DNA demethylation in the hypothalamus promotes transcription of Agtr1a and Slc12a2 and hypertension development.

Increased expression of angiotensin II AT1A receptor (encoded by Agtr1a) and Na+-K+-Cl- cotransporter-1 (NKCC1, encoded by Slc12a2) in the hypothalamic paraventricular nucleus (PVN) contributes to hypertension development. However, little is known about their transcriptional control in the PVN in hypertension. DNA methylation is a critical epigenetic mechanism that regulates gene expression. Here, we determined whether transcriptional activation of Agtr1a and Slc12a2 results from altered DNA methylation in spontaneously hypertensive rats (SHR). Methylated DNA immunoprecipitation and bisulfite sequencing-PCR showed that CpG methylation at Agtr1a and Slc12a2 promoters in the PVN was progressively diminished in SHR compared with normotensive Wistar-Kyoto rats (WKY). Chromatin immunoprecipitation-quantitative PCR revealed that enrichment of DNA methyltransferases (DNMT1 and DNMT3A) and methyl-CpG binding protein 2, a DNA methylation reader protein, at Agtr1a and Slc12a2 promoters in the PVN was profoundly reduced in SHR compared with WKY. By contrast, the abundance of ten-eleven translocation enzymes (TET1-3) at Agtr1a and Slc12a2 promoters in the PVN was much greater in SHR than in WKY. Furthermore, microinjecting of RG108, a selective DNMT inhibitor, into the PVN of WKY increased arterial blood pressure and correspondingly potentiated Agtr1a and Slc12a2 mRNA levels in the PVN. Conversely, microinjection of C35, a specific TET inhibitor, into the PVN of SHR markedly reduced arterial blood pressure, accompanied by a decrease in Agtr1a and Slc12a2 mRNA levels in the PVN. Collectively, our findings suggest that DNA hypomethylation resulting from the DNMT/TET switch at gene promoters in the PVN promotes transcription of Agtr1a and Slc12a2 and hypertension development.

Acorus tatarinowii Schott extract reduces cerebral edema caused by ischemia-reperfusion injury in rats: involvement in regulation of astrocytic NKCC1/AQP4 and JNK/iNOS-mediated signaling.

BACKGROUND: This study aimed to evaluate the effects of the Acorus tatarinowii Schott [Shi Chang Pu (SCP)] extract administered at the start of 2 h of middle cerebral artery occlusion (MCAo), followed by 3 d of reperfusion, and to determine mechanisms involved in anti-edema effects in the penumbra of the cerebral cortex. METHOD: Rats were intraperitoneally administered the SCP extract at a dose of 0.25 g/kg (SCP-0.25 g), 0.5 g/kg (SCP-0.5 g), or 1 g/kg (SCP-1 g) at the start of MCAo. RESULT: SCP-0.5 g and SCP-1 g treatments effectively reduced the cerebral infarct size, ameliorated cerebral edema, reduced blood-brain barrier permeability, and restored neurological function. SCP-0.5 g and SCP-1 g treatments markedly downregulated the levels of glial fibrillary acidic protein, Na+-K+-2Cl- cotransporter type 1 (NKCC1), aquaporin 4 (AQP4), phospho-c-Jun N-terminal kinase (p-JNK)/JNK, inducible nitric oxide synthase (iNOS), 3-nitrotyrosine, intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor-A (VEGF-A), and zonula occluden-1 (ZO-1) and upregulated ZO-3 expression in the penumbra of the cerebral cortex 3 d after reperfusion. CONCLUSIONS: SCP-0.5 g and SCP-1 g treatments exert neuroprotective effects against cerebral infarction and cerebral edema partially by mitigating astrocytic swelling and blood-brain barrier disruption. Moreover, the anti-cerebral edema effects of SCP extract treatments are possibly associated with the downregulation of astrocytic NKCC1/AQP4 and JNK/iNOS-mediated ICAM-1/MMP-9 signaling in the penumbra of the cerebral cortex 3 d after reperfusion.

Blockade of Cell Volume Regulatory Protein NKCC1 Increases TMZ-Induced Glioma Apoptosis and Reduces Astrogliosis.

