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Na-K-Cl Cotransporter Contributes to Glutamate-Mediated Excitotoxicity

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Na-K-Cl Cotransporter Contributes to Glutamate-Mediated Excitotoxicity The Journal of Neuroscience, June 15, 2003 • 23(12):5061–5068 • 5061 Na-K-Cl Cotransporter Contributes to Glutamate-Mediated Excitotoxicity Joe Beck,1,3 Brett Lenart,1 Douglas B. Kintner,1 and Dandan Sun1,2 Departments of 1Neurological Surgery, 2Physiology, and 3Pathology, University of Wisconsin Medical School, Madison, Wisconsin 53792 Ϫ 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 ␮M 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 ␮M) prevented both OGD- and glutamate-mediated cell death. Moreover, blocking of NKCC1 activity with bumetanide (5–10 ␮M) abolished glutamate- or OGD-induced neurotoxicity. Bumetanide was ineffective if added ϩ 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 ϩ Ϫ 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. Ϫ ϩ Key words: excitotoxicity; active Cl transport; Na entry; cell volume; necrosis; bumetanide Ϫ Introduction lular Cl in neurons and contributes to GABA-mediated depo- Cl Ϫ movement has been shown to be a central component of the larization in immature neurons (Misgeld et al., 1986; Sun and acute excitotoxic response in neurons (Rothman, 1985; Olney et Murali, 1998; Sung et al., 2000; Alvarez-Leefmans, 2001). Thus, NKCC1 may affect neuronal excitability through regulation of al., 1986; Nicklas et al., 1987). The acute excitotoxicity is thought Ϫ to be mediated by excessive depolarization of the postsynaptic [Cl ]i (Sung et al., 2000; Jang et al., 2001). In addition, activation of membrane. This results in an osmotic imbalance when countered the ionotropic glutamate NMDA receptor, the AMPA receptor, and by an influx of Cl Ϫ,Naϩ, and water, leading to eventual lysis. the metabotropic glutamate receptor (group I) significantly stimu- Removal or reduction of Cl Ϫ from extracellular medium during lates NKCC1 activity in cortical neurons (Schomberg et al., 2001). excitatory amino acid exposure completely eliminates the acute Our recent study reveals an upregulation of NKCC1 protein excitotoxic response in hippocampal (Rothman, 1985) and reti- expression in the cortex at 24 hr reperfusion after focal ischemia nal (Olney et al., 1986; Nicklas et al., 1987) neurons. A significant (Yan et al., 2001). Pharmacological inhibition of NKCC1 with Ϫ Ϫ bumetanide results in a significant reduction of infarction vol- increase in intracellular Cl concentration ([Cl ]i) is observed in hippocampal neurons during OGD (Inglefield and Schwartz- ume (Yan et al., 2001). In addition, water content increase is 70% Bloom, 1998). Although the mechanisms underlying the Cl Ϫ- less in the bumetanide-treated brains (Yan et al., 2001). These dependent excitotoxicity are not yet understood, blockage of Cl Ϫ results strongly suggest that NKCC1 is important in ischemic Ϫ Ϫ neuronal damage. However, the cellular mechanisms by which entry through the Cl /HCO3 exchanger or GABA receptor effec- tively protects cells against the acute excitotoxicity (Zeevalk et al., NKCC1 contributes to ischemic cell death have not been defined. 1989; Chen et al., 1998; Inglefield and Schwartz-Bloom, 1998). In this study, we report that inhibition of NKCC1 activity abol- We hypothesized that Na-K-Cl cotransporter isoform 1 ished glutamate-mediated neurotoxicity and significantly attenu- (NKCC1) may also contribute to the Cl Ϫ movement during ex- ated OGD-induced neuronal death. Thus, NKCC1 may be involved citotoxicity. The electroneutral NKCC1 transports Na ϩ,Kϩ, and in ischemic cell death through an association with excitotoxicity. Cl Ϫ into cells under physiological conditions (Russell, 2000). A preliminary report was presented at the Ninth International NKCC1 is characteristically inhibited by the sulfamoylbenzoic Symposium on Pharmacology of Cerebral Ischemia (Beck et al., acid “loop” diuretics, such as bumetanide and furosemide (Rus- 2002). sell, 2000). NKCC1 is important in the maintenance of intracel- Materials and Methods Cortical neuron and astrocyte cultures. Primary rat neurons were cultured Received Dec. 17, 2002; revised April 4, 2003; accepted April 7, 2003. using techniques established in our laboratory as described previously This work was supported in part by National Institutes of Health Grant RO1NS38118 and National Science Foun- (Schomberg et al., 2001). Embryonic day (E) 17 pregnant rats were anes- dation Career Award IBN9981826 to