'4 0026-895X/93/061227-05503.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics AIl rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 44:1227-1231 Amphetamine Derivatives Interact with Both Plasma Membrane and Secretory Vesicle Biogenic Amine Transporters SHIMON SCHULDINER, SONIA STEINER-MORDOCH, RODRIGO YELIN, STEPHEN C. WALL, and GARY RUDNICK Departmentof Pharmacology,YaleUniversitySchool of Medicine,New Haven,CT 06510 (S.C.W.,G.R.) andAlexanderSilbermanInstituteof Life Sciences,TheHebrew University,GivatRam, Jerusalem,Israel (S.S.,S.S.-M., R.Y.) ReceivedJune 7, 1993; Accepted September 16, 1993 SUMMARY The interaction of fenfluramine, 3,4-methylenedioxymethamphe- porter, fenfiuramine inhibited serotonin transport and dissipated tamine (MDMA), and p-chloroamphetamine (PCA) with the plate- the transmembrane pH difference (ApH) that drives amine up- let plasma membrane serotonin transporter and the vesicular take. The use of [3H]reserpine-binding measurements to deter- amine transporter were studied using both transport and binding mine drug interaction with the vesicular amine transporter al- measurements. Fenfluramine is apparently a substrate for the lowed assessment of the relative ability of MDMA, PCA, and plasma membrane transporter, and consequently inhibits both fenfluramine to bind to the substrate site of the vesicular trans- serotonin transport and imipramine binding. Moreover, fenflura- porter. These measurements permit a distinction between inhi- mine exchanges with internal [3H]serotonin in a plasma mem- bition of vesicular serotonin transport by directly blocking vesic- brane transporter-mediated reaction that requires NaCI and is ular amine transport and by dissipating ApH. The results indicate blocked by imipramine. These properties are similar to those of that MDMA and fenfluramine inhibit by both mechanisms but MDMA and PCA as previously described. In adrenal chromaffin PCA dissipates ApH without blocking vesicular amine transport granule membrane vesicles containing the vesicular amine trans- directly, Amphetamine and its derivatives are well-known as sym- brane vesicles containing the serotonin transporter (8, 9). In pathomimetic amines that act indirectly by releasing endoge- the present study, we extend these results to fenfluramine, nous biogenic amines (1). This release has been attributed to which demonstrates a similar ability to serve as a substrate for the ability of extraceUular amphetamines to exchange with the serotonin transporter. cytoplasmic biogenic amines (2, 3). Exchange was proposed to The extent of serotonin depletion by MDMA, PCA, and be catalyzed by the transporters that normally serve to inacti- fenfiuramine indicates that serotonin stored in synaptic vesi- cles, as well as cytoplasmic serotonin, is mobilized by these vate transmitter action by reuptake of norepinephrine, dopa- agents. Serotonin is accumulated within synaptic vesicles by a mine, and serotonin. An additional mechanism has been pro- vesicular amine transporter that utilizes the ApH generated by posed in which biogenic amine efflux is stimulated through the vacuolar ATPase (10). Previous studies with amphetamine dissipation of the transmembrane :XpH that drives biogenic derivatives have demonstrated their ability to dissipate this amine uptake into synaptic vesicles (4, 5). ApH by nonionic diffusion and to block serotonin transport (4, A number of amphetamine derivatives preferentially release 5, 8, 9). As amines, however, the amphetamine derivatives are , serotonin and also cause the degeneration of serotonergic nerve potential substrates and inhibitors of the vesicular amine trans- endings. These include the designer drug MDMA (also known porter. [3H]Reserpine binding to chromaffin granule mem- as "ecstasy"), PCA, and the appetite suppressant fenfiuramine, branes is blocked by substrates for the transporter and has Serotonin release by these agents does not require Ca 2+, and is been used as an indicator of its substrate binding site. In the present study, we demonstrate that MDMA and fenfluramine, inhibited by serotonin transport blockers, leading to the pro- but not PCA, inhibit [3H]reserpine binding to the vesicular posal that these agents exchange with intracellular serotonin amine transporter. These results point out differences between through the action of the plasma membrane serotonin trans- serotonin-releasing amphetamine derivatives that may be im- porter (6, 7). We have demonstrated in vitro exchange of portant to their pharmacologic and behavioral effects. serotonin for MDMA and PCA using platelet plasma mere- Experimental Procedures This work was supported by United States Public Health Service grants DA 07295 (G.R.), NS 16708 (S.S.), and a grant from the National Institute for Materials. [3H]serotonin (12.6 Ci/mmol)and [3H]imipramine (40.4 Psychobiology,Israel (S.S.). Ci/mmol) were purchased from DuPont-New England Nuclear (Bos- ABBREVIATIONS: MDMA, 3,4-methylenedioxymethamphetamine; PCA, p-chloroamphetamine; ApH, transmembrane pH difference; Ap.