A combination of phenobarbital and the bumetanide derivative bumepamine prevents neonatal seizures and subsequent hippocampal neurodegeneration in a rat model of birth asphyxia.

OBJECTIVES: Bumetanide was suggested as an adjunct to phenobarbital for suppression of neonatal seizures. This suggestion was based on the idea that bumetanide, by reducing intraneuronal chloride accumulation through inhibition of the Na-K-2Cl cotransporter NKCC1, may attenuate or abolish depolarizing γ-aminobutyric acid (GABA) responses caused by birth asphyxia. However, a first proof-of-concept clinical trial failed. This could have had several reasons, including bumetanide's poor brain penetration, the wide cellular NKCC1 expression pattern in the brain, and problems with the general concept of NKCC1's role in neonatal seizures. We recently replicated the clinical failure of bumetanide to potentiate phenobarbital's effect in a novel rat model of birth asphyxia. In this study, a clinically relevant dose (0.3 mg/kg) of bumetanide was used that does not lead to NKCC1-inhibitory brain levels. The aim of the present experiments was to examine whether a much higher dose (10 mg/kg) of bumetanide is capable of potentiating phenobarbital in this rat model. Furthermore, the effects of the two lipophilic bumetanide derivatives, the ester prodrug N,N-dimethylaminoethylester of bumetanide (DIMAEB) and the benzylamine derivative bumepamine, were examined at equimolar doses. METHODS: Intermittent asphyxia was induced for 30 min by exposing male and female P11 rat pups to three 7 + 3 min cycles of 9% and 5% O2 at constant 20% CO2 . All control pups exhibited neonatal seizures after the asphyxia. RESULTS: Even at 10 mg/kg, bumetanide did not potentiate the effect of a submaximal dose (15 mg/kg) of phenobarbital on seizure incidence, whereas a significant suppression of neonatal seizures was determined for combinations of phenobarbital with DIMAEB or, more effectively, bumepamine, which, however, does not inhibit NKCC1. Of interest, the bumepamine/phenobarbital combination prevented the neurodegenerative consequences of asphyxia and seizures in the hippocampus. SIGNIFICANCE: Both bumepamine and DIMAEB are promising tools that may help to develop more effective lead compounds for clinical trials.

Surfactant Attenuates Air Embolism-Induced Lung Injury by Suppressing NKCC1 Expression and NF-κB Activation.

Excessive amounts of air can enter the lungs and cause air embolism (AE)-induced acute lung injury (ALI). Pulmonary AE can occur during diving, aviation, and iatrogenic invasive procedures. AE-induced lung injury presents with severe hypoxia, pulmonary hypertension, microvascular hyper-permeability, and severe inflammatory responses. Pulmonary AE-induced ALI is a serious complication resulting in significant morbidity and mortality. Surfactant is abundant in the lungs and its function is to lower surface tension. Earlier studies have explored the beneficial effects of surfactant in ALI; however, none have investigated the role of surfactant in pulmonary AE-induced ALI. Therefore, we conducted this study to determine the effects of surfactant in pulmonary AE-induced ALI. Isolated-perfused rat lungs were used as a model of pulmonary AE. The animals were divided into four groups (n = 6 per group): sham, air embolism (AE), AE + surfactant (0.5 mg/kg), and AE+ surfactant (1 mg/kg). Surfactant pretreatment was administered before the induction of pulmonary AE. Pulmonary AE was induced by the infusion of 0.7 cc air through a pulmonary artery catheter. After induction of air, pulmonary AE was presented with pulmonary edema, pulmonary microvascular hyper-permeability, and lung inflammation with neutrophilic sequestration. Activation of NF-κB was observed, along with increased expression of pro-inflammatory cytokines, and Na-K-Cl cotransporter isoform 1 (NKCC1). Surfactant suppressed the activation of NF-κB and decreased the expression of pro-inflammatory cytokines and NKCC1, thereby attenuating AE-induced lung injury. Therefore, AE-induced ALI presented with pulmonary edema, microvascular hyper-permeability, and lung inflammation. Surfactant suppressed the expressions of NF-κB, pro-inflammatory cytokines, and NKCC1, thereby attenuating AE-induced lung injury.

