RESUMO
Chronic obstructive pulmonary disease (COPD) is one of the prevalent causes of worldwide mortality and encompasses two major clinical phenotypes, i.e., chronic bronchitis (CB) and emphysema. The most common cause of COPD is chronic tobacco inhalation. Research focused on the chronic bronchitic phenotype of COPD has identified several pathological processes that drive disease initiation and progression. For example, the lung's mucociliary clearance (MCC) system performs the critical task of clearing inhaled pathogens and toxic materials from the lung. MCC efficiency is dependent on: (1) the ability of apical plasma membrane ion channels such as the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na(+) channel (ENaC) to maintain airway hydration; (2) ciliary beating; and (3) appropriate rates of mucin secretion. Each of these components is impaired in CB and likely contributes to the mucus stasis/accumulation seen in CB patients. This review highlights the cellular components responsible for maintaining MCC and how this process is disrupted following tobacco exposure and with CB. We shall also discuss existing therapeutic strategies for the treatment of chronic bronchitis and how components of the MCC can be used as biomarkers for the evaluation of tobacco or tobacco-like-product exposure.
Assuntos
Bronquite Crônica/fisiopatologia , Água Extravascular Pulmonar/fisiologia , Modelos Biológicos , Depuração Mucociliar/fisiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fumar/efeitos adversos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Canais de Sódio/metabolismoRESUMO
An important issue for in vivo gene therapy for cystic fibrosis (CF) is the percentage of cells within the CF airway that will require correction. In this study, we mixed populations of a CF airway cell line expressing either the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA (corrected cells) or a reporter gene in defined percentages. As few as 6-10% corrected cells within an epithelial sheet generated C1-transport properties similar to sheets comprised of 100% corrected cells. Cell-cell coupling may serve as the mechanism for amplification of the functional effects of corrected cells. These data suggest that in vivo correction of all CF airway cells may not be mandatory.
Assuntos
Fibrose Cística/terapia , Terapia Genética , Transfecção , Comunicação Celular , Células Cultivadas , Cloretos/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística , Epitélio/metabolismo , Expressão Gênica , Humanos , Transporte de Íons , Proteínas de Membrana/genética , Sistema Respiratório/metabolismoRESUMO
We have used in situ hybridization and immunocytochemistry to characterize the cellular distribution of cystic fibrosis (CF) gene expression in human bronchus. The cystic fibrosis transmembrane conductance regular (CFTR) was primarily localized to cells of submucosal glands in bronchial tissues from non-CF individuals notably in the serous component of the secretory tubules as well as a subpopulation of cells in ducts. Normal distribution of CFTR mRNA was found in CF tissues while expression of CFTR protein was genotype specific, with delta F508 homozygotes demonstrating no detectable protein and compound heterozygotes expressing decreased levels of normally distributed protein. Our data suggest mechanisms whereby defects in CFTR expression could lead to abnormal production of mucus in human lung.
