Neurobiology of Disease Na–K–Cl Cotransporter-Mediated Intracellular Na Accumulation Affects Ca Signaling in Astrocytes in an In Vitro Ischemic Model

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 Rb 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 Ca 2 signaling through the Na /Ca 2 exchanger (NCX). A rise in [Ca 2 ]i was detected after OGD/REOX in the presence of a sarcoplasmic-endoplasmic reticulum (ER) Ca 2 -ATPase inhibitor thapsigargin. Moreover, OGD/REOX led to a significant increase in Ca 2 release from ER Ca 2 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 Ca 2 stores. OGD/REOX-mediated Ca 2 accumulation in ER Ca 2 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 Ca 2 accumulation by intracellular Ca 2 stores.