Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 195
Filtrar
1.
Nat Commun ; 11(1): 78, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911626

RESUMO

The SLC12A cation-Cl- cotransporters (CCC), including NKCC1 and the KCCs, are important determinants of brain ionic homeostasis. SPAK kinase (STK39) is the CCC master regulator, which stimulates NKCC1 ionic influx and inhibits KCC-mediated efflux via phosphorylation at conserved, shared motifs. Upregulation of SPAK-dependent CCC phosphorylation has been implicated in several neurological diseases. Using a scaffold-hybrid strategy, we develop a novel potent and selective SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide ("ZT-1a"). ZT-1a inhibits NKCC1 and stimulates KCCs by decreasing their SPAK-dependent phosphorylation. Intracerebroventricular delivery of ZT-1a decreases inflammation-induced CCC phosphorylation in the choroid plexus and reduces cerebrospinal fluid (CSF) hypersecretion in a model of post-hemorrhagic hydrocephalus. Systemically administered ZT-1a reduces ischemia-induced CCC phosphorylation, attenuates cerebral edema, protects against brain damage, and improves outcomes in a model of stroke. These results suggest ZT-1a or related compounds may be effective CCC modulators with therapeutic potential for brain disorders associated with impaired ionic homeostasis.


Assuntos
Encéfalo/metabolismo , Inibidores Enzimáticos/administração & dosagem , Hidrocarbonetos Clorados/administração & dosagem , Nitrilos/administração & dosagem , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/genética , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo
2.
Elife ; 82019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31545168

RESUMO

Prenatal exposure to ethanol induces aberrant tangential migration of corticopetal GABAergic interneurons, and long-term alterations in the form and function of the prefrontal cortex. We have hypothesized that interneuronopathy contributes significantly to the pathoetiology of fetal alcohol spectrum disorders (FASD). Activity-dependent tangential migration of GABAergic cortical neurons is driven by depolarizing responses to ambient GABA present in the cortical enclave. We found that ethanol exposure potentiates the depolarizing action of GABA in GABAergic cortical interneurons of the embryonic mouse brain. Pharmacological antagonism of the cotransporter NKCC1 mitigated ethanol-induced potentiation of GABA depolarization and prevented aberrant patterns of tangential migration induced by ethanol in vitro. In a model of FASD, maternal bumetanide treatment prevented interneuronopathy in the prefrontal cortex of ethanol exposed offspring, including deficits in behavioral flexibility. These findings position interneuronopathy as a mechanism of FASD symptomatology, and posit NKCC1 as a pharmacological target for the management of FASD.


Assuntos
Consumo de Bebidas Alcoólicas/efeitos adversos , Bumetanida/administração & dosagem , Transtornos do Espectro Alcoólico Fetal/prevenção & controle , Complicações na Gravidez/prevenção & controle , Inibidores de Simportadores de Cloreto de Sódio e Potássio/administração & dosagem , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Animais , Córtex Cerebral/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Transtornos do Espectro Alcoólico Fetal/fisiopatologia , Neurônios GABAérgicos/efeitos dos fármacos , Camundongos , Córtex Pré-Frontal/efeitos dos fármacos , Gravidez , Complicações na Gravidez/fisiopatologia , Resultado do Tratamento , Ácido gama-Aminobutírico/metabolismo
3.
Endocrinology ; 160(11): 2529-2542, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31415088

RESUMO

Prenatal testosterone (T)-treated female sheep display reproductive deficits similar to women with polycystic ovarian syndrome (PCOS), including an increase in LH pulse frequency due to actions of the central GnRH pulse generator. In this study, we used multiple-label immunocytochemistry to investigate the possibility of changes in the γ-aminobutyric acid (GABA) neurotransmitter system at two key components of the GnRH pulse generator in prenatal T-treated sheep: kisspeptin/neurokinin B/dynorphin (KNDy) neurons of the arcuate nucleus, and GnRH neurons in the preoptic area (POA) and mediobasal hypothalamus (MBH). We observed a significant decrease and increase, respectively, in the number of GABAergic synapses onto POA and MBH GnRH neurons in prenatal T-treated ewes; additionally, there was a significant increase in the number of GABAergic inputs onto KNDy neurons. To determine the actions of GABA on GnRH and KNDy neurons, we examined colocalization with the chloride transporters NKCC1 and KCC2, which indicate stimulatory or inhibitory activation of neurons by GABA, respectively. Most GnRH neurons in both POA and MBH colocalized NKCC1 cotransporter whereas none contained the KCC2 cotransporter. Most KNDy neurons colocalized either NKCC1 or KCC2, and 28% of the KNDy population contained NKCC1 alone. Therefore, we suggest that, as in the mouse, GABA in the sheep is stimulatory to GnRH neurons, as well as to a subset of KNDy neurons. Increased numbers of stimulatory GABAergic inputs to both MBH GnRH and KNDy neurons in prenatal T-treated animals may contribute to alterations in steroid feedback control and increased GnRH/LH pulse frequency seen in this animal model of PCOS.


