NKCC1 Inhibitor Research Articles

TRPV1 activation stimulates NKCC1 and increases hydrostatic pressure in the mouse lens.

The porcine lens response to a hyperosmotic stimulus involves an increase in the activity of an ion cotransporter sodium-potassium/two-chloride cotransporter 1 (NKCC1). Recent studies with agonists and antagonists pointed to a mechanism that appears to depend on activation of transient receptor potential vanilloid 1 (TRPV1) ion channels. Here, we compare responses in lenses and cultured lens epithelium obtained from TRPV1-/- and wild type (WT) mice. Hydrostatic pressure (HP) in lens surface cells was determined using a manometer-coupled microelectrode approach. The TRPV1 agonist capsaicin (100 nM) caused a transient HP increase in WT lenses that peaked after ∼30 min and then returned toward baseline. Capsaicin did not cause a detectable change of HP in TRPV1-/- lenses. The NKCC inhibitor bumetanide prevented the HP response to capsaicin in WT lenses. Potassium transport was examined by measuring Rb+ uptake. Capsaicin increased Rb+ uptake in cultured WT lens epithelial cells but not in TRPV1-/- cells. Bumetanide, A889425, and the Akt inhibitor Akti prevented the Rb+ uptake response to capsaicin. The bumetanide-sensitive (NKCC-dependent) component of Rb+ uptake more than doubled in response to capsaicin. Capsaicin also elicited rapid (<2 min) NKCC1 phosphorylation in WT but not TRPV1-/- cells. HP recovery was shown to be absent in TRPV1-/- lenses exposed to hyperosmotic solution. Bumetanide and Akti prevented HP recovery in WT lenses exposed to hyperosmotic solution. Taken together, responses to capsaicin and hyperosmotic solution point to a functional role for TRPV1 channels in mouse lens. Lack of NKCC1 phosphorylation and Rb+ uptake responses in TRPV1-/- mouse epithelium reinforces the notion that a hyperosmotic challenge causes TRPV1-dependent NKCC1 activation. The results are consistent with a role for the TRPV1-activated signaling pathway leading to NKCC1 stimulation in lens osmotic homeostasis.

The Role of Chloride in the Breathing‐related Response to Central Acidosis in Developing Birds

Rhythmic spontaneous neural activity (rSNA) is thought to contribute to the organization and development of motor circuits. In spinal motor networks, rSNA has a role in synapse refinement, axon trajectory, and the formation of neuromuscular junctions. In the zebra finch embryo, extracellular recordings of hindbrain breathing‐related motor activity also show that rSNA burst pattern, shape, and neurotransmitter signaling change in an age‐dependent manner. Further, the role of chloride‐mediated neurotransmission exhibits a switch in polarity such that GABAergic and glycinergic signaling transitions from excitatory to inhibitory over the course of 24–48 hours. Despite recognizing these ontogenetic changes in hindbrain rSNA, we are just beginning to explore whether the changing physiochemical environment within the eggshell shapes rhythmic motor activity. As a bird embryo grows within the egg, it experiences progressive alterations in respiratory gases (↓PO2, ↑PCO2), an emerging acidosis, as well as modifications to the anatomical structures that facilitate gas exchange. Data show that lowering and raising the pH of the superfusate bathing the isolated avian hindbrain influences breathing‐linked rSNA as a function of age. Embryos employing diffusive gas exchange within the egg show a decreased rate of rSNA, while freely breathing birds employing pulmonary gas exchange show an increased rate of cranial nerve motor output. Given these findings, our objective here was to test mechanisms contributing to this pH response. We speculate that chloride is an important player as a result of three observations: 1) chloride‐mediated neurotransmission shifts in polarity at nearly the same embryonic age as the switch in the rSNA response to low pH; 2) chloride transport is involved in pH homeostasis; 3) GABAA receptors conduct both chloride and hydrogen carbonate, which are tied to pH regulation. To explore the first point, we recorded rSNA and breathing‐related motor rhythms while lowering pH under chloride free conditions. Results show that the removal of chloride eliminates rSNA in embryonic days (E) 4–6 embryos. On E7‐10, chloride removal decreases the rate of motor output compared with low pH alone. Related to the second point, we treated the hindbrain with low pH and bumetanide, an antagonist for the transmembrane sodium‐potassium‐chloride cotransporter (NKCC1). Data show that, compared to controls, the instantaneous frequency increases in E7‐10 birds. To address the third point, we applied the GABAA receptor antagonist picrotoxin (PTX) during low pH treatments. Results show that PTX and low pH treatment decreases rSNA frequency to 49 ± 11% of control in &lt;E11 birds and increases frequency to 129 ± 20% of control at &gt;E11. Together, our results suggest that interfering with chloride gradients and blocking GABAA receptor activity during low pH treatment has consequences for rSNA that are similar to lowering pH alone. In contrast, the inhibition of NKCC1 with bumetanide seems to disrupt the rhythmic response to central acidosis in the zebra finch hindbrain, at least prior to the onset of breathing.Support or Funding InformationIdaho IDeA Network of Biomedical Research and Excellence Program Grant # P20 GM103408 and the National Institute of Neurological Disorders and Stroke via the AREA Program Grant # 1R15NS087521‐01A1.

