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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.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.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.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.

Made available in DSpace on 2019-10-06T17:04:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-06-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Development and application of a new, simple, inexpensive, and eco-friendly method is presented for the quantification of bumetanide in human urine samples for the detection of doping in sports. The method involves the use of a paper platform and digital image detection and is based on the reaction between bumetanide, p-dimethylaminocinnamaldehyde (p-DAC), and HCl in a methanolic medium, yielding a colored compound in a delimited area on a qualitative filter paper. The concentrations of p-DAC and HCl were optimized by a chemometric experimental design. After the addition of the reagents to the paper, digital images were obtained by scanning in a multifunctional printer and analyzed using the RGB pattern. The linear bumetanide concentration range was 6.90 × 10 −5 –1.37 × 10 −3 mol L −1 with R 2 = 0.993 and the limits of detection (LOD) and quantification (LOQ) were 3.60 × 10 −4 and 1.09 × 10 −4 mol L −1 , respectively. A SPE clean-up step for the samples were required to eliminate the interference of urinary urea. The recoveries obtained varied from 94% to 105%, indicating the absence of significant matrix effects or interferences in urine samples after clean-up step. The proposed method was successfully applied to the analysis of bumetanide in human urine samples. Instituto de Química Universidade Estadual Paulista “Júlio de Mesquita Filho” UNESP, R. Prof. Francisco Degni 55, P.O. Box 355 Instituto de Química Universidade Estadual Paulista “Júlio de Mesquita Filho” UNESP, R. Prof. Francisco Degni 55, P.O. Box 355 CAPES: 001 FAPESP: 2015/21733-1

A novel coordination compound based on Zn(II) and bumetanide (Hbum) of general formulae [Zn(bum)(HO)]·2HO has been synthesized and fully characterized by elemental analyses and single crystal X-ray diffraction. The results indicate that compound 1 possesses a monomeric structure in which the main intermolecular interactions stabilizing the structure are hydrogen bonds. Due to the fully filled d orbitals of Zn(II), this coordination compound could be an ideal candidate to show strong emission. That is the reason why, photoluminescence properties have been studied showing that this compound presents ligand derived emission ruled by ligand-centred π-π* electronic transitions. Additionally, because of the well-recognised therapeutic properties of bumetanide, antiparasitic efficacy against three leishmania spp. strains has been studied for the corresponding ligand and coordination compound., This work has been funded by Junta de Andalucía (FQM-394), the Spanish Ministry of Economy and Competitiveness (MCIU/AEI/FEDER, UE) (PGC2018-102052-A-C22, PGC2018-102052-B-C21 and PGC2018-102047-B-I00), Red Guipuzcoana de Ciencia, Tecnología e Innovación (OF188/2017), University of the Basque Country (GIU 17/13). E.E. is grateful to the Government of the Basque Country for the predoctoral fellowship. The authors thank for technical and human support provided by SGIker of UPV/EHU and European funding (ERDF and ESF).

Bumetanide is a potent loop diuretic that acts as an inhibitor of sodium-potassium-chloride cotransporter 2 (NKCC2) and its isoform NKCC1. Although the expression of NKCC2 is limited to the kidney, NKCC1 is widely expressed in various cells, where it participates in a variety of physiological functions including ion transport, alveolar fluid secretion, and cell volume regulation. We investigated the role of NKCC1 in modulation of host immunity. Lipopolysaccharide (LPS) stimulated the expression and phosphorylation of NKCC1 in RAW264.7 cells in vitro and activated these cells to produce inflammatory cytokines. Enlarging the cell volume in a low-osmotic microenvironment amplified the LPS-induced inflammatory responses and phagocytosis activity of RAW264.7 cells. Pretreatment with the NKCC1 inhibitor bumetanide attenuated LPS-induced activation of inflammatory cells and cell volume-related function. Mice treated with an intratracheal bumetanide spray showed greater resistance to LPS-induced tissue inflammation and acute lung injury in vivo. Our studies suggest that NKCC1 plays a unique role as an amplifier of LPS-induced macrophage functions and that NKCC1 might be a novel target for treating sepsis-related acute respiratory distress syndrome.

The regulatory role of taurocholic acid (TC) and taurolithocholic acid (TLC) in ion transport was investigated with the short-circuit current (Isc) technique in the intestine of Senegalese sole (Solea senegalensis). Under voltage clamp conditions, Isc was stable for over 3 h and averaged −15.42 ± 2.75 μA/cm2 and −65.28 + 7.69 μA/cm2 in the anterior and posterior intestine, respectively. Only bumetanide (200 μM), a loop diuretic that inhibits Na+/K+/2Cl− co-transporters, inhibited the absorptive Isc, and apical application of NPPB (200 μM), EIPA (200 μM), glibenclamide (200 μM) and DIDS (200 μM) were without effect in the absorptive current. Apical application of TC and TLC induced rapid (

Recent studies have revealed an altered expression of NKCC1 and KCC2 in prefrontal cortex (PFC) and hippocampus of schizophrenic patients. Despite extensive considerations, the alteration of NKCC1 and KCC2 co-transporters at different stages of development has not been fully studied. Therefore, we evaluated the expression of these transporters in PFC and hippocampus at time points of four, eight, and twelve weeks in post-weaning social isolation rearing rat model. For this purpose, 23-25 days-old rats were classified into social- or isolation-reared groups. The levels of NKCC1 and KCC2 mRNA expression were evaluated at hippocampus or PFC regions at the time-points of four, eight, and twelve weeks following housing. Post-weaning isolation rearing decreased the hippocampal KCC2 mRNA expression level, but does not affect the NKCC1 mRNA expression. However, no significant difference was observed in the PFC mRNA levels of NKCC1 and KCC2 in the isolation-reared group compared to the socially-reared group during the course of modeling. Further, we assessed the therapeutic effect of selective NKCC1 inhibitor bumetanide (10 mg/kg), on improvement of prepulse inhibition (PPI) test on twelve weeks isolation-reared rats. Intraperitoneal administration of bumetanide (10 mg/kg) did not exert beneficial effects on PPI deficit. Our findings show that isolation rearing reduces hippocampal KCC2 expression level and may underlie hippocampal GABA excitatory. In addition, 10 mg/kg bumetanide is not effective in improving the reduced PPI of twelve weeks isolation-reared rats. Collectively, our findings show that hippocampal chloride transporter KCC2 contributes to excitatory GABA dysregulation in the developmental rat model of schizophrenia.

Traumatic brain injury (TBI) can lead to long-term motor and cognitive dysfunction, which can be at least partly attributed to blood-brain barrier (BBB) disruption. The mechanisms underlying post-TBI BBB disruption, however, are poorly understood thus far. Na + -K + -2Cl - cotransporter isoform 1 (NKCC1) is a universally expressed ion transporter that maintains intracellular ion homeostasis by increasing intracellular K + and Cl − . Having been characterized in stroke models, NKCC1 is activated in various cell types in the ischemic brain, and is thought to mediate BBB disruption, brain edema, and neuronal cell death. In this study, we tested the hypothesis that inhibition of NKCC1 may improve neurological outcomes via protecting against BBB disruption in a TBI mouse model. Adult male C57BL/6 J mice or NKCC1 deficient mice were subjected to controlled cortical impact (CCI). As an alternative to the genetic-based NKCC1 depletion, bumetanide, a selective NKCC1 inhibitor, was administrated (25 mg/kg, i.p .) 15 min after CCI and then every 6 h up to 48 h. Short-term sensorimotor function recovery was determined by rotarod, cylinder test, grid walking and foot fault test. BBB integrity was examined at 48 h post-CCI by measuring Evans blue extravasation, brain water content, and expression levels of tight junction proteins. Our results revealed that administration of bumetanide or genetic depletion of NKCC1 improved short-term neurological recovery against TBI. Bumetanide treatment markedly decreased brain water content and BBB leakage, correlated with reduction of MMP-9 expression and preventing the degradation of tight junction proteins. These findings suggest an important role of NKCC1 activation in mediating BBB disruption after TBI. Thus, NKCC1 inhibition may offer the potential for improving neurological outcomes in clinical TBI.

