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PubMed1999

Use of Ca2+ modulation to evaluate biliary excretion in sandwich-cultured rat hepatocytes.

Previous work in our laboratory has indicated that biliary excretion of a substrate in sandwich-cultured hepatocytes can be quantitated by measurement of substrate accumulation in the presence and absence of extracellular Ca2+. The present study was designed to examine the effects of Ca2+ on taurocholate accumulation and tight junction integrity in cultured hepatocytes. Kinetic modeling was used to characterize taurocholate disposition in the hepatocyte monolayers in the presence and absence of extracellular Ca2+. The accumulation of taurocholate in freshly isolated hepatocytes, which lack an intact canalicular network, was the same in the presence and absence of extracellular Ca2+. Electron microscopy studies showed that Ca2+ depletion increased the permeability of the tight junctions to ruthenium red, demonstrating that tight junctions were the major diffusional barrier between the canalicular lumen and the extracellular space. Cell morphology and substrate accumulation studies in the monolayers indicated that Ca2+ depletion disrupted the tight junctions in 1 to 2 min. The integrity of the disrupted tight junctions was not re-established completely after reincubation in the presence of Ca2+ for 1 h. The accumulation of taurocholate was described best by a two-compartment model (cytosol and bile) with Michaelis-Menten kinetics for both uptake and biliary excretion. In summary, Ca2+ depletion does not alter hepatocyte transport properties of taurocholate. Ca2+ modulation may be a useful approach to study biliary excretion of substrates in sandwich-cultured hepatocytes.

Animals↗
PubMed1986

[Ultrastructure of capillary permeability in human brain tumors. 2: Mechanisms of contrast enhancement in gliomas].

In order to elucidate the mechanism of contrast enhancement on computerized tomography (CT), the alteration of capillary permeability was studied in 3 cases of medulloblastoma, 2 cases of ependymoma, 5 cases of glioblastoma and 5 cases of astrocytoma. The surgical specimens were studied with conventional ultrathin section and freeze-fracture replica techniques. Contrast enhancement on CT scan defined in order of medulloblastoma, ependymoma, glioblastoma and astrocytoma. In the medulloblastomas and glioblastomas, the cell junctions of the capillaries were short, elongated, and, in fact open. Other capillary abnormalities included endothelial hyperplasia with extensive vesicular formation, surface infolding of endothelial cells, irregularity of the basal lamina, and a large extravascular space. Tight junctions were seen as one or twe strands. These tight junctions presented irregular lining of intramembranous particles. In the astrocytomas, the blood vessels appeared relatively normal, and the tight junction were seen as networks of seven strands. The particles lines were not disrupted. In the ependymomas, the tight junctions in one area were seen as a network of six strands, but in the other areas as three strands, and the fenestrae were observed in the replicas but not confirmed in the ultrathin sections. The conclusion drawn from this study is that osmotic opening of the tight junctions that have fewer strands play an important role in the marked contrast enhancement of the gliomas, in addition to transcellular transport of increased pinocytotic vesicles and fenestrations. The irregular basal lamina and large perivascular space also increase extravasation of contrast medium.

Adult↗
PubMed1998

Structural and functional aspects of intercellular junctions in vascular endothelium.

Cell-to-cell-junctions of endothelial cells are specialized and differentiated areas of the plasma membrane. The main functions include the separation of the intravascular and extravascular compartments, the mechanical connection of the cells, and the maintenance of the cell polarity. Although a wide heterogeneity of endothelial cell-to-cell junctions exists in situ, they should be considered in general as adherens type junctions in which gap and tight junctions are morphologically inserted. Under certain pathological conditions, such as wound healing, angiogenesis and many types of inflammation, the interendothelial junctions have to be dissociated and reorganized in which proteins of the junctions are crucially involved. These important mechanisms predict a sophisticated regulation of junctional proteins. The present paper describes the organization and functional aspects of the occludin/ZO-1 complex typically found in tight junctions, the cadherin/catenin complex of the adherens junctions and the connection of these protein complexes to the dense peripheral band via actin filaments. In addition, special attention has been drawn on the function of junction-associated proteins with respect to their role under fluid shear stress and interendothelial gap formation during inflammation.

Animals↗
PubMed1982

Further observations on the Sertoli cell junctions of the mouse testis after metal contract freeze-fracture, and comparisons with cellular junctions of other epithelial cells.

