Optical imaging reveals cation--Cl(-) cotransporter-mediated transient rapid decrease in intracellular Cl(-) concentration induced by oxygen--glucose deprivation in rat neocortical slices.
In brain slices from young (postnatal day (P) 10--15) rat somatosensory cortex, real-time neuronal intracellular Cl(-) concentration ([Cl(-)](i)) recordings were made by an optical technique measuring 6-methoxy-N-ethlquinolinium iodide (MEQ) fluorescence. Oxygen--glucose deprivation (in vitro model of ischemia) induced a long-lasting [Cl(-)](i) increase preceded by a rapid, transient [Cl(-)](i) decrease that could not be inhibited by blockers of Cl(-) pumps, Cl(-) channels, or Cl(-) antiporters, but was sensitive to cation-Cl(-) cotransporter inhibitors (bumetanide and furosemide). Use of low external Na(+) or high external K(+) revealed that the Na(+),K(+)-2Cl(-) cotransporter was inhibited by bumetanide and furosemide, whereas the K(+)-Cl(-) cotransporter was preferentially inhibited by furosemide under our experimental conditions. With a reduced inward driving force for Na(+) (reducing Na(+),K(+)-2Cl(-) cotransport), the transient [Cl(-)](i) decrease was only rarely induced by oxygen-glucose deprivation. In contrast, with a reduced outward driving force for K(+) (reducing K(+)-Cl(-) cotransport), the transient [Cl(-)](i) decrease still occurred. These results suggest that the transient [Cl(-)](i) decrease was primarily mediated by a rapid inhibition of the inwardly directed Na(+),K(+)-2Cl(-) cotransporter. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments suggested that the isoform involved is NKCC1. We hypothesize that the initial rapid Cl(-) efflux might effectively delay the irreversible Cl(-) influx that mediates neuronal injury.
Department of Pediatrics, Nagoya City University Medical School, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. y-yamada@med.nagoya-cu.ac.jp
Y
Y
Yamada
A
A
Fukuda
M
M
Tanaka
Y
Y
Shimano
H
H
Nishino
K
K
Muramatsu
H
H
Togari
Y
Y
Wada
D000818:Animals; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D015687:Cell Hypoxia / Q000502:physiology; D002712:Chlorides / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D005947:Glucose / Q000172:deficiency; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D011188:Potassium / Q000378:metabolism*; D017966:Pyramidal Cells / Q000187:drug effects / Q000378:metabolism*; D011806:Quinolinium Compounds / Q000493:pharmacokinetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters; D013003:Somatosensory Cortex / Q000187:drug effects / Q000378:metabolism*
D002352:Carrier Proteins; D002712:Chlorides; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; D012333:RNA, Messenger; C415467:SLC12A9 protein, human; D028021:Sodium-Potassium-Chloride Symporters; C070311:6-methoxy-N-ethylquinolinium; D012964:Sodium; D005947:Glucose; D011188:Potassium