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Clinical data indicate that hydroalcoholic extracts of Hypericum perforatum might be as valuable as conventional antidepressants in mild-to-moderate depression, with fewer side effects. One clinical trial using two extracts with different hyperforin contents indicated it as the main active principle responsible for the antidepressant activity. Behavioural models in rodents confirm the antidepressant-like effect of Hypericum extracts and also of pure hyperforin and hypericin. A hydroalcoholic extract lacking hyperforin also lacks the antidepressant-like effect. According to pharmacokinetic data and binding studies, it appears that the antidepressant effect of Hypericum extract is unlikely be due to an interaction of hypericin with central neurotransmitter receptors. The main in vitro effects of hyperforin (at concentrations of 0.1-1 microM) are non-specific presynaptic effects, resulting in the non-selective inhibition of the uptake of many neurotransmitters, and the interaction with dopamine D1 and opioid receptors. However, it is still not clear whether these mechanisms can be activated in vivo, since after administration of Hypericum extract brain concentrations of hyperforin are well below those active in vitro. In the rat, Hypericum extract might indirectly activate sigma receptors in vivo (through the formation of an unknown metabolite or production of an endogenous ligand), suggesting a new target for its antidepressant effects.

Stable d-Fenfluramine binding activity was obtained in high yields, in cholate extracts of bovine cerebral cortex crude membrane preparations. Dissociation constant (Kd 17 nM), stereoselectivity and the rank order of potencies of various serotonin uptake inhibitors were similar to those measured in native membranes. The inhibitory effect of Na+ ions was also maintained in the soluble state, since the presence of 100 mM Na+ leads to an even greater reduction of the binding than in membrane-associated binding sites. Photoaffinity labeling of soluble binding sites with p-[125I]d-Fenfluramine has led to the identification of a single specific band of molecular weight around 40-50 kDa. This suggests that d-Fenfluramine binding sites are separate molecular entities from the serotonin transporter, that belongs to a family of integral membrane proteins of 68-73 kDa molecular weight.

Synaptosomes from rat brain cortex and spinal cord were preloaded with [ 3 H]serotonin ( 3 H]5-HT), super/fused and exposed to fluoxetine and/or 15 mM K + . In both regions 10 μM, but not 1 μM fluoxetine evoked a marked tritium overflow, about 2 min later than the immediate [ 3 H]5-HT release induced by K + , and mainly (73%) due to the efflux of a tritiated metabolite of 5-HT, possibly [ 3 H]5-hydroxy-indoleacetic acid. These findings confirm previous data in the rat hippocampus and are probably due to fluoxetine interacting with the 5-HT storage vesicles. One μM fluoxetine significantly reduced the d-fenfluramine-induced [ 3 H]5-HT overflow, in accordance with its action as 5-HT uptake blocker, but did not affect the K + -induced [ 3 H]5-HT overflow. This latter finding does not confirm that fluoxetine inhibits the depolarization-induced Ca 2+ -influx, suggested to involve a drug interaction with the L-type Ca 2+ -channels. Thus, the overflow induced by 10 μM fluoxetine was additive with the depolarization-induced overflow, when the two stimuli were applied together. When 10 μM fluoxetine was added 7 min before 15 mM K + , there was no depolarization-induced overflow. Such inhibition might be only apparent and due either to the fluoxetine-induced loss of vesicular 5-HT or to a fluoxetine-induced alteration of synaptic vesicles. The in vivo relevance of the fluoxetine releasing effect remains to be assessed.

Serotonin (5-HT) added in vitro (10 microM) increased [3H]-glutamate specific binding in the rat hippocampus, reaching statistical significance in layers rich in N-Methyl-D-Aspartate sensitive glutamate receptors. This effect was explained by a significant increase in the apparent affinity of [3H]-glutamate when 5-HT is added in vitro. Two days after lesion of serotonergic afferents to the hippocampus with 5,7-Dihydroxytryptamine [3H]-glutamate binding was significantly decreased in the CA3 region and stratum lacunosum moleculare of the hippocampus, this reduction being reversed by in vitro addition of 10 microM 5-HT. The decrease observed is due to a significant reduction of quisqualate-insensitive (radiatum CA3) and kainate receptors (strata oriens, radiatum, pyramidal of CA3). Five days after lesion [3H]-glutamate binding increased significantly in the CA3 region of the hippocampus but was not different from sham animals in the other hippocampal layers. Two weeks after lesion [3H]-glutamate binding to quisqualate-insensitive receptors was increased in all the hippocampal layers, while kainate and quisqualate-sensitive receptors were not affected. These data are consistent with the possibility that 5-HT is a direct positive modulator of glutamate receptor subtypes.

