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PubMedDate not supplied

A survey of the effects of loop diuretics on the zonulae occludentes of the perilymph-endolymph barrier by freeze fracture.

The acute (30 min) effects of the loop diuretics piretanide, ethacrynic acid, bumetanide, furosemide, and azosemide, and the chronic (8 days) effects of furosemide and bumetanide on the zonulae occludentes (tight junctions) of the perilymph-endolymph barrier in the stria vascularis and in Reissner's membrane of the basal cochlear turns were studied by freeze-fracture. Quantitative analysis of their effects indicated that the structural integrity of the barrier was modified by either as increase or a decrease in the number, depth, and density of the strands of the tight junctions of the strial marginal cells. Only azosemide appeared to modify the tight junctions between the epithelial cells of Reissner's membrane, but it had little effect on the strial junctions. The tight junctions between the basal cells of the stria appeared to be the least affected by the various loop diuretics, although piretanide appeared to increase randomly the intercellular spaces lying between the strands.

Animals↗
PubMed1985

Tracer study on a paracellular route in experimental hydrocephalus.

Considering the possibility of a paracellular pathway for edema resolution, we studied the intracerebral movement of proteins and ionic lanthanum in rats with experimental hydrocephalus. Hydrocephalus was induced by injection of kaolin suspension into the cisterna magna. After induction of hydrocephalus, horseradish peroxidase (HRP), microperoxidase (MP), or lanthanum chloride (LaCl3) were perfused into the ventricle system. HRP and MP were localized mainly in the intercellular spaces between ependymal cells, glial cells, and in perivascular spaces and were restricted by endothelial tight junctions. Ionic lanthanum (La3+), however, penetrated these tight junctions and moved between the blood and CSF cavities by paracellular pathways. These findings indicate that in obstructive hydrocephalus, the tight junctions may constitute part of a paracellular pathway for the transendothelial movement of small solutes, although they prevent the movement of larger molecules.

Animals↗
PubMed1997

Junctions between the sinus endothelial cells of rat spleen.

Junctions between the sinus endothelial cells of rat spleen were examined by electron microscopy, using both freeze-fracture and detergent-extraction techniques. Adherens and tight junctions were observed. Adherens junctions were the predominant junctional structures between endothelial cells and were located on basolateral and lateral surfaces. At the basolateral adherens junctions, actin filaments were associated with the junctional membranes and were continuous with the actin filaments in stress fibers. Cross-bridges were present in the interspaces of the adherens junctions and spacing of the bridges was fairly regular. A form of tight junction, the macula occludens, was also observed between the endothelial cells, but it was not observed at every cellular apposition. Electron-dense material, adjoining the cytoplasmic surfaces of membranes in the tight junctions, separated the junctional membranes from masses of thin filaments. At basolateral tight junctions, the actin filaments were continuous with those in the stress fibers. Based on these observations, the two intercellular junctions were considered to play important roles in sinus functions.

Animals↗
PubMed1992

A freeze-fracture study of the perineurium in galactose neuropathy: morphological changes associated with endoneurial oedema.

Feeding rats with galactose as 40% of their diet results in peripheral nerve oedema related to the intrafascicular accumulation of galactitol and sodium. In this study, associated changes in the perineurium were examined by the freeze-fracture replication technique. Perineurial cells are linked by tight junctions (zonulae occludentes). In normal animals these are made up of anastomosing strands organized in a belt-like arrangement along the margins of continuous cells. The majority of the tight junctions in the galactose-fed animals displayed structural abnormalities. These ranged from slight separation of the strands to fragmentation and dispersal, with looping of isolated strands. Some of the tight junctions contained large dilated compartments within the junctional network. Short lengths of intramembranous particles, probably representing assembly or disassembly of tight junctional strands, were also observed. The membranes of perineurial cells normally possess numerous openings of caveolae. A quantitative assessment showed that the mean density of these caveolae openings was increased in the galactose-fed rats as compared with controls. The alterations in the tight junctions resemble those that have been produced experimentally in epithelia by subjecting them to abnormal osmotic gradients. They also resemble those seen in human diabetic neuropathy in which osmotic disturbances involving the perineurium have been considered to occur. If the alterations involve the inner layers of the perineurium, they are likely to impair its barrier function. The increased number of caveolae openings in galactose neuropathy may represent a reaction to the endoneurial oedema and indicate that the pinocytotic-like vesicles have a transport function.

Animals↗
PubMed1978

The oligodendrocytic junctional complex.

