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2.
Pharm Res ; 31(9): 2539-48, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24633418

RESUMEN

PURPOSE: To mechanistically analyze effects of the medium-chain fatty acid laurate on transepithelial permeability in confluent monolayers of the intestinal epithelial cell line HT-29/B6, in context with an application as an absorption enhancer improving transepithelial drug permeation. METHODS: Transepithelial resistance and apparent permeability for paracellular flux markers was measured using Ussing-type chambers. Two-path impedance spectroscopy was employed to differentiate between transcellular and paracellular resistance, and confocal imaging and Western blotting was performed. RESULTS: Laurate resulted in a substantial and reversible decrease in transepithelial resistance by 50% which was attributed to a decrease in paracellular resistance. Simultaneously, an increase in permeability for fluorescein (330 Da) was detected, while permeabilities for 4 kDa FITC-dextran and sulpho-NHS-SS-biotin (607 Da) remained unaltered. Confocal laser-scanning microscopy revealed a marked reduction of claudin-5, while other tight junction proteins including tricellulin, a protein preventing the paracellular passage of macromolecules, were not affected. CONCLUSIONS: Laurate induces an increase in paracellular permeability for molecules up to a molecular mass of 330 Da by retrieval of claudin-5 from tight junctions without affecting tricellular contacts and the paracellular passage of macromolecules. We hereby provide, for the first time, a mechanistical explanation of laurate-induced permeability enhancement on molecular level.


Asunto(s)
Claudina-5/metabolismo , Mucosa Intestinal/efectos de los fármacos , Lauratos/farmacología , Permeabilidad/efectos de los fármacos , Uniones Estrechas/efectos de los fármacos , Biotina/análogos & derivados , Biotina/farmacocinética , Permeabilidad de la Membrana Celular/efectos de los fármacos , Dextranos/farmacocinética , Fluoresceína/farmacocinética , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Células HT29 , Humanos , Mucosa Intestinal/metabolismo , Succinimidas/farmacocinética , Uniones Estrechas/metabolismo
3.
Biomaterials ; 34(1): 275-82, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23069717

RESUMEN

Sodium caprate is a promising candidate for inducing drug absorption enhancement. The mechanism of that uptake-enhancing effect is not fully understood so far. We investigated how caprate acts in an established human intestinal cell line, HT-29/B6, on the transient opening of transcellular (across the cell membranes) and paracellular (across the tight junction) pathways. Sodium caprate (10 mm) caused a rapid and reversible decrease of transepithelial resistance which is based, as measured by two-path impedance spectroscopy, exclusively on resistance changes of the paracellular pathway. Measurements of paracellular marker fluxes revealed an increased permeability for fluorescein (330 Da) and FITC-dextran (4 and 10 kDa), indicating an opening of the paracellular barrier. Confocal microscopy revealed a marked reduction of tricellulin in tricellular tight junctions and of claudin-5 in bicellular tight junctions. This was not due to altered protein expression, as occludin, claudins or tricellulin were not significantly changed in Western blots. Visualization of the translocation site of the cell membrane-impermeable marker molecule sulpho-NHS-SS-biotin (607 Da) indicated the tricellular tight junction to be the predominant pathway. We suggest that caprate's known enhancing effect on intestinal drug uptake is based on increased permeability in tricellular cell contacts, mediated by reversible removal of tricellulin from the tricellular tight junction.


Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Ácidos Decanoicos/farmacología , Intestinos/citología , Sustancias Macromoleculares/metabolismo , Uniones Estrechas/metabolismo , Actinas/metabolismo , Biomarcadores/metabolismo , Supervivencia Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Impedancia Eléctrica , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Células HT29 , Humanos , L-Lactato Deshidrogenasa/metabolismo , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/efectos de los fármacos , Factores de Tiempo
4.
Ann N Y Acad Sci ; 1258: 86-92, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22731720

RESUMEN

A variety of chemical compounds are currently being discussed as novel drug delivery strategies. One promising strategy is to selectively open the paracellular pathway of epithelia for the passage of macromolecules. A prerequisite for this effect is a rapid and reversible action of these compounds, to allow a marked translocation of a drug, but also to avoid unwanted adverse effects, such as the translocation of noxious agents. Bioactive molecules that elevate paracellular permeability include Ca(2+) chelators, bacterial toxins, and other compounds, some of which perturb the structural basis of epithelial barrier function--the tight junction. Within the tight junction, organ- and tissue-specific barrier properties are determined mainly by claudins. The majority of members of the claudin protein family seal the paracellular pathway. This paper focuses on recent approaches concerning absorption-enhancing effects, with regard to selectivity and mechanism.


