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PubMed2001

Morphological examination of the effects of defibrotide on experimentally induced bladder injury and its relation to interstitial cystitis.

This morphological study aims to investigate the effects of defibrotide, a deoxyribonucleic acid derivative drug with cytoprotective, immunosuppressive and vasorelaxant effects, on protamine sulfate induced bladder injury. Wistar albino female rats were catheterized and intravesically infused with phosphate buffered solution (control group) or, either protamine sulfate (bladder injury group) or protamine sulfate+defibrotide (bladder injury+defibrotide group) dissolved in phosphate buffered solution. The morphology of the urinary bladder was investigated using light and electron microscopy. The number of mast cells in the mucosa, mucosal alterations, intercellular junctions, surface topography and the glycosaminoglycan (GAG) layer as well as microvillus formation on the luminal surface were evaluated. In the bladder injury group, ulcerated areas, irregularity of the GAG layer, increased number of mast cells, vacuole formation, dilated perinuclear cistern, formation of pleomorphic and uniform microvilli and dilatations in the intercellular spaces in the urothelium were observed. In the bladder injury+defibrotide group a relatively normal urothelial topography, GAG layer and a few mast cells in the mucosa, some dilatations between the intercellular areas, less uniform microvilli, regular perinuclear cistern and tight junctions were observed. These results show that defibrotide can inhibit PS induced bladder damage.

Animals↗
PubMed2002

IL-1beta-induced production of metalloproteinases by synovial cells depends on gap junction conductance.

Synovial cells can form networks connected by gap junctions. The purpose of this study was to obtain evidence for a necessary role of gap junction intercellular communication in protein secretion by synovial cells. We developed a novel assay to measure the enzymatic activity of metalloproteinases (MMPs) produced by synovial cells in response to interleukin-1beta (IL-1beta) and employed the assay to explore the biological function of gap junctions. IL-1beta produced a dose-dependent increase in MMP activity that was blocked by exposure to the gap junction inhibitors 18alpha-glycyrrhetinic acid and octanol for as few as 50 min. The inhibitors produced an immediate and marked reduction in intercellular communication, as assessed by transient current analysis using the nystatin perforated-patch method. These observations suggest that communication through gap junctions early in IL-1beta signal transduction is critical to the process of cytokine-regulated secretion of MMPs by synovial cells.

Animals↗
PubMed1999

Inhibition of gap junction communication in alveolar epithelial cells by 18alpha-glycyrrhetinic acid.

Cultured alveolar epithelial cells exhibit gap junction intercellular communication (GJIC) and express regulated levels of connexin (Cx) 43 mRNA and protein. Newly synthesized radiolabeled Cx43 protein equilibrates with phosphorylated Cx43 isoforms; these species assemble to form both connexons and functional gap junction plaques. The saponin 18alpha-glycyrrhetinic acid (GA) rapidly and reversibly blocks GJIC at low concentrations (5 microM). Extended exposure to 18alpha-GA at higher concentrations causes inhibition of GJIC and time- and dose-dependent reductions in both Cx43 protein and mRNA expression. The latter toxic effects are paralleled by disassembly of gap junction plaques and are reversed less readily than acute effects on GJIC. These observations demonstrate 18alpha-GA-sensitive regulation of intercellular communication in epithelial cells from the mammalian lung and suggest a role for Cx43 expression and phosphorylation in acute and chronic regulation of GJIC between alveolar epithelial cells.

Animals↗
PubMed2006

Gap junctions modulate apoptosis and colony growth of human embryonic stem cells maintained in a serum-free system.