Assuntos
Brônquios/química , Fibrose Cística/metabolismo , Glândulas Exócrinas/química , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Regulador de Condutância Transmembrana em Fibrose Cística , Células Epiteliais , Epitélio/química , Humanos , Hibridização In Situ , Modelos Biológicos , Mucosa/química , Pleura/química , Sondas RNA , RNA Antissenso , RNA Mensageiro/análiseRESUMO
The relationships between airway epithelial Cl(-) secretion-Na(+) absorption balance, airway surface liquid (ASL) homeostasis, and lung disease were investigated in selected transgenic mice. 1) To determine if transgenic overexpression of wild-type (WT) human CFTR (hCFTR) accelerated Cl(-) secretion and regulated Na(+) absorption in murine airways, we utilized a Clara cell secretory protein (CCSP)-specific promoter to generate mice expressing airway-specific hCFTR. Ussing chamber studies revealed significantly (â¼2.5-fold) elevated basal Cl(-) secretory currents in CCSP-hCFTR transgenic mouse airways. Endogenous murine airway Na(+) absorption was not regulated by hCFTR, and these mice exhibited no lung disease. 2) We tested whether hCFTR, transgenically expressed on a transgenic mouse background overexpressing the ß-subunit of the epithelial Na(+) channel (ß-ENaC), restored ion transport balance and ASL volume homeostasis and ameliorated lung disease. Both transgenes were active in CCSP-hCFTR/ß-ENaC transgenic mouse airways, which exhibited an elevated basal Cl(-) secretion and Na(+) hyperabsorption. However, the airway disease characteristic of ß-ENaC mice persisted. Confocal studies of ASL volume homeostasis in cultured tracheal cells revealed ASL autoregulation to a height of â¼6 µm in WT and CCSP-hCFTR cultures, whereas ASL was reduced to <4 µm in ß-ENaC and CCSP-hCFTR/ß-ENaC cultures. We conclude that 1) hCFTR overexpression increases basal Cl(-) secretion but does not regulate Na(+) transport in WT mice and 2) transgenic hCFTR produces increased Cl(-) secretion, but not regulation of Na(+) channels, in ß-ENaC mouse airways and does not ameliorate ß-ENaC mouse lung disease.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Canais Epiteliais de Sódio/metabolismo , Transporte de Íons/genética , Pneumopatias/metabolismo , Mucosa Respiratória/metabolismo , Canais de Sódio/metabolismo , Animais , Células Cultivadas , Cloretos/metabolismo , Canais Epiteliais de Sódio/genética , Genótipo , Pulmão/metabolismo , Pneumopatias/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras Genéticas , Mucosa Respiratória/citologia , Mucosa Respiratória/patologia , Sistema Respiratório , Sódio/metabolismo , Canais de Sódio/genética , Traqueia/metabolismo , Uteroglobina/genéticaRESUMO
INTRODUCTION: The effect of SARS-CoV-2 severity or the trimester of infection in pregnant mothers, placentas, and infants is not fully understood. METHODS: A retrospective, observational cohort study in Chapel Hill, NC of 115 mothers with SARS-CoV-2 and singleton pregnancies from December 1, 2019 to May 31, 2021 via chart review to document the infants' weight, length, head circumference, survival, congenital abnormalities, hearing loss, maternal complications, and placental pathology classified by the Amsterdam criteria. RESULTS: Of the 115 mothers, 85.2% were asymptomatic (n = 37) or had mild (n = 61) symptoms, 13.0% had moderate (n = 9) or severe (n = 6) COVID-19, and 1.74% (n = 2) did not have symptoms recorded. Moderate and severe maternal infections were associated with increased C-section, premature delivery, infant NICU admission, and were more likely to occur in Type 1 (p = 0.0055) and Type 2 (p = 0.0285) diabetic mothers. Only one infant (0.870%) became infected with SARS-CoV-2, which was not via the placenta. Most placentas (n = 63, 54.8%) did not show specific histologic findings; however, a subset showed mild maternal vascular malperfusion (n = 26, 22.6%) and/or mild microscopic ascending intrauterine infection (n = 28, 24.3%). The infants had no identifiable congenital abnormalities, and all infants and mothers survived. DISCUSSION: Most mothers and their infants had a routine clinical course; however, moderate and severe COVID-19 maternal infections were associated with pregnancy complications and premature delivery. Mothers with pre-existing, non-gestational diabetes were at greatest risk of developing moderate or severe COVID-19. The placental injury patterns of maternal vascular malperfusion and/or microscopic ascending intrauterine infection were not associated with maternal COVID-19 severity.
