NA-K-Cl cotransporter-1 in the mechanism of cell swelling in cultured astrocytes after fluid...

, , ,

, , ,

*Department of Pathology, University of Miami School of Medicine, Miami, FL, USA

�Department of Biochemistry & Molecular Biology, University of Miami School of Medicine, Miami, FL, USA

�South Florida Foundation for Research & Education Inc., Veterans Affairs Medical Center, Miami, FL, USA

§Diet, Genomics, & Immunology Laboratory, Beltsville Human Nutrition Research Center, United States Department of Agriculture,

Beltsville, MD, USA

¶Laboratory of Integrative Physiology in Veterinary Sciences, Osaka Prefecture University, Osaka, Japan

Traumatic brain injury (TBI) affects approximately 1.5million Americans each year and is a leading cause ofmorbidity and mortality, accounting for as many as 56 000deaths and hundreds of thousands of admissions to intensivecare units per year (Dutton and McCunn 2003). Approxi-mately 200 000 people in the US live with disabilitiesresulting from TBI with immense socioeconomic conse-quences (Kraus 1996; Maas et al. 2000; Marshall 2000;Enriquez and Bullock 2004).

The early development of cerebral edema is a particularlyimportant clinical consequence of TBI since its developmentwill lead to increased intracranial pressure, brain herniationand ultimately death (Marmarou 2003, 2007). While mech-anisms involved in post-traumatic brain edema (TBE) remain

poorly understood, both vasogenic and cytotoxic edemaappear to be involved (Bullock et al. 1991; Ito et al. 1996;Barzo et al. 1997; Unterberg et al. 1997, 2004). During the

Received October 31, 2010; revised manuscript received December 16,2010; accepted January 25, 2011.Address correspondence and reprint requests to Michael D.

Norenberg, MD, Department of Pathology (D-33), University of MiamiSchool of Medicine, P.O. Box 016960, Miami, FL 33101, USA.E-mail: [email protected] used: BCA, bicinchoninic acid; FPI, fluid percussion

injury; ITSs, ion transporting systems; L-NAME, N-nitro-L-argininemethyl ester; NO, nitric oxide; NOS, nitric oxide synthase; ONS, oxi-dative/nitrosative stress; PBN, N-tert-butyl-a-phenylnitrone; p-NKCC1,phosphorylated NKCC1; RT-qPCR, quantitative real-time PCR; TBE,traumatic brain edema; TBI, traumatic brain injury.

Abstract

Brain edema and associated increased intracranial pressure

are major consequences of traumatic brain injury (TBI). An

important early component of the edema associated with TBI

is astrocyte swelling (cytotoxic edema). Mechanisms for such

swelling, however, are poorly understood. Ion channels/

transporters/exchangers play a major role in cell volume

regulation, and a disturbance in one or more of these systems

may result in cell swelling. To examine potential mechanisms

in TBI-mediated brain edema, we employed a fluid percussion

model of in vitro barotrauma and examined the role of the ion

transporter Na+-K+-2Cl)-cotransporter 1 (NKCC1) in trauma-

induced astrocyte swelling as this transporter has been

strongly implicated in the mechanism of cell swelling in various

neurological conditions. Cultures exposed to trauma (3, 4, 5

atm pressure) caused a significant increase in NKCC1 activity

(21%, 42%, 110%, respectively) at 3 h. At 5 atm pressure,

trauma significantly increased NKCC1 activity at 1 h and it

remained increased for up to 3 h. Trauma also increased the

phosphorylation (activation) of NKCC1 at 1 and 3 h. Inhibition

of MAPKs and oxidative/nitrosative stress diminished the

trauma-induced NKCC1 phosphorylation as well as its activity.

Bumetanide, an inhibitor of NKCC1, significantly reduced the

trauma-induced astrocyte swelling (61%). Silencing NKCC1

with siRNA led to a reduction in trauma-induced NKCC1

activity as well as in cell swelling. These findings demonstrate

the critical involvement of NKCC1 in the astrocyte swelling

following in vitro trauma, and suggest that blocking NKCC1

activity may represent a useful therapeutic strategy for the

cytotoxic brain edema associated with the early phase of TBI.

Keywords: astrocytes, bumetanide, cell swelling, Na+-K+-

2Cl)-cotransporter-1, trauma.

J. Neurochem. (2011) 117, 437–448.

JOURNAL OF NEUROCHEMISTRY | 2011 | 117 | 437–448 doi: 10.1111/j.1471-4159.2011.07211.x

� 2011 The AuthorsJournal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 437–448 437

early hours (1–6 h) following trauma cytotoxic brain edemaprevails, with astrocytes being the cells predominantlyaffected (Bullock et al. 1991; Kimelberg 1992; Unterberget al. 1997, 2004). This is in contrast to vasogenic edemawhich usually does not begin until 12–24 h after trauma.Therapy is often not initiated before 6 h, potentially toodelayed to reverse a process that once initiated, progresseswith an inexorably malignant course. This failure to targetthe early cytotoxic swelling may perhaps explain thefrequently reported therapeutic failures of TBE (for review,see Merenda and Bullock 2006).

Various ion transporting systems (ITSs) are largelyresponsible for the maintenance of normal cell volume(Hoffmann 1985; Haas and Forbush 1998; Russell 2000;Kahle et al. 2009). Conversely, several ITSs have beenidentified whose dysfunction appears to mediate astrocyteswelling in several neurological conditions (for reviews, seeChen and Sun 2005; Liang et al. 2007; Kahle et al. 2009).Among these ITSs, the Na+-K+-2Cl) cotransporter-1(NKCC1) plays an important role in cell swelling and brainedema in various neurological conditions, including ischemiaand acute liver failure (for reviews, see Kahle et al. 2009;Jayakumar and Norenberg 2010).

NKCC is a member of a class of membrane proteins thatplays a critical role in the maintenance of intracellular levels ofNa+, K+, and Cl) (Haas 1994). An important function of thiscotransporter is the uptake of K+ from the extracellular space,and it appears to be a major contributor to the regulatoryvolume increase following cell shrinkage (Zhao et al. 2004).However, in conditions associated with elevated extracellularlevels ofK+, such as after ischemia (Chen and Sun 2005; Kahleet al. 2009), TBI (Reinert et al. 2000), and acute hepaticencephalopathy (Sugimoto et al. 1997; Larsen et al. 2001),activation of the cotransporter leads to astrocyte swelling/brainedema. Such edema has been shown to bemarkedly reduced bythe cotransporter inhibitor bumetanide (Su et al. 2002a; Chenand Sun 2005; Jayakumar and Norenberg 2010; Jayakumaret al. 2011). Cultured astrocytes from NKCC1 knockout micehave been reported to exhibit less K+-induced swellingfollowing ischemia, as compared with astrocytes derived fromwild-type mice (Su et al. 2002b). Altogether, these findingsindicate that activation of NKCC1 is an important factor in themediation of cell swelling in various neurological conditions.

Since astrocytes are the cells principally involved in theearly cytotoxic phase of TBE, and since mechanisms forastrocyte swelling in this condition are poorly understood,we investigated the role of NKCC1 in traumatized astrocytesusing an in vitro model of fluid percussion injury (FPI). Ourstudies show that NKCC1 activity is increased after traumato cultured astrocytes, and that blockade of NKCC1 withbumetanide or silencing NKCC1 with siRNA attenuated cellswelling. Additionally, trauma-induced NKCC1 activity andprotein phosphorylation were diminished by inhibitors ofMAPKs and by the antioxidant N-tert-butyl-a-phenylnitrone

(PBN), as well as by the nitric oxide synthase (NOS)inhibitor N-nitro-L-arginine methyl ester (L-NAME), indicat-ing a role of oxidative/nitrosative stress (ONS) and MAPKsin the stimulation of trauma-induced NKCC1 activity. Apreliminary account of these findings has been presented(Jayakumar et al. 2008b).

