RESUMO
BACKGROUND: Soluble epoxide hydrolase inhibitors (sEHi) have anti-arrhythmic effects, and we previously found that the novel sEHi t-AUCB (trans-4[-4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid) significantly inhibited ventricular arrhythmias after myocardial infarction (MI). However, the mechanism is unknown. It's known that microRNA-29 (miR-29) participates in the occurrence of arrhythmias. In this study, we investigated whether sEHi t-AUCB was protective against ischemic arrhythmias by modulating miR-29 and its target genes KCNJ12 and KCNIP2. METHODS: Male 8-week-old C57BL/6 mice were divided into five groups and fed distilled water only or distilled water with t-AUCB of different dosages for seven days. Then, the mice underwent MI or sham surgery. The ischemic region of the myocardium was obtained 24 hours after MI to detect miR-29, KCNJ12, and KCNIP2 mRNA expression levels via real-time PCR and KCNJ12 and KCNIP2 protein expression levels via western blotting. RESULTS: MiR-29 expression levels were significantly increased in the ischemic region of MI mouse hearts and the mRNA and protein expression levels of its target genes KCNJ12 and KCNIP2 were significantly decreased. T-AUCB prevented these changes dose-dependently. CONCLUSION: The sEHi t-AUCB regulates the expression levels of miR-29 and its target genes KCNJ12 and KCNIP2, suggesting a possible mechanism for its potential therapeutic application in ischemic arrhythmia.
Assuntos
Regulação da Expressão Gênica , Proteínas Interatuantes com Canais de Kv/genética , MicroRNAs/genética , Infarto do Miocárdio/genética , Miocárdio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Animais , Western Blotting , Modelos Animais de Doenças , Regulação para Baixo , Proteínas Interatuantes com Canais de Kv/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/biossíntese , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/patologia , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , RNA/genética , RNA/metabolismoRESUMO
Purpose: Diabetic retinopathy (DR) is a leading cause of visual impairment. Müller cells in DR are dysfunctional due to downregulation of the inwardly rectifying potassium channel Kir4.1. Metformin, a commonly used oral antidiabetic drug, is known to elicit its action through 5' adenosine monophosphate-activated protein kinase (AMPK), a cellular metabolic regulator; however, its effect on Kir4.1 channels is unknown. For this study, we hypothesized that metformin treatment would correct circadian rhythm disruption and Kir4.1 channel dysfunction in db/db mice. Methods: Metformin was given orally to db/db mice. Wheel-running activity, retinal levels of Kir4.1, and AMPK phosphorylation were determined at study termination. In parallel, rat retinal Müller cell line (rMC-1) cells were treated using metformin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to assess the effect of AMPK activation on the Kir4.1 channel. Results: The wheel-running activity of the db/db mice was improved following the metformin treatment. The Kir4.1 level in Müller cells was corrected after metformin treatment. Metformin treatment led to an upregulation of clock regulatory genes such as melanopsin (Opn4) and aralkylamine N-acetyltransferase (Aanat). In rMC-1 cells, AMPK activation via AICAR and metformin resulted in increased Kir4.1 and intermediate core clock component Bmal-1 protein expression. The silencing of Prkaa1 (gene for AMPKα1) led to decreased Kir4.1 and Bmal-1 protein expression. Conclusions: Our findings demonstrate that metformin corrects abnormal circadian rhythm and Kir4.1 channels in db/db mouse a model of type 2 diabetes. Metformin could represent a critical pharmacological agent for preventing Müller cell dysfunction observed in human DR.
Assuntos
Ritmo Circadiano/fisiologia , Diabetes Mellitus Experimental/tratamento farmacológico , Retinopatia Diabética/tratamento farmacológico , Regulação da Expressão Gênica , Metformina/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Células Ganglionares da Retina/metabolismo , Animais , Células Cultivadas , Ritmo Circadiano/efeitos dos fármacos , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Modelos Animais de Doenças , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologiaRESUMO
BACKGROUND: We aimed to clarify the role of potassium voltage-gated channel subfamily J member 15 (KCNJ15) in esophageal squamous cell carcinoma (ESCC) cells and its potential as a prognosticator in ESCC patients. METHODS: KCNJ15 transcription levels were evaluated in 13 ESCC cell lines and polymerase chain reaction (PCR) array analysis was conducted to detect coordinately expressed genes with KCNJ15. The biological functions of KCNJ15 in cell invasion, proliferation, migration, and adhesion were validated through small interfering RNA-mediated knockdown experiments. Cell proliferation was further evaluated through the forced expression experiment. KCNJ15 expression was detected in 200 ESCC tissues by quantitative real-time reverse transcription PCR (qRT-PCR) and analyzed in 64 representative tissues by immunohistochemistry. Correlations between KCNJ15 expression levels and clinicopathological features were also analyzed. RESULTS: The KCNJ15 expression levels varied widely in ESCC cell lines and correlated with COL3A1, JAG1, and F11R. Knockdown of KCNJ15 expression significantly repressed cell invasion, proliferation, and migration of ESCC cells in vitro. Furthermore, overexpression of KCNJ15 resulted in increased cell proliferation. Patients were stratified using the cut-off value of KCNJ15 messenger RNA (mRNA) levels in 200 ESCC tissues using receiver operating characteristic curve analysis; the high KCNJ15 expression group had significantly shorter overall and disease-free survival times. In multivariable analysis, high expression of KCNJ15 was identified as an independent poor prognostic factor. Staining intensity of in situ KCNJ15 protein expression tended to be associated with KCNJ15 mRNA expression levels. CONCLUSIONS: KCNJ15 is involved in aggressive tumor phenotypes of ESCC cells and its tissue expression levels may be useful as a prognosticator of patients with ESCC.
