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Martha E Odonnell - One of the best experts on this subject based on the ideXlab platform.
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ischemia induced stimulation of cerebral microvascular endothelial cell na k cl cotransport involves p38 and jnk map kinases
American Journal of Physiology-cell Physiology, 2012Co-Authors: Breanna K Wallace, Karen A Jelks, Martha E OdonnellAbstract:Previous studies have provided evidence that, in the early hours of ischemic stroke, a luminal membrane blood-brain barrier (BBB) Na-K-Cl Cotransporter (NKCC) participates in ischemia-induced cerebral edema formation. Inhibition of BBB NKCC activity by intravenous bumetanide significantly reduces edema and infarct in the rat permanent middle cerebral artery occlusion model of ischemic stroke. We demonstrated previously that the BBB Cotransporter is stimulated by hypoxia, aglycemia, and AVP, factors present during cerebral ischemia. However, the underlying mechanisms have not been known. Ischemic conditions have been shown to activate p38 and JNK MAP kinases (MAPKs) in brain, and the p38 and JNK inhibitors SB-239063 and SP-600125, respectively, have been found to reduce brain damage following middle cerebral artery occlusion and subarachnoid hemorrhage, respectively. The present study was conducted to determine whether one or both of these MAPKs participates in ischemic factor stimulation of BBB NKCC activity. Cultured cerebral microvascular endothelial cell NKCC activity was evaluated as bumetanide-sensitive 86Rb influx. Activities of p38 and JNK were assessed by Western blot and immunofluorescence methods using antibodies that detect total vs. phosphorylated (activated) p38 or JNK. We report that p38 and JNK are present in cultured cerebral microvascular endothelial cells and in BBB endothelial cells in situ and that hypoxia (7% O2 and 2% O2), aglycemia, AVP, and O2-glucose deprivation (5- to 120-min exposures) all rapidly activate p38 and JNK in the cells. We also provide evidence that SB-239063 and SP-600125 reduce or abolish ischemic factor stimulation of BBB NKCC activity. These findings support the hypothesis that ischemic factor stimulation of the BBB NKCC involves activation of p38 and JNK MAPKs.
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arginine vasopressin stimulation of cerebral microvascular endothelial cell na k cl Cotransporter activity is v1 receptor and ca dependent
American Journal of Physiology-cell Physiology, 2005Co-Authors: Martha E Odonnell, Vicki Duong, Jimmy Suvatne, Shahin Foroutan, Denise M JohnsonAbstract:Ischemia-induced brain edema formation is mediated by increased transport of Na and Cl across an intact blood-brain barrier (BBB). Our previous studies have provided evidence that a luminally locat...
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bumetanide inhibition of the blood brain barrier na k cl Cotransporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke
Journal of Cerebral Blood Flow and Metabolism, 2004Co-Authors: Martha E Odonnell, Lien Tran, Steven E AndersonAbstract:Increased transport of Na+ across an intact blood-brain barrier (BBB) participates in edema formation during the early hours of cerebral ischemia. In previous studies, the authors showed that the BBB Na-K-Cl Cotransporter is stimulated by factors present during ischemia, suggesting that the Cotransporter may contribute to the increased brain Na+ uptake in edema. The present study was conducted to determine (1) whether the Na-K-Cl Cotransporter is located in the luminal membrane of the BBB, and (2) whether inhibition of the BBB Cotransporter reduces brain edema formation. Perfusion-fixed rat brains were examined for Cotransporter distribution by immunoelectron microscopy. Cerebral edema was evaluated in rats subjected to permanent middle cerebral artery occlusion (MCAO) by magnetic resonance diffusion-weighted imaging and calculation of apparent diffusion coefficients (ADC). The immunoelectron microscopy studies revealed a predominant (80%) luminal membrane distribution of the Cotransporter. Magnetic reson...
