Emanuela Frittoli

Comparative studies on the anorectic activity of d-fenfluramine in mice, rats, and guinea pigs

SummaryThe present study compares the anorectic activity of d-fenfluramine and its metabolite d-norfenfluramine in three animal species. d-Fenfluramine and d-norfenfluramine show anorectic activity at increasing doses (ED50) in rats, guinea pigs, and mice, d-norfenfluramine being more active than d-fenfluramine in all three species. Equiactive anorectic activities are reached with different brain levels of d-fenfluramine and d-norfenfluramine, guinea pigs being the most sensitive species, followed by rats then mice. The metabolite most probably plays a major role in the anorectic effect of d-fenfluramine in guinea pigs, contributes to the anorectic activity in rats, but adds little to the action of the parent drug in mice. The different sensitivity to d-fenfluramine and d-norfenfluramine in these three species does not appear to be explained by a number of biochemical parameters, including serotonin uptake or release, receptor subtypes, or 3H-d-fenfluramine binding and uptake.

Releasing activities of d-fenfluramine and fluoxetine and fluoxetine on rat hippocampal synaptosomes preloaded with [3H]serotonin

SummaryRat hippocampal synaptosomes preloaded with [3H]serotonin and maintained in a superfusion apparatus were exposed for 3 min to d-fenfluramine or fluoxetine. Both drugs evoked a tritium overflow which was reserpine-sensitive requiring the presence of intact synaptic vesicles. However the two drugs displayed different characteristics: 1) the overflow was immediate with dfenfluramine whereas the releasing activity of fluoxetine showed a delay of about 2 min; 2) d-fenfluramine-induced overflow was already apparent at 0.15 μmol/l whereas the minimal effective concentration of fluoxetine was 2.5 μmol/l. Their concentration-effect curves were differently shaped, the effect of d-fenfluramine being saturable at 5–20 μmol/l (EC50 about 1 gmol/l) while no saturation was observed with fluoxetine up to 10 μmol/l; 3) only 1907o of the tritium overflow evoked by fluoxetine (2.5–10 μmol/l) consisted of true [3H]serotonin, compared with 7001o when 0.5 μmol/l d-fenfluramine was used; 4) the releasing action of 0.5 μmol/l d-fenfluramine was completely Ca++-dependent, while at higher dfenfluramine concentrations the Ca++-independent overflow became more important. The fluoxetine induced overflow was mainly. (70010) Ca++-independent; 5) the releasing acitvity of d-fenfluramine was mainly (80%) blocked by the serotonin uptake blockers indalpine, midalcipram and also fluoxetine whereas fluoxetine-induced overflow was insensitive to inhibition of the serotonin carrier.In conclusion, the releasing activity of d-fenfluramine is already present at a very low concentration (0.5 μmol/l) and at this concentration its mechanism of action was Ca++-dependent, together with the requirement of a functional serotonin carrier. These data therefore do not support the hypothesis of a simple. “displacement” of 5-HT from its storage vesicles but suggest an exocytotic release possibly triggered by interaction of d-fenfluramine with intracellular receptors. A direct releasing activity is also shown for fluoxetine, very marked at 5–10 μmol/l; such effect is different from that of d-fenfluramine and is probably due to the overflow of 5-hydroxyindoleacetic acid, formed in the synaptosomes after the fluoxetine-induced “displacement” of serotonin from its storage vesicles. The active concentrations of fluoxetine on serotonin release are compatible with those found in rat brain at doses inducing an anorectic activity.

Evidence of an exocytotic-like release of [3H]5-hydroxytryptamine induced by d-fenfluramine in rat hippocampal synaptosomes