Objectives This study examined the dose-dependent actions of hydrogen sulfide donor sodium hydrosulphide (NaHS) on isometric contractions and ion transport in rat aorta smooth muscle cells (SMC). Methods Isometric contraction was measured in ring aortas segments from male Wistar rats. Activity of Na+/K+-pump and Na+,K+,2Cl-cotransport was measured in cultured endothelial and smooth muscle cells from the rat aorta as ouabain-sensitive and ouabain-resistant, bumetanide-sensitive components of the 86Rb influx, respectively. Results NaHS exhibited the bimodal action on contractions triggered by modest depolarization ([K+]o=30 mM). At 10−4 M, NaHS augmented contractions of intact and endothelium-denuded strips by ~ 15% and 25%, respectively, whereas at concentration of 10−3 M it decreased contractile responses by more than two-fold. Contractions evoked by 10−4 M NaHS were completely abolished by bumetanide, a potent inhibitor of Na+,K+,2Cl-cotransport, whereas the inhibition seen at 10−3 M NaHS was suppressed in the presence of K+ channel blocker TEA. In cultured SMC, 5×10−5 M NaHS increased Na+,K+,2Cl- - cotransport without any effect on the activity of this carrier in endothelial cells. In depolarized SMC, 45Ca influx was enhanced in the presence of 10−4 M NaHS and suppressed under elevation of [NaHS] up to 10−3 M. 45Ca influx triggered by 10−4 M NaHS was abolished by bumetanide and L-type Ca2+ channel blocker nicardipine. Conclusions Our results strongly suggest that contractions of rat aortic rings triggered by low doses of NaHS are mediated by activation of Na+,K+,2Cl-cotransport and Ca2+ influx via L-type channels., Highlights • NaHS induces rat aorta contractions through activation of Na+,K+,2Cl-cotransport and Ca2+ influx via L-type channels. • NaHS increased Na+,K+,2Cl--cotransport activity in smooth muscle cells but not in endothelial cells. • Low doses of NaHS enhanced 45Ca influx in smooth muscle cells, but high doses of NaHS suppressed it.

Purpose Review and discuss medications efficacious for seizure control, despite primary indications for other diseases, as treatment options in patients who have failed therapy with traditional antiepileptic drugs (AEDs). Methods Literature searches were conducted utilizing PubMed and MEDLINE databases employing combinations of search terms including, but not limited to, "epilepsy", "refractory", "seizure", and the following medications: acetazolamide, amantadine, bumetanide, imipramine, lidocaine, verapamil, and various stimulants. Results Data from relevant case studies, retrospective reviews, and available clinical trials were gathered, analyzed, and reported. Experience with acetazolamide, amantadine, bumetanide, imipramine, lidocaine, verapamil, and various stimulants show promise for cases of refractory epilepsy in both adults and children. Many medications lack large scale, randomized clinical trials, but the available data is informative when choosing treatment for patients that have failed traditional epilepsy therapies. Conclusions All neurologists have encountered a patient that failed nearly every AED, diet, and surgical option. For these patients, we often seek fortuitous discoveries within small series and case reports, hoping to find a treatment that might help the patient. In the present review, we describe medications for which antiepileptic effect has been ascribed after they were introduced for other indications.

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 2 h. To simulate stress after anesthesia, the animals were exposed to a single maternal separation for 3 h at P10. 3–7 days 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.

Hypoxia-ischemia brain damage (HIBD) is one of prevalent causes of neonatal mortality and morbidity. Our data demonstrated that hypoxia-ischemia (HI) induced Na+-K+-Cl--co-transporter 1 (NKCC1) increasing in hippocampus. Previous studies demonstrated that NKCC1 regulates various stages of neurogenesis. In this study, we studied the role of increased NKCC1 in regulating of HI-induced neurogenesis. HIBD model was established in 7days old Sprague-Dawley rat pup, and the expression of NKCC1 was detected by western blot and qPCR. Brain electrical activity in freely rats was monitored by electroencephalography (EEG) recordings. HI-induced neurogenesis was detected by immunofluorescence staining. Neurobehavioral test was to investigate the neuro-protective role of bumetanide, an inhibitor of NKCC1, on neonatal rats after HI. The results showed that bumetanide treatment significantly reduced brain electrical activity and the seizure stage of epilepsy induced by pentylenetetrazol (PTZ) in vivo after HI. In addition, bumetanide restored aberrant hippocampal neurogenesis and associated cognitive function. Our data demonstrated that bumetanide reduces the susceptibility of epilepsy induced by PTZ in rats suffering from HI injury during neonatal period via restoring the ectopic newborn neurons in dentate gyrus (DG) and cognitive function.

Malformations of cortical development (MCD) represent a group of rare diseases with severe clinical presentation as epileptic and pharmacoresistant encephalopathies. Morphological studies in tissue from MCD patients have revealed reduced GABAergic efficacy and increased intracellular chloride concentration in neuronal cells as important pathophysiological mechanisms in MCD. Also, in various animal models, alterations of GABAergic inhibition have been postulated as a predominant factor contributing to perilesional hyperexcitability. Along with this line, the NKCC1 inhibitor bumetanide has been postulated as a potential drug for treatment of epilepsy, mediating its antiepileptic effect by reduction of the intracellular chloride and increased inhibitory efficacy of GABAergic transmission. In the present study, we focused on the focal freeze-lesion model of MCD to compare antiepileptic drugs with distinct mechanisms of action, including NKCC1 inhibition by bumetanide. For this purpose, we combined electrophysiological and optical methods in slice preparations and assessed the properties of seizure like events (SLE) induced by 4-aminopyridine. In freeze-lesioned but not control slices, SLE onset was confined to the perilesional area, confirming that this region is hyperexcitable and likely triggers pathological activity. Bumetanide selectively reduced epileptic activity in lesion-containing slices but not in slices from sham-treated control rats. Moreover, bumetanide caused a shift in the SLE onset site away from the perilesional area. In contrast, effects of other antiepileptic drugs including carbamazepine, lacosamide, acezatolamide and zonisamide occurred mostly independently of the lesion and did not result in a shift of the onset region. Our work adds evidence for the functional relevance of chloride homeostasis in the pathophysiology of microgyrus formation as represented in the focal freeze-lesion model. Further studies in different MCD models and human tissue will be required to validate the effects across different MCD subtypes and species and to assess the translational value of our findings.