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bumetanide inhibition of the blood brain barrier na k cl Cotransporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke
Journal of Cerebral Blood Flow and Metabolism, 2004Co-Authors: Martha E Odonnell, Lien Tran, Tina I Lam, Xiao Bo Liu, Steven E AndersonAbstract:Increased transport of Na+ across an intact blood-brain barrier (BBB) participates in edema formation during the early hours of cerebral ischemia. In previous studies, the authors showed that the BBB Na-K-Cl Cotransporter is stimulated by factors present during ischemia, suggesting that the Cotransporter may contribute to the increased brain Na+ uptake in edema. The present study was conducted to determine (1) whether the Na-K-Cl Cotransporter is located in the luminal membrane of the BBB, and (2) whether inhibition of the BBB Cotransporter reduces brain edema formation. Perfusion-fixed rat brains were examined for Cotransporter distribution by immunoelectron microscopy. Cerebral edema was evaluated in rats subjected to permanent middle cerebral artery occlusion (MCAO) by magnetic resonance diffusion-weighted imaging and calculation of apparent diffusion coefficients (ADC). The immunoelectron microscopy studies revealed a predominant (80%) luminal membrane distribution of the Cotransporter. Magnetic resonance imaging studies showed ADC ratios (ipsilateral MCAO/contralateral control) ranging from 0.577 to 0.637 in cortex and striatum, indicating substantial edema formation. Intravenous bumetanide (7.6-30.4 mg/kg) given immediately before occlusion attenuated the decrease in ADC ratios for both cortex and striatum (by 40-67%), indicating reduced edema formation. Bumetanide also reduced infarct size, determined by TTC staining. These findings suggest that a luminal BBB Na-K-Cl Cotransporter contributes to edema formation during cerebral ischemia.
Bliss Forbush - One of the best experts on this subject based on the ideXlab platform.
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molecular cloning and functional expression of the k cl Cotransporter from rabbit rat and human a new member of the cation chloride Cotransporter family
Journal of Biological Chemistry, 1996Co-Authors: Christopher M Gillen, John A Payne, Susan Brill, Bliss ForbushAbstract:Abstract We report the cloning, sequence analysis, tissue distribution, and functional expression of the K-Cl cotransport protein, KCC1. KCC1 was identified by searching the human expressed sequence tag data base, based on the expectation that it would be distantly related to the Na-K-Cl Cotransporter. Rabbit KCC1 (rbKCC1) and rat KCC1 (rtKCC1) were cloned by screening rabbit kidney and rat brain cDNA libraries using homologous cDNA probes. Human KCC1 (hKCC1) was obtained from I.M.A.G.E. clones and in part by reverse transcription-polymerase chain reaction; it exhibits 97% identity with rbKCC1. KCC1 encodes a 1085-residue polypeptide with substantial sequence homology (24–25% identity) to the bumetanide-sensitive Na-K-Cl Cotransporter (NKCC or BSC) and the thiazide-sensitive Na-Cl Cotransporter (NCC or TSC). Hydropathy analysis of KCC1 indicates structural homology to NKCC, including 12 transmembrane domains, a large extracellular loop with potential N-linked glycosylation sites, and cytoplasmic N- and C-terminal regions. Northern blot analysis revealed a ubiquitously expressed 3.8-kilobase transcript. Much of the genomic sequence of hKCC1 is in the data base, and the gene has been previously localized to 16q22.1 (Larsen, F., Solhein, J., Kristensen, T., Kolsto, A. B., and Prydz, H. (1993) Hum. Mol. Genet. 2, 1589-1595). Epitope-tagged rbKCC1 was stably expressed in human embryonic kidney (HEK 293) cells, resulting in production of a ∼150-kDa glycoprotein. The initial rate of 86Rb efflux from cells expressing rbKCC1 was more than 7 times greater than efflux from control cells and was inhibited by 2 mM furosemide; 86Rb efflux was stimulated by cell swelling. Uptake of 86Rb into rbKCC1 cells after a 15-min pretreatment with 1 mM N-ethylmaleimide was dependent on external chloride but not on external sodium, and was inhibited by furosemide with a Ki of ∼40 μM and by bumetanide with a Ki of ∼60 μM. These data demonstrate that the KCC1 cDNAs encode a widely expressed K-Cl Cotransporter with the characteristics of the K-Cl transporter that has been characterized in red cells.