The junctional complex of oligodendrocytes was studied by means of different electron microscopical techniques. This complex is composed of the following junctional membrane formations: 1) tight junctional domains in the oligodendrocytic membrane near the some of the cells, 2) fasciae occludentes or focal tight junctions on the outer oligodendrocytic loop of myelin and on the outermost myelin membrane, 3) gap junctions of considerable size variations, either on membranes near the soma or on peripheral oligodendrocytic processes, and 4) non-paranodal transverse bands. The different types of oligodendrocytic junctions are discussed in terms of their functional implications.

Animals↗
PubMed2000

The response of junctional complexes to induced desquamation in mouse bladder urothelium.

During desquamation, the cells of mouse urinary bladder epithelium undergo detachment. In this process we examined the disconnection of cell adhesion molecules. Two proteins of cell junctions were studied: ZO1 of tight junctions and desmoplakin of desmosomes. Desquamation was induced by intravesical injection of LPS, constant illumination of mouse for 96 h, application of a combination of stress hormones hydrocortisone and norepinephrine or by removal of calcium with EGTA. All the inducers caused penetration of lanthanum tracer through the tight junctions, indicating paracellular permeability. Dilatation of extracellular spaces between neighboring cells was seen whenever desquamation was induced in bladders containing urine. Desquamation of single cells as well as groups of cells was observed. Contrary to obvious disconnection of cell junctions, as a precondition for desquamation, the distribution of junctional proteins did not change either in urothelial tissue or in desquamated cells. This study demonstrates that all the inducers of desquamation cause first an extensive dysfunction of a blood urine barrier and after that an occasional mechanical disconnection of adhesive junctions which consequently leads to desquamation.

Animals↗
PubMed1985

Epithelial water transport in a balanced gradient system.

The relationship between epithelial fluid transport, standing osmotic gradients, and standing hydrostatic pressure gradients has been investigated using a perturbation expansion of the governing equations. The assumptions used in the expansion are: (a) the volume of lateral intercellular space per unit volume of epithelium is small; (b) the membrane osmotic permeability is much larger than the solute permeability. We find that the rate of fluid reabsorption is set by the rate of active solute transport across lateral membranes. The fluid that crosses the lateral membranes and enters the intercellular cleft is driven longitudinally by small gradients in hydrostatic pressure. The small hydrostatic pressure in the intercellular space is capable of causing significant transmembrane fluid movement, however, the transmembrane effect is countered by the presence of a small standing osmotic gradient. Longitudinal hydrostatic and osmotic gradients balance such that their combined effect on transmembrane fluid flow is zero, whereas longitudinal flow is driven by the hydrostatic gradient. Because of this balance, standing gradients within intercellular clefts are effectively uncoupled from the rate of fluid reabsorption, which is driven by small, localized osmotic gradients within the cells. Water enters the cells across apical membranes and leaves across the lateral intercellular membranes. Fluid that enters the intercellular clefts can, in principle, exit either the basal end or be secreted from the apical end through tight junctions. Fluid flow through tight junctions is shown to depend on a dimensionless parameter, which scales the resistance to solute flow of the entire cleft relative to that of the junction. Estimates of the value of this parameter suggest that an electrically leaky epithelium may be effectively a tight epithelium in regard to fluid flow.

Animals↗
PubMed1994

Different effects of protein kinase inhibitors on the localization of junctional proteins at cell-cell contact sites.

The protein kinase inhibitor H-7 prevents the assembly of tight junctions in cultured Madin Darby Canine Kidney (MDCK) epithelial cells (Balda et al. (1991) J. Membr. Biol. 122, 193-202; Nigam et al. (1991) Biochem. Biophys. Res. Commun. 181, 548-553); however, its mechanism of action is unknown. To understand the basis of the activity of H-7 and other inhibitors we compared the effect of H-7 on the localization of proteins belonging to tight junctions and adherens-type junctions (zonula adhaerens and desmosome), and on the organization of actin microfilaments. Junction assembly was induced in MDCK cells either by the 'Ca2+ switch' procedure or by incubating trypsinized cells at normal extracellular Ca2+, and the cells were then immunofluorescently labeled with antibodies against cingulin, ZO-1, E-cadherin and desmoplakin, and with FITC-phalloidin. Here we show by measuring the transepithelial resistance that, in addition to H-7, H-8 and staurosporine can also significantly block the assembly of tight junctions, whereas HA1004 is poorly active. H-7 inhibited the accumulation of cingulin and ZO-1 in junctional areas most effectively when added during assembly at normal extracellular Ca2+. On the other hand, H-7 did not have major effects on the accumulation of E-cadherin and desmoplakin in the regions of cell-cell contact using either assembly protocol. Electron microscopy confirmed that H-7 does not abolish the formation of adherens-type junctions, suggesting that phosphorylation plays a different role in the assembly of tight junctions versus adherens-type junctions. Finally, in both protocols of junction assembly H-7 caused a major disorganization of actin microfilaments, suggesting that H-7 may prevent TJ assembly through its effect on the cytoskeleton.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2004

Paracellular Cl- permeability is regulated by WNK4 kinase: insight into normal physiology and hypertension.

