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Home , Fenfluramine, Substituted amphetamine, Tryptamine

EuropeanJournalof Pharmacology,215(1992)153-160 153 1992ElsevierSciencePublishersB.V.All rightsreserved0014-2999/92/$05.00

EdP52429

The substituted 3,4-methylenedioxymethamphetamine, , p-chloroamphetamine and fenfiuramine induce 5-hydroxytryptamine release via a common mechanism blocked by and

Urs V. Berger, Xi F. Gu and Efrain C. Azmitia Departmenoft Biology,NewYorkUnicersity,100WashingtonSquareEast,NewYork,NY10003,U.S.A.

Received10October1991,revisedMSreceived5 February 1992,accepted 18February 1992

The abilities of the substituted amphetamines 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine, p-chloro- (PCA) and to induce synaptosomal [3H] (5-HT) release were compared using a novel microassaysystem. The rank order of release potencies was found to be (+)PCA = ( +)-fenfluramine > (+)-MDMA >>( +)- methamphetamine.Combination of two drugs at their ECs0 did not cause more release than either drug alone at an equivalent concentration.In addition, the 5-HT uptake blockers fluoxetine and cocaine inhibited the release induced by MDMA, metharaphetamine,PCA and fenfiuramine to the same percentage. However, threshold concentrations of the substituted amphetaminesknown to inhibit uptake did not attenuate the release caused by higher concentrations of these compounds. These resultssuggests that MDMA, methamphetamine, PCA and fenfiuramine cause 5-HT release via a common mechanism. Farthermore,these results indicate-that the 5-HT uptake blockade induced by these substituted amphetamines in vitro is differentfromthatinducedbyeitherfluoxetineorcocaine. ' a

Amphetamine analogues; Monoamine release; Synaptosomes; 5-HT (5-hydroxytryptamine, serotonin) ._,

I.Introduction phetamines, such as methamphetamine (Ricaurte et ' '

al., 1980; Axt et al., 1990), another drug of abuse; ...... _ 3,4-Methylenedioxymethamphetamine (MDMA), a p-chloroamphetamine (PCA), a very potent serotoner- _ _ .,_o_ derivative, is a drug of abuse gic neurotoxin (Fuller, 1978; Sanders-Bush and Ster ...... _} thathas been subject of recent investigation because of anka, 1978), and the compound fenfluramine _,_-: itsneurotoxic effects in experimental animals (Stone et (Molliver and Molliver, 1988; Appel et al., 1989). The al.,1986; Schmidt, 1987; O'Hearn et al., 1988) and chemical structures of MDMA, PCA, fenfluramine and possiblyin humans (Price et al., 1989). The neurotoxic- methamphetamine are shown in fig. 1. Because of their ityofMDMA consists of a long-lasting degeneration of similar long-term effects on 5-HT axons, these four ser0tonin (5-hydroxytryptamine, 5-HT) axon terminals substituted amphetamines may produce in forebrain regions, as evidenced by decreases in via the same mechanism. levelsof 5-HT and its principal metabolite 5-hydroxyin- A common feature of MDMA, methamphetamine, d01eacetic acid (Stone et al., 1986; Schmidt et al., PCA and fenfluramine is that they cause the release of 1987),decreases in 5-HT synthesis (Stone et al., 1986) 5-HT from presynaptie terminals (Wong et al., 1973; and5-HT uptake activity (Battaglia et al., 1987), and Schmidt and Gibb, 1985; Nichols et al., 1982; Buczko decreases in immunocytoehemical staining of 5-HT ax- et al., 1975). This release is blocked by 5-HT uptake 0ns(O'Hearn et al., 1988; Scalier et al., 1988). The inhibitors (Schmidt and Gibb, 1985; Ross, 1976; Hek- neurotoxic effects of MDMA in animals are similar to matpanah and Peroutka, 1990; Maura et al., 1982; thoseof a number of closely related substituted am- Schmidt et al., 1987), suggesting that it is mediated by the 5-HT uptake carrier. Since the long-term neuro- toxic effects of the substituted amphetamines are also Correspondenceto: E.C.Azmitia,Departmentof Biology,NewYork blocked by 5-HT uptake inhibitors (Azmitia et al., University,100 Washington Square East, New York, NY 10003, 1990; Fuller et al,, 1975; Schmidt, 1987; Hotchkiss and U.S.A.Tel.1.212.9988250, fax 1.212.9954015. . Gibb, 1980), or by other treatments that prevent 5-HT ! - 154

release is the same for each compound. There are studies. Cr O_O i_NI'k,,,CH, CH3 Cl [_N_cHNI_s However, it is unclear whether the mechanism for this with free a( M_ PCs several reports describing differences among the ac. synaptosom, i tions of these substituted amphetamines on serotonin volumes pe_

