 Ϯ 0.13 87.8 Ϯ 3.8 Phenethylamine Substrate 4.33 Ϯ 1.33 86.7 Ϯ 5.8 2.8 Ϯ 0.32 80.7 Ϯ 2.0 (Ϯ)-MDMA Substrate 43.1 Ϯ 7.6 98.9 Ϯ 6.0 27 Ϯ 7 65.3 Ϯ 5.3 (ϩ)-MDMA Substrate 69.2 Ϯ 29.7 104 Ϯ 18 22 Ϯ 3 65.3 Ϯ 3.0 (Ϫ)-MDMA Substrate 32.0 Ϯ 7.9 78.8 Ϯ 6.3 47 Ϯ 5 65.1 Ϯ 2.8 (Ϯ)-Methamphetamine Substrate 14.2 Ϯ 3.0 84.5 Ϯ 4.8 20 Ϯ 3.9 59.9 Ϯ 3.6 (ϩ)-Methamphetamine Substrate 24.1 Ϯ 3.6 88.9 Ϯ 4.0 11 Ϯ 1.5 65.5 Ϯ 2.9 Ϫ Ϯ Ϯ Ϯ Ϯ ( )-Methamphetamine Substrate 27.4 7.6 104 93410 53.6 5.7 Downloaded from (Ϯ)-Fenfluramine Substrate 20.4 Ϯ 4.0 109 Ϯ 6 6.6 Ϯ 0.7 65.3 Ϯ 1.7 (ϩ)-Fenfluramine Substrate 38.0 Ϯ 4.6 116 Ϯ 512Ϯ 1 68.1 Ϯ 2.0 (Ϫ)-Fenfluramine Substrate 18.8 Ϯ 2.9 104 Ϯ 5 3.3 Ϯ 0.4 62.6 Ϯ 1.8 (Ϯ)-Norfenfluramine Substrate 13.7 Ϯ 2.0 99.6 Ϯ 3.9 7.5 Ϯ 1.0 65.3 Ϯ 2.3 (ϩ)-Norfenfluramine Substrate 19.1 Ϯ 1.4 109 Ϯ 2 6.4 Ϯ 0.9 67.6 Ϯ 2.4 (Ϫ)-Norfenfluramine Substrate 27.5 Ϯ 3.1 105 Ϯ 433Ϯ 8 77.1 Ϯ 6.5 (Ϯ)-Amphetamine Substrate 10.5 Ϯ 2.3 95.8 Ϯ 5.3 4.3 Ϯ 0.5 65.7 Ϯ 1.7 ϩ Ϯ Ϯ Ϯ Ϯ ( )-Amphetamine Substrate 18.0 3.8 111 7 2.5 0.2 63.1 1.2 jpet.aspetjournals.org (Ϫ)-Amphetamine Substrate 12.8 Ϯ 2.4 83.1 Ϯ 4.1 57 Ϯ 5 75.2 Ϯ 2.7 Parachloroamphetamine Substrate 7.9 Ϯ 1.3 96.1 Ϯ 4.4 5.0 Ϯ 4.2 74.4 Ϯ 1.5 mCPP Substrate 27.4 Ϯ 4.1 105 Ϯ 547Ϯ 6 104 Ϯ 5 TFMPP Substrate 18.5 Ϯ 3.9 99.9 Ϯ 6.0 35 Ϯ 9 82.5 Ϯ 7.1 (Ϫ)-Cocaine Inactive Ͼ100 Ͼ100 1-Naphthyl-2-aminopropane Substrate 1.25 Ϯ 0.23 99.4 Ϯ 4.2 0.95 Ϯ 0.07 77.8 Ϯ 1.2 Benzphetamine Substrate 8.69 Ϯ 2.25 100 Ϯ 614Ϯ 3 57.0 Ϯ 3.9 Phendimetrazine Inactive Ͼ100 Ͼ100 (ϩ)-Phenmetrazine Inactive Ͼ100 Ͼ100 at ASPET Journals on January 21, 2020 (Ϫ)-Phenmetrazine Inactive Ͼ100 Ͼ100 (ϩ)-Pseudophenmetrazine Inactive Ͼ100 Ͼ100 (Ϫ)-Pseudophenmetrazine Substrate 19.2 Ϯ 4 105 Ϯ 6 12.1 Ϯ 2.5 72.5 Ϯ 3.9 1-Benzylpiperazine Inactive Ͼ100 Ͼ100 Diethylpropion Inactive Ͼ100 Ͼ100 (ϩ)-Diethylnorephedrine Substrate 63.9 Ϯ 20.3 97.0 Ϯ 11.8 Ͼ100 (Ϫ)-Diethylnorephedrine Substrate 71.6 Ϯ 8.5 77.5 Ϯ 3.6 Ͼ100 Phentermine Inactive Ͼ100 Ͼ100 1-Methyl-4-phenylpyridine Substrate 20.7 Ϯ 4.4 90.4 Ϯ 5.5 11.7 Ϯ 1.8 78.4 Ϯ 3.1 (Ϫ)-Epinephrine Substrate 1.23 Ϯ 0.14 99.0 Ϯ 2.0 0.94 Ϯ 0.12 101 Ϯ 2 a Italics indicate suspected activity.

