Specific combinations of ion channel inhibitors reduce excessive Ca²⁺ influx as a consequence of oxidative stress and increase neuronal and glial cell viability in vitro

Abstract

Combinations of Ca²⁺ channel inhibitors have been proposed as an effective means to prevent excess Ca²⁺ flux and death of neurons and glia following neurotrauma in vivo. However, it is not yet known if beneficial outcomes such as improved viability have been due to direct effects on intracellular Ca2+ concentrations. Here, the effects of combinations of Lomerizine (Lom), 2,3-dioxo-7-(1H-imidazol-1-yl)6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]acetic acid monohydrate (YM872), 3,5-dimethyl-1-adamantanamine (memantine (Mem)) and/or adenosine 5'-triphosphate periodate oxidized sodium salt (oxATP) to block voltage-gated Ca²⁺ channels, Ca²⁺ permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, NMDA receptors and purinergic P2X7 receptors (P2X7R) respectively, on Ca²⁺ concentration and viability of rat primary mixed cortical (MC) cultures exposed to hydrogen peroxide (H2O2) insult, were assessed. The contribution of ryanodine-sensitive intracellular stores to intracellular Ca2+ concentration was also assessed. Live cell calcium imaging revealed that a 30min H2O2 insult induced a slow increase in intracellular Ca²⁺, in part from intracellular sources, associated with loss of cell viability by 6h. Most combinations of inhibitors that included oxATP significantly decreased Ca²⁺ influx and increased cell viability when administered simultaneously with H2O2. However, reductions in intracellular Ca²⁺ concentration were not always linked to improved cell viability. Examination of the density of specific cell subpopulations demonstrated that most combinations of inhibitors that included oxATP preserved NG²⁺ non-oligodendroglial cells, but preservation of astrocytes and neurons required additional inhibitors. Olig2+ oligodendroglia and ED-1⁺ activated microglia/macrophages were not preserved by any of the inhibitor combinations. These data indicate that following H2O2 insult, limiting intracellular Ca2+ entry via P2X7R is generally associated with increased cell viability. Protection of NG²⁺ non-oligodendroglial cells by Ca²⁺ channel inhibitor combinations may contribute to observed beneficial outcomes in vivo.