Glioma is one of the most common primary malignant tumors of the central nervous system accounting for approximately 40% of all intracranial tumors. Temozolomide is a conventional chemotherapy drug for adjuvant treatment of patients with high-risk gliomas, including grade II to grade IV. Our bioinformatic analysis of The Cancer Genome Atlas and Chinese Glioma Genome Atlas datasets and immunoblotting assay show that SLC12A2 gene and its encoded Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) protein are abundantly expressed in grade II-IV gliomas. NKCC1 regulates cell volume and intracellular Cl- concentration, which promotes glioma cell migration, resistance to temozolomide, and tumor-related epilepsy in experimental glioma models. Using mouse syngeneic glioma models with intracranial transplantation of two different glioma cell lines (GL26 and SB28), we show that NKCC1 protein in glioma tumor cells as well as in tumor-associated reactive astrocytes was significantly upregulated in response to temozolomide monotherapy. Combination therapy of temozolomide with the potent NKCC1 inhibitor bumetanide reduced tumor proliferation, potentiated the cytotoxic effects of temozolomide, decreased tumor-associated reactive astrogliosis, and restored astrocytic GLT-1 and GLAST glutamate transporter expression. The combinatorial therapy also led to suppressed tumor growth and prolonged survival of mice bearing GL26 glioma cells. Taken together, these results demonstrate that NKCC1 protein plays multifaceted roles in the pathogenesis of glioma tumors and presents as a therapeutic target for reducing temozolomide-mediated resistance and tumor-associated astrogliosis.

Repeated anodal trans-spinal direct current stimulation results in long-term reduction of spasticity in mice with spinal cord injury.

KEY POINTS: Spasticity is a disorder of muscle tone that is associated with lesions of the motor system. This condition involves an overactive spinal reflex loop that resists the passive lengthening of muscles. Previously, we established that application of anodal trans-spinal direct current stimulation (a-tsDCS) for short periods of time to anaesthetized mice sustaining a spinal cord injury leads to an instantaneous reduction of spasticity. However, the long-term effects of repeated a-tsDCS and its mechanism of action remained unknown. In the present study, a-tsDCS was performed for 7 days and this was found to cause long-term reduction in spasticity, increased rate-dependent depression in spinal reflexes, and improved ground and skill locomotion. Pharmacological, molecular and cellular evidence further suggest that a novel mechanism involving Na-K-Cl cotransporter isoform 1 mediates the observed long-term effects of repeated a-tsDCS. ABSTRACT: Spasticity can cause pain, fatigue and sleep disturbances; restrict daily activities such as walking, sitting and bathing; and complicate rehabilitation efforts. Thus, spasticity negatively influences an individual's quality of life and novel therapeutic interventions are needed. We previously demonstrated in anaesthetized mice that a short period of trans-spinal subthreshold direct current stimulation (tsDCS) reduces spasticity. In the present study, the long-term effects of repeated tsDCS to attenuate abnormal muscle tone in awake female mice with spinal cord injuries were investigated. A motorized system was used to test velocity-dependent ankle resistance and associated electromyographical activity. Analysis of ground and skill locomotion was also performed, with electrophysiological, molecular and cellular studies being conducted to reveal a potential underlying mechanism of action. A 4 week reduction in spasticity was associated with an increase in rate-dependent depression of spinal reflexes, and ground and skill locomotion were improved following 7 days of anodal-tsDCS (a-tsDCS). Secondary molecular, cellular and pharmacological experiments further demonstrated that the expression of K-Cl co-transporter isoform 2 (KCC2) was not changed in animals with spasticity. However, Na-K-Cl cotransporter isoform 1 (NKCC1) was significantly up-regulated in mice that exhibited spasticity. When mice were treated with a-tsDCS, down regulation of NKCC1 was detected, and this level did not significantly differ from that in the non-injured control mice. Thus, long lasting reduction of spasticity by a-tsDCS via downregulation of NKCC1 may constitute a novel therapy for spasticity following spinal cord injury.

Differential regulation of chloride homeostasis and GABAergic transmission in the thalamus.

The thalamus is important for sensory integration with the ventrobasal thalamus (VB) as relay controlled by GABAergic projections from the nucleus reticularis thalami (NRT). Depending on the [Cl-]i primarily set by cation-chloride-cotransporters, GABA is inhibitory or excitatory. There is evidence that VB and NRT differ in terms of GABA action, with classical hyperpolarization in VB due to the expression of the Cl- extruder KCC2 and depolarizing/excitatory GABA action in the NRT, where KCC2 expression is low and Cl- accumulation by the Cl- inward transporter NKCC1 has been postulated. However, data on NKCC1 expression and functional analysis of both transporters are missing. We show that KCC2-mediated Cl- extrusion set the [Cl-]i in VB, while NKCC1 did not contribute substantially to Cl- accumulation and depolarizing GABA action in the NRT. The finding that NKCC1 did not play a major role in NRT neurons is of high relevance for ongoing studies on the therapeutic use of NKCC1 inhibitors trying to compensate for a disease-induced up-regulation of NKCC1 that has been described for various brain regions and disease states like epilepsy and chronic pain. These data suggest that NKCC1 inhibitors might have no major effect on healthy NRT neurons due to limited NKCC1 function.