Ϫ 3 phetamine and ( )-MDMA had EMAX values for [ H]tyra- lower EMAX values indicate a lower degree of access to the mine release in the range of 80%. Therefore, only the crystallized pool. [3H]tyramine release assay was able to distinguish substrate One prediction of this hypothesis is that the capacity of from inhibitor. The [3H]dopamine release assay gave ambig- the vesicle for [3H]dopamine uptake should exceed that of uous results. [3H]tyramine, because [3H]dopamine is being sequestered 3 To explain the different EMAX values observed for [ H]do- in the crystallized pool. To test this hypothesis, we deter- 3 3 pamine and [ H]tyramine release, we hypothesized the exis- mined the VMAX values of the vesicles for [ H]tyramine 3 3 tence of two pools for [ H]dopamine: “free” dopamine and and [ H]dopamine uptake. The Km and VMAX values for dopamine tightly associated with the ATP and protein [3H]tyramine and [3H]dopamine are reported in Table 3. 3 present in the vesicles, metaphorically “crystallized” out. We The results indicate that the VMAX for [ H]tyramine up- further hypothesized that [3H]tyramine would mostly be free take is 44% lower than that of [3H]dopamine uptake. A or loosely associated with the ATP/protein complex, but tyra- second prediction of this hypothesis is that whereas dopa- mine would still be able to displace [3H]dopamine from the mine will completely release [3H]dopamine and [3H]tyra- ATP/protein complex (Cooper et al., 2003). According to this mine, tyramine will completely release [3H]tyramine but hypothesis, substrates fully release [3H]tyramine, because it only partially release [3H]dopamine. Consistent with this 3 3 is mostly free, and differ in their ability to release [ H]dopa- prediction, the EMAX of tyramine versus [ H]tyramine was mine depending on the access of the agent to the crystallized 100%, but 87% versus [3H]dopamine (Table 2). A third 3 pool. Stated somewhat differently, the EMAX value of an prediction of this hypothesis is that preloaded [ H]dopa- agent at [3H]dopamine release reflects the degree to which it mine will efflux more slowly than [3H]tyramine, because can displace or exchange for [3H]dopamine in the crystallized the net efflux rate of [3H]dopamine will be strongly influ- pool. High EMAX values mean a higher degree of access, and enced by its presumably slower “dissociation” from the 242 Partilla et al. Downloaded from jpet.aspetjournals.org at ASPET Journals on January 21, 2020

Fig. 2. Release of preloaded vesicular [3H]amine by test agents. Release assays were conducted as described under Materials and Methods. substrates (ϩ)-fenfluramine (A) and (ϩ)-MDMA (B) that deplete rat serotonin partially release [3H]dopamine but not [3H]tyramine. ϩ Serotonin transporter substrates mCPP (C) and TFMPP (D) that do not deplete rat brain serotonin have higher EMAX values than ( )-fenfluramine ϩ Ϯ ϭ and ( )-MDMA. The EC50 and EMAX values are reported in Table 3. Each value is the mean S.D. (n 3).

