RESUMEN
BACKGROUND AND AIMS: The structures that contribute to shunt resistance (Rs) in esophageal epithelium are incompletely understood, with 35-40% of Rs known to be calcium-dependent, reflecting the role of e-cadherin. Two calcium-independent candidates for the remaining approximately 60% of Rs have been identified: the glycoprotein matrix (GPM) within stratum corneum of esophageal epithelium, and the lateral cell membranes (LCMs) from neighboring cells. METHODS: To determine the contribution of GPM and LCMs to Rs, rabbit esophageal epithelium was mounted in Ussing chambers so that transepithelial resistance (R(T)), a marker of Rs, could be monitored during luminal exposure to either glycosidases for disruption of the GPM or to hypertonic urea for separation of the LCMs. RESULTS: Glycosidases had no effect on R(T). In contrast, hypertonic urea reduced R(T), increased fluorescein flux and widened the intercellular spaces. That urea reduced R(T), and so Rs, by widening the intercellular spaces, and not by altering the e-cadherin-dependent apical junctional complex, was supported by the ability of: (a) calcium-free solution to reduce R(T) beyond that produced by urea, (b) hypertonic urea to reduce R(T) beyond that produced by calcium free solution, (c) hypertonic sucrose to collapse the intercellular spaces and raise R(T), and (d) empigen, a zwitterionic detergent, to non-osmotically widen the intercellular spaces and reduce R(T). CONCLUSION: These data indicate that the LCMs from neighboring cells are a major contributor to shunt resistance in esophageal epithelium. As resistor, they are distinguishable from the apical junctional complex by their sensitivity to (luminal) hypertonicity and insensitivity to removal of calcium.
Asunto(s)
Cadherinas/metabolismo , Epitelio/metabolismo , Esófago/metabolismo , Análisis de Varianza , Animales , Cadherinas/farmacología , Permeabilidad de la Membrana Celular/fisiología , Modelos Animales de Enfermedad , Impedancia Eléctrica , Electrofisiología , Epitelio/efectos de los fármacos , Epitelio/patología , Enfermedades del Esófago/metabolismo , Esófago/efectos de los fármacos , Glicósido Hidrolasas/farmacología , Hexosaminidasas/farmacología , Soluciones Hipertónicas/metabolismo , Soluciones Hipertónicas/farmacología , Masculino , Potenciales de la Membrana , Neuraminidasa/farmacología , Probabilidad , Conejos , Distribución Aleatoria , Valores de Referencia , Sensibilidad y Especificidad , Sacarosa/farmacologíaRESUMEN
Epithelial tight junctions (TJs) provide an important route for passive electrolyte transport across airway epithelium and provide a barrier to the migration of toxic materials from the lumen to the interstitium. The possibility that TJ function may be perturbed by airway inflammation originated from studies reporting (1) increased levels of the proinflammatory cytokines interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and IL-1beta in airway epithelia and secretions from cystic fibrosis (CF) patients and (2) abnormal TJ strands of CF airways as revealed by freeze-fracture electron microscopy. We measured the effects of cytokine exposure of CF and non-CF well-differentiated primary human airway epithelial cells on TJ properties, including transepithelial resistance, paracellular permeability to hydrophilic solutes, and the TJ proteins occludin, claudin-1, claudin-4, junctional adhesion molecule, and ZO-1. We found that whereas IL-1beta treatment led to alterations in TJ ion selectivity, combined treatment of TNF-alpha and IFN-gamma induced profound effects on TJ barrier function, which could be blocked by inhibitors of protein kinase C. CF bronchi in vivo exhibited the same pattern of expression of TJ-associated proteins as cultures exposed in vitro to prolonged exposure to TNF-alpha and IFN-gamma. These data indicate that the TJ of airway epithelia exposed to chronic inflammation may exhibit parallel changes in the barrier function to both solutes and ions.