Aims The present study aims to target the quorum sensing (QS) accessory gene regulator A (AgrA) of Staphylococcus aureus to curtail bacterial virulence through drug repurposing approach. Main methods In silico screening of chemical ligands that bind specifically to the S. aureus C-LytTR domain of AgrA (AgrAC) was carried out. AgrA inhibition and downregulation of virulence genes linked to QS system of S. aureus were determined. Efficacy, dermal toxicity and drug tolerance induction were tested in Balb/C mice dermonecrosis model. Key findings Bumetanide bound to the conserved amino acid Tyr-229 of AgrA and showed 70% AgrA inhibition at 0.1 μM. Highly significant reduction in the expression of representative virulence genes such as alpha-hemolysin (~5 log2-fold), phenol-soluble modulins (~4 log2-fold) and panton-valentine leukocidin (~3 log2-fold) was noted in vitro. In vivo studies signified bumetanide to be highly effective in controlling the ulcer development and promoted wound healing. Also, the tested substance did not have dermal toxicity and no tolerance induction as well. Significance Targeting the QS regulators could be a possible alternative approach to curtail virulence in S. aureus. In addition, if the QS inhibitors are repurposed it could accelerate the drug development process and reduce the cost. The identified drug bumetanide inhibited AgrA and the results were in comparable to that of a known virulence inhibitor, diflunisal. The newly reported results of bumetanide in this study are expected to mark the drug's visibility for antibiotic adjunctive therapy and topical drug formulations for skin infections research.

Prolonged ketamine exposure in neonates at anesthetic doses is known to cause long-term impairments of learning and memory. A current theoretical mechanism explains this phenomenon as being neuro-excitotoxicity mediated by compensatory upregulation of N-methyl-D-aspartate receptors (NMDARs), which then initiates widespread neuroapoptosis. Additionally, the excitatory behavior of GABAergic synaptic transmission mediated by GABAA receptors (GABAARs), occurring during the early neuronal development period, is proposed as contributing to the susceptibility of neonatal neurons to ketamine-induced injury. This is due to differential developmental expression patterns of Na+-K+-2Cl− co-transporter (NKCC1) and K+-Cl− co-transporter. Studies have shown that bumetanide, an NKCC1 inhibitor, allows neurons to become inhibitory rather than excitatory early in development. We thus hypothesized that bumetanide co-administration during ketamine treatment would reduce over excitation and protect the neurons from excitotoxicity. In this initial study, the Morris Water Maze test was used to assess the effects of co-administration of ketamine and bumetanide to neonatal Sprague-Dawley rats on long-term learning and memory changes seen later in life. It was revealed that bumetanide, when co-treated with ketamine neonatally, significantly impeded behavioral deficits typically seen in animals exposed to ketamine alone. Therefore, these findings suggest a new mechanism by which neonatal ketamine induced learning impairments can be prevented.

Patients with multiple sclerosis (MS) often complain of neuropathic pain. According to the Gate Control Theory of Pain, spinal networks of GABAergic inhibitory interneurons are important in modulating nociceptive inputs from the periphery. Na+-K+-2Cl- co-transporter 1 (NKCC1) and K+-Cl- co-transporter 2 (KCC2) generally dictate the tone of GABA/glycine inhibition by regulating intracellular chloride concentrations. In this study, we investigated the role of NKCC1 and KCC2 in neuropathic pain observed in the animal model, experimental autoimmune encephalomyelitis (EAE), a commonly used model to study the pathophysiology of MS. Quantitative real-time polymerase chain reactions (qRT-PCR) analysis revealed no change in NKCC1 mRNA transcripts in dorsal root ganglia throughout EAE disease course. However, NKCC1 and KCC2 mRNA levels in the dorsal spinal cord were significantly reduced at disease onset and peak only to recover by the chronic time point. Similarly, Western blot data revealed a significant downregulation of NKCC1 and KCC2 in the dorsal spinal cord at disease onset but an upregulation of NKCC1 protein in the dorsal root ganglia at this time point. Treatment with bumetanide, an NKCC inhibitor, had no effect on mechanical hypersensitivity seen in mice with EAE even though it reversed the changes in the levels of NKCC1 and KCC2. We noted that bumetanide treatment, while effective at reversing the changes in monomeric KCC2 levels was ineffective at reversing the changes in oligomeric KCC2 which remained repressed. These results indicate that mechanical hypersensitivity in EAE is not mediated by altered levels of NKCC1.

The ubiquitin ligase Nedd4-like (Nedd4L, or Nedd4-2) binds to and regulates stability of the epithelial Na+ channel (ENaC) in salt-absorbing epithelia in the kidney, lung, and other tissues. Its role in the distal colon, which also absorbs salt and fluid and expresses ENaC, is unknown. Using a conditional knock-out approach to knock out Nedd4L in mice intestinal epithelium (Nedd4Lf/f ;Vil-CreERT2 ) we show here that Nedd4L depletion leads to a higher steady-state short circuit current (Isc) in mouse distal colon tissue relative to controls. This higher Isc was partially reduced by the addition of apical amiloride and strongly reduced by basolateral bumetanide as well as by depletion of basolateral Cl-, suggesting that Na+/K+/2Cl- (NKCC1/SLC12A2) co-transporter and ENaC are targets of Nedd4L in the colon. In accordance, NKCC1 (and γENaC) protein abundance in the colon of the Nedd4L knock-out animals was increased, indicating that Nedd4L normally suppresses these proteins. However, we did not observe co-immunoprecipitation between Nedd4L and NKCC1, suggesting that Nedd4L indirectly suppresses NKCC1 expression. Low salt diet resulted in a strong increase in β and γ (but not α) ENaC mRNA and protein expression and ENaC activity. Although salt restriction also increased NKCC1 protein and mRNA abundance, it did not lead to its elevated activity (Isc). These results identify NKCC1 as a novel target for Nedd4L-mediated down-regulation in vivo, which modulates ion and fluid transport in the distal colon together with ENaC.

Chloride ion (Cl−) is one of the most abundant anions in our body. Increasing evidence suggests that Cl− plays fundamental roles in various cellular functions. We have previously reported that electroneutral cation-chloride cotransporters, such as Na+-K+-2Cl− cotransporter 1 (NKCC1) and K+-Cl− cotransporter 1 (KCC1), are involved in neurite outgrowth during neuronal differentiation. In the present study, we studied if there is correlation between intracellular Cl− concentrations ([Cl−]i) and the length of growing neurites. We measured [Cl−]i in the cell body and growing neurite tips using halide-sensitive fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), revealing that [Cl−]i in the tip of growing neurite was higher than that in cell body in a single cell. Importantly, there was a significant positive correlation between the length of growing neurite and [Cl−]i in neurite tip. Bumtanide (BMT), an inhibitor of NKCC1, significantly inhibited neurite outgrowth and decreased [Cl−]i in neurite tip. The results obtained in the present study and our previous studies together strongly suggest that high [Cl−]i in neurite tip region is crucial for efficient neurite outgrowth.