The effect and possible mechanism of sevoflurane exposure on electroencephalographic seizures and long-term cognitive function in neonatal rats

Objective To investigate the effect of sevoflurane anesthesia on electroencephalographic (EEG) seizures and long-term behavior and possible mechanism in neonatal rats. Methods A total of 141 postnatal days 4-6 Sprague-Dawley rats (66 male, 75 female) were divided into 3 groups (n=47 in each group) according to random number table method: control group, sevoflurane group, and NKCC1 inhibitor group, with 22 males and 25 females in each group. Rats in the control group were fed in normal cage without anesthesia; rats in the sevoflurane group were anesthetized with 2.1% sevoflurane for 6 hours; rats in the NKCC1 blocker group received intraperitoneal injection of 1.82 mg / kg bumetanide 30 minutes before anesthesia with 2.1% sevoflurane. The rats in the control group and sevoflurane group were injected subcutaneously with the same dose of DMSO at the same time when the NKCC1 blocker group received the drug intervention, so as to eliminate the influence caused by the solvent. The rats were observed for 30 minutes after recovery from anesthesia and then continued to breastfeed normally. Some of the new born rats received EEG monitoring from 9 to 11 days after being raised; the other rats received EPM and PPI respectively at 60 and 70 days after being raised. Results The results of EEG showed that, compared with the control group, the number of epileptic waves((0.429±0.787), (1.571±0.787), t=2.753, P 0.05). Compared with male rats, the average duration of female rats in sevoflurane group decreased ((6.392±3.374), (2.515±2.992), t=3.044, P 0.05). Comparison between male and female rats: compared with male rats in sevoflurane group, the female rats in sevoflurane group had a longer open arm stay time in EPM experiment (P<0.05), the difference was statistically significant. Conclusion Sevoflurane anesthesia for 6 hours can significantly increase the generation of epileptic waves in EEG of male newborn rats, and cause behavioral abnormalities in adult male rats, which may be related to NKCC1.And male rats are more vulnerable to the negative effects of sevoflurane anesthesia on brain nerve development. Key words: Sevoflurane; Neonatal anesthesia; Bumetanide; Gender; Electroencephalo-graphic; Behaviour; Rat

Asthmatic Airway Vagal Hypertonia Involves Chloride Dyshomeostasis of Preganglionic Neurons in Rats.

Airway vagal hypertonia is closely related to the severity of asthma; however, the mechanisms of its genesis are unclear. This study aims to prove that asthmatic airway vagal hypertonia involves neuronal Cl– dyshomeostasis. The experimental airway allergy model was prepared with ovalbumin in male adult Sprague-Dawley rats. Plethysmography was used to evaluate airway vagal response to intracisternally injected γ-aminobutyric acid (GABA). Immunofluorescent staining and Western-blot assay were used to examine the expression of microglia-specific proteins, Na+-K+-2Cl– co-transporter 1 (NKCC1), K+-Cl– co-transporter 2 (KCC2) and brain-derived nerve growth factor (BDNF) in airway vagal centers. Pulmonary inflammatory changes were examined with hematoxylin and eosin staining of lung sections and ELISA assay of ovalbumin-specific IgE in bronchoalveolar lavage fluid (BALF). The results showed that histochemically, experimental airway allergy activated microglia, upregulated NKCC1, downregulated KCC2, and increased the content of BDNF in airway vagal centers. Functionally, experimental airway allergy augmented the excitatory airway vagal response to intracisternally injected GABA, which was attenuated by intracisternally pre-injected NKCC1 inhibitor bumetanide. All of the changes induced by experimental airway allergy were prevented or mitigated by chronic intracerebroventricular or intraperitoneal injection of minocycline, an inhibitor of microglia activation. These results demonstrate that experimental airway allergy augments the excitatory response of airway vagal centers to GABA, which might be the result of neuronal Cl– dyshomeostasis subsequent to microglia activation, increased BDNF release and altered expression of Cl– transporters. Cl– dyshomeostasis in airway vagal centers might contribute to the genesis of airway vagal hypertonia in asthma.