Asunto(s)
Fibrosis Quística/metabolismo , Citocinas/metabolismo , Células Epiteliales/metabolismo , Uniones Estrechas/metabolismo , Western Blotting , Movimiento Celular , Células Cultivadas , Cloro/metabolismo , Citocinas/biosíntesis , Electrofisiología , Ensayo de Inmunoadsorción Enzimática , Humanos , Interferón gamma/metabolismo , Interleucina-1/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Microscopía Electrónica , Microscopía Fluorescente , Permeabilidad , Fosfoproteínas/metabolismo , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sodio/farmacología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo , Proteína de la Zonula Occludens-1RESUMEN
Peptide and protein drugs have become the new generation of therapeutics, yet most of them are only available as injections, and reports on oral local intestinal delivery of peptides and proteins are quite limited. The aim of this work was to develop and evaluate a water-in-oil (w/o) microemulsion system in vitro and in vivo for local intestinal delivery of water-soluble peptides after oral administration. A fluorescent labeled peptide, 5-(and-6)-carboxytetramethylrhodamine labeled HIV transactivator protein TAT (TAMRA-TAT), was used as a model peptide. Water-in-oil microemulsions consisting of Miglyol 812, Capmul MCM, Tween 80, and water were developed and characterized in terms of appearance, viscosity, conductivity, morphology, and particle size analysis. TAMRA-TAT was loaded and its enzymatic stability was assessed in modified simulated intestinal fluid (MSIF) in vitro. In in vivo studies, TAMRA-TAT intestinal distribution was evaluated using fluorescence microscopy after TAMRA-TAT microemulsion, TAMRA-TAT solution, and placebo microemulsion were orally gavaged to mice. The half-life of TAMRA-TAT in microemulsion was enhanced nearly three-fold compared to that in the water solution when challenged by MSIF. The treatment with TAMRA-TAT microemulsion after oral administration resulted in greater fluorescence intensity in all intestine sections (duodenum, jejunum, ileum, and colon) compared to TAMRA-TAT solution or placebo microemulsion. The in vitro and in vivo studies together suggested TAMRA-TAT was better protected in the w/o microemulsion in an enzyme-containing environment, suggesting that the w/o microemulsions developed in this study may serve as a potential delivery vehicle for local intestinal delivery of peptides or proteins after oral administration.
Asunto(s)
Sistemas de Liberación de Medicamentos , Mucosa Intestinal/metabolismo , Péptidos/administración & dosificación , Animales , Estabilidad de Medicamentos , Conductividad Eléctrica , Emulsiones , Femenino , Absorción Intestinal , Ratones , Ratones Endogámicos C57BL , Péptidos/farmacocinética , Rodaminas/administración & dosificación , Viscosidad , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/administración & dosificación , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacocinéticaRESUMEN
Protein kinase A (PKA) signaling is targeted by interactions with A-kinase anchoring proteins (AKAPs) via a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins [AKAP-associated sperm protein (ASP), ropporin (ROPN1), sperm protein 17 (SP17) and calcium binding tyrosine-(Y)-phosphorylation regulated protein (CABYR)] share this highly conserved RII dimerization/docking (R2D2) domain. ASP and ROPN1 are 41% identical in sequence, interact with a variety of AKAPs in a manner similar to PKA, and are expressed in ciliated and flagellated human cells. To test the hypothesis that these proteins regulate motility, we developed mutant mouse lines lacking ASP or ROPN1. Both mutant lines produced normal numbers of cilia with intact ciliary ultrastructure. Lack of ROPN1 had no effect on ciliary motility. However, the beat frequency of cilia from mice lacking ASP is significantly slower than wild type, indicating that ASP signaling may regulate ciliary motility. This is the first demonstration of in vivo function for ASP. Similar localization of ASP in mice and humans indicates that these findings may translate to human physiology, and that these mice will be an excellent model for future studies related to the pathogenesis of human disease.