Bumepamine, a brain-permeant benzylamine derivative of bumetanide, does not inhibit NKCC1 but is more potent to enhance phenobarbital's anti-seizure efficacy.

Based on the potential role of Na-K-Cl cotransporters (NKCCs) in epileptic seizures, the loop diuretic bumetanide, which blocks the NKCC1 isoforms NKCC1 and NKCC2, has been tested as an adjunct with phenobarbital to suppress seizures. However, because of its physicochemical properties, bumetanide only poorly penetrates through the blood-brain barrier. Thus, concentrations needed to inhibit NKCC1 in hippocampal and neocortical neurons are not reached when using doses (0.1-0.5 mg/kg) in the range of those approved for use as a diuretic in humans. This prompted us to search for a bumetanide derivative that more easily penetrates into the brain. Here we show that bumepamine, a lipophilic benzylamine derivative of bumetanide, exhibits much higher brain penetration than bumetanide and is more potent than the parent drug to potentiate phenobarbital's anticonvulsant effect in two rodent models of chronic difficult-to-treat epilepsy, amygdala kindling in rats and the pilocarpine model in mice. However, bumepamine suppressed NKCC1-dependent giant depolarizing potentials (GDPs) in neonatal rat hippocampal slices much less effectively than bumetanide and did not inhibit GABA-induced Ca2+ transients in the slices, indicating that bumepamine does not inhibit NKCC1. This was substantiated by an oocyte assay, in which bumepamine did not block NKCC1a and NKCC1b after either extra- or intracellular application, whereas bumetanide potently blocked both variants of NKCC1. Experiments with equilibrium dialysis showed high unspecific tissue binding of bumetanide in the brain, which, in addition to its poor brain penetration, further reduces functionally relevant brain concentrations of this drug. These data show that CNS effects of bumetanide previously thought to be mediated by NKCC1 inhibition can also be achieved by a close derivative that does not share this mechanism. Bumepamine has several advantages over bumetanide for CNS targeting, including lower diuretic potency, much higher brain permeability, and higher efficacy to potentiate the anti-seizure effect of phenobarbital.

Postnatal Changes in K+/Cl- Cotransporter-2 Expression in the Forebrain of Mice Bearing a Mutant Nicotinic Subunit Linked to Sleep-Related Epilepsy.

The Na+/K+/Cl- cotransporter-1 (NKCC1) and the K+/Cl- cotransporter-2 (KCC2) set the transmembrane Cl- gradient in the brain, and are implicated in epileptogenesis. We studied the postnatal distribution of NKCC1 and KCC2 in wild-type (WT) mice, and in a mouse model of sleep-related epilepsy, carrying the mutant ß2-V287L subunit of the nicotinic acetylcholine receptor (nAChR). In WT neocortex, immunohistochemistry showed a wide distribution of NKCC1 in neurons and astrocytes. At birth, KCC2 was localized in neuronal somata, whereas at subsequent stages it was mainly found in the somatodendritic compartment. The cotransporters' expression was quantified by densitometry in the transgenic strain. KCC2 expression increased during the first postnatal weeks, while the NKCC1 amount remained stable, after birth. In mice expressing ß2-V287L, the KCC2 amount in layer V of prefrontal cortex (PFC) was lower than in the control littermates at postnatal day 8 (P8), with no concomitant change in NKCC1. Consistently, the GABAergic excitatory to inhibitory switch was delayed in PFC layer V of mice carrying ß2-V287L. At P60, the amount of KCC2 was instead higher in mice bearing the transgene. Irrespective of genotype, NKCC1 and KCC2 were abundantly expressed in the neuropil of most thalamic nuclei since birth. However, KCC2 expression decreased by P60 in the reticular nucleus, and more so in mice expressing ß2-V287L. Therefore, a complex regulatory interplay occurs between heteromeric nAChRs and KCC2 in postnatal forebrain. The pathogenetic effect of ß2-V287L may depend on altered KCC2 amounts in PFC during synaptogenesis, as well as in mature thalamocortical circuits.

Actions of Quercetin, a Polyphenol, on Blood Pressure.