Paracellular ion flux across epithelia occurs through selective and regulated pores in tight junctions; this process is poorly understood. Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring hypertension and hyperkalemia. Whereas WNK4 is known to regulate several transcellular transporters and channels involved in NaCl and K+ homeostasis, its localization to tight junctions suggests it might also regulate paracellular flux. We performed electrophysiology on mammalian kidney epithelia with inducible expression of various WNK4 constructs. Induction of wild-type WNK4 reduced transepithelial resistance by increasing absolute chloride permeability. PHAII-mutant WNK4 produced markedly larger effects, whereas kinase-mutant WNK4 had no effect. The electrochemical and pharmacologic properties of these effects indicate they are attributable to the paracellular pathway. The effects of WNK4 persist when induction is delayed until after tight-junction formation, demonstrating a dynamic effect. WNK4 did not alter the flux of uncharged solutes, or the expression or localization of selected tight-junction proteins. Transmission and freeze-fracture electron microscopy showed no effect of WNK4 on tight-junction structure. These findings implicate WNK signaling in the coordination of transcellular and paracellular flux to achieve NaCl and K+ homeostasis, explain PHAII pathophysiology, and suggest that modifiers of WNK signaling may be potent antihypertensive agents.

Amino Acid Substitution↗
PubMed1999

Cadherin function in junctional complex rearrangement and posttranslational control of cadherin expression.

The role of E-cadherin, a calcium-dependent adhesion protein, in organizing and maintaining epithelial junctions was examined in detail by expressing a fusion protein (GP2-Cad1) composed of the extracellular domain of a nonadherent glycoprotein (GP2) and the transmembrane and cytoplasmic domains of E-cadherin. All studies shown were also replicated using an analogous cell line that expresses a mutant cadherin construct (T151) under the control of tet repressor. Mutant cadherin was expressed at approximately 10% of the endogenous E-cadherin level and had no apparent effect on tight junction function or on distributions of adherens junction, tight junction, or desmosomal marker proteins in established Madin-Darby canine kidney cell monolayers. However, GP2-Cad1 accelerated the disassembly of epithelial junctional complexes and delayed their reassembly in calcium switch experiments. Inducing expression of GP2-Cad1 to levels approximately threefold greater than endogenous E-cadherin expression levels in control cells resulted in a decrease in endogenous E-cadherin levels. This was due in part to increased protein turnover, indicating a cellular mechanism for sensing and controlling E-cadherin levels. Cadherin association with catenins is necessary for strong cadherin-mediated cell-cell adhesion. In cells expressing low levels of GP2-Cad1, protein levels and stoichiometry of the endogenous cadherin-catenin complex were unaffected. Thus effects of GP2-Cad1 on epithelial junctional complex assembly and stability were not due to competition with endogenous E-cadherin for catenin binding. Rather, we suggest that GP2-Cad1 interferes with the packing of endogenous cadherin-catenin complexes into higher-order structures in junctional complexes that results in junction destabilization.

Amino Acid Sequence↗
PubMed2001

DLG-1 is a MAGUK similar to SAP97 and is required for adherens junction formation.

Cellular junctions are critical for intercellular communication and for the assembly of cells into tissues. Cell junctions often consist of tight junctions, which form a permeability barrier and prevent the diffusion of lipids and proteins between cell compartments, and adherens junctions, which control the adhesion of cells and link cortical actin filaments to attachment sites on the plasma membrane. Proper tight junction formation and cell polarity require the function of membrane-associated guanylate kinases (MAGUKs) that contain the PDZ protein-protein interaction domain. In contrast, less is known about how adherens junctions are assembled. Here we describe how the PDZ-containing protein DLG-1 is required for the proper formation and function of adherens junctions in Caenorhabditis elegans. DLG-1 is a MAGUK protein that is most similar in sequence to mammalian SAP97, which is found at both synapses of the CNS, as well as at cell junctions of epithelia. DLG-1 is localized to adherens junctions, and DLG-1 localization is mediated by an amino-terminal domain shared with SAP97 but not found in other MAGUK family members. DLG-1 recruits other proteins and signaling molecules to adherens junctions, while embryos that lack DLG-1 fail to recruit the proteins AJM-1 and CPI-1 to adherens junctions. DLG-1 is required for the proper organization of the actin cytoskeleton and for the morphological elongation of embryos. In contrast to other proteins that have been observed to affect adherens junction assembly and function, DLG-1 is not required to maintain cell polarity. Our results suggest a new function for MAGUK proteins distinct from their role in cell polarity.

