Abstract
Background: Subnanomolar ouabain binding to the Na,K-ATPase triggers intracellular signaling, prevents an overload of neurons with Ca2+, and their apoptosis caused by glutamate receptor agonists. Elevated plasma homocysteine (HCY), known as hyperhomocysteinemia, represents a risk factor for stroke and can exacerbate many neuronal disorders. HCY acts as a persistent N-methyl-D-aspartate receptor (NMDAR) agonist, which, in contrast to glutamate, desensitizes NMDARs containing GluN2B subunits. Mechanisms of HCY neurotoxicity remain not clearly understood since GluN2B-containing NMDARs provide a major contribution to excitotoxicity among glutamate receptors.Methods: Using fluorescent tools combined with the confocal microscopy, we compared 0.1 - 1 nM ouabain effects on the intracellular Ca2+ signaling, on the mitochondrial inner membrane voltage and the cell viability in primary cultures of rat cortical neurons in glutamate and HCY neurotoxic insults. We also studied an apoptosis-related protein expression and the involvement of some kinases in ouabain mediated effects.Results: In short insults HCY was less potent than glutamate as a neurotoxic agent. This amino acid induced the voltage loss (∆φmit) of 0.2 of the total mitochondrial inner membrane voltage (φmit) instead of ∆φmit = 0.7 for glutamate. We have found that subnanomolar ouabain exhibited rapid and postponed neuroprotective effects on neurons and (1) rapidly reduced the Ca2+ overload of neurons and the voltage loss of inner mitochondrial membranes evoked by glutamate and HCY, and (2) prevented neuronal apoptosis during 24 h treatments with glutamate or HCY. Using a set of specific kinase inhibitors such as PKA inhibitor, chelerythrine, and KN93, we demonstrated the role of multi-kinase signaling pathways involving PKC and PKA in neuronal survival caused by ouabain in hyperhomocysteinemia. Conclusions: Subnanomolar ouabain prevents neurodegeneration caused by glutamate and HCY. For both amino acids, ouabain evokes an acceleration of Ca2+ export by sodium-calcium exchangers from neurons preventing the voltage loss by mitochondrial inner membranes that rescue neurons in short insults. In prolonged insults, ouabain triggers intracellular neuroprotective cascades, including activation of PKA and PKC for HCY, but not for glutamate. This suggests that different appropriate pharmacology for hyperhomocysteinemia and glutamate excitotoxicity could be applied for clinical treatments.
Dopamine D1 receptor signaling system regulates ryanodine receptor expression after intermittent exposure to methamphetamine in primary cultures of midbrain and cerebral cortical neurons
