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

Abstract

Na-K-Cl cotransporter isoform 1 (NKCC1) plays an important role in maintenance of intracellular Naϩ , K ϩ , and Cl Ϫ 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 86 Rb influx was significantly increased in astrocytes from NKCC1 wild-type (NKCC1 ϩ/ϩ ) and heterozygous mutant (NKCC1 ϩ/Ϫ ) mice. Phosphorylated NKCC1 protein was increased in NKCC1 ϩ/ϩ astrocytes at 2 hr of OGD. Two hours of OGD and 1 hr of reoxygenation (OGD/REOX) triggered an ϳ3. IntroductionNa-K-Cl cotransporter isoform 1 (NKCC1) belongs to the cation-dependent Cl Ϫ transporter family and transports Na ϩ , K ϩ , and Cl Ϫ into cells under physiological conditions (Russell, 2000). NKCC1 is expressed in rat cortical astrocytes (Yan et al., 2001), oligodendrocytes (Hoppe and Kettenmann, 1989;Wang et al., 2003), and Schwann cells (Alvarez-Leefmans, 2001). Functions of NKCC1 in these cells include K ϩ and Na ϩ uptake (Su et al., 2000(Su et al., , 2002b and accumulation of Cl Ϫ above its electrochemical equilibrium (Hoppe and Kettenmann, 1989;Wang et al., 2003). In recent studies, we found that NKCC1 contributes to K ϩ uptake, swelling, and swelling-induced glutamate release in astrocytes in the presence of high extracellular K ϩ (Su et al., 2002a,b). Disruption of Naϩ and Ca 2ϩ homeostasis plays an important role in ischemic cell damage (Siesjö, 1992). A steep inwardly directed Na ϩ gradient is essential for glial functions, such as glutamate reuptake and regulation of intracellular ion concentrations by other secondary ion transporters (Walz, 1989;Longuemare et al., 1999). Breakdown of the Na ϩ gradient is one of the key elements in promoting cellular damage in astrocytes during energy failure (Longuemare et al., 1999). An increase in intracellular Na ϩ concentration ([Na ϩ ] i ) was found in rat spinal cord astrocytes (Rose et al., 1998), rat cortical astrocytes (Longuemare et al., 1999), and mouse cortical astrocytes (Silver et al., 1997) when these cells were exposed to glucose deprivation, NaN 3 -mediated chemical hypoxia, and simulated ischemia, respectively. Mechanisms of the rise in [Na ϩ ] i under reduced energy production conditions are not well understood. Inhibition of Na ϩ /K ϩ -ATPase activity via limited energy production results in Na ϩ accumulation (Silver et al., 1997). However, blocking voltage-gated Na ϩ channels had no effect on the rise of [Na ϩ ] i in rat spinal astrocytes during chemical hypoxia (Rose et al., 1998 reverse-mode operation of the Na ϩ /Ca 2ϩ exchanger (NCX) in neurons and causes irreversible injury during anoxia and ischemia (Li et al., 2000). An increase in intracellular Ca 2ϩ via the reverse-mode operation of NCX occurs in rat astrocytes when [Na ϩ ] i was raised by inhibition of Na ϩ /K ϩ -ATPase activity or activation of AMPA channels (Goldman et al., 1994;Smith et al., 2000). However, it is unknown whether accumulation of intracellular Na ϩ during a...

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