We investigated the gap junctional properties of human embryonic stem cells (hESC) cultivated in a serum-free system using sphingosine-1-phosphate and platelet-derived growth factor (S1P/PDGF). We compared this condition to hESC grown on Matrigel in mouse embryonic fibroblast conditioned medium (MEF-CM) or unconditioned medium (UM). We show that in all culture systems, hESC express connexins 43 and 45. hESC maintained in S1P/PDGF conditions and hESC grown in presence of MEF-CM are coupled through gap junctions while hESC maintained on Matrigel in UM do not exhibit gap junctional intercellular communication. In this latter condition, coupling was retrieved by addition of noggin, suggesting that BMP-like activity in UM inhibits gap junctional communication. Last, our data indicate that the closure of gap junctions by the decoupling agent alpha-glycyrrhetinic acid increases cell apoptosis and inhibits hESC colony growth. Altogether, these results suggest that gap junctions play an important role in hESC maintenance.

Animals↗
PubMed1973

Permeability of muscle capillaries to exogenous myoglobin.

Whale skeletal muscle myoglobin (mol wt 17,800; molecular dimensions 25 x 34 x 42 A) was used as a probe molecule for the pore systems of muscle capillaries. Diaphragms of Wistar-Furth rats were fixed in situ at intervals up to 4 h after the intravenous injection of the tracer, and myoglobin was localized in the tissue by a peroxidase reaction. Gel filtration of plasma samples proved that myoglobin molecules remained in circulation in native monomeric form. At 30-35 s postinjection, the tracer marked approximately 75% of the plasmalemmal vesicles on the blood front of the endothelium, 15% of those located inside and none of those on the tissue front. At 45 s, the labeling of vesicles in the inner group reached 60% but remained nil for those on the tissue front. Marked vesicles appeared on the latter past 45 s and their frequency increased to approximately 80% by 60-75 s, concomitantly with the appearance of myoglobin in the pericapillary spaces. Significant regional heterogeneity in initial labeling was found in the different segments of the endothelium (i.e., perinuclear cytoplasm, organelle region, cell periphery, and parajunctional zone). Up to 60 s, the intercellular junctions and spaces of the endothelium were free of myoglobin reaction product; thereafter, the latter was detected in the distal part of the intercellular spaces in concentration generally equal to or lower than that prevailing in the adjacent pericapillary space. The findings indicate that myoglobin molecules cross the endothelium of muscle capillaries primarily via plasmalemmal vesicles. Since a molecule of this size is supposed to exit through both pore systems, our results confirm the earlier conclusion that the plasmalemmal vesicles represent the large pore system; in addition, they suggest that the same structures are, at least in part, the structural equivalent of the small pore system of this type of capillaries.

Animals↗
PubMed2000

Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium.

Gap junction number and size vary widely in cardiac tissues with disparate conduction properties. Little is known about how tissue-specific patterns of intercellular junctions are established and regulated. To elucidate the relationship between gap junction channel protein expression and the structure of gap junctions, we analyzed Cx43 +/- mice, which have a genetic deficiency in expression of the major ventricular gap junction protein, connexin43 (Cx43). Quantitative confocal immunofluorescence microscopy revealed that diminished Cx43 signal in Cx43 +/- mice was due almost entirely to a reduction in the number of individual gap junctions (226 +/- 52 vs. 150 +/- 32 individual gap junctions/field in Cx43 +/+ and +/- ventricles, respectively; P < 0.05). The mean size of an individual gap junction was the same in both groups. Immunofluorescence results were confirmed with electron microscopic morphometry. Thus when connexin expression is diminished, ventricular myocytes become interconnected by a reduced number of large, normally sized gap junctions, rather than a normal number of smaller junctions. Maintenance of large gap junctions may be an adaptive response supporting safe ventricular conduction.

Animals↗
PubMed1987

Characterization of retinal pigment epithelial cells cultured on microporous filters.