Ecm29-mediated proteasomal distribution modulates excitatory GABA responses in the developing brain.

Neuronal GABAergic responses switch from excitatory to inhibitory at an early postnatal period in rodents. The timing of this switch is controlled by intracellular Cl- concentrations, but factors determining local levels of cation-chloride cotransporters remain elusive. Here, we report that local abundance of the chloride importer NKCC1 and timely emergence of GABAergic inhibition are modulated by proteasome distribution, which is mediated through interactions of proteasomes with the adaptor Ecm29 and the axon initial segment (AIS) scaffold protein ankyrin G. Mechanistically, both the Ecm29 N-terminal domain and an intact AIS structure are required for transport and tethering of proteasomes in the AIS region. In mice, Ecm29 knockout (KO) in neurons increases the density of NKCC1 protein in the AIS region, a change that positively correlates with a delay in the GABAergic response switch. Phenotypically, Ecm29 KO mice showed increased firing frequency of action potentials at early postnatal ages and were hypersusceptible to chemically induced convulsive seizures. Finally, Ecm29 KO neurons exhibited accelerated AIS developmental positioning, reflecting a perturbed AIS morphological plastic response to hyperexcitability arising from proteasome inhibition, a phenotype rescued by ectopic Ecm29 expression or NKCC1 inhibition. Together, our findings support the idea that neuronal maturation requires regulation of proteasomal distribution controlled by Ecm29.

ИССЛЕДОВАНИЕ РОЛИ NA+, K+, 2CL- - КОТРАНСПОРТА В РЕГУЛЯЦИИ ТОНУСА ЛЕГОЧНОЙ АРТЕРИИ

Purpose. We studied the role of Na+, K+, 2Cl- cotransport (NKCC) in contractile activity of pulmonary artery seg- ments with intact endothelium induced by incubation in nonisosmotic solutions. Materials and methods. The influence of nonisosmotic solutions and blocker of NKCC on vascular segments mechanical tension was studied in isometric regime with organ bath technique performed with the 4-channel Myobath II and software complex LAB-TRAX-4/16 (Germany). Hyperosmotic shrinkage was induced by adding 50-300 mM su- crose to the Krebs solution, the hypoosmotic environment was created by reducing the concentration of NaCl from 70 mM to 40 mM. Bumetanide was used as an NKCC blocker. The amplitude of the contractile responses was evaluated as a percentage of the control contraction in highpotassium solution. Results. The addition of 50-300 mM sucrose to the Krebs incubation solution caused a development of dose- dependent of sustained contractile responses. The amplitude of hyperosmotic striction increased with inhibition of NKCC. Incubation of segments in hypoosmotic solution caused the development of transient contractile responses, the amplitude of which decreased in the presence of NKCC inhibitor. Conclusion. Iincubation of pulmonary artery vascular segments in hyper- and hyposmotic solutions induces con- tractile responses, the amplitude of which depends on the NKCC activity.

CATION-CHLORIDE CO-TRANSPORTER 1 (CCC1) Mediates Plant Resistance against Pseudomonas syringae.