D.S. Correspondence should be addressed to Dr. Dandan Sun, Department of Neurological Surgery, Medical School, thetized with halothane and killed. Fetuses were removed and rinsed in University of Wisconsin, H4/332, Clinical Sciences Center, 600 Highland Avenue, Madison, WI 53792. E-mail: cold HBSS. The cortices were removed, minced, and rinsed in HBSS. The [email protected]. tissues were treated with 0.2 mg/ml trypsin at 37°C for 25 min. The cells Copyright © 2003 Society for Neuroscience 0270-6474/03/235061-08$15.00/0 were centrifuged briefly, and the pellet was washed and triturated thor- 5062 • J. Neurosci., June 15, 2003 • 23(12):5061–5068 Beck et al. • NKCC1 in Excitotoxicity ␮ oughly in HBSS. Cell suspension was filtered through a 70 m cell ice-cold washing buffer containing (in mM): 118 NaCl, 26 NaHCO3, 1.8 strainer. The cell suspension was diluted in Eagle’s minimal essential CaCl2, pH 7.40. Radioactivity of the cellular extract in 1% SDS was ana- media (EMEM) containing 100 U/ml penicillin and 100 ␮g/ml strepto- lyzed by liquid scintillation counting (Packard 1900CA, Downers Grove, mycin, 10 mM glutamine, 10% fetal bovine serum, and 10% horse serum. IL). In each experiment, specific activities (counts per micromole per 5 Live cells (6.25 ϫ 10 ) were added to each well of a poly-D-lysine-coated minute) of 36Cl were determined for each assay condition and used to 24-well plate and incubated at 37°C in an incubator (model 3110, calculate intracellular Cl Ϫ content (micromole per milligram per pro- Thermo Forma, Marietta, OH) with 5% CO2 and atmospheric air. After tein). Protein content was measured in each sample using the bicincho- 96 hr in culture, cultures were refed with EMEM containing 4 ␮M cyto- ninic acid method (Schomberg et al., 2003). sine arabinofuranoside (Ara-c) for 72 hr. Ara-c is a mitotic inhibitor that Intracellular Naϩ measurement. Intracellular Na ϩ concentration ϩ prevents growth of glial cells. After the Ara-c treatment, cultures were ([Na ]i) was measured with the fluorescent dye sodium binding benzo- refed with EMEM containing both 10% fetal bovine and 10% horse sera. furan isophthalate (SBFI-AM) as described previously (Su et al., 2002a). Astrocytes were prepared from the same cortical cell suspension as Cultured neurons grown on coverslips were loaded with 2.5 ␮M described above with the following changes. The cell suspension was SBFI-AM at room temperature in HEPES–MEM containing 0.05% plu- diluted in EMEM containing 100 U/ml penicillin and 100 ␮g/ml strep- ronic acid for 45 min. The coverslips were placed in an open-bath imag- 4 tomycin, 10 mM glutamine, and 10% fetal bovine serum; 1.2 ϫ 10 live ing chamber (Model RC24, Warner Instruments, Hamden, CT) contain- cells per well were seeded in collagen-coated 24-well plates. ing HEPES–MEM at ambient temperature. To reduce phototoxicity, the OGD treatment. DIV 7–8 and DIV 14–15 neuronal cultures were HEPES–MEM in these experiments was supplemented with (in mM): 1.0 grown in 24-well plates. The cells were rinsed twice with an isotonic OGD pyruvate, 0.4 ascorbic acid, 0.01 troloxC, and 0.1 butylated hydroxyan- solution, pH 7.4, containing (in mM): 0 glucose, 20 NaHCO3, 120 NaCl, isole (Dent et al., 1999). Using a Nikon TE 300 inverted epifluorescence 5.36 KCl, 0.33 Na2HPO4, 0.44 KH2PO4, 1.27 CaCl2, 0.81 MgSO4. Cells microscope and a 40ϫ Super Fluor oil immersion objective lens, neurons were incubated in 0.5 ml of the OGD solution in a hypoxic incubator were excited every 30 sec at 345 and 385 nm, and the emission fluores- (model 3130, Thermo Forma) containing 94% N2,1%O2, and 5% CO2. cence at 510 nm was recorded. Images were collected and analyzed with The oxygen level in the medium of cultured cells in 24-well plates was the MetaFluor image-processing software (Universal Imaging Corpora- monitored with an oxygen probe (model M1-730, Microelectrodes, Bed- tion) as described previously (Su et al., 2002a). The ratios of 340/380 were ford, NH) and decreased to ϳ2–3% after 60 min in the hypoxic incuba- recorded for 10 min in HEPES–MEM, HEPES–MEM plus 1 ␮M MK801, tor. The OGD incubation was 3 hr for DIV 14–15 neurons or 8 hr for DIV or HEPES–MEM plus 5 ␮M bumetanide to establish baselines, and then 7–8 neurons. For reoxygenation, the cells were incubated for 24 hr in 0.5 the bath chamber buffer was changed to HEPES–MEM with 60 ␮M ml of EMEM containing 5.5 mM glucose at 37°C in the incubator with 5% NMDA, 60 ␮M NMDA plus 1 MK801, or 60 ␮M NMDA plus 5 ␮M CO and atmospheric air.
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