TABLE 3 Via what is termed the “weak base” effect, amphetamine- Michaelis-Menton parameters for ͓3H͔dopamine and ͓3H͔tyramine type agents can deplete vesicular biogenic amine content by vesicular uptake degrading the pH gradient that powers the transporter (Sul- ͓3H͔Tyramine or ͓3H͔dopamine uptake inhibition curves were generated with tyra- mine or dopamine, respectively. The data of three experiments (n ϭ 48 points) were zer and Rayport, 1990). To determine whether this effect pooled and fit to the Michaelis-Menton equation for the best-fit estimates of the VMAX and K . occurred under our assay conditions, we compared the effects M ϩ 3 of ( )-amphetamine and NH4Cl on vesicular [ H]dopamine ͓3H͔Amine K V M MAX release. As reported in Fig. 4, (ϩ)-amphetamine, at micromo- ␮ ␮ M mol/min/mg protein lar concentrations, reduced retained [3H]dopamine in a dose- Ϯ Ϯ Dopamine 0.82 0.13 0.86 0.36 dependent manner, reaching a plateau at approximately 40% Tyramine 0.20 Ϯ 0.02* 0.38 Ϯ 0.05* of control. In contrast, NH Cl, at millimolar concentrations, *P Ͻ 0.01 compared with ͓3H͔dopamine. 4 reduced retained [3H]dopamine below the “nonspecific” level crystallized pool. As reported in Fig. 3, dopamine (1 ␮M)- determined with 100 ␮M dopamine. (ϩ)-Amphetamine (100 3 ␮ ␮ stimulated [ H]tyramine efflux was approximately 10-fold M) and NH4Cl (100 M and 5 mM) did not alter the pH of faster than that observed for [3H]dopamine efflux. the buffer. Amphetamines and VMAT2 243

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). Amphet-

amines can inhibit VMAT2 function via competitive block- ade (Gonzalez et al., 1994) and also deplete vesicular bio- genic 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 (Flecken- stein 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 Downloaded from 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 Fig. 3. Dopamine-induced efflux of preloaded vesicular [3H]amine. Rat examined the interaction of a wide range of compounds on 3 3 brain vesicles were preloaded with [ H]tyramine and [ H]dopamine as VMAT2 function, using three major endpoints: 1) inhibi- jpet.aspetjournals.org described under Materials and Methods. At time 0, 1 ␮M dopamine was tion of [3H]DHTBZ binding, 2) inhibition of [3H]dopamine 3 added, and the amount of retained [ H]amine was measured at various 3 times. The data of three experiments (n ϭ 24 points) were combined and uptake, and 3) release of preloaded [ H]dopamine and [3H]tyramine. In undertaking these experiments, we also fit to the monoexponential decay equation for the best-fit estimate of koff. Each value is the mean Ϯ S.D. (n ϭ 3). were interested to see whether differential interactions at

the VMAT2 might distinguish between serotonin trans- porter substrates that produce long-term serotonin deple-

tion (fenfluramine and MDMA) and serotonin transporter at ASPET Journals on January 21, 2020 substrates that do not (mCPP and TFMPP). Our results show that most amphetamine-like agents tested are sub-

strates for the VMAT2, with EC50 values in the range of 5 ␮ to 50 M; 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 ␮M indatraline to block any resid- ual 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 (ϩ)-amphetamine inhibited [3H]DHTBZ bind- ␮ ing with an IC50 value greater than 10 M (Rostene et al., 1992; Zucker et al., 2001). Moreover, Teng et al. (1998) re- ported that (ϩ)-amphetamine released [3H]dopamine from ϭ ␮ purified vesicles with an EC50 2.2 M, a value almost identical to what we observed (2.5 ␮M; Table 2).