Perturbations of γ-aminobutyric acid (GABA) neurotransmission in the human prefrontal cortex have been implicated in the pathogenesis of schizophrenia (SCZ), but the mechanisms are unclear. NKCC1 (SLC12A2) is a Cl(-)-importing cation-Cl(-) cotransporter that contributes to the maintenance of depolarizing GABA activity in immature neurons, and variation in SLC12A2 has been shown to increase the risk for schizophrenia via alterations of NKCC1 mRNA expression. However, no disease-causing mutations or functional variants in NKCC1 have been identified in human patients with SCZ. Here, by sequencing three large French-Canadian (FC) patient cohorts of SCZ, autism spectrum disorders (ASD), and intellectual disability (ID), we identified a novel heterozygous NKCC1 missense variant (p.Y199C) in SCZ. This variant is located in an evolutionarily conserved residue in the critical N-terminal regulatory domain and exhibits high predicted pathogenicity. No NKCC1 variants were detected in ASD or ID, and no KCC3 variants were identified in any of the three neurodevelopmental disorder cohorts. Functional experiments show Y199C is a gain-of-function variant, increasing Cl(-)-dependent and bumetanide-sensitive NKCC1 activity even in conditions in which the transporter is normally functionally silent (hypotonicity). These data are the first to describe a functional missense variant in SLC12A2 in human SCZ, and suggest that genetically encoded dysregulation of NKCC1 may be a risk factor for, or contribute to the pathogenesis of, human SCZ.

The Na(+)-K(+)-Cl(-) cotransporter NKCC1 plays a major role in the regulation of intraneuronal Cl(-) concentration. Abnormal functionality of NKCC1 has been implicated in several brain disorders, including epilepsy. Bumetanide is the only available selective NKCC1 inhibitor, but also inhibits NKCC2, which can cause severe adverse effects during treatment of brain disorders. A NKCC1-selective bumetanide derivative would therefore be a desirable option. In the present study, we used the Xenopus oocyte heterologous expression system to compare the effects of bumetanide and several derivatives on the two major human splice variants of NKCCs, hNKCC1A and hNKCC2A. The derivatives were selected from a series of ~5000 3-amino-5-sulfamoylbenzoic acid derivatives, covering a wide range of structural modifications and diuretic potencies. To our knowledge, such structure-function relationships have not been performed before for NKCC1. Half maximal inhibitory concentrations (IC50s) of bumetanide were 0.68 (hNKCC1A) and 4.0μM (hNKCC2A), respectively, indicating that this drug is 6-times more potent to inhibit hNKCC1A than hNKCC2A. Side chain substitutions in the bumetanide molecule variably affected the potency to inhibit hNKCC1A. This allowed defining the minimal structural requirements necessary for ligand interaction. Unexpectedly, only a few of the bumetanide derivatives examined were more potent than bumetanide to inhibit hNKCC1A, and most of them also inhibited hNKCC2A, with a highly significant correlation between IC50s for the two NKCC isoforms. These data indicate that the structural requirements for inhibition of NKCC1 and NKCC2 are similar, which complicates development of bumetanide-related compounds with high selectivity for NKCC1.

Experimental studies have indicated that prolonged ketamine exposure in neonates at anesthetic doses causes neuronal apoptosis, which contributes to long-term impairments of learning and memory later in life. The neuronal excitotoxicity mediated by compensatory upregulation of N-methyl- d -aspartate receptors (NMDARs) is proposed to be the underlying mechanism. However, this view does not convincingly explain why excitotoxicity-related apoptotic injury develops selectively in immature neurons. We proposed that the GABAA receptors (GABAARs)-mediated excitatory synaptic signaling due to high expression of the Na+-K+-2Cl− co-transporter (NKCC1), occurring during the early neuronal development period, plays a distinct role in the susceptibility of immature neurons to ketamine-induced injury. Using whole-cell patch-clamp recordings from the forebrain slices containing the anterior cingulate cortex, we found that in vivo repeated ketamine administration significantly induced neuronal hyperexcitability in neonatal, but not adolescent, rats. Such hyperexcitability was accompanied by the increase both in GABAAR- and NMDAR-mediated synaptic transmissions. An interference with the NKCC1 by bumetanide treatment completely reversed these enhanced effects of ketamine exposure and blocked GABAAR-mediated postsynaptic current activity. Thus, these findings were significant as they showed, for the first time, that GABAAR-mediated excitatory action may contribute distinctly to neuronal excitotoxic effects of ketamine on immature neurons in the developing brain.

Because of its potent inhibitory effect on the Na+-K+-2Cl- symporter isotype 1 (NKCC1) in brain neurons, bumetanide has been tested with varying results for treatment of seizures that potentially evolve as a consequence of abnormal NKCC1 activity. However, because of its physicochemical properties, bumetanide only poorly penetrates into the brain. We previously demonstrated that NKCC1 can be also inhibited by azosemide and torasemide, which lack the carboxyl group of bumetanide and thus should be better brain-permeable. Here we studied the brain distribution kinetics of azosemide and torasemide in comparison with bumetanide in mice and used pharmacokinetic-pharmacodynamic modelling to determine whether the drugs reach NKCC1-inhibitory brain concentrations. All three drugs hardly distributed into the brain, which seemed to be the result of probenecid-sensitive efflux transport at the blood-brain barrier. When fractions unbound in plasma and brain were determined by equilibrium dialysis, only about 6-17% of the brain drug concentration were freely available. With the systemic doses (10 mg/kg i.v.) used, free brain concentrations of bumetanide and torasemide were in the NKCC1-inhibitory concentration range, while levels of azosemide were slightly below this range. However, all three drugs exhibited free plasma levels that would be sufficient to block NKCC1 at the apical membrane of brain capillary endothelial cells. These data suggest that azosemide and torasemide are interesting alternatives to bumetanide for treatment of seizures involving abnormal NKCC1 functionality, particularly because of their longer duration of action and their lower diuretic potency, which is an advantage in patients with seizures.

Introduction Congestion is the primary driver of acute decompensated heart failure (ADHF). Unfortunately, diuretic resistance and/or under dosing of diuretics is common, contributing to the large proportion of patients discharged with residual congestion. Given that natriuresis from a dose of IV loop diuretic is nearly complete by 6 h, diuretic titration should occur more frequently than once daily. As such, we designed an automated diuretic titration protocol that would allow nurses to titrate loop diuretic every 6 h. Hypothesis A nurse-driven diuretic titration protocol will provide rapid, effective, and safe titration of loop diuretics. Methods The Yale Diuretic Pathway (YDP) begins with providers ordering the YDP through an EMR order set and specifying (or accepting default) safety parameters such as change in blood pressure (default 0.5 mg/dL increase) and the starting diuretic dose (default 2 mg bumetanide). At 9 AM, the first dose of diuretic is administered, and 2 hours later a spot urine sample is collected, which is used to predict sodium excretion. Based on the YDP algorithm, every 6 h bumetanide is either doubled, administered at the same dose, or held to achieve the daily goal sodium output. The initial EMR order will provide the next AM's diuretic dose (based on the algorithm) thus giving the provider until 3pm to re-order the YDP if they want automated diuresis to continue. For our analysis, we compared data from 3 days before and 3 days after the start of the YDP. Results 161 hospitalized patients with ADHF received the YDP (age 69±12 years, eGFR 50±2 mL/min/1.73m2). Median time from admission to start of the YDP was 2 (1-4) days, and median dose of IV furosemide equivalents before YDP was 120 (80-140) mg. With the first YDP dose, suboptimal sodium output was displayed with a median calculated 6-hour sodium output of 37 (16-84) mmol. With subsequent doses, mean daily urine output (1817±90 mL pre-YDP vs. 3034±82 mL on-YDP), net fluid output (-1083±86 mL pre-YDP vs. -2071±89 mL on-YDP), and delta weight (pre-YDP -.31±.26 kg vs. -2.54±.34 kg on-YDP) improved substantially (p 0.5 mg/dL. Conclusion The YDP appears to allow fast, safe, and effective titration of loop with significantly improved decongestion following initiation. Further research to formally assess the YDP in randomized setting, and impact of this strategy on clinical outcomes, is warranted.