j[_]_cH3NH'",,,CH3 FaC ]_'_NI_,,,,_CH3 neurons. For example, fenfluramine, but not PCA, is followed by c_ able to inhibit the 5-HT depletion induced by H75/12, second sedi] an agent whose 5-HT-depleting action requires uptake Xg. The P: METH FEN into 5-HT terminals (Fattacini et al., 1991). This find. brain weight

neuronal uptake of H75/12 into serotonin neurons in 1.2, NaHCC C.,x o CH%. vivo.ingsuggestsFurther thatdifferencesfenfluramine,exist butbetweennot PCA,PCAblocksand fenthe- Naasynapto120,someI_ 0 .0 o_C___K_ fluramine in two pharmacological effects that are re. min at 37°C

through 5-HT release (Pawlowski et al., 1980). In the total uptake, j[_/_ NH -''*cl''l_ _C _.0_ garded to result from stimulation, possibly nM). Non-sp moxa_ ca:am flexor reflex test in the spinal cord-transsected rat, the fiuoxetine. T, 5-HT uptake blocker prevents potentiation spun down Fig. l. Chemicalstructuresof MDMA,methamphetamine(METH), by PCA but not that induced by fenfluramine. Simi. buffer and si PCA, fenfluramine (FEN), fluoxetine and cocaine. larly, zimelidine inhibits the in rats in- pellet was re.. u ducedby PCA but not that inducedby fenfluramine, ofKrebsbuf release (Berger et al., 1989), it appears that 5-HT Differences between PCA- and fenfiuramine-induced our release release plays an important role in the sequence of hyperthermia in rats have also been described by Frey dency of the events that lead to neurotoxicity. (1975). experiments, f' Apart from the involvement of the uptake carrier, In the present study, we used a novel microassay pended in a the mechanism bywhich the substituted amphetamines system to investigate whether MDMA, metham, waseither re · cause 5-HT release is not entirely clear. These drugs phetamine, PCA and fenfluramine cause 5-HT release or in which c_ may enter the serotonergic terminal and displace 5-HT via the same mechanism. We first characterized this mM ethylene from its intraneuronal binding sites which may be system, then we compared the 5-HT-releasing action of induced by K J followed by transport of the displaced 5-HT out of the these drugs using three approaches. In the first series first set of exl ,, terminal by the uptake carrier acting in reverse of experiments, we determined whether a combinati0n The releas_ (Sanders-Bush and Martin, 1982). However, it is not of two drugs releases more 5-HT than does either drug of 200 t_l in known how, or in fact whether, the substituted am- alone at an equivalent concentration. We hypothesized mark) that ha J. phetamines enter the terminal. They may be taken up that if these drugs caused release via different mecha- cally, drug eft J by the uptake carrier and/or they may passively diffuse nisms, then a combination of two of them may causea twoplates we] 'f,, through the membrane due to their high lipophilicity, significant higher percentage of release than each one In the firs! r' The release-inhibiting effect of uptake blockers could alone at an equivalent concentration. In a second set of with 150 tzl p j thus be due to either preventing the entry of the experiments, we determined whether 5-HT uptake the releasing_ releasing drug into the terminal or blocking the trans- blockade inhibits the 5-HT-releasing action of the four 20rain in a Ti port of 5-HT out of the terminal (Fuller, 1980). The substituted amphetamines to the same extent. 5-HT was started b_. substituted amphetamines may have some interaction uptake was inhibited using fluoxetine, a specific 5-HT *ames (approx with the uptake carrier because (1) PCA, MDMA and uptake blocker (Wong et al., 1983), and cocaine, a and third set fenfluramine also inhibit the accumulation of 5-HT more general monoamine uptake blocker (Richelson 130 tzl of Kr into synaptosomes or brain slices (Wong et al., 1973; and Pfenning, 1984). Both fluoxetine and cocaine do uptake inhibitc Steele et al., 1987; Belin et al., 1976; Kannengiesser et not contain the amphetamine structure and are not 50/_1 of synap al., 1976; Knapp and Mandell, 1976) and (2) this up- considered substituted amphetamines (fig. 1). Finally, Incubated furtl take inhibiting effect, in particular that of fenflu- in a third series of experiments, we tested whether uptake blocke] ramine, is observed at lower concentrations than the fenfluramine or one of the other substituted am- rain, the relea releasing effect (Carruba et al., 1977; Borroni et al., phetamines could act as an uptake blocker and attenu- releasing drugs 1983; Ross et al., 1977; Kannengiesser et al., 1976; ate the releasing effect of another substituted am- After 20 m: Knapp and Mande!l, 1976).Severalstudies have how- phetamine, spinningthe [ ever been unable to demonstratedirectlythat PCA, Supernatants fenfluramineor MDMAare substratesforuptake(Ross Somalpellet I: and Ask, 1980; Ross, 1976; Belin et al., 1976; Sanders- 2. Materials and methods 100%ethanol radioactivity re Bush and Steranka, 1978; Azmitia, unpublished obser- 2.1. Synaptosomal [3H]5-HT release vations). _tlof ethanol MDMA, methamphetamine, PCA and fenfluramine Male Sprague-Dawley rats (200-300 g, Tac0nic fluidand radio: all appear to cause carrier-mediated 5-HT release. Farms, NY), maintained on a 12-h light/dark cycle liquidscintillati 155 withfree access to food and , were used for the 2.2. Statistical analysis _L studies. Crude whole brain (excluding cerebellum) 'y' synaptosomes were prepared by homogenization in 10 Results are expressed as percent of control of the volumes per brain weight of 0.32 M ice-cold sucrose, radioactivity remaining in the synaptosomes. Each ex- ,d_, followed by a first sedimentation at 350 ×g, and a periment was repeated at least twice. The Sigmaplot second sedimentation of the S1 supernatant at 12 500 4.0 curve-fitting program and the logistic function of ×g. The P2 pellet was resuspended in 10 volumes/g De Lean et al. (1978) were used to fit concentration-re- brainweight of Krebs-Ringer buffer containing (mM): sponse curves and calculate ECso values. Statistical NaCI120, KCI 1.86, CaCI 2 1.2, MgSO4 1.2, KH2PO 4 significance of the release-inhibiting effects of fluoxe- '_-,) 1.2,NaHCO3 25, glucose 11.1 and 0.1. The tine and cocaine was determined on the raw data using synaptosomes were then loaded by incubation for 25 the two-tailed Student's t-test. Differences between the 1 mia at 37°C with [3H]5-HT (final concentration 100 means for the percent inhibition caused by the uptake nM).Non-specific uptake, which accounted for 10% of blockers were tested using one-way analysis of vari- W totaluptake, was determined in presence of 10 -4 M ance. fluoxetine. To terminate the uptake, synaptosomes were spun down for 5 min at 2400 ×g, resuspended in 2.3. Drugs bufferand spun again for 5 min at 2400 × g. The final pelletwas resuspended in 12.5 volumes/g brain weight Drugs were obtained from the following sources: ofKrebs buffer. In initial experiments to characterize (+)-PCA' HCI from Sigma Chemical Company (St. _ ' ourrelease system, we determined the Ca2+ depen- Louis, MO); (+)-fenfluramine · HCI from A.H. Robins ,,J dencyof the release induced by 15 mM KCl. In these Company (Richmond, VA); (+)-MDMA' HCI, (+)- experiments, the final synaptosomal pellet was resus- methamphetamine. HCI and (-)-cocaine' HCl from _ O/ pended in a modified Krebs-Buffer in which CaCl 2 the National Institute of Drug Abuse; fluoxetine · HCl waseither replaced with the same amount of MgSO 4, from Eli Lilly Company (Indianapolis, IN), and [3H]5- orinwhich calcium was chelated by the addition of 1.5 HT (specific activity 25.2 Ci/mmol) from New England . mMethyleneglycoltetraacetate (EGTA). The release Nuclear (Boston, MA). lt5 inducedby KCI was determined as described for the :__, firstset of experiments (see below). '_ 3 The release assay was performed in a final volume 3. Results ' _:_,.A of200 /al in 96-well cell culture plates (Nunc, Den- _q mark)that had been coated with poly-D-. Typi- 3.1. Calcium dependency of KCl-induced release _ cally,drug effects were studied in quadruplicates and ..... _r_, tWOplateswere processed simultaneously. A 20-rain incubation with 15 mM KCI caused 20- . _>_ In the first set of experiments, plates were filled 25% [3H]5-HT release in our system. This potassium ...... with150/al per well of Krebs buffer, with or without stimulated release was Ca2+-dependent. In Ca2+-free ...... _a_ ';_

