In vitro studies on the mechanism by which (+)-norfenfluramine induces serotonin and dopamine release from the vesicular storage pool

(+)-Norfenfluramine is the main metabolite of the serotoninergic anorectic agent (+)-fenfluramine. Both compounds inhibit 5-HT reuptake and stimulate its release, although they induce release from different pools, with (+)-norfenfluramine acting primarily on the cytoplasmic pool. Moreover, (+)-norfenfluramine was more potent than the parent drug in inducing dopamine release. In order to investigate whether (+)-norfenfluramine induces a Ca2+-dependent vesicular release, like some amphetamine derivatives, in the present study we preloaded synaptosomes with the [3H]neurotransmitter ([3H]5-HT or [3H]dopamine), superfused (washed) them for 47 min in the absence of pargyline and then exposed them to the releasing stimulus. With this protocol, the cytoplasmic pool should be absent and the [3H]neurotransmitter should mainly be stored in synaptic vesicles, where (+)-norfenfluramine should act to induce release. This was confirmed by a significant decrease of (+)-norfenfluramine-induced [3H]5-HT and [3H]dopamine release after reserpine pretreatment. The dose-response curves of (+)-norfenfluramine-induced [3H]5-HT release were superimposable in hippocampus and hypothalamus, and also superimposable on the curve for (+)-fenfluramine-induced [3H]5-HT release; the dopamine releasing potency of (+)-norfenfluramine in the striatum was more than ten times lower. The [3H]5-HT release induced by (+)-norfenfluramine was partly (about 50%) but significantly Ca2+-dependent, and it was also markedly (68%) inhibited by Cd2+, a non-specific blocker of voltage-dependent Ca2+ channels, suggesting that the Ca2+-dependent release is mediated by entry of Ca2+ into the synaptosomes through these channels. The [3H]dopamine release induced by 5 microM (+)-norfenfluramine was completely Ca2+-independent whereas at higher concentrations (10 and 20 microM) it was only slightly (20%) Ca2+-dependent. We have no clear explanation why (+)-norfenfluramine has these different effects on serotoninergic and dopaminergic synaptosomes.