Materials and methods

Astrocyte culturesAstrocyte cultures were prepared from brains of 1- to 2-day-old rat

pups by the method of Ducis et al. (1990). Briefly, cerebral corticeswere freed of meninges, minced, dissociated by trituration and

vortexing, passed through sterile nylon sieves, placed in Dulbecco’s

modified Eagle’s medium containing penicillin, streptomycin, and

fetal bovine serum, and incubated at 37�C in a humidified chamber

provided with 5% CO2 and 95% air. After 10 days in culture, bovine

serum was replaced with 10% horse serum. After 14 days, cultures

were treated and maintained with dibutyryl-cAMP (Sigma, St Louis,

MO, USA) so as to enhance cell differentiation (Juurlink and Hertz

1985). Cultures consisted of at least 98% astrocytes, as determined

by glial fibrillary acidic protein and glutamine synthetase immuno-

cytochemistry. The remaining cells consisted of microglia. Exper-

iments were carried out in 3- to 4-week-old cells. Procedures

followed guidelines established by the National Institutes of HealthGuide for the Care and use of Laboratory Animals and were

approved by our Institutional Animal Care and Use Committee.

FPI model of in vitro traumaThe fluid percussion instrument employed was initially described by

Sullivan et al. 1976; and subsequently modified for cell culture by

Shepard et al. (1991) and Panickar et al. (2002). Briefly, the fluid

percussion device (60 cm long plexiglass cylinder) was sealed at

one end with a stopper covered on its exposed end with a rubber

pad. A second plexiglass cylinder (7.2 cm long) was connected to

the first cylinder on one end and to a hollow metal injury screw on

the other end. The cylinders were filled with saline. The injury

chamber was coupled to the fluid percussion device with non-

distensible Tygon tubing. Injury was produced when a 4.8-kg

weight attached to a pendulum fell from a specific height. The arc of

the pendulum is marked off in degrees from 0� to 90� and the impact

was measured by a high-resolution pressure transducer at the

coupler of the fluid percussion device (PowerLab system; AD-

Instruments, Inc., Colorado Springs, CO, USA). Pressure within the

piston was recorded and converted to atmospheres (1 atm =

760 mmHg = 14.7 pounds per square inch). Astrocyte cultures

grown in individual 35 mm culture dishes were placed in the injury

chamber, and exposed to different pressure levels of trauma (2, 3, 4,

5 atms), which was created by briefly striking the piston (two

strikes, each 25-ms duration). Sham controls were treated exactly

like the traumatized cells, except that fluid percussion was not

performed.

NKCC1 activityNKCC1 activity was measured as the bumetanide-sensitive K+

influx, using 86Rb as a tracer for K+ by a modification of the method

of Sun and Murali (1999). Briefly, primary astrocyte cultures were

Journal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 437–448� 2011 The Authors

438 | A. R. Jayakumar et al.

traumatized for different time periods (1–3 h). At the end of trauma,

cultures were equilibrated and pre-incubated with or without 50 lM

bumetanide for 15–30 min at 37�C in an isotonic HEPES-minimal

essential medium (140 mM NaCl, 5.36 mM KCl, 0.81 mM MgSO4,

1.27 mM CaCl2, 0.44 mM KH2PO4, 0.33 mM Na2HPO4, 5.55 mM

glucose, and 20 mM HEPES). Cultures were then exposed to 1 lCi/mL 86Rb for 3 min and rinsed with ice-cold 0.1 M MgCl2. Cells

were then extracted in 1% sodium dodecyl sulfate, and the

radioactivity was analyzed by liquid scintillation. The 86Rb influx

rate was calculated by subtracting the influx (with bumetanide) from

total influx (without bumetanide) as described previously (Sun and

Murali 1999), and expressed as nmol of 86Rb/mg of protein/min.

Quadruplicate determinations were obtained throughout the study,

and the protein content was determined by the bicinchoninic acid

(BCA) method (Pierce, Rockford, IL, USA).

Cell volume determinationCell volume was estimated by measuring the intracellular water

space by the method of Kletzien et al. (1975), as modified by

Bender and Norenberg (1998). Briefly, 1 mM 3-O-methylglucose

and 0.5 lCi/mL 3H-labeled 3-O-methylglucose were added to the

culture 6 h before the volume assay. At the end of the incubation

period, the culture medium was aspirated, and an aliquot was saved

for radioactivity determination. Cells were rapidly washed six times

with ice-cold buffer containing 229 mM sucrose, 1 mM Tris nitrate,

0.5 mM calcium nitrate, and 0.1 mM phloretin, pH 7.4. Cells were

then harvested into 0.5 mL of 1 N sodium hydroxide. Radioactivity

in the cell extracts and medium were determined, and an aliquot of

the cell extract was used for protein estimation (BCA method).

Values were normalized to protein level, and the cell volume was

expressed as lL/mg of protein.

Transfection of cultures with siRNAsRat NKCC1 specific siRNA (ON-TARGET plus SMART pool,

catalog# L-096782-01, Gene ID, 83629; Accession nos.: NM

031798; Thermo-Scientific Dharmacon, Lafayette, CO, USA) was

used in this study. Scrambled siRNA (ON-TARGET plus Non-

Targeting Pool, catalog# D-001810-10) was used as control.

Transfection of siRNAs in astrocyte cultures was carried out using

‘TransIT-TKO’ transfection reagent (catalog# MIR 2150; Mirus,

Madison, WI, USA). TransIT-TKO transfection reagent (20 lL) wasdiluted in 250 lL Dulbecco’s modified Eagle’s medium (catalog#

11885-092; Invitrogen-Gibco, Carlsbad, CA, USA) without serum,

and incubated at 37�C for 30 min. The siRNA was diluted in

Dulbecco’s modified Eagle’s medium containing transfection

reagent and incubated in 37�C for another 30 min. The siRNA

and the transfection reagent complex were then diluted in 1.75 mL

of medium, with 10% horse serum (catalog# 26050-088; Invitrogen-

Gibco), and added to the culture plates. Time of transfection and

concentration of siRNA for optimal transfection were determined by

employing different concentrations of siRNA (10–200 nM) and

incubating the transfected cultures for different time periods (24–

96 h). The extent of NKCC1 silencing was confirmed by quanti-

tative real-time PCR (RT-qPCR) and western blots.

RNA isolation and RT-qPCRIsolation of mRNA was performed using RNAqueous�-4PCR kit

(#AM1914; Ambion, Austin, TX, USA) according to manufacturer’s

directions. Following RNA extraction from astrocyte cultures,

cDNA was generated with a High Capacity cDNA Reverse

Transcription Kit (catalog# 4368814; Applied Biosystems, Foster

City, CA, USA), according to manufacturer’s instructions, using

2 lg RNA as a template. The cDNA was diluted 1 : 20 with

Nuclease-Free Water (catalog# 10977-015; Invitrogen-Gibco), ali-

quoted, and stored at )20�C until used. RT-qPCR was done using

10 lL diluted cDNA on the Mx3005P Multiplex Quantitative PCR

System (catalog# 401513; Stratagene/Agilent Technologies, Wil-

mington, DE, USA) using RT-qPCR SYBR GREEN Reagents

(Brilliant� II SYBR� Green QPCR Master Mix; Agilent Technol-

ogies) with ROX reference dye (final reaction volume 25 lL). A2 lM stock solution containing both forward and reverse primer pairs

were reconstituted in Nuclease-Free Water (catalog# 10977-015;

Invitrogen-Gibco) and stored at )20�C. RT-qPCR cycling conditions

were as follows: an initial 95�C for 10 min, followed by 40 cycles of

95�C for 30 s, 58�C for 30 s, 72�C for 15 s. MxPro-Mx3005P v4.10

software (Stratagene/Agilent Technologies, Wilmington, DE, USA)

was used to determine the crossing points for each amplification

reaction. Results were exported to Microsoft Excel for analysis.

NKCC1 mRNA primer pairs (NM_031798; Forward: 5¢-TGAGCC-CTGGCGCATCACAG-3¢, Reverse: 5¢-CGCTGGACACAGCAC-CCTTCC-3¢). All RT-qPCR data were normalized against ribosomal

protein large subunit 13a mRNA (RPL13a, NM_173340; Forward:

5¢-GGCTGAAGCCTACCAGAAAG-3¢, Reverse: 5¢-CTTTGCCTT-TTCCTTCCGTT-3¢).