Preliminary study of analgesic effect of bumetanide on neuropathic pain in patients with spinal cord injury.

BACKGROUND/OBJECTIVE: The current study evaluated the analgesic effects of bumetanide as an adjunctive treatment in managing neuropathic pain following spinal cord injury. The peripheral expression level of Na-K-Cl-cotransporter-1 (NKCC1) and K-Cl-cotransporter-2 (KCC2) genes in polymorphonuclear lymphocytes (PMLs) assessed as a possible biomarker indicating central underlying mechanisms. METHODS: This open-label, single-arm, pilot trial of bumetanide (2 mg/day) is an add-on treatment conducted in 14 SCI patients for 19 weeks. The whole duration consisted of three phases: pre-treatment (1 month), titration (3 weeks), and active treatment (4 months). Ultimately, nine patients completed the study. The primary outcome variables were the endpoint pain score measured by the numeric rating scale (NRS), and the short-form McGill Pain Questionnaire. Secondary endpoints included the Short-Form Health Survey that measures the quality of life. Blood samples were collected and used for determining the expression of NKCC1 and KCC2 genes in transcription and translation levels. RESULTS: Bumetanide treatment significantly reduced average pain intensity according to the NRS and the short form of the McGill Pain Questionnaire scores. The baseline expression of KCC2 protein was low between groups and increased significantly following treatment (P < 0.05). Through the current study, pain improvement accompanied by the more significant mean change from the baseline for the overall quality of life. CONCLUSION: These data might be a piece of preliminary evidence for the analgesic effect of bumetanide on neuropathic pain and could support the potential role of the upregulation of KCC2 protein and involvement of GABAergic disinhibition in producing neuropathic pain.

Preventing neuronal edema increases network excitability after traumatic brain injury.

Edema is an important target for clinical intervention after traumatic brain injury (TBI). We used in vivo cellular resolution imaging and electrophysiological recording to examine the ionic mechanisms underlying neuronal edema and their effects on neuronal and network excitability after controlled cortical impact (CCI) in mice. Unexpectedly, we found that neuronal edema 48 hours after CCI was associated with reduced cellular and network excitability, concurrent with an increase in the expression ratio of the cation-chloride cotransporters (CCCs) NKCC1 and KCC2. Treatment with the CCC blocker bumetanide prevented neuronal swelling via a reversal in the NKCC1/KCC2 expression ratio, identifying altered chloride flux as the mechanism of neuronal edema. Importantly, bumetanide treatment was associated with increased neuronal and network excitability after injury, including increased susceptibility to spreading depolarizations (SDs) and seizures, known agents of clinical worsening after TBI. Treatment with mannitol, a first-line edema treatment in clinical practice, was also associated with increased susceptibility to SDs and seizures after CCI, showing that neuronal volume reduction, regardless of mechanism, was associated with an excitability increase. Finally, we observed an increase in excitability when neuronal edema normalized by 1 week after CCI. We conclude that neuronal swelling may exert protective effects against damaging excitability in the aftermath of TBI and that treatment of edema has the potential to reverse these effects.

Molecular characterization of Na+/K+/2Cl- cotransporter 1 alpha from Trachinotus ovatus (Linnaeus, 1758) and its expression responses to acute salinity stress.

