title: Na-K-Cl cotransporter contributes to glutamate-mediated excitotoxicity.
abstract: We hypothesized that cation-dependent Cl- transport protein Na-K-Cl cotransporter isoform 1 (NKCC1) plays a role in the disruption of ion homeostasis in cerebral ischemia. In the current study, a role for NKCC1 in neuronal death was elucidated in neurotoxicity induced by glutamate and oxygen and glucose deprivation (OGD). Incubation of cortical neurons cultured for 14-15 d in vitro (DIV) with 100 microm glutamate for 24 hr resulted in 50% cell death. Three hours of OGD followed by 21 hr of reoxygenation led to 70% cell death. Inhibition of NMDA receptors with dizocilpine hydrogen maleate (1 microm) prevented both OGD- and glutamate-mediated cell death. Moreover, blocking of NKCC1 activity with bumetanide (5-10 microm) abolished glutamate- or OGD-induced neurotoxicity. Bumetanide was ineffective if added after 10-120 min of glutamate incubation or 3-6 hr of OGD treatment. Accumulation of intracellular Na+ and 36Cl content after NMDA receptor activation was inhibited by bumetanide. Blockage of NKCC1 significantly attenuated cell swelling after OGD or NMDA receptor activation. This neuroprotection was age dependent. Inhibition of NKCC1 did not protect DIV 7-8 neurons against OGD-mediated cell death. In contrast, cell death in DIV 7-8 neurons was prevented by the protein-synthesis inhibitor, cycloheximide. Taken together, the results suggest that NKCC1 activity is involved in the acute excitotoxicity as a result of excessive Na+ and Cl- entry and disruption of ion homeostasis.
authors.affiliation: Department of Neurological Surgery, University of Wisconsin Medical School, Madison, Wisconsin 53792, USA.
authors.forename: Joe
authors.initials: J
authors.lastname: Beck
authors.forename: Brett
authors.initials: B
authors.lastname: Lenart
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Kintner
authors.forename: Dandan
authors.initials: D
authors.lastname: Sun
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002034:Bumetanide / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D015687:Cell Hypoxia / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D004232:Diuretics / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005947:Glucose / Q000172:deficiency / Q000378:metabolism; D018698:Glutamic Acid / Q000633:toxicity*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009498:Neurotoxins / Q000633:toxicity*; D010100:Oxygen / Q000494:pharmacology; D051381:Rats; D012964:Sodium / Q000378:metabolism; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters / Q000502:physiology*
chemical_list: D002712:Chlorides; D004232:Diuretics; D018691:Excitatory Amino Acid Antagonists; D009498:Neurotoxins; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D018698:Glutamic Acid; D012964:Sodium; D005947:Glucose; D010100:Oxygen