Na-K-Cl Cotransporter-Mediated Intracellular Na+ Accumulation Affects Ca2+ Signaling in Astrocytes in an In Vitro Ischemic Model

摘要

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.