Western blotsAstrocyte cultures were solubilized in lysis buffer [125 mM Tris-

HCl, pH 6.8, 4% sodium dodecyl sulfate, phosphatase inhibitors

(Sigma)], and a protease inhibitor mixture (Roche Products,

Indianapolis, IN, USA), and protein levels were measured by the

BCA method. Equal amounts of protein were subjected to gel

electrophoresis, as previously described (Jayakumar et al. 2008a),and transferred to nitrocellulose membranes. Following blocking

with non-fat dry milk, membranes were incubated with respective

antibodies. Total NKCC1 antibody (T4) was obtained from the

Developmental Studies Hybridoma Bank developed under the

auspices of the NICHD, National Institutes of Health, and

maintained by the University of Iowa Department of Biological

Sciences (Iowa City, IA, USA). Primary antibody to detect

phosphorylated NKCC1 (p-NKCC1, R5) was a gift from Dr Biff

Forbush, Yale University. Anti-a-tubulin antibody was obtained

from Oncogene (San Diego, CA, USA). Primary antibodies to detect

total and p-NKCC1 as well as the a-tubulin were used at 1 : 1000

dilution. Anti-rabbit and anti-mouse horseradish peroxidase-conju-

gated secondary antibodies (Vector Laboratories, Burlingame, CA,

USA) were used at 1 : 1000 dilution. Optical density of the bands

were determined with the Chemi-Imager (Alpha Innotech, San

Leandro, CA, USA) digital imaging system, and the results were

quantified with the Sigma Scan Pro (Jandell Scientific, San Jose,

CA, USA) program as a proportion of the signal of a housekeeping

protein band (a-tubulin).

Determination of NKCC1 oxidation and nitrationNKCC1 oxidative adducts were determined in NKCC1-immuno-

precipitated samples with the OxyBlotTM protein oxidation detection

kit (S7150; Millipore Corporation, Billerica, MA, USA) as

� 2011 The AuthorsJournal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 437–448

NKCC1 in trauma-induced astrocyte swelling | 439

described previously (Jayakumar et al. 2008a). Lysates were

immunoprecipitated with NKCC1 (T4) antibody and subjected to

gel electrophoresis and immunoblotting as described before. Anti-

nitrotyrosine (mouse monoclonal antibody (1 : 1000 dilution;

catalog# 487923; EMD Biosciences, San Diego, CA, USA) was

used to detect nitrated NKCC1 (T4).

Statistical analysisEach group consisted of four to five culture dishes per experiment

for each time point studied for the cell swelling and NKCC1 activity.

At least two to four plates were used for western blot analysis.

Experiments were performed from 4 to 7 separate seeding. Extent of

cell swelling and NKCC1 activity obtained were normalized to

protein values and subjected to analysis of variance (ANOVA)

followed by Tukey’s post hoc comparisons. Intensity unit values

obtained from optical density of the bands in western blots were also

subjected to ANOVA followed by Tukey’s post hoc comparison test.

At each time point, the experimental cultures were compared with

their respective control.

Results

Effects of different levels of FPI on NKCC1 activityThe activity of NKCC1 was determined by measuring thebumetanide-sensitive K+ influx, using 86Rb as a tracer for K+.Cultures exposed to 2 atm pressure showed a slight, but notstatistically significant increase in NKCC1 activity at 3 h(Fig. 1a). A significant increase in NKCC1 activity wasobserved 3 h after cultures were exposed to 3, 4, 5 atms oftrauma (Fig. 1a).

Time course of NKCC1 activity following FPISince 5 atm pressure gave the most efficient stimulation inNKCC1 activity 3 h after trauma, we chose this pressurelevel in further studies. Astrocyte cultures were exposed toFPI for different time periods (30 min to 3 h). Trauma didnot alter NKCC1 activity at 30 min (Fig. 1b); however,significant increases in NKCC1 activity were observed at 1,2, and 3 h after trauma (38%, 56%, and 121%, respectively,p < 0.05 vs. control; Fig. 1b). Increased NKCC1 activitywas also observed at 6 and 24 h after trauma (71% and74%, respectively, p < 0.05 vs. control) (data not shown inFig. 1).

Phosphorylation of NKCC1 in cultured astrocytes after FPIAs phosphorylation of NKCC1 protein is associated with itsactivation (Gimenez and Forbush 2003), we examinedwhether altered protein phosphorylation contributes to itsincreased activity. We found that NKCC1 phosphorylationwas increased in cultured astrocytes 1 and 3 h after 5 atms ofFPI (Fig. 2).

Oxidation and nitration of NKCC1 by traumaWe previously documented that cultured astrocytes exposedto trauma caused an increase in free radical production(Panickar et al. 2002), and that treatment of cultures withantioxidants or NOS inhibitors reduced trauma-inducedastrocyte swelling (Jayakumar et al. 2008c). Additionally,we previously reported that exposure of cultured astrocytesto ammonia increased the oxidation/nitration of NKCC1 andthat inhibition of NKCC1 oxidation/nitration led to areduction in cell swelling (Jayakumar et al. 2008a). Thesestudies suggested that ONS is a factor in the activation ofNKCC1 and in the mechanism of cell swelling. To examinewhether ONS influences NKCC1 activity following trauma,we examined whether oxidation or nitration of NKCC1occurs in traumatized cultured astrocytes. Cultures exposedto trauma displayed an increase in both oxidized (carbony-lated) and nitrated (nitrotyrosinated) NKCC1 (Fig. 3).Increased oxidation of NKCC1 was detected at 1 h (180%

0

0

20

40

60

80

100

120

C

C 30′ 1 h 2 h 3 h

2 3 4

Atm

5

50NK

CC

1 ac

tivity

(%

con

trol

)N

KC

C1

activ

ity(n

mol

/mg

prot

ein/

min

)

100

150*

**

*†

*

*

200

250(a)

(b)

Fig. 1 (a) Na+-K+-2Cl)-cotransporter 1 (NKCC1) activity increased as

a function of severity of trauma (3 h). (b) Time-dependent NKCC1

activity in traumatized cultured astrocytes (5 atm pressure). NKCC1

activity was first observed at 1 h and it progressively increased for 2

and 3 h after trauma. ANOVA, n = 6 for time-dependent NKCC1 activity

measurement, and n = 5 for NKCC1 activity measurement after dif-

ferent levels of trauma. *p < 0.05 versus control. �p < 0.05 versus 2 h

after trauma. C, control. Error bars represent mean ± SEM.



as compared with control, p < 0.05), which persisted for upto 3 h (Fig. 3a and b). Increase in nitrated NKCC1 was alsoobserved at 1 h (135% vs. control) after which there was a

decline at 2–3 h (Fig. 3c and d). Astrocytes co-treated(immediately after trauma) with the antioxidant PBN(100 lmol/L) or with the NOS inhibitor L-NAME(250 lmol/L) showed a reduction in NKCC1 activity 3 hpost-trauma (Fig. 3e). The concentration of PBN andL-NAME used were selected based on their maximalinhibitory effect on astrocyte swelling following trauma(Jayakumar et al. 2008c). These studies indicate that oxida-tion/nitration of NKCC1 influence its activity after trauma.

Inhibition of trauma-induced p-NKCC1 protein expressionby antioxidants and L-NAMETreatment of cultures (immediately after trauma) with theantioxidants PBN (100 lM), a-tocopherol (Vit-E; 150 lM),and dimethylthiourea (100 lM) significantly decreasedtrauma-induced increase in p-NKCC1 levels after 3 h(76.4%, 71.5%, and 74.2%, respectively; p < 0.05; Fig. 4).Cultures treated with the NOS inhibitor L-NAME (250 lM)also significantly diminished the trauma-induced increase inp-NKCC1 level after 3 h (67.01%; p < 0.05; Fig. 4). Thesefindings strongly suggest that ONS contributes to the trauma-induced NKCC1 phosphorylation.