20therainreleasingin a Titertekdrugs, andmicroplatewere equilibratedincubator. Theat 37°Creleasefor buffera very (CaClsmall 2releasereplaced(93.1by MgSOn),+ 2.4 versus15 mM76.4KCI+ 0.8%causedin _ . "_._} [' wasstarted by adding 50 /al of the loaded synapto- normal buffer, % of corresponding controls + S.E.M.). tl 's0mes(approximately 80/ag of protein). In the second Similarly, in presence of 1.5 mM EGTA, 15 mM KCI andthird set of experiments, plates were filled with caused no release (97.6 + 3.5 versus 80.6 + 1.2% in 130/zi of Krebs buffer, with or without the 5-HT normal buffer). uptakeinhibitors, and again equilibrated at 37°C. Then 50/zlof synaptosomes were added and the trays were 3.2. Release potencies and effects of drug combinations incubatedfurther in order to allow association of the uptakeblockers to the transporter protein. After 10 To determine the potencies of MDMA, metham- rain,the release was started by adding 20 /al of the phetamine, PCA and fenfluramine to cause 5-HT re- releasingdrugs, lease in our system,concentration-responsecurves _, After 20 min at 37°C, release was terminated by were established that consisted of seven points in the spinningthe plates at 6°C for 10 min at 2500xg. range of 10-7-10 -3 M. Representative curves from Supematantswere carefully aspirated and the synapto- one experiment are shown in fig. 2. Table 1 shows s0malpellet blow-dried. Twohundred microliters of mean values, obtained from three to four different l_/o ethanol were added to each well to extract the experiments, of the drug concentrations that caused radioactivityretained in synaptosomes. After 1 h, 150 50% release (ECs0). (+)-PCA and (+)-fenfiuramine glof ethanol extract were added to 3 mi scintillation were very similar in their potency to cause [3H]5-HT

