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1.
Nat Commun ; 12(1): 447, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469018

RESUMO

Cerebrospinal fluid (CSF) provides vital support for the brain. Abnormal CSF accumulation, such as hydrocephalus, can negatively affect perinatal neurodevelopment. The mechanisms regulating CSF clearance during the postnatal critical period are unclear. Here, we show that CSF K+, accompanied by water, is cleared through the choroid plexus (ChP) during mouse early postnatal development. We report that, at this developmental stage, the ChP showed increased ATP production and increased expression of ATP-dependent K+ transporters, particularly the Na+, K+, Cl-, and water cotransporter NKCC1. Overexpression of NKCC1 in the ChP resulted in increased CSF K+ clearance, increased cerebral compliance, and reduced circulating CSF in the brain without changes in intracranial pressure in mice. Moreover, ChP-specific NKCC1 overexpression in an obstructive hydrocephalus mouse model resulted in reduced ventriculomegaly. Collectively, our results implicate NKCC1 in regulating CSF K+ clearance through the ChP in the critical period during postnatal neurodevelopment in mice.


Assuntos
Líquido Cefalorraquidiano/metabolismo , Plexo Corióideo/patologia , Hidrocefalia/patologia , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Animais , Animais Recém-Nascidos , Plexo Corióideo/diagnóstico por imagem , Plexo Corióideo/crescimento & desenvolvimento , Plexo Corióideo/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Hidrocefalia/congênito , Hidrocefalia/diagnóstico , Hidrocefalia/fisiopatologia , Injeções Intraventriculares , Pressão Intracraniana/fisiologia , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Transgênicos , Membro 2 da Família 12 de Carreador de Soluto/genética
2.
Elife ; 92020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33315011

RESUMO

N-Glycanase 1 (NGLY1) is a cytoplasmic deglycosylating enzyme. Loss-of-function mutations in the NGLY1 gene cause NGLY1 deficiency, which is characterized by developmental delay, seizures, and a lack of sweat and tears. To model the phenotypic variability observed among patients, we crossed a Drosophila model of NGLY1 deficiency onto a panel of genetically diverse strains. The resulting progeny showed a phenotypic spectrum from 0 to 100% lethality. Association analysis on the lethality phenotype, as well as an evolutionary rate covariation analysis, generated lists of modifying genes, providing insight into NGLY1 function and disease. The top association hit was Ncc69 (human NKCC1/2), a conserved ion transporter. Analyses in NGLY1-/- mouse cells demonstrated that NKCC1 has an altered average molecular weight and reduced function. The misregulation of this ion transporter may explain the observed defects in secretory epithelium function in NGLY1 deficiency patients.


Assuntos
Defeitos Congênitos da Glicosilação/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Animais , Modelos Animais de Doenças , Drosophila melanogaster , Camundongos , Camundongos Knockout , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Fenótipo
3.
PLoS One ; 15(5): e0232967, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32413057

RESUMO

The pivotal role of KCC2 and NKCC1 in development and maintenance of fast inhibitory neurotransmission and their implication in severe human diseases arouse interest in posttranscriptional regulatory mechanisms such as (de)phosphorylation. Staurosporine (broad kinase inhibitor) and N-ethylmalemide (NEM) that modulate kinase and phosphatase activities enhance KCC2 and decrease NKCC1 activity. Here, we investigated the regulatory mechanism for this reciprocal regulation by mass spectrometry and immunoblot analyses using phospho-specific antibodies. Our analyses revealed that application of staurosporine or NEM dephosphorylates Thr1007 of KCC2, and Thr203, Thr207 and Thr212 of NKCC1. Dephosphorylation of Thr1007 of KCC2, and Thr207 and Thr212 of NKCC1 were previously demonstrated to activate KCC2 and to inactivate NKCC1. In addition, application of the two agents resulted in dephosphorylation of the T-loop and S-loop phosphorylation sites Thr233 and Ser373 of SPAK, a critical kinase in the WNK-SPAK/OSR1 signaling module mediating phosphorylation of KCC2 and NKCC1. Taken together, these results suggest that reciprocal regulation of KCC2 and NKCC1 via staurosporine and NEM is based on WNK-SPAK/OSR1 signaling. The key regulatory phospho-site Ser940 of KCC2 is not critically involved in the enhanced activation of KCC2 upon staurosporine and NEM treatment, as both agents have opposite effects on its phosphorylation status. Finally, NEM acts in a tissue-specific manner on Ser940, as shown by comparative analysis in HEK293 cells and immature cultured hippocampal neurons. In summary, our analyses identified phospho-sites that are responsive to staurosporine or NEM application. This provides important information towards a better understanding of the cooperative interactions of different phospho-sites.