Assuntos
Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Canais de Potássio Corretores do Fluxo de Internalização , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Humanos , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização/genética , PrognósticoRESUMO
Purpose: Diabetic patients often are affected by vision problems. We previously identified diabetic retinopathy (DR) as a disease of clock gene dysregulation. TNF-α, a proinflammatory cytokine, is known to be elevated in DR. Müller cells maintain retinal water homeostasis and K+ concentration via Kir4.1 channels. Notably, Kir4.1 expression is reduced in diabetes; however, the interplay of TNF-α, Kir4.1, and clock genes in Müller cells remains unknown. We hypothesize that the Kir4.1 in Müller cells is under clock regulation, and increase in TNF-α is detrimental to Kir4.1. Methods: Long-Evans rats were made diabetic using streptozotocin (STZ). Retinal Kir4.1 expression was determined at different time intervals. Rat Müller (rMC-1) cells were transfected with siRNA for Per2 or Bmal1 and in parallel treated with TNF-α (5-5000 pM) to determine Kir4.1 expression. Results: Kir4.1 expression exhibited a diurnal rhythm in the retina; however, with STZ-induced diabetes, Kir4.1 was reduced overall. Kir4.1 rhythm was maintained in vitro in clock synchronized rMC-1 cells. Clock gene siRNA-treated rMC-1 exhibited a decrease in Kir4.1 expression. TNF-α treatment of rMCs lead to a profound decrease in Kir4.1 due to reduced colocalization of Kir4.1 channels with synapse-associated protein (SAP97) and disorganization of the actin cytoskeleton. Conclusions: Our findings demonstrate that Kir4.1 channels possess a diurnal rhythm, and this rhythm is dampened with diabetes, thereby suggesting that the increase in TNF-α is detrimental to normal Kir4.1 rhythm and expression.
Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética/genética , Células Ependimogliais/metabolismo , Regulação da Expressão Gênica , Canais de Potássio Corretores do Fluxo de Internalização/genética , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/uso terapêutico , Animais , Western Blotting , Células Cultivadas , Ritmo Circadiano , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/metabolismo , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/patologia , Imuno-Histoquímica , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Ratos , Ratos Long-Evans , Reação em Cadeia da Polimerase em Tempo Real , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologiaRESUMO
Purpose: To test the effects of rearing light intensity on retinal function and morphology in the retinoschisis knockout (Rs1-KO) mouse model of X-linked retinoschisis, and whether it affects functional outcome of RS1 gene replacement. Methods: Seventy-six Rs1-KO mice were reared in either cyclic low light (LL, 20 lux) or moderate light (ML, 300 lux) and analyzed at 1 and 4 months. Retinal function was assessed by electroretinogram and cavity size by optical coherence tomography. Expression of inward-rectifier K+ channel (Kir4.1), water channel aquaporin-4 (AQP4), and glial fibrillary acidic protein (GFAP) were analyzed by Western blotting. In a separate study, Rs1-KO mice reared in LL (n = 29) or ML (n = 27) received a unilateral intravitreal injection of scAAV8-hRs-IRBP at 21 days, and functional outcome was evaluated at 4 months by electroretinogram. Results: At 1 month, no functional or structural differences were found between LL- or ML-reared Rs1-KO mice. At 4 months, ML-reared Rs1-KO mice showed significant reduction of b-wave amplitude and b-/a-wave ratio with no changes in a-wave, and a significant increase in cavity size, compared to LL-reared animals. Moderate light rearing increased Kir4.1 expression in Rs1-KO mice by 4 months, but not AQP4 and GFAP levels. Administration of scAAV8-hRS1-IRBP to Rs1-KO mice showed similar improvement of inner retinal ERG function independent of LL or ML rearing. Conclusions: Rearing light conditions affect the development of retinal cavities and post-photoreceptor function in Rs1-KO mice. However, the effect of rearing light intensity does not interact with the efficacy of RS1 gene replacement in Rs1-KO mice.
