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: Brooks B Pond, Ken Berglund, Thomas Kuner, Guoping Feng, George J Augustine, Rochelle D Schwartz-Bloom
• Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience (J Neurosci) Vol. 26 Issue 5 Pg. 1396-406 (Feb 01 2006) ISSN: 1529-2401 [Electronic] United States
• PMID: 16452663 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Chlorides
• Culture Media
• Recombinant Fusion Proteins
• Slc12a2 protein, mouse
• Sodium Potassium Chloride Symporter Inhibitors
• Sodium-Potassium-Chloride Symporters
• Solute Carrier Family 12, Member 2
• clomeleon
• Bumetanide
• Picrotoxin
• Furosemide
• Glucose

Topics

• Animals
• Brain Ischemia (metabolism, pathology)
• Bumetanide (pharmacology)
• Chlorides (analysis, metabolism)
• Culture Media
• Furosemide (pharmacology)
• Glucose (metabolism)
• Hippocampus (cytology, metabolism)
• Hydrogen-Ion Concentration
• In Vitro Techniques
• Mice
• Mice, Inbred C57BL
• Mice, Transgenic
• Neurons (metabolism, pathology)
• Picrotoxin (pharmacology)
• Recombinant Fusion Proteins (genetics)
• Sodium Potassium Chloride Symporter Inhibitors (pharmacology)
• Sodium-Potassium-Chloride Symporters (physiology)
• Solute Carrier Family 12, Member 2