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 Amphetamines and Related Compounds at the Vesicular Monoamine Transporter 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 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 February 26, 2006; accepted July 7, 2006 Downloaded from ABSTRACT Amphetamine-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 neurotransmitter and an increase in cytoplasmic data strongly suggest that amphetamine-type agents deplete neurotransmitter. Some compounds, such as reserpine, “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 base 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/protein complex that helps store amine. at ASPET Journals on January 21, 2020 3 including 1) inhibition of [ H]dihydrotetrabenazine binding, 2) Finally, the VMAT2 assays we have developed should prove 3 inhibition of vesicular [ H]dopamine uptake, and 3) release of useful for guiding the synthesis and evaluation of novel VMAT2 preloaded [3H]dopamine and [3H]tyramine. Several important agents as possible treatment agents for addictive disorders. The vesicular monamine transporter type 2 (VMAT2) biogenic amine transporters, as uptake inhibitors or as sub- pumps its substrates dopamine, serotonin, norepinephrine, strates, also termed releasers (Rothman et al., 2001). Re- epinephrine, and histamine into vesicular storage vesicles uptake inhibitors bind to transporter proteins, but they are against a gradient. This process is powered by the vesicular not themselves transported. These drugs 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 synapse. 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, reuptake 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)piperazine; 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 dopamine transporter 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 indatraline, and 0.1 mM pargyline, 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 tissue 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 neurotransmitters 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 brains 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