European Journal of Pharmacology, 90 (1983) 293-296
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Elsevier
Short communication I N H I B I T O R Y EFFECT OF 5 - M E T H O X Y - N , N - D I M E T H Y L T R Y P T A M I N E O N T H E S Y N A P T O S O M A L UPTAKE OF 5 - H Y D R O X Y T R Y P T A M I N E O D D - G E I R B E R G E *, D I N A H C H A C H O and K JELL HOLE
Department of Phystology, Umverstty of Bergen, ,4rstadvelen 19, N- 5000 Bergen, Norway Received 28 March 1983, accepted 6 April 1983
O -G BERGE, D CHACHO and K HOLE, Inhtbztory effect of 5-methoxy-N,N-dtmethyltryptamme on the synaptosomal uptake of 5-hydroxytryptamme, European J Pharmacol 90 (1983) 293-296 5-Methoxy-N,N-dlmethyltryptamane (5-MeODMT) in concentratlons of 0 5-500 #M produced a significant lnhtbltlon of [14C]5-hydroxytryptamme ([14C]5-HT) uptake in stnatal, luppocampal and hypothalamlc crude synaptosomes from rat brmn Higher concentrations of 5-MeODMT (10 #M) also lnl'nblted the uptake of [3H]dopamme ([3H]DA) and induced the release of [14C]5-HT and [3H]DA from preloaded synaptosomes It appears that the 5-HT agonist properties of 5-MeODMT may involve reuptake lntUbmon in addmon to the previously documented direct receptor stimulation 5-Methoxy-N,N-dimethyltryptarmne
5-Hydroxytryptanune
1. Introduction In behavioral stu&es, the hallucinogenic substance 5 - m e t h o x y - N , N - & m e t h y l t r y p t a m m e (5M e O D M T ) acts as a 5-hydroxytryptamlne (5-HT) receptor agonlst (Grahame-Smlth, 1971; Fuxe et al., 1972; Jacobs, 1976, Berge, 1982) The drug has proved to be a useful tool for investigating 5-HT mechamsms m the central nervous system. It ~s uncertain, however, whether the 5-HT agonlst properties of the drug are due to direct receptor stimulation only, or if other aspects of serotoner~c funcUons such as the reuptake of transmitter are affected as well (Uebelhack et al., 1978). In the present study, we have investigated the effect of 5 - M e O D M T on uptake of [14C]5-HT m crude synaptosomes prepared from different rat brain structures. For the sake of comparison, we also investigated the effect on the u p t a k e of [3H]dopamme ([3H]DA). Under the conditions of the present experiments, an increase in release may
* To whom all correspondence should be addressed 0014-2999/83/$03 00 © 1983 Elsevier Soence Pubhshers B V
Catecholammes
Synaptosomal uptake
be interpreted as an mhtbltlon of uptake. The release of [14C]5HT and [3H]DA from synaptosomal preparations was therefore studied under the same conditions as the uptake.
