RESUMEN
BACKGROUND: The cystic fibrosis (CF) sweat gland is defective in ß-adrenergically-stimulated sweat secretion in the coil and chloride reabsorption in the duct. Whereas chloride reabsorption is regularly assessed by quantitative pilocarpine iontophoresis (QPIT), the measurement of ß-adrenergic sweat secretion is not yet established in clinical practice. METHODS: A novel sweat bubble imaging protocol was developed that determines sweat secretion rates by automatic recording, processing and quality control of the kinetics of sweat droplet formation. RESULTS: Treatment of CF patients with the CFTR modulators elexacaftor, tezacaftor and ivacaftor reduced the sweat chloride concentration measured in QPIT in the majority of patients to values in the intermediate or normal range. In contrast, the ß-adrenergically-stimulated sweat secretion rate assayed by the automated bubble sweat test was normalized in only 3 patients, slightly increased in 12 patients and remained undetectable in 8 patients. CONCLUSIONS: ß-adrenergic sweat stimulation in the coil is apparently rather stringent in its requirements for a wild type CFTR conformation whereas chloride reabsorption in the duct tolerates residual structural and functional deficits of native or pharmacologically rescued mutant CFTR in the apical membrane.
Asunto(s)
Fibrosis Quística , Adrenérgicos/análisis , Adrenérgicos/uso terapéutico , Aminofenoles , Benzodioxoles , Agonistas de los Canales de Cloruro , Cloruros/análisis , Fibrosis Quística/diagnóstico , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Combinación de Medicamentos , Humanos , Indoles , Pirazoles , Piridinas , Pirrolidinas , Quinolonas , Sudor/químicaAsunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Fibrosis Quística/genética , Adulto , Fibrosis Quística/epidemiología , Fibrosis Quística/cirugía , Femenino , Asesoramiento Genético , Humanos , Recién Nacido , Tomografía Computarizada por Rayos X/métodos , Turquía/epidemiologíaRESUMEN
Parathyroid hormone (PTH) enhances cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anion secretion by the human intestinal epithelial cell line Caco-2. With the patch-clamp and Ussing chamber techniques, we investigated how PTH stimulates CFTR activity in Caco-2 cells. Cell-attached recordings revealed that PTH stimulated the opening of CFTR-like channels, while impedance analysis demonstrated that PTH increased apical membrane capacitance, a measure of membrane surface area. Using ion substitution experiments, the PTH-stimulated increase in short-circuit current (Isc), a measure of transepithelial ion transport, was demonstrated to be Cl-- and HCO3--dependent. However, the PTH-stimulated increase in Isc was unaffected by the carbonic anhydrase inhibitor acetazolamide, but partially blocked by the intermediate-conductance Ca2+-activated K+ channel (IKCa) inhibitor clotrimazole. TRAM-34, a related IKCa inhibitor, failed to directly inhibit CFTR Cl- channels in cell-free membrane patches, excluding its action on CFTR. In conclusion, PTH enhances CFTR-mediated anion secretion by Caco-2 monolayers by increasing the expression and function of CFTR in the apical membrane and IKCa activity in the basolateral membrane.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Mucosa Intestinal/metabolismo , Hormona Paratiroidea/metabolismo , Aniones/metabolismo , Células CACO-2 , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Mucosa Intestinal/citología , Transporte Iónico , Regulación hacia ArribaRESUMEN
Abstract Objective: Cystic fibrosis diagnosis is dependent on the chloride ion concentration in the sweat test (≥ 60 mEq/mL - recognized as the gold standard indicator for cystic fibrosis diagnosis). Moreover, the salivary glands express the CFTR protein in the same manner as sweat glands. Given this context, the objective was to verify the correlation of saliva chloride concentration and sweat chloride concentration, and between saliva sodium concentration and sweat sodium concentration, in patients with cystic fibrosis and healthy control subjects, as a tool for cystic fibrosis diagnosis. Methods: There were 160 subjects enrolled: 57/160 (35.70%) patients with cystic fibrosis and two known CFTR mutations and 103/160 (64.40%) healthy controls subjects. Saliva ion concentration was analyzed by ABL 835 Radiometer® equipment and, sweat chloride concentration and sweat sodium concentration, respectively, by manual titration using the mercurimetric procedure of Schales & Schales and flame photometry. Statistical analysis was performed by the chi-squared test, the Mann -Whitney test, and Spearman's correlation. Alpha = 0.05. Results: Patients with cystic fibrosis showed higher values of sweat chloride concentration, sweat sodium concentration, saliva chloride concentration, and saliva sodium concentration than healthy controls subjects (p-value < 0.001). The correlation between saliva chloride concentration and sweat chloride concentration showed a positive Spearman's Rho (correlation coefficient) = 0.475 (95% CI = 0.346 to 0.587). Also, the correlation between saliva sodium concentration and sweat sodium concentration showed a positive Spearman's Rho = 0.306 (95% CI = 0.158 to 0.440). Conclusions: Saliva chloride concentration and saliva sodium concentration are candidates to be used in cystic fibrosis diagnosis, mainly in cases where it is difficult to achieve the correct sweat amount, and/or CFTR mutation screening is difficult, and/or reference methods for sweat test are unavailable to implement or are not easily accessible by the general population.