Assuntos
COVID-19 , Complicações Infecciosas na Gravidez , Nascimento Prematuro , Feminino , Humanos , Imunoglobulina G , Lactente , Transmissão Vertical de Doenças Infecciosas , Mães , Placenta/patologia , Gravidez , Complicações Infecciosas na Gravidez/patologia , Nascimento Prematuro/epidemiologia , Nascimento Prematuro/patologia , Estudos Retrospectivos , SARS-CoV-2RESUMO
BACKGROUND: Anaerobic bacteria are increasingly regarded as important in cystic fibrosis (CF) pulmonary infection. The aim of this study was to determine the effect of antibiotic treatment on aerobic and anaerobic microbial community diversity and abundance during exacerbations in patients with CF. METHODS: Sputum was collected at the start and completion of antibiotic treatment of exacerbations and when clinically stable. Bacteria were quantified and identified following culture, and community composition was also examined using culture-independent methods. RESULTS: Pseudomonas aeruginosa or Burkholderia cepacia complex were detected by culture in 24/26 samples at the start of treatment, 22/26 samples at completion of treatment and 11/13 stable samples. Anaerobic bacteria were detected in all start of treatment and stable samples and in 23/26 completion of treatment samples. Molecular analysis showed greater bacterial diversity within sputum samples than was detected by culture; there was reasonably good agreement between the methods for the presence or absence of aerobic bacteria such as P aeruginosa (κ=0.74) and B cepacia complex (κ=0.92), but agreement was poorer for anaerobes. Both methods showed that the composition of the bacterial community varied between patients but remained relatively stable in most individuals despite treatment. Bacterial abundance decreased transiently following treatment, with this effect more evident for aerobes (median decrease in total viable count 2.3×10(7) cfu/g, p=0.005) than for anaerobes (median decrease in total viable count 3×10(6) cfu/g, p=0.046). CONCLUSION: Antibiotic treatment targeted against aerobes had a minimal effect on abundance of anaerobes and community composition, with both culture and molecular detection methods required for comprehensive characterisation of the microbial community in the CF lung. Further studies are required to determine the clinical significance of and optimal treatment for these newly identified bacteria.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Fibrose Cística/microbiologia , Infecções Oportunistas/tratamento farmacológico , Adolescente , Adulto , Antibacterianos/uso terapêutico , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias Aeróbias/classificação , Bactérias Aeróbias/efeitos dos fármacos , Bactérias Aeróbias/isolamento & purificação , Bactérias Anaeróbias/classificação , Bactérias Anaeróbias/efeitos dos fármacos , Bactérias Anaeróbias/isolamento & purificação , Infecções Bacterianas/complicações , Contagem de Colônia Microbiana , Fibrose Cística/complicações , Fibrose Cística/fisiopatologia , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Infecções Oportunistas/complicações , Polimorfismo de Fragmento de Restrição , Escarro/microbiologia , Adulto JovemRESUMO
Airway epithelia are positioned at the interface between the body and the environment, and generate complex signaling responses to inhaled toxins and other stresses. Luminal mechanical stimulation of airway epithelial cells produces a propagating wave of elevated intracellular Ca(2+) that coordinates components of the integrated epithelial stress response. In polarized airway epithelia, this response has been attributed to IP(3) permeation through gap junctions. Using a combination of approaches, including enzymes that destroy extracellular nucleotides, purinergic receptor desensitization, and airway cells deficient in purinoceptors, we demonstrated that Ca(2+) waves induced by luminal mechanical stimulation in polarized airway epithelia were initiated by the release of the 5' nucleotides, ATP and UTP, across both apical and basolateral membranes. The nucleotides released into the extracellular compartment interacted with purinoceptors at both membranes to trigger Ca(2+) mobilization. Physiologically, apical membrane nucleotide-release coordinates airway mucociliary clearance responses (mucin and salt, water secretion, increased ciliary beat frequency), whereas basolateral release constitutes a paracrine mechanism by which mechanical stresses signal adjacent cells not only within the epithelium, but other cell types (nerves, inflammatory cells) in the submucosa. Nucleotide-release ipsilateral and contralateral to the surface stimulated constitutes a unique mechanism by which epithelia coordinate local and distant airway defense responses to mechanical stimuli.