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/deficiencia , Cilios/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de Unión al GTP rho/deficiencia , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Genotipo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Transducción de Señal , Motilidad Espermática/fisiología , Espermatozoides/metabolismo , Espermatozoides/fisiología , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Barrett's esophagus (BE) is a specialized columnar epithelium (SCE) that develops as replacement for damaged squamous epithelium (SqE) in subjects with reflux disease, and as such it is apparently more acid resistant than SqE. How SCE resists acid injury is poorly understood; one means may involve altered tight junctions (TJs) since the TJ in SqE is an early target of attack and damage by acid in reflux disease. To assess this possibility, quantitative RT-PCR for 21 claudins was performed on endoscopic biopsies on SCE of BE and from healthy SqE from subjects without esophageal disease. In SCE, Cldn-18 was the most highly expressed at the mRNA level and this finding is paralleled by marked elevation in protein expression on immunoblots. In contrast in SqE, Cldn-18 was minimally expressed at the mRNA level and undetectable at the protein level. Immunofluorescence studies showed membrane localization of Cldn-18 and colocalization with the tight junction protein, zonula occludens-1. When Cldn-18 was overexpressed in MDCK II cells and mounted as monolayers in Ussing chambers, it raised electrical resistance and, as shown by lower dilution potentials to a NaCl gradient and lower diffusion potentials to acidic gradients, selectively reduced paracellular permeability to both Na(+) and H(+) compared with parental MDCK cells. We conclude that Cldn-18 is the dominant claudin in the TJ of SCE and propose that the change from a Cldn-18-deficient TJ in SqE to a Cldn-18-rich TJ in SCE contributes to the greater acid resistance of BE.
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Esófago de Barrett/metabolismo , Esófago/química , Esófago/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Uniones Estrechas/química , Uniones Estrechas/metabolismo , Adolescente , Adulto , Anciano , Células Cultivadas , Claudinas , Femenino , Humanos , Concentración de Iones de Hidrógeno , Masculino , Persona de Mediana EdadRESUMEN
Palmitoylation of integral membrane proteins can affect intracellular trafficking, protein-protein interactions and protein stability. The goal of the present study was to determine whether claudins, transmembrane-barrier-forming proteins of the tight junction, are palmitoylated and whether this modification has functional implications for the tight-junction barrier. Claudin-14, like other members of the claudin family, contains membrane-proximal cysteines following both the second and the fourth transmembrane domains, which we speculated could be modified by S-acylation with palmitic acid. We observed that [(3)H]-palmitic acid was incorporated into claudin-14 expressed by transfection in both cultured epithelial cells and fibroblasts. Mutation of cysteines to serines following either the second or the fourth transmembrane segments decreased the incorporation of [(3)H]-palmitic acid, and mutation of all four cysteines eliminated palmitoylation. We previously reported that expression of claudin-14 in epithelial monolayers results in a fivefold increase in electrical resistance. By contrast, expression of the mutant claudin-14 resulted in smaller increases in resistance. The mutants localized less well to tight junctions and were also found in lysosomes, suggesting an alteration in trafficking or stability. However, we observed no change in protein half-life and only a small shift in fractionation out of caveolin-enriched detergent-resistant membranes. Although less well localized to the tight junction, palmitoylation-deficient claudin-14 was still concentrated at sites of cell-cell contact and was competent to assemble into freeze-fracture strands when expressed in fibroblasts. These results demonstrate that palmitoylation of claudin-14 is required for efficient localization into tight junctions but not stability or strand assembly. Decreased ability of the mutants to alter resistance is probably the result of their less efficient localization into the barrier.