Trachinotus ovatus is widely cultured in the ponds and gulf on the southeast coast of China. The dramatic salinity decrease caused by heavy rainfall could cause mass mortality of T. ovatus in aquaculture. It is very important to understand the osmoregulatory mechanism of T. ovatus. Na+/K+/2Cl- cotransporter 1a (NKCC1a) is involved in the osmoregulation of fish and plays a crucial role in cell volume homeostasis and maintenance of the electrolyte content. In this study, we characterized nkcc1a (designed as Tonkcc1a) from T. ovatus and investigated its expression responses to acute salinity changes. Tonkcc1a is approximately 70 kb in length and contains 26 exons and 25 introns. The phylogenetic analysis confirmed that ToNKCC1a belonged to the NKCC1a subclade. Quantitative real-time (qRT-PCR) analysis indicated that Tonkcc1a was ubiquitously expressed in all examined tissues, with the highest mRNA levels observed in gills, and the lowest level in liver. When T. ovatus were transferred from seawater (30‰) into fresh water, the expression levels of Tonkcc1a mRNA were significantly downregulated in gills and kidney, whereas its expression level was markedly upregulated in intestine. When transferred from seawater (30‰) to 10‰ sea water, the expression levels of Tonkcc1a mRNA were clearly increased in gills and kidney. When transferred from seawater (30‰) to 20‰ sea water, the expression of Tonkcc1a mRNA increased to some extent in gills, kidney, and intestine. When transferred from seawater (30‰) to 40‰ sea water, the expression levels of Tonkcc1a mRNA were dramatically upregulated in gills and intestine compared to that in the control. These results suggested that Tonkcc1a was involved in the response to acute salinity changes.

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.

Effect of infusion speed of 7.5% hypertonic saline on brain edema in patients with craniocerebral injury: An experimental study.

This study firstly used a rat traumatic brain injury model to compare the therapeutic effects of different intravenous infusion speed of 7.5% hypertonic saline (HS). Then the authors applied different delivery rate of 7.5% HS to two groups of patients to figure out the optimal infusion rates. A total of 100 rats were randomly divided into control group, group A (7.5% HS 6 mL/h), group B (7.5% HS 3 mL/h), and group C (7.5% HS 2 mL/h). All rats were established for the brain injury model. A total of 30 patients were selected and randomly divided into group A (250 mL/h) and group B (125 mL/h), with 15 cases in each group. Urine amount was recorded per hour; furthermore, blood was extracted from the patients to measure the levels of AQP4, NKCC1, tumour necrosis factor-α (TNF-a), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6). Compared with other groups, the expression levels of NKCC1 and AQP4 mRNA in group A was the lowest (P < 0.05). NKCC1 and AQP4 protein expression levels were the lowest in all the groups (P < 0.05). On the aspect of patients, group A displayed more significant difference compared with B group in terms of AQP4, NKCC1, TNF-a, IL-1ß, and IL-6 (P < 0.05). In the two groups, a significant difference was noted in the urine amount at 4 h after administration (P < 0.05). In our study, infusion of hypertonic saline (250 mL/h) at the optimal rate of 7.5% HS decreased the intracranial pressure, brain tissue edema, and inflammatory cytokine expression; moreover, it can promote brain tissue protection.

Single cell analysis of Crohn's disease patient-derived small intestinal organoids reveals disease activity-dependent modification of stem cell properties.

BACKGROUND: Intestinal stem cells (ISCs) play indispensable roles in the maintenance of homeostasis, and also in the regeneration of the damaged intestinal epithelia. However, whether the inflammatory environment of Crohn's disease (CD) affects properties of resident small intestinal stem cells remain uncertain. METHODS: CD patient-derived small intestinal organoids were established from enteroscopic biopsy specimens taken from active lesions (aCD-SIO), or from mucosa under remission (rCD-SIO). Expression of ISC-marker genes in those organoids was examined by immunohistochemistry, and also by microfluid-based single-cell multiplex gene expression analysis. The ISC-specific function of organoid cells was evaluated using a single-cell organoid reformation assay. RESULTS: ISC-marker genes, OLFM4 and SLC12A2, were expressed by an increased number of small intestinal epithelial cells in the active lesion of CD. aCD-SIOs, rCD-SIOs or those of non-IBD controls (NI-SIOs) were successfully established from 9 patients. Immunohistochemistry showed a comparable level of OLFM4 and SLC12A2 expression in all organoids. Single-cell gene expression data of 12 ISC-markers were acquired from a total of 1215 cells. t-distributed stochastic neighbor embedding analysis identified clusters of candidate ISCs, and also revealed a distinct expression pattern of SMOC2 and LGR5 in ISC-cluster classified cells derived from aCD-SIOs. Single-cell organoid reformation assays showed significantly higher reformation efficiency by the cells of the aCD-SIOs compared with that of cells from NI-SIOs. CONCLUSIONS: aCD-SIOs harbor ISCs with modified marker expression profiles, and also with high organoid reformation ability. Results suggest modification of small intestinal stem cell properties by unidentified factors in the inflammatory environment of CD.