Platelets of Spontaneous Hypertensive (SH) rats show a significant reduction in serotonin uptake, compared with that of rats with normal blood pressure. The difference was due to the Vmax, which was about 40% of normals, while the Km values were almost constant. The possible analogy between blood platelets and serotonergic nerve terminals in SH rats is considered in the evaluation of the results.

100 μM guanine nucleotide Gpp (NH)p reduces the affinity of the serotonergic antagonist metergoline for 3 H-5HT binding sites in rat cerebral cortex. This effect is present both in inhibition binding and in saturation experiments. The hypothesis that the interaction of some serotonergic antagonists with 3 H-5HT binding sites is regulated by guanine nucleotides is discussed.

In the present paper we confirm and extend previous studies showing heterogeneous 3H-imipramine (3H-IMI) binding sites. Inhibition curves of various drugs (serotonin, imipramine, desmethyl-imipramine, d-fenfluramine, d-norfenfluramine and indalpine, a potent serotonin uptake inhibitor) obtained using 2 nM 3H-IMI and in presence of 120 mM NaCl, confirmed the presence of at least three 3H-IMI binding sites: two of these (high and low affinities) were serotonin-insensitive while the third one was selectively inhibited by serotonin and indalpine with nanomolar affinities. Moreover, this last component was found to be selectively modulated by chronic imipramine treatment thus suggesting a closer relation to serotonin uptake mechanism. These data indicate that the use of a more selective inhibitors of the serotonin-sensitive component (like indalpine or serotonin itself) to define non specific 3H-IMI, may be of help in understanding its relation with serotonin uptake system.

Premazepam (PRZ) in vitro competitively displaced 3H-diazepam (DIA), 3H-flunitrazepam (FLU) and 3H-RO 15-1788 from their binding sites on rat brain synaptosomes, with a potency intermediate to other benzodiazepines (BDZs), and Hill coefficients near 1 in different brain regions. Incubation at 37 degrees C reduced premazepam's affinity for BDZ receptors to a lower extent than other benzodiazepines and had no effect on the Hill coefficient. The IC50 of PRZ on 3H-RO 15-1788 and 3H-FLU binding was markedly reduced by GABA in rat cortex, like those of reference classical BDZs, but was GABA-independent in the cerebellum. The IC50 of the BDZ antagonist, RO 15-1788 was unaffected by GABA in both brain areas. The possibility that PRZ behaves as a partial agonist in the cortex and as an antagonist in the cerebellum is discussed.

The data relating the pharmacological activities of benzodiazepines to in vivo occupancy of their receptor sites is reviewed, in the light of the complexity of evaluating pharmacodynamic parameters in living animals. Methodological problems in the measurement of in vivo receptor occupancy and species differences in benzodiazepine metabolism are considered in detail.

Denzimol, a new anticonvulsant drug with a pharmacological profile similar to that of phenytoin, enhances the ataxic and antimetrazol activity of diazepam in rats without affecting its activity against picrotoxin-induced seizures. In vivo and ex vivo denzimol enhances the binding of 3 H-flunitrazepam in cortex and in hippocampus but not in cerebellum. The possibility of this increase in the number of benzodiazepine binding sites contributing in some way to enhancement of the depressive and anticonvulsant activity of diazepam is discussed.

Binding to several receptors was compared in brain regions of 3 and. 21–23 month-old rats. In crude membrane preparations of aged rats the number of dopamine antagonist receptors in striatum was much reduced (−53%). β-Noradrenergic receptors (cortex) and benzodiazepine receptors (hippocampus and cerebellum) were less but significantly reduced and serotonergic receptors, α1 noradrenergic receptors (both in cortex) and dopamine agonist receptors (striatum) were unchanged. For each receptor binding the KD values were the same in young and old animals. GABA receptor binding (hippocampus and cerebellum) evaluated at only one 3H-GABA concentration (8 nM) was similar in both groups when expressed per protein content but significantly reduced in aged rats when expressed per tissue wet weight because of the partial purification of the synaptic membranes used for 3H-GABA binding. In our experimental conditions age-related changes of specific binding sites in the central nervous system were selective for some receptors studied and did not seem to be due to general non-specific modification of brain tissue composition.