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PubMed2002

Regulation of RPE intercellular junction integrity and function by hepatocyte growth factor.

PURPOSE: To evaluate the effect of hepatocyte growth factor (HGF) on the integrity and function of tight junctions and adherens junctions in the retinal pigment epithelial (RPE) monolayer. METHODS: Fresh bovine eyes were dissected to obtain 2- to 3-mm(2) explants of intact RPE with underlying choroid and sclera. Explants were cultured with or without HGF (20 ng/mL) for various periods (20 minutes to 72 hours). Junction integrity was assessed by transmission and scanning electron microscopy; localization, expression, and phosphorylation of junction proteins; and measurement of transepithelial resistance (TER), diffusion of fluorescent labeling in the plasma membrane, and the migration of RPE cells from the monolayer. RESULTS: Untreated explants consisted of polarized cells with apical microvilli and well-developed tight and adherens junctions. After HGF treatment, the explants showed loss of tight and adherens junctions ultrastructurally, diffusion of fluorescent label from apical to lateral membrane domains, and increased chemotactic migration of RPE cells from the monolayer. Primary cultures of confluent RPE cells showed a progressive decrease in TER. Western blot analysis showed rapid tyrosine phosphorylation of ZO-1, occludin, and beta-catenin within 20 minutes of stimulation. There was a marked loss of ZO-1 protein within 1 hour of HGF treatment. After 6 hours of treatment with HGF, occludin, claudin-1, and beta-catenin were redistributed from the membrane to the cytoplasm. CONCLUSIONS: Treatment of RPE explants with HGF results in rapid disassembly of tight and adherens junctions associated with loss or redistribution of junctional proteins, decreased TER, and increased migration of RPE cells from the monolayer.

Animals↗
PubMed1981

Fine structure and intercellular junctions of a freshwater teleost (Pimelodus maculatus) hepatocytes by freeze-fracture replicas.

The Pt-C replicas of a freshwater teleost hepatic cells were studied by electron microscopy. The surface of hepatocytes bordering the perisinusoidal space is studied with numerous microvilli. Cell-to-cell attachments are made through gap junctions and spot desmosomes. Tight junctions are found between hepatocytes close to the bile canaliculi. Some of the numerous corpuscular elements found in the cytoplasm can be identified as organelles, specially if their interiors are partially exposed by cleaving.

Animals↗
PubMed1986

The electrotonic location of low-resistance intercellular junctions between a pair of giant neurones in the snail Lymnaea.

The passive electrotonic properties of neurones VD1 and RPD2 in the brain of the snail Lymnaea can be represented by a soma-finite cable model with closed-circuit axon termination. There is a considerable individual variation in input resistance, membrane time constant, electrotonic length and axon-soma conductance ratio, but the average values for these parameters are similar in the two neurones. The cells are tightly coupled by an electrotonic synapse giving an average steady-state coupling coefficient of 0.68 and an average resistance measured between recording sites in the cell bodies of 20 M omega. Calculations using a model consisting of a symmetrical pair of cells with standard values for the electrotonic parameters show that in this system, for a soma-soma resistance of 20 M omega, the junction cannot be more than 0.16 length constants from the cell bodies. Reduction in coupling due to membrane current losses in such short proximal axon segments is insignificant. Intra-axonal recordings indicate that most of the coupling resistance is located at the junction between VD1 and RPD2, which must therefore be closer to the cell bodies than the limiting value of 0.16 length constants assuming an electrical equivalent model which includes the standard electrotonic parameters. If all the soma-soma resistance is located at the junction, then it could be physically a single array of gap-junction particles. Despite its low conductance (1/20 M omega = 50 nS) and possibly small physical dimensions, the electrotonic synapse is more than sufficient to ensure spike synchrony in the two cells.

Action Potentials↗
PubMed1982

The freeze-fractured median eminence. I. Development of intercellular junctions in the ependyma of the 3rd ventricle of the rat.