Role of MAPKs in the phosphorylation of NKCC1Several signaling kinases have been proposed to mediateNKCC1 phosphorylation. We recently documented theactivation of mitogen-activated protein kinases (MAPKs)[extracellular signal-regulated kinase 1/2 (ERK1/2),p38MAPK, and c-Jun N-terminal kinase 1 (JNK1)] inastrocyte cultures after FPI (Jayakumar et al. 2008c), and

Trauma (5 atm)

C 30 min

350

300

250

200

150

50

0 0

0T T + PBN T + L-N

20

40

60

80

100

120

140*

NK

CC

1 ac

tivity

(% o

ver

cont

rol)

100

200

300

400

500

600

C 30 min1 h 2 h 3 h C 30 min1 h 2 h 3 h

**

*

†*

†

††

†100

1 h 2 h 3 h

Trauma (5 atm)

C 30 min 1 h 2 h 3 h

Oxidized NKCC1

Oxi

datio

n of

NK

CC

1

Nitr

atio

n of

NK

CC

1

Nitrated NKCC1

α-tubulin α-tubulin

(a) (c)

(d)

(e)

(b)

Fig. 3 Effect of trauma on Na+-K+-2Cl)-

cotransporter 1 (NKCC1) oxidation/nitration.

Cultured astrocytes were traumatized

(5 atm pressure) for different time periods

and oxidized/nitrated NKCC1 protein was

determined. (a) Western blots show a sig-

nificant increase in oxidized NKCC1 at 1, 2,

and 3 h after trauma (quantification in b). (c)

A significant increase in nitrated NKCC1

protein was observed at 1 h after trauma

(quantification in d). (e) NKCC1 activity in

astrocytes exposed to 5 atm trauma along

with the antioxidant N-tert-butyl-a-phenyl-

nitrone (PBN, 100 lmol/L) and a nitric oxide

synthase inhibitor N-nitro-L-arginine methyl

ester (L-NAME, 250 lmol/L). PBN and

L-NAME significantly reduced NKCC1

activity 3 h after trauma. ANOVA, n = 4 for

oxidation and nitration measurements, and

n = 5 for NKCC1 activity. *p < 0.05 versus

control. �p < 0.05 versus 1 h trauma in (b)

and (d), and 3 h trauma in (e). C, control; T,

Trauma. Error bars represent mean ± SEM.

Contro

l

Traum

a-1 h

Traum

a-3 h

p-NKCC1

α-tubulin

500

*

*

400

300

200

100

01 h

p-N

KC

C1

expr

essi

on (

% o

ver

cont

rol)

3 h

(a)

(b)

Fig. 2 (a) Western blots show a significant increase in phosphorylated

Na+-K+-2Cl)-cotransporter 1 (p-NKCC1) protein expression when

cultures were exposed to 5 atm trauma. (b) Quantification of trauma-

induced changes in p-NKCC1. NKCC1 levels were normalized against

a-tubulin. ANOVA, n = 5. *p < 0.05 versus control. Error bars represent

mean ± SEM.



inhibition of such activation attenuated cell swelling. Wetherefore examined whether activation of MAPKs contributeto increased phosphorylation of NKCC1. Cultures exposed totrauma caused an increase in NKCC1 phosphorylation 3 hafter percussion injury, and such phosphorylation wassignificantly reduced by SB239063 (10 lmol/L) andSP600125 (5 lmol/L) inhibitors of p38-MAPK and JNK,respectively (Fig. 5a and b). Likewise, trauma-inducedincrease in NKCC1 activity was significantly reduced byinhibitors of p38-MAPK and JNK (Fig. 5c). While theERK1/2 inhibitor UO126 inhibited both NKCC1 phosphor-ylation and its activity (24% and 19% reduction, respec-tively), that inhibition was not statistically significantdifferent from that observed after trauma alone. Theconcentrations of MAPK inhibitors were selected based ondose–response studies performed for their maximal inhibi-tory effect on MAPK activation and astrocyte swelling(Jayakumar et al. 2006, 2008c).

NKCC1 protein expression in cultured astrocytes after FPIIn addition to the involvement of phosphorylation inthe activation of NKCC1, increased NKCC1 proteinexpression has also been shown to contribute to its increasedactivity (Su et al. 2002a; Abbruscato et al. 2004; Jayakumaret al. 2008a). However, astrocytes exposed to trauma did notshow any change in total NKCC1 protein expression(Fig. 6), although a significant increase (1.2-fold) in NKCC1mRNA level was observed 3 h after trauma (data notshown).

Contro

l

Trauma

T + P

BN

T + L

-NAM

E

T + V

itE

T + D

MTU

p-NKCC1

α-tubulin

0PBN LN VE DMTU

Trauma

100

p-N

KC

C1

expr

essi

on (

% o

ver

cont

rol)

200

300

*

††

††

Fig. 4 Effect of antioxidants and L-NAME on phosphorylated Na+-K+-

2Cl)-cotransporter 1 (p-NKCC1) protein expression. Astrocytes trea-

ted with PBN, VitE and DMTU all significantly decreased trauma-in-

duced increase in p-NKCC1 levels after 3 h. Cultures treated with the

nitric oxide synthase (NOS) inhibitor L-NAME also significantly dimin-

ished p-NKCC1 levels 3 h after trauma. Data were subjected to ANOVA

(n = 3; *p < 0.05 vs. control; �p < 0.05 vs. trauma). Error bars repre-

sent mean ± SEM. T, Trauma; PBN, N-tert-butyl-a-phenylnitrone; LN

(L-NAME), N-nitro-L-arginine methyl ester; VE (vitamin E, a-tocoph-

erol); DMTU, dimethylthiourea.

Control

Trauma

T + SB

T + UO

T + SP

p-NKCC1

α-tubulin

300 *

*

† †

†

†

250

200

150

100

50

0

0

20

40

60

80

100

120

140

p-N

KC

C1

expr

essi

on(%

ove

r co

ntro

l)N

KC

C1

activ

ity(%

ove

r co

ntro

l)

T T + SB T + UO T + SP

T T + SB T + UO T + SP

(a)

(b)

(c)

Fig. 5 (a) Western blots show phosphorylated Na+-K+-2Cl)-cotrans-

porter 1 (p-NKCC1) expression when cultures were exposed to 5 atm

trauma with MAPK inhibitors. (b) Quantification of trauma-induced

changes in p-NKCC1 protein expression 3 h following trauma. (c)

NKCC1 activity after astrocytes exposed to 5 atm trauma with MAPKs

inhibitors. MAPKs inhibitors significantly reduced NKCC1 activity 3 h

following trauma. Phosphorylated NKCC1 levels were normalized

against a-tubulin. ANOVA, n = 4. *p < 0.05 versus control. �p < 0.05

versus trauma. Error bars represent mean ± SEM. T, trauma; SB,

SB239063 (inhibitor of p38-MAPK); SP, SP600125 (inhibitor of JNK);

UO, UO126 (inhibitor of MEK1/2).



Astrocyte swelling after FPI in cultured astrocytesWe recently documented cell swelling after FPI (5 atmpressure) in cultured astrocytes (Jayakumar et al. 2008c).Cell swelling was observed at 1 h and it continued for up to24 h with the peak increase occurring at 3 h. This timeperiod of cell swelling is similar to that observed inexperimental animals and humans following TBI (Bullocket al. 1991; Kimelberg 1992; Unterberg et al. 1997, 2004).In this study, we first examined the effect of different levelsof traumatic pressure on astrocyte swelling. Cultures exposedto 2 atm pressure and examined 3 h later showed a slight, butnot statistically significant, increase in cell swelling (Fig. 7a).However, a significant increase in cell swelling was observedat 3 h after cultures were exposed to 3, 4, and 5 atm trauma(Fig. 7a). The extent of astrocyte swelling correlated with theseverity of trauma on NKCC1 activity (r = 0.76, p < 0.05;Fig. 1a), as well as with levels of NKCC1 activity (r = 0.79,p < 0.05; Fig. 1b).

