title: Ionic mechanism of ouabain-induced concurrent apoptosis and necrosis in individual cultured cortical neurons.
abstract: Energy deficiency and dysfunction of the Na+, K+-ATPase are common consequences of many pathological insults. The nature and mechanism of cell injury induced by impaired Na+, K+-ATPase, however, are not well defined. We used cultured cortical neurons to examine the hypothesis that blocking the Na+, K+-ATPase induces apoptosis by depleting cellular K+ and, concurrently, induces necrotic injury in the same cells by increasing intracellular Ca2+ and Na+. The Na+, K+-ATPase inhibitor ouabain induced concentration-dependent neuronal death. Ouabain triggered transient neuronal cell swelling followed by cell shrinkage, accompanied by intracellular Ca2+ and Na+ increase, K+ decrease, cytochrome c release, caspase-3 activation, and DNA laddering. Electron microscopy revealed the coexistence of ultrastructural features of both apoptosis and necrosis in individual cells. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) blocked >50% of ouabain-induced neuronal death. Potassium channel blockers or high K+ medium, but not Ca2+ channel blockade, prevented cytochrome c release, caspase activation, and DNA damage. Blocking of K+, Ca2+, or Na+ channels or high K+ medium each attenuated the ouabain-induced cell death; combined inhibition of K+ channels and Ca2+ or Na+ channels resulted in additional protection. Moreover, coapplication of Z-VAD-FMK and nifedipine produced virtually complete neuroprotection. These results suggest that the neuronal death associated with Na+, K+-pump failure consists of concurrent apoptotic and necrotic components, mediated by intracellular depletion of K+ and accumulation of Ca2+ and Na+, respectively. The ouabain-induced hybrid death may represent a distinct form of cell death related to the brain injury of inadequate energy supply and disrupted ion homeostasis.
authors.affiliation: Department of Neurology and Center for the Study of Nervous System Injury, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: Ai Ying
authors.initials: AY
authors.lastname: Xiao
authors.forename: Ling
authors.initials: L
authors.lastname: Wei
authors.forename: Shuli
authors.initials: S
authors.lastname: Xia
authors.forename: Steven
authors.initials: S
authors.lastname: Rothman
authors.forename: Shan Ping
authors.initials: SP
authors.lastname: Yu
mesh_terms: D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D006706:Homeostasis / Q000187:drug effects; D006822:Hybrid Cells / Q000166:cytology / Q000187:drug effects; D017136:Ion Transport / Q000187:drug effects; D051379:Mice; D009336:Necrosis; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D010042:Ouabain / Q000633:toxicity*; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors
chemical_list: D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D010042:Ouabain; D012964:Sodium; D020169:Caspases; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium; D002118:Calcium