Assuntos
Trifosfato de Adenosina/metabolismo , Comunicação Celular/fisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Uridina Trifosfato/metabolismo , Animais , Apirase/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Brônquios/citologia , Cálcio/metabolismo , Polaridade Celular/fisiologia , Células Cultivadas , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Knockout , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y2 , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Estresse MecânicoRESUMO
We recently showed that the COOH terminus of the cystic fibrosis transmembrane conductance regulator associates with the submembranous scaffolding protein EBP50 (ERM-binding phosphoprotein 50 kD; also called Na(+)/H(+) exchanger regulatory factor). Since EBP50 associates with ezrin, this interaction links the cystic fibrosis transmembrane conductance regulator (CFTR) to the cortical actin cytoskeleton. EBP50 has two PDZ domains, and CFTR binds with high affinity to the first PDZ domain. Here, we report that Yes-associated protein 65 (YAP65) binds with high affinity to the second EBP50 PDZ domain. YAP65 is concentrated at the apical membrane in airway epithelia and interacts with EBP50 in cells. The COOH terminus of YAP65 is necessary and sufficient to mediate association with EBP50. The EBP50-YAP65 interaction is involved in the compartmentalization of YAP65 at the apical membrane since mutant YAP65 proteins lacking the EBP50 interaction motif are mislocalized when expressed in airway epithelial cells. In addition, we show that the nonreceptor tyrosine kinase c-Yes is contained within EBP50 protein complexes by association with YAP65. Subapical EBP50 protein complexes, containing the nonreceptor tyrosine kinase c-Yes, may regulate apical signal transduction pathways leading to changes in ion transport, cytoskeletal organization, or gene expression in epithelial cells.
Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Trocadores de Sódio-Hidrogênio , Quinases da Família src , Sequência de Aminoácidos , Animais , Sítios de Ligação , Brônquios , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Membrana Celular/ultraestrutura , Colo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-yes , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
An airway epithelial cell line (CF/T43) was developed by infecting cultured airway epithelial cells from patients with cystic fibrosis (CF) with the pZIPneoSV(X)1/SV40T retrovirus and selecting for G418 resistance and ion transport properties. The distinctive chloride secretory phenotypes of the CF cell line CF/T43 and a normal cell line (NL/T4) were not perturbed by SV40T-induced cell transformation. Epithelial cell lines generated from CF cells with the SV40T gene can be used to test candidate CF genes and to evaluate the molecular mechanisms responsible for the CF phenotype.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Cloretos/fisiologia , Fibrose Cística/fisiopatologia , Proteínas de Membrana/fisiologia , Vírus 40 dos Símios/imunologia , Transformação Genética , Amilorida/farmacologia , Calcimicina/farmacologia , Linhagem Celular , Membrana Celular/fisiologia , Canais de Cloreto , Colforsina/farmacologia , Fibrose Cística/patologia , Condutividade Elétrica , Epitélio/efeitos dos fármacos , Epitélio/patologia , Epitélio/fisiologia , Éteres/farmacologia , Técnica de Fratura por Congelamento , Humanos , Junções Intercelulares , Canais Iônicos/fisiologia , Ionomicina , Microscopia Eletrônica , Pólipos NasaisRESUMO
The effect of the number of cystic fibrosis (CF) alleles on cholera toxin (CT)-induced intestinal secretion was examined in the CF mouse model. CF mice that expressed no CF transmembrane conductance regulator (CFTR) protein did not secrete fluid in response to CT. Heterozygotes expressed 50 percent of the normal amount of CFTR protein in the intestinal epithelium and secreted 50 percent of the normal fluid and chloride ion in intestinal epithelium and secreted 50 percent of the normal fluid and chloride ion and fluid secretion suggests that CF heterozygotes might possess a selective advantage of resistance to cholera.