Assuntos
Etilmaleimida/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Estaurosporina/farmacologia , Simportadores/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo
4.
PLoS Genet ; 16(4): e1008643, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32294086

RESUMO

Hereditary hearing loss is challenging to diagnose because of the heterogeneity of the causative genes. Further, some genes involved in hereditary hearing loss have yet to be identified. Using whole-exome analysis of three families with congenital, severe-to-profound hearing loss, we identified a missense variant of SLC12A2 in five affected members of one family showing a dominant inheritance mode, along with de novo splice-site and missense variants of SLC12A2 in two sporadic cases, as promising candidates associated with hearing loss. Furthermore, we detected another de novo missense variant of SLC12A2 in a sporadic case. SLC12A2 encodes Na+, K+, 2Cl- cotransporter (NKCC) 1 and plays critical roles in the homeostasis of K+-enriched endolymph. Slc12a2-deficient mice have congenital, profound deafness; however, no human variant of SLC12A2 has been reported as associated with hearing loss. All identified SLC12A2 variants mapped to exon 21 or its 3'-splice site. In vitro analysis indicated that the splice-site variant generates an exon 21-skipped SLC12A2 mRNA transcript expressed at much lower levels than the exon 21-included transcript in the cochlea, suggesting a tissue-specific role for the exon 21-encoded region in the carboy-terminal domain. In vitro functional analysis demonstrated that Cl- influx was significantly decreased in all SLC12A2 variants studied. Immunohistochemistry revealed that SLC12A2 is located on the plasma membrane of several types of cells in the cochlea, including the strial marginal cells, which are critical for endolymph homeostasis. Overall, this study suggests that variants affecting exon 21 of the SLC12A2 transcript are responsible for hereditary hearing loss in humans.


Assuntos
Perda Auditiva Neurossensorial/congênito , Perda Auditiva Neurossensorial/genética , Mutação , Domínios Proteicos/genética , Membro 2 da Família 12 de Carreador de Soluto/química , Membro 2 da Família 12 de Carreador de Soluto/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cloretos/metabolismo , Cóclea/metabolismo , Cóclea/patologia , Surdez/congênito , Surdez/genética , Éxons/genética , Feminino , Expressão Gênica , Células HEK293 , Humanos , Lactente , Macaca fascicularis , Masculino , Linhagem , Splicing de RNA , RNA Mensageiro/análise , RNA Mensageiro/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo
5.
Free Radic Res ; 54(4): 231-243, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32295440

RESUMO

Amphotericin B has been the gold standard for the treatment of invasive mycosis for many years. Its resistance mechanisms are reported to be mainly related to the decrease of ergosterol content or the changes of cell wall. Previous study has shown that Saccharomyces cerevisiae strain lack of BSC2 was sensitive significantly to Amphotericin B. In the present study, the role of BSC2 on Amphotericin B resistance were investigated. We found that BSC2 enhanced the resistance of yeast cells to Amphotericin B, which was not related to cellular ergosterol content. BSC2 can maintain the permeability of mitochondrial membrane and cell membrane integrity by inhibiting the accumulation of intercellular reactive oxygen species and alleviating the production of lipid peroxidation and superoxide radical. These alterations were attributed to the enhancement of the activities of superoxide dismutase, catalase and glutathione peroxidase, and the increased glutathione content. Taken together, BSC2 inhibits oxidative damage induced by Amphotericin B through increasing activities of antioxidant enzymes and levels of GSH to alleviate the accumulation of reactive oxygen species, lipid peroxidation and superoxide radical, resulting in the maintenance of mitochondrial membrane potential and cell membrane integrity. However, Amphotericin B resistance mediated by BSC2 is independent of Yap1p, GSH1 and Hog1p. The results demonstrate for the first time that BSC2 enhances cell resistance to Amphotericin B by inhibiting oxidative damage in yeast. Our findings improve current understanding of the mechanism of Amphotericin B resistance and provide potential strategy for reducing Amphotericin B resistance.