Platinum-based drugs, used in treating tumors, cause numerous undesirable effects in patients, like neuropathic pain, hypersensitivity, reddening, pruritus and rash. Changes in Na+ transport modify local osmolality and contribute to the initiation of hypersensitivity and allergy. They are also associated with stimulation of C-fibres and hyperalgesia. Cl- transport is essential for regulation of sweat composition and the migration of immunocompetent cells. The aim of the conducted study was to assess the effect of a cisplatin solution on the electrophysiological parameters of the isolated rabbit skin specimens. The difference in transepithelial electrical potential (PD) and resistance (R) in stationary conditions and during 15 s mechanical-chemical stimulation (PDmin and PDmax), were measured. Measurement of R revealed that tissue samples were live, and their permeability to ions were stable. Control specimens had PD -0.22 mV (median). The PD of specimens treated by cisplatin was -0.55 mV (median), to for cisplatin and bumetanide 0 mV (median). Treatment with cisplatin did not change the continuous transport of Na+ and K+ ions, but did change that of Cl- ions. Stimulation of samples with the transport blockers of Cl-, Na+ and both induced repeatable and measurable reactions in the transport of the appropriate ions. It was shown that absorption of Na+ ions and release of Cl- ions was intensified than in the untreated specimens. It was proven in the study that cisplatin influences the Na+ and Cl- transport in the skin cells. Restoring the balance in ion flow can prevent side effects of use cisplatin-based drugs.

Na+-K+-2Cl- cotransporter (NKCC) is expressed at exceptionally high levels in gastric parietal cells. Bumetanide, a potent loop diuretic that blocks NKCC, usually causes a decrease in gastric acid secretion. Endotoxaemia causes hypochlorhydria in vivo, in which lipopolysaccharide (LPS) plays an important role. This study aimed to investigate the effect of NKCC2 on gastric acid secretion and its alteration in LPS-treated mice. The scanning ion-selective electrode technique and real-time pH titration combined with RNA interference were used to determine the effects of bumetanide on gastric acid secretion. Immunochemistry and Western blotting were performed to investigate the changes in NKCC2 expression in LPS-treated mice. Immunoreactivity of NKCC1 and NKCC2 was mainly observed near the basolateral and apical membranes of parietal cells, respectively. Pretreatment with bumetanide reduced the histamine-stimulated H+ flux in the mouse gastric mucosa. The apical, but not the basolateral, addition of bumetanide inhibited forskolin- or histamine/3-isobutyl-1-methylxanthine(IBMX)-induced gastric acid secretion. In vivo treatment with NKCC2 siRNA inhibited forskolin-induced acid secretion. Upon histamine stimulation, the majority of NKCC2 was targeted to the apical membrane in the gastric mucosa and in primary cultured parietal cells. The expression of NKCC2 and vesicle-associated membrane protein-2 (VAMP2), but not that of H+/K+-ATPase, was decreased in the gastric mucosa of LPS-treated mice. Blocking apical NKCC2, but not basolateral NKCC1, by bumetanide inhibited secretagogue-induced gastric acid secretion, during which the membrane trafficking of NKCC2 may be necessary. The downregulation of NKCC2 and VAMP2 may be related to the reduced gastric acid secretion induced by LPS.

Herein, we describe the design and synthesis of new benzenesulfonamide derivatives as selective COX-2 inhibitors based on bumetanide scaffold. Benzenesulfonamides bearing both the pyrazole 6b and the triazoles 9a, 9c were good inhibitors of COX-2 with IC50 values of 0.32, 0.28 and 0.17 µM, respectively. These benzenesulfonamides 6b, 9a and 9c exhibited a higher selectivity index than the reference drug celecoxib. Molecular modeling study showed that incorporation of bumetanide led to a unique binding mode that is most likely the reason for the observed significant COX-2 selectivity. The anti-inflammatory activity of synthesized compounds revealed that triazoles 9a and 9c demonstrated higher efficacy than celecoxib upon using in vivo carrageenan-induced rat paw edema model. Most of the prepared compounds possess low ulcerogenic potential when administered orally. Therefore, these compounds have a great potential to be developed as safe therapeutics for inflammation, pain, and other diseases where COX-2 plays important role in their pathophysiology.

Diarrhea is one of the major public health problems. In this study, the role of the decoction extract of Tunisian chamomile (Matricaria recutita) in the modulation of gastrointestinal motility, its antisecretory effects as well as its part in the regulation of the net ion fluxes transported across the epithelium (mainly Na+ and Cl-) were studied. Chamomile decoction extract (CDE) significantly inhibited the intestinal motility and the gastric emptying in a dose-dependent manner. The physiological study also showed that CDE had a direct effect on short-circuits current (Isc) intensity using bumetanide. These results suggested that this decrease in Isc using CDE could be interpreted as a reduction in electrogenic Cl- secretion together with a stimulation of neutral NaCl absorption. CDE can also modulate the gastric and intestinal motilities as well as the intestinal epithelium transport of water and electrolytes. Hence, this study confirmed that CDE is a potential beverage, due to the high amount of biologically active compounds that can prevent digestive diseases as well as maintain intestinal comfort.

There are reports indicating that diuretics including chlorothiazide, furosemide, ethacrynic acid, amiloride and bumetanide can have anticonvulsant properties. Intracellular acidification appears to be a mechanism for the anticonvulsant action of some diuretics. This study was conducted to investigate whether or not triamterene, a K(+)-sparing diuretic, can generate protection against seizures induced by intravenous or intraperitoneal pentylenetetrazole (PTZ) models. And to see if, triamterene can withstand maximal electroshock seizure (MES) in mice. We also investigated to see if there is any connection between triamterene's anti-seizure effect and ATP-sensitive K(+) (KATP) channels. Five days triamterene oral administration (10, 20 and 40 mg/kg), significantly increased clonic seizure threshold which was induced by intravenous pentylenetetrazole. Triamterene (10, 20 and 40 mg/kg) treatment also increased the latency of clonic seizure and decreased its frequency in intraperitoneal PTZ model. Administration of triamterene (20 mg/kg) also decreased the incidence of tonic seizure in MES-induced seizure. Co-administration of a KATP sensitive channel blocker, glibenclamide, in the 6th day, 60 min before intravenous PTZ blocked triamterene's anticonvulsant effect. A KATP sensitive channel opener, diazoxide, enhanced triamterene's anti-seizure effect in both intravenous PTZ or MES seizure models. At the end, triamterene exerts anticonvulsant effect in 3 seizure models of mice including intravenous PTZ, intraperitoneal PTZ and MES. The anti-seizure effect of triamterene probably is induced through KATP channels.