Retinal pigment epithelium (RPE) cultured on microporous filter supports was compared to RPE cultured on plastic and evaluated for features characteristic of RPE in vivo. RPE cells grown on filters were cuboidal, formed junctional complex structures between cells, and had elaborate microvilli and basal infoldings similar to RPE in vivo, while RPE grown on plastic also formed intercellular junctions but appeared squamous and had few microvilli and basal infoldings. RPE grown on filters or plastic secreted an extracellular matrix at the basal surface and ingested isolated rat rod outer segments at the apical surface. RPE grown on filters coated with laminin or fibronectin became confluent more rapidly than RPE grown on uncoated filters, while RPE grown at the same density on filters coated with collagen type I did not become confluent. The laminin and fibronectin coatings did not alter the RPE cell morphology; however, cells seeded on collagen-coated filters grew in large disorganized clusters. RPE grown on laminin-coated filters formed functional tight junctions as evidenced by the capacity of RPE monolayers to prevent the bulk flow of medium and the passage of trypan blue across the filter. Radiolabeled sucrose and inulin were used to measure the paracellular flux through the tight junctions between cells. The passage of these tracers was linear over time, with the lower molecular weight tracer, sucrose, passing through the monolayer more readily than inulin. Values for the flux of radiolabeled bovine serum albumin across RPE monolayers fell between values for sucrose and inulin. The results from these studies show that RPE monolayers cultured on laminin-coated filters maintain a morphology similar to that of RPE in vivo, are capable of ingesting rod outer segments, and form a selectively permeable barrier to various tracers. This culture system should be useful for studies of transepithelial transport, secretion, endocytosis and exocytosis that require independent control of the extracellular environment at the apical and basolateral cell surfaces.

Animals↗
PubMedDate not supplied

Transport and function of cx26 mutants involved in skin and deafness disorders.

We examined the subcellular localization and function of several Cx26 mutants that exhibit both sensorineural deafness and various skin disease phenotypes. To facilitate these aims, all Cx26 mutants were tagged at the carboxyl-terminal with green fluorescent protein (GFP), which has previously been shown not to affect Cx26 transport, assembly or function. In this article we focus on two point mutations (R75W and DeltaE42) that occur in the first extracellular loop region of Cx26, a region hypothesized to be critical for correct hemichannel docking between contacting cells. In gap junctional intercellular communication (GJIC)-deficient HeLa cells, both R75W-GFP and DeltaE42-GFP were transported to the cell surface and assembled into gap junction-like structures. Neither R75W-GFP nor DeltaE42-GFP formed gap junctions that were permeable to Lucifer Yellow suggesting they are loss-of-function mutations. We also examined the phenotype of these two mutations in a rat epidermal keratinocyte (REK) cell line that is capable of undergoing differentiation. Using antibodies against several members of the connexin family reportedly expressed by epidermal keratinocytes, we found these cells endogenously expressed Cx43 and Cx26 but not Cx30, Cx32, or Cx37. When expressed in REK cells, similar to in HeLa cells, R75W-GFP and DeltaE42-GFP were assembled at the cell surface into structures that resembled gap junctions. Future experiments will examine the effect of the Cx26 mutants on the function and differentiation of these epidermal keratinocytes.

Animals↗
PubMed1995

Alveolar lining layer is thin and continuous: low-temperature scanning electron microscopy of rat lung.

The low-temperature electron microscope, which preserves aqueous structures as solid water at liquid nitrogen temperature, was used to image the alveolar lining layer, including surfactant and its aqueous subphase, of air-filled lungs frozen in anesthetized rats at 15-cmH2O transpulmonary pressure. Lining layer thickness was measured on cross fractures of walls of the outermost subpleural alveoli that could be solidified with metal mirror cryofixation at rates sufficient to limit ice crystal growth to 10 nm and prevent appreciable water movement. The thickness of the liquid layer averaged 0.14 micron over relatively flat portions of the alveolar walls, 0.89 micron at the alveolar wall junctions, and 0.09 micron over the protruding features (9 rats, 20 walls, 16 junctions, and 146 areas), for an area-weighted average thickness of 0.2 micron. The alveolar lining layer appears continuous, submerging epithelial cell microvilli and intercellular junctional ridges; varies from a few nanometers to several micrometers in thickness, and serves to smooth the alveolar air-liquid interface in lungs inflated to zone 1 or 2 conditions.