Assuntos
Terapia Genética/métodos , Luz , Segmento Interno das Células Fotorreceptoras da Retina/patologia , Retinosquise/terapia , Animais , Western Blotting , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Modelos Animais de Doenças , Eletrorretinografia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Seguimentos , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização/genética , RNA/genética , Segmento Interno das Células Fotorreceptoras da Retina/efeitos da radiação , Retinosquise/diagnóstico , Retinosquise/genética , Retinosquise/fisiopatologia , Fatores de Tempo , Tomografia de Coerência ÓpticaRESUMO
To elucidate the complex molecular mechanisms of anaplastic thyroid carcinoma (ATC), the mRNA and miRNA expression profiles of ATC were systematically explored. A total of 55 common differentially expressed genes (DEGs) were obtained from two mRNA expression datasets including 23 ATC samples and 24 paired normal samples. Gene expression levels of three randomly selected DEGs, VCAN, COL5A1 and KCNJ16, were examined using RT-PCR in 10 ATC samples. Notably, the ATC and normal samples were clearly classified into two groups based on their common DEGs. Moreover 23 common DEGs, such as TG, NKX2-1, KCNJ16 and CTHRC1, were predicted to be the potential targets of 17 identified miRNAs in ATC. Meanwhile, several miRNA target genes were associated with biological processes related to tumor progression such as angiogenesis, cell migration or growth and potassium channel regulation. In summary, the poor prognosis of ATC is possibly caused via complex biological processes. Firstly, angiogenesis was activated by the high expression of CTHRC1, VCAN and POSTN, providing necessary nutrition for tumor cells. Then tumor distant metastasis was induced via stimulation of cell migration and cell growth or regulation of cell-cell interaction. Moreover, intracellular potassium concentration changes promoted ATC progression indirectly. Hence, identification of these critical DEGs was valuable in understanding the molecular mechanisms of ATC.
Assuntos
Moléculas de Adesão Celular/biossíntese , Colágeno Tipo V/biossíntese , Proteínas da Matriz Extracelular/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Carcinoma Anaplásico da Tireoide/genética , Versicanas/biossíntese , Moléculas de Adesão Celular/genética , Comunicação Celular , Movimento Celular/genética , Proliferação de Células/genética , Colágeno Tipo V/genética , Proteínas da Matriz Extracelular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , MicroRNAs/biossíntese , MicroRNAs/genética , Neovascularização Patológica/genética , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Prognóstico , Carcinoma Anaplásico da Tireoide/patologia , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Versicanas/genéticaRESUMO
DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.
Assuntos
Astrócitos/fisiologia , Metilação de DNA , Canais de Potássio Corretores do Fluxo de Internalização/genética , Animais , Astrócitos/metabolismo , Citosina/metabolismo , DNA/genética , DNA/metabolismo , Fosfatos de Dinucleosídeos/genética , Fosfatos de Dinucleosídeos/metabolismo , Expressão Gênica , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Regiões Promotoras Genéticas , Ratos Transgênicos , Ativação Transcricional , TranscriptomaRESUMO
Müller cells are principal glial cells in rat retina and have attracted much attention in glaucoma studies. However, it is not clear whether adenosine and adenosine receptor (AR) antagonists play any roles in the regulation of potassium channels in Müller cells and subsequently in the promotion of glutamine synthetase (GS) and L-Glutamate/L-Aspartate Transporter (GLAST) functions. We found that chronic ocular hypertension (COH) in rat down-regulated Müller cells Kir2.1, Kir4.1, TASK-1, GS and GLAST expressions and attenuated the peak of inward potassium current. Retinal ganglion cells (RGC) count was lower in the COH rats than that in the sham operation animals. Intravitreal injection of selective A2A AR antagonist SCH442416 up-regulated Müller cell Kir4.1, TASK-1, GS and GLAST expressions and enhanced inward potassium currents compared with those in the COH rats with vehicle control. Meanwhile, the RGC count was higher following intravitreal injection of SCH442416 in the COH rats than that after vehicle injection. The fact that PKA inhibitor H-89 blocked these SCH442416 effects suggested that the PKA signaling pathway was involved in the observed ocular responses following the intravitreal SCH442416 injection.
