Endoplasmic reticulum Ca2+ dysregulation and endoplasmic reticulum stress following in vitro neuronal ischemia: role of Na+-K+-Cl- cotransporter.
We investigated the role of Na(+)-K(+)-Cl(-) cotransporter (NKCC1) in conjunction with Na(+)/Ca(2+) exchanger (NCX) in disruption of endoplasmic reticulum (ER) Ca(2+) homeostasis and ER stress development in primary cortical neurons following in vitro ischemia. Oxygen-glucose deprivation (OGD) and reoxygenation (REOX) caused a rise in [Na(+)](cyt) which was accompanied by an elevation in [Ca(2+)](cyt). Inhibition of NKCC1 with its potent inhibitor bumetanide abolished the OGD/REOX-induced rise in [Na(+)](cyt) and [Ca(2+)](cyt). Moreover, OGD significantly increased Ca(2+)(ER) accumulation. Following REOX, a biphasic change in Ca(2+)(ER) occurred with an initial release of Ca(2+)(ER) which was sensitive to inositol 1,4,5-trisphosphate receptor (IP(3)R) inhibition and a subsequent refilling of Ca(2+)(ER) stores. Inhibition of NKCC1 activity with its inhibitor or genetic ablation prevented the release of Ca(2+)(ER). A similar result was obtained with inhibition of reversed mode operation of NCX (NCX(rev)). OGD/REOX also triggered a transient increase of glucose regulated protein 78 (GRP78), phospho-form of the alpha subunit of eukaryotic initiation factor 2 (p-eIF2alpha), and cleaved caspase 12 proteins. Pre-treatment of neurons with NKCC1 inhibitor bumetanide inhibited upregulation of GRP78 and attenuated the level of cleaved caspase 12 and p-eIF2alpha. Inhibition of NKCC1 reduced cytochrome C release and neuronal death. Taken together, these results suggest that NKCC1 and NCX(rev) may be involved in ischemic cell damage in part via disrupting ER Ca(2+) homeostasis and ER function.
Chen
Xinzhi
X
Neuroscience Training Program, and Department of Neurological Surgery, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
Kintner
Douglas B
DB
Luo
Jing
J
Baba
Akemichi
A
Matsuda
Toshio
T
Sun
Dandan
D
D000818:Animals; D002545:Brain Ischemia / Q000378:metabolism* / Q000473:pathology; D002118:Calcium / Q000378:metabolism*; D015687:Cell Hypoxia / Q000502:physiology; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000473:pathology / Q000502:physiology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005260:Female; D005947:Glucose / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000378:metabolism / Q000473:pathology*; D018384:Oxidative Stress / Q000502:physiology*; D011247:Pregnancy; D028021:Sodium-Potassium-Chloride Symporters / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D064506:Solute Carrier Family 12, Member 2
D000091342:Endoplasmic Reticulum Chaperone BiP; C000717248:Hspa5 protein, mouse; C578878:Slc12a2 protein, mouse; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D005947:Glucose; D002118:Calcium