Bumetanide treatment during early development rescues maternal separation-induced susceptibility to stress.

Stress is a major risk factor for psychiatric disorders, such as depression, posttraumatic stress disorder, and schizophrenia. Early life stress, such as maternal separation, can have long-term effects on the development of the central nervous system and pathogenesis of psychiatric disorders. In the present study, we found that maternal separation increased the susceptibility to stress in adolescent rats, increased the expression of Na+/K+/2Cl- cotransporter 1 (NKCC1) on postnatal day 14, and increased the expression of K+/2Cl- cotransporter 2 (KCC2) and γ-aminobutyric acid A (GABAA) receptor subunits on postnatal day 40 in the hippocampus. NKCC1 inhibition by the U.S. Food and Drug Administration-approved drug bumetanide during the first two postnatal weeks rescued the depressive- and anxiety-like behavior that was induced by maternal separation and decreased the expression of NKCC1, KCC2 and GABAA receptor α1 and ß2,3 subunits in the hippocampus. Bumetanide treatment during early development did not adversely affect body weight or normal behaviors in naive rats, or affect serum osmolality in adult rats. These results suggest that bumetanide treatment during early development may prevent the maternal separation-induced susceptibility to stress and impairments in GABAergic transmission in the hippocampus.

Actions of quercetin, a flavonoid, on ion transporters: its physiological roles.

Flavonoids keep us healthy by controlling various body and cellular functions. It is well known that cations, such as Na+ , K+ , and Ca2+ , play important roles in the regulation of body and cellular functions, including generation of action potentials and the resting membrane potential of neural and muscle cells and signal transduction as intracellular second messengers. However, we have little information on the physiological roles of anions, particularly Cl- , in body and cellular functions. Quercetin, a flavonoid, stimulates Na+ -K+ -2Cl- cotransporter 1 (NKCC1), which is one of the most important ion transporters regulating the cytosolic Cl- concentration ([Cl- ]c ). Here, we introduce the molecular mechanism by which flavonoids, specifically quercetin, act through elevation of [Cl- ]c via activation of NKCC1 on important factors controlling various body and cellular functions, such as (1) antihypertensive actions controlling blood volume dependent on the amounts of renal Na+ reabsorption via expression of the epithelial Na+ channel, (2) neurite-elongating actions via polymerization of tubulin by inhibiting GTPase activity, and (3) antibacterial and antiviral infective actions through stimulation of epithelial Cl- secretion by increasing the driving force for epithelial Cl- secretion.

Astaxanthin protects astrocytes against trauma-induced apoptosis through inhibition of NKCC1 expression via the NF-κB signaling pathway.

BACKGROUND: Astaxanthin (ATX) is a carotenoid pigment with pleiotropic pharmacological properties that is seen as a possible drug for treating cerebral ischemic injury and subarachnoid hemorrhage. Na+-K+-2Cl- co-transporter-1 (NKCC1), an intrinsic membrane protein expressed by many cell types, is activated by various insults, leading to the formation of cell swelling and brain edema. We previously established that ATX attenuated brain edema and improved neurological outcomes by modulating NKCC1 expression after traumatic brain injury in mice. This paper explored the molecular mechanism of ATX-mediated inhibition of NKCC1 utilizing an in vitro astrocyte stretch injury model. RESULTS: Stretch injury in cultured astrocytes lowered cell viability time-dependently, which was substantially reducing by pretreating with ATX (50 µmol/L). Stretch injury increased Bax level and cleaved caspase-3 activity, and decreased Bcl-2 level and pro-caspase 3 activity, resulting in the apoptosis of astrocytes. Additionally, stretch injury substantially raised the gene and protein expressions of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α and prompted the expression and nuclear translocation of NF-κB. Pretreatment with ATX remarkably prevented the trauma-induced initiation of NF-κB, expressions of pro-inflammatory cytokines, and cell apoptosis. Moreover, stretch injury markedly elevated the gene and protein expression of NKCC1, which was partly blocked by co-treatment with ATX (50 µmol/L) or an NF-κB inhibitor (PDTC, 10 µmol/L). Cleaved caspase-3 activity was partially reduced by PDTC (10 µmol/L) or an NKCC1 inhibitor (bumetanide, 50 µmol/L). CONCLUSIONS: ATX attenuates apoptosis after stretch injury in cultured astrocytes by inhibiting NKCC1 expression, and it acts by reducing the expression of NF-κB-mediated pro-inflammatory factors.