As with the biogenic amine transporters, VMAT2 can be assessed both by binding and functional assays. Binding 3 ϩ Fig. 4. VMAT2-mediated [ H]dopamine release. ( )-Amphetamine re- assays label the transporter with a compound that inhibits 3 ␮ leased [ H]dopamine to the level produced by 100 M dopamine, whereas 3 3 the transporter ([ H]DHTBZ), and functional assays mea- NH4Cl released [ H]dopamine to levels below that of “nonspecific” activ- ity. Each value is the mean Ϯ S.D. (n ϭ 3). sure the ability of the transporter to translocate a substrate ([3H]dopamine) across the . Our previous work Discussion with the biogenic amine transporters indicated that trans- porter inhibitors had similar potencies in both types of as-

The VMAT2 is a long-studied transporter that serves to says, whereas substrates were much more potent in the func- concentrate its substrates, the biogenic amines, in storage tional assay than the binding assay. Indeed, this seemed to

vesicles (Schuldiner et al., 1998; Cooper et al., 2003; Sulzer be the case for the VMAT2 as well (Table 1). Compounds 244 Partilla et al.

3 known to be VMAT2 substrates (dopamine, norepinephrine, than that for [ H]dopamine uptake (Table 3), consistent with 3 3 and serotonin), inhibited [ H]dopamine uptake, with IC50 [ H]dopamine being sequestered into a location to which values between 590 and 1700 nM, whereas they were inac- [3H]tyramine has limited access. Second, whereas dopamine 3 3 tive in the [ H]DHTBZ assay. In contrast, known VMAT2 completely released [ H]tyramine, tyramine partially re- 3 ϭ uptake inhibitors (tetrabenazine, ketanserin, and reserpine) leased [ H]dopamine (EMAX 87%). Three, preloaded had similar potencies in both assays. Using this approach to [3H]dopamine effluxed more slowly than [3H]tyramine, be- define transporter activity, the data indicated that all of the cause the net efflux rate of [3H]dopamine was reduced by its active test agents examined (Table 1) were putative sub- presumably slower dissociation from the crystallized pool strates. Among the VMAT2 substrates, 1-napthyl-2-amino- (Fig. 3). These experiments support the hypothesis that 3 propane was the most potent at inhibiting [ H]dopamine VMAT2 substrates have EMAX values of approximately 100% ϭ 3 uptake (IC50 500 nM). with the [ H]tyramine release assay and that EMAX values in A number of test drugs displayed no activity in the [3H]do- the [3H]dopamine release assay reflect the degree of access to pamine uptake inhibition assay (Table 1). For example, (ϩ)- the crystallized pool. Ϫ ϭ ϭ phenmetrazine and ( )-phenmetrazine, the major metabo- Dopamine (pKa 8.9) and tyramine (pKa 9.74) are lites of phendimetrazine (Rothman et al., 2002), were primary amines, and like the other primary (amphetamine, Ϫ ϭ essentially inactive. Interestingly, ( )-pseudophenmetrazine pKa 9.8) and secondary amines, these agents would be and (ϩ)-pseudophenmetrazine, which were inhibitor and positively charged at the acidic pH present in the vesicles. 3 substrate, respectively, at the dopamine transporter, also Thus, the finding that [ H]dopamine is apparently seques- Downloaded from 3 differed in their interaction at the VMAT2, because only tered into a different compartment than [ H]tyramine cannot (Ϫ)-pseudophenmetrazine was active in the [3H]dopamine arise from differences in net charge. Moreover, it is unlikely ϩ 3 release assay. ( )-Pseudophenmetrazine had an IC50 value that the slower release of [ H]dopamine, compared with of 92 ␮M in the [3H]dopamine uptake inhibition assay, and it [3H]tyramine, results from some of the [3H]dopamine under- was inactive in the [3H]dopamine release assay, suggesting going oxidation, because oxidation is limited both by the ϩ that ( )-pseudophenmetrazine may be a VMAT2 inhibitor. presence of ascorbic acid and the inhib- jpet.aspetjournals.org However, the very low potency of (ϩ)-pseudophenmetrazine itor pargyline in the assay buffer. in these assays rules out the possibility of a definitive exper- An intriguing observation to emerge from this study is that iment to establish this point. agents differ in their ability to be sequestered in the vesicle We next developed release assays similar to those used for via a tight association with the ATP/protein complex. As the plasma membrane biogenic amine transporters (Roth- noted above, our data suggest that the EMAX value of a man et al., 2001). In this procedure, [3H]substrate is incu- substrate for releasing [3H]dopamine reflects its access to