Resumo Objetivo: O diagnóstico da fibrose cística depende do valor da concentração de íons de cloreto no teste do suor (≥ 60 mEq/mL - reconhecido como o indicador-padrão para o diagnóstico da doença). Além disso, as glândulas salivares expressam a proteína RTFC igualmente às glândulas sudoríparas. Nesse contexto, nosso objetivo foi verificar a correlação da concentração de cloreto na saliva e a concentração de cloreto no suor e entre a concentração de sódio na saliva e a concentração de sódio no suor em pacientes com fibrose cística e indivíduos controles saudáveis, como uma ferramenta para diagnóstico de fibrose cística. Métodos: Contamos com a participação de 160 indivíduos [57/160 (35,70%) com fibrose cística e duas mutações no gene RTFC conhecidas e 103/160 (64,40%) indivíduos controles saudáveis]. A concentração de íons na saliva foi analisada pelo equipamento ABL 835 da Radiometer® e a concentração de cloreto no suor e sódio no suor, respectivamente, por titulação manual utilizando o método mercurimétrico de Schales & Schales e fotometria de chama. A análise estatística foi realizada pelo teste qui-quadrado, pelo teste de Mann-Whitney e pela correlação de Spearman. Alpha = 0,05. Resultados: Os pacientes com fibrose cística apresentaram maiores valores na concentração de cloreto no suor, concentração de sódio no suor, concentração de cloreto na saliva e concentração de sódio na saliva do que os indivíduos-controle saudáveis (valor de p < 0,001). A correlação entre as concentrações de cloreto na saliva e cloreto no suor mostrou Rho de Spearman (coeficiente de correlação) positivo = 0,475 (IC de 95% = 0,346 a 0,587). Além disso, a correlação entre concentração de sódio na saliva e concentração de sódio no suor mostrou Rho de Spearman positivo = 0,306 (IC de 95% = 0,158 a 0,440). Conclusões: A concentração de cloreto na saliva e a concentração de sódio na saliva são candidatas a ser usadas como diagnóstico de fibrose cística, principalmente em casos em que é difícil atingir a quantidade correta de suor, e/ou o exame da mutação RTFC é difícil e/ou o método de referência para o teste do suor não se encontra disponível ou não é de fácil acesso ao público em geral.
Asunto(s)
Humanos , Masculino , Femenino , Recién Nacido , Lactante , Preescolar , Niño , Adolescente , Adulto , Persona de Mediana Edad , Adulto Joven , Saliva/química , Sodio/química , Sudor/química , Cloruros/análisis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Fibrosis Quística/diagnóstico , Sodio/metabolismo , Biomarcadores/análisis , Estudios de Casos y Controles , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , GenotipoRESUMEN
Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del-Na+/H+-exchange regulatory factor-1 complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR- or rPhe508del-containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 down-regulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an appealing target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.