Pharmacological inhibition of NKCC1 activity attenuatestrauma-induced astrocyte swellingThe precise significance of NKCC1 activation in cellswelling after trauma is unclear. To examine its potentialrole in trauma-induced astrocyte swelling, we performed a

dose–response curve (25, 50, 100 lM) to establish bumeta-nide’s effect on cell swelling. Cultured astrocytes wereexposed to FPI and then co-treated with or withoutbumetanide. A significant increase in cell volume wasobserved 3 h after trauma (46% as compared with control;p < 0.05). Cell swelling was attenuated by treatment withbumetanide (25, 50, 100 lM; Fig. 7b), with the highestreduction observed with 50 and 100 lM (62% and64%; p < 0.05; Fig. 7b). We previously demonstrated that

Contro

l30

min

1 h 2 h 3 h

Trauma

Total NKCC1

α-tubulin

120

100

80

60

40

20

Tota

l NK

CC

1 pr

otei

n ex

pres

sion

(% in

tens

ity)

0C 30 min 1 h 2 h 3 h

(a)

(b)

Fig. 6 (a) Western blots show no change in total Na+-K+-2Cl)-co-

transporter 1 (NKCC1) protein expression when cultures were ex-

posed to 5 atm trauma. (b) Quantification of trauma-induced changes

in total NKCC1 protein expression. NKCC1 levels were normalized

against a-tubulin. ANOVA, n = 4. C, control. Error bars represent

mean ± SEM.

0

0

10

20

30

40

50

60

Sham

*

†

† †

T 25 µM 50 µM 100 µM

T + bumetanide

2 3 4 5

Atm

20

40

60

80

Cel

l vol

ume

(% o

ver

cont

rol)

Cel

l vol

ume

(% o

ver

cont

rol)

100

*

*

*

120

140

160

180(a)

(b)

Fig. 7 Effect of the severity of trauma and bumetanide on cell swelling

in cultured astrocytes. (a) Cell swelling occurred as a function of

severity of trauma. Cell swelling was first detected at 3 h after expo-

sure to 3 atm barotrauma, and cell volume progressively increased

with greater severity of trauma. (b) Cultures exposed to 5 atm trauma

showed an increase in cell volume at 3 h. Treatment of cultures with

bumetanide significantly reduced the trauma-induced cell swelling.

ANOVA, n = 5. *p < 0.05 versus control; �p < 0.05 versus trauma. T,

Trauma. Error bars represent mean ± SEM.



bumetanide alone (25, 50, 100 lM) had no effect on cellvolume in cultured astrocytes (Jayakumar et al. 2008a). The50 lM bumetanide used in this study was shown to bespecific to NKCC1 (Rose and Ransom 1996).

Effect of silencing NKCC1 gene expression on trauma-induced astrocyte swellingIn addition to pharmacological inhibition, we also inhibitedNKCC1 protein expression using siRNA specific to NKCC1mRNA, and determined whether such reduction inNKCC1 protein expression altered the trauma-inducedNKCC1 activity as well as astrocyte swelling. Astrocytecultures were exposed to different concentrations of NKCC1siRNA (10–200 nM/L final concentration) for different timeperiods (24–96 h), and the extent of mRNA silencing andprotein expression were measured. Cultures exposed toNKCC1 siRNA (25–100 nM) significantly reduced NKCC1mRNA level (Fig. 8), with maximal silencing observed at96 h with 100 nM siRNA (approximately 80% reduction).Similar to mRNA levels, maximal inhibition of NKCC1protein expression was observed with 100 nM siRNA treatedfor 96 h (approximately 60% reduction; Fig. 9). Since100 nM NKCC1 siRNA at the 96 h time point gave themost efficient reduction in NKCC1, we chose this dose andtime point in further studies examining the effects of NKCC1siRNA on NKCC1 activity and cell swelling after trauma. Toestablish whether long-term exposure to transfection reagentscaused cell injury, we also measured the release of lactatedehydrogenase (Koh and Choi 1987) in cultures that wereexposed to 100 nM NKCC1 siRNA or to the transfectionreagent alone for 96 h. Exposure to transfection reagents for96 h showed no lactate dehydrogenase release as comparedwith control cultures (data not shown).

Non-transfected cells exposed to 5 atm trauma caused asignificant increase in NKCC1 activity as well as in cellswelling 3 h after trauma (110% and 41%, respectively, ascompared with sham controls). In contrast, cells transfected

with 100 nM NKCC1 siRNA for 96 h showed a significantreduction in NKCC1 activity (61%), as well as in cellswelling (69%) 3 h after cultures were exposed to trauma(Fig. 10a and b).

Discussion

Our study demonstrates that FPI to cultured astrocytes causeda significant increase in NKCC1 activity as well as enhancedNKCC1 phosphorylation (activation) without any change intotal NKCC1 protein expression. Trauma significantlyincreased astrocyte swelling and such swelling correspondedwell with the level of NKCC1 activity. Bumetanide, aninhibitor of NKCC1, or treatment of cultures with siRNA toNKCC1 significantly reduced trauma-induced astrocyteswelling. These findings are in substantial agreement with areport by Lu et al. 2006, who showed an increase in NKCC1protein expression in the choroid plexus of rat brain aftertrauma, and that bumetanide attenuated the trauma-inducedbrain edema. We additionally found that the antioxidant PBNand the NOS inhibitor L-NAME, as well as inhibitors ofMAPKs, significantly reduced NKCC1 activity, suggestingthat ONS and activation of MAPKs are important factors inthe activation of NKCC1 following trauma to culturedastrocytes. Our findings support a key role of NKCC1 in themechanism of astrocyte swelling/brain edema in TBI.

C0.0

0.2

0.4

NK

CC

1 m

RN

Are

lativ

e fo

ld c

hang

e

0.6

0.8

1.0

1.2

3 day

**

**

*

*

4 day

Scr 25 50 100 C Scr 25 50 100nM siRNA nM siRNA

Fig. 8 Effect of Na+-K+-2Cl)-cotransporter 1 (NKCC1) silencing on

trauma-induced NKCC1 mRNA. A significant decline in NKCC1 mRNA

level was observed at 3 and 4 days after cultures exposed to different

concentrations of NKCC1 siRNA. ANOVA, n = 4. *p < 0.05 versus Scr

(scrambled siRNA). C, control. Error bars represent mean ± SEM.

C Scr

140

* *

*

120

100

80

60

40

20

0C Scr 25 50 100

nM siRNA (4 day)

25 50 100

NKCC1 protein

α-tubulin

nM siRNA (4 day)

NK

CC

1 pr

otei

n ex

pres

sion

(% c

ontr

ol)

(a)

(b)


trauma-induced NKCC1 protein expression. (a) Western blots show a

significant decline in NKCC1 protein expression 4 days after exposure

to different concentrations of NKCC1 siRNA. (b) Quantification of

NKCC1 protein expression after exposure of cultured astrocytes to

NKCC1 siRNA. ANOVA, n = 4. *p < 0.05 versus Scr (scrambled siRNA).

C, control. Error bars represent mean ± SEM.



An important aspect of TBI is the early development ofcytotoxic brain edema (Bullock et al. 1991; Ito et al. 1996;Barzo et al. 1997; Unterberg et al. 1997, 2004). Cytotoxicbrain edema is characterized by sustained intracellular wateraccumulation, predominantly involving astrocytes (Bullocket al. 1991; Kimelberg 1992; Unterberg et al. 1997, 2004;Mongin and Kimelberg 2005). Magnetic resonance imagingin experimental TBI models has shown that cytotoxic edemais clinically significant, especially in the early phase of TBI(2–6 h) (Barzo et al. 1997; Unterberg et al. 2004). While themechanisms responsible for cytotoxic edema in TBI areunclear, various mediators have been implicated, includingglutamate, lactate, Ca2+, nitric oxide (NO), arachidonic acid

and its metabolites, free oxygen/nitrogen radicals, themitochondrial permeability transition, histamine, kinins,and stress responsive kinases (Bullock et al. 1999; Unterberget al. 2004; Mongin and Kimelberg 2005; Marmarou 2007;Norenberg et al. 2007). How all of these factors areintegrated to bring about cell swelling is not clear.