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primary structure functional expression and chromosomal localization of the bumetanide sensitive na k cl Cotransporter in human colon
Journal of Biological Chemistry, 1995Co-Authors: John A Payne, Melanie Haas, Christian Lytle, David C Ward, Bliss ForbushAbstract:By moving chloride into epithelial cells, the Na-K-Cl Cotransporter aids transcellular movement of chloride across both secretory and absorptive epithelia. Using cDNA probes from the recently identified elasmobranch secretory Na-K-Cl Cotransporter (sNKCC1) (Xu, J. C., Lytle, C. Zhu, T. T., Payne, J. A., Benz, E., and Forbush, B., III (1994) Proc. Natl. Acad. Sci. 91, 2201-2205), we have identified the human homologue. By screening cDNA libraries of a human colonic carcinoma line, T84 cell, we identified a sequence of 4115 bases from overlapping clones. The deduced protein is 1212 amino acids in length, and analysis of the primary structure indicates 12 transmembrane segments. The primary structure is 74% identical to sNKCC1, 91% identical to a mouse Na-K-Cl Cotransporter (mNKCC1), 58% identical to rabbit and rat renal Na-K-Cl Cotransporters (NKCC2), and 43% identical to the thiazide-sensitive Na-Cl Cotransporters from flounder urinary bladder and rat kidney. Similar to sNKCC1 and mNKCC1, the 5'-end of the human colonic Cotransporter is rich in G + C content. Interestingly, a triple repeat (GCG)7 occurs within the 5'-coding region and contributes to a large alanine repeat (Ala15). Two sites for N-linked glycosylation are predicted on an extracellular loop between putative transmembrane segments 7 and 8. A single potential site for phosphorylation by protein kinase A is present in the predicted cytoplasmic C-terminal domain. Northern blot analysis revealed a 7.4-7.5-kilobase transcript in T84 cells and shark rectal gland and a approximately 7.2-kilobase transcript in mammalian colon, kidney, lung, and stomach. Metaphase spreads from lymphocytes were probed with biotin-labeled cDNA and avidin fluorescein (the Cotransporter gene was localized to human chromosome 5 at position 5q23.3). Human embryonic kidney cells stably transfected with the full-length cDNA expressed a approximately 170-kDa protein recognized by anti-Cotransporter antibodies. Following treatment with N-glycosidase F, the molecular mass of the expressed protein was similar to that predicted for the core protein from the cDNA sequence (132-kDa) and identical to that of deglycosylated T84 Cotransporter (approximately 135-kDa). The stably transfected cells exhibited a approximately 15-fold greater bumetanide-sensitive 86Rb influx than control cells, and this flux required external sodium and chloride. Flux kinetics were consistent with an electroneutral cotransport of 1Na:1K:2Cl. Preincubation in chloride-free media was necessary to activate fully the expressed Cotransporter, suggesting a [Cl]-dependent regulatory mechanism.
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molecular cloning and functional expression of the bumetanide sensitive na k cl Cotransporter
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Christian Lytle, John A Payne, Tracey T Zhu, Edward J Benz, Bliss ForbushAbstract:By mediating the coupled movement of Na, K, and Cl ions across the plasma membrane of most animal cells, the bumetanide-sensitive Na-K-Cl Cotransporter (NKCC) plays a vital role in the regulation of ionic balance and cell volume. The transporter is a central element in the process of vectorial salt transport in secretory and absorptive epithelia. A cDNA encoding a Na-K-Cl cotransport protein was isolated from a shark rectal gland library by screening with monoclonal antibodies to the native shark Cotransporter. The 1191-residue protein predicted from the cDNA sequence has 12 putative transmembrane domains flanked by large cytoplasmic N and C termini. Regulatory phosphoacceptor residues in isolated peptides are identified as Thr-189 and Thr-1114 in the predicted sequence. Northern blot analysis identified a 7.4-kb mRNA in rectal gland and most other shark tissues; a 5.2-kb mRNA was restricted to shark kidney. Homology with an uncharacterized gene from Caenorhabditis elegans and with the thiazide-sensitive Na-Cl Cotransporter of flounder urinary bladder was found over most of the coding region; shorter stretches of homology were found with a C. elegans cDNA and with an uncharacterized gene of cyanobacterium. Human HEK-293 cells have been stably transfected with the shark cDNA and shown to express Na-K-Cl cotransport activity with the bumetanide sensitivity of the shark protein. The expressed transporter is functionally quiescent in the host cells and can be activated by depleting the cells of chloride.
