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PubMed1982

Permeability and structural studies of heart cell gap junctions under normal and altered ionic conditions.

The permeability and ultrastructure of communicating junctions of cultured neonatal rat ventricular cells are examined under control conditions and during treatments which raise intracellular Ca2+. Lucifer Yellow (487 mol wt) is used to examine junctional permeability. Under normal ionic conditions dye transfer from an injected muscle cell to neighboring muscle cells occurs rapidly (in less than 6 sec) while transfer to neighboring fibroblasts occurs more slowly. Application of monensin, which results in a partial contracture with superimposed asynchrony, or A23187, which results in a partial contracture, do not inhibit the transfer of dye between the muscle cells. A23187 did result in junctional blockade between muscle cells and fibroblasts. Freeze-fractured gap junctions from control and monensin-treated cells exhibit no distinguishable differences. Center-to-center spacing was not significantly different, 9.0 nm +/- 1.4 SD versus 9.2 nm +/- 1.3 SD, respectively; and particle diameters were virtually unchanged, 8.69 nm +/- 0.9 SD versus 8.61 nm +/- 1.07 SD, respectively. These results suggest that concentrations of intracellular Ca2+ sufficient to support a partial contracture and asynchronous contractile activity do not result in a block of intercellular junctions in cultured myocardial cells. These results are discussed in terms of intracellular Ca2+ -buffering and junctional sensitivity to Ca2+.

Animals↗
PubMed1997

Interendothelial junctions in normal human Schlemm's canal respond to changes in pressure.

PURPOSE: To determine if changes in the structure and complexity of junctions between endothelial cells lining Schlemm's canal (SC) occur in normal human eyes with changes in perfusion pressure. METHODS: Twelve normal human eyes were either perfusion-fixed (at 15 or 45 mm Hg) or immersion-fixed (0 mm Hg) in modified Karnovsky's fluid. 'Outflow facility was measured continually during the perfusion fixation. The intercellular junctions of the endothelial cells of SC were ultrastructurally examined in thin sections, including serial sections and freeze-fracture replicas. Morphometric data on the number of junctional strands per total length of tight junction were documented and categorized by the number of strands (one, two, or three or more). The length of endothelial cell overlap was measured on thin sections. RESULTS: In freeze-fracture replicas, perfusion-fixed eyes demonstrated less complex junctions. At 15 mm Hg, 18.06% of the total junctional length was represented by three or more strands; at 45 mm Hg, this percentage decreased to 8.59%. In immersion-fixed eyes, 24.17% of the total junctional length was represented by three or more strands. These differences were statistically significant (P < 0.0012). In sections, the amount of endothelial cell overlap, and thus the length of paracellular pathway, was reduced in perfusion-fixed versus immersion-fixed eyes (P < 0.02). Extensive serial sectioning demonstrated that giant vacuoles were formed, either by individual endothelial cells or by two or more adjacent endothelial cells. CONCLUSIONS: When compared with specimens fixed at zero pressure, overlap between endothelial cells of SC is reduced significantly when this cell layer is under conditions of flow similar to those encountered in vivo. The tight junctions between cells of the inner wall of SC become less complex with increasing pressure. Our data suggest that the paracellular pathway into SC in the normal eye is sensitive to modulation within a range of physiologically relevant pressures.

Aged↗
PubMed1980

Malignant schwannoma: a light microscopic and ultrastructural study.

The light microscopic and ultrastructural features of 3 cases of malignant schwannoma were studied and compared with those of other types of soft-tissue sarcoma. The tumor in 1 of these cases originated in an intercostal nerve and was composed of compactly arranged spindle-shaped tumor cells. The other 2 cases showed osteogenic areas in addition to exhibiting prominent nuclear palisading, focal myxoid changes, and a rosette-like arrangement of tumor cells. The tumor in 1 of these latter cases occurred at the site of a preexisting neurofibroma. The Schwann cell origin of these tumors is strongly supported by the ultrastructural findings of basement membranes and conspicuous intercellular junctions in all 3 cases and dense-core granules in 1.

Basement Membrane↗
PubMed1992

Inhibition of intercellular communication in human keratinocytes by fractionated asphalt fume condensates.