The monoamine releasing activity of d-fenfluramine was investigated with an in vitro model consisting of synaptosomes preloaded 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 microM) induced only [3H]5-hydroxytryptamine ([3H]5-HT) release from hippocampal synaptosomes whereas 10 microM also induced some overflow from hippocampal synaptosomes preloaded with [3H]noradrenaline or from striatal synaptosomes preloaded with [3H]dopamine, although the overflow was much lower than from 5-HTergic synaptosomes. We then focused on the [3H]5-HT release induced by 0.5 microM d-fenfluramine, which was previously shown to be Ca2+ dependent. The same finding was confirmed in the present study with other experimental protocols, indicating the requirement for extracellular Ca2+ 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-induced [3H]5-HT release was not altered by 1 microM nitrendipine (blocking the L-type Ca2+ channels) but was slightly decreased (20%) by 0.5 microM omega-conotoxin (blocking the N-type Ca2+ channels). It was also inhibited by 0.5 microM clonidine, interacting with alpha 2-adrenergic heteroreceptors, and by 10 nM tetanus toxin, known to affect the exocytosis of different neurotransmitters including 5-HT. These compounds had very similar effects on the Ca(2+)-dependent, exocytotic release of [3H]5-HT induced by depolarization, i.e. by 15 mM K+.(ABSTRACT TRUNCATED AT 250 WORDS)

The modulation of [3H]noradrenaline and [3H]serotonin release from rat brain synaptosomes is not mediated by the α2B-adrenoceptor subtype

SummaryThe present study aimed at relating the presynaptic α2-adrenoceptors, known to modulate noradrenaline and serotonin release, with the recently described α2A- and α2B-adrenoceptor subtypes. The effects of the agonist oxymetazoline (selective for α2A subtype) and of three adrenoceptor antagonists (idazoxan, 1-(2-pyrimidinyl)piperazine (PmP) and prazosin, the last one known to be α2B selective) were evaluated on [3H]noradrenaline and [H]serotonin release in superfused synaptosomes from rat brain cortex. These drugs were also tested in [3H]yohimbine binding to human platelet membranes (containing only α2A receptors) and to neonatal rat lung membranes (containing only α2B receptors).The affinity pattern of these compounds at α2A-adrenoceptors in binding studies was oxymetazoline > = idazoxan > PmP > prazosin; at α2B-adrenoceptors it was idazoxan > = prazosin > PmP = oxymetazoline. Oxymetazoline inhibited with high and similar potencies the K+-evoked [3H]noradrenaline and [3H]serotonin release, IC50 18 and 7 nM, respectively; in the same conditions, the IC50 values of noradrenaline were 42 and 168 nM, respectively. The antagonist affinity pattern (antagonism against noradrenaline) was idazoxan > PmP > prazosin, either on [3H]serotonin release.These results indicate that presynaptic α2 auto- or heteroreceptors do not belong to the α2B subtype and suggest that the modulation of noradrenaline and serotonin release may be mediated by the α2A-adrenoceptor subtype.

Antagonist properties of 1-(2-pyrimidinyl) piperazine at presynaptic α2-adrenoceptors in the rat brain

The effect of 1-(2-pyrimidinyl)piperazine (PmP), the main metabolite of buspirone, was tested on K(+)-evoked [3H]noradrenaline and [3H]serotonin release from superfused synaptosomes obtained from rat cerebral cortex. PmP had no effects per se, but antagonized the effect of noradrenaline, producing a parallel shift to the right of the noradrenaline inhibition curves. From these data pA2 (apparent affinity) values of 6.8 and 7.3 were calculated for PmP on presynaptic alpha 2-adrenoceptors located on noradrenergic and serotonergic nerve terminals, respectively.

In vitro and in vivo effects of the anorectic agent dexfenfluramine on the central serotoninergic neuronal systems of non-human primates. A comparison with the rat

The effects of repeated subcutaneous (s.c) injections of dexfenfluramine (d-F; 10 mg/kg, twice daily, for 4 days) on the contents of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the brain were assessed in primates (cynomolgus and rhesus monkeys) and compared with the regional brain concentrations of unchanged drug and its active metabolite, dexnorfenfluramine (d-NF). This four-day, high-dose, regimen caused a large depletion of 5-HT (more than 95%) and of 5-HIAA (80–90%) in all brain areas studied (cortex, hippocampus, putamen, caudate nucleus and hypothalamus) 2 h after the last injection of d-F. Analysis of the plasma and brain contents of d-F and d-NF confirmed that both compounds were concentrated as in other species, in regions of the primate brain. However, d-NF was concentrated to a greater extent than d-F, and there were differences between the two primate species. Unlike in the rat brain, concentrations of d-NF greatly exceeded those of d-F in the primate brain suggesting that in these primates the d-NF may play a major role in the overall neurochemical response. The effects of d-F and d-NF on different in vitro parameters of serotoninergic neuronal function did not show appreciable differences between cynomolgus or rhesus monkeys when compared to rats, the ability of the two compounds to inhibit 5-HT reuptake, to enhance its release, and to affect the binding of [3H] -d-F or of [3H] -mesulergine (a ligand for 5-HT2c receptors) being similar. Kinetic differences in the disposition of d-F appear to have more relevance than biochemical effects in providing an explanation for the more marked brain depletion induced by d-F in primates than in rodents.