Plasma membrane (PM) depolarization functions as an initial step in plant defense signaling pathways. However, only a few ion channels/transporters have been characterized in the context of plant immunity. Here, we show that the Arabidopsis (Arabidopsis thaliana) Na+:K+:2Cl- (NKCC) cotransporter CCC1 has a dual function in plant immunity. CCC1 functions independently of PM depolarization and negatively regulates pathogen-associated molecular pattern-triggered immunity. However, CCC1 positively regulates plant basal and effector-triggered resistance to Pseudomonas syringae pv. tomato (Pst) DC3000. In line with the compromised immunity to Pst DC3000, ccc1 mutants show reduced expression of genes encoding enzymes involved in the biosynthesis of antimicrobial peptides, camalexin, and 4-OH-ICN, as well as pathogenesis-related proteins. Moreover, genes involved in cell wall and cuticle biosynthesis are constitutively down-regulated in ccc1 mutants, and the cell walls of these mutants exhibit major changes in monosaccharide composition. The role of CCC1 ion transporter activity in the regulation of plant immunity is corroborated by experiments using the specific NKCC inhibitor bumetanide. These results reveal a function for ion transporters in immunity-related cell wall fortification and antimicrobial biosynthesis.

CSIG-01. NA-K-CL COTRANSPORTER PROTEIN IN THE PATHOGENESIS OF LOW-GRADE GLIOMAS

Abstract Low-grade gliomas (LGGs) present a high incidence of epilepsy, infiltrative growth, resistance to therapy, and high risk of transforming into high-grade gliomas (HGGs). Temozolomide (TMZ) is a conventional chemotherapy drug for adjuvant treatment of patients with high-risk LGGs. Na+-K+-2Cl- co-transporter 1 (NKCC1) regulates cell volume and intracellular Cl- concentration which promotes glioma cell migration, resistance to TMZ, and tumor-associated epilepsy. Our new bioinformatic analysis of TCGA and CGGA datasets shows that LGGs expressed higher SLC12A2 gene mRNA (encoding NKCC1 protein) than HGGs, which was confirmed at NKCC1 protein levels. In syngeneic mouse glioma models with intracranial transplantation of two different mouse glioma cell lines (GL26 and SB28), we detected upregulated NKCC1 protein expression in glioma tumor cells as well as in peri-tumor reactive astrocytes in response to TMZ monotherapy. This provided a rationale for us to test the efficacy of combining NKCC1 pharmacological blockade with TMZ on improving therapeutic outcomes. 5-day combination therapy of TMZ with potent NKCC1 inhibitor bumetanide reduced tumor proliferation, potentiated TMZ-mediated apoptosis, and decreased peri-tumor reactive astrocyte formation, which collectively led to suppressed tumor growth and prolonged survival of tumor-bearing mice. Taken together, these results demonstrate that NKCC1 protein plays multifaceted roles in regulating the pathogenesis of LGGs and its blockade presents therapeutic potentials for reducing TMZ-mediated resistance.

Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury.

Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain. We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons. To this aim, rats with persistent hyperalgesia were randomly divided into four groups. Rats in the control group received no treatment, and the rat sciatic nerve was only exposed in the sham group. Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide, an inhibitor of NKCC1, based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group. In the experiment measuring thermal withdrawal latency, bumetanide (15 mg/kg) was intravenously administered. In the patch clamp experiment, bumetanide (10 µg/µL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour, or bumetanide (5 µg/µL) was intrathecally injected. The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats. We found that the thermal withdrawal latency of rats was significantly decreased on days 7, 14, and 21 after model establishment. After intravenous injection of bumetanide, the reduction in thermal retraction latency caused by model establishment was significantly inhibited. Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7, 14, and 21 after model establishment. No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment. The Cl- (chloride ion) fluorescent probe technique was used to evaluate the change of Cl- concentration in dorsal root ganglion neurons of chronic constriction injury model rats. We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl- fluorescent probe whose fluorescence intensity decreases as Cl- concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment. The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased, and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment. After bumetanide administration, the above indicators were significantly suppressed. These results confirm that CCI can induce abnormal overexpression of NKCC1, thereby increasing the Cl- concentration in dorsal root ganglion neurons; this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia. In addition, bumetanide can achieve analgesic effects. All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital, College of Medicine, Shihezi University, China on February 22, 2017 (approval No. A2017-169-01).

