It is well established that a long-term action of L-glutamate (Glu), an excitatory neurotransmitter in the CNS, or L-homocysteine (HCY), an endogenous sulfur-containing amino acid involved in synthesis of methionine and cysteine, on cortical neurons in primary cultures induces neurodegeneration by necrosis and apoptosis which represents a widely used model of excitotoxicity and hyperhomocysteinemia, correspondently. Using a fluorescent viability assay (FVA, Mironova et al., 2007), and on-line monitoring of intracellular Ca2+ (Ca2+ imaging experiments with Fluo-3) and mitochondrial membrane potential (using rhodamine123) with confocal microscopy, here neuroprotective effects of ouabain at concentrations 0.1 nM – 1 nM against excitotoxic stress were investigated in vitro. During short- (4 h) as well as long- (24 h) term treatments with Glu (100 µM) or HCY (100 µM) in the presence of 30 µM glycine at both 0.1 nM and 1 nM ouabain prevented apoptosis of cortical neurons. In short-scale experiments this neuroprotective effects of ouabain appeared to be determined by an enchancement of Ca2+ extrusion from neurons by Na+/Ca2+-exchanger so that neither an accumulation of Ca2+ in neurons nor a drop of mitochondrial membrane potential were observed. In long-scale experiments in case of HCY 0.1 nM and 1 nM ouabain triggered neuroprotective cascades that involved activation of PKA and PKC, since proteinkinase A (PKA) inhibitor and proteinkinase C (PKC) inhibitor — chelerythrine abolished the neuroprotection and more than a half of neuronal population exhibited apoptosis. However, these inhibitors did not affect ouabain-caused neuroprotection against Glu excitotoxicity.
Thus, the data reveal that different intracellular cascades are involved in ouabain- triggered neuroprotection against Glu- and CHY-caused neurodegeneration that may suggest an existence of different possible targets for therapy in excitotoxicity and hyperhomocysteinemia