Anorectic effect and brain concentrations of D-fenfluramine in the marmoset: relationship to the in vivo and in vitro effects on serotonergic mechanisms

SummaryThe present study investigated the anorectic activity of d-fenfluramine (d-F) and the relationship with brain levels of unchanged drug and its metabolite d-norfenfluramine (d-NF) in marmosets, relating them to neurochemical effects on the serotoninergic system. d-F and d-NF were equally active in reducing food intake (ED50 about 3 mg/kg, p.o.). However, the brain concentrations of the metabolite required to reduce food intake after synthetic d-NF were more than twice those after d-F, indicating that d-NF contributes to but does not completely explain the anorectic effect of d-F. At this dose d-F did not appreciably modify the serotonin (5-HT) and 5-hydroxyindoleacetic (5-HIAA) contents of the brain regions examined, except for a slight enhancement of 5-HIAA in hippocampus. In vitro in brain cortical synaptosomes d-F inhibited [3H]5-HT uptake more potently than d-NF, as in other species. d-F and d-NF showed similar potency in stimulating [3H]5-HT release, in a Ca++ dependent manner. The tritium released by d-F and d-NF appeared to be mainly unmetabolized [3H]5-HT.Like in other species the marmoset too has saturable and specific [3H]d-F binding sites, for which d-NF has lower affinity. d-F and d-NF have low affinities for 5-HT receptor subtypes, except that d-NF has appreciable affinity for 5-HT1Cand 5-HT1Dreceptors. Unlike in rodents but similarly to primates in the striatum the pharmacology of 5-HT receptors seems to correspond to the 5-HT1D subtype. Brain concentrations of d-F and d-NF at anorectic doses exceeded the concentrations required in vitro to influence the serotoninergic system. Therefore the effect of d-F on food intake might possibly be explained by an interaction with the 5-HT system, particularly uptake and release mechanisms, and that of d-NF by an action on 5-HT1C and 5-HT1D receptor subtypes.

Tetanus toxin inhibits depolarization-induced [3H]serotonin release from rat brain cortex synaptosomes

The effects of tetanus toxin on depolarization-induced [3H]serotonin release from superfused rat brain cortex synaptosomes was investigated. Two hours preincubation of the synaptosomes with tetanus toxin resulted in a concentration-dependent decrease of K(+)-stimulated release, with an IC50 of about 30 nM (4.5 micrograms/ml); this inhibitory effect was blocked by a previous incubation of the tetanus toxin with antitoxin serum. Tetanus toxin had no effect on reserpine-induced release, a model of Ca(2+)-independent release. These results indicate that tetanus toxin is able to alter the exocytotic machinery of serotoninergic terminals, in agreement with results obtained with other neurotransmitters. They also indicate that serotoninergic terminals possess the receptor for tetanus toxin. These findings are in line with in vivo observations suggesting a role for serotoninergic system in tetanus intoxication.

Involvement of P-type Ca2+ channels in the K+- and d-fenfluramine-induced [3H]5-HT release from rat hippocampal synaptosomes

The Ca2(+)-dependent [3H]5-HT release induced by depolarization or by 0.5 microM d-fenfluramine in rat hippocampal synaptosomes, was significantly reduced (35-42%) by three different P-type Ca2+ channels blockers (omega-Agatoxin-IVA, 100 nM, funnel-web spider toxin, FTX, 0.05 microliters/ml, and its synthetic analogue, sFTX, 1 mM), indicating the major role of these channels in the Ca2+ influx preceding neurotransmitter release.