Assuntos
Anfotericina B/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Antifúngicos/farmacologia , Antioxidantes/farmacologia , Farmacorresistência Fúngica , Ergosterol/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/sangue , Membro 2 da Família 12 de Carreador de Soluto/genética , Fatores de Transcrição/metabolismo
6.
Fluids Barriers CNS ; 17(1): 15, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32046744

RESUMO

BACKGROUND: The classical view of cerebrospinal fluid (CSF) production posits the choroid plexus as its major source. Although previous studies indicate that part of CSF production occurs in the subarachnoid space (SAS), the mechanisms underlying extra-choroidal CSF production remain elusive. We here investigated the distributions of aquaporin 1 (AQP1) and Na+/K+/2Cl- cotransporter 1 (NKCC1), key proteins for choroidal CSF production, in the adult rodent brain and spinal cord. METHODS: We have accessed AQP1 distribution in the intact brain using uDISCO tissue clearing technique and by Western blot. AQP1 and NKCC1 cellular localization were accessed by immunohistochemistry in brain and spinal cord obtained from adult rodents. Imaging was performed using light-sheet, confocal and bright field light microscopy. RESULTS: We determined that AQP1 is widely distributed in the leptomeningeal vasculature of the intact brain and that its glycosylated isoform is the most prominent in different brain regions. Moreover, AQP1 and NKCC1 show specific distributions in the smooth muscle cell layer of penetrating arterioles and veins in the brain and spinal cord, and in the endothelia of capillaries and venules, restricted to the SAS vasculature. CONCLUSIONS: Our results shed light on the molecular framework that may underlie extra-choroidal CSF production and we propose that AQP1 and NKCC1 within the leptomeningeal vasculature, specifically at the capillary level, are poised to play a role in CSF production throughout the central nervous system.


Assuntos
Aquaporina 1/metabolismo , Sistema Nervoso Central/metabolismo , Transporte de Íons/fisiologia , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Animais , Plexo Corióideo/metabolismo , Imuno-Histoquímica/métodos , Camundongos Endogâmicos C57BL , Roedores/metabolismo
7.
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 , Nitrilas/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
8.
Biochem Pharmacol ; 171: 113738, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31786261

RESUMO

Dysregulation of alveolar macrophage activation has been recognized as the major mechanism in the pathogenesis of acute lung injury. The aim of the present study was to investigate the role of NKCC1 regulating mechanism in modulating macrophage activation. Knockout (SPAK-/- and WNK4-/-) and knockin (WNK4D561A/+) mice were used in this study. LPS induced expression of p-NKCC1 and activation of NFκB in the primary culture of alveolar macrophages. WNK4 or SPAK knockout suppressed p-NKCC1 expression and inflammation cascade activation, whereas WNK4 knockin enhanced these responses. Intrapulmonary administration of LPS induced in vivo expression and phosphorylation of NKCC1 in alveolar inflammation cells and caused a shift in the cell population from macrophage to neutrophil predominance. WNK4 or SPAK knockout attenuated the LPS-induced alveolar cell-population shifting, macrophage NKCC1 phosphorylation, and acute lung injury, whereas WNK4 knockin augmented the inflammatory response. In summary, our results demonstrated the presence of NKCC1 in alveolar macrophage, which is inducible by lipopolysaccharide. Our results also showed showed that the WNK4-SPAK-NKCC1 cascade plays an important role in modulating macrophage activation to regulate LPS-induced lung inflammation and lung injury.