Ependymal junctions in both the ventro-lateral wall and infundibular floor of the 3rd ventricle were examined in adult, young, neonatal, and fetal rats in freeze-fracture preparations. During late fetal and early postnatal life, tight junctions developed in both ventro-lateral and floor ependyma with, first, the appearance of an area of waffle-like or honeycomb texture in the membrane where tight junctions will appear; second, the decoration of P-face crests of this honeycomb with individual particles; and lastly, fusion of these particles into smooth strands. In adults, tight junctions were rare in the lateral ependyma, but persisted on the floor as a loose network. Many small ependymal gap junctions were already present at the earliest fetal stage (17 days) examined. They occupied significantly larger proportions of the membranes on the ventro-lateral wall than on the floor throughout the time course due to an increase in their size but not in their numbers. The smallest gap junctions were regarded as the newest in a process of renewal throughout life. The development of both junctional types parallels what is known on the onset of neuroendocrine functions in the median eminence. In the rat, this is apparently just before, at, or just after birth.

Animals↗
PubMed1984

Intercellular junctions in the cells of the human enamel organ as revealed by freeze-fracture.

Examined by thin sections and freeze-fracture replication techniques, secretory ameloblasts possessed two sets of the junctional complexes at both proximal and distal ends of the cell bodies, which consisted of tight junctions and occasional gap junctions and desmosomes. The proximal tight junction was fascia occludens, whereas the distal tight junction was zonula occludens. Between adjacent ameloblasts, mature gap junctions were frequent. The stratum-intermedium cells were connected to each other and to the stellate-reticulum cells and ameloblasts by well-developed desmosomes, gap junctions and fascia or macula-type tight junctions. Stellate-reticulum cells were inter-connected by many extensive cytoplasmic processes, in which well-developed desmosomes, small gap junctions and occasional macula-type tight junctions appeared. Thus fascia or macula-type tight junctions as well as many desmosomes seem to serve in mechanical, cell-to-cell adhesion during tooth formation. Frequent and large gap junctions between adjacent stratum-intermedium cells and between the stratum intermedium and the base of the ameloblast suggest that, in relation to enamel formation, these two cell layers form a functional unit.

Ameloblasts↗
PubMed1997

Ultrastructural characterization of the early changes in intercellular junctions in response to cadmium (Cd2+) exposure in LLC-PK1 cells.

Previous studies have shown that Cd2+ can disrupt the Ca2+-dependent junctions between LLC-PK1 cells. The objective of the present studies was to further characterize the early junctional effects of Cd2+ in LLC-PK1 cells and to identify the initial site of injury. LLC-PK1 cells were grown on permeable membrane supports and were exposed to 10 microM Cd2+ from the basolateral compartment. The integrity of cell junctions was assessed by light and electron microscopy and by measuring the transepithelial electrical resistance. After as little as 15 min of Cd2+ exposure, there was an increase in the amount of light transmitted between the cells. The transepithelial resistance began to decline by 30 min and continued to fall until reaching zero after about 6 hr. Ultrastructural analysis showed that the initial disruption of cell-cell junctions coincided with a decrease in the density of intracellular plaques associated with the adhering junctions (zonulae adherens). This effect increased with time and paralleled an increase in the space between cells and a change in the shape of the cells from squamous to rounded. Sectioning the cells horizontally, in a plane parallel to the membrane support, allowed us to see large areas of zonulae adherens. Cd2+ caused the formation of gaps within the zonulae which increased with time of exposure. No significant changes in most occluding junctions (zonulae occludens) were seen until 6-8 hr after Cd2+ exposure. These results indicate that the adhering junctions and associated cytoplasmic components are primary sites of early Cd2+ injury in LLC-PK1 cells and they suggest that these effects may result from the interaction of Cd2+ with target sites associated with the basolateral cell surface.

Animals↗
PubMed1995

Rapid disruption of epithelial barrier function by Salmonella typhimurium is associated with structural modification of intercellular junctions.

Short-term infection of MDCK II monolayers with Salmonella typhimurium SL1344 caused a progressive decrease in transepithelial electrical resistance concomitant with decreased cation permselectivity and increased paracellular inulin flux. Cytochemical staining of F-actin, E-cadherin, and ZO-1 revealed the concentration of each junctional protein in invaded cells as a result of contraction at their apical poles and resultant distortion of adjacent uninvaded cells.

Actins↗
PubMed1996

Determination of gap junctional intercellular communication by capacitance measurements.