Assuntos
Adenosina/farmacologia , Células Ependimogliais/metabolismo , Glaucoma/patologia , Canais de Potássio/efeitos dos fármacos , Antagonistas de Receptores Purinérgicos P1/farmacologia , Sistema X-AG de Transporte de Aminoácidos/biossíntese , Animais , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Glutamato-Amônia Ligase/biossíntese , Isoquinolinas/farmacologia , Masculino , Proteínas do Tecido Nervoso , Técnicas de Patch-Clamp , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Canais de Potássio de Domínios Poros em Tandem/biossíntese , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacologiaRESUMO
Gastric acid secretion is mediated by the K(+)-dependent proton pump (H(+),K(+)-ATPase), which requires a continuous supply of K(+) at the luminal side of the apical membrane. Several K(+) channels are implicated in gastric acid secretion. However, the identity of the K(+) channel(s) responsible for apical K(+) supply is still elusive. Our previous studies have shown the translocation of KCNJ15 from cytoplasmic vesicles to the apical membrane on stimulation, indicating its involvement in gastric acid secretion. In this study, the stimulation associated trafficking of KCNJ15 was observed in a more native context with a live cell imaging system. KCNJ15 molecules in resting live cells were scattered in cytoplasm but exhibited apical localization after stimulation. Furthermore, knocking down KCNJ15 expression with a short hairpin RNA adenoviral construct abolished histamine-stimulated acid secretion in rabbit primary parietal cells. Moreover, KCNJ15, like H(+),K(+)-ATPase, was detected in all of the parietal cells by immunofluorescence staining, whereas only about half of the parietal cells were positive for KCNQ1 under the same condition. Consistently, the endogenous protein levels of KCNJ15, analyzed by Western blotting, were higher than those of KCNQ1 in the gastric mucosa of human, mouse, and rabbit. These results provide evidence for a major role of KCNJ15 in apical K(+) supply during stimulated acid secretion.
Assuntos
Ácido Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Histamina/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Adolescente , Animais , Feminino , Mucosa Gástrica/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos , Células Parietais Gástricas/metabolismo , CoelhosRESUMO
Potassium inwardly rectifying channel, subfamily J, member 1 (KCNJ1), as an ATP-dependent potassium channel, plays an essential role in potassium balance. KCNJ1 variation is associated with multiple diseases, such as antenatal Bartter syndrome and diabetes. However, the role of KCNJ1 in clear cell renal cell carcinoma (ccRCC) is still unknown. Here, we studied the expression and function of KCNJ1 in ccRCC. The expression of KCNJ1 was evaluated in ccRCC tissues and cell lines by quantitative real-time PCR (qRT-PCR), Western blot, and immunohistochemistry analysis. The relationship between KCNJ1 expression and clinicopathological characteristics was analyzed. p3xFLAG-CMV-14 vector containing KCNJ1 was constructed and used for transfecting ccRCC cell lines 786-O and Caki-2. The effects of KCNJ1 on cell proliferation, invasion, and apoptosis were detected in ccRCC cell lines using cell proliferation assay, transwell assay, and flow cytometry, respectively. We found that KCNJ1 was low-expressed in ccRCC tissues samples and cell lines, and its expression level was significantly associated with tumor pathology grade (P = 0.002) and clinical stage (P = 0.023). Furthermore, the KCNJ1 expression was a prognostic factor of ccRCC patient's survival (P = 0.033). The re-expression of KCNJ1 in 786-O and Caki-2 significantly inhibited cancer cell growth and invasion and promoted cancer cell apoptosis. Moreover, knockdown of KCNJ1 in HK-2 cells promoted cell proliferation. Collectively, these data highlight that KCNJ1, low-expressed in ccRCC and associated with poor prognosis, plays an important role in ccRCC cell growth and metastasis.
Assuntos
Apoptose/genética , Carcinoma de Células Renais/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Prognóstico , Adulto , Idoso , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Canais de Potássio Corretores do Fluxo de Internalização/biossínteseRESUMO
Neutrophils are phagocytic cells that play a critical role in innate immunity by destroying bacterial pathogens. Channels belonging to the inward rectifier potassium channel subfamily 2 (Kir2 channels) have been described in other phagocytes (monocytes/macrophages and eosinophils) and in hematopoietic precursors of phagocytes. Their physiological function in these cells remains unclear, but some evidence suggests a role in growth factor-dependent proliferation and development. Expression of functional Kir2 channels has not been definitively demonstrated in mammalian neutrophils. Here, we show by RT-PCR that neutrophils from mouse bone marrow and liver express mRNA for the Kir2 subunit Kir2.1 but not for other subunits (Kir2.2, Kir2.3, and Kir2.4). In electrophysiological experiments, resting (unstimulated) neutrophils from mouse bone marrow and liver exhibit a constitutively active, external K(+)-dependent, strong inwardly rectifying current that constitutes the dominant current. The reversal potential is dependent on the external K(+) concentration in a Nernstian fashion, as expected for a K(+)-selective current. The current is not altered by changes in external or internal pH, and it is blocked by Ba(2+), Cs(+), and the Kir2-selective inhibitor ML133. The single-channel conductance is in agreement with previously reported values for Kir2.1 channels. These properties are characteristic of homomeric Kir2.1 channels. Current density in short-term cultures of bone marrow neutrophils is decreased in the absence of growth factors that are important for neutrophil proliferation [granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF)]. These results demonstrate that mouse neutrophils express functional Kir2.1 channels and suggest that these channels may be important for neutrophil function, possibly in a growth factor-dependent manner.
