RESUMEN
Major depressive disorder is one of the most common neuropsychiatric disorders worldwide. The treatment of choice that shows good efficacy in mood stabilization is based on selective serotonin reuptake inhibitors (SSRIs). Their primary mechanism of action is considered to be the increased synaptic concentration of serotonin through blockade of the serotonin transporter (SERT). In this study, we described an alternative mode of action of fluoxetine (FLX), which is a representative member of the SSRI class of antidepressants. We observed that FLX robustly decreases both glutamatergic and gamma-Aminobutyric acid (GABA)-ergic synaptic release in a SERT-independent manner. Moreover, we showed that this effect may stem from the ability of FLX to change the levels of main components of the SNARE (solubile N-ethylmaleimide-sensitive factor attachment protein receptor) complex. Our data suggest that this downregulation of SNARE fusion machinery involves diminished activity of protein kinase C (PKC) due to FLX-induced blockade of P/Q type of voltage-gated calcium channels (VGCCs). Taken together, by virtue of its inhibition at SERT, fluoxetine increases extracellular serotonin levels; however, at the same time, by reducing SNARE complex function, this antidepressant reduces glutamate and GABA release.
Asunto(s)
Fluoxetina/farmacología , Ácido Glutámico/metabolismo , Proteínas SNARE/metabolismo , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismo , Animales , Canales de Calcio/metabolismo , Exocitosis/efectos de los fármacos , Femenino , Humanos , Modelos Neurológicos , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/fisiología , Proteína Quinasa C/metabolismo , Ratas WistarRESUMEN
Riluzole is a potent neuroprotective agent which primarily inhibits excitatory neurotransmission interfering with presynaptic release, uptake and postsynaptic actions of glutamate by mechanisms that are not well understood. Riluzole and related prodrugs with improved blood brain barrier penetrance, are shown to be effective for the treatment of amyotrophic lateral sclerosis, ataxias, epilepsy and mood disorders. Our study was undertaken to decipher molecular and subcellular mechanisms of riluzole's antiglutamatergic effect, particularly focusing on presynaptic active zone structure and function. Applying multifarious live cell imaging techniques and amperometric glutamate recordings, we measured the impact of riluzole on presynaptic activity, synaptic vesicle recycling and glutamate release. Our in vitro and in vivo data revealed a unique mechanism whereby riluzole reduces the efficacy of glutamatergic transmission by selectively lowering the size of the readily releasable pool. This effect was correlated with the inhibition of protein kinase C-dependent Munc18-1 phosphorylation which is known to interfere with neurotransmitter release.
Asunto(s)
Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/metabolismo , Fármacos Neuroprotectores/farmacología , Riluzol/farmacología , Transmisión Sináptica/efectos de los fármacos , Vesículas Sinápticas/efectos de los fármacos , Animales , Antígeno CD146/metabolismo , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Masculino , Ratones Endogámicos C57BL , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Proteína Quinasa C/metabolismo , Ratas Wistar , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismoRESUMEN
The presynaptic action of Bothriopsis bilineata smaragdina (forest viper) venom and Bbil-TX, an Asp49 PLA2 from this venom, was examined in detail in mouse phrenic nerve-muscle (PND) preparations in vitro and in a neuroblastoma cell line (SK-N-SH) in order to gain a better insight into the mechanism of action of the venom and associated Asp49 PLA2. In low Ca(2+) solution, venom (3µg/ml) caused a quadriphasic response in PND twitch height whilst at 10µg/ml the venom additionally induced an abrupt and marked initial contracture followed by neuromuscular facilitation, rhythmic oscillations of nerve-evoked twitches, alterations in baseline and progressive blockade. The venom slowed the relaxation phase of muscle twitches. In low Ca(2+), Bbil-TX [210nM (3µg/ml)] caused a progressive increase in PND twitch amplitude but no change in the decay time constant. Venom (10µg/ml) and Bbil-TX (210nM) caused minor changes in the compound action potential (CAP) amplitude recorded from sciatic nerve preparations, with no significant effect on rise time and latency; tetrodotoxin (3.1nM) blocked the CAP at the end of the experiments. In mouse triangularis sterni nerve-muscle (TSn-m) preparations, venom (10µg/ml) and Bbil-TX (210nM) significantly reduced the perineural waveform associated with the outward K(+) current while the amplitude of the inward Na(+) current was not significantly affected. Bbil-TX (210nM) caused a progressive increase in the quantal content of TSn-m preparations maintained in low Ca(2+) solution. Venom (3µg/ml) and toxin (210nM) increased the calcium fluorescence in SK-N-SH neuroblastoma cells loaded with Fluo3 AM and maintained in low or normal Ca(2+) solution. In normal Ca(2+), the increase in fluorescence amplitude was accompanied by irregular and frequent calcium transients. In TSn-m preparations loaded with Fluo4 AM, venom (10µg/ml) caused an immediate increase in intracellular Ca(2+) followed by oscillations in fluorescence and muscle contracture; Bbil-TX did not change the calcium fluorescence in TSn-m preparations. Immunohistochemical analysis of toxin-treated PND preparations revealed labeling of junctional ACh receptors but a loss of the presynaptic proteins synaptophysin and SNAP25. Together, these data confirm the presynaptic action of Bbil-TX and show that it involves modulation of K(+) channel activity and presynaptic protein expression.