Asphalt fume condensate (AFC) and chromatographically separated fractions have been shown to cause cancer in mouse skin. The levels of known carcinogenic initiators in these complex mixtures, however, are considered too low to account for their carcinogenic potency. It has been proposed that AFC may contain co-carcinogenic or tumor-promoting agents in addition to carcinogenic initiators. Modulation of gap junctional intercellular communication (GJIC) has been implicated as an important effect of tumor promoters. In this study, we examined the effect of five chromatographically generated fractions of AFC on GJIC in cultured human epidermal keratinocytes (HEK). HEK cells were exposed overnight to medium containing DMSO extracts of AFC fractions. GJIC was evaluated by dye-coupling of microinjected Lucifer Yellow CH. All AFC fractions produced a concentration-dependent inhibition of GJIC. The apparent potency of each fraction correlated with its relative polarity based on HPLC elution characteristics. Cells with reduced GJIC as a result of AFC fraction exposure were found to exclude propidium iodide, suggesting that inhibition of GJIC occurred in the absence of cell killing. However, significantly reduced culture DNA content was found following the overnight exposure to the highest concentrations of AFC fractions C, D and E.

Cell Communication↗
PubMed1987

Chemical synapses, particle arrays, pseudo-gap junctions and gap junctions of neurons and glia in the buccal ganglion of Helisoma.

The nervous system of the snail, Helisoma trivolvis, has been utilized for a wide range of studies of neuronal plasticity; however, the ultrastructural features of this tissue were previously unknown. The present study examined the nature of synaptic interactions of neurons and glia and considered several plasma membrane specializations of these cells. The symmetrical pair of buccal ganglia consisted of a ring of unipolar neurons surrounding a central neuropil. The neurons were separated by two morphologically distinct types of glia: type I were most numerous and possessed an electron-dense homogeneous cytoplasm, whereas type II glia were of lower electron density, possessed a heterogeneous cytoplasm, and appeared to be phagocytic. Gap junctions were abundant between glia and were occasionally found between neuronal processes, including those of neurons 19 injected with horseradish peroxidase (HRP). Comparison of neuron and glial gap junction widths (16.4 and 17.6 nm, respectively) in thin sections and their intramembrane particle diameters (13.1 and 13.7 nm, respectively) by freeze fracture, did not elucidate significant differences. A heterogeneous population of putative chemical synapses, similar to those reported in other molluscs, was also observed between axonal collaterals in the neuropil. Additionally, examination of freeze-fractured neuropil revealed rhombic arrays of particles localized on neuronal membranes; these arrays do not appear to form intercellular junctions but may represent postsynaptic receptor sites. Freeze fracture also revealed small, square arrays consisting of 7-9 nm diameter particles on glial membranes which may correspond to pentalaminar membrane contacts (pseudo-gap junctions) seen in thin sections between glia situated around dilated extracellular spaces (lacunae).

Animals↗
PubMed1979

Transcapillary transport after thermal injury.

The pathophysiology of the burn wound is characterized by an inflammatory reaction leading to rapid edema formation due to (1) dilatation of resistance vessels with increased effective transcapillary filtration pressure, (2) increased extravascular osmotic activity created in damaged tissue, and (3) increased microvascular permeability to macromolecules. In extensive burns increased microvascular permeability was found also in tissues remote from the thermal injury. These reactions are due to direct heat effect on the microvasculature and to chemical mediators of inflammation. Important is the increased biosynthesis of prostaglandins at the site of tissue injury which may partly explain vasodilatation, increased microvascular permeability and accumulation of polymorphonuclear leucocytes observed following thermal injury. The morphological interpretations of the changes in the functional ultrastructure of the blood-lymph barrier following thermal injury seem to be a remarkable and persistant increase in the numbers of vacuoles and many open endothelial intercellular junctions. Further less explored changes of the interstitial tissue after severe burn trauma seem to be of great importance.

Animals↗
PubMed2006

Junctional expression of the prion protein PrPC by brain endothelial cells: a role in trans-endothelial migration of human monocytes.

The conversion of prion protein (PrP(C)) to its protease-resistant isoform is involved in the pathogenesis of prion diseases. Although PrP(C) is highly expressed in neurons and other cell types, its physiological function still remains elusive. Here, we describe how we evaluated its expression, subcellular localization and putative function in brain endothelial cells, which constitute the blood-brain barrier. We detected its expression in microvascular endothelium in mouse brain sections and at intercellular junctions of freshly isolated brain microvessels and cultured brain endothelial cells of mouse, rat and human origin. PrP(C) co-localized with the adhesion molecule platelet endothelial cell adhesion molecule-1 (PECAM-1); moreover, both PrP(C) and PECAM-1 were present in raft membrane microdomains. Using mixed cultures of wild-type and PrP(C)-deficient mouse brain endothelial cells, we observed that PrP(C) accumulation at cell-cell contacts was probably dependent on homophilic interactions between adjacent cells. Moreover, we report that anti-PrP(C) antibodies unexpectedly inhibited transmigration of U937 human monocytic cells as well as freshly isolated monocytes through human brain endothelial cells. Significant inhibition was observed with various anti-PrP(C) antibodies or blocking anti-PECAM-1 antibodies as control. Our results strongly support the conclusion that PrP(C) is expressed by brain endothelium as a junctional protein that is involved in the trans-endothelial migration of monocytes.