Biff Forbush - One of the best experts on this subject based on the ideXlab platform.
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molecular motions involved in na k cl Cotransporter mediated ion transport and transporter activation revealed by internal cross linking between transmembrane domains 10 and 11 12
Journal of Biological Chemistry, 2014Co-Authors: Michelle Y Monette, Suma Somasekharan, Biff ForbushAbstract:We examined the relationship between transmembrane domain (TM) 10 and TM11/12 in NKCC1, testing homology models based on the structure of AdiC in the same transporter superfamily. We hypothesized that introduced cysteine pairs would be close enough for disulfide formation and would alter transport function: indeed, evidence for cross-link formation with low micromolar concentrations of copper phenanthroline or iodine was found in 3 of 8 initially tested pairs and in 1 of 26 additionally tested pairs. Inhibition of transport was observed with copper phenanthroline and iodine treatment of P676C/A734C and I677C/A734C, consistent with the proximity of these residues and with movement of TM10 during the occlusion step of ion transport. We also found Cu2+ inhibition of the single-cysteine mutant A675C, suggesting that this residue and Met382 of TM3 are involved in a Cu2+-binding site. Surprisingly, cross-linking of P676C/I730C was found to prevent rapid deactivation of the transporter while not affecting the dephosphorylation rate, thus uncoupling the phosphorylation and activation steps. Consistent with this, (a) cross-linking of P676C/I730C was dependent on activation state, and (b) mutants lacking the phosphoregulatory domain could still be activated by cross-linking. These results suggest a model of NKCC activation that involves movement of TM12 relative to TM10, which is likely tied to movement of the large C terminus, a process somehow triggered by phosphorylation of the regulatory domain in the N terminus.
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loop diuretic and ion binding residues revealed by scanning mutagenesis of transmembrane helix 3 tm3 of na k cl Cotransporter nkcc1
Journal of Biological Chemistry, 2012Co-Authors: Suma Somasekharan, Jessica E Tanis, Biff ForbushAbstract:The Na-K-Cl Cotransporter (NKCC) plays central roles in cellular chloride homeostasis and in epithelial salt transport, but to date little is known about the mechanism by which the transporter moves ions across the membrane. We examined the functional role of transmembrane helix 3 (TM3) in NKCC1 using cysteine- and tryptophan-scanning mutagenesis and analyzed our results in the context of a structural homology model based on an alignment of NKCC1 with other amino acid polyamine organocation superfamily members, AdiC and ApcT. Mutations of residues along one face of TM3 (Tyr-383, Met-382, Ala-379, Asn-376, Ala-375, Phe-372, Gly-369, and Ile-368) had large effects on translocation rate, apparent ion affinities, and loop diuretic affinity, consistent with a proposed role of TM3 in the translocation pathway. The prediction that Met-382 is part of an extracellular gate that closes to form an occluded state is strongly supported by conformational sensitivity of this residue to 2-(trimethylammonium)ethyl methanethiosulfonate, and the bumetanide insensitivity of M382W is consistent with tryptophan blocking entry of bumetanide into the cavity. Substitution effects on residues at the intracellular end of TM3 suggest that this region is also involved in ion coordination and may be part of the translocation pathway in an inward-open conformation. Mutations of predicted pore residues had large effects on binding of bumetanide and furosemide, consistent with the hypothesis that loop diuretic drugs bind within the translocation cavity. The results presented here strongly support predictions of homology models of NKCC1 and demonstrate important roles for TM3 residues in ion translocation and loop diuretic inhibition.