fluidand radioactivity was counted for 1 min (Beckman release (ECs0 around 3 /aM), while (+)-MDMA was. t2 liquidscintillation counter), slightly less (8 /aM), and (+)-methamphetamine con- 256

· tosomesin our system(data not shown).Thus, wefirst loo _2o [ ..... determined those uptake blocker concentrations which I _, loo J- * o· _PcNs would attenuate the release induced by one of the _ 80 | _ · UDUA substituted amphetamines (i.e. PCA), but which would i ! , urm not cause an extensive decrease in counts on their own o . 80 _- These concentrations were found to be around 80 nM _ eo _'_ / _ for fluoxetine and 5 tzM for cocaine, causing a small g 8 eo [- decrease in synaptosomal counts of 15-25% compared -g_ | to control. Subsequently,we determined whetherflu- 40 , · 40_- oxetine or cocaine at these concentrations would atten.

i [ uate the [3H]5-HT-releasing action of all four subst[. 1 _' 2o tuted amphetamines. We tested different concentra, mo

tions of each substituted amphetamine that produced _ B o 5-HT release in the range of 30-60%. In fig. 4, results _ 8o

Fig. 2. Concentration-response curves for the synaptosomal [3H]5- tions which produced a comparable amount of release __ 60 HT release induced by MDMA (filled triangles), metbamphetamine

cles).(openThediamamountonds), PCAoft0-?[3H](filledt0-51O--HT3Druconcesqrelguares)10-seaentrsaet(ordiaionn1d0-[Iainatfenfluraminea e) is expressed(openincir-% of typical70 PCAexperiments.... are shown forMDMAthose concentra-_o l° 40 { _', I of controls of the amount of radioactivity remaining in the synapto- _\. P_ somes after a 20-min incubation at 37°C with the releasing drug at the approximate concentration (abscissa). Control values were in the Fig.4. Attenuation of s range of 20000-25000 counts per rain/well. _o _ 0 _ _h0wreleasetheeffects80nMbYofflu°x_the _atchedbars show their - - -_- - - 0 -._ batedfor 10 rain with th_ siderably less (23 gM) potent. Each drug reduced the so --0- ! 13 of controls of the r_ synaptosomal counts to approximately 10% of control _ _ _er drug treatment. 'I within two orders of magnitude of its ECso. i t -_ -v ...... To test whether the [3H]5-HT-releasing effects were _ 40 -v-- , :0ncentrationsdent'sin izM.t-te,* additive, two drugs were combined at their approxi- mate ECso concentration (3/zM for PCA and fenflu- o _o , , ' ' ' .... ram(ne, 10 /zM for MDMA and methamphetamine) _ _ PC, UDU^_N U_'H Pcs UDOArE. uEr, (about 40-50%). Fit and compared to the effects of each of these drugs -' § alone at ECso and at 2 x ECs0. The results are shown ·_ ,_ 7o ZEN...... 4ETH -- foureachsubstitutedblocked signifami in four separate graphs in fig. 3. Each graph shows the _. _ To determine wh [3H]5-HT-releasing effect of the tested substituted am- _ ' tine and cocaine i phetamine either alone at its ECs0 and at 2 x ECs0, or _' .... 4_- in combination with one of the other drugs. In no case 6o _ · _ amphetamine, we c_ did the combination of two drugs at the ECs0 exceed _ -_- .... t- i ti0ntrationfor andeachdetermirtested the releasing effect of the single drugs at 2 × ECs0. so - - -0- -_- _ _ /seven experiments

3.3. Effects of 5-HT uptake blockade using fluoxetine 40 -_- -_- -_ - - - _ ccaine,ocainetableand 802). nMTh< and cocaine 0ne-way analysis ol