Assuntos
Núcleo Arqueado do Hipotálamo/fisiopatologia , Neurônios GABAérgicos/fisiologia , Hormônio Liberador de Gonadotropina/metabolismo , Síndrome do Ovário Policístico/fisiopatologia , Área Pré-Óptica/fisiopatologia , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Modelos Animais de Doenças , Dinorfinas/metabolismo , Feminino , Kisspeptinas/metabolismo , Neurocinina B/metabolismo , Síndrome do Ovário Policístico/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Área Pré-Óptica/metabolismo , Ovinos , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/metabolismo , Testosterona
4.
Biomed Res Int ; 2019: 1602895, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31179315

RESUMO

The aim was to investigate the effect of dichloroacetate (DCA) on thymus weight, Hassall's corpuscle number (HCs), and NKCC1 RNA expression in Wistar rats aged 4-5 weeks. They were investigated in the controls and DCA-treated gonad-intact and castrated males and females. The treatment lasted 4 weeks with DCA 200 mg/kg/day. At the end of the experiment, rat thymus was weighted, and its lobe was taken for the expression of NKCC1 RNA determined by the PCR method and of Hassall's corpuscles by immunohistochemistry. DCA caused a thymus weight decrease in DCA-treated gonad-intact rats of both genders as compared with their controls (p < 0.05), and no such impact was found in castrated DCA-treated males and females. DCA caused an increase of the HCs in gonad-intact males (p < 0.05), and no such increase in the DCA-treated gonad-intact females was found. There was gender-related difference in the HCs when comparing DCA-treated gonad-intact males and females: males showed significantly higher HCs (p < 0.05); no gender-related differences were found in the castrated DCA-treated groups. The Slc12a2 gene RNA expression level was found to be significantly decreased only in gonad-intact and in castrated DCA-treated males. The authors discuss the gender-related DCA effects on the thymus.


Assuntos
Ácido Dicloroacético/farmacologia , Células Epiteliais/efeitos dos fármacos , RNA/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Timo/efeitos dos fármacos , Animais , Castração , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Imuno-Histoquímica , Masculino , Orquiectomia , Ratos , Ratos Wistar , Timo/patologia
5.
Biomed Res Int ; 2019: 8941046, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31240228

RESUMO

Chloride (Cl-) homeostasis is an essential process involved in neuronal signalling and cell survival. Inadequate regulation of intracellular Cl- interferes with synaptic signalling and is implicated in several neurological diseases. The main inhibitory neurotransmitter of the central nervous system is γ-aminobutyric acid (GABA). GABA hyperpolarises the membrane potential by activating Cl- permeable GABAA receptor channels (GABAAR). This process is reliant on Cl- extruder K+-Cl- cotransporter 2 (KCC2), which generates the neuron's inward, hyperpolarising Cl- gradient. KCC2 is encoded by the fifth member of the solute carrier 12 family (SLC12A5) and has remained a poorly understood component in the development and severity of many neurological diseases for many years. Recent advancements in next-generation sequencing and specific gene targeting, however, have indicated that loss of KCC2 activity is involved in a number of diseases including epilepsy and schizophrenia. It has also been implicated in neuropathic pain following spinal cord injury. Any variant of SLC12A5 that negatively regulates the transporter's expression may, therefore, be implicated in neurological disease. A recent whole exome study has discovered several causative mutations in patients with epilepsy. Here, we discuss the implications of KCC2 in neurological disease and consider the evolving evidence for KCC2's potential as a therapeutic target.