Assuntos
Medula Óssea/metabolismo , Fígado/metabolismo , Neutrófilos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: Neovascularization and vaso-obliteration are vision-threatening events that develop by interactions between retinal vascular and glial cells. A high-salt diet is causal in cardiovascular and renal disease, which is linked to modulation of the renin-angiotensin-aldosterone system. However, it is not known whether dietary salt influences retinal vasculopathy and if the renin-angiotensin-aldosterone system is involved. We examined whether a low-salt (LS) diet influenced vascular and glial cell injury and the renin-angiotensin-aldosterone system in ischemic retinopathy. APPROACH AND RESULTS: Pregnant Sprague Dawley rats were fed LS (0.03% NaCl) or normal salt (0.3% NaCl) diets, and ischemic retinopathy was induced in the offspring. An LS diet reduced retinal neovascularization and vaso-obliteration, the mRNA and protein levels of the angiogenic factors, vascular endothelial growth factor, and erythropoietin. Microglia, which influence vascular remodeling in ischemic retinopathy, were reduced by LS as was tumor necrosis factor-α. Macroglial Müller cells maintain the integrity of the blood-retinal barrier, and in ischemic retinopathy, LS reduced their gliosis and also vascular leakage. In retina, LS reduced mineralocorticoid receptor, angiotensin type 1 receptor, and renin mRNA levels, whereas, as expected, plasma levels of aldosterone and renin were increased. The aldosterone/mineralocorticoid receptor-sensitive epithelial sodium channel alpha (ENaCα), which is expressed in Müller cells, was increased in ischemic retinopathy and reduced by LS. In cultured Müller cells, high salt increased ENaCα, which was prevented by mineralocorticoid receptor and angiotensin type 1 receptor blockade. Conversely, LS reduced ENaCα, angiotensin type 1 receptor, and mineralocorticoid receptor expression. CONCLUSIONS: An LS diet reduced retinal vasculopathy, by modulating glial cell function and the retinal renin-angiotensin-aldosterone system.
Assuntos
Dieta Hipossódica , Canais Epiteliais de Sódio/fisiologia , Microglia/fisiologia , Sistema Renina-Angiotensina/fisiologia , Neovascularização Retiniana/dietoterapia , Complexo 1 de Proteínas Adaptadoras/análise , Aldosterona/sangue , Aldosterona/fisiologia , Animais , Animais Recém-Nascidos , Aquaporina 4/biossíntese , Aquaporina 4/genética , Peso Corporal , Células Cultivadas , Modelos Animais de Doenças , Comportamento de Ingestão de Líquido , Células Ependimogliais/química , Células Ependimogliais/patologia , Eritropoetina/análise , Gliose/etiologia , Gliose/fisiopatologia , Hematócrito , Transporte de Íons , Isquemia/fisiopatologia , Glomérulos Renais/patologia , Sistema de Sinalização das MAP Quinases , Fosforilação , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização/genética , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/metabolismo , Neovascularização Retiniana/fisiopatologia , Neovascularização Retiniana/prevenção & controle , Retinopatia da Prematuridade , Sódio/metabolismo , Cloreto de Sódio na Dieta/efeitos adversos , Fator de Necrose Tumoral alfa/biossíntese , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
At birth, asphyxial stressors such as hypoxia and hypercapnia are important physiological stimuli for adrenal catecholamine release that is critical for the proper transition to extrauterine life. We recently showed that chronic opioids blunt chemosensitivity of neonatal rat adrenomedullary chromaffin cells (AMCs) to hypoxia and hypercapnia. This blunting was attributable to increased ATP-sensitive K(+) (KATP) channel and decreased carbonic anhydrase (CA) I and II expression, respectively, and involved µ- and δ-opioid receptor signaling pathways. To address underlying molecular mechanisms, we first exposed an O2- and CO2-sensitive, immortalized rat chromaffin cell line (MAH cells) to combined µ {[d-Arg(2),Ly(4)]dermorphin-(1-4)-amide}- and δ ([d-Pen(2),5,P-Cl-Phe(4)]enkephalin)-opioid agonists (2 µM) for â¼7 days. Western blot and quantitative real-time PCR analysis revealed that chronic opioids increased KATP channel subunit Kir6.2 and decreased CAII expression; both effects were blocked by naloxone and were absent in hypoxia-inducible factor (HIF)-2α-deficient MAH cells. Chronic opioids also stimulated HIF-2α accumulation along a time course similar to Kir6.2. Chromatin immunoprecipitation assays on opioid-treated cells revealed the binding of HIF-2α to a hypoxia response element in the promoter region of the Kir6.2 gene. The opioid-induced regulation of Kir6.2 and CAII was dependent on protein kinase A, but not protein kinase C or calmodulin kinase, activity. Interestingly, a similar pattern of HIF-2α, Kir6.2, and CAII regulation (including downregulation of CAI) was replicated in chromaffin tissue obtained from rat pups born to dams exposed to morphine throughout gestation. Collectively, these data reveal novel mechanisms by which chronic opioids blunt asphyxial chemosensitivity in AMCs, thereby contributing to abnormal arousal responses in the offspring of opiate-addicted mothers.