Assuntos
NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/genética , Lesão Pulmonar/metabolismo , Macrófagos Alveolares/citologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/genética , Fosforilação/efeitos dos fármacos , Pneumonia/induzido quimicamente , Pneumonia/genética , Pneumonia/metabolismo , Proteínas Serina-Treonina Quinases/genética , Membro 2 da Família 12 de Carreador de Soluto/genética
9.
Am J Physiol Regul Integr Comp Physiol ; 318(1): R17-R29, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31617750

RESUMO

The present study provides molecular and functional characterization of Na+-K+-2Cl- cotransporter (NKCC1/Slc12a2) in the gills of sea lamprey (Petromyzon marinus), the most basal extant vertebrate with an osmoregulatory strategy. We report the full-length peptide sequence for the lamprey Na-K-Cl cotransporter 1 (NKCC1), which we show groups strongly with and occupies a basal position among other vertebrate NKCC1 sequences. In postmetamorphic juvenile lamprey, nkcc1 mRNA was present in many tissues but was fivefold higher in the gill than any other examined tissue, and NKCC1 protein was only detected in the gill. Gill mRNA and protein abundances of NKCC1 and Na+-K+-ATPase (NKA/Atp1a1) were significantly upregulated (20- to 200-fold) during late metamorphosis in fresh water, coinciding with the development of salinity tolerance, and were upregulated an additional twofold after acclimation to seawater (SW). Immunohistochemistry revealed that NKCC1 in the gill is found in filamental ionocytes coexpressing NKA, which develop during metamorphosis in preparation for SW entry. Lamprey treated with bumetanide, a widely used pharmacological inhibitor of NKCC1, exhibited higher plasma Cl- and osmolality as well as reduced muscle water content after 24 h in SW; there were no effects of bumetanide in freshwater-acclimated lamprey. This work provides the first functional characterization of NKCC1 as a mechanism for branchial salt secretion in lampreys, providing evidence that this mode of Cl- secretion has been present among vertebrates for ~550 million years.


Assuntos
Brânquias/fisiologia , Osmorregulação/fisiologia , Petromyzon/fisiologia , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Sequência de Aminoácidos , Animais , Bumetanida/farmacologia , Regulação da Expressão Gênica , Filogenia , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Membro 2 da Família 12 de Carreador de Soluto/genética
10.
Mol Neurobiol ; 57(1): 1-10, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31493242

RESUMO

The cation-chloride cotransporters Na+-K+-2Cl--1 (NKCC1) and K+-2Cl--2 (KCC2) critically regulate neuronal responses to gamma-aminobutyric acid (GABA). NKCC1 renders GABA excitatory in immature neurons while expression of KCC2 signals GABA maturation to its inhibitory role. Imbalances in NKCC1/KCC2 alter GABA neurotransmission, which may contribute to hyperexcitability and blunted inhibition in neurocircuitry after neonatal exposure to anesthesia. Thus, we hypothesized that anesthetics may dysregulate NKCC1 and/or KCC2 in developing brain. We exposed postnatal day (PND) 7 mice to sevoflurane or carrier gases and assessed NKCC1 and KCC2 expression across three brain regions 6 h and 24 h after initial exposure. To test differences in behavior, we challenged pups receiving sevoflurane or carrier gases on PND7 with propofol on PND8 and recorded parameters of anesthesia induction and maintenance. Sevoflurane exposure increased cortical NKCC1 at 6 h (p = 0.03) and decreased cortical and hippocampal KCC2 at 24 h (p = 0.009 and p = 0.007, respectively). NKCC1/KCC2 ratio was significantly increased at both 6 h (p = 0.02) and 24 h (p = 0.03) in cortex and at 24 h (p = 0.02) in hippocampus. After propofol challenge on PND8, pups previously exposed to sevoflurane on PND7 regained righting reflex significantly faster than their non-exposed cohort (p < 0.001). Disturbing NKCC1/KCC2 balance may underlie circuit hyperexcitability and contribute to neurodevelopmental impairments we have observed in previous studies of neonatal anesthesia exposure. Human infants previously exposed to anesthesia may require higher concentrations of anesthetic drugs, potentially compounding their susceptibility for neurodevelopmental sequalae.