Disorder of blood pressure control causes serious diseases in the cardiovascular system. This review focuses on the anti-hypertensive action of quercetin, a flavonoid, which is one of the polyphenols characterized as the compounds containing large multiples of phenol structural units, by varying the values of various blood pressure regulatory factors, such as vascular compliance, peripheral vascular resistance, and total blood volume via anti-inflammatory and anti-oxidant actions. In addition to the anti-inflammatory and anti-oxidant actions of quercetin, we especially describe a novel mechanism of quercetin's action on the cytosolic Cl- concentration ([Cl-]c) and novel roles of the cytosolic Cl- i.e.: (1) quercetin elevates [Cl-]c by activating Na⁺-K⁺-2Cl- cotransporter 1 (NKCC1) in renal epithelial cells contributing to Na⁺ reabsorption via the epithelial Na⁺ channel (ENaC); (2) the quercetin-induced elevation of [Cl-]c in renal epithelial cells diminishes expression of ENaC leading to a decrease in renal Na⁺ reabsorption; and (3) this reduction of ENaC-mediated Na⁺ reabsorption in renal epithelial cells drops volume-dependent elevated blood pressure. In this review, we introduce novel, unique mechanisms of quercetin's anti-hypertensive action via activation of NKCC1 in detail.

Astaxanthin alleviates cerebral edema by modulating NKCC1 and AQP4 expression after traumatic brain injury in mice.

BACKGROUND: Astaxanthin is a carotenoid pigment that possesses potent antioxidative, anti-inflammatory, antitumor, and immunomodulatory activities. Previous studies have demonstrated that astaxanthin displays potential neuroprotective properties for the treatment of central nervous system diseases, such as ischemic brain injury and subarachnoid hemorrhage. This study explored whether astaxanthin is neuroprotective and ameliorates neurological deficits following traumatic brain injury (TBI). RESULTS: Our results showed that, following CCI, treatment with astaxanthin compared to vehicle ameliorated neurologic dysfunctions after day 3 and alleviated cerebral edema and Evans blue extravasation at 24 h (p < 0.05). Astaxanthin treatment decreased AQP4 and NKCC1 mRNA levels in a dose-dependent manner at 24 h. AQP4 and NKCC1 protein expressions in the peri-contusional cortex were significantly reduced by astaxanthin at 24 h (p < 0.05). Furthermore, we also found that bumetanide (BU), an inhibitor of NKCC1, inhibited trauma-induced AQP4 upregulation (p < 0.05). CONCLUSIONS: Our data suggest that astaxanthin reduces TBI-related injury in brain tissue by ameliorating AQP4/NKCC1-mediated cerebral edema and that NKCC1 contributes to the upregulation of AQP4 after TBI.

A novel GABA-mediated corticotropin-releasing hormone secretory mechanism in the median eminence.

Corticotropin-releasing hormone (CRH), which is synthesized in the paraventricular nucleus (PVN) of the hypothalamus, plays an important role in the endocrine stress response. The excitability of CRH neurons is regulated by γ-aminobutyric acid (GABA)-containing neurons projecting to the PVN. We investigated the role of GABA in the regulation of CRH release. The release of CRH was impaired, accumulating in the cell bodies of CRH neurons in heterozygous GAD67-GFP (green fluorescent protein) knock-in mice (GAD67(+/GFP)), which exhibited decreased GABA content. The GABAA receptor (GABAAR) and the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), but not the K(+)-Cl(-) cotransporter (KCC2), were expressed in the terminals of the CRH neurons at the median eminence (ME). In contrast, CRH neuronal somata were enriched with KCC2 but not with NKCC1. Thus, intracellular Cl(-) concentrations ([Cl(-)]i) may be increased at the terminals of CRH neurons compared with concentrations in the cell body. Moreover, GABAergic terminals projecting from the arcuate nucleus were present in close proximity to CRH-positive nerve terminals. Furthermore, a GABAAR agonist increased the intracellular calcium (Ca(2+)) levels in the CRH neuron terminals but decreased the Ca(2+) levels in their somata. In addition, the increases in Ca(2+) concentrations were prevented by an NKCC1 inhibitor. We propose a novel mechanism by which the excitatory action of GABA maintains a steady-state CRH release from axon terminals in the ME.

Mechanism involved in Danshen-induced fluid secretion in salivary glands.