Osmotically induced removal of lens epithelial cells to prevent PCO after pediatric cataract surgery: Pilot study to assess feasibility

Development of an osmotic-shock technique to remove human lens epithelial cells (LECs) as a preventive measure to address posterior capsule opacification (PCO) after pediatric cataract surgery. Department of Genetics, UCL Institute of Ophthalmology, London, England, and Department of Ophthalmology, Ruijin Hospital, Jiao Tong University, Shanghai, China. Laboratory study. Various tissue preparations of human LECs (cultured on coverslips/collagen-coated membrane inserts, human lens capsule biopsies, and lens organ cultured PCO models) were subjected to a single or incremental hyperosmotic shock (NaCl, 350-4000 mOsm/L) in the presence of inhibitors of the Na+-K+-2Cl- cotransporter (NKCC) (to disable the regulatory volume increase [RVI] process). The integrity of the cell monolayer was determined by phase-contrast microscopy, viability assays, and measurement of transepithelial resistance. Hyperosmotic shock (400 mOsm/L) caused rapid cell shrinkage (<5minutes) in all the LEC models studied. In the absence of the NKCC inhibitor, the shrunk cells gradually returned to their original cell volume and architecture over time, while still exposed to the hyperosmotic shock. However, inhibition of the RVI process disabled the ability for restoration of cell volume leading to persistent cell shrinkage, subsequently resulting in cell detachment from the underlying support medium. Hyperosmotic shock in the presence of inhibitors of the RVI process was effective in rapidly detaching LECs from their basement membranes. This technique could potentially facilitate removal of residual LECs left on the lens capsule after cataract surgery, thus decreasing or eliminating the risk for aggressive cell proliferation and the development of PCO.

Correction to: A Preliminary Study Evaluating the Safety and Efficacy of Bumetanide, an NKCC1 Inhibitor, in Patients with Drug-Resistant Epilepsy

The correct name of the co-author should be ''Vajiheh Aghamollaii'', and not ''Vajihe Aghamollaii'' as given in the original publication of the article.

Abstract 255: Antagonizing Na-K-Cl cotransporter NKCC1 impairs medulloblastoma cell viability

Abstract Medulloblastoma, a common pediatric brain cancer, is comprised of four molecular subgroups: Wnt-activated; Shh-activated; group 3; and group 4. Group 3 subgroup patients have the lowest survival rate and the highest rates of relapse and metastasis [1]. The standard-of-care for medulloblastoma consists of surgical removal of the tumor, radiation, and chemotherapy [1]. However, the standard-of-care poses many risk factors including hearing loss, permanent damage to endocrine and neurocognitive function, and secondary tumors [1]. There is a high demand for new treatment options for medulloblastoma. Interestingly, group 3 medulloblastoma tumors have high expression of GABRA5 and SLC12A2, which code for the α5-subunit of the ligand-gated ionotropic γ-aminobutyric acid type A receptor (GABAAR) and the sodium-potassium-chloride cotransporter isoform-1 (NKCC-1), respectively [2]. Several recent studies have provided evidence that ion channel activity contributes to brain cancer progression [3]. If so, then modulating ion channel(s) function may impair cancer cell viability. We have already shown that use of a positive allosteric modulator of the GABAAR can impair viability of group 3 medulloblastoma cells in vitro and in vivo and with greater specificity and potency than standard-of-care chemotherapeutic [4, 5]. Others have shown that inhibition or down-regulation of NKCC-1 can reduce cell viability, increase apoptosis, and decrease migration and invasion of cells derived from various cancer types [3]. We have examined how modulating NKCC-1 and GABAAR alone and simultaneously impacts the viability of group 3 medulloblastoma cells. We find that there is a synergistic effect in the inhibition of NKCC-1 and positive modulation of GABAAR. We will report on these observations and our model for the mechanism of action.

Role of estradiol in mediation of etomidate-caused seizure-like activity in neonatal rats

BackgroundThe goal of this study was to investigate the effect of estradiol in mediation of electroencephalogram (EEG) abnormality induced by etomidate in neonatal rats. MethodsSprague-Dawley rats were anesthetized using intraperitoneal etomidate for 2 h on postnatal days (P) 4, 5, or 6 and recorded electroencephalogram in two ways. First, pups were recorded EEG two and a half hours under etomidate anesthesia, in subgroups, estradiol receptor antagonist ICI182780 and estradiol synthase inhibitor formestane were given subcutaneously in male rats 15 min prior to etomidate. Second, pups were anesthetized with etomidate for 2 h on P4,5 or 6 and then recovered from anesthesia, EEG were recorded for one hour in two postnatal periods of P9-P11 and P14-P16. Subgroup rats that received bumetanide, NKCC1 inhibitor, to test the NKCC1-GABAAR signaling effect on neonatal brain development, negative control groups and maternally separated for 2 h on P4, 5, or 6 were studied in 16 groups. Each group’s n was = 8. ResultsMale pups showed more severe seizure-like activities than female pups in P4-P6 under etomidate anesthesia. Pups pretreated with ICI182780 and formestane showed a less abnormalities of EEG in male rats. Etomidate caused seizure-like activity in P4-P6 could extend to P9-P11, but not seen in P14-P16, Pretreated with bumetanide only alleviated abnormalities in male pups other than female in P9-P11. ConclusionsEstradiol involves in the NKCC1-GABAAR mediated seizure-like activity caused by etomidte in neonatal rats and these the abnormality lasts near two weeks.