Assuntos
Encéfalo/metabolismo , Cátions/metabolismo , Cloretos/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/metabolismo , Animais , Animais Recém-Nascidos , Hipocampo/metabolismo , Camundongos , Neurônios/metabolismo , Reflexo de Endireitamento , Sevoflurano/farmacologia
11.
Mediators Inflamm ; 2019: 7583760, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31582903

RESUMO

Diabetic heart dysfunctions during cardiac surgeries have revealed several clinical problems associated with ion imbalance. However, the mechanism of ion imbalance mediated by cardioplegia and a diabetic heart is largely unclear. We hypothesized that ion transporters might be regulated differently in the diabetic heart and that the differentially regulated ion transporters may involve in ion imbalance of the diabetic heart after cardioplegic arrest. In this study, we modified the Langendorff-free cardioplegia method and identified the involved ion transporters after cardioplegia-induced arrest between wild type and db/db heart. Enhanced expression of Na+-K+-2Cl- cotransporter 1 (NKCC1) was observed in the db/db heart compared to the wild type heart. Enhanced NKCC1 activity was observed in the left ventricle of db/db mice compared to that of wild type after cardioplegia-induced arrest. The expression and activity of Slc26a6, a dominant Cl-/HCO3 - exchanger in cardiac tissues, were enhanced in left ventricle strips of db/db mice compared to that of wild type. The Cl- transporting activity in left ventricle strips of db/db mice was dramatically increased as compared to that of wild type. Interestingly, expression of Slc26a6, as well as carbonic anhydrase IV as a supportive enzyme of Slc26a6, was increased in db/db cardiac strips compared to wild type cardiac strips. Thus, the enhanced Cl- transporting activity and expression by NKCC1 and Slc26a6 in db/db cardiac tissues after cardioplegia-induced arrest provide greater insight into enhanced acidosis and Cl- movement-mediated db/db heart dysfunction. Thus, we suggested that enhanced Cl- influx and HCO3 - efflux through NKCC1 and Slc26a6 offer more acidic circumstances in the diabetic heart after cardioplegic arrest. These transporters should be considered as potential therapeutic targets to develop the next generation of cardioplegia solution for protection against ischemia-reperfusion injury in diabetic hearts.


Assuntos
Antiporters/metabolismo , Parada Cardíaca Induzida/efeitos adversos , Parada Cardíaca/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Transportadores de Sulfato/metabolismo , Animais , Antiporters/genética , Coração , Parada Cardíaca/etiologia , Concentração de Íons de Hidrogênio , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Membro 2 da Família 12 de Carreador de Soluto/genética , Transportadores de Sulfato/genética
12.
Am J Physiol Cell Physiol ; 317(6): C1153-C1160, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31532720

RESUMO

The nonselective anion exchanger Slc26a6, also known as putative anion transporter 1 and chloride/formate exchanger, is thought to play a major role in HCO3- transport in exocrine glands. In this study, Slc26a6 null mice were used to explore the function of Slc26a6 in the exocrine pancreas. Slc26a6 primarily localized to the apical membrane of pancreatic exocrine acinar cells. The volume of stimulated juice secretion by the ex vivo pancreas was significantly reduced ~35% in Slc26a6-/- mice, but no changes occurred in the gross structure or gland weights of Slc26a6 null mice. The secretion of pancreatic juice by Slc26a6+/+ mice was dependent on HCO3- while, in contrast, fluid secretion by Slc26a6-/- mice was independent of HCO3-, suggesting that Slc26a6 mediates the HCO3--dependent component of fluid secretion. Consistent with these observations, disruption of Slc26a6 also significantly reduced HCO3- secretion by the pancreas ~35%. Taken together, these results demonstrate that the apical Slc26a6 anion exchanger in acinar cells is involved in HCO3--dependent fluid secretion but that another major HCO3--independent pathway is the primary driver of the fluid secretion process in the mouse pancreas.