Assuntos
Canais de Cloreto/metabolismo , Neurônios/metabolismo , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Ácido gama-Aminobutírico/farmacologia , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Cloretos/metabolismo , Epilepsia/metabolismo , Marcação de Genes , Homeostase , Humanos , Potenciais da Membrana , Neuralgia/metabolismo , Neurotransmissores/farmacologia , Fosforilação , Esquizofrenia/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Traumatismos da Medula Espinal , Simportadores/genética
6.
Ideggyogy Sz ; 72(5-6): 181-186, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31241262

RESUMO

Background and purpose: Methylation is a key epigenetic modification of DNA and regarding its impact on epilepsy, it is argued that "DNA methylation may play an important role in seizure susceptibility and maintenance of the disorder". DNA methylation status of KCC2 (SCL12A5) and NKCC1 (SCL12A2) associated with refractory temporal lobe epilepsy was investigated in our study. Methods: Thirty-eight patients with temporal lobe epilepsy (TLE) who were diagnosed by video EEG monitoring and 32 healthy control subjects were included in the study. Twenty-three patients in TLE group were men and the remaining 15 were women. Among them, 27 had unilateral temporal focus (9 with right; 18 with left) and 11 patients had bilateral TLE. We analyzed promoter region methylation status of the KCC2 (SCL12A5) and NKCC1 (SCL12A2) genes in the case and control groups. Gene regions of interest were amplified through PCR and sequencing was accomplished with pyro-sequencing. Results: We found a significant relationship between TLE and methylation on the NKCC1. However, there was no association between TLE and methylation on the KCC2 gene. Also, we found no association between right or left and unilateral or bilateral foci of TLE. There was no relationship between TLE and methylation on the NKCC1and KCC2 genes in terms of mesial temporal sclerosis in cranial MRI, head trauma or febrile convulsions. Conclusion: The methylation of NKCC1 can be a mecha-nism of refractory temporal lobe epilepsy. There are limited findings about DNA methylation in TLE. Therefore, further studies with large sample sizes are necessary.


Assuntos
Metilação de DNA , Epilepsia do Lobo Temporal/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/metabolismo , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Estudos de Casos e Controles , Eletroencefalografia , Epilepsia do Lobo Temporal/diagnóstico , Epilepsia do Lobo Temporal/genética , Feminino , Humanos , Imagem por Ressonância Magnética , Masculino , Regiões Promotoras Genéticas , Membro 2 da Família 12 de Carreador de Soluto/genética , Simportadores/genética
7.
Respir Physiol Neurobiol ; 264: 40-50, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30999061

RESUMO

Twenty-five years ago, Filiano and Kinney (1994) proposed that a critical period of postnatal development constitutes one of the three risk factors for sudden infant death syndrome (SIDS). The underlying mechanism was poorly understood. In the last 17 years, much has been uncovered on this period in the rat. Against several expected trends of development, abrupt neurochemical, metabolic, ventilatory, and electrophysiological changes occur in the respiratory system at P12-13. This results in a transient synaptic imbalance with suppressed excitation and enhanced inhibition, and the response to acute hypoxia is the weakest at this time, both at the cellular and system's levels. The basis for the synaptic imbalance is likely to be contributed by a reduced expression of brain-derived neurotrophic factor (BDNF) and its TrkB receptors in multiple brain stem respiratory-related nuclei during the critical period. Exogenous BDNF or a TrkB agonist partially reverses the synaptic imbalance, whereas a TrkB antagonist accentuates the imbalance. A transient down-regulation of pituitary adenylate cyclase-activating polypeptide (PACAP) at P12 in respiratory-related nuclei also contributes to the vulnerability of this period. Carotid body denervation during this time or perinatal hyperoxia merely delays and sometimes prolongs, but not eliminate the critical period. The rationale for the necessity of the critical period in postnatal development is discussed.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Hipóxia/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptor trkB/metabolismo , Fenômenos Fisiológicos Respiratórios , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Animais , Ratos , Receptor trkB/agonistas , Receptor trkB/antagonistas & inibidores
8.
Br J Anaesth ; 122(4): 490-499, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30857605

RESUMO

BACKGROUND: The factors determining peak susceptibility of the developing brain to anaesthetics are unclear. It is unknown why postnatal day 7 (P7) male rats are more vulnerable to anaesthesia-induced memory deficits than littermate females. Given the precocious development of certain regions in the female brain during the neonatal critical period, we hypothesised that females are susceptible to anaesthetic brain injury at an earlier time point than previously tested. METHODS: Female rats were exposed to isoflurane (Iso) 1 minimum alveolar concentration or sham anaesthesia at P4 or P7. Starting at P35, rats underwent a series of behavioural tasks to test their spatial and recognition memory. Cell death immediately after anaesthesia was quantified by Fluoro-Jade C staining in select brain regions, and developmental expression of the chloride transporters KCC2 and NKCC1 was analysed by immunoblotting in male and female rats at P4 and P7. RESULTS: Female rats exposed to Iso at P4 displayed impaired spatial, object-place, -context, and social recognition memory, and increased cell death in the hippocampus and laterodorsal thalamus. Female rats exposed at P7 exhibited only decreased performance in object-context compared with control. The ratio of NKCC1/KCC2 expression in cerebral cortex was higher in P4 females than in P7 females, and similar to that in P7 males. CONCLUSIONS: Female rats exposed to Iso at P4 are sensitive to anaesthetic injury historically observed in P7 males. This is consistent with a comparably immature developmental state in P4 females and P7 males. The window of anaesthetic vulnerability correlates with sex-specific cortical expression of chloride transporters NKCC1 and KCC2. These findings suggest that both sex and developmental age play important roles in determining the outcome after early anaesthesia exposure.