Animals↗
PubMed2005

Cellular fluid mechanics and mechanotransduction.

Mechanotransduction, the transformation of an applied mechanical force into a cellular biomolecular response, is briefly reviewed focusing on fluid shear stress and endothelial cells. Particular emphasis is placed on recent studies of the surface proteoglycan layer (glycocalyx) as a primary sensor of fluid shear stress that can transmit force to apical structures such as the plasma membrane or the actin cortical web where transduction can take place or to more remote regions of the cell such as intercellular junctions and basal adhesion plaques where transduction can also occur. All of these possibilities are reviewed from an integrated perspective.

Actin Cytoskeleton↗
PubMed1983

A simplified method for the rapid isolation of cardiac intercalated discs.

A simple and rapid method for the isolation of intercalated discs from a single rat heart is described. The outstanding features of this method are (i) a large quantity of starting material is not necessary, (ii) ultracentrifugation and separating gradient steps are not required, (iii) the disc fraction is relatively pure as verified by electron microscopy, and (iv) the starting tissue is rapidly homogenized into buffer, reducing possible damage to membrane structures after animal death. The fraction is rich in intercellular junctions, with gap junctions, fasciae adherentes and maculae adherentes all appearing well preserved. It should prove a useful starting base for further purification of these junctions and of sarcolemma from a specific portion of the cell surface membrane, namely the major area of structural interaction between adjacent cardiac cells.

Animals↗
PubMed1987

The ultrastructure of membranes in sympathetic ganglia.

This review presents some recent observations made on membranes in the mammalian sympathetic ganglia after application of freeze-fracturing and histochemistry. After freeze-fracture clear differences have been found between the neuronal and the satellite cell plasmalemma. The satellite cell plasma membrane exhibits specialized intramembrane particles not found in the neuronal membrane. Freeze-fracture cytochemistry reveals a further difference between the neurons and satellite cells i.e., a higher density of beta-hydroxysterols is present in the satellite cell membrane than in the neuronal membrane. Histochemical methods to localize 5'-nucleotidase, Ca2+-ATPase and Na+/K+-ATPase in the membranes have been utilized. The cytochemical reaction products of 5'-nucleotidase and Ca2+-ATPase were found on the external aspect of the plasma membranes. The Na+/K+-ATPase reaction product was located on the cytoplasmic aspect of the membranes, with most activity seen in the satellite cell membrane. With respect to the intercellular junctions, the presynaptic membranes have been analyzed with the freeze-fracture technique under stimulated and unstimulated conditions and ultrastructural differences have been observed which might be correlated to neurotransmission. Furthermore, gap junctions and tight junctional elements occur between the satellite cells surrounding the neuronal perikarya and their processes.

5'-Nucleotidase↗
PubMed1981

Paracellular route of aqueous outflow in the trabecular meshwork and canal of Schlemm. A freeze-fracture study of the endothelial junctions in the sclerocorneal angel of the macaque monkey eye.

The intercellular junctions of the endothelial cells of the trabecular meshwork and canal of Schlemm were examined with the electron microscope in the macaque monkey eye by both thin-sectioned specimens and the freeze-fracturing technique. The endothelial cells that line the beams of the meshwork are joined by gap junctions and short, isolated strands of tight junction; zonulae occludentes are absent. Thus aqueous humor can freely traverse the patent endothelial clefts of the trabecular meshwork. The endothelial cells of the canal of Schlemm are joined by zonulae occludentes and a small number of minute gap junctions. In 57% of their length, the tight junctions consist of one or two strands; the strands are rarely more than four. They remain preferentially associated with the E-face of the membrane, run parallel to one another, and only exceptionally branch or anastomose. Thus they are provided with free endings and do not form a bidimensional network. As a result of this organization, the zonula occludens is traversed by meandering channels of extracellular space or split pores, which connect the open endothelial clefts on the luminal and tissue fronts of the junction. The frequency of slit pores is 0.134 per micrometer of zonula occludens. They occupy 0.87% of the intercellular boundary and 0.0015% of the area of the endothelium. Estimates of the fluid conductance of the zonulae occludentes indicate that the intercellular clefts of the endothelium of Schlemm's canal filter but a small fraction of the amount of aqueous humor that leaves the anterior chamber through the conventional route.