Asunto(s)
Aminopiridinas/farmacología , Benzodioxoles/farmacología , Calpaína/antagonistas & inhibidores , Membrana Celular/efectos de los fármacos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Calpaína/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Biología Computacional , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Variación Genética/efectos de los fármacos , Humanos , Proteómica , TemperaturaAsunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Fibrosis Quística/patología , Mucosa Intestinal/patología , Organoides/patología , Animales , Bioensayo/métodos , Fibrosis Quística/diagnóstico , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Proteínas Mutantes/análisis , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Organoides/metabolismo , Cultivo Primario de Células/métodosRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is the secretory chloride channel in epithelial tissues that has a central role in cystic fibrosis (CF) lung and gastrointestinal disease. A recent publication demonstrates a close association between CFTR and TMEM16A, the calcium-activated chloride channel. Thus, no CFTR chloride currents could be detected in airways and large intestine from mice lacking epithelial expression of TMEM16A. Here, we demonstrate that another plasma membrane-localized TMEM16 paralogue, TMEM16F, can compensate for the lack of TMEM16A. Using TMEM16 knockout mice, human lymphocytes, and a number of human cell lines with endogenous protein expression or heterologous expression, we demonstrate that CFTR can only function in the presence of either TMEM16A or TMEM16F. Double knockout of intestinal epithelial TMEM16A/F expression did not produce offsprings, suggesting a lethal phenotype in utero. Plasma membrane-localized TMEM16A or TMEM16F is required for exocytosis and expression of CFTR in the plasma membrane. TMEM16A/F proteins may therefore have an impact on disease severity in CF. KEY MESSAGES: ⢠Cystic fibrosis is caused by the defective Cl- channel cystic fibrosis transmembrane conductance regulator (CFTR). ⢠A close relationship exists between CFTR and the calcium-activated chloride channels TMEM16A/TMEM16F. ⢠In conditional airway and intestinal knockout mice, lymphocytes from Scott disease patients and in overexpressing cells, CFTR is not functional in the absence of TMEM16A and TMEM16F. ⢠TMEM16A and TMEM16F support membrane exocytosis and are essential for plasma membrane insertion of CFTR.
Asunto(s)
Anoctamina-1/metabolismo , Anoctaminas/metabolismo , Membrana Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Animales , Anoctamina-1/análisis , Anoctamina-1/genética , Anoctaminas/análisis , Anoctaminas/genética , Línea Celular , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Exocitosis , Células HEK293 , Humanos , Ratones Noqueados , Proteínas de Transferencia de Fosfolípidos/análisis , Proteínas de Transferencia de Fosfolípidos/genéticaRESUMEN
The sperm produced in the seminiferous tubules pass through the rete testis, efferent ducts, and epididymis. The epididymis has three distinct regions known as caput, corpus, and cauda. The transit through the epididymis is an essential process in sperm maturation. The lumen of each epididymal region has a unique fluid composition regulated by many ion channels and transporters in the epithelial cells. The objective of this study was to map the sites of localization of ion channels ENaC and CFTR along the length of the mouse and rat epididymis using confocal microscopic imaging. The integrity of the fine structure of the tissues was verified by fluorescent phalloidin staining of actin filaments visualized by high-resolution confocal microscopy. The 2D and 3D images showed preservation of the stereocilia. Based on these images we determined morphometric parameters of the epithelial cells and ducts. ENaC and CFTR immunofluorescence appeared almost continuously on the apical membrane of caput and in smooth muscle myoid cells. In cauda, CFTR expression was observed continuously in long stretches of epithelium interrupted by clusters of cells that showed no CFTR expression. Similar patterns of localization were observed in both mouse and rat samples. Mutations in the CFTR gene are known to result in male infertility. Based on the widespread presence of ENaC along the epididymis we suggest that mutations in ENaC subunits may also be associated with male infertility. The diverse phenotypes associated with CFTR mutations may be due to malfunction of CFTR at specific subcellular locations in the male reproductive system.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Epidídimo/química , Canales Epiteliales de Sodio/análisis , Animales , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/química , Canales Epiteliales de Sodio/genética , Técnica del Anticuerpo Fluorescente , Infertilidad Masculina/genética , Espacio Intracelular/química , Masculino , Ratones , Microscopía Confocal , Mutación , Ratas , Distribución TisularRESUMEN