Electrical coupling between cells is usually measured using the double-patch-clamp technique with cell pairs. Here, a single patch-clamp technique that is not limited to cell pairs is described to determine electrical coupling between cells. Capacitance measurements in clusters of normal rat kidney (NRK) fibroblasts were used to study intercellular communication. In the whole-cell patch-clamp configuration capacitive transients were evoked by applying small voltage pulses. Total membrane capacitance was calculated from these capacitive transients after determination of access resistance, membrane conductance, and the decay constant of the transients, or alternatively by integrating the current transient. We found that in clusters of one to ten cells, membrane capacitance increased linearly with cell number, showing that the cells are electrically coupled. Membrane conductance of the cluster of cells also increased, as expected for cells that are well coupled. In subconfluent and confluent cultures, high membrane conductances together with large capacitive transients were observed, indicative of electrical coupling. Capacitance could only be determined qualitatively under these conditions, due to space clamp problems. In the presence of the gap junctional inhibitors halothane, heptanol or octanol, capacitance of all clusters of cells fell to single-cell levels, showing a complete uncoupling of the cells. The tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also uncoupled the cells completely, with 10 min. We conclude that capacitance measurements can provide a useful tool to study changes in intercellular communication in clusters of cells.

Alcohols↗
PubMed1983

Studies of the guinea pig epididymis. II. Intercellular junctions of principal cells.

The junctional complexes of the principal cells in the guinea pig epididymis were analyzed using freeze fracture and ultrathin section goniometric techniques. Replicas of the seven regions (I to VII) investigated reveal a continuous decrease in the number of tight junctional strands, ranging from 15.73 +/- 3.54 in zone I (proximal) to 4.39 +/- 0.78 in zone VII (distal tubule). The distance from the adluminal to the basolateral strand also diminishes from proximal, 0.73 +/- 0.02 micron to distal, 0.19 +/- 0.03 micron. The junctional strands appear on the P-face and anastomose forming compartments which are larger in the basolateral areas than those in the apical. The network of strands frequently form terminal loops and blind endings towards the more basal parts of the lateral membrane. Freeze fracture images also exhibit randomly distributed particulate aggregations which correspond to maculae adhaerentes, the highest number of which are found in zone IV, V, and VII. Desmosomal figures are found not only below, but also adjacent and intermingled among the tight junctional strands. This special junctional arrangement is confirmed upon goniometric analysis of ultrathin sections from zones IV, V, and VII. Electron dense desmosomal plaques are seen parallel and directly subjacent to the membranes of the tight junctions, following the strands in both directions to finally converge on the punctiform connections. Goniometry also reveals a dense feltwork of material closely applied along the lateral cell border. These zonulae adhaerentes are seen to be of greatest length and density in zones I, VI, and VII.

Animals↗
PubMed1991

Perineurium of sciatic nerve in normal and diabetic rodents: freeze-fracture study of intercellular junctional complexes.

A comparative study has been carried out using the freeze-fracture technique on the perineurium of the sciatic nerve from normal and diabetic mice (C57Bl/Ks, BALB/c and CD1 strains) and rats of various ages. The replicas showed that tight junctions connected perineurial cells both within the same cell layer (zonulae occludentes) and between adjacent layers (maculae occludentes). In neonates, a number of zonulae occludentes were characterized by short, incomplete or fragmented ridges at various intervals from each other; in adults, tight junctions appeared as 'mature' networks of interconnected, branching and/or anastomosing strands. Zonulae occludentes of diabetic mice also exhibited frequent interruption of the strands and reduction in the branching of strands. Gap junctions occurred in both zonulae and maculae occludentes of normal and diabetic rats at all ages. In the C57Bl/Ks strain such junctions occurred more frequently in zonulae occludentes of diabetic animals. It is suggested that perineurial cells are coupled by gap junctions to allow fast transfer of ions and small-sized molecules across the layers; under pathological conditions, such as diabetes, the increase in cell-to-cell signalling may be important in controlling the abnormal metabolic situation.

Aging↗
PubMed1985

Intercellular junctions in the developing rat corneal endothelium.