Animals↗
PubMed2001

Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta1 (TGF-beta1) alter connexin 43 phosphorylation in MC3T3-E1 Cells.

BACKGROUND: Bone morphogenetic proteins (BMPs) and transforming growth factor-betas (TGF-betas) are important regulators of bone repair and regeneration. BMP-2 and TGF-beta1 have been shown to inhibit gap junctional intercellular communication (GJIC) in MC3T3-E1 cells. Connexin 43 (Cx43) has been shown to mediate GJIC in osteoblasts and it is the predominant gap junctional protein expressed in these murine osteoblast-like cells. We examined the expression, phosphorylation, and subcellular localization of Cx43 after treatment with BMP-2 or TGF-beta1 to investigate a possible mechanism for the inhibition of GJIC. RESULTS: Northern blot analysis revealed no detectable change in the expression of Cx43 mRNA. Western blot analysis demonstrated no significant change in the expression of total Cx43 protein. However, significantly higher ratios of unphosphorylated vs. phosphorylated forms of Cx43 were detected after BMP-2 or TGF-beta1 treatment. Immunofluorescence and cell protein fractionation revealed no detectable change in the localization of Cx43 between the cytosol and plasma membrane. CONCLUSIONS: BMP-2 and TGF-beta1 do not alter expression of Cx43 at the mRNA or protein level. BMP-2 and TGF-beta1 may inhibit GJIC by decreasing the phosphorylated form of Cx43 in MC3T3-E1 cells.

Animals↗
PubMedDate not supplied

[Characterization of the desmoglein in renal cells in culture].

Desmosome junctions are found in epithelial tissues. They both link cells externally and anchor cytoplasmic intermediate filaments to the plasma membrane. Quantitative and qualitative abnormalities in intercellular junctions have been described in a broad spectrum of human and animal cancers. Current efforts are aimed at exploring the possibility that some of these defects may account for the hallmarks of malignancy, namely tumour invasion and metastasis. Desmosomes are constituted by several proteins, one of them is desmoglein-1 (DG-1), a transmembrane glycoprotein who glycosylated portion is major component of the adhesion mediating desmoglia. In order to know the similarity between tissue DG-1 and cultured renal cells DG-1 was used antisera raised against DG-1 to identify cross-reacting components. Anti DG-1 antibodies stained cell-cell boundaries in a punctate fashion in epithelial tissue and on densely grown monolayers of renal cells. Radioimmunoprecipitation and immunoelectrotransference show positive reaction with anti DG-1 antibodies with desmosomes obtained from epithelial tissue and renal cells monolayers, but last one was less positive. Results suggest some minor differences between DG-1 extracted from diverse sources but they have a commun immunodominant epitope.

Animals↗
PubMed1994

Cadherin expression in carcinomas: role in the formation of cell junctions and the prevention of invasiveness.

It has been realized that the loss of epithelial differentiation in carcinomas, which is accompanied by higher mobility and invasiveness of the tumor cells, is often a consequence of reduced intercellular adhesion. A variety of recent reports have indicated that the primary cause for the 'scattering' of the cells in invasive carcinomas is a disturbance of the integrity of intercellular junctions, often involving loss of a functional cell-cell adhesion molecule E-cadherin. It has also been suggested that during invasion, carcinoma cells convert to a sort of mesenchymal stage, as do normal epithelial cells during development. In the present review, permanent and transient molecular mechanisms are discussed which lead to the impairment of junction integrity of the epithelial cells and thus to the progression of carcinomas towards a more metastatic state. Furthermore, the now extensive literature on the down-regulation of E-cadherin expression in human and animal carcinomas is reviewed in detail.

Animals↗
PubMed2006

JAM-C regulates tight junctions and integrin-mediated cell adhesion and migration.