Recently, it has been suggested that bumetanide, an inhibitor of the Na-K-2Cl co-transporter (NKCC1), may be useful in the treatment of central nervous system (CNS) disorders. However, from a physicochemical perspective, bumetanide may not cross the blood-brain barrier to the extent that is necessary for it to be an effective brain NKCC1 inhibitor in vivo. High plasma-protein binding, potentially high brain-tissue binding and putative efflux transporters including organic anion transporter 3 (OAT3) contribute to the poor pharmacokinetic profile of bumetanide. Bidirectional permeability assays are an in vitro method to determine the impact of plasma-protein/brain tissue binding, as well as efflux transport, on the permeability of a compound. We established and validated a cell line stably overexpressing human OAT3 using lentiviral cloning techniques for use in in vitro bidirectional permeability assays. Using efflux transport studies, we show that bumetanide is a transported substrate of human OAT3, exhibiting a transport ratio of ≥1.5, which is attenuated by OAT3 inhibitors. Bidirectional permeability assays were carried out in the presence and absence of either albumin or brain homogenate to elucidate the effect of plasma-protein/brain tissue binding. These tests confirmed the pharmacokinetic limitations for brain delivery of bumetanide. In this experiment, bumetanide is 53% bound to albumin, 77% bound to brain tissue and accumulates in brain cells. Moreover, we conclusively established that bumetanide is a transported substrate of OAT3. Taken together, these bidirectional permeability studies highlight the potential of efflux transporter inhibition as an augmentation strategy for enhanced delivery of bumetanide to the CNS.

The Malpighian (renal) tubules play important roles in ionic and osmotic homeostasis in insects. In Lepidoptera, the Malpighian tubules are structurally regionalized and the concentration of Na(+) and K(+) in the secreted fluid varies depending on the segment of tubule analyzed. In this work, we have characterized fluid and ion (Na(+), K(+), H(+)) transport by tubules of the larval stage of the cabbage looper Trichoplusia ni; we have also evaluated the effects of fluid secretion inhibitors and stimulants on fluid and ion transport. Ramsay assays showed that fluid was secreted by the iliac plexus but not by the yellow and white regions of the tubule. K(+) and Na(+) were secreted by the distal iliac plexus (DIP) and K(+) was reabsorbed in downstream regions. The fluid secretion rate decreased>50% after 25μM bafilomycin A1, 500μM amiloride or 50μM bumetanide was added to the bath. The concentration of K(+) in the secreted fluid did not change, whereas the concentration of Na(+) in the secreted fluid decreased significantly when tubules were exposed to bafilomycin A1 or amiloride. Addition of 500μM cAMP or 1μM 5-HT to the bath stimulated fluid secretion and resulted in a decrease in K(+) concentration in the secreted fluid. An increase in Na(+) concentration in the secreted fluid was observed only in cAMP-stimulated tubules. Secreted fluid pH and the transepithelial electrical potential (TEP) did not change when tubules were stimulated. Taken together, our results show that the secretion of fluid is carried out by the upper regions (DIP) in T. ni Malpighian tubules. Upper regions of the tubules secrete K(+), whereas lower regions reabsorb it. Stimulation of fluid secretion is correlated with a decrease in the K(+)/Na(+) ratio.

Background 1.5 million children under 12 months of age are exposed to general anesthesia annually in the United States alone. Human and especially animal studies provide evidence that exposure to general anesthesia during the early postnatal period may lead to long-term neurocognitive abnormalities via poorly understood mechanisms. We investigated whether an immature stress response system and γ-aminobutyric acid (GABA) type A receptor activities are involved in mediating these abnormalities. Methods Sprague–Dawley rats at postnatal days 4, 5 or 6 were anesthetized with 2.1% sevoflurane for 6 h; maternally separated and house reared rats served as controls. Results Sevoflurane anesthesia markedly increased corticosterone levels in rat pups of both genders. In adulthood, these rats responded to stress with heightened secretion of corticosterone and a greater increase in corticosterone levels in males versus females. Only male rats, previously exposed to neonatal sevoflurane, had a higher frequency of miniature inhibitory postsynaptic currents in CA1 neurons, spent a shorter time in open arms of the elevated plus maze (EPM) and exhibited impaired prepulse inhibition (PPI) of startle. Pretreatment of male rats prior to sevoflurane with the Na + -K + -2Cl − cotransporter inhibitor, bumetanide, or the mineralocorticoid receptor antagonist, RU28318, normalized endocrine responses to stress and the EPM behavior in adulthood, while only those pretreated with bumetanide exhibited normalized PPI of startle responses. Neither bumetanide nor RU28318 altered the effect of sevoflurane on synaptic activity. Conclusions Sevoflurane-enhanced neuronal excitation and elevated corticosteroid levels at the time of anesthesia contribute to the mechanisms initiating neonatal sevoflurane-induced long-term endocrine and neurobehavioral abnormalities.

The cellular mechanisms of calcification in sea urchin larvae are still not well understood. Primary mesenchyme cells within the larval body cavity form a syncytium to secrete CaCO3 spicules from intracellular amorphous CaCO3 (ACC) stores. We studied the role of Na(+)K(+)2Cl(-) cotransporter (NKCC) in intracellular ACC accumulation and larval spicule formation of Strongylocentrotus droebachiensis. First, we incubated growing larvae with three different loop diuretics (azosemide, bumetanide, and furosemide) and established concentration-response curves. All loop diuretics were able to inhibit calcification already at concentrations that specifically inhibit NKCC. Calcification was most effectively inhibited by azosemide (IC50=6.5 μM), while larval mortality and swimming ability were not negatively impacted by the treatment. The inhibition by bumetanide (IC50=26.4 μM) and furosemide (IC50=315.4 μM) resembled the pharmacological fingerprint of the mammalian NKCC1 isoform. We further examined the effect of azosemide on the maintenance of cytoplasmic cords and on the occurrence of calcification vesicles using fluorescent dyes (calcein, FM1-43). Fifty micromolars of azosemide inhibited the maintenance of cytoplasmic cords and resulted in increased calcein fluorescence within calcification vesicles. The expression of NKCC in S. droebachiensis was verified by PCR and Western blot with a specific NKCC antibody. In summary, the pharmacological profile of loop diuretics and their specific effects on calcification in sea urchin larvae suggest that they act by inhibition of NKCC via repression of cytoplasmic cord formation and maintenance.

GABAA receptor-mediated inhibition is active and may contribute to epileptiform synchronization. The efficacy of inhibition relies on low levels of intracellular Cl(-), which are controlled by KCC2 activity. This evidence has led us to analyze with field potential recordings the effects induced by the KCC2 blockers VU0240551 (10 μM) or bumetanide (50 μM) and by the KCC2 enhancer CLP257 (100 μM) on the epileptiform discharges generated by piriform and entorhinal cortices (PC and EC, respectively) in an in vitro brain slice preparation. Ictal- and interictal-like discharges along with high-frequency oscillations (HFOs, ripples: 80-200 Hz, fast ripples: 250-500 Hz) were recorded from these two regions during application of 4-aminopyridine (4AP, 50 μM). Blocking KCC2 activity with either VU024055 or high doses of bumetanide abolished ictal discharge in both PC and EC; in addition, these experimental procedures decreased the interval of occurrence and duration of interictal discharges. In contrast, enhancing KCC2 activity with CLP257 increased ictal discharge duration in both regions. Finally, blocking KCC2 activity decreased the duration and amplitude of pharmacologically isolated synchronous GABAergic events whereas enhancing KCC2 activity led to an increase in their duration. Our data demonstrate that in vitro ictogenesis is abolished or facilitated by inhibiting or enhancing KCC2 activity, respectively. We propose that these effects may result from the reduction of GABAA receptor-dependent increases in extracellular K(+) that are known to rest on KCC2 function.