Animals↗
PubMed1998

Cadherin mediated cell-cell adhesion is regulated by tyrosine phosphatases in human keratinocytes.

Normal Human Keratinocytes express on their cell surface E- and P-cadherins, two Ca2+ dependent homophilic cell adhesion molecules that mediate keratinocyte-keratinocyte adherens junctions. In other cell types, adherens-type junctions are reported to be major subcellular targets for tyrosine specific protein phosphorylation (Volberg et al. (1991) Cell Regul., 2, 105-120) involving tyrosine kinases and tyrosine phosphatases. We investigated the role of tyrosine phosphatases in the regulation of cadherin mediated keratinocyte-keratinocyte adhesion. We report the results of a wide tyrosine phosphatase inhibition using pervanadate, a modified vanadate derivative known to inhibit most tyrosine phosphatases. Keratinocytes treated with pervanadate, exhibit an important change in cellular morphology and cadherins/catenins localization as shown by phase contrast microscopy and immunocytochemistry. In this conditions, cadherins and catenins no longer colocalize with the actin cytoskeleton of cells and the amount of E-cadherin bound to the cytoskeleton decreases. A more intense phosphotyrosine labelling is seen at the edges of the treated cells, suggesting that an increase in the phosphorylation rate of some cadherin-catenin complex proteins induces a diminished intercellular adhesion. Finally immunoprecipitation experiments of the E-cadherin/catenin complex from pervanadate treated keratinocytes reveal an increase in the tyrosine phosphorylation rate of E-cadherin, beta catenin and probably gamma catenin. These data suggest an essential role for the protein tyrosine phosphatases in keratinocyte intercellular junctions.

Actins↗
PubMed1999

Astrocytes induce several blood-brain barrier properties in non-neural endothelial cells.

The passage of immunocompetent cells across the blood-brain barrier (BBB) is regulated at the level of the cerebral capillaries which have specific morphological and biochemical properties. We have developed and characterized an in vitro model of the BBB using immortalized human endothelial cells (ECV 304) induced by rat astrocytes. In this model, endothelial cells are attached together by continuous intercellular junctions with numerous tight junctions, develop a permeability barrier having a significant transcellular electrical resistance, possess high activities of gamma-glutamyl transpeptidase (gamma-GTP) and express the brain-type glucose transporter 1 (GLUT-1). These parameters are also characteristic of brain capillary endothelial cells. Under the culture conditions used, the ECV 304 cells express the intercellular adhesion molecule-1 (ICAM-1) on the external plasma membrane at concentrations which could permit lymphocyte adhesion to be studied.

Animals↗
PubMed2003

Lamellipodium extension and cadherin adhesion: two cell responses to cadherin activation relying on distinct signalling pathways.

Cell adhesion molecules of the cadherin family contribute to the regulation of cell shape and fate by mediating strong intercellular adhesion through Ca2+-dependent interaction of their ectodomain and association of their cytoplasmic tail to actin. However, the mechanisms co-ordinating cadherinmediated adhesion with the reorganization of the actin cytoskeleton remain elusive. Here, the formation of de novo contacts was dissected by spreading cells on a highly active N-cadherin homophilic ligand. Cells responded to N-cadherin activation by extending lamellipodium and organizing cadherin-catenin complexes and actin filaments in cadherin adhesions. Lamellipodium protrusion, associated with actin polymerization at the leading edge sustained the extension of cadherin contacts through a phosphoinositide 3-kinase (PI 3-kinase)-Rac1 pathway. Cadherin adhesions were formed by PI 3-kinase-independent, Rac1-dependent co-recruitment of adhesion complexes and actin filaments. The expression and localization of p120 at the plasma membrane, associated with an increase in membrane-associated Rac1 was required for both cell responses, consistent with a major role of p120 in signalling pathways initiated by cadherin activation and contributing to Rac1-dependent contact extension and maturation. These results provide additional information on the mechanisms by which cadherin coordinates adhesion with dynamic changes in the cytoskeleton to control cell shape and intercellular junction organization.