Ion channels, exchangers, and transporters representimportant systems involved in the mechanism of cell volumeregulation (Garty and Benos 1988; Haas and Forbush 1998;Liang et al. 2007; Jayakumar and Norenberg 2010). Struc-tural and functional changes in these systems will result inthe loss of ion homeostasis (Annunziato et al. 2004;Iwamoto 2004; Zima and Blatter 2006) and the subsequentaccumulation of intracellular water. In particular, the activa-tion of the Na+-K+-Cl) cotransporter has been shown tocontribute to cell swelling when inappropriately activated(Kahle et al. 2009; Jayakumar and Norenberg 2010).

Astrocyte cultures exposed to oxygen-glucose deprivationor to a pathophysiological concentration of ammonia havebeen shown to stimulate NKCC1 activity (Kintner et al.2007; Jayakumar et al. 2008a), and cultured astrocytesderived from NKCC knockout mice exhibit less K+-inducedswelling following ischemia as compared with wild-typemice astrocytes (Su et al. 2002b). Increased NKCC1 activitywas observed in edematous rat brains after ischemia andacute liver failure, and such cell swelling/brain edema wasreduced by pre-treatment with the NKCC1 inhibitor, bumet-anide (Yan et al. 2001; Jayakumar and Norenberg 2010).Taken together, these findings support a key role of NKCC1in the mechanism of astrocyte swelling in various neurolog-ical conditions.

While the specific stimuli responsible for NKCC1 activa-tion are incompletely understood, phosphorylation ofNKCC1 is known to increase its activity by promoting itstranslocation to the plasma membrane (Gimenez and Forbush2003). Such phosphorylation was shown to be mediated, inpart, through increased activity of several protein kinases(Wong et al. 2001; Piechotta et al. 2003; Andersen et al.2004; Moriguchi et al. 2005; Akimova et al. 2006; Gagnonet al. 2006; Vitari et al. 2006; Flatman 2008). Among thesekinases included MAPKs, a kinase known to be activated inrat brain after TBI (Otani et al. 2002; Raghupathi et al. 2003;Raghupathi 2004). Additionally, we recently demonstratedthe activation of MAPKs in traumatized astrocyte cultures(Jayakumar et al. 2008c). We now document the phosphor-ylation of NKCC1 in traumatized cultured astrocytes, andthat pharmacological inhibition of p38-MAPK and JNKsignificantly reduced the phosphorylation of NKCC1 as wellas its activity. These findings suggest that activation ofMAPKs is an important factor in the phosphorylation andactivation of NKCC1 in traumatized astrocyte cultures.

Although UO126, an inhibitor of ERK1/2, significantlyreduced cell swelling after trauma (Jayakumar et al. 2008c),inhibition of ERK1/2 did not influence NKCC1 activity. The

0

0

20

40

60

80

100

120

140

160

C

*

* *

*

†

†

Scr T 100 nMsiRNA

C Scr T 100 nMsiRNA

+5 atm Trauma

+5 atm Trauma

20

NK

CC

1 ac

tivity

(nm

ol/m

g pr

otei

n/m

in)

Cel

l vol

ume

(% c

ontr

ol)

40

60

80

100(a)

(b)


trauma-induced NKCC1 activity and cell swelling. (a) Cultures

exposed to trauma caused an increase in NKCC1 activity 3 h after

trauma, and such activation was diminished in NKCC1 siRNA-treated

cells (4 days with 100 nM). (b) Cultures showed an increase in

astrocyte swelling 3 h after trauma. Such swelling was diminished in

NKCC1 siRNA-treated cells (4 days with 100 nM). ANOVA, n = 4.

*p < 0.05 versus Scr (scrambled siRNA); �p < 0.05 versus trauma. C,

control; T, Trauma. Error bars represent mean ± SEM.



reason for failure of UO126 to inhibit NKCC1 activity is notknown. It is possible, however, that activation of ERK1/2may have stimulated various ITSs, other than NKCC1, thatmight have evoked cell swelling after trauma.

In addition to phosphorylation, ONS has been implicatedin the regulation of NKCC1 activity (for review, seeJayakumar and Norenberg 2010). ONS has been hypothe-sized to play an important role in the pathogenesis of TBI(Hall and Braughler 1993; Cherian et al. 2004; Unterberget al. 2004; Darwish et al. 2007; Eghwrudjakpor and Allison2010). Consistent with these observations, we previouslyshowed that cultured astrocytes exposed to trauma caused anincrease in free radical production (Panickar et al. 2002). Wenow show an increase in protein oxidation/nitration ofNKCC1 in traumatized astrocytes, and that treatment ofcultures with antioxidants or with an NOS inhibitor(L-NAME) reduced trauma-induced NKCC1 activity as wellas its phosphorylation.

While increased oxidation of NKCC1 was observed at 1–3 h after trauma, the increase in nitrated NKCC1 wasdetected only at 1 h, after which a decline was observed at 2–3 h. The reason for such decline is not clear. It is possible,however, that trauma-induced nitration may be negativelyregulated by NO after 1 h. This possibility is plausible asVaziri and Wang (1999) showed that increased proteintyrosine nitration negatively regulates NOS thereby resultingin reduced NO concentration. This observation is consistentwith our previous study showing that NO levels peaked at1 h and decreased after 2 and 3 h following trauma incultured astrocytes (Jayakumar et al. 2008c). Additionally,we now show that the NOS inhibitor L-NAME significantlyreduced NKCC1 phosphorylation, as well as its activity 3 hafter trauma, suggesting that increased nitration at 1 hcontributes to the increase in NKCC1 activity, even thoughthe level of nitrated NKCC1 was reduced at later time points(2 and 3 h). It has also been shown that protein nitrationstimulates protein phosphorylation and that such phosphor-ylation persists despite the absence of nitration after its acuterise (Pacher et al. 2007).

The mechanism by which oxidation/nitration of NKCC1leads to its activation is unclear. It is possible that oxidation/nitration causes structural changes in NKCC1, and that suchchanges result in increased NKCC1 activity. Alternatively,oxidation/nitration may facilitate the ability of signalingkinases to phosphorylate NKCC1 (Jayakumar et al. 2008a).It is also possible that ONS mediates the activation ofsignaling kinases that may phosphorylate (activate) NKCC1(Kyriakis and Avruch 2001; Norenberg et al. 2009).

Increased NKCC1 protein expression is also known tocontribute to its increased activity (for review, see Jaya-kumar and Norenberg 2010). However, we did not observeany change in NKCC1 protein expression 3 h after trauma.It appears that increased oxidation and nitration, and/orphosphorylation, represent the primary factors responsible

for the increased NKCC1 activity in traumatized culturedastrocytes.

We earlier showed that astrocyte cultures exposed totrauma induced ONS and activation of MAPK, and thatinhibition of ONS/MAPK reduced cell swelling in culturedastrocytes (Jayakumar et al. 2008c). We now show thattrauma to astrocytes increases oxidation/nitration of NKCC1,and that inhibition of ONS and MAPK reduced NKCC1phosphorylation as well as its activity. Taken together, thesestudies suggest that oxidation/nitration of NKCC1 andactivation of MAPK contributes to the phosphorylation ofNKCC1 as well as to its activity, and that such activationleads to cell swelling in cultured astrocytes after trauma.

In summary, our findings indicate that NKCC1 activity isincreased after trauma to cultured astrocytes, and that suchactivation was mediated, at least in part, through ONS andMAPKs. Additionally, pharmacological inhibition of trauma-induced NKCC1 activation with bumetanide or silencingNKCC1 with siRNA significantly reduced trauma-inducedastrocyte swelling. Our study suggests that activation ofNKCC1 is an important factor in the mediation of astrocyteswelling (cytotoxic brain edema) after trauma, and thatNKCC1 represents a potential therapeutic target for the earlycytotoxic brain edema associated with TBI.

Acknowledgements

This work was supported by a Merit Review from the Department of

Veterans Affairs. We thank Alina Fernandez-Revuelta for the

preparation of cell cultures.