The Na-K-Cl co-transporter 1 (NKCC1; a member of the cation-chloride co-transporter family) mediates the coupled movement of Na(+) and/or K(+) with Cl(-) across the plasma membrane of cells (Haas and Forbush, 2000, Annu. Rev. Physiol., 62, 515-534; Russell, 2000, Physiol. Rev., 80, 211-276). Although it acts as an important regulator of cell volume, secretion, and modulator of cell apoptosis and proliferation (Chen et al., 2005, J. Cereb. Blood Flow Metab., 25, 54-66; Kahle et al., 2008, Nat. Clin. Pract. Neurol., 4, 490-503; Kidokoro et al., 2014, Am. J. Physiol. Ren. Physiol., 306, F1155-F1160; Wang et al., 2011, Cell. Physiol. Biochem., 28, 703-714), NKCC1׳s effects on oligodendrocyte precursor cells (OPCs) have not been characterized. The aim of this study was to investigate whether and to what extent inhibition of NKCC1 alters oxygen glucose deprivation (OGD)-induced cell cycle progression. In the present study, we demonstrated that inhibition of NKCC1 with bumetanide attenuates the decrease in OGD-induced DNA synthesis in cultured OPCs. Western blots showed that NKCC1 inhibition led to an increased expression of cyclin D1, CDK 4, and cyclin E in OGD-treated cells. Furthermore, our results showed bumetanide attenuated the decrease in OGD-induced proliferation and arrest of cell cycle progression via the P-38 MAPK signaling cascade. Thus, NKCC1 plays important roles in the proliferation of OPCs under OGD-induced stress.

The Mongolian gerbil, Meriones unguiculatus, has been widely employed as a model for studies of the inner ear. In spite of its established use for auditory research, no robust protocols to induce ototoxic hair cell damage have been developed for this species. In this paper, we demonstrate the development of an aminoglycoside-induced model of hair cell loss, using kanamycin potentiated by the loop diuretic furosemide. Interestingly, we show that the gerbil is relatively insensitive to gentamicin compared to kanamycin, and that bumetanide is ineffective in potentiating the ototoxicity of the drug. We also examine the pathology of the spiral ganglion after chronic, long-term hair cell damage. Remarkably, there is little or no neuronal loss following the ototoxic insult, even at 8 months post-damage. This is similar to the situation often seen in the human, where functioning neurons can persist even decades after hair cell loss, contrasting with the rapid, secondary degeneration found in rats, mice and other small mammals. We propose that the combination of these factors makes the gerbil a good model for ototoxic damage by induced hair cell loss., Highlights • We have explored different paradigms of ototoxicity in the gerbil model. • The gerbil sensory hair cells seem relatively resiliant to gentamicin. • Hair cell damage is not followed by significant loss of spiral ganglion neurons. • This generates a simple model for hair cell loss that resembles human lesions. • This model should be ideal to study regenerative strategies and cochlear implantation.

The regulatory role of arginine vasotocin (AVT) and isotocin (IT) in Cl− secretion was investigated with the short circuit current (Isc) technique in opercular epithelia of killifish (Fundulus heteroclitus) and gilthead sea bream (Sparus aurata). Sea bream operculum showed ~ 4-fold lower number of Na/K-ATPase immunoreactive cells and ~ 12-fold lower secretory current than the killifish. In sea bream opercular membranes, the basolateral addition of AVT (10− 6 M) significantly stimulated Cl− secretion, while IT (10− 6 M) was without effect. In killifish, IT produced an immediate dose-dependent stimulation of Cl− secretion with significant effect at doses ≥ 10− 7 M and stimulation maxima (∆Isc ~ 25 μA ⋅ cm− 2) at 10− 6 M. The basolateral addition of bumetanide (200 μM) abolished > 75% of the effect of IT on Cl− secretion. In turn, AVT had a dual effect on killifish opercular Isc: an immediate response (~ 3 min) with Isc reduction in an inverted bell-shaped dose–response manner with higher current decrease (− 22 μA ⋅ cm− 2) at 10− 8 M AVT, and a sustained dose-dependent stimulation of Cl− secretion (stable up to 1 h), with a threshold significant effect at 10− 8 M and maximal stimulation (~ 20 μA ⋅ cm− 2) at 10− 6 M. Both effects of AVT appear receptor type specific. The V1-receptor antagonist SR 49059 abolished Isc reduction in response to AVT, while the specific V2-receptor antagonist (Tolvaptan, 1 μM) abolished the stimulatory action of AVT on Cl− secretion. According to these results, we propose a modulatory role for AVT and IT in Cl− (NaCl) secretion across the opercular epithelium of marine teleost., JAM-S was funded by Ministry of Education (Spain) through the program “Formación de Profesorado Universitario” (Ref: AP2008-01194). This work was partially supported by Ministry of Science and Education, Spain by Project AGL2010-14876 to JMM and by the Ministry of Science and Higher Education and European Social Funds through the Portuguese National Science Foundation by Projects PTDC/MAR/104008/2008 and PTDC/MAR-BIO/3811/2012 to JF.

Neonatal seizures caused by perinatal asphyxia and hypoxic-ischemic encephalopathy can be refractory to conventional anticonvulsants. This may be due to the depolarizing effects of gamma-aminobutyric acid (GABA) achieved by the activity of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1). The aim of this study is to evaluate the long-term effects of bumetanide, a NKCC1 inhibitor, on hippocampal neurogenesis and seizure susceptibility in hypoxia-induced neonatal seizure model. Wistar rats were subjected to hypoxia-induced neonatal seizures at postnatal day 10 (P10). Following acute seizures, the rats were treated with intraperitoneal injection (i.p.) of bumetanide at a dose of 0.5mg/kg for 3 weeks. In later adulthood, hypoxia-induced seizures increased the number of newborn dentate gyrus cells (DGCs), promoted mossy fiber sprouting (MFS) and reduced the apical dendritic complexity of newborn DGCs 1 month after the insults. In addition, these seizures resulted in long-lasting consequences, such as spontaneous electroencephalography (EEG) seizures, though spatial learning impairments were not seen. Bumetanide treatments significantly enhanced cell proliferation and dendritic development of newborn DGCs after neonatal seizures, accompanied by the decreased seizure activity. However, systemic administration of bumetanide resulted in much lower brain concentrations, and was incompatible with NKCC1 inhibition in blood-brain barrier (BBB)-protected brain tissue. Our results suggested that bumetanide might have long-term effects in suppressing seizure activity, and altering the neurogenesis after neonatal seizures. These effects of bumetanide may be mediated by the targets outside the BBB-protected central nerve system (CNS) or CNS-located target(s) other than NKCC1.