2. Materials and methods Male Wlstar rats (Mqllegaard, Denmark) weigtung 250-300 g were lolled by decapitation and the brains rapidly removed. The following structures were dissected out: H i p p o c a m p u s (weight of each. 60-66 rag), stnatum (each: 50-60 mg), and hypothalamus including nelghbourlng tissue (about 150 mg)
2 1 Uptake of [14C]5-HT and [3H]DA mto crude synaptosomal preparattons The brain tissue was homogenized in 10 vol of 0.25 M sucrose, the homogenate centrifuged at 1 000 × g for 10 mln and the sediment &scarded. One hundred #1 of the supernatant and 20 #l of a
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solution of 5 - M e O D M T (Sigma) in concentrations ranging from 0.1 to 500/~M were added to 1.88 ml of a modified Krebs-Rlnger bicarbonate buffer, p H 7.4, containing 0.2 m g / m l ascorblc acid, 20 /~g/ml pargyhne and 465 g / m l EDTA, and saturated with 95% O 2 and 5% CO 2. After premcubatlon of the mixture for 5 mln at 37°C, the isotopes were added [ 14C]5-HT (51.0 m C t / m m o l ) and [3H]DA (15.4 C t / m m o l , The Radlochemlcal Centre, Amersham) were incubated together m a final concentration of 20 nM In prehnunary studies, it was found that accumulation of isotopes under these conditions was linear between 1 and 5 mln. After 5 nun the incubation with isotope was ternunated by coohng, and the tubes were centrifuged at 40000 × g for 15 man The pellets were washed with ice-cold 0.9% NaC1 and dissolved in Protosol (New England Nuclear). The radioactwity was counted in a Packard Trl-Carb 460 CD liquid scintillation system [14 C]5-HT and [3H]DA activities were separated as described by Ross et al (1972) The uptake in the presence of the uptake inhibitor cocaine (0 3 mM) was subtracted from the total uptake
2 2 Release of[14C]5-HTand [3H]DA from strtatal crude synaptosomal preparattons Crude synaptosomal preparattons (prepared as above) were loaded with [14C]5-HT and [3H]DA by incubation with the isotopes (20 nM) for 5 man. After centrlfugatIon at 40 000 × g for 15 m m the supernatant was discarded and the surface of the pellet washed once with ice-cold NaC1 0.9%. Modified Krebs-Rmger bicarbonate buffer (37°C) and various concentrations of 5 - M e O D M T were added to the preloaded pellets. The pellet was then dissolved by agataUon, and remcubated for 5 nun at 37°C After cooling, the tubes were centrifuged at 40000 × g for 15 mln. The pellet was dissolved in Protosol and the radioactwIty measured as described above.
2 3 Stattstwal analysts Statements of statistical significance are based on two-way analysis of variance, at the 5% probabihty level
3. Results
3 1. Uptake in synaptosomal preparattons As shown in fig. 1, 5 - M e O D M T inhibited the uptake of [14C]5-HT in striatal (IC50 2.8 /~M) as well as in hypothalanuc (ICs0 2 2 /~M) preparations. For both structures an lnlubltlOn was observed for 5 - M e O D M T concentrations of 0.5 /~M and higher, increasing to a maximum of about 90% inhibition with increasing concentrations. The uptake of [3H]DA was also inhabited, but to a lesser extent than [14C]5-HT uptake. The inhabttion required higher doses of 5 - M e O D M T (5 /~M or higher) and was considerably weaker in the stnatum (maximum inhibition 31%) than in the hypothalamus (ICs0 12/~M). In happocampal synaptosomes (data not shown) the 5-MeODMT-Induced Inhibition of uptake of [14C]5-HT and [3H]DA was similar to the lnhtbltaon in hypothalamlc synaptosomes shown m fig 1
3 2 Release from synaptosomal preparatwns 5 - M e O D M T in concentrations above 10 # M significantly increased the release of [14C]5-HT and [3H]DA from preloaded striatal synaptosomes (fig. 2). There was no slgmficant difference between the two isotopes.