Assuntos
Células Acinares/metabolismo , Antiporters/genética , Bicarbonatos/metabolismo , Líquidos Corporais/metabolismo , Pâncreas Exócrino/metabolismo , Transportadores de Sulfato/genética , Células Acinares/citologia , Animais , Anoctamina-1/genética , Anoctamina-1/metabolismo , Antiporters/deficiência , Aquaporina 5/genética , Aquaporina 5/metabolismo , Peso Corporal , Linhagem Celular , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Transporte de Íons , Masculino , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Tamanho do Órgão , Pâncreas Exócrino/citologia , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Transportadores de Sulfato/deficiência
13.
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
14.
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
15.
Biophys J ; 117(4): 767-779, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31400920

RESUMO

Pacemaker depolarization in interstitial cells of Cajal (ICCs) is believed to be induced by Ca2+ transients and activation of anoctamin-1 (Ano1) channels in the plasma membrane. However, block of store-operated calcium entry (SOCE) or the Na-K-2Cl cotransporter (NKCC1) terminates pacemaker activity in ICC, indicating these transporters are involved in the initiation or maintenance of pacemaker activity. We hypothesized that SOCE contributes to pacemaker depolarization by maintaining [Ca2+] in the endoplasmic reticulum, which is the underlying source of Ca2+ transients for activation of Ano1. NKCC1 maintains the Cl- gradient supporting the driving force for inward current mediated by Ano1. Currently mechanisms sustaining release of Ca2+ and activation of Ano1 channels during the plateau phase of slow waves are unknown, but the reverse mode of the Na+/Ca2+ exchange may contribute. We generated a mathematical model of pacemaker activity based on current empirical observations from ICC of mouse small intestine that incorporates functions of SOCE and NKCC1. This model reproduces experimental findings, suggesting roles for SOCE and Ano1 channels: blocking of either NKCC1 or SOCE in our model terminates pacemaker activity. Direct contribution of NKCC1 to pacemaker activity in a beat-to-beat manner is not predicted by our model. Instead, NKCC1 plays a maintenance role supporting the driving force for Cl- efflux. Incorporation of SOCE allows the model to drive pacemaker activity without a diastolic depolarization, as observed in cardiac pacemaking. Further biological experiments are necessary to validate and further refine the roles of NKCC1, Na+/Ca2+ exchange, and Ano1 in the pacemaker mechanism of ICC.


Assuntos
Relógios Biológicos , Sinalização do Cálcio , Células Intersticiais de Cajal/metabolismo , Modelos Neurológicos , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Potenciais de Ação , Animais , Cálcio/metabolismo , Humanos , Células Intersticiais de Cajal/fisiologia
16.
Chronobiol Int ; 36(11): 1592-1598, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31441327

RESUMO

The presence of a circadian clock in the retinal pigment epithelium (RPE) was discovered recently. However, little is known about mechanisms or processes regulated by the RPE clock. We cultured ARPE-19 monolayers in a transwell culture system, and we found rhythmic mRNA expression of the sodium-potassium-chloride co-transporter SLC12A2. We localized the corresponding protein product, NKCC1, on the apical membrane of ARPE-19 cells. We found that concentrations of sodium, potassium, and chloride oscillated in apical supernatants. The ion concentration gradients between supernatants strongly correlated with SLC12A2 mRNA expression. Our results suggest that the circadian clock regulates ion transport by the RPE via NKCC1 expression.