Asunto(s)
Células Epiteliales/citología , Modelos Teóricos , Animales , Calcio/metabolismo , Quelantes/metabolismo , Quitosano/metabolismo , Enterotoxinas/metabolismo , Ácidos Grasos/metabolismo , Humanos , Uniones Estrechas
5.
Ann N Y Acad Sci ; 1258: 100-7, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22731722

RESUMEN

The aim of this study was to analyze the influence of quercetin on intestinal barrier function using the human colonic epithelial cell line HT-29/B6 and rat small and large intestine in vitro. Rat native ileum and late distal colon were incubated in Ussing chambers, and the total resistance (R(T) ) was measured, and expression of tight junction proteins was characterized in immunoblots. By simulating inflammatory conditions with TNF-α, we examined the barrier-preventive effects of quercetin. Incubation with TNF-α led to a decrease of R(T) in HT-29/B6 cell monolayers, which could be partially inhibited by quercetin. In accordance with cell culture experiments, quercetin increased mucosal resistance of rat ileum and late distal colon. Thus, barrier disturbance in late distal colon specimens induced by TNF-α and IFN-γ could be partially prevented by coincubation with quercetin. These findings demonstrate that quercetin enhances barrier function in rat small and large intestine and possesses protective effects on cytokine-induced barrier damage.


Asunto(s)
Colon/efectos de los fármacos , Quercetina/farmacología , Animales , Línea Celular , Claudinas/genética , Colon/metabolismo , Humanos , Técnicas In Vitro , Masculino , Regiones Promotoras Genéticas , Ratas , Ratas Wistar , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo
6.
PLoS One ; 6(3): e18107, 2011 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-21479205

RESUMEN

BACKGROUND AND PURPOSE: The root extract of the African Uzara plant is used in traditional medicine as anti-diarrheal drug. It is known to act via inhibition of intestinal motility, but malabsorptive or antisecretory mechanisms are unknown yet. EXPERIMENTAL APPROACH: HT-29/B6 cells and human colonic biopsies were studied in Ussing experiments in vitro. Uzara was tested on basal as well as on forskolin- or cholera toxin-induced Cl(-) secretion by measuring short-circuit current (I(SC)) and tracer fluxes of (22)Na(+) and (36)Cl(-). Para- and transcellular resistances were determined by two-path impedance spectroscopy. Enzymatic activity of the Na(+)/K(+)-ATPase and intracellular cAMP levels (ELISA) were measured. KEY RESULTS: In HT-29/B6 cells, Uzara inhibited forskolin- as well as cholera toxin-induced I(SC) within 60 minutes indicating reduced active chloride secretion. Similar results were obtained in human colonic biopsies pre-stimulated with forskolin. In HT-29/B6, the effect of Uzara on the forskolin-induced I(SC) was time- and dose-dependent. Analyses of the cellular mechanisms of this Uzara effect revealed inhibition of the Na(+)/K(+)-ATPase, a decrease in forskolin-induced cAMP production and a decrease in paracellular resistance. Tracer flux experiments indicate that the dominant effect is the inhibition of the Na(+)/K(+)-ATPase. CONCLUSION AND IMPLICATIONS: Uzara exerts anti-diarrheal effects via inhibition of active chloride secretion. This inhibition is mainly due to an inhibition of the Na(+)/K(+)-ATPase and to a lesser extent to a decrease in intracellular cAMP responses and paracellular resistance. The results imply that Uzara is suitable for treating acute secretory diarrhea.


Asunto(s)
Antidiarreicos/farmacología , Apocynaceae/química , Cloruros/metabolismo , Medicina Tradicional , Extractos Vegetales/farmacología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Adulto , Anciano , Transporte Biológico/efectos de los fármacos , Carbacol/farmacología , Toxina del Cólera/farmacología , Colforsina/farmacología , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , AMP Cíclico/metabolismo , Impedancia Eléctrica , Femenino , Células HT29 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Masculino , Persona de Mediana Edad , Nistatina/farmacología , Ouabaína/farmacología , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo
7.
J Nutr ; 141(5): 783-9, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21430244