The surface events which occur during the morphogenesis of the corneal endothelium were studied in the rat with special attention to intercellular contacts, using scanning and transmission electron microscopy, and freeze-fracture technique. Confluency was reached at the 16th-17th day of fetal development. As soon as adjacent cells make contact, marginal interdigitated finger-like processes developed and apical cell junctions appeared, namely tight and gap junctions. Tight junction networks progressively developed, girdling the apical side of lateral membranes, nevertheless, they remained very irregular in their apicobasal thicknesses. At every stage studied, neutralized lanthanum used as an ultrastructural tracer was seen filling the intercellular clefts and junction domains. Hence, the apical junctions do not constitute an impermeable barrier to this tracer in the developing corneal endothelium.

Animals↗
PubMed1980

Thin section and freeze-fracture studies of hexachlorophene induced alterations in the rat liver with special regard to intercellular junctions.

The ultrastructural changes in the liver cells of male Wistar rats induced by the oral and intraperitoneal application of hexachlorophene (HCP) were studied by using the freeze-fracture technique and by producing ultrathin sections. The rats were given the HCP once at a dose of 30 mg/kg body weight and were sacrificed 60 hours after treatment. The enlargement of bile canaliculi, the disorganization of tight junctions and the increase in number of gap junctions were the most prominent alterations observed in the liver cells. Furthermore, an increase in the smooth and rough endoplasmic reticulum and the microbodies were found. Pathological alterations in the mitochondria were also seen. In general, the effects of the intraperitoneal application on the liver were more pronounced than the effects of the oral application.

Animals↗
PubMed1976

Freeze-fracture observations on the intercellular junctions of Sertoli cells and of Leydig cells in the human testis.

Normal and feminized human testes were examined by means of freeze-fracture. In both cases, the junctional complexes between adjacent Sertoli cells show a unique characteristic feature. Many parallel linear occluding junctions are located circumferentially around the cell surface at the level of the nucleus. They are more than forty in number over one Sertoli cell surface. In the A face, the particles are not prominent on the ridge of the junctions. Instead, they are clearly seen in the center of the groove of the junctions on the B face. Gap junctions consisting of particle aggregation are not found between the Sertoli cells. The gap junctions between the Leydig cells are frequently observed in both normal and feminized testes. They are round or elliptic in contour and about several micrometers in diameter. The particles, about 8 nm in diameter, are closely packed to form a quasi-hexagonal pattern. The pits are found on the B face corresponding to the particle aggregation.

Adult↗
PubMed1982

Unorthodox pattern of microvilli and intercellular junctions in regular retinular cells of the porcellanid crab Petrolisthes.

1. Retinular fine structures in compound eyes of the porcellanid crab Petrolisthes differs significantly from two paguroid anomurans Clibanarius and Pagurus which basically conform to the usual conservative decapod crustacean retinular pattern. 2. Bidirectional orientation of microvilli has been discovered in rhabdomeres of retinular cells R1-R7 in Petrolisthes. Distally the regular rhabdom has mainly a typical banded microvillus structure (Figs. 7,8). Proximally rhabdom banding continues but uniquely all seven regular retinular cells contribute sets of alternately orthogonal microvilli to each band (Figs. 5, 6, 12). This unorthodox pattern should reduce polarization sensitivity and enhance sensitivity to unpolarized light. 3. In this special region microvillus layers are strongly elliptical in cross section with the minor axis parallel to the microvilli (Fig. 12). Hence the ends of the major axes protrude considerably from the central area of overlap (Fig. 6). 4. Retinular cell eight has bidirectional microvilli (Figs. 5-7) as usual in brachyuran crabs. Unlike the latter as well as paguroid crabs, Petrolisthes has square facets and a rectangular retinular array (Figs. 1, 3) similar to other galatheids and macruran decapods generally. It also resembles macrurans (shrimps and lobsters) in having perirhabdomal vacuoles absent or much reduced. 5. Tight junctions occur widely between adjacent retinular cells (Figs. 14, 17) especially basally immediately distal to longitudinal zonular adherentes (Figs. 6, 16) typical of compound eyes. Freeze fracture reveals in addition numerous rectangular arrays of particles on the protoplasmic face of retinular cell membrance near, but not part of, the rhabdom (Figs. 19, 20). Other authors have hypothesized polarized transfer functions for similar particle aggregates in certain vertebrate cells.