Competing interests

The authors declare that they have no conflicts of interest either

financially or non-financially in this study.

References

Abbruscato T. J., Lopez S. P., Roder K. and Paulson J. R. (2004) Reg-ulation of blood-brain barrier Na,K,2Cl-cotransporter throughphosphorylation during in vitro stroke conditions and nicotineexposure. J. Pharmacol. Exp. Ther. 310, 459–468.

Akimova O. A., Grygorczyk A., Bundey R. A., Bourcier N., Gekle M.,Insel P. A., Orlov S. N. (2006) Transient activation and delayedinhibition of Na+, K+, Cl) cotransport in ATP-treated C11-MDCKcells involve distinct P2Y receptor subtypes and signaling mech-anisms. J. Biol. Chem. 281, 31317–31325.

Andersen G. O., Skomedal T., Enger M., Fidjeland A., Bratteliet J., LevyF. O., Osnes J. B. (2004) 1-AR-mediated activation of NKCC in ratcardiomyocytes involves ERK-dependent phosphorylation of thecotransporter. Am. J. Physiol. Heart Circ. Physiol. 286, H1354–H1360.

Annunziato L., Pignataro G. and Di Renzo G. F. (2004) Pharmacology ofbrain Na+/Ca2+exchanger: from molecular biology to therapeuticperspectives. Pharmacol. Rev. 56, 633–654.

Barzo P., Marmarou A., Fatouros P., Hayasaki K, Corwin F (1997)Contribution of vasogenic and cellular edema to traumatic brain



swelling measured by diffusion-weighted imaging. J. Neurosurg.87, 900–907.

Bender A. S. and Norenberg M. D. (1998) Effect of benzodiazepines andneurosteroids on ammonia-induced swelling in cultured astrocytes.J. Neurosci. Res. 54, 673–680.

Bullock R., Maxwell W. L., Graham D. I., Teasdale G. M., Adams J. H.(1991) Glial swelling following human cerebral contusion: anultrastructural study. J. Neurol. Neurosurg. Psychiatry 54, 427–434.

Bullock M. R., Lyeth B. G. and Muizelaar J. P. (1999) Current status ofneuroprotection trials for traumatic brain injury: lessons fromanimal models and clinical studies. Neurosurgery 45, 207–217.

Chen H. and Sun D. (2005) The role of Na–K–Cl co-transporter incerebral ischemia. Neurol. Res. 27, 280–286.

Cherian L., Hlatky R. and Robertson C. S. (2004) Nitric oxide in trau-matic brain injury. Brain Pathol. 14, 195–201.

Darwish R. S., Amiridze N. and Aarabi B. (2007) Nitrotyrosine as anoxidative stress marker: evidence for involvement in neuro-logic outcome in human traumatic brain injury. J. Trauma 63, 439–442.

Ducis I., Norenberg L. O. B. and Norenberg M. D. (1990) The benzo-diazepine receptor in cultured astrocytes from genetically epilepsy-prone rats. Brain Res. 531, 318–321.

Dutton R. P. and McCunn M. (2003) Traumatic brain injury. Curr. Opin.Crit. Care 9, 503–509.

Eghwrudjakpor P. O. and Allison A. B. (2010) Oxidative stress fol-lowing traumatic brain injury: enhancement of endogenous anti-oxidant defense systems and the promise of improved outcome.Niger. J. Med. 19, 14–21.

Enriquez P. and Bullock R. (2004) Molecular and cellular mechanisms inthe pathophysiology of severe head injury. Curr. Pharm. Des. 10,2131–2143.

Flatman P. W. (2008) Cotransporters, WNKs and hypertension: an up-date. Curr. Opin. Nephrol. Hypertens. 17, 186–192.

Gagnon K. B., England R. and Delpire E. (2006) Characterization ofSPAK and OSR1, regulatory kinases of the Na–K–2Cl cotrans-porter. Mol. Cell. Biol. 26, 689–698.

Garty H. and Benos D. J. (1988) Characteristics and regulatory mech-anisms of the amiloride-blockable Na+ channel. Physiol. Rev. 68,309–373.

Gimenez I. and Forbush B. (2003) Short-term stimulation of the renalNa–K–Cl cotransporter (NKCC2) by vasopressin involves phos-phorylation and membrane translocation of the protein. J. Biol.Chem. 278, 26946–26951.

Haas M. (1994) The Na–K–Cl cotransporters. Am. J. Physiol. 267,C869–C885.

Haas M. and Forbush B. III (1998) The Na–K–Cl co-transporters.J. Bioenerg. Biomembr. 30, 161–172.

Hall E. D. and Braughler J. M. (1993) Free radicals in CNS injury. Res.Publ. Assoc. Res. Nerv. Ment. Dis. 71, 81–105.

Hoffmann E. K. (1985) Role of separate K+ and Cl) channels and ofNa+/Cl) cotransport in volume regulation in Ehrlich cells. Fed.Proc. 44, 2513–2519.

Ito J., Marmarou A., Barzo P., Fatouros P., Corwin F. (1996) Charac-terization of edema by diffusion-weighted imaging in experimentaltraumatic brain injury. J. Neurosurg. 84, 97–103.

Iwamoto T. (2004) Forefront of Na+/Ca2+exchanger studies: molecularpharmacology of Na+/Ca2+exchange inhibitors. J. Pharmacol. Sci.96, 27–32.

Jayakumar A. R. and Norenberg M. D. (2010) The Na-K-Cl Co-trans-porter in astrocyte swelling. Metab. Brain Dis. 25, 31–38.

Jayakumar A. R., Panickar K. S., Murthy Ch. R., Norenberg M. D.(2006) Oxidative stress and mitogen-activated protein kinasephosphorylation mediate ammonia-induced cell swelling and glu-

tamate uptake inhibition in cultured astrocytes. J. Neurosci. 26,4774–4784.

Jayakumar A. R., Liu M., Moriyama M., Ramakrishnan R., Forbush B.3rd, Reddy P. V., Norenberg M. D. (2008a) Na–K–Cl Cotrans-porter-1 in the mechanism of ammonia-induced astrocyte swelling.J. Biol. Chem. 283, 33874–33882.

Jayakumar A. R., Panickar K. S., Moriyama M., Liu M., Norenberg M.D. (2008b) Ion transporting systems in the mechanism of astrocyteswelling after trauma. J. Neurochem. 104, 135–135.

Jayakumar A. R., Rao K. V., Panickar K. S., Moriyama M., Reddy P. V.,Norenberg M. D. (2008c) Trauma-induced cell swelling in culturedastrocytes. J. Neuropathol. Exp. Neurol. 67, 417–427.

Jayakumar A. R., Valdes V. and Norenberg M. D. (2011) The Na-K-Clcotransporter in the brain edema of acute liver failure. J. Hepatol.54, 272–278.

Juurlink B. H. and Hertz L. (1985) Plasticity of astrocytes in primarycultures: an experimental tool and a reason for methodologicalcaution. Dev. Neurosci. 7, 263–277.

Kahle K. T., Simard J. M., Staley K. J., Nahed B. V., Jones P. S., SunD. (2009) Molecular mechanisms of ischemic cerebral edema:role of electroneutral ion transport. Physiology (Bethesda) 24,257–265.

Kimelberg H. K. (1992) Astrocytic edema in CNS trauma. J. Neuro-trauma 9, S71–S81.

Kintner D. B., Luo J., Gerdts J., Ballard A. J., Shull G. E., Sun D. (2007)Role of Na+, K+, Cl) cotransport and Na+/Ca2+exchange in mito-chondrial dysfunction in astrocytes following in vitro ischemia.Am. J. Physiol. Cell Physiol. 292, C1113–C1122.

Kletzien R. F., Pariza M. W., Becker J. E., Potter V. R. (1975) Amethod using 3-O-methyl-D-glucose and phloretin for the deter-mination of intracellular water space of cells in monolayerculture. Ann. Biochem. 68, 537–544.

Koh J. Y. and Choi D. W. (1987) Quantitative determination ofglutamate mediated cortical neuronal injury in cell culture bylactate dehydrogenase efflux assay. J. Neurosci. Methods 20, 83–90.