Assuntos
Anestésicos Inalatórios/toxicidade , Disfunção Cognitiva/induzido quimicamente , Isoflurano/toxicidade , Fatores Etários , Anestésicos Inalatórios/administração & dosagem , Animais , Animais Recém-Nascidos , Comportamento Animal/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Córtex Cerebral/metabolismo , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , Esquema de Medicação , Feminino , Isoflurano/administração & dosagem , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Ratos Sprague-Dawley , Fatores Sexuais , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/metabolismo
9.
Am J Physiol Cell Physiol ; 316(4): C545-C558, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30865516

RESUMO

Na+-K+-2Cl- cotransporter-1 (NKCC1) mediates the electroneutral transport of Na+, K+, and Cl- and is normally localized to the basolateral membrane of polarized epithelial cells. We recently reported the first known solute carrier family 12 member 2 ( SLC12A2) mutation (we call NKCC1-DFX) that causes epithelial dysfunction in an undiagnosed disease program case. The heterozygous mutation leads to truncation of the COOH-terminal tail of the cotransporter, resulting in both mutant and wild-type cotransporters being mistrafficked to the apical membrane of polarized epithelial cells. Here we demonstrate by using consecutive truncations and site-directed mutagenesis of the COOH-terminal domain of NKCC1 that truncation of NKCC1 COOH domain uncouples the cotransporter from the lateral membrane. We identify a dileucine motif that, when mutated, leads to cotransporter accumulation in the cytoplasm and mistrafficking to the apical/subapical region of epithelial cells, thereby recapitulating the phenotype observed with the patient mutation. We show that truncation deletion and LL substitution mutants are trafficked out of the endoplasmic reticulum and trans-Golgi network but accumulate in early and late endosomes where they are degraded.


Assuntos
Membrana Celular/metabolismo , Leucina/metabolismo , Fragmentos de Peptídeos/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Animais , Cães , Leucina/genética , Células Madin Darby de Rim Canino , Fragmentos de Peptídeos/genética , Transporte Proteico/fisiologia , Membro 2 da Família 12 de Carreador de Soluto/genética
10.
Neuroreport ; 30(6): 457-462, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30920433

RESUMO

The ionic driving force for the chloride-permeable GABAA receptor is subject to spatial control and distribution of chloride transporters. NKCC1 and KCC2 are mostly expressed in neurons in a specific manner. In the striatum, the localization of these transporters in identified neurons is unknown. In this study, the expression of these transporters was found to be different between projection neurons and interneurons. NKCC1 immunoreactivity was observed in the soma of adult BAC-D1-eGFP+ and D2-eGFP+ striatal projection neurons (SPNs). KCC2 was not expressed in either projection neuron and immunoreactivity to this transporter was observed only in the neuropile. However, NKCC1 and KCC2 co-transporters were not localized in intracellular biocytin-injected dendrites of SPNs of the direct or indirect pathways (D1-SPNs and D2-SPNs). Experiments with PV Cre transgenic mice transfected with Cre-dependent adeno-associated viruses containing tdTomato in the striatum showed a cell-type-specific distribution of KCC2 chloride transporter co-expression associated with PV interneurons. Thus, depolarizing actions of GABA responses in adult projection neurons can be explained by the expression and somatic localization of the NKCC1 transporters. A somato/dendritic distribution of KCC2 expression was observed only in striatal interneurons and corresponds to the hyperpolarizing action of GABA recorded in these cells. This correlates the different roles for GABA actions in striatal neuronal excitability with the expression of specific chloride transporters.


