Coumarin br Materials and methods br

Materials and methods
Results
Discussion l-glutamate is the major excitatory neurotransmitter in the brain, and is functionally involved in many processes of the nervous system which have been connected with several neurological diseases [4]. These conditions are characterized by the time-dependent damage of many cellular components, leading to cell death. Glutamate-mediated neurotoxicity is triggered as a result of glutamate binding to the NMDA receptor, and to a minor extent other receptors, including AMPA/kainate and voltage sensitive Ca2+ Coumarin [30]. Activation of the NMDA receptor involves opening of the ion channel to allow the influx of Ca2+. During this triggering phase, the neuron is exposed to an excess of glutamate, causing the [Ca2+]i to rise to micromolar levels. This phase can either result in acute necrosis, both during and immediately after the exposure, or it can be followed by the latent and final phases. The latent phase can be characterized by the recovery of the [Ca2+]i to a basal level, and the final (delayed) phase can be described by a gradual rise in [Ca2+]i that reaches a sustained plateau, with Ca2+ dependent apoptosis as a consequence [31]. The latent and final phases can proceed even after the removal of extracellular glutamate [32]. The intensity of the glutamate exposure determines which of the two routes to neuronal cell death is followed. Whilst exposure to glutamate at the concentration of 1 μM results in no significant degree of immediate necrosis or delayed apoptosis in cortical neurons, higher concentrations of glutamate will ultimately lead to cell death, due to either delayed apoptosis (30–100 μM) or acute necrosis (1–3 mM). The treatment with glutamate at the concentrations of 3 μM and 10 μM for 24 h has been shown to activate caspase-3, and induce apoptotic cell death in the culture of hippocampal neurones (IC50 = 3.6 μM, determined by MTT assay) [32,33]. The affinity of the NMDA receptor toward glutamate has been determined to be in the low micromolar range (EC50 = 2.3 μM) [34]. In our study, two concentrations of glutamate were used: “acute” 20 μM (capable of triggering a detectable Ca2+ influx and decreasing the viability of cortical neurons to 30% after 24 h) and “chronic” 5 μM (Ca2+ influx is under the detection level and drops the viability of cortical neurons to 50% after 24 h). We demonstrated that, with exception of two compounds, all neurosteroids under investigation significantly reduced glutamate-mediated Ca2+ influx during the triggering phase of glutamate-induced excitotoxicity. These results are in good agreement with their ability to inhibit NMDA currents in a system of recombinant NMDARs expressed in HEK293 cells [22]. The very low inhibitory activity of 12 is likely caused by its low membrane permeability [20] as previously demonstrated in Caco-2 intestinal permeability assay. Likewise, the low inhibitory activity of compound 17 can be explained by limited solubility [22]. Note that ten of the tested neurosteroids (3, 5–9, 13–16) displayed better inhibitory activity than memantine (1) in the glutamate-induced Ca2+ influx assay. Moreover, compounds 6, 13, and 16 proved to be more potent inhibitors than pregnanolone sulfate (3), and the activity of compound 13 was comparable with that of MK-801 (2) in preventing glutamate-induced [Ca2+]i increase. Based on the comparative study with glutamate and NMDA as Ca2+ inducers, all tested neurosteroids were shown to act predominantly on the NMDA receptor. The massive glutamate-induced influx of extracellular Ca2+ increases the cytosolic free Ca2+ that is associated with mitochondrial dysfunction and activation of proteases, nucleases and oxidizing enzymes that form ROS. Mitochondria, neuronal nitric oxide synthase (nNOS) are xanthine oxidase (XOD) are mainly implicated in ROS production in this a cascade-like process leading to cell death [35,36]. Generation of ROS starts within minutes after glutamate treatment [37]. To confirm participation of apoptosis in this cascade, the effect of neurosteroids on caspase-3 activation was investigated. The activity of caspase-3 was measured at its maximum around 3 h after glutamate (20 μM). A similar time frame was previously reported for glutamate-induced toxicity in neuronal cells at the concentration of 30 μM [38]. A significant decrease in ROS levels was observed for the studied neurosteroids 3 h after co-treatment with glutamate (20 μM). There was also a correlation with their ability to attenuate Ca2+ influx. An analogous correlation was observed for caspase-3 activation and therefore, the same trend of improved cell viability is expected.