title: 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 P2X7receptors (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 H2O2insult 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 H2O2insult, 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.
authors.affiliation: Experimental and Regenerative Neurosciences, The University of Western Australia, Crawley, Western Australia, Australia; School of Animal Biology, The University of Western Australia, Crawley, Western Australia, Australia; School of Anatomy, Physiology and Human Biology, The University of Western Australia, Crawley, Western Australia, Australia.
authors.forename: Ryan L
authors.initials: RL
authors.lastname: O'Hare Doig
authors.affiliation: Experimental and Regenerative Neurosciences, The University of Western Australia, Crawley, Western Australia, Australia; School of Animal Biology, The University of Western Australia, Crawley, Western Australia, Australia.
authors.forename: Carole A
authors.initials: CA
authors.lastname: Bartlett
authors.affiliation: Experimental and Regenerative Neurosciences, The University of Western Australia, Crawley, Western Australia, Australia; School of Animal Biology, The University of Western Australia, Crawley, Western Australia, Australia; School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Western Australia, Australia.
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Smith
authors.affiliation: Experimental and Regenerative Neurosciences, The University of Western Australia, Crawley, Western Australia, Australia; School of Anatomy, Physiology and Human Biology, The University of Western Australia, Crawley, Western Australia, Australia.
authors.forename: Stuart I
authors.initials: SI
authors.lastname: Hodgetts
authors.affiliation: Experimental and Regenerative Neurosciences, The University of Western Australia, Crawley, Western Australia, Australia; School of Animal Biology, The University of Western Australia, Crawley, Western Australia, Australia.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Dunlop
authors.affiliation: School of Anatomy, Physiology and Human Biology, The University of Western Australia, Crawley, Western Australia, Australia; Victor Chang Cardiac Research Institute, Sydney, New South Wales, Australia.
authors.forename: Livia
authors.initials: L
authors.lastname: Hool
authors.affiliation: Experimental and Regenerative Neurosciences, The University of Western Australia, Crawley, Western Australia, Australia; School of Animal Biology, The University of Western Australia, Crawley, Western Australia, Australia. Electronic address: lindy.fitzgerald@uwa.edu.au.
authors.forename: Melinda
authors.initials: M
authors.lastname: Fitzgerald
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism; D018920:Coculture Techniques; D006861:Hydrogen Peroxide / Q000633:toxicity; D007093:Imidazoles / Q000494:pharmacology; D008559:Memantine / Q000494:pharmacology; D009457:Neuroglia / Q000187:drug effects* / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology*; D018377:Neurotransmitter Agents / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology; D010879:Piperazines / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D017981:Receptors, Neurotransmitter / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D002412:Cations; D007093:Imidazoles; D018696:Neuroprotective Agents; D018377:Neurotransmitter Agents; D010879:Piperazines; D011810:Quinoxalines; D017981:Receptors, Neurotransmitter; C110629:YM 872; D006861:Hydrogen Peroxide; C052424:lomerizine; D002118:Calcium; D008559:Memantine
keywords: Ca(2+) channel inhibitors; Cell viability; NG2-glia; intracellular Ca(2+) concentration; oligodendroglia