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PubMedDate not supplied

Distinct effects of protein kinase C on the barrier function at different developmental stages.

We show here, that activation of protein kinase C by the phorbol ester PMA improves barrier function in colon carcinoma (HT 29) cells. By contrast, in canine kidney (MDCK I) cells it caused increased permeability and opening of tight junctions; the latter has also been noticed in other studies. Thus, with PMA confluent HT 29 cells responded with a reduced passage of 330 kDa sodium fluorescein, increased transepithelial electrical resistance, and a change in the cell shape of the HT 29 cells from an irregular to a regular, hexagonal form. Confocal imaging revealed parallel distinct changes in the staining of occludin and caludin-1, viz. a translocation from cytoplasmic clusters to apical cell-cell contacts. Interestingly, in both cell lines protein kinase A activation caused a decreased in the threonine phosphorylation of occludin that correlated with tight junction assembly in HT 29 cells and tight junction disassembly in MDCK I cells. We conclude that protein kinase C regulation of the epithelial barrier involves specific molecular mechanisms and achieves distinct effects at different developmental stages.

Animals↗

PubMedDate not supplied

Ultrastructural characteristics of endothelial permeability in chronic hypertension.

This study examined characteristics of paracellular and pinocytotic permeability pathways across the middle cerebral artery endothelium of 12- to 16- month-old spontaneously hypertensive rats (SHR). Interendothelial junctions in SHR, like those of age-matched Wistar-Kyoto controls, were impermeable to lanthanum and horseradish peroxidase (HRP) tracers. Freeze-fracture preparations revealed that interendothelial junctions of chronically hypertensive rats are characterized by a twofold increase over controls in the number of tight junctional strands and the mean apical-basal depth. It is believed that this tight junctional hypertrophy may function to increase adhesive forces between neighboring endothelial cells, and may play a role inthe prevention of hypertension-induced paracellular permeability increases. Morphological and tracer studies of pinocytotic pathways indicated that, it the middle cerebral artery, endothelial vesicular transport activity is not increased during chronic hypertension. No evidence was found to indicate the presence of transendothelial permeability channels across control or hypertensive arterial endothelium. Thus, increased transendothelial permeability, commonly observed in acute hypertension, does not appear to occur during chronic hypertension, at least in the middle cerebral artery. Our findings suggest that the arterial endothelium may undergo structural (tight junctional) adaptation in response to prolonged hypertension.

Animals↗

PubMed1975

Nitrogenous cations as probes of permeation channels.

Nitrogenous cations may provide information-rich probes of cation-selective channels. Hence, for 52 nitrogenous cations we have used dilution potentials and biionic potentials to measure relative permeability coefficients (P's) across gallbladder epithelia of frog and rabbit, and have also determined the free-solution mobilities. Measured P's of most cations are uninfluenced by the presence of the netral form. The main permeation pathway for most hydrophilic cations is across the tight junctions. P's decrease with molecular size and increase with number of donor protons available for hydrogen-bond formation. Selectivity isotherms have been constructed from variation in P's due to pH or due to differences among individual animals. Both types of variation are consistent with the pattern expected from variation in electrostatic field strength of cation-binding sites. The isotherms permit P's to be re-expressed in a way that largely eliminates effects of species differences in field strength. Remaining species differences in P's are well fitted by a model of steric restriction, provided that one takes into account the effect of hydrogen bonding on molecular size. Rabbit gallbladder behaves as if it has narrower permeation channels than frog gallbladder. After correction for these steric effects, P is found to increase with number of donor protons nH up to four protons, with a steeper slope in rabbit than in frog gallbladder, but is independent of nH from four to at least nine. Two groups of cations appear to permeate significantly via pathways other than tight junctions: oxycations, via polar pathways in epithelial cell membranes of rabbit but not frog gallbladder; and lipid-soluble cations, via membrane lipid. The results suggest that the cation-binding sites of gallbladder tight junction are acidic proton-acceptors that discriminate more sharply among proton donors than does water. Proton-rich solutes tend to be more permeant for two reasons: stronger binding energies to membrane proton-acceptor sites, and smaller effective size in a proton-acceptor environment. As deduced from comparisons of nitrogenous cation selectivity patterns, the permeation channel through gallbladder tight junction differs from nerve's sodium channel and artificial carriers and channels in its higher hydration and lower range of selectivity. Based on the steric analysis of nitrogenous cation permeation, one can correct alkali cation permeability coefficients for the effect of steric restriction.