Assuntos
Corpo Estriado/metabolismo , Neurônios/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
11.
Biol Pharm Bull ; 42(3): 501-506, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30828081

RESUMO

The mechanism underlying the increased pharmacological effects of phenobarbital in rats with glycerol-induced acute renal failure (ARF) was examined. In the experiments, a surgical cannula was inserted in the lateral ventricle of the rats for phenobarbital infusion, and the ARF induction was performed by intramuscular administration of 50% glycerol. The onset time of anesthesia by phenobarbital was determined with the tail flick method. In addition, cerebral microsomes were prepared from excised cerebral cortices of sham and ARF rats, and the cerebral expression of the γ-aminobutyric acid (GABA)A receptor and two cation-chloride transporters, KCC2 and NKCC1, was evaluated by Western blotting, as their functions are involved in the anesthetic effects of phenobarbital. When phenobarbital was infused in the ventricle, anesthesia was induced 2.2-times faster in ARF rats than in sham rats, and there was no detectable increase in the cerebral expression of the GABAA receptor in ARF rats. It was additionally noted that the cerebral expression of KCC2 decreased, whereas that of NKCC1 was unaltered in ARF rats. These findings indicated that the anesthetic effects of phenobarbital are potentiated in ARF rats, probably due to imbalanced cerebral expression of KCC2 and NKCC1, suggesting that altered cation-chloride handling in nerve cells is associated.


Assuntos
Lesão Renal Aguda/induzido quimicamente , Glicerol/toxicidade , Fenobarbital/farmacologia , Lesão Renal Aguda/metabolismo , Anestésicos Intravenosos/farmacologia , Animais , Bumetanida/farmacologia , Diuréticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipnóticos e Sedativos/farmacologia , Masculino , Distribuição Aleatória , Ratos , Ratos Wistar , Receptores de GABA-A/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/genética , Simportadores/metabolismo , Uretana/farmacologia
12.
Neurol Sci ; 40(5): 1007-1013, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30759289

RESUMO

The etiology of juvenile myoclonic epilepsy (JME) is still unknown and the process of elaboration of multiple genetic mechanisms is ongoing. The aim of this study was to investigate the potential role of NKCC1 (SCL12A2) and KCC2 (SCL12A5) in JME by comparing their DNA methylation status in patients with JME versus healthy controls. Forty-nine patients with JME and 39 healthy individuals were compared for DNA methylation at the 5CpG islands. A total of 71 (81%) samples were found to have methylation in the NKCC1 gene, 36 (73%) from patients and 35 (90%) from healthy individuals. Out of the KCC2 samples, 50 (57%) were found to have methylation, 33 (67%) from patients and 17 (44%) from healthy individuals. In patients with JME, methylation of NKCC1 (73%) was lower than its methylation in the controls (90%) (p = 0.047). On the other hand, methylation of KCC2 in patients with JME (67%) was greater than the methylation in the controls (44%) (p = 0.022). Twenty-eight patients were treated with VPA and ongoing medications were not found to be associated with methylation (p > 0.05). In the present study, we determined significantly lower NKCC1 DNA methylation and significantly higher KCC2 DNA methylation levels in patients with JME compared with the healthy controls. This implies that NKCC1 expression can be higher and KCC2 expression can be reduced in affected people. Further studies that investigate the potential effect of DNA methylation mechanisms regulating gene expression on seizure activity and how they change JME network activity will be helpful.


Assuntos
Metilação de DNA , Epilepsia Mioclônica Juvenil/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/metabolismo , Adulto , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Epilepsia Mioclônica Juvenil/tratamento farmacológico , Epilepsia Mioclônica Juvenil/genética , Regiões Promotoras Genéticas , Membro 2 da Família 12 de Carreador de Soluto/genética , Simportadores/genética
13.
PLoS One ; 14(1): e0210660, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30629699

RESUMO

After intracerebral hemorrhage (ICH), brain edema commonly occurs and can cause death. Along with edema, there are significant alterations in the concentrations of key ions such as sodium, potassium, and chloride, which are essential to brain function. NKCC1, a cation-chloride cotransporter, is upregulated after brain damage, such as traumatic injury and ischemic stroke. NKCC1 brings sodium and chloride into the cell, possibly worsening ion dyshomeostasis. Bumetanide, a specific NKCC1 antagonist, blocks the transport of chloride into cells, and thus should attenuate the increases in chloride, which should lessen brain edema and improve neuronal functioning post-ICH, as with other injuries. We used the collagenase model of ICH to test whether bumetanide treatment for three days (vs. vehicle) would improve outcome. We gave bumetanide beginning at two hours or seven days post-ICH and measured behavioural outcome, edema, and brain ion content after treatment. There was some evidence for a minor reduction in edema after early dosing, but this did not improve behaviour or lessen injury. Contrary to our hypothesis, bumetanide did not normalize ion concentrations after late dosing. Bumetanide did not improve behavioural outcome or affect lesion volume. After ICH, bumetanide is safe to use in rats but does not improve functional outcome in the majority of animals.


