Na ϩ /Hϩ exchanger isoform 1 (NHE1) is a major acid extrusion mechanism after intracellular acidosis. We hypothesized that stimulation of NHE1 after cerebral ischemia contributes to the disruption of Na ϩ homeostasis and neuronal death. In the present study, expression of NHE1 was detected in cultured mouse cortical neurons. Three hours of oxygen and glucose deprivation (OGD) followed by 21 h of reoxygenation ( ϩ/ϩ mice. NHE1 ϩ/ϩ mice treated with HOE 642 or NHE1 heterozygous mice exhibited a ϳ33% decrease in infarct size ( p Ͻ 0.05). These results imply that NHE1 activity disrupts Na ϩ and Ca 2ϩ homeostasis and contributes to ischemic neuronal damage.

We reported previously that inhibition of Na(+)-K(+)-Cl(-) cotransporter isoform 1 (NKCC1) by bumetanide abolishes high extracellular K(+) concentration ([K(+)](o))-induced swelling and intracellular Cl(-) accumulation in rat cortical astrocytes. In this report, we extended our study by using cortical astrocytes from NKCC1-deficient (NKCC1(-/-)) mice. NKCC1 protein and activity were absent in NKCC1(-/-) astrocytes. [K(+)](o) of 75 mM increased NKCC1 activity approximately fourfold in NKCC1(+/+) cells (P < 0.05) but had no effect in NKCC1(-/-) astrocytes. Intracellular Cl(-) was increased by 70% in NKCC1(+/+) astrocytes under 75 mM [K(+)](o) (P < 0.05) but remained unchanged in NKCC1(-/-) astrocytes. Baseline intracellular Na(+) concentration ([Na(+)](i)) in NKCC1(+/+) astrocytes was 19.0 +/- 0.5 mM, compared with 16.9 +/- 0.3 mM [Na(+)](i) in NKCC1(-/-) astrocytes (P < 0.05). Relative cell volume of NKCC1(+/+) astrocytes increased by 13 +/- 2% in 75 mM [K(+)](o), compared with a value of 1.0 +/- 0.5% in NKCC1(-/-) astrocytes (P < 0.05). Regulatory volume increase after hypertonic shrinkage was completely impaired in NKCC1(-/-) astrocytes. High-[K(+)](o)-induced (14)C-labeled D-aspartate release was reduced by approximately 30% in NKCC1(-/-) astrocytes. Our study suggests that stimulation of NKCC1 is required for high-[K(+)](o)-induced swelling, which contributes to glutamate release from astrocytes under high [K(+)](o).

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...

We hypothesized that high extracellular K(+) concentration ([K(+)](o))-mediated stimulation of Na(+)-K(+)-Cl(-) cotransporter isoform 1 (NKCC1) may result in a net gain of K(+) and Cl(-) and thus lead to high-[K(+)](o)-induced swelling and glutamate release. In the current study, relative cell volume changes were determined in astrocytes. Under 75 mM [K(+)](o,) astrocytes swelled by 20.2 +/- 4.9%. This high-[K(+)](o)-mediated swelling was abolished by the NKCC1 inhibitor bumetanide (10 microM, 1.0 +/- 3.1%; P < 0.05). Intracellular (36)Cl(-) accumulation was increased from a control value of 0.39 +/- 0.06 to 0.68 +/- 0.05 micromol/mg protein in response to 75 mM [K(+)](o). This increase was significantly reduced by bumetanide (P < 0.05). Basal intracellular Na(+) concentration ([Na(+)](i)) was reduced from 19.1 +/- 0.8 to 16.8 +/- 1.9 mM by bumetanide (P < 0.05). [Na(+)](i) decreased to 8.4 +/- 1.0 mM under 75 mM [K(+)](o) and was further reduced to 5.2 +/- 1.7 mM by bumetanide. In addition, the recovery rate of [Na(+)](i) on return to 5.8 mM [K(+)](o) was decreased by 40% in the presence of bumetanide (P < 0.05). Bumetanide inhibited high-[K(+)](o)-induced (14)C-labeled D-aspartate release by ~50% (P < 0.05). These results suggest that NKCC1 contributes to high-[K(+)](o)-induced astrocyte swelling and glutamate release.

We hypothesized that cation-dependent Cl- transport protein Na-K-Cl cotransporter isoform 1 (NKCC1) plays a role in the disruption of ion homeostasis in cerebral ischemia. In the current study, a role for NKCC1 in neuronal death was elucidated in neurotoxicity induced by glutamate and oxygen and glucose deprivation (OGD). Incubation of cortical neurons cultured for 14-15 d in vitro (DIV) with 100 microm glutamate for 24 hr resulted in 50% cell death. Three hours of OGD followed by 21 hr of reoxygenation led to 70% cell death. Inhibition of NMDA receptors with dizocilpine hydrogen maleate (1 microm) prevented both OGD- and glutamate-mediated cell death. Moreover, blocking of NKCC1 activity with bumetanide (5-10 microm) abolished glutamate- or OGD-induced neurotoxicity. Bumetanide was ineffective if added after 10-120 min of glutamate incubation or 3-6 hr of OGD treatment. Accumulation of intracellular Na+ and 36Cl content after NMDA receptor activation was inhibited by bumetanide. Blockage of NKCC1 significantly attenuated cell swelling after OGD or NMDA receptor activation. This neuroprotection was age dependent. Inhibition of NKCC1 did not protect DIV 7-8 neurons against OGD-mediated cell death. In contrast, cell death in DIV 7-8 neurons was prevented by the protein-synthesis inhibitor, cycloheximide. Taken together, the results suggest that NKCC1 activity is involved in the acute excitotoxicity as a result of excessive Na+ and Cl- entry and disruption of ion homeostasis.