title: Na-K-Cl cotransporter-mediated intracellular Na+ accumulation affects Ca2+ signaling in astrocytes in an in vitro ischemic model.
abstract: Na-K-Cl cotransporter isoform 1 (NKCC1) plays an important role in maintenance of intracellular Na+, K+, and Cl- levels in astrocytes. We propose that NKCC1 may contribute to perturbations of ionic homeostasis in astrocytes under ischemic conditions. After 3-8 hr of oxygen and glucose deprivation (OGD), NKCC1-mediated 86Rb influx was significantly increased in astrocytes from NKCC1 wild-type (NKCC1+/+) and heterozygous mutant (NKCC1+/-) mice. Phosphorylated NKCC1 protein was increased in NKCC1+/+ astrocytes at 2 hr of OGD. Two hours of OGD and 1 hr of reoxygenation (OGD/REOX) triggered an 3.6-fold increase in intracellular Na+ concentration ([Na+]i) in NKCC1+/+ astrocytes. Inhibition of NKCC1 activity by bumetanide or ablation of the NKCC1 gene significantly attenuated the rise in [Na+]i. Moreover, NKCC1+/+ astrocytes swelled by 10-30% during 20-60 min of OGD. Either genetic ablation of NKCC1 or inhibition of NKCC1 by bumetanide-attenuated OGD-mediated swelling. An NKCC1-mediated increase in [Na+]i may subsequently affect Ca2+ signaling through the Na+/Ca2+ exchanger (NCX). A rise in [Ca2+]i was detected after OGD/REOX in the presence of a sarcoplasmic-endoplasmic reticulum (ER) Ca2+-ATPase inhibitor thapsigargin. Moreover, OGD/REOX led to a significant increase in Ca2+ release from ER Ca2+ stores. Furthermore, KB-R7943 (2-[2-[4(4-nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate), an inhibitor of reverse-mode operation of NCX, abolished the OGD/REOX-induced enhancement in filling of ER Ca2+ stores. OGD/REOX-mediated Ca2+ accumulation in ER Ca2+ stores was absent when NKCC1 activity was ablated or pharmacologically inhibited. These findings imply that stimulation of NKCC1 activity leads to Na+ accumulation after OGD/REOX and that subsequent reverse-mode operation of NCX contributes to increased Ca2+ accumulation by intracellular Ca2+ stores.
authors.affiliation: Department of Neurosurgery, University of Wisconsin Medical School, Madison, Wisconsin 53792, USA.
authors.forename: Brett
authors.initials: B
authors.lastname: Lenart
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Kintner
authors.forename: Gary E
authors.initials: GE
authors.lastname: Shull
authors.forename: Dandan
authors.initials: D
authors.lastname: Sun
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001253:Astrocytes / Q000378:metabolism / Q000502:physiology*; D001920:Bradykinin / Q000494:pharmacology; D002545:Brain Ischemia / Q000378:metabolism / Q000503:physiopathology*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D016923:Cell Death / Q000502:physiology; D015687:Cell Hypoxia / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D005947:Glucose / Q000172:deficiency; D051379:Mice; D018345:Mice, Knockout; D008817:Mice, Mutant Strains; D008928:Mitochondria / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000502:physiology; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000502:physiology; D028021:Sodium-Potassium-Chloride Symporters / Q000096:biosynthesis / Q000502:physiology*; D064506:Solute Carrier Family 12, Member 2
chemical_list: D002712:Chlorides; D009419:Nerve Tissue Proteins; C578878:Slc12a2 protein, mouse; D019831:Sodium-Calcium Exchanger; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D000255:Adenosine Triphosphate; D012964:Sodium; D005947:Glucose; D011188:Potassium; D001920:Bradykinin; D002118:Calcium