Amines↗

PubMed1997

Organization of cell junctions and cytoskeleton in the reticular lamina in normal and ototoxically damaged organ of Corti.

The reticular lamina creates an ion barrier, withstands mechanical stress in the organ of Corti and is able to maintain its integrity during and after severe hair cell loss. Tight junctions maintain the ionic gradient whereas adherens junctions and the cytoskeleton are responsible for the integrity and mechanical resistance of tissues. In this study we used immunofluorescence and electron microscopy to examine the distribution of proteins of tight junctions (cingulin), adherens junctions (E-cadherin, alpha- and beta-catenin) and the cytoskeleton (actin, cytokeratin and tubulin) in whole-mounts of the normal and ototoxically damaged organ of Corti. In normal ears the proteins of adherens junctions were found in all cell types of the reticular lamina. We now demonstrate that all cells forming the reticular lamina partially overlap each other organizing extensive cell contacts with a complex three-dimensional shape. During scar formation, the tight junctions as well as adherens junctions between hair and supporting cells appeared in two distinct focal planes, which could help to preserve the ionic barrier and tissue integrity during hair cell degeneration. During scar formation all cytoskeletal structures in the reticular lamina were reorganized in a specific spatio-temporal pattern. We present a three-dimensional model of cell contact organization in the reticular lamina of normal ears and during scar formation.

Actins↗

PubMed2003

Do cell culture conditions influence the carrier-mediated transport of peptides in Caco-2 cell monolayers?

Despite the fact that different laboratories have reported large differences in permeability for actively transported substrates, Caco-2 cell monolayers are widely used as in vitro model to study small intestinal drug transport. Therefore, we investigated the effect of cell culture conditions, such as time in culture, membrane support, seeding density and supplements to the medium, on the morphology, the formation of tight junctions, as well as the expression of two peptide transporters (PepT1, HPT1) and the efflux pump, P-glycoprotein (Pgp), in Caco-2 cell monolayers. Tight junction formation was assessed by transepithelial electrical resistance measurements; multi-cell layer formation by confocal laser scanning microscopy, the expression of transporters by RT-PCR and the permeability of the PepT1 substrate, cephradine. Both morphology and the expression of carrier-mediated transporters, varied strongly as a function of culture conditions. An increase of differentiation, as documented by tight, homogeneous cell monolayer formation displaying a strong expression of all carrier-mediated transporters, was found up to 3 weeks post seeding. One week later, multi-layer structures were observed and the expression of Pgp decreased. Polyester and polyethylene terephthalate membrane supports decreased the paracellular transport rates substantially, while collagen-coating of PC inserts showed no influence on the morphology and even increased carrier-mediated transporter expression. An average seeding density of 6x10(4) cells/cm(2) seemed to be most favorable, since lower seeding densities led to thin monolayers with altered tight junctions and higher seeding densities to the formation of multilayers. In summary, the expression of carrier-mediated transporters was strongly affected by the culture conditions. The full differentiation was reached after 21 days on collagen-coated polycarbonate inserts at an initial seeding density of 6x10(4) cells/cm(2).

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Uncoupling of gate and fence functions of MDCK cells by the actin-depolymerizing reagent mycalolide B.

The tight junction serves as a paracellular gate to seal the paracellular space of apposing cells and as a molecular fence to prevent diffusion of membrane proteins and lipids in epithelial cells. Although involvement of the actin cytoskeleton has been considered to be important in these two functions, it remains to be elucidated whether both functions are regulated in a coupled manner or differentially by actin. Treatment of highly polarized MDCK cells with mycalolide B (MB), a recently developed actin-depolymerizing reagent, induced a decrease of transepithelial resistance in a dose- and time-dependent manner with reversibility when the reagent was washed out. Changes in cytoskeletal actin, such as a reduction of cortical actin, irregularity of stress fibers, and punctated actin aggregates, were observed after MB treatment. However, the fence function, as studied by diffusion of apically labeled sphingomyelin/BSA complex, remained intact in the MB-treated MDCK cells. Localization of junctional molecules and apical marker proteins such as E-cadherin, ZO-1, and 114-kDa protein was shown to be unaffected. Furthermore, freeze-fracture study showed apparent tight junction strands. Collectively, MB treatment abolished the paracellular gate but not the fence function of MDCK cells, suggesting that cytoskeletal actin may play differential roles in the gate and fence functions of the tight junction.