Assuntos
Células Cromafins/metabolismo , Canais KATP/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Córtex Suprarrenal/citologia , Córtex Suprarrenal/metabolismo , Medula Suprarrenal/citologia , Medula Suprarrenal/metabolismo , Analgésicos Opioides/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Anidrase Carbônica I/biossíntese , Anidrase Carbônica II/biossíntese , Hipóxia Celular , Linhagem Celular , Células Cromafins/citologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dopamina/metabolismo , D-Penicilina (2,5)-Encefalina/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Hipercapnia , Indóis/farmacologia , Isoquinolinas/farmacologia , Canais KATP/genética , Maleimidas/farmacologia , Morfina/farmacologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Norepinefrina/metabolismo , Oligopeptídeos/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Gravidez , Regiões Promotoras Genéticas , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Sulfonamidas/farmacologiaRESUMO
OBJECTIVE: Activating mutations in the KCNJ11 gene, encoding the Kir6.2 subunit of the KATP channel, result in permanent neonatal diabetes mellitus. They also may cause neurologic symptoms such as mental retardation and motor problems (iDEND syndrome) and epilepsy (DEND syndrome). Sulphonylurea (SU) treatment is reported to alleviate both the neurologic symptoms and diabetes in such cases. The study aimed to establish the magnitude and functional basis of the effect of SUs on the neurologic phenotype in children with iDEND using neuroimaging before and after insulin replacement with glibenclamide. RESEARCH DESIGN AND METHODS: To localize and quantify the effect of glibenclamide administration, we performed single-photon emission computed tomography in seven patients with different mutations in KCNJ11. In five patients, measurements before and after initiation of SU treatment were performed. RESULTS Significant changes in single-photon emission computed tomography signal intensity after transfer to SU therapy were restricted to the cerebellum, consistent with previous data showing high Kir6.2 expression in this brain region. Cerebellar perfusion improved for both left (P = 0.006) and right (P = 0.01) hemispheres, with the mean improvement being 26.7 ± 7.1% (n = 5). No patients showed deterioration of cerebellar perfusion on SU therapy. Electrophysiological studies revealed a good correlation between the magnitude of KATP channel dysfunction and the clinical phenotype; mutant channels with the greatest reduction in adenosine 5'-triphosphate inhibition were associated with the most severe neurologic symptoms. CONCLUSIONS: We conclude it is likely that at least some of the beneficial effects of SU treatment on neurodevelopment in iDEND patients result from improved cerebellar perfusion.
Assuntos
Cerebelo/metabolismo , Diabetes Mellitus/tratamento farmacológico , Canais de Potássio Corretores do Fluxo de Internalização/genética , Compostos de Sulfonilureia/uso terapêutico , Adolescente , Cerebelo/irrigação sanguínea , Cerebelo/efeitos dos fármacos , Criança , Pré-Escolar , Feminino , Glibureto/uso terapêutico , Humanos , Lactente , Recém-Nascido , Masculino , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
BACKGROUND: Neuromodulation is used to restore neural function in disorders that stem from an imbalance in the activity of specific neural networks when they prove refractory to pharmacological therapy. The Kir2.1 gene contributes to stabilizing the resting potential below the threshold of activation of voltage-gated sodium channels and action potentials. Therefore, the delivery of the Kir2.1 gene to neuronal cells could reduce the probability of action potential generation, inhibiting excessive neural activity. OBJECTIVE: To address the hypothesis that overexpression of the inwardly rectifying potassium channel 2.1 (Kir2.1) gene could inhibit motor neuron activity and therefore be therapeutically used in gene-based neuromodulation. METHODS: To induce expression of Kir2.1, the inducible RheoSwitch promoter was used and controlled by ligand. In vivo gene expression was accomplished by an adenoviral vector to deliver unilaterally into the lumbar spinal cord of rats. RESULTS: Behavioral assays demonstrated that neuromuscular inhibition was exclusive to rats that received the ligand. Histological analysis also showed evidence of some motor neuron loss in these animals. Behavioral effects of Kir2.1 expression were completely reversible, arguing that the behavioral effect did not result from motor neuron death. CONCLUSION: Delivery of the gene for Kir2.1 inhibits neurons by resisting depolarization to the action potential threshold. Regulated neuronal expression of Kir2.1 may provide an elegant means for neuromodulation in a selected neuronal population.