A Novel Na + -K + -Cl − Cotransporter 1 Inhibitor STS66* Reduces Brain Damage in Mice After Ischemic Stroke

Background and Purpose- Inhibition of brain NKCC1 (Na+-K+-Cl- cotransporter 1) with bumetanide (BMT) is of interest in ischemic stroke therapy. However, its poor brain penetration limits the application. In this study, we investigated the efficacy of 2 novel NKCC1 inhibitors, a lipophilic BMT prodrug STS5 (2-(Dimethylamino)ethyl 3-(butylamino)-4-phenoxy-5-sulfamoyl-benzoate;hydrochloride) and a novel NKCC1 inhibitor STS66 (3-(Butylamino)-2-phenoxy-5-[(2,2,2-trifluoroethylamino)methyl]benzenesulfonamide), on reducing ischemic brain injury. Methods- Large-vessel transient ischemic stroke in normotensive C57BL/6J mice was induced with 50-min occlusion of the middle cerebral artery and reperfusion. Focal, permanent ischemic stroke in angiotensin II (Ang II)-induced hypertensive C57BL/6J mice was induced by permanent occlusion of distal branches of middle cerebral artery. A total of 206 mice were randomly assigned to receive vehicle DMSO, BMT, STS5, or STS66. Results- Poststroke BMT, STS5, or STS66 treatment significantly decreased infarct volume and cerebral swelling by ≈40% to 50% in normotensive mice after transient middle cerebral artery occlusion, but STS66-treated mice displayed better survival and sensorimotor functional recovery. STS5 treatment increased the mortality. Ang II-induced hypertensive mice exhibited increased phosphorylatory activation of SPAK (Ste20-related proline alanine-rich kinase) and NKCC1, as well as worsened infarct and neurological deficit after permanent distal middle cerebral artery occlusion. Conclusions- The novel NKCC1 inhibitor STS66 is superior to BMT and STS5 in reducing ischemic infarction, swelling, and neurological deficits in large-vessel transient ischemic stroke, as well as in permanent focal ischemic stroke with hypertension comorbidity.

Targeted treatment of anaerobic cancer. Patent evaluation of US2016279084 and US2017056350

ABSTRACTIntroduction: Based on the initial studies of J. Folkman in 1970s, which led to the proposal of the antiangiogenic therapy, many drugs targeting VEGF or its receptors have been developed with some of them approved for cancer treatment. However, these molecules so far have shown only a limited effect on survival benefits in patients. Thus, new approaches are needed to treat this disease. Considering that cancer utilizes both aerobic and anaerobic glycolytic pathway, authors of patents US2016279084 and US2017056350 propose a method to eradicate the disease, able to affect both metabolic pathways.Areas covered: Patent US2016279084 describes a method consisting of the utilization of either a pharmaceutical cocktail containing antiglycolytic agents (a lactate transporter inhibitor and a NKCC inhibitor) and an angiogenesis inhibitor or a pharmaceutical cocktail containing a lactate transporter inhibitor and an angiogenesis inhibitor in combination with blood vessel occlusion. Patent US2017056350 is strictly related to US2016279084; indeed, it proposes a method consisting of blood vessel occlusion and treatment with a pharmaceutical cocktail, containing the carbonic anhydrase inhibitor bumetanide in presence or absence of an angiogenesis inhibitor.Expert opinion: Although the proposed methodology is very interesting and promising, further studies are necessary to assess the clinical applicability of the inventions.

Elevation of extracellular osmolarity improves signs of myotonia congenita in vitro: a preclinical animal study.