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intramolecular and intermolecular fluorescence resonance energy transfer in fluorescent protein tagged na k cl Cotransporter nkcc1 sensitivity to regulatory conformational change and cell volume
Journal of Biological Chemistry, 2008Co-Authors: Meike Pedersen, Monica Carmosino, Biff ForbushAbstract:To examine the structure and function of the Na-K-Cl Cotransporter, NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Fluorescent protein tags were added at the N-terminal residue between the regulatory domain and the membrane domain and within a poorly conserved region of the C terminus. Both singly and doubly tagged NKCC1s were appropriately trafficked to the cell membrane and were fully functional; regulation was normal except when YFP was inserted near the regulatory domain, in which case activation occurred only upon incubation with calyculin A. Quenching of YFP fluorescence by Cl(-) provided a ratiometric indicator of intracellular [Cl(-)]. All of the CFP/YFP NKCC pairs exhibited some level of FRET, demonstrating the presence of dimers or higher multimers in functioning NKCC1. With YFP near the regulatory domain and CFP in the C terminus, we recorded a 6% FRET change signaling the regulatory phosphorylation event. On the other hand, when the probe was placed at the extreme N terminus, such changes were not seen, presumably due to the length and predicted flexibility of the N terminus. Substantial FRET changes were observed contemporaneous with cell volume changes, possibly reflective of an increase in molecular crowding upon cell shrinkage.
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pask proline alanine rich ste20 related kinase a regulatory kinase of the na k cl Cotransporter nkcc1
Journal of Biological Chemistry, 2003Co-Authors: Brian F X Dowd, Biff ForbushAbstract:Abstract Although the phosphorylation-dependent activation of the Na-K-Cl Cotransporter (NKCC1) has been previously well documented, the identity of the kinase(s) responsible for this regulation has proven elusive. Recently, Piechotta et al. (Piechotta, K., Lu, J., and Delpire, E. (2002) J. Biol. Chem. 277, 50812–50819) reported the binding of PASK (also referred as SPAK (STE20/SPS1-related proline-alanine-rich kinase)) and OSR1 (oxidative stress response kinase) to cation-chloride Cotransporters KCC3, NKCC1, and NKCC2. In this report, we show that overexpression of a kinase inactive, dominant negative (DN) PASK mutant drastically reduces both shark (60 ± 5%) and human (80 ± 3%) NKCC1 activation. Overexpression of wild type PASK causes a small (sNKCC1 22 ± 8% p < 0.05, hNKCC1 12 ± 3% p < 0.01) but significant increase in shark and human Cotransporter activity in HEK cells. Importantly, DNPASK also inhibits the phosphorylation of two threonines, contained in the previously described N-terminal regulatory domain. We additionally show the near complete restoration of NKCC1 activity in the presence of the protein phosphatase type 1 inhibitor calyculin A, demonstrating that DNPASK inhibition results from an alteration in kinase/phosphatase dynamics rather than from a decrease in functional Cotransporter expression. Coimmunoprecipitation assays confirm PASK binding to NKCC1 in transfected HEK cells and further suggest that this binding is not a regulated event; neither PASK nor NKCC1 activity affects the association. In cells preloaded with 32Pi, the phosphorylation of PASK, but not DNPASK, coincides with that of NKCC1 and increases 5.5 ± 0.36-fold in low [Cl]e. These data conclusively link PASK with the phosphorylation and activation of NKCC1.
Steven E Anderson - One of the best experts on this subject based on the ideXlab platform.
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bumetanide inhibition of the blood brain barrier na k cl Cotransporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke
Journal of Cerebral Blood Flow and Metabolism, 2004Co-Authors: Martha E Odonnell, Lien Tran, Steven E AndersonAbstract:Increased transport of Na+ across an intact blood-brain barrier (BBB) participates in edema formation during the early hours of cerebral ischemia. In previous studies, the authors showed that the BBB Na-K-Cl Cotransporter is stimulated by factors present during ischemia, suggesting that the Cotransporter may contribute to the increased brain Na+ uptake in edema. The present study was conducted to determine (1) whether the Na-K-Cl Cotransporter is located in the luminal membrane of the BBB, and (2) whether inhibition of the BBB Cotransporter reduces brain edema formation. Perfusion-fixed rat brains were examined for Cotransporter distribution by immunoelectron microscopy. Cerebral edema was evaluated in rats subjected to permanent middle cerebral artery occlusion (MCAO) by magnetic resonance diffusion-weighted imaging and calculation of apparent diffusion coefficients (ADC). The immunoelectron microscopy studies revealed a predominant (80%) luminal membrane distribution of the Cotransporter. Magnetic reson...