Animals↗
PubMed1986

Reassembly of the occluding junctions in a renal cell line with characteristics of proximal tubular cells.

LLC-PK1 cells from trypsin-treated confluent cultures formed a continuous monolayer when plated at high cell density on collagen-coated Nuclepore filters. These monolayers developed a significant transepithelial electrical resistance that reached a maximum at 20 h. At 48 h, the resistance decreased to a value usually one-half the value obtained at 20 h. These changes were associated with an increase in the cell density of the monolayers. The drop in electrical resistance at 48 h was not observed when cell growth was arrested with excess thymidine. A hyperbolic relationship was demonstrated between cell density and electrical resistance. Although the increase in cell density was associated with an increase in the unidirectional flux of mannitol across the monolayers, selectivity studies indicated that the intrinsic properties of the occluding junctions were similar in the high and low cell density monolayers. These results indicate that, when cell growth is not arrested, changes in transepithelial electrical resistance observed after plating correspond to an increase in cell density and not to changes in the intrinsic properties of the occluding junctions. The development of transepithelial electrical resistance was delayed when the cells were in exponential growth. No such delay was observed, however, when exponential growth was synchronized. These findings and results obtained with the antimicrotubular agent Nocodazole indicate that the delay in the development of transepithelial electrical resistance is due to the asynchronous transit of the cells through the mitotic phase of the cell cycle: a time when most of the intercellular junctions are probably disrupted. Further investigation revealed that inhibition of protein but not mRNA synthesis blocked the development of occluding junctions in cells from confluent and exponentially growing cultures alike. These results indicate that, in contrast to MDCK cells, regulation of the occluding junctions in exponentially growing LLC-PK1 cells occurs at the translational not at the transcriptional level of protein synthesis.

Animals↗
PubMed1983

The blood-testis barrier in the toad (Bufo arenarum Hensel): a freeze-fracture and lanthanum tracer study.

Intercellular junctions between Sertoli cells in the toad testis were studied by freeze-fracture and electron-opaque intercellular markers. These junctional specializations are characterized in thin sections by a series of focal fusions on the outer leaflets of both adjacent cell plasmalemmas, associated with bundles of fine filaments in the subjacent Sertoli cell cytoplasms. However, the wide subsurface cisterna of the endoplasmic reticulum, a component constantly associated with Sertoli cell junctions in mammals, is absent in the toad. The intravascularly injected lanthanum hydroxide, used as a tracer compound, gains access to the seminiferous tubules and surrounds spermatogonia and leptotene spermatocytes, but is persistently excluded from germ cells in later stages of development. This indicates that, as is the case in the mammalian testis, a permeability barrier to lanthanum is established which isolates all germ cells beyond leptotene spermatocytes. Freeze-fracture reveals the characteristic occluding junctions between Sertoli cells, but a variation in their geometric patterns was clearly observed in different regions of the toad seminiferous epithelium. The membrane-fractured faces of Sertoli cells embracing differentiating spermatids exhibit a deep junctional complex: up to 50 rows of particles between adjacent Sertoli cells separate these late germ cells from the periphery of the seminiferous tubules. Sertoli cells surrounding early germ cells generally exhibit, instead, a discontinuous, poorly developed network of interconnected rows of particles with few widely spaced strands. This seems to permit the percolation of the intercellular marker in areas of the seminiferous epithelium containing spermatogonia and leptotene spermatocytes.

Animals↗
PubMed2003

The conditional inactivation of the beta-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility.

Using the Cre/loxP system we conditionally inactivated beta-catenin in endothelial cells. We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos; however, vascular patterning in the head, vitelline, umbilical vessels, and the placenta was altered. In addition, in many regions, the vascular lumen was irregular with the formation of lacunae at bifurcations, vessels were frequently hemorrhagic, and fluid extravasation in the pericardial cavity was observed. Cultured beta-catenin -/- endothelial cells showed a different organization of intercellular junctions with a decrease in alpha-catenin in favor of desmoplakin and marked changes in actin cytoskeleton. These changes paralleled a decrease in cell-cell adhesion strength and an increase in paracellular permeability. We conclude that in vivo, the absence of beta-catenin significantly reduces the capacity of endothelial cells to maintain intercellular contacts. This may become more marked when the vessels are exposed to high or turbulent flow, such as at bifurcations or in the beating heart, leading to fluid leakage or hemorrhages.

Actins↗