OBJECTIVE: Cystic fibrosis diagnosis is dependent on the chloride ion concentration in the sweat test (≥60mEq/mL - recognized as the gold standard indicator for cystic fibrosis diagnosis). Moreover, the salivary glands express the CFTR protein in the same manner as sweat glands. Given this context, the objective was to verify the correlation of saliva chloride concentration and sweat chloride concentration, and between saliva sodium concentration and sweat sodium concentration, in patients with cystic fibrosis and healthy control subjects, as a tool for cystic fibrosis diagnosis. METHODS: There were 160 subjects enrolled: 57/160 (35.70%) patients with cystic fibrosis and two known CFTR mutations and 103/160 (64.40%) healthy controls subjects. Saliva ion concentration was analyzed by ABL 835 Radiometer® equipment and, sweat chloride concentration and sweat sodium concentration, respectively, by manual titration using the mercurimetric procedure of Schales & Schales and flame photometry. Statistical analysis was performed by the chi-squared test, the Mann-Whitney test, and Spearman's correlation. Alpha=0.05. RESULTS: Patients with cystic fibrosis showed higher values of sweat chloride concentration, sweat sodium concentration, saliva chloride concentration, and saliva sodium concentration than healthy controls subjects (p-value<0.001). The correlation between saliva chloride concentration and sweat chloride concentration showed a positive Spearman's Rho (correlation coefficient)=0.475 (95% CI=0.346 to 0.587). Also, the correlation between saliva sodium concentration and sweat sodium concentration showed a positive Spearman's Rho=0.306 (95% CI=0.158 to 0.440). CONCLUSIONS: Saliva chloride concentration and saliva sodium concentration are candidates to be used in cystic fibrosis diagnosis, mainly in cases where it is difficult to achieve the correct sweat amount, and/or CFTR mutation screening is difficult, and/or reference methods for sweat test are unavailable to implement or are not easily accessible by the general population.
Asunto(s)
Cloruros/análisis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Fibrosis Quística/diagnóstico , Saliva/química , Sodio/química , Sudor/química , Adolescente , Adulto , Biomarcadores/análisis , Estudios de Casos y Controles , Niño , Preescolar , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Femenino , Genotipo , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Sodio/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Newborn screening for cystic fibrosis (NBSCF) was introduced in the Dutch NBS program in 2011 with a novel strategy. METHODS: Dutch NBSCF consisted of four steps: immuno-reactive trypsin (IRT), Pancreatitis-associated Protein (PAP), DNA analysis by Inno-LiPa (35 mutations), extended gene analysis (EGA) as fourth step and as safety net. Only samples with two CFTR-variants were considered screen-positive, but samples with one disease-causing variant were considered also screen-positive from April 2013. The first 5â¯years of NBSCF were evaluated during a follow-up ranging from 2 to 6.8â¯years for sensitivity, specificity, positive predictive value (PPV), ratio of CF/Cystic Fibrosis Screen Positive infants with an Inconclusive Diagnosis (CFSPID) and median age at diagnosis, and were compared to other novel strategies for NBSCF and European Cystic Fibrosis Society (ECFS) Best Practice Standards of Care. RESULTS: NBSCF achieved a sensitivity of 90% (95% CI 82%-94%), specificity of 99.991% (95% CI 99.989%-99.993%), PPV of 63% (95% CI 55%-69%), CF/CFSPID ratio of 4/1, and median age at diagnosis of 22â¯days, if samples with two variants as well as samples with one disease-causing variant were considered screen-positive. CONCLUSION: The program achieved the goal to minimize the number of false positives and showed a favourable performance but sensitivity and CF/CFSPID ratio did not meet criteria of EFCS Best Standards of Care. Changed cut-off values for PAP and IRT and classification of R117H-7T/9T to non-pathogenic may improve sensitivity to ≥95% and CF/CFSPID ratio to 10/1. PPV is estimated to be around 60%.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/diagnóstico , Tamización de Portadores Genéticos/métodos , Guías como Asunto , Mutación , Tamizaje Neonatal/normas , Sistema de Registros , Biomarcadores/sangre , Fibrosis Quística/sangre , Fibrosis Quística/epidemiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Análisis Mutacional de ADN , Femenino , Humanos , Recién Nacido , Masculino , Países Bajos/epidemiología , Curva ROC , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND/AIMS: Cystic fibrosis (CF) is a lethal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR). ΔF508, the most common mutation, is a misfolded protein that is retained in the endoplasmic reticulum and degraded, precluding delivery to the cell surface [
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Qa-SNARE/metabolismo , Línea Celular , Fibrosis Quística/genética , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/patología , Silenciador del Gen , Humanos , Transporte de Proteínas , Proteolisis , Proteínas Qa-SNARE/análisis , Proteínas Qa-SNARE/genéticaRESUMEN