Assuntos
Bumetanida/uso terapêutico , Hemorragia Cerebral/tratamento farmacológico , Animais , Hemorragia Cerebral/metabolismo , Cloretos/metabolismo , Colagenases/metabolismo , Modelos Animais de Doenças , Masculino , Espectrometria de Massas , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Resultado do Tratamento
14.
FASEB J ; 33(1): 1051-1061, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30148674

RESUMO

The Kelch-like 3 ( KLHL3) mutations contributed to the most common causative genes in patients with pseudohypoaldosteronism type II (PHAII); however, the molecular mechanisms of PHAII-causing mutations in BTB domain of KLHL3 in vivo have not been investigated. We generated and analyzed Klhl3 knock-in (KI) mice carrying a missense M131V mutation in the BTB domain (corresponding to human KLHL3 M78V mutation). Klhl3M131V/+ KI mice exhibited typical PHAII phenotype with an exaggerated diuretic response to hydrochlorothiazide. Their kidney tissues showed an unchanged KLHL3, decreased cullin 3 (Cul3), and increased with-no-lysine kinases (WNKs) WNK1 and WNK4 along with an enhanced downstream ste20-related proline/alanine-rich kinase/oxidative stress response kinase 1-N(K)CC phosphorylation. Their Cul3 protein in the cytosol of distal convoluted tubule cells was also significantly attenuated on immunogold-labeling electron microscopy. In microdissected renal tubules, Klhl3M131V/+ KI mice expressed high levels of Wnk4 mRNA in the distal nephron. In vitro coimmunoprecipitation showed the KLHL3 BTB domain mutation retained intact interaction with WNKs but reduced binding to Cul3, thus leading to the increased abundance of total WNKs. In summary, Klhl3M131V/+ KI mice feature typical PHAII with a simultaneous increase of WNK1 and WNK4 through the impaired KLHL3 BTB domain binding to Cul3.-Lin, C.-M., Cheng, C.-J., Yang, S.-S., Tseng, M.-H., Yen, M.-T., Sung, C.-C., Lin, S.-H. Generation and analysis of a mouse model of pseudohypoaldosteronism type II caused by KLHL3 mutation in BTB domain.


Assuntos
Domínio BTB-POZ , Proteínas dos Microfilamentos/genética , Mutação de Sentido Incorreto , Pseudo-Hipoaldosteronismo/genética , Animais , Proteínas Culina/metabolismo , Modelos Animais de Doenças , Furosemida/administração & dosagem , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Hidroclorotiazida/administração & dosagem , Túbulos Renais/metabolismo , Camundongos , Fenótipo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pseudo-Hipoaldosteronismo/metabolismo , RNA Mensageiro/genética , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo
15.
J Cell Physiol ; 234(2): 1630-1642, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30159893

RESUMO

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.


Assuntos
Neoplasias Encefálicas/enzimologia , Transição Epitelial-Mesenquimal , Glioblastoma/enzimologia , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos Nus , Invasividade Neoplásica , Transdução de Sinais , Membro 2 da Família 12 de Carreador de Soluto/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética
16.
Am J Physiol Cell Physiol ; 316(4): C525-C544, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30576237

RESUMO

Choroid plexus epithelial cells (CPECs) secrete cerebrospinal fluid (CSF). They express Na+-K+-ATPase and Na+-K+-2Cl- cotransporter 1 (NKCC1) on their apical membrane, deviating from typical basolateral membrane location in secretory epithelia. Given this peculiarity, the direction of basal net ion fluxes mediated by NKCC1 in CPECs is controversial, and cotransporter function is unclear. Determining the direction of basal NKCC1-mediated fluxes is critical to understanding the function of apical NKCC1. If NKCC1 works in the net efflux mode, it may be directly involved in CSF secretion. Conversely, if NKCC1 works in the net influx mode, it would have an absorptive function, contributing to intracellular Cl- concentration ([Cl-]i) and cell water volume (CWV) maintenance needed for CSF secretion. We resolve this long-standing debate by electron microscopy (EM), live-cell-imaging microscopy (LCIM), and intracellular Na+ and Cl- measurements in single CPECs of NKCC1+/+ and NKCC1-/- mouse. NKCC1-mediated ion and associated water fluxes are tightly linked, thus their direction is inferred by measuring CWV changes. Genetic or pharmacological NKCC1 inactivation produces CPEC shrinkage. EM of NKCC1-/- CPECs in situ shows they are shrunken, forming large dilations of their basolateral extracellular spaces, yet remaining attached by tight junctions. Normarski LCIM shows in vitro CPECs from NKCC1-/- are ~17% smaller than NKCC1+/+. CWV measurements in calcein-loaded CPECs show that bumetanide (10 µM) produces ~16% decrease in CWV in NKCC1+/+ but not in NKCC1-/- CPECs. Our findings suggest that under basal conditions apical NKCC1 is continuously active and works in the net inward flux mode maintaining [Cl-]i and CWV needed for CSF secretion.