Role of environmental stressors in determining the developmental outcome of neonatal anesthesia.

BACKGROUND: The majority of studies evaluating neurocognition in humans who had procedures under anesthesia early in life found long-term deficits even though the typical anesthesia duration normalized to the human life span is much shorter than that shown to induce developmental abnormalities in rodents. Therefore, we studied whether subsequent environmental stressors contribute to deficiencies programmed by a brief neonatal etomidate exposure. METHODS: Postnatal days (P) 4, 5, or 6, Sprague-Dawley rats, pretreated with vehicle or the Na+-K+-2Cl- (NKCC1) inhibitor, bumetanide, received two injections of etomidate resulting in anesthesia for 2h. To simulate stress after anesthesia, the animals were exposed to a single maternal separation for 3h at P10. 3-7days after exposure to etomidate the rats had increased hypothalamic NKCC1 mRNA and corticotropin releasing hormone (CRH) mRNA and decreased K+-2Cl- (KCC2) mRNA levels with greater changes in males. In rats neonatally exposed to both etomidate and maternal separation, these abnormalities persisted into adulthood. These animals also exhibited extended corticosterone responses to restraint stress with increases in total plasma corticosterone more robust in males, as well as behavioral abnormalities. Pretreatment with the NKCC1 inhibitor ameliorated most of these effects. CONCLUSIONS: Post-anesthesia stressors may exacerbate/unmask neurodevelopmental abnormalities even after a relatively short anesthetic with etomidate, leading to dysregulated stress response systems and neurobehavioral deficiencies in adulthood. Amelioration by bumetanide suggests a mechanistic role for etomidate-enhanced gamma-aminobutyric acid type A receptor-mediated depolarization in initiating long-lasting alterations in gene expression that are further potentiated by subsequent maternal separation.

Inhibition of Na(+)-K(+)-2Cl(-) Cotransporter-1 attenuates traumatic brain injury-induced neuronal apoptosis via regulation of Erk signaling.

Traumatic brain injury (TBI) is the leading cause of mortality and morbidity worldwide and is characterized by immediate brain damage and secondary injuries, such as brain edema and ischemia. However, the exact pathological mechanisms that comprise these associated secondary injuries have not been fully elucidated. This study aimed to investigate the role of the Na(+)-K(+)-2Cl(-) cotransporter-1 (NKCC1) in the disruption of ion homeostasis and neuronal apoptosis in TBI. Using a traumatic neuron injury (TNI) model in vitro and a controlled cortex injury (CCI) model in vivo, the present study investigated changes in the expression and effects of NKCC1 in TBI using western blot, RNA interference, a lactate dehydrogenase (LDH) release assay, TdT-mediated dUTP Nick end-labeling (TUNEL) analysis, sodium imaging, brain water content, and neurological severity scoring. TBI induced the expression of NKCC1 to be significantly upregulated in the cortex, both in vitro and in vivo. Pharmacological inhibitor bumetanide (Bume) or NKCC1 RNA interference significantly attenuated TBI-induced intracellular Na(+) increase, inhibited neuronal apoptosis, and improved brain edema and neurological function. Furthermore, NKCC1 inhibition also significantly inhibited TBI-induced extracellular signal-regulated kinase (Erk) activation. Erk inhibition significantly protected neurons from TBI injury; however, Erk inhibition had no effect on NKCC1 expression or the neuroprotective effect of NKCC1 inhibition against TBI. This study demonstrates the role of NKCC1 in TBI-induced brain cortex injury, establishing that NKCC1 may play a neurotoxic role in TBI and that the inhibition of NKCC1 may protect neurons from TBI via the regulation of Erk signaling.

Effect of vasopressin on Na(+)-K(+)-2Cl(-) cotransporter (NKCC) and the signaling mechanisms on the murine late distal colon.

It has been demonstrated that the antidiuretic hormone vasopressin is able to regulate the expression of Na-K-Cl cotransporters (NKCC1 and NKCC2) in the kidney. The present study investigated the effects of long- and short-term administration of vasopressin on NKCC and the possible signaling mechanism of vasopressin in the mouse distal colon using the siRNA, real-time PCR, western blotting and Ussing chambers method. The results showed the presence of NKCC2 expression in the colon, which was verified with a siRNA technique. The mRNA and protein expression level of NKCC2 significantly increased by about 40% and 90% respectively in response to restricting water intake to 1ml/day/20g for 7 days. In contrast, the NKCC1 expression level was unchanged in the colon. To determine the short-term activation of NKCC2 by vasopressin in vitro, we found that the administration of vasopressin caused a 3-fold increase in mouse colon NKCC2 phosphorylation, which was detected with phosphospecific antibody R5. In addition, the Ussing chamber results showed that NKCC2, cAMP and Ca(2+) signaling pathway may be involved in the vasopressin-induced response. Further, adenylate cyclase inhibitor MDL-12330A and PKA inhibitor H89 and Ca(2+) chelator BAPTA-AM reversed the vasopressin induced NKCC2 phosphorylation level increase by about 35%, 28% and 42% respectively suggesting vasopressin stimulate NKCC2 phosphorylation increase mediated by cAMP-PKA and Ca(2+) signaling in the colon. Collectively, these data suggest that the expression and phosphorylation of NKCC2 are increased in the colon by vasopressin stimulation, in association with enhanced activity of the vasopressin/cAMP and Ca(2+) pathways.

Bile deficiency induces changes in intestinal Cl(-) and HCO3 (-) secretions in mice.

AIMS: Biliary tract obstruction is a common clinical lesion. However, the effect of biliary tract obstruction on intestinal secretion is poorly understood. In this study, we made an investigation on intestinal HCO3 (-) and Cl(-) secretions in an experimental model of murine biliary duct ligation. METHODS: Murine intestinal mucosal HCO3 (-) and Cl(-) secretions were examined in vitro in Ussing chambers by pH-stat and short-circuit current (Isc ) techniques. The mRNA and protein expressions of the cystic fibrosis transmembrane conductance regulator (CFTR) and the Na(+) -K(+) -2Cl(-) cotransporter (NKCC1) were analysed by real-time PCR, western blot and immunohistochemistry. RESULTS: Basal Cl(-) secretion and forskolin-stimulated duodenal and jejunal mucosal HCO3 (-) and Cl(-) secretions in mice with common biliary duct ligation were markedly elevated, compared with controls (P < 0.05 and P < 0.01). Further experiments showed that basal Cl(-) secretion and forskolin-stimulated duodenal and jejunal mucosal HCO3 (-) and Cl(-) secretions in mice with external bile drainage were also markedly elevated. CFTRinh -172 inhibited forskolin-stimulated HCO3 (-) and Cl(-) secretions. The mRNA and protein expression levels of CFTR and NKCC1 in the intestinal mucosa with both biliary duct ligation and external bile drainage were markedly higher than those in controls (P < 0.001). Bile acid administration restored the changes in function and expression of CFTR and NKCC1 in the intestinal mucosa. CONCLUSION: Bile deficiency in the intestine up-regulates the expressions of intestinal mucosal CFTR and NKCC1 and enhances intestinal mucosal HCO3 (-) and Cl(-) secretion capacity, which contributes to the understanding of intestinal physiological function for patients with biliary duct obstruction.