Cystic fibrosis (CF) is caused by mutations in the gene that encodes the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to almost total absence of CFTR at the plasma membrane, a defect potentially corrected via drug-based therapies. Herein, we report the first proof-of-principle study of a noninvasive imaging probe able to detect CFTR at the plasma membrane. We radiolabeled the CFTR inhibitor, CFTRinh -172a, with technetium-99m via a pyrazolyl-diamine chelating unit, yielding a novel 99m Tc(CO)3 complex. A non-radioactive surrogate showed that the structural modifications introduced in the inhibitor did not affect its activity. The radioactive complex was able to detect plasma membrane CFTR, shown by its significantly higher uptake in wild-type versus mutated cells. Furthermore, assessment of F508del CFTR pharmacological correction in human cells using the radioactive complex revealed differences in corrector versus control uptake, recapitulating the biochemical correction observed for the protein.
Asunto(s)
Benzoatos/química , Membrana Celular/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Radiofármacos/química , Tecnecio/química , Tiazolidinas/química , Línea Celular , Membrana Celular/genética , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Diaminas/química , Humanos , Mutación , Pirazoles/químicaRESUMEN
Simultaneous and point-of-care detection of multiple protein biomarkers has significant impact on patient care. Spinal Muscular Atrophy (SMA), Cystic Fibrosis (CF) and Duchenne Muscular Dystrophy (DMD) are well known progressive hereditary disorders associated with increased morbidity as well as mortality. Therefore, rapid detection of biomarkers specific for these three disorders in newborns offers new opportunities for early diagnosis, delaying symptoms and effective treatment. Here, we report the development of a disposable carbon nanofiber (CNF)-based electrochemical immunosensor for simultaneous detection of survival motor neuron 1 (SMN1), cystic fibrosis transmembrane conductance regulator (CFTR) and DMD proteins. The CNF-modified array electrodes were first functionalized by electroreduction of carboxyphenyl diazonium salt. Then, the immunosensor was fabricated by the covalent immobilization of the three antibodies on the working electrodes of the array sensor via carbodiimide (EDC/NHS) chemistry. Simultaneous detection of CFTR, DMD and SMN1 was achieved with high sensitivity and detection limits of 0.9â¯pg/ml, 0.7â¯pg/ml and 0.74â¯pg/ml, respectively. The multiplexed immunosensor has also shown strong selectivity against non-specific proteins. Moreover, high recovery percentage was obtained when the immunosensor was applied in spiked whole blood samples. This voltammetric immunosensor offers cost effective, easy to use, rapid and high throughput potential screening method for these three hereditary disorders using only few drops of blood.
Asunto(s)
Análisis Químico de la Sangre/instrumentación , Análisis Químico de la Sangre/métodos , Enfermedades Genéticas Congénitas/diagnóstico , Nanofibras/química , Tamizaje Neonatal/métodos , Carbono/química , Fibrosis Quística/sangre , Fibrosis Quística/diagnóstico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/sangre , Enfermedades Genéticas Congénitas/sangre , Humanos , Recién Nacido , Límite de Detección , Proteínas Musculares/análisis , Proteínas Musculares/sangre , Atrofia Muscular Espinal/diagnóstico , Distrofia Muscular de Duchenne/sangre , Distrofia Muscular de Duchenne/diagnóstico , Proteína 1 para la Supervivencia de la Neurona Motora/análisis , Proteína 1 para la Supervivencia de la Neurona Motora/sangreRESUMEN
Anesthetic agents have been implicated in the causation of neurological and cognitive deficits after surgery, the exacerbation of chronic neurodegenerative disease, and were recently reported to promote the onset of the neurologic respiratory disease Congenital Central Hypoventilation Syndrome (CCHS), related to misfolding of the transcription factor Phox2B. To study how anesthetic agents could affect neuronal function through alterations to protein folding, we created neuronal cell models emulating the graded disease severity of CCHS. We found that the gas anesthetic isoflurane and the opiate morphine potentiated aggregation and mislocalization of Phox2B variants, similar to that seen in CCHS, and observed transcript and protein level changes consistent with activation of the endoplasmic reticulum (ER) unfolded protein response. Attenuation of ER stress pathways did not result in a correction of Phox2B misfolding, indicating a primary effect of isoflurane on protein structure. We also observed that isoflurane hindered the folding and activity of proteins that rely heavily on ER function, like the CFTR channel. Our results show how anesthetic drugs can alter protein folding and induce ER stress, indicating a mechanism by which these agents may affect neuronal function after surgery.
