Na-K-Cl Cotransporter-Mediated Intracellular Na+Accumulation Affects Ca2+Signaling in Astrocytes in anIn VitroIschemic Model

Open Access
Publisher Website
Google Scholar
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.