Using a metal contact freezing methods, the junctional complexes of Sertoli cells and other epithelial cells (intestine, liver, and epididymis) in the mouse were studied by freeze-fracture in unfixed and in aldehyde-fixed specimens. The tight junctions of the Sertoli cells without fixation consists of discrete rows of particles located preferentially on the E face. Continuous furrows with or without tight-junctional particles are found on both fractured faces. The majority of the tight-junctional particles of th Sertoli cells are situated at one side of the furrow rather than in the center. The "aisle" configuration of the gap junction is seen in developing Sertoli cells prepared without fixation. Tight junctions of epithelial cells other than Sertoli cells consists of discrete rows of particles on the E face and furrows on the P face when prepared without fixation. The tight and gap junctions of the Sertoli cells are not affected by fixation, but the tight junctions of other types of epithelial cells are effected as previously suggested. A distribution pattern of the gap-junctional particles of the Sertoli cells and of other cells shows a pleomorphism. Changes in gap junctions produced by fixation could not be detected clearly. Characteristics of the Sertoli-cell junctions are discussed. Desmosomes between the intestinal epithelial cells without fixation are presented.

Animals↗
PubMed1975

[Junction zones of luteal cells in the luteotrophic phase in humans].

Three different types of junctions were revealed studying human corpus luteum during luteotrophic phasis : tight-junctions, gap-junctions and septate junctions. Tight-junctions were characterized by a discontinuous central leaflet formed by osmiophilous punctiform components separated by osmiophobic spaces. Lanthanum nitrate permeability of these junctions lead us to think that they may be leaky. They were only a very few gap-junctions, they were found to be as classically described, made up of seven leaflets. Septate junctions were about 250 A long, some septa were arrayed in a discordely fashion and were separated by lanthanum nitrate permeable spaces. The location of these different junctions was determined with accuracy: tight and gap junctions were located on the lateral faces and around cellular interdigitations, septate junctions were shown to bind either the microvillosities of a same cell or the microvillosities of two bordering cells. The fonctionnal signification of these junctions is discussed. Les auteurs remercient vivement M. le Professeur SOUTOUL et M. le Docteur LANSAC du Service de Gynécologie-Obstétrique (C.H.U. Bretonneau) pour leur aimable collaboration.

Adult↗
PubMed1989

Morphological changes in the junctional complex of cells in the arachnoid layer of the rat after cold injury.

The junctional complexes of cells in the outer arachnoid layer overlying the cerebral cortex of 2-week-old rats were examined with freeze-fracture electron microscopy up to 60 min after transcranial cold injury to the dorsal surface of the brain. Within 30 min after injury, areas of gap and tight junctions with morphological features characteristic of junction formation and/or junction disruption were found scattered among normal junctional complexes in some arachnoid cells. Within 60 min after injury, tight junctions with features typical of less leaky zonulae occludentes were present in all arachnoid cells examined. These morphological features include increases in the number of tight junctional strands and the number of strand-to-strand anastomoses. Gap junctions were interspersed among the tight junctional strands, and many were completely encircled by the strands. The increase in the number and complexity of the tight junctional strands in response to brain injury may be the morphological basis for the maintenance of the cerebrospinal fluid-blood dural barrier.

Animals↗
PubMed2002

The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost.

Membrane-associated guanylate kinase (Maguk) proteins are scaffold proteins that contain PSD-95-Discs Large-zona occludens-1 (PDZ), Src homology 3, and guanylate kinase domains. A subset of Maguk proteins, such as mLin-2 and protein associated with Lin-7 (Pals)1, also contain two L27 domains: an L27C domain that binds mLin-7 and an L27N domain of unknown function. Here, we demonstrate that the L27N domain targets Pals1 to tight junctions by binding to a PDZ domain protein, Pals1-associated tight junction (PATJ) protein, via a unique Maguk recruitment domain. PATJ is a homologue of Drosophila Discs Lost, a protein that is crucial for epithelial polarity and that exists in a complex with the apical polarity determinant, Crumbs. PATJ and a human Crumbs homologue, CRB1, colocalize with Pals1 to tight junctions, and CRB1 interacts with PATJ albeit indirectly via binding the Pals1 PDZ domain. In agreement, we find that a Drosophila homologue of Pals1 participates in identical interactions with Drosophila Crumbs and Discs Lost. This Drosophila Pals1 homologue has been demonstrated recently to represent Stardust, a crucial polarity gene in Drosophila. Thus, our data identifies a new multiprotein complex that appears to be evolutionarily conserved and likely plays an important role in protein targeting and cell polarity.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Differential regulation of junctional complex assembly in renal epithelial cell lines.

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and beta-catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner.

3T3 Cells↗
PubMed1997

ZO1 in corneal epithelium: association to the zonula occludens and adherens junctions.

The corneal epithelium, like other stratifying epithelium, does not present a well formed junctional complex as compared to that of simple epithelia. However, the resistance barrier of the corneal epithelium is to a great extent generated by zonula occludens (ZO, tight junction), which are formed between the cells of the apical-most strata. The tight junction provides a continuous seal around the apical aspect of adjoining epithelial cells, thereby preventing the free passage of molecules between adjacent epithelial cells (paracellular pathway). We have examined rabbit corneal epithelia with monoclonal antibody against the tight junction associated protein ZO1. With this antibody, we resolved two distinct patterns of ZO1 expression, one being the lateral boundary of the apical cell, which appeared as a true zonula around these cells. The second pattern of expression for ZO1 was at a set of punctate spots that correspond to the connection of the most apical portion of the basal corneal epithelial cells, with the above wing cells. En face, confocal analyses revealed that these areas consisted of 5-6 distinct spots per basal cell at or near the contact points with the immediate wing cells above. ImmunoEM revealed that the mid-epithelial accumulations of ZO1 were not tight junctions, but rather a form of adherens junction. The expression of ZO1 in the mid-epithelial level of the cornea is neither correlated with the presence of tight junction, nor with the established barrier functions. Interestingly, these junctions in the corneal epithelium also contain paxillin, a focal adhesion associated phosphoprotein which is a target of pp125 focal adhesion kinase, erbB-2 kinase and p21Obcr/abl oncogene. We postulated that the ZO1/paxillin adherens junction within stratified epithelium, such as the corneal epithelium, may function to reinforce attachments at the level of the basal cell to wing cell junction and be regulated by reversible phosphorylation. We speculate that the regulated phosphorylation of tyrosine residues on paxillin may perform a critical role in controlling epithelial cell-cell interactions as it does in cell-matrix adhesion.

Animals↗
PubMed1982

Giant hypertrophic gastropathy (Ménétrier's disease): pharmacologic effects on protein leakage and mucosal ultrastructure.

Seven patients with giant hypertrophic gastropathy participated in a gastric intubation perfusion study to investigate the route and mechanism of protein leakage associated with this disease. All patients had gastric tight junctions wider than those in healthy controls. Acute administration of propantheline bromide reduced gastric albumin leakage (-50.7%, p less than 0.05) and concurrently decreased width of tight junctions (p less than 0.05) in all patients. Another antisecretory agent, cimetidine, had no consistent effect on protein leakage or on the width of tight junctions. Pentagastrin and bethanechol chloride increased protein loss but had no effect on the width of tight junctions. These results are consistent with the hypothesis that proteins may take a paracellular route via the tight junctions as they traverse the gastric mucosa and that this may have a cholinergic mechanism.

Adult↗
PubMed2005

Abrogation of IFN-gamma mediated epithelial barrier disruption by serine protease inhibition.

The intestinal barrier function is often impaired in a variety of diseases including chronic inflammatory bowel disease. Increased intestinal permeability during episodes of active disease correlates with destruction or rearrangement of the tight junction protein complex. IFN-gamma has been widely studied for its effect on barrier function and tight junction structures but its mode of action remains unclear. Since the claudin family of tight junction proteins is proposed to be involved in barrier maintenance we studied the effect of IFN-gamma on claudin expression in relation to epithelial barrier function. Cycloheximide and protease inhibitors were used to study mechanisms of IFN-gamma mediated barrier disruption. Intestinal epithelial cells were exposed to IFN-gamma and permeability was evaluated by horse radish peroxidase (HRP) and 4 kD FITC-dextran fluxes. Occludin and claudin-1, -2, -3, and -4 tight junction protein expression was determined by Western blotting. Occludin and claudin-2 protein expression was dramatically reduced after IFN-gamma exposure, which correlated with increased permeability for HRP and FITC-dextran. Interestingly, cleavage of claudin-2 was observed after incubation with IFN-gamma. Serine protease inhibitor AEBSF completely abrogated IFN-gamma mediated barrier disruption which was associated with preservation of claudin-2 expression. Moreover, IFN-gamma induced loss of barrier integrity was found to affect claudin-2 and occludin expression through different mechanisms. Since inhibition of serine protease activity abrogates IFN-gamma mediated barrier disruption this may be an important target for therapeutic intervention.

Blotting, Western↗
PubMed1975

[Role of cations and surface membrane proteins in the adhesion of rat liver parenchymal cells. I. Effect of dirrerent perfusion periods on the structure of cellular junctions].

Electronmicroscopic study of the structure of the rat liver cell junctions in the normal conditions and after 90 min. perfusion by the physiological solution showed 4 kinds of specialized junctions between the hepatocytes with typical structure: tight junction, gap junction, intermediate junction, and desmosomes. The long perfusion does not cause visible changes in the contact structure or the sizes of the intercellular space. The preservation of the tight junction after the long perfusion is demonstrated by the lanthanum tracer: lanthanum is never found in the tight junction and in the bile capillary. Lanthanum reveales globular structure of the middle layer of the gap junction.

Animals↗
PubMed2004

The cytoplasmic domain of CEACAM1-L controls its lateral localization and the organization of desmosomes in polarized epithelial cells.

Two CEACAM1 isoforms with different cytoplasmic domains, CEACAM1-L and CEACAM1-S, are unequally distributed in polarized epithelial MDCK cells. CEACAM1-S is exclusively apical whereas CEACAM1-L occurs both in apical and lateral cell surfaces. Using confocal microscopy and CEACAM1-L mutants, we identified several amino acids in the cytoplasmic domain that were instrumental for the lateral localization. Tyr515, but not Tyr488, constituted a prominent lateral targeting signal. Pervanadate-stimulated Tyr phosphorylation induced rapid phosphatidylinositol 3-kinase-dependent disappearance of lateral CEACAM1-L, whereas staurosporine, a Ser/Thr kinase inhibitor, resulted in slower phosphatidylinositol 3-kinase-independent disappearance. Both drugs caused accumulation of CEACAM1-L in a late endosome/lysosome compartment. Colocalization studies of occludin, ZO-1, E-cadherin, beta-catenin and desmoplakin indicated that laterally localized CEACAM1-L was present in adherens junctions but not in tight junctions or desmosomes. Overexpressed CEACAM1-L did not affect the organization of tight junction or adherens junction proteins, but perturbed the arrangement of desmosomes. The abundance of desmosomes in the lateral cell surfaces decreased significantly and the submembraneous cytokeratin filaments became disorganized. The signal for desmosomal perturbance resided within amino acids 484-518 in the C-terminal part of the cytoplasmic domain, among which an intact Tyr515 was indispensable.

Amino Acid Sequence↗
PubMed1987

Filipin and digitonin studies of cell membrane changes during junction breakdown in the dystrophic rat retinal pigment epithelium.

We have previously found that a breakdown of tight junctions in retinal pigment epithelial cells of Royal College of Surgeons' rats is associated with a redistribution of intramembrane particles and Na-K-ATPase activity. Changes in the lipid and sterol composition of membranes can alter their fluidity, permeability and enzyme activity, and may contribute to changes in cell barrier function in the dystrophic epithelium. We have now used filipin and digitonin, which bind to membrane sterols and produce membrane deformations recognizable by freeze-fracture and thin-section electron microscopy, to study the distribution of cholesterol and related 3-B-hydroxysterols in the dystrophic epithelium. The results of these studies show that in the normal pigment epithelium and prior to tight junction breakdown in the dystrophic epithelium, filipin- and digitonin-sterol complexes are rare in the membranes between tight junctions and adhering junctions, and in areas of attachment between the plasma membrane and basal lamina. Complexes are more numerous in the basal infoldings, and most densely packed in the lateral and apical microvillous membranes. During junction breakdown, complexes increase substantially in apical, basal, junctional, and nuclear membranes. Later, after the junctions disappear, complexes decrease. These results indicate that alterations in the expression of membrane sterols accompany the changes in structure and function of tight junctions in the dystrophic retinal pigment epithelium.

Animals↗
PubMed1997

Is the pial microvessel a good model for blood-brain barrier studies?

Pial microvessels have commonly been used as model systems for studying blood-brain barrier (BBB) properties instead of cerebral cortical microvessels. Since pial microvessels are relatively accessible they have been especially employed in electrophysiological and pharmacological studies. Measurements of electrical resistance across endothelial cells (EC) as a measure of their barrier properties have been made exclusively from pial microvessels in in vivo BBB studies. Similarly the observed responses of microvessels to the application of pharmacological agents have commonly been made on pial microvessels as representative of BBB vasculature. In this review the properties of pial and cerebral microvessels are compared to determine whether the use of the pial microvessel as a model for BBB studies is valid. Similarities are described in their ultrastructural features, permeability to electron dense tracers and molecular characteristics. Measurements of electrical resistance from pial microvessels are compared with measurements from cerebral EC monolayers in tissue culture and indirect determinations for cerebral microvessels in situ. Two notable differences between pial and cerebral microvessels are described in the adult nervous system. Tight junctions between cerebral EC appear to consist of a uniform population. In pial microvessels however tight junctions consist of two populations in one the inter-EC tight junctions resemble those between cerebral EC, with fusion of adjacent EC membranes. In the second population the inter-EC tight junctions differ with a discernible gap between adjacent EC membranes. The distribution of the endothelial barrier antigen (EBA) is uniform between EC of cerebral microvessels. By contrast EC of pial microvessels from a heterogeneous population for EBA expression which is related to the proximity of the EC to the astrocytic glia limitans. The role of astrocytes in the induction and maintenance of the BBB characteristics is briefly reviewed. The possible significance of the lack of an astrocytic ensheathment of pial microvessels is assessed. In summary, caution is urged in employing pial microvessels in BBB studies and the need for more information on possible pial microvessel heterogeneity is stressed.

Animals↗
PubMed2001

Effects of high-cholesterol diet on the interendothelial clefts and the associated junctional complexes in rat aorta.

The arterial endothelial intercellular cleft (AEC) and its associated junctional complex (JC) are the determinants of permeability to macromolecules. This study analyzed frequencies of AEC and JC profile types in the rat thoracic aorta at 1 and 12 months after feeding the animals with a normal or a high-cholesterol diet. Rats on either a normal diet or high-cholesterol diet for 12 months showed more of the simple 'end to end' or 'overlap' types (P < 0.01) but fewer complex 'interdigitating' type (P < 0.01) of AEC compared to the 1 month group. With regard to JC, the frequencies of gap junctions were decreased (P < 0.01) while the tight junctions and the normal junctionless complex were increased (P < 0.01) after 12 months of normal diet as compared with 1 month on the normal diet. These changes in frequencies for gap junction and tight junction were even greater for the high-cholesterol diet than for the normal diet treatment. Moreover, the incidence of open junctions was also noticeably increased after 12 months of high-cholesterol diet. These findings suggest that the proportions of the AEC and JC were highly responsive to aging whereas those of JC were more susceptible to the high-cholesterol diet treatment.

Aging↗
PubMed1994

Serum-free culture and characterization of renal epithelial cells isolated from human fetal kidneys of varying gestational age.

Cell cultures were initiated from seven human fetal kidneys that varied in gestational age from 90 days to newborn. The growth medium utilized was a 1:1 mixture of Dulbecco's modified Eagle's and Ham's F12 supplemented with selenium (5 ng/ml), insulin (5 micrograms/ml), transferrin (5 micrograms/ml), hydrocortisone (36 ng/ml), triiodothyronine (4 pg/ml), and epidermal growth factor (10 ng/ml). For all the kidney isolates, initial cell attachment occurred within 12 h through multicell spheroids, and by 24 h a rapidly growing population of cells was obtained. Confluency was reached within 3 to 6 days. A combination of light microscopy, immunohistochemistry, and ultrastructural evaluation was utilized to characterize the resulting cultures as epithelial and homogeneous within each isolate and among the isolates. That is, regardless of gestational age of the fetal kidney used as starting material, an identical or highly similar population of cells was obtained. By light microscopy, the cultures were noted to form very few domes, the number being an indication of transport activity. However, ultrastructural examination revealed that the cells were noted to form domes composed of only a few cells or "micro-domes" that would not be visible by light microscopy. Within the micro-domes as well as other areas of the monolayer an apparent absence of tight junctions was noted by routine transmission electron microscopy. However, by freeze fracture analysis cells were shown to possess sealing strands, the structural component of tight junctions. It is postulated that the tight junctions of fetal epithelial cells are structurally altered as compared to tight junctions in adult renal epithelial cell cultures.

Cells, Cultured↗
PubMed2005

Renal clear-cell carcinoma: an ultrastructural study on the junctional complexes.

Junctional complexes such as tight junctions, adherens junctions, and desmosomes play crucial roles in the structure and function of epithelial cells. These junctions are involved in increasing cell-cell contact and as well serve as signaling centers regulating multiple functions in epithelial cells. Carcinoma cell lines cultured in the laboratory generally lack junctional complexes. However, studies directed towards understanding the distribution of junctional complexes in human cancer tissues are lacking. In this study, we analyzed by electron microscopy the distribution of junctional complexes in patients diagnosed with renal clear-cell carcinoma. We found that both tight junctions and adherens junctions were drastically reduced in patients with cancer compared to normal tissues. Desmosomes were not detected in normal proximal tubules while distinctly present in cancer tissues. These results suggest that analysis of junctional complexes in human tumors should provide valuable information that might have prognostic and diagnostic value.

Aged↗