At high concentrations, both fluoxetine and cocaine 30 . , ..... cause a decrease in the [3H]5-HT content of the synap- ,CA _0U^_, urrH .c^ uouA_, _ Fig. 3. Release of [3H]5-HT from preloaded synaptosomes inducec _ABLE2 TABLE I by the substituted amphetamines alone or in combinations of ts0 gleanvalues (+_S.E.M.) expressed in % of controls of the radioactivity remaining in synaplc* :mphetamine-induced re Mean ECs0 values (4.S.EM.) for the synaptosomal [3HI5-HT re- somes (ordinate) after a 20-rain incubation period. Each drug_a,. %e-wayanalysis of var lease induced by the four substituted amphetamines. The data from used at the approximate ECso concentration (3 lzM for PCAand : _tweenthese values. n = 3-4 experiments were combined and the ECso values were calculated using Sigmaplot 4.0 and the logistic function of De Lean circlesfenfluramine,show the10effects/_M forof theMDMAdrugs aloneand methamphetamine).at their ECs0 v_hieOpes0_::.'. Flu et al. (1978). triangles show their effects in combination with the test drug thatt, _lD_--_ 10., Isomer ECso (/.tM) n indicated in the upper left corner. The two dashed lines indicatethe Slethamphetamine 9. values for the test drugs at ECso and at 2× ECs0. The valuesf0t_i _2& 11. Fenfiuramine + 2.924.0.35 3 ;. . PCA 4- 3.43 + 0.45 4 drug combinations fall either in between the two dashed lines,orare .,nfiuramme 10. MDMA + 7.964.1.77 4 not significantly different from them, indicating that the combinatioe ----- Methamphetamine + 23.634.6.76 3 of two drugs doesalonenotat releasean equivalentmore concentration.5-HT than each of the dru._, ''_bstituted% inhibitionamphetamine.of release, 157

100 aus, we first mo ttionswhich A

which would _oo * ** _ 80 ,ntheirown. _ ** · § aund 80 nM _ 60 singa small _ 60 % compared whetherwouldatten-flu- ,0 _r.s0)r.. O) uou^(5) ..t, 0o, _i _1 _1 _l four substi- I nl 40 !10.4lia 10.114 a I(i0.5 8 I 115 20 PCA Fen MDMA Meth tat produced B t concentra- mo1 ] Fig. 5. Inabilityof prior exposure to a threshold concentrationof fig. 4, results _u 80**_ fenfluramine(Fen, 0.1 t.*M)to block5-HT release inducedby PCA, nt of release _ ** * *** tions (/_M). Ordinate: % of controlsof radioactivityremaining in 60 synaptosomesafter drug treatment. Open bars showthe effects of the releasingdrugs on controlsynaptosomes;hatched barsshowtheir _. effects on synaptosomes that were preincubated for 10rain with 0.1 e concentra-' ' _ 4o_** MDMA or methamphetaminetzMfenfluramine,(Meth) at the indicatedconcentra- i___. PCA (1) Fen (3) MDMA (5) Meth (20) Fig.4.Attenuationof substitutedamphetamine-induc[3edH]5-l-r[' releaseby 80 nM fluoxetine(A) or 5 p.M cocaine (B). Open bars showthe effectsof the releasing drugs on control synaptosomes; significant differences between the four substituted hatchedbarsshowtheir effectson synaptosomesthat were preincu- amphetamines. 4- 0 hatedfor 10minwiththe uptake blocker.The releaseisexpressedin %of controlsof the radioactivityremaining in the synaptosomes 3.4. Effects of 5-HT uptake blockade using substituted -v afterdrug treatment. The numbers in parentheses indicate drug mcentrationsin gM. * P < 0.05, ** P < 0.01, * **P < 0.001,Stu- amphetamines dent's t-test,performedon the raw data. In this experiment, we tested whether concentra- , , , tionsof substitutedamphetaminesthat blockeduptake MDMFENA Mm (about40-50%). Fluoxetine, 80 nM, and 5 gM Cocaine would be able to attenuate the releasing effects of one eachblocked significantly the releasing action of all of these drugs. The synaptosomes were first preincu-