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bumetanide inhibition of the blood brain barrier na k cl Cotransporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke
Journal of Cerebral Blood Flow and Metabolism, 2004Co-Authors: Martha E Odonnell, Lien Tran, Tina I Lam, Xiao Bo Liu, Steven E AndersonAbstract:Increased transport of Na+ across an intact blood-brain barrier (BBB) participates in edema formation during the early hours of cerebral ischemia. In previous studies, the authors showed that the BBB Na-K-Cl Cotransporter is stimulated by factors present during ischemia, suggesting that the Cotransporter may contribute to the increased brain Na+ uptake in edema. The present study was conducted to determine (1) whether the Na-K-Cl Cotransporter is located in the luminal membrane of the BBB, and (2) whether inhibition of the BBB Cotransporter reduces brain edema formation. Perfusion-fixed rat brains were examined for Cotransporter distribution by immunoelectron microscopy. Cerebral edema was evaluated in rats subjected to permanent middle cerebral artery occlusion (MCAO) by magnetic resonance diffusion-weighted imaging and calculation of apparent diffusion coefficients (ADC). The immunoelectron microscopy studies revealed a predominant (80%) luminal membrane distribution of the Cotransporter. Magnetic resonance imaging studies showed ADC ratios (ipsilateral MCAO/contralateral control) ranging from 0.577 to 0.637 in cortex and striatum, indicating substantial edema formation. Intravenous bumetanide (7.6-30.4 mg/kg) given immediately before occlusion attenuated the decrease in ADC ratios for both cortex and striatum (by 40-67%), indicating reduced edema formation. Bumetanide also reduced infarct size, determined by TTC staining. These findings suggest that a luminal BBB Na-K-Cl Cotransporter contributes to edema formation during cerebral ischemia.
Jason H Huang - One of the best experts on this subject based on the ideXlab platform.
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nkcc1 up regulation contributes to early post traumatic seizures and increased post traumatic seizure susceptibility
Brain Structure & Function, 2017Co-Authors: Fushun Wang, Xiaowei Wang, Lee A Shapiro, Maria L Cotrina, Weimin Liu, Ernest W Wang, Wei Wang, Jason H HuangAbstract:Traumatic brain injury (TBI) is not only a leading cause for morbidity and mortality in young adults (Bruns and Hauser, Epilepsia 44(Suppl 10):210, 2003), but also a leading cause of seizures. Understanding the seizure-inducing mechanisms of TBI is of the utmost importance, because these seizures are often resistant to traditional first- and second-line anti-seizure treatments. The early post-traumatic seizures, in turn, are a contributing factor to ongoing neuropathology, and it is critically important to control these seizures. Many of the available anti-seizure drugs target gamma-aminobutyric acid (GABAA) receptors. The inhibitory activity of GABAA receptor activation depends on low intracellular Cl−, which is achieved by the opposing regulation of Na+–K+–Cl− Cotransporter 1 (NKCC1) and K+–Cl−–Cotransporter 2 (KCC2). Up-regulation of NKCC1 in neurons has been shown to be involved in neonatal seizures and in ammonia toxicity-induced seizures. Here, we report that TBI-induced up-regulation of NKCC1 and increased intracellular Cl− concentration. Genetic deletion of NKCC1 or pharmacological inhibition of NKCC1 with bumetanide suppresses TBI-induced seizures. TGFβ expression was also increased after TBI and competitive antagonism of TGFβ reduced NKKC1 expression, ameliorated reactive astrocytosis, and inhibited seizures. Thus, TGFβ might be an important pathway involved in NKCC1 up-regulation after TBI. Our findings identify neuronal up-regulation of NKCC1 and its mediation by TGFβ, as a potential and important mechanism in the early post-traumatic seizures, and demonstrate the therapeutic potential of blocking this pathway.