Assuntos
Relógios Circadianos/fisiologia , Regulação da Expressão Gênica/fisiologia , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , cis-trans-Isomerases/metabolismo , Análise de Variância , Técnicas de Cultura de Células , Linhagem Celular , Células Cultivadas , Ritmo Circadiano , Humanos , Imuno-Histoquímica , Transporte de Íons , Potássio/metabolismo , Sódio/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/genética , cis-trans-Isomerases/genética
17.
Nature ; 572(7770): 488-492, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31367042

RESUMO

Cation-chloride cotransporters (CCCs) mediate the electroneutral transport of chloride, potassium and/or sodium across the membrane. They have critical roles in regulating cell volume, controlling ion absorption and secretion across epithelia, and maintaining intracellular chloride homeostasis. These transporters are primary targets for some of the most commonly prescribed drugs. Here we determined the cryo-electron microscopy structure of the Na-K-Cl cotransporter NKCC1, an extensively studied member of the CCC family, from Danio rerio. The structure defines the architecture of this protein family and reveals how cytosolic and transmembrane domains are strategically positioned for communication. Structural analyses, functional characterizations and computational studies reveal the ion-translocation pathway, ion-binding sites and key residues for transport activity. These results provide insights into ion selectivity, coupling and translocation, and establish a framework for understanding the physiological functions of CCCs and interpreting disease-related mutations.


Assuntos
Microscopia Crioeletrônica , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/ultraestrutura , Peixe-Zebra , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cátions Monovalentes/metabolismo , Cloretos/metabolismo , Citosol/metabolismo , Síndrome de Gitelman/genética , Humanos , Transporte de Íons , Modelos Moleculares , Simulação de Dinâmica Molecular , Potássio/metabolismo , Domínios Proteicos , Sódio/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/química , Membro 2 da Família 12 de Carreador de Soluto/genética , Peixe-Zebra/genética
18.
J Neurosci ; 39(37): 7244-7259, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31308096

RESUMO

Early life, systemic inflammation causes long-lasting changes in behavior. To unmask possible mechanisms associated with this phenomenon, we asked whether the intrinsic membrane properties in hippocampal neurons were altered as a consequence of early life inflammation. C57BL/6 mice were bred in-house and both male and female pups from multiple litters were injected with lipopolysaccharide (LPS; 100 µg/kg, i.p.) or vehicle at postnatal day (P)14, and kept until adolescence (P35-P45) or adulthood (P60-P70), when brain slices were prepared for whole-cell and perforated-patch recordings from CA1 hippocampal pyramidal neurons. In neurons of adult male mice pretreated with LPS, the number of action potentials elicited by depolarizing current pulses was significantly increased compared with control neurons, concomitant with increased input resistance, and a lower action potential threshold. Although these changes were not associated with changes in relevant sodium channel expression or differences in capacitance or dendritic architecture, they were linked to a mechanism involving intracellular chloride overload, revealed through a depolarized GABA reversal potential and increased expression of the chloride transporter, NKCC1. In contrast, no significant changes were observed in neurons of adult female mice pretreated with LPS, nor in adolescent mice of either sex. These data uncover a potential mechanism involving neonatal inflammation-induced plasticity in chloride homeostasis, which may contribute to early life inflammation-induced behavioral alterations.SIGNIFICANCE STATEMENT Early life inflammation results in long-lasting changes in many aspects of adult physiology. In this paper we reveal that a brief exposure to early life peripheral inflammation with LPS increases excitability in hippocampal neurons in a sex- and age-dependent manner through a chloride homeostasis disruption. As this hyperexcitability was only seen in adult males, and not in adult females or adolescent animals of either sex, it raises the possibility of a hormonal interaction with early life inflammation. Furthermore, as neonatal inflammation is a normal feature of early life in most animals, as well as humans, these findings may be very important for the development of animal models of disease that more appropriately resemble the human condition.


Assuntos
Região CA1 Hipocampal/metabolismo , Homeostase/fisiologia , Inflamação/metabolismo , Células Piramidais/metabolismo , Caracteres Sexuais , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Fatores Etários , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Feminino , Homeostase/efeitos dos fármacos , Inflamação/induzido quimicamente , Lipopolissacarídeos/toxicidade , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células Piramidais/efeitos dos fármacos , Fatores Sexuais
19.
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
20.
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 , Imageamento por Ressonância Magnética , Masculino , Regiões Promotoras Genéticas , Membro 2 da Família 12 de Carreador de Soluto/genética , Simportadores/genética
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