Asunto(s)
Proteínas de la Membrana/metabolismo , Ácido Palmítico/metabolismo , Uniones Estrechas/fisiología , Animales , Línea Celular , Membrana Celular/metabolismo , Claudinas , Perros , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Humanos , Proteínas de la Membrana/genética , Mutación , Factores de TiempoRESUMEN
Ciliogenesis in human fetal airway epithelium occurs from 11 to 24 gestational weeks. Using genetic and antigenic markers specific for human axonemal dynein heavy chain 9, we characterized temporal aspects of axonemal dynein expression associated with large airway epithelial ciliogenesis during human fetal development. Late in the first trimester, an undifferentiated columnar epithelium is characteristic of the large airways, and immunocytochemical studies exhibited focal localization of axonemal dynein antigen on luminal epithelial cell borders at sites consistent with emergent ciliary beds. From 12 to 22 wk, immunocytochemical labeling of new ciliary beds was prominent, and localization within the cytoplasm of epithelial cells suggested avid synthesis of axonemal dynein in advance of ciliogenic events. Quantitative RT-PCR of tracheal RNA and in situ hybridization studies compared favorably with immunocytochemical findings with the earliest expression of axonemal dynein at 9-10 wk gestation. These studies have documented that axonemal dynein is expressed early in human fetal life during airway epithelial maturation and well before histological or ultrastructural evidence of ciliogenesis is apparent.
Asunto(s)
Dineínas/genética , Expresión Génica , Tráquea/embriología , Dineínas Axonemales , Cilios/fisiología , Cilios/ultraestructura , Dineínas/metabolismo , Desarrollo Embrionario y Fetal , Epitelio/embriología , Femenino , Feto/citología , Feto/metabolismo , Feto/fisiología , Feto/ultraestructura , Humanos , Inmunohistoquímica , Microscopía Electrónica , Embarazo , Primer Trimestre del Embarazo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Airway epithelial tight junctions (TJs) serve to separate the external and internal environments of the lung. However, the members of the claudin family that mediate this function have not been fully delineated. We characterized the claudin expression in normal airways removed from human donors during lung transplantation and determined the contribution of each claudin to airway barrier function. Stable cell lines in NIH/3T3 and human airway (IB3.1) cells were constructed expressing the claudin components found in the human airway, claudin-1, -3, or -5. The effects of claudin expression on transepithelial resistance, permeability coefficients, and claudin-claudin interactions were assessed. Claudin-1 and -3 decreased solute permeability, whereas claudin-5 increased permeability. We also detected oligomerization of claudin-5 in cell lines and in freshly excised human airways. Coimmunoprecipitation studies revealed heterophilic interactions between claudin species in both cell lines and human airway epithelium. These suggest that airway TJs are regulated by claudinclaudin interactions that confer the selectivity of the junction.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Uniones Estrechas/fisiología , Células 3T3 , Animales , Bronquios/fisiología , Claudina-1 , Claudina-3 , Claudina-5 , Humanos , Ratones , Proteínas Recombinantes/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/fisiología , TransfecciónRESUMEN
Exposure to complex mixtures of air pollutants produces inflammation in the upper and lower respiratory tract. Because the nasal cavity is a common portal of entry, respiratory and olfactory epithelia are vulnerable targets for toxicological damage. This study has evaluated, by light and electron microscopy and immunohistochemical expression of nuclear factor-kappa beta (NF-kappaB) and inducible nitric oxide synthase (iNOS), the olfactory and respiratory nasal mucosae, olfactory bulb, and cortical and subcortical structures from 32 healthy mongrel canine residents in Southwest Metropolitan Mexico City (SWMMC), a highly polluted urban region. Findings were compared to those in 8 dogs from Tlaxcala, a less polluted, control city. In SWMMC dogs, expression of nuclear neuronal NF-kappaB and iNOS in cortical endothelial cells occurred at ages 2 and 4 weeks; subsequent damage included alterations of the blood-brain barrier (BBB), degenerating cortical neurons, apoptotic glial white matter cells, deposition of apolipoprotein E (apoE)-positive lipid droplets in smooth muscle cells and pericytes, nonneuritic plaques, and neurofibrillary tangles. Persistent pulmonary inflammation and deteriorating olfactory and respiratory barriers may play a role in the neuropathology observed in the brains of these highly exposed canines. Neurodegenerative disorders such as Alzheimer's may begin early in life with air pollutants playing a crucial role.