AIM: Danshen's capability to induce salivary fluid secretion and its mechanisms were studied to determine if it could improve xerostomia. METHODS: Submandibular glands were isolated from male Wistar rats under systemic anesthesia with pentobarbital sodium. The artery was cannulated and vascularly perfused at a constant rate. The excretory duct was also cannulated and the secreted saliva was weighed in a cup on an electronic balance. The weight of the accumulated saliva was measured every 3 s and the salivary flow rate was calculated. In addition, the arterio-venous difference in the partial oxygen pressure was measured as an indicator of oxygen consumption. In order to assess the mechanism involved in Danshen-induced fluid secretion, either ouabain (an inhibitor of Na(+)/K(+) ATPase) or bumetanide (an inhibitor of NKCC1) was additionally applied during the Danshen stimulation. In order to examine the involvement of the main membrane receptors, atropine was added to block the M3 muscarinic receptors, or phentolamine was added to block the α1 adrenergic receptors. In order to examine the requirement for extracellular Ca(2+), Danshen was applied during the perfusion with nominal Ca(2+) free solution. RESULTS: Although Danshen induced salivary fluid secretion, 88.7 ± 12.8 µL/g-min, n = 9, (the highest value around 20 min from start of DS perfusion was significantly high vs 32.5 ± 5.3 µL/g-min by carbamylcholine, P = 0.00093 by t-test) in the submandibular glands, the time course of that secretion differed from that induced by carbamylcholine. There was a latency associated with the fluid secretion induced by Danshen, followed by a gradual increase in the secretion to its highest value, which was in turn followed by a slow decline to a near zero level. The application of either ouabain or bumetanide inhibited the fluid secretion by 85% or 93%, and suppressed the oxygen consumption by 49% or 66%, respectively. These results indicated that Danshen activates Na(+)/K(+) ATPase and NKCC1 to maintain Cl(-) release and K(+) release for fluid secretion. Neither atropine or phentolamine inhibited the fluid secretion induced by Danshen (263% ± 63% vs 309% ± 45%, 227% ± 63% vs 309% ± 45%, P = 0.899, 0.626 > 0.05 respectively, by ANOVA). Accordingly, Danshen does not bind with M3 or α1 receptors. These characteristics suggested that the mechanism involved in DS-induced salivary fluid secretion could be different from that induced by carbamylcholine. Carbamylcholine activates the M3 receptor to release inositol trisphosphate (IP3) and quickly releases Ca(2+) from the calcium stores. The elevation of [Ca(2+)]i induces chloride release and quick osmosis, resulting in an onset of fluid secretion. An increase in [Ca(2+)]i is essential for the activation of the luminal Cl(-) and basolateral K(+) channels. The nominal removal of extracellular Ca(2+) totally abolished the fluid secretion induced by Danshen (1.8 ± 0.8 µL/g-min vs 101.9 ± 17.2 µL/g-min, P = 0.00023 < 0.01, by t-test), suggesting the involvement of Ca(2+) in the activation of these channels. Therefore, IP3-store Ca(2+) release signalling may not be involved in the secretion induced by Danshen, but rather, there may be a distinct signalling process. CONCLUSION: The present findings suggest that Danshen can be used in the treatment of xerostomia, to avoid the systemic side effects associated with muscarinic drugs.

Ammonia exposure increases the expression of Na(+):K (+):2Cl (-) cotransporter 1a in the gills of the giant mudskipper, Periophthalmodon schlosseri.

The giant mudskipper, Periophthalmodon schlosseri, is an obligate air-breathing teleost that can actively excrete ammonia against high concentrations of environmental ammonia. This study aimed to clone and sequence the Na (+) :K (+) :2Cl (-) cotransporter 1 (nkcc1) from the gills of P. schlosseri, and to determine the effects of ammonia exposure on its mRNA expression and protein abundance after pre-acclimation to slightly brackish water (salinity 3; SBW) for 2 weeks. The complete coding cDNA sequences of nkcc1a consisted of 3453 bp, coding for 1151 amino acid with an estimated molecular mass of 125.4 kDa. Exposure to 75 mmol l(-1) NH4Cl in SBW had no effect on the mRNA expression of nkcc1a. However, western blotting revealed a significant increase in the protein abundance of multiple T4-immunoreactive bands of molecular mass 170-250 kDa in the gills of P. schlosseri exposed to ammonia. Furthermore, immunofluorescence microscopy demonstrated the colocalization of the increased T4-immunoreactive protein with Na(+)/K(+)-ATPase (Nka) α-subunit to the basolateral membrane of certain ionocytes in the gills of the ammonia-exposed fish. As Nkcc1 is known to have a basolateral localization, it can be concluded that ammonia exposure led to an increase in the expression of glycosylated Nkcc1, the molecular masses of which were reduced upon enzymatic deglycosylation, in the gills of P. schlosseri. The dependency on post-transcriptional and post-translational regulation of branchial Nkcc1 in P. schlosseri would facilitate prompt responses to changes in environmental condition. As NH4 (+) can replace K(+), NH4 (+) could probably enter ionocytes through the basolateral Nkcc1a during active ammonia excretion, but increased influx of Na(+), NH4 (+) and 2Cl(-) would alter the transmembrane Na(+) gradient. Consequently, exposure of P. schlosseri to ammonia would also result in an increase in branchial activity of Nka with decreased NH4 (+) affinity so as to maintain intracellular Na(+) and K(+) homeostasis as reported elsewhere.