Assuntos
Líquidos Corporais/metabolismo , Cloretos/metabolismo , Toxina da Cólera/toxicidade , Fibrose Cística/genética , Mucosa Intestinal/metabolismo , Proteínas de Membrana/genética , Alelos , Animais , Canais de Cloreto/metabolismo , Cruzamentos Genéticos , AMP Cíclico/metabolismo , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística , Feminino , Heterozigoto , Intestino Delgado/metabolismo , Masculino , Proteínas de Membrana/metabolismo , CamundongosRESUMO
Cystic fibrosis results from defects in the gene encoding a cyclic adenosine monophosphate-dependent chloride ion channel known as the cystic fibrosis transmembrane conductance regulator (CFTR). To create an animal model for cystic fibrosis, mice were generated from embryonic stem cells in which the CFTR gene was disrupted by gene targeting. Mice homozygous for the disrupted gene display many features common to young human cystic fibrosis patients, including failure to thrive, meconium ileus, alteration of mucous and serous glands, and obstruction of glandlike structures with inspissated eosinophilic material. Death resulting from intestinal obstruction usually occurs before 40 days of age.
Assuntos
Fibrose Cística/genética , Modelos Animais de Doenças , Proteínas de Membrana/genética , Animais , Fibrose Cística/patologia , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística , Sistema Digestório/metabolismo , Sistema Digestório/patologia , Glândulas Exócrinas/patologia , Vesícula Biliar/patologia , Genitália Masculina/patologia , Genótipo , Crescimento , Obstrução Intestinal/etiologia , Obstrução Intestinal/patologia , Fígado/patologia , Masculino , Mecônio/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Muco/metabolismo , Mutagênese , Pâncreas/patologia , RNA Mensageiro/metabolismo , Glândulas Salivares/patologiaRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an adenosine 3',5'-monophosphate (cyclic AMP)-activated chloride channel. In cystic fibrosis (CF) patients, loss of CFTR function because of a genetic mutation results in defective cyclic AMP-mediated chloride secretion across epithelia. Because of their potential role as an animal model for CF, mice with targeted disruption of the murine CFTR gene [CFTR(-/-)] were tested for abnormalities in epithelial chloride transport. In both freshly excised tissue from the intestine and in cultured epithelia from the proximal airways, the cyclic AMP-activated chloride secretory response was absent in CFTR(-/-) mice as compared to littermate controls. Thus, disruption of the murine CFTR gene results in the chloride transport abnormalities predicted from studies of human CF epithelia.
Assuntos
Cloretos/metabolismo , Fibrose Cística/metabolismo , Modelos Animais de Doenças , Proteínas de Membrana/fisiologia , Amilorida/farmacologia , Animais , Transporte Biológico , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/farmacologia , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística , Epitélio/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Membrana/genética , Camundongos , Mutação , Mucosa Nasal/metabolismo , Traqueia/metabolismoRESUMO
Cystic fibrosis transmembrane regulator (CFTR), the gene product that is mutated in cystic fibrosis (CF) patients, has a well-recognized function as a cyclic adenosine 3',5'-monophosphate (cAMP)-regulated chloride channel, but this property does not account for the abnormally high basal rate and cAMP sensitivity of sodium ion absorption in CF airway epithelia. Expression of complementary DNAs for rat epithelial Na+ channel (rENaC) alone in Madin Darby canine kidney (MDCK) epithelial cells generated large amiloride-sensitive sodium currents that were stimulated by cAMP, whereas coexpression of human CFTR with rENaC generated smaller basal sodium currents that were inhibited by cAMP. Parallel studies that measured regulation of sodium permeability in fibroblasts showed similar results. In CF airway epithelia, the absence of this second function of CFTR as a cAMP-dependent regulator likely accounts for abnormal sodium transport.
