Association of alpha-dystrobrevin with reorganizing tight junctions.

Alpha-dystrobrevin (alpha-DB) has been described primarily as a cytoplasmic component of the dystrophin-glycoprotein complex in skeletal muscle cells. Isoforms of alpha-DB show different localization in cells and tissues; at basolateral membranes in epithelial cells, dystrobrevins mediate contact with the extracellular matrix, peripheral and transmembrane proteins and the filamentous actin cytoskeleton. Beside their structural role, alpha-DBs are assumed to be important in cell signalling and cell differentiation. We have primarily assessed the role of alpha-DB in two epithelial cell lines (MDCK I, HT 29), which represent different developmental stages and exhibit distinct permeability characteristics. Using a polyclonal anti-alpha-DB antibody, we have investigated its expression, localization and association with tight junction (TJ)- associated proteins (ZO-1, occludin) before and after protein kinase C (PKC) activation with phorbol myristate acetate. Distinct subsets of alpha-DB isoforms were detected in the two cell lines by immunoblotting. In both cell lines there was submembranous localization of alpha-DB both apically and basolaterally, shown with confocal imaging. PKC activation caused a reorganization of TJ, which was parallel to increased localization of alpha-DB to TJ areas, most pronounced in MDCK I cells. Moreover, actin and ZO-1 co-immunoprecipitated with a-DB, as displayed with immunoblotting. Our findings suggest that a-dystrobrevin specifically is associated with the tight junctions during their reorganization.

Altered impedance during pigment aggregation in Xenopus laevis melanophores.

Melanophores are dark-brown pigment cells located in the skin of amphibia, fish and many invertebrates. The skin colour of these organisms is regulated by the translocation of pigment organelles, and the pigment distribution can be altered by external stimuli. The ability to change colour in response to stimuli makes these cells of interest for biosensing applications. It was investigated whether pigment aggregation in Xenopus laevis melanophores can be detected by impedance measurements performed in transparent microvials. The results show that cell attachment, cell spreading and pigment aggregation all resulted in impedance changes, seen particularly at the highest frequency tested (10 kHz). The mechanisms behind the impedance changes were investigated by the addition of latrunculin or melatonin, both of which cause pigment aggregation. The latrunculin-induced aggregation was associated with cell area decrease and filamentous actin (F-actin) breakdown, processes that can influence the impedance. Lack of F-actin breakdown and an increase in cell area during melatonin-induced aggregation suggest that some other intracellular process also contributes to the impedance decrease seen for melatonin. It was shown that impedance measurements reflect not only cell attachment and cell spreading, but also intracellular events.

Augmented increase in tight junction permeability by luminal stimuli in the non-inflamed ileum of Crohn's disease.

BACKGROUND: Crohn's disease is associated with deranged intestinal permeability in vivo, suggesting dysfunction of tight junctions. The luminal contents are important for development of neoinflammation following resection. Regulation of tight junctions by luminal factors has not previously been studied in Crohn's disease. AIMS: The aim of the study was to investigate the effects of a luminal stimulus, known to affect tight junctions, on the distal ileum in patients with Crohn's disease. PATIENTS: Surgical specimens from the distal ileum of patients with Crohn's disease (n=12) were studied, and ileal specimens from colon cancer patients (n=13) served as controls. METHODS: Mucosal permeability to 51Cr-EDTA and electrical resistance were studied in Ussing chambers during luminal exposure to sodium caprate (a constituent of milk fat, affecting tight junctions) or to buffer only. The mechanisms involved were studied by mucosal ATP levels, and by electron and confocal microscopy. RESULTS: Baseline permeability was the same in non-inflamed ileum of Crohn's disease and controls. Sodium caprate induced a rapid increase in paracellular permeability--that is, increased permeation of 51Cr-EDTA and decreased electrical resistance--which was more pronounced in non-inflamed ileum of Crohn's disease, and electron microscopy showed dilatations within the tight junctions. Moreover, sodium caprate induced disassembly of perijunctional filamentous actin was more pronounced in Crohn's disease mucosa. Mucosal permeability changes were accompanied by mitochondrial swelling and a fall in epithelial ATP content, suggesting uncoupling of oxidative phosphorylation. CONCLUSIONS: The tight junctions in the non-inflamed distal ileum of Crohn's disease were more reactive to luminal stimuli, possibly mediated via disturbed cytoskeletal contractility. This could contribute to the development of mucosal neoinflammation in Crohn's disease.

Localization of the insulin receptor in caveolae of adipocyte plasma membrane.

The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with beta-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. beta-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.

Epithelial permeability to proteins in the noninflamed ileum of Crohn's disease?

BACKGROUND & AIMS: Crohn's disease (CD) is associated with a disturbed intestinal barrier. Permeability studies have focused on inert molecules, but little is known about transepithelial transport of macromolecules with antigenic potential in humans. The aim of this study was to quantify permeation and to characterize passage routes for macromolecules in ileal mucosa in CD. METHODS: Noninflamed and inflamed ileal mucosa specimens from patients with CD (n = 12) and ileal specimens from patients with colon cancer (n = 7) were studied regarding transmucosal permeation of ovalbumin, dextran (mol wt, 40,000), and 51Cr-EDTA for 90 minutes in vitro in Ussing chambers. Transepithelial passage routes for fluorescent ovalbumin and dextran 40,000 were investigated by confocal microscopy. RESULTS: Noninflamed ileum from CD patients showed increased permeation of ovalbumin compared with ileum from colon cancer patients (P < 0.05). Dextran permeation was equal in the three groups, whereas 51Cr-EDTA permeability was increased in inflamed ileum. Ovalbumin passed both transcellularly and paracellularly, but dextran followed a strictly paracellular route. Both markers were subsequently endocytosed by cells of the lamina propria. CONCLUSIONS: Noninflamed ileal mucosa from patients with CD shows increased epithelial permeability to ovalbumin, probably by augmented transcytosis. This increase in antigen load to the lamina propria could be an initiating pathogenic event in CD.