Actins↗

PubMed2006

Proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma modulate epithelial barrier function in Madin-Darby canine kidney cells through mitogen activated protein kinase signaling.

BACKGROUND: The tight junction is a dynamic structure that is regulated by a number of cellular signaling processes. Occludin, claudin-1, claudin-2 and claudin-3 are integral membrane proteins found in the tight junction of MDCK cells. These proteins are restricted to this region of the membrane by a complex array of intracellular proteins which are tethered to the cytoskeleton. Alteration of these tight junction protein complexes during pathological events leads to impaired epithelial barrier function that perturbs water and electrolyte homeostasis. We examined MDCK cell barrier function in response to challenge by the proinflammatory cytokines tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma). RESULTS: Exposure of MDCK cells to TNFalpha/IFNgamma resulted in a marked sustained elevation of transepithelial electrical resistance (TER) as well as elevated paracellular permeability. We demonstrate that the combination of TNFalpha/IFNgamma at doses used in this study do not significantly induce MDCK cell apoptosis. We observed significant alterations in occludin, claudin-1 and claudin-2 protein expression, junctional localization and substantial cytoskeletal reorganization. Pharmacological inhibition of ERK1/2 and p38 signaling blocked the deleterious effects of the proinflammatory cytokines on barrier function. CONCLUSION: These data strongly suggest that downstream effectors of MAP kinase signaling pathways mediate the TNFalpha/IFNgamma-induced junctional reorganization that modulates MDCK cell barrier function.

Animals↗

PubMed1999

Binding and uptake of cationic lipid:pDNA complexes by polarized airway epithelial cells.

To better understand the barriers associated with cationic lipid-mediated gene transfer to polarized epithelial cells, Fischer rat thyroid (FRT) cells and polarized normal human bronchial epithelial (NHBE) cells grown on filter supports at an air-liquid interface were used to study the binding and uptake of cationic lipid:plasmid DNA (pDNA) complexes. The efficiencies of binding and uptake of cationic lipid:pDNA complexes by these cell systems were monitored using fluorescence microscopy of fluorescently tagged lipid or pDNA probes. Fluorescent probe bound to the cell surface was differentiated from internalized probe by adding trypan blue, which quenched the fluorescence of bound but not internalized probes. For proliferating cells, binding and internalization of the cationic lipid:pDNA complexes were determined to be efficient. In contrast, little binding or internalization of the complexes was observed using polarized epithelial cells. However, after aspirating a small area of cells from the filter support, virtually all of the cells adjoining this newly formed edge bound and internalized the cationic lipid:pDNA complexes. To determine if their uptake in edge cells was related to the ability of the complexes to access the basolateral membranes of these cells, the binding and uptake of complexes was monitored in polarized NHBE cells that had been pretreated with EGTA or Ca2+-free media, strategies known to disrupt tight junctions. Cells treated in this manner bound and internalized cationic lipid:pDNA complexes efficiently and also expressed significant levels of transgene product. Control cells with intact tight junctions neither bound complexes nor expressed significant transgene product. These data confirm and extend earlier observations that the polarized apical membranes of airway epithelial cells are resistant to transfection by lipid:pDNA complexes. Further, in contrast to previous studies that have shown the entry step of complexes is not an important barrier for COS and HeLa cells, binding and entry of complexes in polarized NHBE cells appear to be rate limiting. These findings suggest that strategies designed to open the tight junctions of polarized epithelial cells may improve gene delivery to these cells for diseases such as cystic fibrosis (CF).