Junctional Adhesion Molecules (JAMs) have been described as major components of tight junctions in endothelial and epithelial cells. Tight junctions are crucial for the establishment and maintenance of cell polarity. During tumor development, they are remodeled, enabling neoplastic cells to escape from constraints imposed by intercellular junctions and to adopt a migratory behavior. Using a carcinoma cell line we tested whether JAM-C could affect tight junctions and migratory properties of tumor cells. We show that transfection of JAM-C improves the tight junctional barrier in tumor cells devoid of JAM-C expression. This is dependent on serine 281 in the cytoplasmic tail of JAM-C because serine mutation into alanine abolishes the specific localization of JAM-C in tight junctions and establishment of cell polarity. More importantly, the same mutation stimulates integrin-mediated cell migration and adhesion via the modulation of beta1 and beta3 integrin activation. These results highlight an unexpected function for JAM-C in controlling epithelial cell conversion from a static, polarized state to a pro-migratory phenotype.

Amino Acid Sequence↗
PubMed2005

Lipopolysaccharide decreases connexin 43 expression on nasal epithelial cells in vitro.

Decreased connexin 43 (Cx43) expression as a result of application of lipopolysaccharide (LPS) may limit the diffusion of intercellular signaling molecules that is essential to the coordinated function of neighboring cells. Therefore, it may be related to a ciliary beating defect in nasal epithelial cells and result in accumulation of harmful substances.Gap junction intercellular communication (GJIC) is altered during inflammation in tracheal epithelial cells. Thus, we aimed to investigate whether LPS affects the expression of Cx43, the elementary protein composing the gap junction of nasal epithelial cells, in vitro.LPS (Pseudomonas aeruginosa serotype 10) was applied to epithelial cells obtained from nasal polyp for 24 h in vitro. As an inflammatory indicator, IL-8 secretion was measured using a commercially available ELISA kit. Cx43 protein was detected semi-quantitatively using Western blotting. The nasal epithelial cells constitutively secreted IL-8 at a concentration of 0.45+/-0.03 ng/microg protein. In the presence of 10(-2) mg/ml LPS, the concentration of IL-8 was significantly increased to 0.55+/-0.05 ng/microg protein (n=8). Expression of the Cx43:beta-actin ratio decreased in a time- and dose-dependent fashion (10(-3)-10(-1) mg/ml LPS).

Adolescent↗
PubMed1996

Effect of tumor-promoting and anti-promoting chemicals on the viability and junctional coupling of human HeLa cells transfected with DNAs coding for various murine connexin proteins.

Gap-junctional intercellular communication is thought to be essential for maintaining cellular homeostasis and growth control. Its perturbation entails toxicological implications and it has been correlated with the in vivo tumor-promoting potential of chemicals. Little is known about the mechanism(s) responsible for the tumor promoters interference with the cellular coupling. Moreover, nongenotoxic carcinogens, as well as connexins (gap-junctional protein subunits), are known to be organ-/tissue-specific; this implies that the effect of different agents should be evaluated on their specific target, that is, connexin. To investigate the role of different connexins in regulating gap-junctional gating and to compare the properties of homotypic junctional channels, we evaluated the effects of tissue-specific tumor promoters and anti-promoters on the viability and intercellular coupling (dye-transfer) of HeLa cells stably transfected with cDNAs coding for connexin(cx)43, cx40, cx26 and cx32. The results demonstrate that the transfectants possess individual junctional permeabilities, differentially affected by the chemicals, they also show different sensitivities to the cytotoxic effect of the compounds. These findings confirm that connexin diversity may be responsible for the different gating properties of gap-junctional channels, being also suggestive for their separate functions and independent regulatory mechanisms.

Animals↗
PubMed2006

Endogenous hemichannels play a role in the release of ATP from Xenopus oocytes.