RESUMEN

TGFß (isoforms 1-3) has barrier-protective effects in the intestine. The mechanisms involved in regulating tight junction protein expression are poorly understood. The aim of this study was to elucidate TGFß-dependent protective effects with special attention to promoter regulation of tight junction proteins using the HT-29/B6 cell model. In addition, the effects of whey protein concentrate 1 (WPC1), a natural source of TGFß in human nutrition, were examined. For this purpose, the claudin-4 promoter was cloned and tested for its activity. It exhibited transactivation in response to TGFß1, which was intensified when Smad-4 was cotransfected, indicating a Smad-4-dependent regulatory component. Shortening and mutation of the promoter altered and attenuated this effect. WPC1 induced an increase in the claudin-4 protein level and resistance of HT-29/B6 cell monolayers. Anti-TGFß(1-3) antibodies blocked these whey protein effects, suggesting that a main part of this function was mediated through TGFß. This effect was observed on intact monolayers as well as when barrier function was impaired by preexposure to IFNγ. In conclusion, TGFß1 affects claudin-4 gene expression via Smad-4-dependent and -independent transcriptional regulation, resulting in barrier protection, a cytokine effect that is also found in whey protein concentrates used in enteral nutrition.


Asunto(s)
Mucosa Intestinal/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de la Leche/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia Arriba , Animales , Bovinos , Claudina-4 , Impedancia Eléctrica , Alimentos Funcionales , Genes Reporteros , Células HT29 , Humanos , Interferón gamma/toxicidad , Proteínas de la Membrana/genética , Proteínas de la Leche/uso terapéutico , Mutagénesis Sitio-Dirigida , Mutación , Regiones Promotoras Genéticas , Sustancias Protectoras/metabolismo , Sustancias Protectoras/uso terapéutico , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes , Transducción de Señal , Proteína Smad4/genética , Proteína Smad4/metabolismo , Activación Transcripcional , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/uso terapéutico , Proteína de Suero de Leche
8.
J Cell Sci ; 123(Pt 23): 4145-55, 2010 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-21062898

RESUMEN

TNFα-mediated tight junction defects contribute to diarrhea in inflammatory bowel diseases (IBDs). In our study, the signaling pathways of the TNFα effect on barrier- or pore-forming claudins were analyzed in HT-29/B6 human colon monolayers. Berberine, a herbal therapeutic agent that has been recently established as a therapy for diabetes and hypercholesterinemia, was able to completely antagonize the TNFα-mediated barrier defects in the cell model and in rat colon. Ussing chamber experiments and two-path impedance spectroscopy revealed a decrease of paracellular resistance after TNFα to 11±4%, whereas transcellular resistance was unchanged. The permeability of the paracellular marker fluorescein was increased fourfold. Berberine alone had no effect while it fully prevented the TNFα-induced barrier defects. This effect on resistance was confirmed in rat colon. TNFα removed claudin-1 from the tight junction and increased claudin-2 expression. Berberine prevented TNFα-induced claudin-1 disassembly and upregulation of claudin-2. The effects of berberine were mimicked by genistein plus BAY11-7082, indicating that they are mediated via tyrosine kinase, pAkt and NFκB pathways. In conclusion, the anti-diarrheal effect of berberine is explained by a novel mechanism, suggesting a therapeutic approach against barrier breakdown in intestinal inflammation.


Asunto(s)
Berberina/farmacología , Proteína Oncogénica v-akt/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Uniones Estrechas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Línea Celular Tumoral , Colon/efectos de los fármacos , Colon/enzimología , Colon/metabolismo , Humanos , Técnicas In Vitro , Masculino , Proteína Oncogénica v-akt/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/genética , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/genética , Quinasa de Factor Nuclear kappa B
9.
J Comp Physiol B ; 180(4): 591-8, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20049600

RESUMEN

In tubular epithelia, barrier function varies in a segment-specific way. The aim of this study was to correlate the presence of tight junction proteins and paracellular barrier properties along rat intestine. Tissue segments of duodenum, jejunum, ileum, and colon were stripped of submucosal cell layers and mounted in Ussing chambers for impedance spectroscopy to measure epithelial resistance (R (epi)). In parallel, expression of tight junction proteins was analysed by Western blots and immune fluorescence confocal microscopy. Colon showed highest R (epi), followed by duodenum, jejunum, and ileum. In small intestine, common transepithelial resistance (R (trans) or TER) overestimated true R (epi) by approximately 60%. In colon, strongest expression of "tightening" claudins 1, 3, 4, 5, and 8 was detected. In accordance with R (epi) the most proximal of the small intestinal segments, duodenum exhibited highest expression of "tightening" claudins and lowest expression of claudins mediating permeability, namely claudin-2, -7, and -12, compared to jejunum and ileum. These results correspond to the specific role of the duodenum as the first segment facing the acidic gastric content.