bated to steady state, and test drugs are then added. Samples this compartment. Compounds such as the endogenous at ASPET Journals on January 21, 2020 are filtered a short time later, and the drug-induced “release” VMAT2 substrates, which have EMAX values of approxi- was calculated based on the amount of [3H]substrate re- mately 100%, tightly associate with the ATP/protein com- tained on the filter. We examined the ability of test agents to plex. Agents such as (ϩ)-methamphetamine, with much 3 3 release both [ H]dopamine and [ H]tyramine. Our initial lower EMAX values (65%), presumably are sequestered to experiments indicated that the substrates dopamine and nor- lesser extent. If this hypothesis is correct, then agents with 3 epinephrine fully released both [ H]substrates (Fig. 1), EMAX values near 100%, such as mCPP, TFMPP, and whereas tetrabenazine and ketanserin, known VMAT2 inhib- 1-napthyl-2-aminopropane, might be sequestered in the ves- itors, were partial releasers. These data suggested that the icle and act as false neurotransmitters. degree of release (EMAX) could be used to distinguish between A number of contrasting results emerged between the substrates and inhibitors. However, the data obtained with [3H]tyramine and [3H]dopamine release assays. For exam- the first set of test agents were not clear-cut. When a broader ple, (ϩ)-norfenfluramine is approximately 3-fold more potent array of compounds were examined, it became clear that, in the [3H]dopamine assay, but (Ϫ)-norfenfluramine has sim- with few exceptions, most agents fully released [3H]tyramine ilar potencies in both assays. (ϩ)-Amphetamine is approxi- but partially released [3H]dopamine. mately 7-fold more potent in the [3H]dopamine assay, but Ϫ To explain the different EMAX values observed with the ( )-amphetamine was approximately 4.4-fold more potent in [3H]dopamine and [3H]tyramine release assays, we hypoth- the [3H]tyramine assay. The reasons for these differences are esized, based on the fact that dopamine tightly associates not clear. One possibility is that the association and dissoci- with the ATP and protein present in the vesicles (Cooper et ation constants of the free [3H]dopamine with the ATP/pro- 3 al., 2003), that [ H]dopamine existed in two pools within the tein complex contribute to the measured EC50 value of a test vesicle: a free and a crystallized out pool. The latter pool agent. Unfortunately, our experimental approach is not sen- presumably represents [3H]dopamine tightly associated with sitive enough to measure the kinetics of this system. the ATP/protein complex. We further hypothesized that Our work also provides some additional insight into the [3H]tyramine would mostly be free or loosely associated with mechanisms of amphetamine-induced neurotransmitter re- the ATP/protein complex but that tyramine would still be lease. According to the weak base hypothesis (Sulzer and able to displace [3H]dopamine from the ATP/protein complex. Rayport, 1990; Schuldiner et al., 1993), amphetamine de- According to this hypothesis, substrates fully release pletes vesicular biogenic amine content by degrading the pH [3H]tyramine, because it is mostly free, and they differ in gradient that powers the transporter. However, our data 3 their ability to release [ H]dopamine depending on the access (Fig. 4) indicate that raising intravesicular pH with NH4Cl in of the agent to the crystallized pool. the presence of 100 ␮M dopamine reduces vesicular [3H]do- Three experiments support this hypothesis. First, the pamine only at millimolar concentrations. It is noteworthy 3 VMAX of vesicular [ H]tyramine uptake is substantially lower that neither amphetamine nor any other test agent besides Amphetamines and VMAT2 245