Assuntos
Plexo Corióideo/efeitos dos fármacos , Plexo Corióideo/fisiologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Animais , Bumetanida/farmacologia , Células Cultivadas , Plexo Corióideo/ultraestrutura , Células Epiteliais/ultraestrutura , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL
17.
Neurosci Lett ; 689: 33-44, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29329909

RESUMO

It is well known that the electrical signaling in neuronal networks is modulated by chloride (Cl-) fluxes via the inhibitory GABAA and glycine receptors. Here, we discuss the putative contribution of Cl- fluxes and intracellular Cl- to other forms of information transfer in the CNS, namely the bidirectional communication between neurons and astrocytes. The manuscript (i) summarizes the generic functions of Cl- in cellular physiology, (ii) recaps molecular identities and properties of Cl- transporters and channels in neurons and astrocytes, and (iii) analyzes emerging studies implicating Cl- in the modulation of neuroglial communication. The existing literature suggests that neurons can alter astrocytic Cl- levels in a number of ways; via (a) the release of neurotransmitters and activation of glial transporters that have intrinsic Cl- conductance, (b) the metabotropic receptor-driven changes in activity of the electroneutral cation-Cl- cotransporter NKCC1, and (c) the transient, activity-dependent changes in glial cell volume which open the volume-regulated Cl-/anion channel VRAC. Reciprocally, astrocytes are thought to alter neuronal [Cl-]i through either (a) VRAC-mediated release of the inhibitory gliotransmitters, GABA and taurine, which open neuronal GABAA and glycine receptor/Cl- channels, or (b) the gliotransmitter-driven stimulation of NKCC1. The most important recent developments in this area are the identification of the molecular composition and functional heterogeneity of brain VRAC channels, and the discovery of a new cytosolic [Cl-] sensor - the Wnk family protein kinases. With new work in the field, our understanding of the role of Cl- in information processing within the CNS is expected to be significantly updated.


Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Comunicação Celular/fisiologia , Cloretos/metabolismo , Neurônios/citologia , Animais , Tamanho Celular , Humanos , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Transdução de Sinais , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Transmissão Sináptica
18.
J Biol Chem ; 294(7): 2519-2528, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30563844

RESUMO

Many erythrocyte processes and pathways, including glycolysis, the pentose phosphate pathway (PPP), KCl cotransport, ATP release, Na+/K+-ATPase activity, ankyrin-band 3 interactions, and nitric oxide (NO) release, are regulated by changes in O2 pressure that occur as a red blood cell (RBC) transits between the lungs and tissues. The O2 dependence of glycolysis, PPP, and ankyrin-band 3 interactions (affecting RBC rheology) are controlled by O2-dependent competition between deoxyhemoglobin (deoxyHb), but not oxyhemoglobin (oxyHb), and other proteins for band 3. We undertook the present study to determine whether the O2 dependence of Na+/K+/2Cl- cotransport (catalyzed by Na+/K+/2Cl- cotransporter 1 [NKCC1]) might similarly originate from competition between deoxyHb and a protein involved in NKCC1 regulation for a common binding site on band 3. Using three transgenic mouse strains having mutated deoxyhemoglobin-binding sites on band 3, we found that docking of deoxyhemoglobin at the N terminus of band 3 displaces the protein with no lysine kinase 1 (WNK1) from its overlapping binding site on band 3. This displacement enabled WNK1 to phosphorylate oxidative stress-responsive kinase 1 (OSR1), which, in turn, phosphorylated and activated NKCC1. Under normal solution conditions, the NKCC1 activation increased RBC volume and thereby induced changes in RBC rheology. Because the deoxyhemoglobin-mediated WNK1 displacement from band 3 in this O2 regulation pathway may also occur in the regulation of other O2-regulated ion transporters, we hypothesize that the NKCC1-mediated regulatory mechanism may represent a general pattern of O2 modulation of ion transporters in erythrocytes.