In about 20-40% of patients, status epilepticus (SE) is refractory to standard treatment with benzodiazepines, necessitating second- and third-line treatments that are not always successful, resulting in increased mortality. Rat models of refractory SE are instrumental in studying the changes underlying refractoriness and to develop more effective treatments for this severe medical emergency. Failure of GABAergic inhibition is a likely cause of the development of benzodiazepine resistance during SE. In addition to changes in GABAA receptor expression, trafficking, and function, alterations in Cl(-) homeostasis with increased intraneuronal Cl(-) levels may be involved. Bumetanide, which reduces intraneuronal Cl(-) by inhibiting the Cl(-) intruding Na(+), K(+), Cl(-) cotransporter NKCC1, has been reported to interrupt SE induced by kainate in urethane-anesthetized rats, indicating that this diuretic drug may be an interesting candidate for treatment of refractory SE. In this study, we evaluated the effects of bumetanide in the kainate and lithium-pilocarpine models of SE as well as a model in which SE is induced by sustained electrical stimulation of the basolateral amygdala. Unexpectedly, bumetanide alone was ineffective to terminate SE in both conscious and anesthetized adult rats. However, it potentiated the anticonvulsant effect of low doses of phenobarbital, although this was only seen in part of the animals; higher doses of phenobarbital, particularly in combination with diazepam, were more effective to terminate SE than bumetanide/phenobarbital combinations. These data do not suggest that bumetanide, alone or in combination with phenobarbital, is a valuable option in the treatment of refractory SE in adult patients.

Bumetanide, which is known as a potent diuretic, is currently under investigation for its potential anti-epileptic effects in neonatal seizures. The purpose of this study was to examine the molecular structure of bumetanide both in the free form and its solutions via vibrational spectra (FT-IR, FT-Raman spectroscopies) and quantum chemical calculations. FT-IR and FT-Raman spectra of the title compound were recorded for the solid phase and the solutions of DMSO and ethanol. Optimized molecular geometry and vibrational wavenumbers of bumetanide were calculated by DFT/B3LYP functional with 6-31G(d,p), 6-31G++(d,p) and 6-311++G(d,p) basis sets. The assignment of the vibrational modes were performed based on total energy distribution (TED). The same calculations were performed for the molecule in DMSO and ethanol solutions using the polarizable conductor continuum model (CPCM) method. Lastly, probable donor–acceptor interactions of the molecule were examined with NBO analysis in different media. In all forms of bumetanide (the free molecule and the other solvents), some significant changes were observed in the dihedral angles and the vibrational frequencies.

In stroke-prone spontaneously hypertensive rats (SHRSP/Izm), ischemia induces swelling of astrocytes, a process that subsequently leads to neuronal death. Following ischemic insult, arginine vasopressin (AVP) can induce edema and l-serine released by astrocytes supports the survival of neuronal cells. The purpose of this study was to examine whether AVP contributed to the regulation of l-serine production following ischemic stroke. Here, we used cultured astrocytes from SHRSP/Izm rats and Wistar Kyoto rats (WKY/Izm) to examine whether AVP changed the production of l-serine and/or altered gene expression levels of the neural amino acid transporter (Slc1a4), 3-phosphoglycerate dehydrogenase (Phgdh) and serine racemase (SRR). Furthermore, using astrocytes from the congenic rat SHRpch1_18 strain having quantitative trait loci (QTL) of stroke, we examined expression of those genes under conditions of hypoxia and reoxygenation (H/R). The expression levels of ASCT1 protein, the genes described above and l-serine levels were determined by Western blotting (WB), RT-PCR, real-time quantitative RT-PCR and HPLC. AVP increased the production of l-serine and the expression of Slc1a4 in WKY/Izm and SHRSP/Izm astrocytes. The production of l-serine and the expression of Slc1a4 were lower in SHRSP/Izm than in WKY/Izm cells. This difference was not seen with Phgdh. In the SHRpch1_18 strain, the expression of Slc1a4 and Phgdh significantly decreased after H/R. AVP-mediated enhanced expression of ASCT1 was blocked by the addition of bumetanide. These results suggest that the AVP-mediated attenuated expression of ASCT1 in astrocytes is associated with reduced l-serine production in SHRSP/Izm astrocytes. We hypothesize that reduction of gene expression by AVP might be related to the induction of stroke in the SHRpch1_18 rat strain.

Introduction In general, clinicians round on patients hospitalized with heart failure (HF) once per day. Additionally, the metrics commonly used to determine response to diuretics, such as fluid and weight loss, are often not recorded after each diuretic dose. Given these limitations, diuretics are generally titrated at best daily. In patients with diuretic resistance, this can translate to lost hospital days with ineffective doses of diuretics. To address this issue, we designed a protocol (herein referred to as the Yale Diuretic Pathway or YDP) to allow for rapid and safe loop diuretic escalation. Hypothesis The nurse driven auto-titrating YDP will lead to rapid dose escalation, without an excessive incidence of hypokalemia, worsening renal function, or hypotension. Methods Advanced heart failure physicians selected 51 patients, largely those who were failing traditional diuretic therapy, for this non-randomized pilot of the YDP. The provider could select the starting dose of diuretic (though the default was 2 mg of bumetanide). Additionally, providers would select safety parameters such as lowest tolerable blood pressure (default was a systolic reading of 90 mmHg or less) and largest allowable increase in creatinine (0.5 mg/dL). Diuretics were titrated based on predicted sodium excretion after each dose, which was predicted using an equation based on a spot urine sample taken 2 h after administration. Based on the predicted sodium output diuretic doses were either held, kept constant, or increased. Patients could receive up to 3 doses daily and a maximum of 12.5 mg bumetanide, equivalent to 500 mg of furosemide per dose and 1500 mg of furosemide per day. Signals for safety such as hypotension, electrolyte abnormalities, and changes in renal function were examined. Results Patients were on the YDP for an average of 3 ± 1.3 days, and the median starting dose was 80 mg of furosemide equivalents (IQR 80-160 mg). The majority of patients required the maximum daily dose of furosemide equivalents over this period (median = 1500mg, IQR 980 to 1500mg) and they reached their peak dose within 1.9 ± 0.7 days. Despite this degree of diuretic resistance, YDP still resulted in a weight loss of 4.0± 3.3 kgs total and fluid output of 9.4L ± 5.1L over the average 3 days of the protocol. With respect to safety, the average rise in creatinine was 0.1 mg/dL. No patients experienced a serum potassium below 3.0 mg/dL and only 3 patients had a serum magnesium below 1.5 mg/dL. Discontinuation of YDP for issues such as a rise in creatinine (10%) and reduction in systolic blood pressure (8%) were relatively uncommon. Conclusion The YDP lead to safe and rapid loop diuretic titration in a population comprised largely of diuretic resistant HF patients. Randomized studies are needed better characterize the safety and efficacy of the YDP.

The treatment of neonatal seizures has not changed significantly over the last 50 years despite advances in antiepileptic drug (AED) development for older children and adults. Recently new drugs have emerged some of which address age-specific challenges or mechanisms and will be discussed in this review. The loop diuretic bumetanide blocks the neuronal NKCC1 co-transporter and is thought specifically to supress seizures in the immature brain. Levetiracetam has been used in children and infants with good efficacy, an excellent safety profile, and near-ideal pharmacokinetic characteristics. Randomised controlled trials are now underway to test the efficacy of some newer AEDs for neonatal seizures. Topiramate has been shown to have neuroprotective properties in addition to its antiepileptic action and trials in babies with hypoxic-ischaemic encephalopathy are now planned. There is an urgent need to develop age-specific AEDs for preterm and term babies. These drugs must be evaluated with multicentre, collaborative trials using innovative methods and high ethical standards to overcome age-specific challenges with the ultimate aim of improving the outcome for neonates with seizures.