Asunto(s)
Anestésicos por Inhalación/efectos adversos , Proteínas de Homeodominio/metabolismo , Hipoventilación/congénito , Isoflurano/efectos adversos , Morfina/efectos adversos , Agregación Patológica de Proteínas/inducido químicamente , Apnea Central del Sueño/inducido químicamente , Factores de Transcripción/metabolismo , Línea Celular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Proteínas de Homeodominio/análisis , Humanos , Hipoventilación/inducido químicamente , Hipoventilación/metabolismo , Hipoventilación/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Agregado de Proteínas/efectos de los fármacos , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Pliegue de Proteína/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Apnea Central del Sueño/metabolismo , Apnea Central del Sueño/patología , Factores de Transcripción/análisis , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function causes cystic fibrosis (CF), predisposing the lungs to chronic infection and inflammation. In young infants with CF, structural airway defects are increasingly recognized before the onset of significant lung disease, which suggests a developmental origin and a possible role in lung disease pathogenesis. The role(s) of CFTR in lung development is unclear and developmental studies in humans with CF are not feasible. Young CF pigs have structural airway changes and develop spontaneous postnatal lung disease similar to humans; therefore, we studied lung development in the pig model (non-CF and CF). CF trachea and proximal airways had structural lesions detectable as early as pseudoglandular development. At this early developmental stage, budding CF airways had smaller, hypo-distended lumens compared to non-CF airways. Non-CF lung explants exhibited airway lumen distension in response to forskolin/IBMX as well as to fibroblast growth factor (FGF)-10, consistent with CFTR-dependent anion transport/secretion, but this was lacking in CF airways. We studied primary pig airway epithelial cell cultures and found that FGF10 increased cellular proliferation (non-CF and CF) and CFTR expression/function (in non-CF only). In pseudoglandular stage lung tissue, CFTR protein was exclusively localized to the leading edges of budding airways in non-CF (but not CF) lungs. This discreet microanatomic localization of CFTR is consistent with the site, during branching morphogenesis, where airway epithelia are responsive to FGF10 regulation. In summary, our results suggest that the CF proximal airway defects originate during branching morphogenesis and that the lack of CFTR-dependent anion transport/liquid secretion likely contributes to these hypo-distended airways.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Pulmón/embriología , Animales , Células Cultivadas , AMP Cíclico/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Femenino , Factor 10 de Crecimiento de Fibroblastos/fisiología , Humanos , Morfogénesis , Porcinos , Tráquea/anomalíasRESUMEN
Cystic fibrosis transmembrane conductance regulator (CFTR) is relevant to sperm quality, sperm capacitation and male fertility. However, it is still unknown whether CFTR can be a potential parameter for fecundity prediction in healthy couples. In this study, 135 healthy couples were divided into groups according to their fertility. We demonstrated that the sperm CFTR expression level of healthy males who never impregnated their partners (49 cases, 38.68 ± 2.71%) was significantly lower than that of fertile men (86 cases, 46.35 ± 2.32%). Sperm CFTR expression level accurately corresponded with fertility through the logistic regression model. Receiver operating characteristic (ROC) curve analysis showed that the cut-off value of sperm CFTR expression level for fecundity prediction was 43.75%. Furthermore, cumulative pregnancy rates (CPRs) of CFTR > 43.75% group and CFTR ≤ 43.75% group during the follow-up periods were 80.6% and 49.3% respectively. Meanwhile, the mean time to pregnancy (TTP) of CFTR ≤ 43.75% group (26.79 ± 2.35) was significantly longer than that of CFTR > 43.75% group (16.46 ± 2.42). Therefore, sperm CFTR expression level is relevant to fecundity of healthy couples and shows potential predictive capacity of fecundity.