Assuntos
AMP Cíclico/metabolismo , Fibrose Cística/metabolismo , Proteínas de Membrana/metabolismo , Canais de Sódio/metabolismo , Células 3T3 , Absorção , Amilorida/farmacologia , Animais , Linhagem Celular , Permeabilidade da Membrana Celular , Canais de Cloreto/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística , DNA Complementar , Cães , Humanos , Camundongos , Técnicas de Patch-Clamp , Ratos , Sódio/metabolismo , TransfecçãoRESUMO
The epithelium of nasal tissue excised from subjects with cystic fibrosis exhibited higher voltage and lower conductance than tissue from control subjects. Basal sodium ion absorption by cystic fibrosis and normal nasal epithelia equaled the short-circuit current and was amiloride-sensitive. Amiloride induced chloride ion secretion in normal but not cystic fibrosis tissue and consequently was more effective in inhibiting the short-circuit current in cystic fibrosis epithelia. Chloride ion-free solution induced a smaller hyperpolarization of cystic fibrosis tissue. The increased voltage and amiloride efficacy in cystic fibrosis reflect absorption of sodium ions across an epithelium that is relatively impermeable to chloride ions.
Assuntos
Cloretos/metabolismo , Fibrose Cística/metabolismo , Mucosa Nasal/metabolismo , Sódio/metabolismo , Absorção , Amilorida/farmacologia , Humanos , Canais Iônicos/metabolismoRESUMO
Purinergic signalling regulates airway defence mechanisms, suggesting that extracellular purines could serve as airway inflammation biomarkers in cystic fibrosis (CF). The purines adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine were measured in sputum from 21 adults (spontaneously expectorated from seven CF patients, induced from 14 healthy controls) to assess normal values and CF-associated changes. Subsequently, purine levels were measured in bronchoalveolar lavage fluid (BALF) from 37 children (25 CF patients, 12 disease controls) and compared with neutrophil counts, presence of airway infection and lung function. To noninvasively assess airway purines, ATP levels were measured using luminometry in exhaled breath condensate (EBC) from 14 children with CF and 14 healthy controls, then 14 CF children during a pulmonary exacerbation. Both ATP and AMP were elevated in sputum and BALF from CF subjects compared with controls. In BALF, ATP and AMP levels were inversely related to lung function and strongly correlated with neutrophil counts. In EBC, ATP levels were increased in CF relative to controls and decreased after treatment of CF pulmonary exacerbation. The purines adenosine triphosphate and adenosine monophosphate are candidate biomarkers of neutrophilic airways inflammation. Measurement of purines in sputum or exhaled breath condensate may provide a relatively simple and noninvasive method to track this inflammation.
Assuntos
Monofosfato de Adenosina/análise , Trifosfato de Adenosina/análise , Líquido da Lavagem Broncoalveolar/imunologia , Fibrose Cística/imunologia , Escarro/imunologia , Adolescente , Adulto , Biomarcadores/análise , Testes Respiratórios/métodos , Estudos de Casos e Controles , Criança , Pré-Escolar , Fibrose Cística/diagnóstico , Feminino , Humanos , Inflamação/imunologia , Mediadores da Inflamação/análise , Contagem de Leucócitos , Pulmão/imunologia , Masculino , Neutrófilos/imunologiaRESUMO
Autoimmune disorders, medical interventions, and aging are all known to be associated with salivary gland hypofunction, which results in the uncomfortable feeling of dry mouth (xerostomia) and significantly diminished oral health. The current therapeutic regimen includes increasing oral hydration using over-the-counter oral comfort agents and the use of systemic cholinergic drugs to stimulate salivary output. However, these approaches produce very transient relief or are associated with uncomfortable side-effects. Thus, new treatments that provide long-lasting relief from discomfort and improve oral health with minimal side-effects would benefit the therapy of this disease. The processes that mediate fluid loss from the oral cavity, such as the absorption of fluid from the oral mucosa, represent novel therapeutic targets for xerostomia. Preventing fluid absorption from the oral cavity is predicted to improve oral hydration and alleviate the clinical symptoms and discomfort associated with dry mouth. Furthermore, therapeutic strategies that prevent fluid absorption should complement current approaches that increase salivary output. This review discusses the current understanding of oral fluid balance and how these processes may be manipulated to provide relief for those suffering from dry mouth.