BDNF modifies hippocampal KCC2 and NKCC1 expression in a temporal lobe epilepsy model.

Excitatory GABA actions, induced by altered expression of chloride transporters (KCC2/NKCC1), can contribute to seizure generation in temporal lobe epilepsy. In the present study, we evaluated whether BDNF administration can affect KCC2/NKCC1 expression, ictogenesis and behavioral alterations in this paradigm. Status epilepticus was induced in male rats with pilocarpine, followed by a treatment of either a single high dose or multiple injections of BDNF during the latent phase of temporal lobe epilepsy. Chloride transporters expression, spontaneous recurrent seizures, and hyperexcitability post-seizural behaviors were evaluated after treatment. NKCC1 protein expression was markedly upregulated, whereas that of KCC2 was significantly downregulated in epileptic hippocampi compared to intact controls. Application of BDNF (both single high dose and multiple injections) increased KCC2 expression in epileptic hippocampi, while NKCC1 expression was downregulated exclusively by the single high dose injection of BDNF. Development of spontaneous recurrent seizures was delayed but not prevented by the treatment, and hyperexcitability behaviors were ameliorated for a short period of time. To prevent GABA-A mediated depolarization and design appropriate treatment strategies for temporal lobe epilepsy, chloride transporters can be considered as a target. Future studies are warranted to investigate any possible therapeutic effects of BDNF via altering chloride transporters expression.

Contributions of the Na⁺/K⁺-ATPase, NKCC1, and Kir4.1 to hippocampal K⁺ clearance and volume responses.

Network activity in the brain is associated with a transient increase in extracellular K(+) concentration. The excess K(+) is removed from the extracellular space by mechanisms proposed to involve Kir4.1-mediated spatial buffering, the Na(+)/K(+)/2Cl(-) cotransporter 1 (NKCC1), and/or Na(+)/K(+)-ATPase activity. Their individual contribution to [K(+)]o management has been of extended controversy. This study aimed, by several complementary approaches, to delineate the transport characteristics of Kir4.1, NKCC1, and Na(+)/K(+)-ATPase and to resolve their involvement in clearance of extracellular K(+) transients. Primary cultures of rat astrocytes displayed robust NKCC1 activity with [K(+)]o increases above basal levels. Increased [K(+)]o produced NKCC1-mediated swelling of cultured astrocytes and NKCC1 could thereby potentially act as a mechanism of K(+) clearance while concomitantly mediate the associated shrinkage of the extracellular space. In rat hippocampal slices, inhibition of NKCC1 failed to affect the rate of K(+) removal from the extracellular space while Kir4.1 enacted its spatial buffering only during a local [K(+)]o increase. In contrast, inhibition of the different isoforms of Na(+)/K(+)-ATPase reduced post-stimulus clearance of K(+) transients. The astrocyte-characteristic α2ß2 subunit composition of Na(+)/K(+)-ATPase, when expressed in Xenopus oocytes, displayed a K(+) affinity and voltage-sensitivity that would render this subunit composition specifically geared for controlling [K(+)]o during neuronal activity. In rat hippocampal slices, simultaneous measurements of the extracellular space volume revealed that neither Kir4.1, NKCC1, nor Na(+)/K(+)-ATPase accounted for the stimulus-induced shrinkage of the extracellular space. Thus, NKCC1 plays no role in activity-induced extracellular K(+) recovery in native hippocampal tissue while Kir4.1 and Na(+)/K(+)-ATPase serve temporally distinct roles.

Functional expression of human NKCC1 from a synthetic cassette-based cDNA: introduction of extracellular epitope tags and removal of cysteines.

The Na-K-Cl cotransporter (NKCC) couples the movement of Na(+), K(+), and Cl(-) ions across the plasma membrane of most animal cells and thus plays a central role in cellular homeostasis and human physiology. In order to study the structure, function, and regulation of NKCC1 we have engineered a synthetic cDNA encoding the transporter with 30 unique silent restriction sites throughout the open reading frame, and with N-terminal 3xFlag and YFP tags. We show that the novel cDNA is appropriately expressed in HEK-293 cells and that the YFP-tag does not alter the transport function of the protein. Utilizing the Cl(-) -sensing capability of YFP, we demonstrate a sensitive assay of Na-K-Cl cotransport activity that measures normal cotransport activity in a fully activated transporter. In addition we present three newly developed epitope tags for NKCC1 all of which can be detected from outside of the cell, one of which is very efficiently delivered to the plasma membrane. Finally, we have characterized cysteine mutants of NKCC1 and found that whereas many useful combinations of cysteine mutations are tolerated by the biosynthetic machinery, the fully "cys-less" NKCC1 is retained in the endoplasmic reticulum. Together these advances are expected to greatly assist future studies of NKCC1.