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Fxg 1 Inlubltlon of uptake into crude synaptosomal preparations by 5 - M e O D M T (3 observations carried out in tnphcate for each point) O O [t4C]5-HT uptake into hypothalamlc synaptosomes, • • [14C]5-HT uptake into stnatal synaptosomes, r, zx [3H]DA uptake into hypothalamxc synaptosomes, • • [ 3H]DA uptake into stnatal synaptosomes
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Fig 2 Release of isotope from stnatal synaptosomes (3 ob-
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4. Discussion The results showed that 5-MeODMT in concentrations of 0 5 /~M and higher inhibited the uptake of [ 14C]5-HT into crude synaptosomal preparations. The susceptibility to inhibition seems to be a general property of 5-HT synaptosomes since the inhibition occurred at the same concentrations and was of similar magnitude in the three structures mvesUgated [3H]DA uptake was also inhibited by 5MeODMT, but only when concentrations of 5/~M and higher were used. Clear regional differences were found as the mhtbltion was considerably lower in the strlatum than in the hypothalamus and hippocampus. The striatum is rich in dopamlnerglc terrmnals but contains almost no noradrenerglc terminals (Starke et al., 1978) while the hlppocampus contains noradrenerglc but few dopaminerglc terminals (Blschoff et al., 1979). The hypothalamus contmns both types of terminals. Since [3H]DA is readily taken up into both kinds of catecholamlnerglc terminals (Snyder and Coyle, 1969), the regional differences found suggest that 5-MeODMT inhibits catecholamlne uptake into noradrenergtc synaptosomes to a greater extent than into dopamlnergic synaptosomes However, since approxamately ten t~mes higher concentrations are required to affect the [3H]DA uptake than the [IaC]5-HT uptake, it seems hkely that the 5-HT uptake inhibition may be of greater functional significance m VlVO
The apparent uptake inhibition could have been partly due to an increase in the release of accumulated labeled amines When the release from preloaded synaptosomes was studied under con&tlons similar to those of the uptake study, it was found that 5-MeODMT increased the release of [14C]5-HT and [3H]DA to approximately the same extent. It should be noted, however, that in the case of [14C]5-HT the concentration of 5M e O D M T required to increase the release was 15 times htgher (10/~M) than the concentration necessary to reduce the uptake. For this amine, the release therefore only contributes a minor part to the apparent uptake intubltlOn. In studies usmg similar methods, we have found that 5-MeODMT in high concentrations inhibits synaptosomal uptake of [14C]chollne to approximately the same extent as [3H]DA uptake in the hypothalamus, whereas [~4C]GABA uptake is almost unaffected (unpubhshed observations) Taken together with the present findings, these observations indicate that 5-MeODMT inhibits 5-HT reuptake with some degree of selectwlty and may therefore enhance the in vivo stimulation of 5-HT receptors by increasing the amount of 5-HT in the synapse However, further stu&es are needed to evaluate the relative importance of this mechanism to the direct 5-HT receptor stimulating properties of the drug documented in previous studies (Berge, 1982).
Acknowledgements This investigation was supported by the Norwegtan Research council for Soence and the Humamtles The authors would like to thank Ms Ida Haugan for expert techmcal assistance
References Berge, O-G, 1982, Effects of 5-HT receptor agonlsts and antagomsts on a reflex response to radiant heat m normal and spinally transected rats, Pare 13, 253 Blschoff, S, B Scattonand J Korf, 1979, Blochermcalevidence for a transmitter role of doparmne m the rat hippocampus, Brain Res 165, 161 Fuxe, K, B Holmstedt and G Jonsson, 1972, Effects of 5-methoxy-N,N-&methyltryptanuneon central monoamme neurons, European J Pharmacol 19, 25
296 Grahame-Srmth, D G , 1971, Inhlbttory effects of chlorpromazme on the syndrome of hyperactwlty produced by L-tryptophan or 5-methoxy-N,N-dlmethyltryptanune in rats treated w~th a monoamlne oxtdase mlubltor, Br J Pharmacol 43, 856 Jacobs, B L, 1976, An ammal behavioral model for studying central serotonerg~c synapses, Life Sol 19, 777 Ross, S B, A L Renyl and S O Ogren, 1972, InhlblUon of the uptake of noradrenahne and 5-hydroxytryptanune by chlorphentermlne and chlorlmlpramme, European J Pharmacol 17, 107
Snyder, S H and J T Coyle, 1969, Reg/onal differences m 3H-norepmephnne and 3H-doparmne uptake into rat brain homogenates, J Pharmacol Exp Ther 165, 78 Starke, K , W Relmann, A Zumstem and G Herttmg, 1978, Effects of dopamme receptor agomsts and antagomsts on release of dopanune m the rabb~t caudate nucleus in wtro, Naunyn-Schrmedeb Arch Pharmacol 305, 27 Uebelhack, R , L Franke and K Seldel, 1978, Wlrkung von LSD und 5-Methoxy-N,N-dlmethyltryptamme auf die active Aufnahme von [3H]-Serotonm m Synaptosomenfraktlonen aus Rattenlurn, Acta Blol Med Germ 37, 1611