Asunto(s)
Claudinas/metabolismo , Regulación de la Expresión Génica/fisiología , Mucosa Intestinal/metabolismo , Uniones Estrechas/metabolismo , Animales , Western Blotting , Impedancia Eléctrica , Electrofisiología , Microscopía Confocal , Microscopía Fluorescente , Ratas , Análisis Espectral/métodos
10.
Scand J Gastroenterol ; 44(10): 1226-35, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19658020

RESUMEN

OBJECTIVE: In Inflammatory bowel disease (IBD), elevated cytokines are responsible for disturbed intestinal transport and barrier function. The mechanisms of cytokine action have usually been studied in cell culture models only; therefore the aim of this study was to establish an in vitro model based on native intestine to analyze distinct cytokine effects on barrier function, mucosal structure, and inherent regulatory mechanisms. MATERIAL AND METHODS: Rat colon was exposed to tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) in Ussing chambers. Transepithelial resistance (R(t)) and (3)H-mannitol fluxes were measured for characterization of the paracellular pathway. Transcellular transport was analyzed by horseradish peroxidase (HRP) flux measurements. Expression and distribution of tight junction proteins were characterized in immunoblots and by means of confocal laser-scanning microscopy (LSM). RESULTS: Colonic viability could be preserved for 20 h in a specialized in vitro set-up. This was sufficient to alter mucosal architecture with crypt surface reduction. R(t) was decreased (101+/-10 versus 189+/-10 Omega x cm(2)) with a parallel increase in mannitol permeability after cytokine exposure. Tight junction proteins claudin-1, -5, -7, and occludin decreased (45+/-10%, 16+/-7%, 42+/-8%, and 42+/-13% of controls, respectively), while claudin-2 increased to 208+/-32%. Occludin and claudin-1 translocated from the plasma membrane to the cytoplasm. HRP flux increased from 0.73+/-0.09 to 8.55+/-2.92 pmol x h(-1) x cm(-2). CONCLUSIONS: A new experimental IBD model with native colon in vitro is presented. One-day exposure to TNFalpha and IFNgamma alters mucosal morphology and impairs epithelial barrier function by up-regulation of the paracellular pore-former claudin-2 and down-regulation of the barrier-builders claudin-1, -5, and -7. These alterations resemble changes seen in IBD and thus underline their prominent role in IBD pathogenicity.


Asunto(s)
Enfermedades Inflamatorias del Intestino/fisiopatología , Interferón gamma/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiopatología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Modelos Animales de Enfermedad , Técnicas In Vitro , Enfermedades Inflamatorias del Intestino/inmunología , Masculino , Ratas , Ratas Wistar
11.
Best Pract Res Clin Gastroenterol ; 23(3): 395-406, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19505667

RESUMEN

The gastrointestinal tract possesses a huge epithelial surface area and performs many different tasks. Amongst them are the digestive and absorptive functions. Disorders of intestinal absorption and secretion comprise a variety of different diseases, e.g. coeliac disease, lactase deficiency or Whipple's disease. In principle, impaired small intestinal function can occur with or without morphological alterations of the intestinal mucosa. Therefore, in the work up of a malabsorptive syndrome an early small intestinal biopsy is encouraged in conjunction with breath tests and stool analysis to guide further management. In addition, there is an array of functional tests, the clinical availability of which becomes more and more limited. In any case, early diagnosis of the underlying pathophysiology is most important, in order to initiate proper therapy. In this chapter, diagnostic procedure of malabsorption is discussed with special attention to specific disease like coeliac disease, Whipple's disease, giardiasis and short bowel syndrome. Furthermore, bacterial overgrowth, carbohydrate malabsorption and specific nutrient malabsorption (e.g. for iron or vitamins) and protein-losing enteropathy are presented with obligatory and optional tests as used in the clinical setting.