Kraus J. F. (1996) Epidemiology of head injury, in Neurotrauma(Narayan R. K., Wilberger J. E. and Povlishock J., eds), pp. 12–39.McGraw-Hill, New York.

Kyriakis J. M. and Avruch J. (2001) Mammalian mitogen-activatedprotein kinase signal transduction pathways activated by stress andinflammation. Physiol. Rev. 81, 807–869.

Larsen F. S., Moller K. and Strauss G. (2001) Specific cerebral efflux ofpotassium in patients with fulminant hepatic failure. Liver Transp. l7, C22.

Liang D., Bhatta S., Gerzanich V. and Simard J. M. (2007) Cytotoxicedema: mechanisms of pathological cell swelling. Neurosurg.Focus 22, E2.

Lu K. T., Wu C. Y., Cheng N. C., Wo Y. Y., Yang J. T., Yen H. H., YangY. L. (2006) Inhibition of the Na+-K+-2Cl)-cotransporter in cho-roid plexus attenuates traumatic brain injury-induced brain edemaand neuronal damage. Eur. J. Pharmacol. 548, 99–105.

Maas A. I., Dearden M., Servadei F, Stocchetti N, Unterberg A (2000)Current recommendations for neurotrauma. Curr. Opin. Crit. Care6, 281–292.

Marmarou A. (2003) Pathophysiology of traumatic brain edema: currentconcepts. Acta Neurochir. 86, 7–10.

Marmarou A. (2007) A review of progress in understanding the patho-physiology and treatment of brain edema. Neurosurg. Focus22(E1), 1–10.

Marshall L. F. (2000) Head injury: recent past, present, and future.Neurosurgery 47, 546–561.

Merenda A. and Bullock R. (2006) Clinical treatments for mitochondrialdysfunctions after brain injury. Curr. Opin. Crit. Care 12, 90–96.



Mongin A. A. and Kimelberg H. K. (2005) Astrocyte swelling inneuropathology, in Neuroglia (Kettenmann H. R. B., ed.), pp.550–562. Oxford University Press, New York.

Moriguchi T., Urushiyama S., Hisamoto N., Iemura S., Uchida S.,Natsume T., Matsumoto K., Shibuya H. (2005) WNK1 regulatesphosphorylation of cation-chloride-coupled cotransporters via theSTE20-related kinases, SPAK and OSR1. J. Biol. Chem. 280,42685–42693.

Norenberg M. D., Jayakumar A. R., Rama Rao K. V., Panickar K. S.(2007) New concepts in the mechanism of ammonia-inducedastrocyte swelling. Metab. Brain Dis. 22, 219–234.

Norenberg M. D., Rama Rao K. V. and Jayakumar A. R. (2009) Sig-naling factors in the mechanism of ammonia neurotoxicity. Metab.Brain Dis. 24, 103–117.

Otani N., Nawashiro H., Fukui S., Nomura N., Yano A., Miyazawa T.,Shima K. (2002) Differential activation of mitogen-activated pro-tein kinase pathways after traumatic brain injury in the rat hippo-campus. J. Cereb. Blood Flow Metab. 22, 327–334.

Pacher P., Beckman J. S. and Liaudet L. (2007) Nitric oxide and per-oxynitrite in health and disease. Physiol. Rev. 87, 315–424.

Panickar K., Jayakumar A. R. and Norenberg M. D. (2002) Differentialresponse of neural cells to trauma-induced free radical production.Neurochem. Res. 27, 161–166.

Piechotta K., Garbarini N., England R., Delpire E. (2003) Character-ization of the interaction of the stress kinase SPAK with the Na+,K+, Cl) cotransporter in the nervous system: evidence for a scaf-folding role of the kinase. J. Biol. Chem. 278, 52848–52856.

Raghupathi R. (2004) Cell death mechanisms following traumatic braininjury. Brain Pathol. 14, 215–222.

Raghupathi R., Muir J. K., Fulp C. T., Pittman R. N., McIntosh T. K.(2003) Acute activation of mitogen-activated protein kinasesfollowing traumatic brain injury in the rat: implications for post-traumatic cell death. Exp. Neurol. 183, 438–448.

Reinert M., Khaldi A., Zauner A., Doppenberg E., Choi S., Bullock R.(2000) High extracellular potassium and its correlates after severehead injury: relationship to high intracranial pressure. Neurosurg.Focus 8, e10.

Rose C. R. and Ransom B. R. (1996) Intracellular Na+ homeostasis incultured rat hippocampal astrocytes. J. Physiol. 491, 291–305.

Russell J. M. (2000) Sodium-potassium-chloride co-transport. Physiol.Rev. 80, 211–276.

Shepard S. R., Ghajar J. B. G. et al. (1991) Fluid percussion barotraumachamber: a new in vitro model for traumatic brain injury. J. Surg.Res. 51, 417–424.

Su G., Kintner D. B. and Sun D. (2002a) Contribution of Na+-K+-Cl)

cotransporter to high-[K(+)](o)-induced swelling and EAA releasein astrocytes. Am. J. Physiol. Cell Physiol. 282, C1136–C1146.

Su G., Kintner D. B., Flagella M., Shull G. E., Sun D. (2002b) Astro-cytes from Na+-K+-Cl– cotransporter-null mice exhibit absence ofswelling and decrease in EAA release. Am. J. Physiol. Cell Physiol.282, C1147–C1160.

Sugimoto H., Koehler R. C., Wilson D. A., Brusilow S. W., TraystmanR. J. (1997) Methionine sulfoximine, a glutamine synthetaseinhibitor, attenuates increased extracellular potassium activityduring acute hyperammonemia. J. Cereb. Blood Flow Metab. 17,44–49.

Sullivan H. G., Martinez J. and Becker D. P. (1976) Fluid-percussionmodel of mechanical brain injury in the cat. J. Neurosurg. 45, 521–534.

Sun D. and Murali S. G. (1999) Na+-K+-2Cl) Cotransporter in immaturecortical neurons: a role in intracellular Cl) regulation. J. Neuro-physiol. 81, 1939–1948.

Unterberg A. W., Stroop R., Thomale U. W., Kiening K. L., Pauser S.and Vollmann W. (1997) Characterisation of brain edema following‘‘controlled cortical impact injury’’ in rats. Acta Neurochir. 70,106–108.

Unterberg A. W., Stover J., Kress B., Kiening K. L. (2004) Edema andbrain trauma. Neuroscience 129, 1021–1029.

Vaziri N. D. and Wang X. Q. (1999) cGMP-mediated negative-feedbackregulation of endothelial nitric oxide synthase expression by nitricoxide. Hypertension 34, 1237–1241.

Vitari A. C., Thastrup J., Rafiqi F. H., DeakM.,Morrice N. A., KarlssonH.K., Alessi D. R. (2006) Functional interactions of the SPAK/OSR1kinases with their upstream activator WNK1 and downstream sub-strate NKCC1. Biochem. J. 397, 223–231.

Wong J. A., Gosmanov A. R., Schneider E. G., Thomason D. B. (2001)Insulin-independent, MAPK-dependent stimulation of NKCCactivity in skeletal muscle. Am. J. Physiol. Regul. Integr. Comp.Physiol. 281, R561–R571.

Yan Y. P., Dempsey R. J. and Sun D. (2001) Na+, K+, Cl) co-transporterin rat focal cerebral ischemia. J. Cereb. Blood Flow Metab. 21,711–721.

Zhao H., Hyde R. and Hundal H. (2004) Signalling mechanismsunderlying the rapid and additive stimulation of NKCC activity byinsulin and hypertonicity in rat L6 skeletal muscle cells. J. Physiol.560, 123–136.

Zima A. V. and Blatter L. A. (2006) Redox regulation of cardiac calciumchannels and transporters. Cardiovasc. Res. 71, 310–321.



Date post:	18-Nov-2023
Category:	Documents
Upload:	miamihand
View:	0 times
Download:	0 times

NA-K-Cl cotransporter-1 in the mechanism of cell swelling in cultured astrocytes after fluid...

Documents