, , - fourTosubstituteddetermine amphetamines,whether the blocking effect of fluoxe- batedat a concentrationwith one of onthethefourthresholdsubstitutedof causingamphetaminesrelease - - 4- o- tine and cocaine is the same for each substituted but within the range for producing uptake inhibition: amphetamine, we calculated the percentage of inhibi- 0.1/aM for PCA, fenfluramine and MDMA, and I/am " _ ti0nfor each tested substituted amphetamine concen- for methamphetamine. After 10 min, the same drug or _ationand determined the means over all experiments one of the other substituted amphetamines was added (sevenexperiments for fluoxetine and three for co- at a concentration causing 25 or 50% release (0.4 and 3 nine, table 2). The blocking effects of both 5 /aM /aM for PCA and fenfluramine, 0.5 and 8 /aM for cocaineand 80 nM fluoxetine were* between 9-12%. MDMA, and 5 and 20 /aM for methamphetamine). 0ne-way analysis of these values did not reveal any Figure 5 shows the results for fenfluramine. Prior ex- posure to a threshold concentration of fenfluramine IdDkFEN4A IdEr}'l did not cause attenuation of release caused by subse- ptosomesinduced TABLE2 quent administration of fenfluramine at a higher con- centration. Likewise, combination of low fenfluramine abinationsof two: Meanvalues(+S.E.M.) for the percent inhibition of substituted _aininginsynapto- _al_hetamine-inducedrelease caused by fiuoxetine and cocaine, with high PGA, MDMA or methamphetamine also did d. Each drugwas 0_c-waanalysiy sof variance revealed no significantdifferences not cause attenuation of release (fig. 5). MDMA, txM for PCA and _tweenthesevalues, methamphetamine and PeA were similarly unable to phetamine).Open b block the releasing effect of itself or the other substi- · EC5o, whilesolid Fluoxetinea n Cocainea n b _etest drugthatis _'_ 10.05+1.51 7 9.06+ 1.13 8 tuted amphetamines (data not shown). We then re- t linesindicatethe _{ethamphetamine9.93± 1.53 7 11.78-t-1.13 5 peated these experiments using slightly higher pre-in- The valuesforall _CA 11.46±1.22 12 11.09±1.59 9 cubation concentrations of the drugs: 0.3/aM for PeA lashedlines,oran: kntluramine 10.23±0.86 II 11.39+2.15 8 and fenfluramine, 0.5/aM for MDMA, and 5/aM for at the combination each of the drugs '%_ion of release, b number of data points tested for each methamphetamine. These concentrations, which caused on. _titatedamphetamine. 15-25% release on their own, were equally unable to 158 attenuate the release induced by subsequent adminis- PCA and fenfluramine display the same rank order in however, in agre tration of higher concentrations of the substituted am- their affinity to the 5-HT uptake carrier (Poblete et al.. found that pretr phetamines (data not shown). 1989, and unpublished observation) as in their poten, blockthe 5-HT-( ciesforrelease. 1988). In the second set of experiments, we determined The finding t 4. Discussion whether blockade of the 5-HT uptake carrier would contrast to fluo_ block similarly the release caused by all four substi, the5-HT release In this study, we compared directly the release of tuted amphetamines. The results show that 5 gM lngeffects of the [3H]5-HT from preloaded synaptosomes induced by cocaine and 80 nM fluoxetine block the release in. entfrom those c the substituted amphetamines MDMA, metham- duced by each of the four substituted amphetaminest0 that the substitt phetamine, PCA and fenfluramine. For this purpose, approximately the same extent, i.e. 9-12% (table 2). the 5-HT uptak we used a novel microassay system in which the release This capacity of the two uptake blockers to inhibit the hibitory effects t was determined in 96-well tissue culture plates in a release induced by all four drugs to the same percent, uptake carrier. total volume of 200 /xl. In agreement with earlier age suggests that the uptake carrier is involved to the may inhibit the studies, we could demonstrate that all four drugs cause same extent in the 5-HT-releasing action of these drugs, carrier on the ol [3H]5-HT release and that the release is blocked by The relatively low values for the percent release inhibi, this notion is th 5-HT uptake inhibitors. Also, we found ECso values tion are due to the low concentrations of uptake block- substrate for for the release that are in the same range or lower than ers used. In additional experiments, we observed a (B6nisch _nd T1 those reported in earlier studies (Ross, 1976; Kan- more pronounced inhibition with higher uptake blocker caine andxfluo_ nengiesser et al., 1976; Borroni et al., 1983; Ross et al., concentrations. Yet, at those higher concentrations, amphetamine c 1977; Knapp and Mandell, 1976; Schmidt et al., 1987; cocaine and fluoxetine caused also a greater reduction bulkier molecuh Tessel and Rutledge, 1976). Only two recent studies in synaptosomal [3H]5-HT contents on their own, which and are less lik (McKenna et al., 1991; Hekmatpanah and Peroutka, obscured the blocking effect on the release induced by ever, direct exp_ 1990) have described ECso values that are about one- low substituted amphetamine concentrations. Whether amphetamines a tenth of ours. This discrepancy may be due to differ- this release induced by uptake blockers is genuine, as stilllacking (Ro: ences in assay protocols. A very low concentration of suggested by Peyer et al. (1982), or whether it is the Bush and Stera [3H]5-HT was used (9 nM) in these studies, and the result of inhibiting the reuptake of spontaneously re- unpublished ob_ synaptosomes were not washed after the loading step. leased [3H]5-HT needs to be explored in further stud- fore required t Furthermore, the KCl-induced [3H]5-HT release in ies. inhibition cause these two studies was not Ca2+-dependent (see Finally, we investigated whether any of the four tine and cocain_ McKenna et al., 1991), whereas in our system the substituted amphetamines which are known to act as In conclusior