3 NH4Cl, released [ H]dopamine to a level below that produced 42 mostly amphetamine-related agents with VMAT2 has led by 100 ␮M dopamine, indicating that even extraordinarily to several important findings. First, our work indicates that

high concentrations of amphetamine-type drugs do not de- most agents are VMAT2 substrates. Second, our data plete vesicular amine via the free-base effect. For these strongly suggest that amphetamine-type agents deplete ve- agents to do so would probably require millimolar concentra- sicular neurotransmitter via a carrier-mediated exchange tions, which are far beyond the range they might achieve mechanism rather than via the weak base effect, although in vivo. this conclusion needs to be confirmed via direct measurement Some evidence suggests that the ability of MDMA to re- of vesicular pH. Third, our data fail to reveal differential

lease neuronal serotonin (Mlinar and Corradetti, 2003) and VMAT2 interactions among agents that do and do not pro- amphetamine to release neuronal dopamine (Jones et al., duce long-term 5-HT depletion. Fourth, the data revealed the 1998) is dependent on release of vesicular amine. Consistent presence of two pools of [3H]amine within the vesicle, one with these in vivo data, our results (Table 2) indicate that pool that is free and one pool that is tightly associated with (Ϯ)-amphetamine and (Ϯ)-MDMA release vesicular dopa- the ATP/protein complex that helps store amine. Finally, the

mine at the pharmacologically relevant EC50 values of 4.3 VMAT2 assays we have developed should prove useful for ␮ and 27 M, respectively. In contrast, other amphetamine- guiding the synthesis and evaluation of novel VMAT2 agents type agents, such as phentermine, phenmetrazine, and as possible treatment agents for addictive disorders (Miller et 1-benzylpiperazine, are potent releasers of neuronal dopa- al., 2004).

mine (Baumann et al., 2000, 2005; Rothman et al., 2002), but Downloaded from References they are inactive at VMAT2. Agents such as these may prove to be valuable control compounds for determining the impor- Baumann MH, Ayestas MA, Dersch CM, Brockington A, Rice KC, and Rothman RB (2000) Effects of phentermine and fenfluramine on extracellular dopamine and tance of vesicular release for the in vivo actions of amphet- serotonin in rat : therapeutic implications. Synapse 36:102– amine-type agents. 113. Baumann MH, Ayestas MA, Dersch CM, and Rothman RB (2001) 1-(m-Chlorophe- Certain serotonin transporter substrates, such as fenflura- nyl)piperazine (mCPP) dissociates in vivo serotonin release from long-term sero- tonin depletion in rat brain. 24:492–501.

mine (McCann et al., 1997) and MDMA (Green et al., 2003), jpet.aspetjournals.org Baumann MH, Clark RD, Budzynski AG, Partilla JS, Blough BE, and Rothman RB are described as “neurotoxic” based on their ability to pro- (2004) Effects of “legal x” piperazine analogs on dopamine and serotonin release in duce, when administered at high doses, persistent decreases rat brain. Ann NY Acad Sci 1025:189–197. Baumann MH, Clark RD, Budzynski AG, Partilla JS, Blough BE, and Rothman RB in markers of the presynaptic serotonin nerve terminal., (2005) N-Substituted abused by mimic the molecular mech- although recent data strongly suggest that these agents may anism of 3,4-methylenedioxymethamphetamine (MDMA, or “Ecstasy”). Neuropsy- chopharmacology 30:550–560. not cause axotomy in the rat (Rothman et al., 2003; Wang et Cooper JR, Bloom FE, and Roth RH (2003) The Biochemical Basis of Neuropharma- al., 2004). As reviewed previously, being a serotonin trans- cology, Oxford University Press, New York. Fleckenstein AE and Hanson GR (2003) Impact of psychostimulants on vesicular

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