Asunto(s)
Enfermedades Gastrointestinales/diagnóstico , Intestinos/fisiopatología , Pruebas Respiratorias , Metabolismo de los Hidratos de Carbono/fisiología , Enfermedad Celíaca/diagnóstico , Enfermedad Celíaca/terapia , Intolerancia a la Fructosa/diagnóstico , Intolerancia a la Fructosa/fisiopatología , Enfermedades Gastrointestinales/fisiopatología , Enfermedades Gastrointestinales/terapia , Giardiasis/diagnóstico , Humanos , Absorción Intestinal/fisiología , Síndromes de Malabsorción/diagnóstico , Síndrome del Intestino Corto/diagnóstico , Síndrome del Intestino Corto/microbiología , Síndrome del Intestino Corto/terapia
12.
Ann N Y Acad Sci ; 1165: 211-9, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19538309

RESUMEN

Tight junctions form the paracellular barrier for ions and uncharged solutes not only in "tight" but also in "leaky" epithelia. In the premolecular era of tight junction research, this was believed to be achieved in a perfect or less perfect way, depending mainly on the amount of horizontally oriented tight junction strands. During the past decade it emerged that tight junction molecules, such as claudin-1 and many others, strengthen the barrier, while a few claudins, such as claudin-2 or -10, weaken it. This report focuses on three claudins: one channel former and two barrier builders. Claudin-2 represents the prototype of a paracellular, channel-forming, tight junction protein responsible for specific transfer of solutes across the epithelium without entering the cells. This channel is selective for small cations but nearly impermeable to anions and uncharged solutes of any size. In contrast, claudin-5, a tight junction protein typical for all endothelia but also found in some epithelia, was characterized as a potent barrier builder. Claudin-8, another barrier builder, was demonstrated to be regulated by Na(+) uptake in surface epithelial cells of human colon. Here, aldosterone enhanced Na(+) absorption by dual action: transcellularly by inducing the epithelial sodium channel and paracellularly by preventing back leakage of absorbed Na(+) by upregulating claudin-8.


Asunto(s)
Proteínas de la Membrana/metabolismo , Uniones Estrechas/metabolismo , Animales , Cationes/metabolismo , Permeabilidad de la Membrana Celular , Claudinas , Colon/metabolismo , Células Epiteliales/metabolismo , Humanos , Sodio/metabolismo , Regulación hacia Arriba
13.
Ann N Y Acad Sci ; 1165: 267-73, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19538315

RESUMEN

High dietary intake of fruits and vegetables is associated with a reduced disease risk. Therefore, clinical interest is growing in therapies based on dietary supplements and effects of food components. Immune-modulatory and barrier-protective effects have been described for the amino acid glutamine and the trace element zinc. In Caco-2-cells, zinc is necessary to maintain the expression of proteins like ZO-1 and occludin, and experimental evidence exists that glutamine has enterocyte-protective effects and modulates intestinal barrier function in stressed animals and humans. Polyunsaturated fatty acids (PUFA) improve paracellular permeability after IL-4 incubation. Enhancement of barrier properties by long-chain PUFA is discussed controversially, but a beneficial role preventing the redistribution of occludin and ZO-1 and reduction of epithelial resistance by IFN-gamma and TNF-alpha exists. In addition, a group of secondary plant compounds, the polyphenols, are supposed to be important in this respect. The flavonoid quercetin and its metabolite DHBA increased epithelial resistance of Caco-2-cells to 157 +/- 4% of control values, and DHBA up to 119 +/- 4% of control values, respectively. This is due to a 2.3 +/- 0.1-fold expression rate of the tight junction protein claudin-4.


Asunto(s)
Ácidos Grasos Insaturados/farmacología , Flavonoides/farmacología , Glutamina/farmacología , Mucosa Intestinal/metabolismo , Proteínas de la Membrana/metabolismo , Fenoles/farmacología , Células CACO-2 , Permeabilidad de la Membrana Celular , Claudina-4 , Impedancia Eléctrica , Células Epiteliales/metabolismo , Ácidos Grasos Insaturados/metabolismo , Humanos , Interferón gamma/metabolismo , Interleucina-4/metabolismo , Intestinos/efectos de los fármacos , Ocludina , Polifenoles , Quercetina/metabolismo , Quercetina/farmacología , Uniones Estrechas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo
14.
Ann N Y Acad Sci ; 1165: 294-300, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19538319

RESUMEN

The epithelium in inflamed intestinal segments of patients with Crohn's disease is characterized by a reduction of tight junction strands, strand breaks, and alterations of tight junction protein content and composition. In ulcerative colitis, epithelial leaks appear early due to micro-erosions resulting from upregulated epithelial apoptosis and in addition to a prominent increase of claudin-2. Th1-cytokine effects by interferon-gamma in combination with TNFalpha are important for epithelial damage in Crohn's disease, while interleukin-13 (IL-13) is the key effector cytokine in ulcerative colitis stimulating apoptosis and upregulation of claudin-2 expression. Focal lesions caused by apoptotic epithelial cells contribute to barrier disturbance in IBD by their own conductivity and by confluence toward apoptotic foci or erosions. Another type of intestinal barrier defect can arise from alpha-hemolysin harboring E. coli strains among the physiological flora, which can gain pathologic relevance in combination with proinflammatory cytokines under inflammatory conditions. On the other hand, intestinal barrier impairment can also result from transcellular antigen translocation via an initial endocytotic uptake into early endosomes, and this is intensified by proinflammatory cytokines as interferon-gamma and may thus play a relevant role in the onset of IBD. Taken together, barrier defects contribute to diarrhea by a leak flux mechanism (e.g., in IBD) and can cause mucosal inflammation by luminal antigen uptake. Immune regulation of epithelial functions by cytokines may cause barrier dysfunction not only by tight junction impairments but also by apoptotic leaks, transcytotic mechanisms, and mucosal gross lesions.