KCl-induced release showed Ca e+ dependency. The uptake blocker, could attenuate the releasing effect of we could not de validity of our assay system to study 5-HT release is the other drugs. The same protocol with a preincuba- ing action of tl_ further indicated by the finding that the rank order of tion period was used that could demonstrate the block- therefore prop_ release potencies found in the present study, i.e. (+)- lng activity of cocaine and fluoxetine. In contrast to release via the PCA = (+)-fenfluramine,> (+)-MDMA :_ (+)-meth- cocaine and fluoxetine, the substituted amphetamines the reported d amphetamine (table 1), is very similar to the one found themselves, when used at concentrations on the thres- ramine in some by McKenna et al. (1991). Additional confirmation is hold of causing release, could not block the release 1980;Frey, 197,' that in our system a decreased content in serotonergic induced by another substituted amphetamine (fig. 5 5-HT release b synaptosomes (obtained by in vivo treatment with PCA) and other data not shown). It is feasible that these tions on 5-HT reduces the PCA-induced [3H]5-HT release (data not threshold concentrations used for the preincubati0n terns. Since the shown), may havebeen too lowto cause a marked inhibitionof neuronsmaybe Three sets of experiments were performed in order 5-HT uptake. However, when we used higher concen- al., 1990; Berg_ to determine whether differences exist in the way the trations of the substituted amphetamines (that caused tested substitu four substituted amphetamines cause 5-HT release about 15-25% release and that should have caused mechanism of [ from presynaptic terminals. In the first set of experi- inhibition of uptake), we still could not detect attenua- ments, we found that the combination of two of the tion of release. Thus, in this assay system, we did not substituted amphetamines at their approximate ECs0 observe a protective effect that could be attributed to did not cause more 5-HT release than either drug the uptake inhibitory activity of the substituted am- Acknowledgem_ alone at the equivalent concentration, i.e. at 2 x ECs0 phetamines observed in in vitro studies (Wong et al., (fig. 3). This result suggests that all of these substituted 1973; Steele et al., 1987; Belin et al., 1976; Kan- This work was amphetamines are interchangeable with one another nengiesser et al., 1976; Knapp and Mandell, 1976). postdoctoralfello_ when causing release. In other words, these drugs may Hence, we could not confirm whether there are differ- Foundation.We th compete for the same site on the serotonergic terminal, ences in the uptake inhibitory effect between PCA and manuscript. This site may in fact be the 5-HT uptake carrier itself, fenfluramine, as has been suggested based on an in since we have found that MDMA, methamphetamine, vivo study (Fattacini et al., 1991). Our results are, 159 rder in however,in agreement with another in vivo study that References :et al., f0_ndthat pretreatment with fenfluramine could not poten- blockthe 5-HT-depleting effects of PCA (Fuller et al., Appel, N.M.,J. Contrera and E.B. De Souza, 1989,Fenfluramine 1988). selectively and differentially decreases the density of serotonergic trained The finding that the substituted amphetamines, in nerveterminals in rat brain:evidencefrom immunocytochemical would contrastto fluoxetine and cocaine, did not attenuate studies, J. Pharmacol. Exp. Ther. 249, 928. substi- the5-HT release suggests that the 5-HT uptake block- Axtevidence, K.J.,T.M.for Teunedegenerationand M.E.of serotoneMolliver,rgic 1990,axonsDirectfollowingmorphologicadmin- 5 /.tM lngeffects of the substituted amphetamines are differ- istration of amphetamine derivatives: dl-p-chloroamphetamine, ase in- entfrom those of fluoxetine and cocaine. It is possible d-methamphetamine,and d- and I-fenfluramine,Soc. Neurosci. _inesto thatthe substituted amphetamines are substrates for Abstr.16, 1032. ble 2). the5-HT uptake carrier and cause their uptake in- Azmitia, E.C., R.B. Murphy and P.M. Whitaker-Azmitia,1990, ibit the hbitoryeffects through competition with 5-HT for the MDMA (ecstasy) effectson cultured serotonergic neurons: evi- dence for Ca _ +)-dependent toxicity linked to release, Brain Res. ercent- uptakecarrier. Cocaine and fluoxetine, in contrast, 510, 97. to the mayinhibit the 5-HT uptake by blocking the uptake Battaglia, G.,S.Y.Yeh, E. O'Hearn, M.E. Molliver,M.J.Kuharand : drugs· carrieron the outside of the membrane. In support of E.B. De Souza, 1987, 3,4-Methylenedioxymethamphetamine and inhibi- thisnotion is the finding that amphetamine itself is a 3,4-methylenedioxyamphetamine destroy serotonin terminals in · block- substrate for the uptake carrier rat brain: Quantification of neurodegeneration by measurement of 13H]-labeled serotonin uptake sites, J. Pharmacol. rved a {B6nischand Trendelenburg, 1988).Furthermore, co- Exp.Ther.242,911. f ,. vlocker caineand fluoxetine, neither of which contain the Belin, M.F., J.C. Kouyoumdjian, J. Bardakdjian, J. Duhault and P. _ rations, amphetamine core structure (fig. 1), appear to be Gonnard, 1976, Effects of fenfluramine on accumulation of 5-hy- , :luction bulkiermolecules than the substituted amphetamines droxytryptamine and other neurotransmittersinto synaptosomes , which andare less likely to be substrates for uptake. How- of rat brain, Neuropharmacology 15,613. Berger, U.V., R. Grzanna and M.E. Molliver, 1989, Depletion of _ced by ever,direct experimental evidence that the substituted serotonin using p-chlorophenylalanine (PCPA) and /hether _mphetamines are indeed substrates of 5-HT uptake is protects against the neurotoxiceffects of p-chloroamphetamine Line,as stilllacking(Ross and Ask, 1980; Ross, 1976; Sanders- (PCA)inthe brain,Exp.Neurol.103,111. _.is the Bushand Steranka, 1978; Belin et al., 1976; Azmitia, B6nisch,H. and U. Trendelenburg,1989,The mechanismof action

