Hypericum perforatum L. extract does not inhibit 5-HT transporter in rat brain cortex.

The hydroalcoholic extract of Hypericum perforatum L. is an effective antidepressant, although its mechanism of action is still unknown. It inhibits the synaptosomal uptake of serotonin (5-HT), dopamine and noradrenaline, suggesting a biochemical mechanism similar to the synthetic standard antidepressants. In the present study, further investigating this hypothesis, we confirmed that a hydromethanolic extract of H. perforatum inhibited [3H]5-HT accumulation in rat brain cortical synaptosomes with an IC50 value of 7.9 microg/ml. The IC50 of pure hyperforin was 1.8 microg/ml, so the activity of the total extract is not related only to its hyperforin content (<5%). This inhibitory effect, however, is not due to a direct interaction with, and blockade of, the 5-HT transporters since the extract, like hyperforin, did not inhibit [3H]citalopram binding (IC50 > 100 microg/ml and 10 microg/ml, respectively). We also found that 3-10 microg/ml of the extract, or 0.3-1 microg/ml hyperforin, induced marked tritium release from superfused synaptosomes previously loaded with [3H]5-HT. The releasing effect of the extract resembles the releasing effect of a reserpine-like compound (Ro 04-1284), i.e. it was slightly delayed and was 5-HT carrier- and calcium-independent. These data suggest that the hydromethanolic extract of H. peforatum, similarly to Ro 04-1284, rapidly depletes storage vesicles, raising the cytoplasmic concentration of 5-HT, and this increase is presumably responsible for the apparent inhibition of [3H]5-HT uptake. Therefore, our in vitro data do not confirm that the hydromethanolic extract of H. perforatum acts as a classical 5-HT uptake inhibitor but indicate reserpine-like properties. However, the concentrations of the active component(s) effective in vitro as reserpine-like agent(s) (i.e. corresponding to > or =3 microg/ml of the hydromethanolic extract) do not seem to be achieved in the brain after pharmacologically effective doses of the extract, as indicated by the finding that there were no significant changes of rat brain 5-HT and 5-hydroxyindoleacetic acid levels after a schedule of treatment (3 x 300 mg/kgday, orally) active in an animal model predictive of antidepressant-like activity. These data also suggest that the antidepressant effect of H. perforatum extracts is unlikely to be associated with interaction with GABA, benzodiazepine and 5-HT1 receptors since, in receptor binding studies, we found IC50 values higher than 5 microg/ml. Therefore other, still unknown, mechanisms are possibly involved in H. perforatum antidepressant effects.

The assembly of Ni2+-actin: some peculiarities.

Nickel alters the organisation of highly dynamic cytoskeletal elements. In cultured cells Ni2+ causes microtubule aggregation and bundling as well as microfilament aggregation and redistribution. Here, we have analysed the effect(s) of Ni2+ on in vitro actin polymerisation. Using limited proteolysis by trypsin we have suggested that the regions around Arg-62 and Lys-68 change their conformation following Ni2+ binding to the single high-affinity site for divalent cations in the G-actin molecule. We have found that Ni2+ shortens the lag phase of actin polymerisation and increases the rate of assembly mainly because of an increased elongation rate. Ni2+ has no significant effect on the final plateau of actin polymerisation nor on the actin critical concentration. Electron microscopy revealed that actin filaments polymerised by 2 mM Ni2+ showed some tendency to lateral aggregation, being frequently formed by the cohesion of two or three filaments. Furthermore, they often appeared shorter than those of control as also confirmed by the larger amount of free filament ends as well as the faster depolymerisation rate than control.