High brain ammonia tolerance and down-regulation of Na+:K+:2Cl(-) Cotransporter 1b mRNA and protein expression in the brain of the Swamp Eel, Monopterus albus, exposed to environmental ammonia or terrestrial conditions.

Na(+):K(+):2Cl(-) cotransporter 1 (NKCC1) has been implicated in mediating ischemia-, trauma- or ammonia-induced astrocyte swelling/brain edema in mammals. This study aimed to determine the effects of ammonia or terrestrial exposure on ammonia concentrations in the plasma and brain, and the mRNA expression and protein abundance of nkcc/Nkcc in the brain, of the swamp eel Monopterusalbus. Ammonia exposure led to a greater increase in the ammonia concentration in the brain of M. albus than terrestrial exposure. The brain ammonia concentration of M. albus reached 4.5 µmol g(-1) and 2.7 µmol g(-1) after 6 days of exposure to 50 mmol l(-1) NH4Cl and terrestrial conditions, respectively. The full cDNA coding sequence of nkcc1b from M. albus brain comprised 3276 bp and coded for 1092 amino acids with an estimated molecular mass of 119.6 kDa. A molecular characterization indicated that it could be activated through phosphorylation and/or glycosylation by osmotic and/or oxidative stresses. Ammonia exposure for 1 day or 6 days led to significant decreases in the nkcc1b mRNA expression and Nkcc1b protein abundance in the brain of M. albus. In comparison, a significant decrease in nkcc1b mRNA expression was observed in the brain of M. albus only after 6 days of terrestrial exposure, but both 1 day and 6 days of terrestrial exposure resulted in significant decreases in the protein abundance of Nkcc1b. These results are novel because it has been established in mammals that ammonia up-regulates NKCC1 expression in astrocytes and NKCC1 plays an important role in ammonia-induced astrocyte swelling and brain edema. By contrast, our results indicate for the first time that M. albus is able to down-regulate the mRNA and protein expression of nkcc1b/Nkcc1b in the brain when confronted with ammonia toxicity, which could be one of the contributing factors to its extraordinarily high brain ammonia tolerance.

Activations of GABAergic signaling, HSP70 and MAPK cascades are involved in baicalin's neuroprotection against gerbil global ischemia/reperfusion injury.

Baicalin, a flavonoid compound isolated from the plant Scutellaria baicalensis Georgi, is known as a protective agent against delayed neuronal cell death after ischemia/reperfusion. To investigate the neuroprotective mechanism of baicalin, the present study was conducted to explore whether the alterations of GABAergic signaling, heat shock protein 70 (HSP70) and mitogen-activated protein kinases (MAPKs) were involved in its neuroprotection on gerbils global ischemia. The bilateral carotid arteries were occluded by 5 min and baicalin at the dose of 200 mg/kg was intraperitoneally injected into the gerbils immediately after cerebral ischemia. Seven days after reperfusion, neurological deficit was scored and changes in hippocampal neuronal cell death were assessed by Nissl staining as well as NeuN immunohistochemistry. The mRNA and protein expressions of GABAergic signal molecules (GABA(A)R α1, GABA(A)R γ2, KCC2 and NKCC1) were determined in ischemic hippocampus by real-time RT-PCR and Western blot, respectively. In addition, HSP70 and MAPKs cascades (ERK, JNK and p38) were also detected using western blot assay. Our results illustrated that baicalin treatment significantly facilitated neurological function, suppressed the ischemia-induced neuronal damage. Besides, administration of baicalin also caused a striking increase of GABA(A)R α1, GABA(A)R γ2 and KCC2 together with the decrease of NKCC1 at mRNA and protein levels in gerbils hippocampus following an ischemic insult. Furthermore, the protein expressions of HSP70 and phosphorylated ERK (p-ERK) were evidently augmented while the phosphorylated JNK (p-JNK) and phosphorylated p38 (p-p38) were strikingly diminished in ischemic gerbils with baicalin treatment. These findings suggest that baicalin activates GABAergic signaling, HSP70 and MAPKs cascades in global ischemia, which may be a mechanism underlying the baicalin's neuroprotection.