_sly re- unpublishedobservations). Further studies are there- oI,f eds.indirectlyU. Trendelenburgacting sympathomimeticand N. Weineramines,(Springer-Verlag,in: CatecholaminesBerlin) _'_' :r stud- forerequired to compare the nature of the uptake p. 247. inhibitioncaused by substituted amphetamines, fluoxe- Borroni, E., A. Ceci, S. Garattini and T. Mennini, 1983, Differences le four tineand cocaine in vitro, betweend-fenfluramineand d-norfenfluraminein serotonin , act as In conclusion, in all three experimental paradigms presynaptic mechanisms, J. Neurochem.40, 891. ffect of wecould not detect any differences in the 5-HT-releas- Buczko, W., G. De Gaetano and S. Garattini, 1975, Effect of fenfluramine on 5-hydroxytryptamine uptake and release by rat incuba- lngaction of the four substituted amphetamines. We blood platelets, Br. J. Pharmacol. 53, 563. .f _ :, : block- therefore propose that these drugs all cause 5-HT Carruba, M.O., G.B. Picotti, F. Zambotti and P. Mantegazza, 1977, :.... _rast to releasevia the same mechanism. We hypothesize that Effect of , fenfluramineand chlorimipramine on the _ _s_-_ _amines thereported differences between PeA and fenflu- 5-hydroxytryptamine uptake and storage mechanisms in rat brain: _'_s : thres- faminein some experimental tests (Pawlowski et al., similaritiesand differences,Naunyn-Schmiedeb. Arch. Pharma- _---_,w,-_ col. 300, 227. release 1980;Frey, 1975) are not due to a difference in causing De Lean, A., P.J. Munson and D. Robbard, 1978,Simultaneous (fig. 5 5-HTrelease but may have been caused by drug ac- analysis of familiesof sigmoidal curves: applicationto bioassay, _t these li0nson 5-aT receptors or on other transmitter sys- radioligand assay,and physiological dose-response curves,Am.3. ubation terns.Since the toxicity of PCA and MDMA on 5-HT Physiol.235,E97. ,ition of neuronsmay be related to release of5-HT (Azmitia et Fattaccini, C.M., H. Gozlan and M. Hamon, 1991, Differential effects of d-fenfiuramine and p-chloroamphetamine on H75/ concen- _1.,1990; Berger et al., 1989), we speculate that the 12-induced depletion of 5-hydroxytryptamineand in caused tested substituted amphetamines share a common the rat brain, Neuropharmacology30, 15. caused mechanismof producing neurotoxicity. Frey, H.H.. 1975, Hyperthermia induced by amphetamine, p-chloro- Lttenua- amphetamineand fenfiuraminein the rat, Eur. J. Pharmacol.13, didnot 163. ,utedto Fuller, R.W., 1978,Structure-activityrelationshipsamongthe halo- ed am- &knowledgements genated amphetamines, Ann. N.Y. Acad. Sci. 12, 147. Fuller, R.W., 1980, Mechanism by which uptake inhibitors antago- ; et al.. nize p-chloroamphetamine-induced depletion of brain serotonin, _; Kan- This work was supported by NIDA Contract 271-96-7403 and a Neurochem. Res. 5, 241. 1976). postdoctoralfellowship to U.V.B from the Swiss National Science Fuller, R.W., K.W. Perry and B.B. 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