During myotonia congenita, reduced chloride (Cl- ) conductance results in impaired muscle relaxation and increased muscle stiffness after forceful voluntary contraction. Repetitive contraction of myotonic muscle decreases or even abolishes myotonic muscle stiffness, a phenomenon called 'warm up'. Pharmacological inhibition of low Cl- channels by anthracene-9-carboxylic acid in muscle from mice and ADR ('arrested development of righting response') muscle from mice showed a relaxation deficit under physiological conditions compared to wild-type muscle. At increased osmolarity up to 400mosmolL-1 , the relaxation deficit of myotonic muscle almost reached that of control muscle. These effects were mediated by the cation and anion cotransporter, NKCC1, and anti-myotonic effects of hypertonicity were at least partly antagonized by the application of bumetanide. Low chloride-conductance myotonia is caused by mutations in the skeletal muscle chloride (Cl- ) channel gene type 1 (CLCN1). Reduced Cl- conductance of the mutated channels results in impaired muscle relaxation and increased muscle stiffness after forceful voluntary contraction. Exercise decreases muscle stiffness, a phenomena called 'warm up'. To gain further insight into the patho-mechanism of impaired muscle stiffness and the warm-up phenomenon, we characterized the effects of increased osmolarity on myotonic function. Functional force and membrane potential measurements were performed on muscle specimens of ADR ('arrested development of righting response') mice (an animal model for low gCl- conductance myotonia) and pharmacologically-induced myotonia. Specimens were exposed to solutions of increasing osmolarity at the same time as force and membrane potentials were monitored. In the second set of experiments, ADR muscle and pharmacologically-induced myotonic muscle were exposed to an antagonist of NKCC1. Upon osmotic stress, ADR muscle was depolarized to a lesser extent than control wild-type muscle. High osmolarity diminished myotonia and facilitated the warm-up phenomenon as depicted by a faster muscle relaxation time (T90/10 ). Osmotic stress primarily resulted in the activation of the NKCC1. The inhibition of NKCC1 with bumetanide prevented the depolarization and reversed the anti-myotonic effect of high osmolarity. Increased osmolarity decreased signs of myotonia and facilitated the warm-up phenomenon in different in vitro models of myotonia. Activation of NKCC1 activity promotes warm-up and reduces the number of contractions required to achieve normal relaxation kinetics.

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

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

Differential regulation of chloride homeostasis and GABAergic transmission in the thalamus

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

NKCC1 promotes EMT-like process in GBM via RhoA and Rac1 signaling pathways.

Glioblastoma is the most common and lethal primary intracranial tumor. As the key regulator of tumor cell volume, sodium‐potassium‐chloride cotransporter 1 (NKCC1) expression increases along with the malignancy of the glioma, and NKCC1 has been implicated in glioblastoma invasion. However, little is known about the role of NKCC1 in the epithelial‐mesenchymal transition‐like process in gliomas. We noticed that aberrantly elevated expression of NKCC1 leads to changes in the shape, polarity, and adhesion of cells in glioma. Here, we investigated whether NKCC1 promotes an epithelial–mesenchymal transition (EMT)‐like process in gliomas via the RhoA and Rac1 signaling pathways. Pharmacological inhibition and knockdown of NKCC1 both decrease the expressions of mesenchymal markers, such as N‐cadherin, vimentin, and snail, whereas these treatments increase the expression of the epithelial marker E‐cadherin. These findings indicate that NKCC1 promotes an EMT‐like process in gliomas. The underlying mechanism is the facilitation of the binding of Rac1 and RhoA to GTP by NKCC1, which results in a significant enhancement of the EMT‐like process. Specific inhibition or knockdown of NKCC1 both attenuate activated Rac1 and RhoA, and the pharmacological inhibitions of Rac1 and RhoA both impair the invasion and migration abilities of gliomas. Furthermore, we illustrated that NKCC1 knockdown abolished the dissemination and spread of glioma cells in a nude mouse intracranial model. These findings suggest that elevated NKCC1 activity acts in the regulation of an EMT‐like process in gliomas, and thus provides a novel therapeutic strategy for targeting the invasiveness of gliomas, which might help to inhibit the spread of malignant intracranial tumors.

Bumetanide attenuates acute lung injury by suppressing macrophage activation

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.