Animals↗
PubMed1978

[Structural diversity of communicating intercellular junctions (gap junctions) in the posterior cecal epithelium of the crustacean Orchestis].

Two types of gap junction are described, they are different in the size, the spacing and the polarity of their intramembrane particles. The dense type shows two kinds of particles which rather remain joined to the P fracture face. The loose type shows larger and more spaced out particles which rather are held by the E fracture face. These observations demonstrate that one needs to shade the distinction between P-type gap (Vertebrate) and E-type gap (Arthropoda).

Animals↗
PubMed1979

Fine structure of the ependyma and intercellular junctions in the area postrema of the rat.

Ependymal cells and their junctional complexes in the area postrema of the rat were studied in detail by tracer experiments using horseradish peroxidase (HRP) and colloidal lanthanum and by freeze-etch techniques, in addition to routine electron microscopy. The ependyma of the area postrema is characterized as flattened cells possessing very few cilia, a moderate amount of microvilli, a well-developed Golgi apparatus and rough endoplasmic reticulum. Numerous vesicles or tubular formations with internal dense content were found to accumulate in the basal processes of ependymal cells; the basal process makes contact with the perivascular basal lamina. It is suggested that the dense material in the tubulovesicular formations is synthesized within the ependymal cell and discharged into the perivascular space. The apical junctions between adjacent ependymal cells display very close apposition, with a gap of 2--3 nm, but no fusion of adjacent plasma membranes; they thus represent a transitional form between the zonulae adhaerentes present in the ordinary mural ependyma and the zonulae occludentes in the choroidal epithelium. A direct intercommunication between the ventricular cerebrospinal fluid (CSF) and the blood vascular system indicates that a region exists lacking a blood-ventricular CSF barrier.

Animals↗
PubMed1975

[Intercellular junctions in the guinea pig stria vascularis as shown by freeze-etching (author's transl)].

Freeze-fracture replicas of the guinea pig inner ear were analyzed to study the occurrence and structure of zonulae and fasciae occludentes (tight junctions) and nexuses (gap junctions) in the stria vascularis. Zonulae occludents of the marginal cells, which form a barrier towards the potassium-rich endolymph of the cochlear duct, were demonstrated to be of the "intermediate to tight" type (Claude and Goodenough, 1973). Abundant, extensive cell contacts of the basal cells were identified as fasciae occludentes forming a barrier towards the spiral ligament. Nexuses were found connecting marginal, intermediate and basal cells of the stria vascularis as well as between the basal cells and fibrocytes of the spiral ligament. Frequently, nexuses were seen within the areas of the basal cell fasciae occludentes. It is supposed that nexuses mediate the electrotonic coupling between neighbouring stria vascularis cells and that probably they are an essential factor for the development of the endocochlear potential. It is pointed out that the intercellular spaces of the stria vascularis represent a compartment with high oxygen supply and an important metabolic system. A high intramembrane particle density was observed on some nonjunctional areas of the cell membranes, especially on the A-fracture faces of the marginal cell extensions. It appears that these membranes exhibit a high metabolic activity. Finally, the findings are discussed with respect to the production and maintenance of the endocochlear potential.

Animals↗
PubMed1977

Intercellular junctions, intramembranous particles and cytoskeletal elements of deep cells of the Fundulus gastrula.

The fine structure of motile deep cells of the gastrula stage of Fundulus heteroclitus was studied with transmission electron microscopy, using both thin sectioning and freeze-cleave techniques. Gastrula deep cells form extensive non-junctiona appositions with each other, in which the apposed plasma membranes are parallel and separated by a distance of 26-28 nm. They also form gap junctions. Tight junctions, desmosomes, and extensive interdigitations of apposed plasma membranes were not observed. The plasma membranes of deep cells contain numerous unclustered intramembranous particles. Cytoplasmic microtubules were found, but they appear to be small in number, sparsely distributed, and mainly randomly oriented. Microfilaments are also present and are localized largely in the cortical cytoplasm and in thin cell extensions. The significance of these findings for the contact and locomotory behavior of deep cells is discussed.

Animals↗