A global investigation of gene deletion strains that affect premature stop codon bypass in yeast, Saccharomyces cerevisiae.

Protein biosynthesis is an orderly process that requires a balance between rate and accuracy. To produce a functional product, the fidelity of this process has to be maintained from start to finish. In order to systematically identify genes that affect stop codon bypass, three expression plasmids, pUKC817, pUKC818 and pUKC819, were integrated into the yeast non-essential loss-of-function gene array (5000 strains). These plasmids contain three different premature stop codons (UAA, UGA and UAG, respectively) within the LacZ expression cassette. A fourth plasmid, pUKC815 that carries the native LacZ gene was used as a control. Transformed strains were subjected to large-scale ß-galactosidase lift assay analysis to evaluate production of ß-galactosidase for each gene deletion strain. In this way 84 potential candidate genes that affect stop codon bypass were identified. Three candidate genes, OLA1, BSC2, and YNL040W, were further investigated, and were found to be important for cytoplasmic protein biosynthesis.

Direct control of Na(+)-K(+)-2Cl(-)-cotransport protein (NKCC1) expression with aldosterone.

Sodium/potassium/chloride cotransporter (NKCC1) proteins play important roles in Na(+) and K(+) concentrations in key physiological systems, including cardiac, vascular, renal, nervous, and sensory systems. NKCC1 levels and functionality are altered in certain disease states, and tend to decline with age. A sensitive, effective way of regulating NKCC1 protein expression has significant biotherapeutic possibilities. The purpose of the present investigation was to determine if the naturally occurring hormone aldosterone (ALD) could regulate NKCC1 protein expression. Application of ALD to a human cell line (HT-29) revealed that ALD can regulate NKCC1 protein expression, quite sensitively and rapidly, independent of mRNA expression changes. Utilization of a specific inhibitor of mineralocorticoid receptors, eplerenone, implicated these receptors as part of the ALD mechanism of action. Further experiments with cycloheximide (protein synthesis inhibitor) and MG132 (proteasome inhibitor) revealed that ALD can upregulate NKCC1 by increasing protein stability, i.e., reducing ubiquitination of NKCC1. Having a procedure for controlling NKCC1 protein expression opens the doors for therapeutic interventions for diseases involving the mis-regulation or depletion of NKCC1 proteins, for example during aging.

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.

NKCC1 and KCC2 protein expression is sexually dimorphic in the hippocampus and entorhinal cortex of neonatal rats.

Seizure susceptibility appears to be greater in males than females during the early developmental stages of the brain when the gamma-aminobutyric acid (GABA), acting through its GABA-A receptor, predominantly produces neuronal depolarization. GABA-mediated excitation has been observed when the NKCC1 (chloride importer) expression level is higher than KCC2 (chloride exporter). In this study, the relative protein expression of NKCC1 and KCC2 over ß-actin was evaluated in the hippocampus and entorhinal cortex of male and female rats during postnatal days (PND) 1, 3, 5, 7, 9, 11, 13 and 15 using Western blotting assays. For both cerebral regions in the females, the NKCC1/ß-actin expression ratio was constant during all evaluated ages, whereas the KCC2/ß-actin expression ratio increased gradually until reaching a maximal level at PND9 that was nearly three- and ten-fold higher in the hippocampus and entorhinal cortex, respectively, compared with the initial level. In males, the NKCC1/ß-actin expression ratio was constant during the first week, peaking almost three-fold higher than the initial level at PND9 in the hippocampus and at PND11 in the entorhinal cortex and then returning to the initial values at PND13, whereas the KCC2/ß-actin expression ratio increased gradually to reach a maximal and steady level at PND5, which were nearly two- and four-fold higher in the hippocampus and entorhinal cortex, respectively, compared with the intial level. In conclusion, the NKCC1/ß-actin and KCC2/ß-actin expression ratios displayed a specific expression profile for each gender and cerebral region, which could be related with the differences in seizure susceptibility observed between genders.