Alkaline Phosphatase↗

PubMed1984

Structural features of and cholesterol distribution in M-cell membranes in guinea pig, rat, and mouse Peyer's patches.

Evidence suggests that M cells, which are antigen-sampling epithelial cells that overlie the domes of Peyer's patches, have an apical plasma membrane that differs from that of absorptive cells. We examined the structural features of rat, mouse, and guinea pig M-cell apical membranes and compared them with those of dome and villus absorptive cell apical membranes using electron microscopy of thin sections and freeze-fracture replicas. We also determined the distribution of morphologically detectable cholesterol in M-cell plasma and intracellular membranes in dome epithelium exposed to the polyene antibiotic, filipin. The areal density of P-face intramembrane particles was significantly less on M-cell microvilli than on microvilli of dome absorptive cells and villus absorptive cells. Areal densities of E-face intramembrane particles were similar on M-cell and absorptive-cell microvilli. M-cell apical plasma membranes were rich in cholesterol, displaying numerous filipin-induced membrane lesions in thin sections and freeze-fracture replicas. In contrast, apical membrane endocytic pits and coated vesicles in M cells failed to show filipin-induced membrane lesions. These findings suggest that, compared with the apical membranes of absorptive cells, those of M cells have a low protein to lipid ratio and an abundance of morphologically detectable cholesterol except in domains involved in endocytosis. Additionally, a subpopulation of dome epithelial cells displayed distinctive tight junctions with high strand counts and unusual depth. Although the cell type associated with these modified tight junctions could not be identified with certainty, their interspecies frequency paralleled the interspecies frequency of M cells in dome epithelium. These expanded tight junctions may result from physical tension caused by epithelial shape changes induced by intraepithelial lymphoid trafficking or, alternatively, may help buttress M cells that have attenuated cytoplasmic processes due to the presence of central hollows.

Animals↗

PubMed1972

Permeable junctional complexes. The movement of lanthanum across rabbit gallbladder and intestine.

Ionic lanthanum has been used to study transepithelial ion permeation in in vitro rabbit gallbladder and intestine (ileum) by adding 1 mM La(3+) to only the mucosal bathing solution. Transepithelial fluid transport electrical potential differences (p.d.), and resistances were measured. During La(3+) treatment the gallbladder's rate of active solute-coupled fluid transport remained constant, the resistance increased, and the 2:1 NaCl diffusion p.d. decreased. Mucosa-to-serosa fluxes of (140)La(3+) were measured and indicate a finite permeability of the gallbladder to La(3+). La(3+) also increased the transepithelial resistance and p d. of ileum. Electron microscopic examination of La(3+)-treated gallbladder showed: (a) good preservation of the fine structure, (b) electron-opaque lanthanum precipitates in almost every lateral intercellular space, most frequently near the apical end of the lateral spaces close to or within the junctional complex, (c) lanthanum among the subjacent muscle and connective tissue layers, and (d) lanthanum filling almost the entire length of so-called "tight" junctions. No observations were made which unequivocally showed the penetration of lanthanum into the gallbladder cells. Electron micrographs of similar La(3+)-treated ilea showed lanthanum deposits penetrating the junctional complexes. These results coupled with other physiological studies indicate that the low resistance pathway for transepithelial ion permeation in gallbladder and ileum is through the tight junctions A division of salt-transporting epithelia into two main groups, those with "leaky" junctional complexes and those with tight junctional complexes, has been proposed.

Animals↗

PubMed1981

Endocytosis of cell-cell junctions and spontaneous cell disaggregation in a cultured human ovarian adenocarcinoma. (COLO 316).

Although cultured COLO 316 human ovarian adenocarcinoma cells are joined by extensive tight junctions and numerous demosomes in confluent monolayers, viable cells may be spontaneously released into the nutrient medium. Intracytoplasmic vesicles containing tight junctional and desmosomal elements were identified in freeze-fracture and thin section preparations of the released cells and some vesicles exhibited structural signs of degradation. Possible mechanisms for tight junctional and desmosomal interiorization and the possible relationship between junctional interiorization and certain malignant behaviors are discussed.

Adenocarcinoma↗

PubMed1988

Transepithelial migration of eosinophils in experimental nasal allergy in guinea pigs.