ATP is an electrically charged molecule that functions both in the supply of energy necessary for cellular activity and as an intercellular signaling molecule. Although controlled ATP secretion occurs via exocytosis of granules and vesicles, in some cells, and under certain conditions, other mechanisms control ATP release. Gap junctions, intercellular channels formed by connexins that link the cytoplasm of two adjacent cells, control the passage of ions and molecules up to 1 kDa. The channel is formed by two moieties called hemichannels, or connexons, and it has been suggested that these may represent an alternative pathway for ATP release. We have investigated the release of ATP through hemichannels from Xenopus oocytes that are formed by Connexin 38 (Cx38), an endogenous, specific type of connexin. These hemichannels generate an inward current that is reversibly activated by calcium-free solution and inhibited by octanol and flufenamic acid. This calcium-sensitive current depends on Cx38 expression: it is decreased in oocytes injected with an antisense oligonucleotide against Cx38 mRNA (ASCx38) and is increased in oocytes overexpressing Cx38. Moreover, the activation of these endogenous connexons also allows transfer of Lucifer Yellow. We have found that the release of ATP is coincident with the opening of hemichannels: it is calcium-sensitive, is inhibited by octanol and flufenamic acid, is inhibited in ASCx38 injected oocytes, and is increased by overexpression of Cx38. Taken together, our results suggest that ATP is released through activated hemichannels in Xenopus oocytes.

Adenosine Triphosphate↗
PubMed1976

Specialized junction in the distal convoluted tubule of rat kidney.

The ultrastructure and permeability of the apical junctions between epithelial cells of the distal nephron have been studied in rat kidney using a collidal lanthanum tracer and uranyl acetate staining en bloc. The apical intercellular junctions of the macula densa and juxtaglomerular segment of the early distal convoluted tubule measure up to 0.5 mu in length and about 50 A in width. Lanthanum permeates the occluding portion of these junctions in a discontinuous manner, defining a series of closely spaced and parallel lines of fusion that run in a direction perpendicular to the apical-basal axis of the tubular cells. The apical junctions of the remainder of the distal convoluted and cortical collecting tubules are impermeable bolanthanum. This distinctive apical tight junction can account for the greater permeability to ions of the early distal convoluted vs. late distal convoluted and cortical collecting tubules.

Animals↗
PubMed2004

Cell and toxicant specific phosphorylation of conexin43: effects of lindane and TPA on rat myometrial and WB-F344 liver cell gap junctions.

Previous studies showed that the pesticide lindane (gamma-hexachlorocyclohexane) inhibits gap junction intercellular communication in rat myometrial cells. The present study tested the hypothesis that lindane and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit gap junction communication in rat myometrial and liver WBr-F344 cells by the common mechanism of increasing phosphorylation of the gap junction protein connexin43. We evaluated changes of connexin43 phosphorylation using Western blot of standard SDS-PAGE gels and cell immunostaining, and we monitored gap junction communication using microinjection and transfer of Lucifer yellow dye. Exposure of rat myometrial cells to lindane or TPA nearly abolished dye transfer but did not alter the electrophoretic mobility of connexin43, and neither lindane nor TPA increased phosphorylation of connexin43 as assessed by immunoblot with anti-phospho-connexin43 (S368) antibody. However, TPA increased punctate immunofluorescence staining of phospho-connexin43 (S368) in myometrial cells whereas lindane had no such effect. In WBr-F344 cells, lindane and TPA inhibited dye transfer. Lindane increased immunostaining for phospho-connexin43 (S368) in WBr-F344 cells without altering the abundance, electrophoretic mobility or phosphorylation of connexin43 as detected in immunoblots. TPA intensified a slower migrating connexin43 band and increased phospho-connexin43 (S368) in immunoblots, and intensified phospho-connexin43 immunostaining at WBr-F344 cell interfaces and nuclear regions. These results show that phosphorylation of connexin43 at serine 368 occurred in cell and toxicant specific manners and was independent of changes in electrophoretic mobility in standard SDS-PAGE gels. Moreover, lindane inhibited gap junction communication in myometrial cells by a mechanism that was not be explained by changes in phosphorylation of connexin43.

Animals↗