European Journal of Pharmacology, 238 (1993) 9-17 9 © 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00 EJP 53142 Evidence of an exocytotic-like release of [3H]5-hydroxytryptamine induced by d-fenfluramine in rat hippocampal synaptosomes Marco Gobbi, Emanuela Frittoli, Angela Uslenghi and Tiziana Mennini lstituto di Ricerche Farmacologiche 'Mario Negri', Via Eritrea 62, 20157 Milan, Italy Received 30 December 1992, accepted 6 April 1993 The monoamine releasing activity of d-fenfluraminc was investigated with an in vitro model consisting of synaptosomes preloadcd with the 3H-neurotransmitter and extensively washed in a superfusion apparatus before a 3-min exposure to d-fenfluramine. With this model, the drug-induced release is real and is not confused by inhibition of reuptake by the drug. d-Fenfluramine (0.5/~M) induced only [3H]5-hydroxytryptamine ([3H]5-HT) releasc from hippocampal synaptosomes whereas 10 tzM also induced some overflow from hippocampal synaptosomes preloaded with [3H]noradrenaline or from striatal synapto- somes preloaded with [3H]dopamine, although the overfow was much lower than from 5-HTergic synaptosomes. We then focused on the [3H]5-HT release induced by 0.5 /~M d-fenfluraminc, which was previously shown to be Ca 2+ dependent. The same finding was confirmed in the present study with other experimental protocols, indicating the requirement for cxtracellular Ca 2 ~ ions. By measuring [3H]5-HT uptake into rat hippocampal synaptosomes we confirmed that Ca 2+-ions are not required for the function of the 5-HT uptake carrier or for its interaction with d-fenfluramine, d-Fenfluramine-induccd [3H]5-HT release was not altered by 1 p.M nitrcndipine (blocking the L-type Ca 2+ channels) but was slightly decreased (20%) by 0.5/~M to-conotoxin (blocking the N-type Ca 2+ channels). It was also inhibited by 0.5 ~zM clonidine, interacting with aa-adrencrgic heteroreceptors, and by 10 nM tetanus toxin, known to affect the exocytosis of different neurotransmittcrs including 5-HT. These compounds had very similar effects on the Ca 2 Ldependent, exocytotic release of [3H]5-HT induced by depolarization, i.e. by 15 mM K +. These data suggest that d-fenfluramine, after its carrier-mediated entry into the synaptosomcs, induces an influx of extracellular Ca 2 ~, triggering an exocytotic-like release of 5-HT. 5-HT (5-hydroxytryptamine, serotonin); 5-HT release; Synaptosomes; d-Fenfluramine; Ca 2 + channels I. Introduction d-Fenfluramine is an anorectic compound whose mechanism of action appears to be related to a selec- tive interaction with the 5-hydroxytryptaminergic sys- tem in the central nervous system (see review by Garat- tini et al., 1989). This interaction is supported by data showing that d-fenfluramine inhibits 5-hydroxytrypta- mine (5-HT) uptake (Garattini et ai., 1979; Borroni et al., 1983; Mennini et al., 1985) and induces 5-HT release from nerve terminals (Mennini et al., 1981a; 1985; Langer and Moret, 1982; Maura et al., 1982), thus acting as an indirect 5-HT agonist. The IC50 of d-fenfluramine for inhibiting [3H]5-HT uptake in vitro in rat brain synaptosomes ranges from 0.5 to 1 /~M. Recent data, obtained with rabbit platelets, indicate that the mechanism is different from that of the classic Correspondence to: M. Gobbi, Istituto di Ricerche Farmacologiche 'Mario Negri', Via Eritrea 62, 20157 Milano, Italy. 5-HT uptake inhibitors (i.e. imipraminc, citalopram, fluoxetine) since d-fenfluramine appears to behave as a competitive substrate at the 5-HT uptake carrier (W61fel and Graefe, 1992). The molecular mechanism involved in d-fen- fluramine-induced 5-HT release is still poorly defined. In vitro studies with superfused rat brain synaptosomes showed that d-fenfluramine released 5-HT from a vesicular pool (Mennini et al., 1981a; Borroni et al., 1983) and that the release was inhibited in the pres- ence of 5-HT uptake inhibitors (Maura et al., 1982; Gobbi et al., 1992). To date the only experimentally supported explanation of this finding was proposed by Maura et al. (1982). They proposed that fenfluramine, tested at 5 /~M, enters the nerve ending mainly by diffusion and displaces 5-HT from the synaptic vesi- cles, which then exit by a carrier-mediated outward transport. This is consistent with data showing that the release induced by a relatively high concentration of d-fenfluramine, 10 /xM, is Ca2+-independent (Langer and Moret, 1982; Gandhi and Jones, 1990).