Assuntos
Mucosa Bucal/efeitos dos fármacos , Xerostomia/tratamento farmacológico , Absorção , Água Corporal/efeitos dos fármacos , Água Corporal/metabolismo , Humanos , Transporte de Íons/efeitos dos fármacos , Mucosa Bucal/metabolismo , Saliva/efeitos dos fármacos , Saliva/metabolismo , Taxa Secretória/efeitos dos fármacos , Água/metabolismo , Equilíbrio Hidroeletrolítico/efeitos dos fármacosRESUMO
The response of cultured human nasal epithelia to hypertonic bathing solutions was tested using ion-selective microelectrode and quantitative microscopy. Raised luminal, but not serosal, osmolality (+/- 150 mM mannitol) decreased Na+ absorption but did not induce Cl- secretion. Raised luminal osmolality increased cell Cl- activity, Na+ activity, and transepithelial resistance and decreased both apical and basolateral membrane potentials and the fractional resistance of the apical membrane; equivalent circuit analysis revealed increases in apical, basolateral, and shunt resistances. Prolonged exposure (10 min) to 430 mosM luminal solution elicited no regulation of any parameter. Optical measurements revealed a reduction in the thickness of preparations only in response to luminal hypertonic solutions. We conclude that (a) airway epithelial cells exhibit asymmetric water transport properties, with the apical membrane water permeability exceeding that of the basolateral membrane; (b) the cellular response to volume loss is a deactivation of the basolateral membrane K+ conductance and the apical membrane Cl- conductance; (c) luminal hypertonicity slows the rate of Na+ absorption but does not induce Cl- secretion; and (d) cell volume loss increases the resistance of the paracellular path. We speculate that these properties configure human nasal epithelium to behave as an osmotic sensor, transducing information about luminal solutions to the airway wall.
Assuntos
Mucosa Nasal/metabolismo , Equilíbrio Hidroeletrolítico , Adulto , Amilorida/farmacologia , Permeabilidade da Membrana Celular , Epitélio/metabolismo , Feminino , Humanos , Soluções Hipertônicas , Transporte de Íons , Masculino , Manitol/metabolismo , Pessoa de Meia-IdadeRESUMO
Current hypotheses describing the function of normal airway surface liquid (ASL) in lung defense are divergent. One theory predicts that normal airways regulate ASL volume by modulating the flow of isosmotic fluid across the epithelium, whereas an alternative theory predicts that ASL is normally hyposmotic. These hypotheses predict different values for the osmotic water permeability (P(f)) of airway epithelia. We measured P(f) of cultures of normal and cystic fibrosis (CF) airway epithelia that, like the native tissue, contain columnar cells facing the lumen and basal cells that face a basement membrane. Xz laser scanning confocal microscopy recorded changes in epithelial height and transepithelial volume flow in response to anisosmotic challenges. With luminal hyperosmotic challenges, transepithelial and apical membrane P(f) are relatively high for both normal and CF airway epithelia, consistent with an isosmotic ASL. Simultaneous measurements of epithelial cell volume and transepithelial water flow revealed that airway columnar epithelial cells behave as osmometers whose volume is controlled by luminal osmolality. Basal cell volume did not change in these experiments. When the serosal side of the epithelium was challenged with hyperosmotic solutions, the basal cells shrank, whereas the lumen-facing columnar cells did not. We conclude that (a) normal and CF airway epithelia have relatively high water permeabilities, consistent with the isosmotic ASL theory, and the capacity to restore water on airway surfaces lost by evaporation, and (b) the columnar cell basolateral membrane and tight junctions limit transepithelial water flow in this tissue.