We examined eosinophil-migration through the nasal epithelium into nasal cavity in a guinea pig model exhibiting 8-day passive cutaneous anaphylaxis antibody-dependent nasal allergy. Transmission electron microscopic observation of this process revealed that eosinophils traversed the epithelium through the intercellular space and split the tight-junctions of epithelial lining cells. Freeze-fracture studies of this process showed that the morphology of tight-junction was not changed after eosinophil-migration. These observations may indicate that tight-junctions close after eosinophil-migration.

Animals↗

PubMed2004

Occludin expression decreases with the progression of human endometrial carcinoma.

The tight junctions of the glandular epithelium are crucial for the maintenance of cell polarity, separating the plasma membrane into apical and basolateral domains. Thus abnormalities of the tight junctions may result in the structural disturbances of glandular epithelial neoplasia. In this study we introduced an anti-occludin monoclonal antibody for semiquantitative assay of the occludin expression in tissue sections of human normal and neoplastic endometrial epithelia using the Adobe Photoshop and NIH Image programs. Normal endometrial glands and samples of endometrial hyperplasia and endometrioid carcinoma grade 1 fully expressed occludin at the apical cell border. In endometrioid carcinomas grades 2 and 3, however, occludin disappeared in solid areas of the carcinomatous tissues. Occludin was also found at the apical borders of the cancer cells that formed glandular structures. Occludin expression decreased progressively in parallel with the increase in carcinoma grade, and the decreased occludin expression correlated with myometrial invasion and lymph node metastasis. These results suggest that the loss of tight junctions has a close relationship with structural atypia in the progression of human endometrial carcinomas and their malignant potential.

Adenocarcinoma↗

PubMed1984

Astroglial-axonal interrelationship during regeneration of the optic nerve in goldfish. A freeze-fracture study.

The cytoplasmic membranes of astrocytes in the regenerating optic nerve of the goldfish were investigated by means of freeze-fracture technique. In contrast to avian and mammalian astrocytes the astrocytes in the fish white matter do not reveal the characteristic orthogonal particle assemblies. Whereas astrocytes hitherto were believed to be interconnected mainly by gap junctions, astrocytes in the optic nerve of the goldfish are additionally interconnected by prominent desmosomes and large tight junctions. The tight junctional strands are frequently intercalated by gap junctions. Astroglial membrane domains, which are directly juxtaposed to untreated myelinated optic fibers, are longitudinally deformed. The distance of these longitudinal grooves corresponds to the caliber of fibers associated with them. The intramembranous particles in these membranous domains are distributed randomly. During optic nerve regeneration, membranes of astrocytes associated with regenerating, still unmyelinated fibers are either longitudinally deformed or smooth. If deformed according to the axonal course the membrane shows a non-random distribution of intramembranous particles; if smooth, the membrane shows a random distribution of its particles. The non-random particle distribution in the astrocytic P-face is characterized by an impoverishment of particles along the concave groove and an enrichment of particles along the convexe bulge which corresponds to the wedge between two associated axons. This phenomenon is restricted to the regeneration period and disappears some weeks after recovery of vision. Then the astrocytic membranes which are associated to regenerated fibers are undulated nevertheless revealing a random particle distribution. It is suggested, that these findings reflect a glial-neuronal interrelationship characterized by the neuron as inductor and the glial cell as responder to axonal signals. The astroglial response to axonal signals would include the formation of attractive glial channels. The working hypothesis is proposed that the presence of orthogonal particle assemblies could contribute to a disturbed astroglial response in the mammalian central nervous system.

Animals↗

PubMed2006

Dimerization of the scaffolding protein ZO-1 through the second PDZ domain.