+, trans- membraneelectrochemical potential for H+. 1227 1228 Schuldiner et al. ton, MA). [aH]Reserpine (20 Ci/mmol) was purchased from the Nuclear serotonin. The standard error of replicate assay values was typically Research Centre (Negev, Israel). All other reagents were reagent grade, less than 10% of the mean. _ purchased from commercial sources. ApH measurements. Chromaffin granule membrane vesicles (80 Preparation of membranes. Outdated human platelet concen- _g protein) were incubated at room temperature in a cuvette containing E trates were purchased from the Connecticut Red Cross, Farmington, 2 ml of 10 mM Tris-N-(2-hydroxyethyl)piperazine-N-3-propanesul- _ l_ O. CT. Platelets from 50 to 100 individuals were pooled for each membrane fonic acid (pH 8.5), 150 mM KCI, 6 #M acridine orange, and 5 mM ATP. to preparation. Platelet plasma membrane vesicles were isolated by the The relative fluorescence of the mixture was measured using an exci- method of Barber and Jamieson (11) with the modifications described tation wavelength of 490 nm and an emission wavelength of 526 nm previously (12). Chromaffin granule membrane vesicles were prepared on a Perkin-Elmer LS-bB Luminescence Spectrometer. When the _ s as described by Schuldiner et al. (13) by repeated osmotic lysis of bovine baseline fluorescence stabilized, acidification was initiated by the ad- adrenal medullary chromaffin granules isolated by differential sedi- dition of 6 mM MgS04 and the relative fluorescence was monitored. mentation. Approximately15 minlater, additionsof fenfiuramineweremadeand Binding assays. Imipramine binding was measured at 25° using the increase in fluorescence was measured. After the last addition, the filtration assay described previously (14). Membrane vesicles were NH4CI was added to a final concentration of 10 mM to neutralize the Fig. suspended at a protein concentration of 0.3 mg/mi in an assay buffer vesicle interior and completely reverse the quenching. In replicate tran_ of 200 mM NaCI containing 10 mM lithium phosphate (pH 6.7), 1 mM experiments, the concentration of fenfluramine required for half-max- rate_ MgSO4, and the indicated concentration of [3H]imipramine (19-23 imal reversal of the fluorescence quenching varied by less than 10% of men cpm/fmol). After a 20-min incubation, the reactions (300 _1per assay) the mean value, pres wereterminatedbydilutionwith4-ml,ice-cold,iso-osmoticNaC1and (mca Date filtered through Whatman GF/B filters pretreated with 0.3% polyeth- Results for .' yleneimine. The tube and filter were washed three times with 4 ml of fenfit ice-cold NaC1 solution. Filters were placed in Optifluor (Packard; Fenfiuramine interaction with the plasma membrane pane Downers Grove, IL) and counted after 5 hr. Binding in the absence of Na + or in the presence of 100/_M serotonin was taken as a control for serotonin transporter. Previous results with MDMA (9) and overrami' nonspecific binding. The standard error of replicate assay values was PCA (8) indicated that these amphetamine derivatives inter- abs_ typically less than 5% of the mean. acted directly with the serotonin transporter of platelet plasma pres Measurements of reserpine binding were performed as described membrane as demonstrated by their ability to compete with are (15). Membranes were diluted to a protein concentration of approxi- serotonin for transport and with imipramine for binding. Fur- imip' mately 0.15 mg/ml in a solution containing 0.3 M sucrose, 10 mM K- thermore, these compounds exchange with serotonin in a trans- lathe,tio HEPES (pH 8.5), 2.5 mM MgSO4, 5 mM KCI, 5 mM ATP (where porter-mediated process and are likely to be substrates for the dilut indicated), and 1 nM [3H]reserpine. The mixture was incubated at 37° transporter. To test the generality of this process, we examined con_ and after 30 min (4 hr in samples where ATP was omitted), a 200-_1 the ability of another amphetamine derivative, fenfuramine serc sample of the suspension wasapplied to a 3-mi column of Sephadex G- (N-ethyl-a-methyl-m-(trifluoromethyl)phenethylamine), to in- late( 50 (Pharmacia; Uppsala, Sweden). The column was prepacked in a teract with the serotonin transporter, latio disposable syringe by centrifugation for 90 seconds in a clinical centri- this fuge at 100 x g. After sample application, the column was centrifuged Fig. 1 (left panel) presents data indicating that fenfluramine imer for 2 min at 200 x g and the effluent was assayed for radioactivity,