Assuntos
Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Neurônios/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Medula Espinal/fisiologia , Animais , Linhagem Celular Tumoral , Regulação da Expressão Gênica/fisiologia , Humanos , Fármacos Neuromusculares Despolarizantes/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Distribuição Aleatória , Ratos , Medula Espinal/citologiaRESUMO
The sulfonylurea receptor 1 (Sur1)-regulated NC(Ca-ATP) channel is a nonselective cation channel that is regulated by intracellular calcium and adenosine triphosphate. The channel is not constitutively expressed, but is transcriptionally upregulated de novo in all cells of the neurovascular unit, in many forms of central nervous system (CNS) injury, including cerebral ischemia, traumatic brain injury (TBI), spinal cord injury (SCI), and subarachnoid hemorrhage (SAH). The channel is linked to microvascular dysfunction that manifests as edema formation and delayed secondary hemorrhage. Also implicated in oncotic cell swelling and oncotic (necrotic) cell death, the channel is a major molecular mechanism of 'accidental necrotic cell death' in the CNS. In animal models of SCI, pharmacological inhibition of Sur1 by glibenclamide, as well as gene suppression of Abcc8, prevents delayed capillary fragmentation and tissue necrosis. In models of stroke and TBI, glibenclamide ameliorates edema, secondary hemorrhage, and tissue damage. In a model of SAH, glibenclamide attenuates the inflammatory response due to extravasated blood. Clinical trials of an intravenous formulation of glibenclamide in TBI and stroke underscore the importance of recent advances in understanding the role of the Sur1-regulated NC(Ca-ATP) channel in acute ischemic, traumatic, and inflammatory injury to the CNS.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Sistema Nervoso Central/lesões , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Droga/metabolismo , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/fisiologia , Animais , Barreira Hematoencefálica , Lesões Encefálicas/genética , Lesões Encefálicas/metabolismo , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Morte Celular , Sistema Nervoso Central/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Necrose , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Receptores de Droga/biossíntese , Receptores de Droga/efeitos dos fármacos , Receptores de Droga/fisiologia , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Receptores de Sulfonilureias , Supressão Genética , Regulação para CimaRESUMO
The cardiac electrical impulse depends on an orchestrated interplay of transmembrane ionic currents in myocardial cells. Two critical ionic current mechanisms are the inwardly rectifying potassium current (I(K1)), which is important for maintenance of the cell resting membrane potential, and the sodium current (I(Na)), which provides a rapid depolarizing current during the upstroke of the action potential. By controlling the resting membrane potential, I(K1) modifies sodium channel availability and therefore, cell excitability, action potential duration, and velocity of impulse propagation. Additionally, I(K1)-I(Na) interactions are key determinants of electrical rotor frequency responsible for abnormal, often lethal, cardiac reentrant activity. Here, we have used a multidisciplinary approach based on molecular and biochemical techniques, acute gene transfer or silencing, and electrophysiology to show that I(K1)-I(Na) interactions involve a reciprocal modulation of expression of their respective channel proteins (Kir2.1 and Na(V)1.5) within a macromolecular complex. Thus, an increase in functional expression of one channel reciprocally modulates the other to enhance cardiac excitability. The modulation is model-independent; it is demonstrable in myocytes isolated from mouse and rat hearts and with transgenic and adenoviral-mediated overexpression/silencing. We also show that the post synaptic density, discs large, and zonula occludens-1 (PDZ) domain protein SAP97 is a component of this macromolecular complex. We show that the interplay between Na(v)1.5 and Kir2.1 has electrophysiological consequences on the myocardium and that SAP97 may affect the integrity of this complex or the nature of Na(v)1.5-Kir2.1 interactions. The reciprocal modulation between Na(v)1.5 and Kir2.1 and the respective ionic currents should be important in the ability of the heart to undergo self-sustaining cardiac rhythm disturbances.
Assuntos
Potenciais de Ação , Arritmias Cardíacas/mortalidade , Regulação da Expressão Gênica , Potenciais da Membrana , Proteínas Musculares/biossíntese , Miócitos Cardíacos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Canais de Sódio/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Proteína 1 Homóloga a Discs-Large , Inativação Gênica , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Musculares/genética , Miócitos Cardíacos/patologia , Canal de Sódio Disparado por Voltagem NAV1.5 , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Canais de Sódio/genética , Proteína da Zônula de Oclusão-1RESUMO
AIMS: To investigate the mechanism behind cytotoxic edema formation following subarachnoid hemorrhage (SAH). METHODS: We explored the role of aquaporin-4 (AQP4), inwardly rectifying K(+) 4.1 (Kir4.1) channels and their upstream orchestrators p53 and p38MAPK in this process. A p53 inhibitor, pifithrin-α (PFT-α) was administered intraperitoneally to rats undergoing SAH by endovascular perforation. Totally, 98 male SD rats were categorized into sham, SAH, SAH+ dimethyl sulfoxide (DMSO), SAH+ 0.2 or 2.0 mg/kg PFT-α groups. At 24 h after SAH, MRI (diffusion-weighted imaging [DWI]), immunohistochemistry, and Western blot were used. RESULTS: MRI (DWI) showed a significant cytotoxic edema in the brain following SAH with PFT-α therapy reducing it. Immunohistochemistry and Western blot showed an increased level of p53, phosphorylated-p38MAPK and AQP4 and a reduced level of Kir4.1; all of which could be reversed following PFT-α treatment. Treble labeling staining revealed colocalization of p53 with phosphorylated-p38MAPK and unmatched expression of AQP4 and Kir4.1 within astrocyte cells. CONCLUSION: These results indicated p53 mediates the formation of cytotoxic edema in the brain following SAH; an uncoupling expression of AQP4 and Kir4.1 on astrocytic end feet orchestrated by p38MAPK was partly responsible.