Asunto(s)
Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , Uniones Estrechas/metabolismo , Animales , Apoptosis , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/metabolismo , Epitelio/metabolismo , Humanos , Enfermedades Inflamatorias del Intestino/patología , Interleucina-13/metabolismo , Mucosa Intestinal/patología
15.
Biochem Biophys Res Commun ; 378(1): 45-50, 2009 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-19000657

RESUMEN

In distal colon, the limiting factor for Na(+) absorption is represented by the epithelial sodium channel (ENaC). During absorption, high transepithelial Na(+) gradients are observed. In human colon and in HT-29/B6-GR cells, we investigated whether Na(+) back-leakage is prevented by paracellular sealing. Tissues and cells were incubated with corticosteroids. Barrier properties were analyzed in electrophysiological experiments. Subsequently, analysis of ENaC and tight junction protein expression, localization, and regulation was performed. In colon, nanomolar aldosterone induced sodium absorption via ENaC. Concomitantly, paracellular (22)Na(+) permeability was reduced by half and claudin-8 within the tight junction complex was nearly doubled. Real-time PCR validated an increase of claudin-8 transcripts. Two-path impedance spectroscopy following ENaC induction in HT-29/B6-GR revealed a specific increase of paracellular resistance. These results represent an important physiological implication: Na(+) absorption is paralleled by claudin-8-mediated sealing of the paracellular barrier to prevent Na(+) back-leakage, supporting steep Na(+) gradients in distal colon.


Asunto(s)
Colon/metabolismo , Proteínas de la Membrana/metabolismo , Sodio/metabolismo , Uniones Estrechas/metabolismo , Aldosterona/metabolismo , Aldosterona/farmacología , Línea Celular , Permeabilidad de la Membrana Celular , Células Cultivadas , Claudinas , Colon/efectos de los fármacos , Humanos , Transporte Iónico , Ocludina , Transducción de Señal , Regulación hacia Arriba
16.
J Nutr ; 138(6): 1067-73, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18492835

RESUMEN

Quercetin is the most abundant flavonoid and is assumed to have positive effects on the gastrointestinal mucosa after dietary intake. The aim of the study was to analyze the influence of quercetin on intestinal barrier function using the human colonic epithelial cell line Caco-2. Transepithelial resistance (R(t)), tracer fluxes of [(3)H]-mannitol, 22Na+, and 36Cl- as well as electrogenic ion transport were determined in Ussing chambers. Expression of tight junction (TJ) proteins and mRNA was analyzed in Western blots and quantitative RT-PCR, respectively. Regulation of transcription was analyzed by reporter gene assay. Cellular distribution of TJ proteins was examined by confocal laser scanning microscopy (LSM). Apoptotic rate was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Quercetin induced a dose-dependent increase of R(t) persisting for >2 d. Daily addition of quercetin was able to perpetuate the effect, which was seen whether quercetin was added apically or to the basolateral compartment. Parallel to the R(t) increase, quercetin induced a strong increase of the TJ protein claudin-4 but not of other claudins. Confocal LSM showed a localization of claudin-4 in TJ. Apoptotic rate was not affected by quercetin. Consistent with these changes, fluxes of Na+ and Cl-, but not of mannitol, were reduced. Reporter gene assays revealed a stimulatory effect of quercetin on claudin-4 transcription. The flavonoid quercetin enhances barrier function via transcriptional expression regulation of the TJ protein claudin-4, which represents an important protective effect of this food component against barrier disturbance in intestinal inflammation.


Asunto(s)
Antioxidantes/farmacología , Células Epiteliales/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Quercetina/farmacología , Células CACO-2 , Comunicación Celular , Claudina-4 , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mucosa Intestinal/metabolismo , Proteínas de la Membrana/genética , Factor de Transcripción STAT1
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