GABAergic excitation after febrile seizures induces ectopic granule cells and adult epilepsy.

Temporal lobe epilepsy (TLE) is accompanied by an abnormal location of granule cells in the dentate gyrus. Using a rat model of complex febrile seizures, which are thought to be a precipitating insult of TLE later in life, we report that aberrant migration of neonatal-generated granule cells results in granule cell ectopia that persists into adulthood. Febrile seizures induced an upregulation of GABA(A) receptors (GABA(A)-Rs) in neonatally generated granule cells, and hyperactivation of excitatory GABA(A)-Rs caused a reversal in the direction of granule cell migration. This abnormal migration was prevented by RNAi-mediated knockdown of the Na(+)K(+)2Cl(-) co-transporter (NKCC1), which regulates the excitatory action of GABA. NKCC1 inhibition with bumetanide after febrile seizures rescued the granule cell ectopia, susceptibility to limbic seizures and development of epilepsy. Thus, this work identifies a previously unknown pathogenic role of excitatory GABA(A)-R signaling and highlights NKCC1 as a potential therapeutic target for preventing granule cell ectopia and the development of epilepsy after febrile seizures.

NKCC1 upregulation disrupts chloride homeostasis in the hypothalamus and increases neuronal activity-sympathetic drive in hypertension.

Hypertension is a major risk factor for coronary artery disease, stroke, and kidney failure. However, the etiology of hypertension in most patients is poorly understood. Increased sympathetic drive emanating from the hypothalamic paraventricular nucleus (PVN) plays a major role in the development of hypertension. Na(+)-K(+)-2Cl(-) cotransporter-1 (NKCC1) in the brain is critically involved in maintaining chloride homeostasis and in neuronal responses mediated by GABA(A) receptors. Here we present novel evidence that the GABA reversal potential (E(GABA)) of PVN presympathetic neurons undergoes a depolarizing shift that diminishes GABA inhibition in spontaneously hypertensive rats (SHRs). Inhibition of NKCC1, but not KCC2, normalizes E(GABA) and restores GABA inhibition of PVN neurons in SHRs. The mRNA and protein levels of NKCC1, but not KCC2, in the PVN are significantly increased in SHRs, and the NKCC1 proteins on the plasma membrane are highly glycosylated. Inhibiting NKCC1 N-glycosylation restores E(GABA) and GABAergic inhibition of PVN presympathetic neurons in SHRs. Furthermore, NKCC1 inhibition significantly reduces the sympathetic vasomotor tone and augments the sympathoinhibitory responses to GABA(A) receptor activation in the PVN in SHRs. These findings suggest that increased NKCC1 activity and glycosylation disrupt chloride homeostasis and impair synaptic inhibition in the PVN to augment the sympathetic drive in hypertension. This information greatly improves our understanding of the pathogenesis of hypertension and helps to design better treatment strategies for neurogenic hypertension.

Kinases SPAK and OSR1 are upregulated by estradiol and activate NKCC1 in the developing hypothalamus.

In immature neurons the amino acid neurotransmitter, GABA provides the dominant mode for neuronal excitation by inducing membrane depolarization due to Cl(-) efflux through GABA(A) receptors (GABA(A)Rs). The driving force for Cl(-) is outward because the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) elevates the Cl(-) concentration in these cells. GABA-induced membrane depolarization and the resulting activation of voltage-gated Ca(2+) channels is fundamental to normal brain development, yet the mechanisms that regulate depolarizing GABA are not well understood. The neurosteroid estradiol potently augments depolarizing GABA action in the immature hypothalamus by enhancing the activity of the NKCC1 cotransporter. Understanding how estradiol controls NKCC1 activity will be essential for a complete understanding of brain development. We now report that estradiol treatment of newborn rat pups significantly increases protein levels of two kinases upstream of the NKCC1 cotransporter, SPAK (STE20/SPS1-related proline alanine rich kinase) and OSR1 (oxidative stress response kinase). The estradiol-induced increase is transcription dependent, and its time course parallels that of estradiol-enhanced phosphorylation of NKCC1. Antisense oligonucleotide-mediated knockdown of SPAK, and to a lesser degree of OSR1, precludes estradiol-mediated enhancement of NKCC1 phosphorylation. Functionally, knockdown of SPAK or OSR1 in embryonic hypothalamic cultures diminishes estradiol-enhanced Ca(2+) influx induced by GABA(A)R activation. Our data suggest that SPAK and OSR1 may be critical factors in the regulation of depolarizing GABA-mediated processes in the developing brain. It will be important to examine these kinases with respect to sex differences and developmental brain anomalies in future studies.