The tight junction protein ZO-1 is known to link the transmembrane proteins occludin, claudins, and JAMs to many cytoplasmic proteins and the actin cytoskeleton. Although specific roles for ZO-1 at the tight junction are unknown, it is widely assumed that ZO-1, together with its homologs ZO-2 and ZO-3, serves as a platform to scaffold various transmembrane and cytoplasmic tight junction proteins. Thus the manner in which the zonula occludens (ZO) proteins multimerize has implications for the protein networks they can coordinate. The purpose of our study was to determine whether ZO-1 forms homodimers and to determine the protein interaction region. Using laser light scattering and analytical centrifugation, we show that protein sequences corresponding to the NH(2)-terminal half of ZO-1 form stable homodimers with a submicromolar equilibrium dissociation constant. Analysis of the molecular weight of different truncated forms of ZO-1 revealed that the second PDZ domain is both necessary and sufficient for dimerization. This interaction does not use the beta-finger motif described for other PDZ dimers. Furthermore, ZO-1 does not dimerize via an Src homology 3 to Guk domain interaction as was demonstrated previously for MAGUKs, like PSD-95. Results from immunoprecipitation experiments with polarized Madin-Darby canine kidney epithelial cells stably transfected with full-length GFP-ZO-1 indicate that a substantial portion of ZO-1 forms homodimers in vivo. As described previously, ZO-1 also forms heterodimers with ZO-2 and ZO-3. We conclude that the dimerization of ZO proteins is unlike that of other MAGUKs and that the previously unrecognized ZO-1 homodimers may allow formation of protein networks distinct from those of heterodimers with ZO-2 and ZO-3.

Animals↗

PubMed1993

Effects of modulated and continuous microwave irradiation on pyroantimonate precipitable calcium content in junctional complex of mouse small intestine.

The pyroantimonate precipitable calcium content of intestinal epithelial cells was investigated in mice following total body irradiation with 2450 MHz continuous and low frequency (16 Hz) square modulated waves. In the control animals the reaction products appeared in the intercellular space of adjacent cells including intermediate junctions and desmosomes and were absent in the area of tight junctions. Immediately after low frequency modulated microwave irradiation at 0.5 and 1mW/cm2 power densities, a rapid distribution of pyroantimonate precipitable calcium content was observed. The pyroantimonate deposits were located on the cytoplasmic side of lateral membrane, in the area of junctional complex, including tight junction, and in other parts of lateral plasma membrane. These changes were reversible and 24 hours after the irradiation the distribution of pyroantimonate deposits was similar to the control. Continuous waves with same energy not altered the distribution of precipitable calcium. We conclude the low frequency modulated microwave irradiation can modify the calcium distribution without heat effects.

Animals↗

PubMed1977

Ultrastructure and distribution of intercellular junctions in corneal endothelium.

The intercellular junctions of the corneal endothelium has been studied in rabbit, monkey and human eyes. A union of apposing outer leaflets (tight junction) is usually found near the apical end of the intercellular clefts. However, evidence from lanthanum tracer studies indicates that the tight junctions do not seal the intercellular clefts completely. In addition to tight junctions, the presence of gap junctions or nexuses is demonstrated in all three species studied.

Animals↗

PubMed2000

Regulation of glucose transporters during development of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) separates the outer retina from its blood supply. To satisfy the retina's large requirement for glucose, the RPE expresses high levels of glucose transporters. In most rat cells, the transporter GLUT3 provides a basal level of transport, but the expression of GLUT1 can be regulated. The opposite is true in chicken (P. Wagstaff, H.Y. Kang, D. Mylott, P.J. Robbins, M.K. White, Characterization of the avian GLUT1 glucose transporter: differential regulation of GLUT1 and GLUT3 in chicken embryo fibroblasts, Mol. Biol. Cell 6 (1995) 1575-1589). We examined chick RPE to determine which isoform is regulated during development, and if the neural retina regulates GLUT expression. By RT-PCR, RPE expressed GLUT1 and GLUT3, but not GLUT2. Only the level of GLUT1 increased between E5 and E18. A corresponding increase in GLUT1 protein was observed by immunoblotting. Most of the increase occurred between E14 and E18, which corresponds to the late stage of tight junction development. A culture model of development was used to examine the intermediate phase, which extends from E7 to E14. While medium conditioned by the neural retina decreased paracellular diffusion across the tight junctions, it increased diffusion through the glucose transporters. Unlike mammals, chick upregulates different isoforms in quiescent RPE and proliferating fibroblasts. Further, the upregulation of glucose transport is coordinated with the development of tight junctions in the blood-retinal barrier.

Animals↗