Assuntos
Aquaporina 4/biossíntese , Edema Encefálico/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Hemorragia Subaracnóidea/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/biossíntese , Animais , Benzotiazóis/farmacologia , Edema Encefálico/complicações , Regulação da Expressão Gênica , Hipocampo/metabolismo , Masculino , Canais de Potássio Corretores do Fluxo de Internalização/genética , Ratos , Ratos Sprague-Dawley , Hemorragia Subaracnóidea/complicações , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/genéticaRESUMO
Fetal nicotine exposure causes impaired adrenal catecholamine secretion and increased neonatal mortality during acute hypoxic challenges. Both effects are attributable to upregulation of ATP-sensitive K(+) channels (K(ATP) channels) and can be rescued by pretreatment with the blocker, glibenclamide. Although use of in vitro models of primary and immortalized, fetal-derived rat adrenomedullary chromaffin cells (i.e., MAH cells) demonstrated the involvement of α7 nicotinic ACh receptor (nAChR) stimulation and the transcription factor, HIF-2α, the latter's role was unclear. Using Western blots, we show that chronic nicotine causes a progressive, time-dependent induction of HIF-2α in MAH cells that parallels the upregulation of K(ATP) channel subunit, Kir6.2. Moreover, a common HIF target, VEGF mRNA, was also upregulated after chronic nicotine. All the above effects were prevented during co-incubation with α-bungarotoxin (100 nM), a specific α7 nAChR blocker, and were absent in HIF-2α-deficient MAH cells. Chromatin immunoprecipitation (ChIP) assays demonstrated binding of HIF-2α to a putative hypoxia response element in Kir6.2 gene promoter. Specificity of this signaling pathway was validated in adrenal glands from pups born to dams exposed to nicotine throughout gestation; the upregulation of both HIF-2α and Kir6.2 was confined to medullary, but not cortical, tissue. This study has uncovered a signaling pathway whereby a nonhypoxic stimulus (nicotine) promotes HIF-2α-mediated transcriptional upregulation of a novel target, Kir6.2 subunit. The data suggest that the HIF pathway may be involved in K(ATP) channel-mediated neuroprotection during brain ischemia, and in the effects of chronic nicotine on ubiquitous brain α7 nAChR.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Células Cromafins/metabolismo , Canais KATP/biossíntese , Nicotina/administração & dosagem , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Regulação para Cima/fisiologia , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Linhagem Celular Transformada , Feminino , Exposição Materna/efeitos adversos , Gravidez , Distribuição Aleatória , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Transcrição Gênica/fisiologiaRESUMO
Potassium ions are required for gastric acid secretion. Several potassium channels have been implicated in providing K(+) at the apical membrane of parietal cells. In examining the mRNA expression levels between gastric mucosa and liver tissue, KCNJ15 stood out as the most highly specific K(+) channel in the gastric mucosa. Western blot analysis confirmed that KCNJ15 is abundant in the stomach. Immunofluorescence staining of isolated gastric glands indicated that KCNJ15 was expressed in parietal cells and chief cells, but not in mucous neck cells. In resting parietal cells, KCNJ15 was mainly found in puncta throughout the cytoplasm but was distinct from H(+)-K(+)-ATPase. Upon stimulation, KCNJ15 and H(+)-K(+)-ATPase become colocalized on the apical membranes, as suggested by immunofluorescence staining. Western blot analysis of the resting and the stimulated membrane fractions confirmed this observation. From nonsecreting preparations, KCNJ15-containing vesicles sedimented after a 4-h centrifugation at 100,000 g, but not after a 30-min spin, which did sediment most of the H(+)-K(+)-ATPase-containing tubulovesicles. Most of the KCNJ15 containing small vesicle population was depleted upon stimulation of parietal cells, as indicated by the fact that the KCNJ15 signal was shifted to a large membrane fraction that sedimented at 4,000 g. Our results demonstrate that, in nonsecreting parietal cells, KCNJ15 is stored in vesicles distinct from the H(+)-K(+)-ATPase-enriched tubulovesicles. Furthermore, upon stimulation, KCNJ15 and H(+)-K(+)-ATPase both translocate to the apical membrane for active acid secretion. Thus KCNJ15 can be added to the family of apical K(+) channels in gastric parietal cells.