Assuntos
Brônquios/metabolismo , Fibrose Cística/metabolismo , Água/metabolismo , Brônquios/citologia , Diferenciação Celular , Células Cultivadas , Fibrose Cística/patologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Cinética , Osmose , Permeabilidade , Junções Íntimas/metabolismoRESUMO
Cystic fibrosis airway epithelia exhibit a spectrum of ion transport properties that differ from normal, including not only defective cAMP-mediated Cl- secretion, but also increased Na+ absorption and increased Ca(2+)-mediated Cl- secretion. In the present study, we examined whether adenovirus-mediated (Ad5) transduction of CFTR can correct all of these CF ion transport abnormalities. Polarized primary cultures of human CF and normal nasal epithelial cells were infected with Ad5-CBCFTR at an moi (10(4)) which transduced virtually all cells or Ad5-CMV lacZ as a control. Consistent with previous reports, Ad5-CBCFTR, but not Ad5-CMV lacZ, corrected defective CF cAMP-mediated Cl- secretion. Basal Na+ transport rates (basal Ieq) in CF airway epithelial sheets (-78.5 +/- 9.8 microA/cm2) were reduced to levels measured in normal epithelial sheets (-30.0 +/- 2.0 microA/cm2) by Ad5-CBCFTR (-36.9 +/- 4.8 microA/cm2), but not Ad5-CMV lacZ (-65.8 +/- 6.1 microA/cm2). Surprisingly, a significant reduction in delta Ieq in response to ionomycin, a measure of Ca(2+)-mediated Cl- secretion, was observed in CFTR-expressing (corrected) CF epithelial sheets (-6.9 +/- 11.8 microA/cm2) when compared to uninfected CF epithelial sheets (-76.2 +/- 15.1 microA/cm2). Dose response effects of Ad5-CBCFTR on basal Na+ transport rates and Ca(2+)-mediated Cl- secretion suggest that the mechanism of regulation of these two ion transport functions by CFTR may be different. In conclusion, efficient transduction of CFTR corrects hyperabsorption of Na+ in primary CF airway epithelial cells and restores Ca(2+)-mediated Cl- secretion to levels observed in normal airway epithelial cells. Moreover, assessment of these ion transport abnormalities may represent important endpoints for testing the efficacy of gene therapy for cystic fibrosis.
Assuntos
Cloretos/metabolismo , Fibrose Cística/metabolismo , Proteínas de Membrana/metabolismo , Mucosa Nasal/fisiologia , Sódio/metabolismo , Adenoviridae/genética , Transporte Biológico , Western Blotting , Cálcio/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Fibrose Cística/genética , Fibrose Cística/terapia , Regulador de Condutância Transmembrana em Fibrose Cística , Terapia Genética , Humanos , Imuno-Histoquímica , Potenciais da Membrana , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Mucosa Nasal/citologia , Proteínas Recombinantes/metabolismoRESUMO
The transepithelial chloride permeability of airway and sweat ductal epithelium has been reported to be decreased in patients with cystic fibrosis (CF). In the present study, we investigated whether the airway epithelial defect was in the cell path by characterizing the relative ion permeabilities of the apical membrane of respiratory epithelial cells from CF and normal subjects. Membrane electric potential difference (PD) and the responses to luminal Cl- replacement, isoproterenol, and amiloride were measured with intracellular microelectrodes. The PD across the apical barrier was smaller for CF (-11 mV) than normal (-29 mV) epithelia whereas the PD across the basolateral barrier was similar, (-26 and -34 mV respectively). In contrast to normal nasal epithelium, the apical membrane in CF epithelia was not Cl- permselective and was not responsive to isoproterenol. Amiloride, a selective Na+ channel blocker, induced a larger apical membrane hyperpolarization and a greater increase in transepithelial resistance in CF epithelia. Both reduced apical cell membrane Cl- conductance and increased Na+ conductance appear to contribute to the abnormal function of respiratory epithelia of CF patients.