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fertilidad/fisiología , Índice de Embarazo , Espermatozoides/metabolismo , Adulto , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Femenino , Estudios de Seguimiento , Voluntarios Sanos , Humanos , Masculino , Embarazo , Capacitación Espermática , Adulto JovenRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel belongs to the ATP-binding cassette (ABC) transporter superfamily and regulates Cl- secretion in epithelial cells for water secretion. Loss-of-function mutations to the CFTR gene cause dehydrated mucus on the apical side of epithelial cells and increase the susceptibility of bacterial infection, especially in the airway and pulmonary tissues. Therefore, research on the molecular properties of CFTR, such as its gating mechanism and subcellular trafficking, have been intensively pursued. Dysregulated CFTR trafficking is one of the major pathological hallmarks in cystic fibrosis (CF) patients bearing missense mutations in the CFTR gene. Hormones that activate cAMP signaling, such as catecholamine, have been found to regulate the intracellular trafficking of CFTR. Insulin is one of the hormones that regulate cAMP production and promote trafficking of transmembrane proteins to the plasma membrane. The functional interactions between insulin and CFTR have not yet been clearly defined. In this review article, I review the roles of CFTR in epithelial cells, its regulatory role in insulin secretion, and a mechanism of CFTR regulation by insulin.
Asunto(s)
Cloro/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Insulina/metabolismo , Animales , AMP Cíclico/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Insulina/análisis , Resistencia a la Insulina , Transporte Iónico , Mutación MissenseRESUMEN
Cystic fibrosis transmembrane conductance regulator (CFTR) channel gating is predominantly regulated by protein kinase A (PKA)-dependent phosphorylation. In addition to regulating CFTR channel activity, PKA phosphorylation is also involved in enhancing CFTR trafficking and mediating conformational changes at the interdomain interfaces of the protein. The major cystic fibrosis (CF)-causing mutation is the deletion of phenylalanine at position 508 (F508del); it causes many defects that affect CFTR trafficking, stability, and gating at the cell surface. Due to the multiple roles of PKA phosphorylation, there is growing interest in targeting PKA-dependent signaling for rescuing the trafficking and functional defects of F508del-CFTR. This review will discuss the effects of PKA phosphorylation on wild-type CFTR, the consequences of CF mutations on PKA phosphorylation, and the development of therapies that target PKA-mediated signaling.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Eliminación de Secuencia , Animales , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Descubrimiento de Drogas , Humanos , Fosforilación/efectos de los fármacos , Mutación Puntual/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Eliminación de Secuencia/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
CFTR biogenesis starts with its co-translational insertion into the membrane of endoplasmic reticulum and folding of the cytosolic domains, towards the acquisition of a fully folded compact native structure. Efficiency of this process is assessed by the ER quality control system that allows the exit of folded proteins but targets unfolded/misfolded CFTR to degradation. If allowed to leave the ER, CFTR is modified at the Golgi and reaches the post-Golgi compartments to be delivered to the plasma membrane where it functions as a cAMP- and phosphorylation-regulated chloride/bicarbonate channel. CFTR residence at the membrane is a balance of membrane delivery, endocytosis, and recycling. Several adaptors, motor, and scaffold proteins contribute to the regulation of CFTR stability and are involved in continuously assessing its structure through peripheral quality control systems. Regulation of CFTR biogenesis and traffic (and its dysregulation by mutations, such as the most common F508del) determine its overall activity and thus contribute to the fine modulation of chloride secretion and hydration of epithelial surfaces. This review covers old and recent knowledge on CFTR folding and trafficking from its synthesis to the regulation of its stability at the plasma membrane and highlights how several of these steps can be modulated to promote the rescue of mutant CFTR.