Assuntos
Eritrócitos/metabolismo , Hemoglobinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Eritrócitos/citologia , Camundongos , Fosforilação
19.
Respir Physiol Neurobiol ; 261: 15-23, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30590202

RESUMO

Spinal chloride-dependent synaptic inhibition is critical in regulating breathing and requires neuronal chloride gradients established by cation-chloride cotransporters Na+-K+-2Cl- (NKCC1) and K+-Cl- (KCC2). Spinal transection disrupts NKCC1/KCC2 balance, diminishing chloride gradients in neurons below injury, contributing to spasticity and chronic pain. It is not known if similar disruptions in NKCC1/KCC2 balance occur in respiratory motor neurons after incomplete cervical contusion (C2SC). We hypothesized that C2SC disrupts NKCC1/KCC2 balance in phrenic motor neurons. NKCC1 and KCC2 immunoreactivity was assessed in CtB-positive phrenic motor neurons. Five weeks post-C2SC: 1) neither membrane-bound nor cytosolic NKCC1 expression were significantly changed, although the membrane/cytosolic ratio increased, consistent with net chloride influx; and 2) both membrane and cytosolic KCC2 expression increased, although the membrane/cytosolic ratio decreased, consistent with net chloride efflux. Thus, contrary to our original hypothesis, complex shifts in NKCC1/KCC2 balance occur post-C2SC. The functional significance of these changes remains unclear.


Assuntos
Medula Cervical/lesões , Contusões/metabolismo , Neurônios Motores/metabolismo , Nervo Frênico/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/patologia , Medula Cervical/metabolismo , Medula Cervical/patologia , Vértebras Cervicais , Contusões/patologia , Citosol/metabolismo , Citosol/patologia , Modelos Animais de Doenças , Masculino , Neurônios Motores/patologia , Nervo Frênico/patologia , Distribuição Aleatória , Ratos Endogâmicos Lew , Traumatismos da Medula Espinal/metabolismo
20.
Epilepsia ; 59(11): 2106-2117, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30306542

RESUMO

OBJECTIVE: Dravet syndrome is a rare neurodevelopmental disease, characterized by general cognitive impairment and severe refractory seizures. The majority of patients carry the gene mutation SCN1A, leading to a defective sodium channel that contributes to pathogenic brain excitability. A γ-aminobutyric acid (GABAergic) impairment, as in other neurodevelopmental diseases, has been proposed as an additional mechanism, suggesting that seizures could be alleviated by GABAergic therapies. However, up to now the physiological mechanisms underlying the GABAergic dysfunction in Dravet syndrome are still unknown due to the scarce availability of this brain tissue. Here we studied, for the first time, human GABAA -evoked currents using cortical brain tissue from Dravet syndrome patients. METHODS: We transplanted in Xenopus oocytes cell membranes obtained from brain tissues of autopsies of Dravet syndrome patients, tuberous sclerosis complex patients as a pathological comparison, and age-matched controls. Additionally, experiments were performed on oocytes expressing human α1ß2γ2 and α1ß2 GABAA receptors. GABAA currents were recorded using the two-microelectrodes voltage-clamp technique. Quantitative real-time polymerase chain reaction, immunohistochemistry, and double-labeling techniques were carried out on the same tissue samples. RESULTS: We found (1) a decrease in GABA sensitivity in Dravet syndrome compared to controls, which was related to an increase in α4- relative to α1-containing GABAA receptors; (2) a shift of the GABA reversal potential toward more depolarizing values in Dravet syndrome, and a parallel increase of the chloride transporters NKCC1/KCC2 expression ratio; (3) an increase of GABAA currents induced by low doses of cannabidiol both in Dravet syndrome and tuberous sclerosis complex comparable to that induced by a classical benzodiazepine, flunitrazepam, that still persists in γ-less GABAA receptors. SIGNIFICANCE: Our study indicates that a dysfunction of the GABAergic system, considered as a feature of brain immaturity, together with defective sodium channels, can contribute to a general reduction of inhibitory efficacy in Dravet brain, suggesting that GABAA receptors could be a target for new therapies.


Assuntos
Córtex Cerebral/patologia , Epilepsias Mioclônicas/patologia , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico/metabolismo , Adulto , Animais , Anticonvulsivantes/farmacologia , Canabidiol/farmacologia , Membrana Celular/transplante , Córtex Cerebral/ultraestrutura , Criança , Pré-Escolar , Estimulação Elétrica , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/metabolismo , Feminino , Humanos , Larva , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/genética , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Xenopus , Ácido gama-Aminobutírico/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA