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At least 1,207 records · Page 67Linked to original sources
PubMed1992

IEC-18, a nontransformed small intestinal cell line for studying epithelial permeability.

Small intestinal epithelium is leaky and allows permeation of hydrophilic molecules of various sizes. Passively absorbed hydrophilic permeability probes have been shown to permeate across intestinal epithelium mainly through the paracellular pathways. In this study we introduce microporous filter-grown IEC-18 epithelial cells, a nontransformed small intestinal cell line, as a in vitro model of intestinal epithelium for the study of epithelial permeability. IEC-18 cells, originally derived from native rat ileal crypts, form confluent epithelium when grown on hydrated collagen-coated Millicell-CM permeable inserts (Millipore Corp., Bedford, Mass.). With scanning and transmission electron microscopy, the presence of tight junctions and desmosomes between cells and the development of microvilli at the apical surface were confirmed. Immunofluorescent labeling of ZO-1 proteins and desmoplakins verified the presence of tight-junctional proteins (ZO-1) and desmosomes in the intercellular junctions of confluent IEC-18 epithelium. The net electrical resistance of IEC-18 epithelium (28 omega-cm2) was similar to resistance values obtained from small intestinal tissue with (50 to 100 omega-cm2) or without (20 to 45 omega-cm2) muscularis and serosal layers. Assessment of mannitol and dextran permeation revealed early "maturation" of paracellular pathway, with increasing restriction of permeation to both probes through day 4. Resistance across IEC-18 epithelium also reached plateau levels between 4 and 7 days. Permeability studies with various probes indicate that cross-sectional diameter rather than molecular weight of the probe is the important determinant of permeation rate. IEC-18 epithelium selectively restricted the permeation of probes proportional to probe size; permeation of larger probes such as albumin was negligible. We conclude that cultured IEC-18 epithelial cells, because of their native crypt origin, similarity in resistance to small intestinal epithelia, retention of ability to differentiate into villus-like enterocytes, and permeability characteristics, are a useful model of intestinal epithelium for the study of permeability and paracellular transport.

Animals↗
PubMed2001

Stimulation of intercellular communication of poor-communicating cells by gap-junction-competent cells enhances the HSV-TK/GCV bystander effect in vitro.

We have previously shown that gap-junctional intercellular communication (GJIC) appears to play a role in the bystander effect that is observed in anticancer suicide gene therapy mediated by herpes simplex virus (HSV) thymidine kinase (tk) and ganciclovir (GCV). We now report that when connexin-expressing (Cx+) cells are present within a noncommunicating population of cells (Cx-), there is GJIC between the Cx+ and Cx- cells and that due to this stimulation of GJIC, the bystander effect also occurs when the 2 cell types are mixed. We transfected HeLa cells, which do not express any detectable level of connexin, with Cx43. The Cx+ and Cx- HeLa cells were further transfected with the tk gene, giving 4 phenotypes: Cx+tk-, Cx+tk+, Cx-tk+ and Cx-tk-. We observed GJIC between Cx+ and Cx- cells, but not between Cx- and Cx- cells, regardless of the tk genotype. Similarly, we observed the HSV-tk/GCV bystander effect in Cx+tk-/Cx-tk+ and Cx+tk+/Cx-tk- cocultures. The extent of the bystander effect in cocultures of Cx+tk- and Cx-tk+ cells was stronger than in cocultures of Cx+tk+ and Cx-tk- cells when each mixture had the same ratio of Cx+ and tk+ cells. These results suggest that Cx-expressing HeLa cells stimulate GJIC capacity between them and non-Cx-expressing HeLa cells, which mediates the bystander effect in mixtures of Cx+ cells and Cx- cells in vitro. Thus, Cx expression even in only a limited fraction of tumor cells may enhance the efficacy of the HSV-tk/GCV strategy by inducing a bystander effect.

Antiviral Agents↗
PubMed1976

Ultrastructure and permeability of lymph node microvasculature in the mouse.

The microvasculature of lymph nodes and Peyer's patches consists of arterioles, capillaries and venules. The postcapillary segment comprises high-endothelial venules (HE venules) as well as ordinary venules. In order to study the ultrastructure of the microvasculature, particularly with respect to the nature of intercellular junctions, lanthanum and ruthenium red were used as tracers. Furthermore, to evaluate the permeability properties of the different segments of the microvasculature, intravenously injected horseradish peroxidase (HRP; MW: 40,000) was used. All segments of the microvasculature are permeable to HRP. However, the mechanism of transport across the vascular wall varies in the different segments, apparently correlated with a gradual decrease in number of transport vesicles and a gradual attenuation in the sealing of the endothelial cells. Tight junctions are present in arterioles, and it is assumed that HRP reach the basal lamina exclusively by vesicular transport. Incomplete or focal tight junctions are present in the capillaries, and both intercellular and vesicular pathways are observed. In the venules the intercellular pathway seems to be the dominant one, while vesicular transfer is negligible. However, some micropinocytic vesicles in the HE venule endothelial cells probably represent the initial stage of an intracellular digestion.

Animals↗
PubMed1988

Inhibition of mouse hepatocyte intercellular communication by paraquat-generated oxygen free radicals.

Intercellular communication through gap junctions functions in electrical synapsing, homeostasis, hormonal response, embryogenesis, and growth control. Many neurotoxicants, teratogens, and carcinogens are capable of inhibiting gap junctional intercellular communication and this effect may be related to their toxic activity. In addition, many of these toxic agents are capable of stimulating oxygen free radical production in cells. The purpose of this study was to determine if oxygen free radicals at noncytotoxic levels could inhibit intercellular communication in primary cultured mouse hepatocytes. Intercellular communication was evaluated in 24-hr-old cultures of male B6C3F1/Cr1BR mouse hepatocytes by microinjection of fluorescent Lucifer Yellow CH dye and visualization of dye spread to adjacent hepatocytes (dye-coupling). Dye-coupling was rapidly established in freshly plated primary cultured hepatocytes reaching a level of over 90% after 24 hr of culture. After 24 hr, dye-coupling paralleled hepatocyte survival. Treatment of hepatocyte cultures with noncytotoxic concentrations of paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride; PQ) (0.5-5 mM), hydrogen peroxide (0.5-2 mM), glucose oxidase (0.1 U/ml), or xanthine oxidase (0.2 U/ml plus 1 mM xanthine) for exposure durations of 2-8 hr resulted in concentration-dependent decreases in dye-coupling. Addition of the antioxidants DPPD (N,N-diphenyl-p-phenylenediamine; 25 microM) and vitamin E (D,L-alpha-tocopherol acetate; 100 microM) decreased the inhibitory effect of PQ on dye-coupling. In contrast, addition of the catalase inhibitor 3-amino-1,2,4-triazole or the glutathione depletor diethylmaleate to PQ-treated cultures potentiated PQ-induced inhibition of dye-coupling. PQ stimulated NADPH-dependent mouse liver microsomal superoxide radical production. Thus, one effect of prooxidant compounds appears to be the inhibition of IC. This effect may be important in the sublethal toxicity of oxygen radical generating compounds.

Animals↗
PubMed2004

Alpha-catenin: at the junction of intercellular adhesion and actin dynamics.

Alpha-catenin has often been considered to be a non-regulatory intercellular adhesion protein, in contrast to beta-catenin, which has well-documented dual roles in cell-cell adhesion and signal transduction. Recently, however, alpha-catenin has been found to be important not only in connecting the E-cadherin-beta-catenin complex to the actin cytoskeleton, but also in coordinating actin dynamics and inversely correlating cell adhesion with proliferation. As the number of alpha-catenin-interacting partners increases, intriguing new connections imply even more complex regulatory functions for this protein.

Actins↗
PubMed2000

Occludin modulates transepithelial migration of neutrophils.

Neutrophils cross epithelial sheets to reach inflamed mucosal surfaces by migrating along the paracellular route. To avoid breakdown of the epithelial barrier, this process requires coordinated opening and closing of tight junctions, the most apical intercellular junctions in epithelia. To determine the function of epithelial tight junction proteins in this process, we analyzed neutrophil migration across monolayers formed by stably transfected epithelial cells expressing wild-type and mutant occludin, a membrane protein of tight junctions with four transmembrane domains and both termini in the cytosol. We found that expression of mutants with a modified N-terminal cytoplasmic domain up-regulated migration, whereas deletion of the C-terminal cytoplasmic domain did not have an effect. The N-terminal cytosolic domain was also found to be important for the linear arrangement of occludin within tight junctions but not for the permeability barrier. Moreover, expression of mutant occludin bearing a mutation in one of the two extracellular domains inhibited neutrophil migration. The effects of transfected occludin mutants on neutrophil migration did not correlate with their effects on selective paracellular permeability and transepithelial electrical resistance. Hence, specific domains and functional properties of occludin modulate transepithelial migration of neutrophils.

Actins↗
PubMed1988

[Delayed cerebral radionecrosis associated with brain edema].

In a patient with a delayed cerebral radionecrosis, we examined the ultrastructural changes of capillaries and discussed the cause of the development of severe brain edema which is one of specific features of the radionecrosis. A 42-year-old male was found to have cerebral radionecrosis two and one-half years following split-course radiotherapy which was done after subtotal excision of a left parasagittal meningioma. The surgical specimens were studied with conventional ultrathin section and freeze-fracture replica techniques. The common characteristics of capillary endothelia were attenuation of its electron density, less degree of surface infolding, irregular thin and thick width, increased pinocytotic vesicles which were calculated as 37 per square micron with replica preparations. The discontinuity of endothelia was also revealed. The basal laminae were irregular in thickness. The pericapillary space was markedly enlarged. A fibrin-like substance accumulated in the extravascular space. Inflammatory cells also infiltrated the pericapillary zone. The tight junctions were preserved. The intercellular junctions were composed of 5 strands which appeared to be a continuous array of particles. We concluded that activated pinocytotic vesicles (vesicular transport) played an important role in increasing the permeability in the vessels of delayed cerebral radionecrosis, in addition to a possible leakage through discontinuity of endothelia.

Adult↗
PubMed1997

[Intestinal intercellular tight junctions. I. Structure and molecular mechanisms of regulation].

The existence of tight junctions between epithelial cells has been known for over 100 years, but their exact functions remained elusive until recently. The present paper summarizes the newest knowledge about morphology, molecular components and regulation of tight junctions. In the chapter about morphology the criteria for classification of epithelia as well as the importance of the "freeze fracture" electron-microscopic technique in forming our present ideas about the fine structure of tight junctions will be discussed. The two existing theories about the molecular composition of the resistance-forming barrier ("lipid-model" and "protein-model", resp.) and the important properties of the tight-junction-associated proteins characterized in the past years are discussed in the chapter about molecular components. Finally, in the last part of the paper the factors playing an important role in the regulation of tight junctions, including the cytoskeleton, the protein kinases A and C, the phospholipase C and two categories of the GTP-binding proteins are reviewed.

Animals↗
PubMed1977

Sertoli cell junctions in the germ cell-free testis of the congenic mouse.

Intercellular junctions between the Sertoli cells in the germ cell-free testis of the congenic mouse were studied during ontogeny. In freeze-fracture replicas, these junctions are characterized by a great number of parallel occluding junctions, in which the particles are preferentially located in the center of the grooves in the E face. These particles appear to be arranged in discrete rows in both P and E fracture faces. In the initial stages of junction formation, large gap junctions with hexagonally packed particle aggregates and particle-free regions are observed. In the later stages, short segments of the occluding junctions are found associated with gap junctions. In later stages, the gap junctions diminish in size and number. These junctions are quite similar to those between Sertoli cells in the normal mouse and rat reported previously. The Sertoli cell junctions develop in the absence of the spermatogenic cells of this congenic mouse.

Age Factors↗
PubMed2002

Junctional adhesion molecules and interendothelial junctions.

Similar to epithelia, endothelial cells are linked to each other via intercellular junctional complexes including gap junctions, adherens junctions and tight junctions. While polarized epithelial cells show a high degree of spatial sorting of junctional complexes, endothelia organize their junctions randomly. For this reason the nature of endothelial contacts may be highly adaptable to the need of permeability and leukocyte transmigration. For instance, high endothelial venules (HEVs) in lymphoid organs, where lymphocytes continuously exit the bloodstream, generally show more leaky contacts than brain with its impermeable blood-brain barrier. We recently identified an Ig superfamily molecule named JAM-2 which is specifically expressed in junctions of lymphatic endothelial cells and HEVs. We showed that JAM-2 belongs to the novel CTX molecular family and we now cloned the human equivalent of JAM-2. The presence of JAM-2 at sites of constitutive lymphocyte circulation argues for a role of this molecule in facilitating transmigration. This is supported by the increased transmigration in vitro across endothelial cells overexpressing JAM-2 at intercellular contacts.

Amino Acid Sequence↗
PubMed1981

A biochemical dissection of the cardiac intercalated disk: isolation of subcellular fractions containing fascia adherentes and gap junctions.

In view of our limited knowledge of the biochemical composition of intercellular junctions, a method was developed for the preparation from rats and mice of plasma membranes containing cardiac intercalated disks. When these membranes were extracted with detergents, e.g. N-lauryl sarcosinate or deoxycholate, the detergent-insoluble material contained structures derived mainly from fascia adherentes junctions, but a few gap junctions and maculae adherentes were also present. When the detergent extraction was carried out at an alkaline pH, the maculae adherentes junctions were dissolved. Fractionation of the detergent-insoluble extract on a sucrose gradient yielded a fraction containing fascia adherentes junction of density 1.20-1.26 g/cm3. Gap junctions banded at a lower density, 1.16-1.20 g/cm3. Polyacrylamide gel electrophoresis showed that the major polypeptide bands in the fascia adherentes-enriched fraction were of molecular weights 134000, 108000, 62-64000, 58000, 47000 and 43000. Although fractions with the gap junctions were contaminated by fascia adherentes junctions, the major polypeptides were calculated by subtraction to be of mol. wt 37000, 26000 and 19000. Two glycoproteins corresponding to minor polypeptides visualized by Coomassie Blue staining were present in the fascia adherentes fraction. Comparison of the fascia adherentes-enriched fraction with a Z-disc fraction prepared from rabbit hearts indicated a different morphology and polypeptide composition.

Animals↗
PubMed2005

Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels.

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.

Aorta↗
PubMed1989

The association of gap junctions with large particles in the crypt epithelium of the rat small intestine.

Intercellular junctions in the epithelium of the rat small intestine were studied by the freeze-fracture method. Gap junctions were found between columnar cells of the crypt far more frequently than between absorptive cells of the villus. In addition, in the crypt, large particles 11 to 13 nm in diameter were often associated with the gap junction which primarily consisted of usual gap junctional particles 8 to 9 nm in diameter. Both the usual and large particles possessed a central pit. The intestinal crypt is the site of epithelial cell proliferation and differentiation in the process of physiological cell renewal. The predominant presence of gap junctions in this region suggests that they play a certain role in the proliferation and differentiation of crypt columnar cells. Further studies are required to elucidate the nature of the large particles, a precursor of typical connexons and/or a functionally different gap junction.

Animals↗
PubMed1975

Cell contacts in human islets of Langerhans.

The freeze-fracturing technique was applied to fresh human islets of Langerhans. With this technique, the inside of cellular membranes was revealed, and specific membrane differentiations, hitherto unknown, were observed in the plasma membrane of the endocrine cells. The specific membrane differentiations represent two types of intercellular junctions, namely the tight junction, which determines a closure of the extracellular space and the gap junction which allows molecules and ions to diffuse from one cell to another (sharing the gap junction) without leaking in the extracellular space (intercellular coupling). The presence of such junctions may be important for the secretory behavior of the cells within the islet.

Adult↗
PubMed1999

A dominant mutant of occludin disrupts tight junction structure and function.

The tight junction is the most apical intercellular junction of epithelial cells and forms a diffusion barrier between individual cells. Occludin is an integral membrane protein specifically associated with the tight junction which may contribute to the function or regulation of this intercellular seal. In order to elucidate the role of occludin at the tight junction, a full length and an N-terminally truncated murine occludin construct, both FLAG-tagged at the N terminus, were stably introduced into the murine epithelial cell line CSG 120/7. Both constructs were correctly targeted to the tight junction, as defined by colocalization with another tight junction protein, ZO-1. The construct lacking the N terminus and extracellular domains of occludin was found to exert a dramatic effect on tight junction integrity. Cell monolayers failed to develop an efficient permeability barrier, as demonstrated by low transcellular electrical resistance values and an increased paracellular flux to small molecular mass tracers. Furthermore, gaps were found to have been induced in the P-face associated tight junction strands, as visualized by freeze-fracture electron microscopy. These findings demonstrate an important role for the N-terminal half of occludin in tight junction assembly and maintaining the barrier function of the tight junction.

Animals↗