title: The chloride transporter Na(+)-K(+)-Cl- cotransporter isoform-1 contributes to intracellular chloride increases after in vitro ischemia.
abstract: Ischemic episodes in the CNS cause significant disturbances in neuronal ionic homeostasis. To directly measure changes in intracellular Cl- concentration ([Cl-]i) during and after ischemia, we used Clomeleon, a novel ratiometric optical indicator for Cl-. Hippocampal slices from adult transgenic mice expressing Clomeleon in hippocampal neurons were subjected to 8 min of oxygen-glucose deprivation (OGD) (an in vitro model for ischemia) and reoxygenated in the presence of glucose. This produced mild neuronal damage 3 h later that was prevented when the extracellular [Cl-] was maintained at 10 mm during reoxygenation. OGD induced a transient decrease in fluorescence resonance energy transfer within Clomeleon, indicating an increase in [Cl-]i. During reoxygenation, there was a partial recovery in [Cl-]i, but [Cl-]i rose again 45 min later. To investigate sources of Cl- accumulation, we examined the effects of Cl- transport inhibitors on the rises in [Cl-]i during and after OGD. Bumetanide and furosemide, which inhibit Cl- influx through the Na(+)-K(+)-Cl- cotransporter isoform-1 (NKCC-1) and efflux through the K(+)-Cl- cotransporter isoform-2, were unable to inhibit the first rise in [Cl-]i, yet entirely prevented the secondary rise in [Cl-]i during reoxygenation. In contrast, picrotoxin, which blocks the GABA-gated Cl- channel, did not inhibit the secondary rise in [Cl-]i after OGD. [Cl-]i increases during reoxygenation were accompanied by an increase in phosphorylation of NKCC-1, an indication of increased NKCC-1 activity after OGD. We conclude that NKCC-1 plays an important role in OGD-induced Cl- accumulation and subsequent neuronal damage.
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Brooks B
authors.initials: BB
authors.lastname: Pond
authors.forename: Ken
authors.initials: K
authors.lastname: Berglund
authors.forename: Thomas
authors.initials: T
authors.lastname: Kuner
authors.forename: Guoping
authors.initials: G
authors.lastname: Feng
authors.forename: George J
authors.initials: GJ
authors.lastname: Augustine
authors.forename: Rochelle D
authors.initials: RD
authors.lastname: Schwartz-Bloom
mesh_terms: D000818:Animals; D002545:Brain Ischemia / Q000378:metabolism* / Q000473:pathology; D002034:Bumetanide / Q000494:pharmacology; D002712:Chlorides / Q000032:analysis / Q000378:metabolism*; D003470:Culture Media; D005665:Furosemide / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D010852:Picrotoxin / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters / Q000502:physiology*; D064506:Solute Carrier Family 12, Member 2
chemical_list: D002034:Bumetanide; D002712:Chlorides; D003470:Culture Media; D005665:Furosemide; D005947:Glucose; D010852:Picrotoxin; D011993:Recombinant Fusion Proteins; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; C578878:Slc12a2 protein, mouse; C415371:clomeleon