Actins↗
PubMed2006

Identification of multiple claudins in the rat epididymis.

The luminal environment of the epididymis is highly specialized with specific proteins, ions, pH, etc. required for sperm maturation. Tight junctions between epididymal principal cells are responsible for the formation of the blood-epididymal barrier, which regulates this luminal environment. Claudins (Cldns) are a recently discovered family of transmembrane proteins and are essential components of tight junctions. Previous work from our laboratory has demonstrated the presence and localization of Cldn-1 in all regions of the rat epididymis. The objective of this study was to determine the presence and localization of other Cldns in the epididymis. Using RT-PCR we have identified mRNA transcripts for Cldn-3 through -9 in each region of the adult rat epididymis. Immunolocalization of Cldn-3, Cldn-4, and Cldn-5 were done in adult as well as in 42- and 14-day-old rats. Cldn-5 in adult rats was localized exclusively in blood vessels of the interstitium. Cldn-3 was localized apically in the epididymal epithelium between adjacent principal cells throughout the epididymis, where tight junctions have been reported histologically. There were no differences in the localization of Cldn-3 in epididymides of rats at the different ages. In 14-day-old rats, Cldn-4 was localized all along the lateral plasma membrane between adjacent principal cells. The immunostaining was more pronounced in the proximal regions of the epididymis. In both 42-day-old rats and adults, Cldn-4 was localized primarily to apical tight junctions between principal cells and staining was more pronounced in the proximal region of the epididymis. Cldn-16 transcripts were also identified by RT-PCR. These transcripts were present in both proximal and distal regions of the epididymis of young (Day 14 and 21) animals, but only in the proximal (initial segment) region of the adult epididymis. These data indicate that epididymal tight junctions are composed of several Cldns, suggestive of a complex regulation of the blood-epididymal barrier.

Aging↗
PubMed2003

Molecular anatomy of intercellular junctions in brain endothelial and epithelial barriers: electron microscopist's view.

In this review, we have tried to summarize the current knowledge on the distribution of important molecular components of intercellular junctions-both tight junctions (TJs) and adherens junctions (AJs)-at the level of ultrastructure. For this purpose, immunogold procedure was applied to ultrathin sections of brain samples obtained from mice, rats, and humans and embedded in hydrophilic resin Lowicryl K4M. The results of our observations performed with transmission electron microscopy (EM) are discussed and compared with findings of other authors. Although the main structures responsible for the barrier and fence functions of the blood-brain barrier (BBB) and blood-CSF barrier are TJs present between endothelial cells (ECs) of brain capillaries and epithelial cells of the choroid plexus, their functional characteristics (e.g. tightness of the barrier evaluated by electrical resistance) differ significantly. Therefore, our main attention is focused on the presence and distribution of both intrinsic, i.e. integral membrane (transmembrane), molecules such as occludin, claudins, and junctional adhesion molecule (JAM) in TJs, and cadherins in AJs, as well as peripheral molecules of both types of junctions, e.g. zonula occludens (ZO) proteins and catenins. The latter group of molecules connects transmembrane proteins with the cell cytoskeleton. A close spatial association of the TJ proteins with those of AJs indicates that both junctional types are intermingled in the BBB type of endothelium. One of most important purposes of this work is to find out the junction-associated molecules that can serve as sensitive markers of normal or disturbed function of brain barriers. Understanding the structural-functional relations between molecular components of junctional complexes in physiological and experimental conditions of both barriers can provide important information about the etiology of various pathological conditions of the central nervous system and also help to elaborate new therapeutic approaches.

Animals↗
PubMed2006

Role of aPKC isoforms and their binding partners Par3 and Par6 in epidermal barrier formation.

The skin water barrier, essential for terrestrial life, is formed by a multilayered stratifying epithelium, which shows a polarized distribution of both differentiation and intercellular junction markers. Recently, several reports showed the crucial importance of tight junctions for the in vivo water barrier function of the skin. In simple epithelial cells, intercellular junction formation is closely coupled to the establishment of polarity. However, if and how polarity proteins contribute to epidermal differentiation and junction formation is not yet known. Here, we have characterized the localization and isoform expression of the polarity protein atypical PKC (aPKC) and its binding partners Par3 and Par6 in epidermis and primary keratinocytes of mice. Their distribution is only partially overlapping in the granular layer, the site of functional tight junctions, suggesting that next to a common Par3/Par6/aPKC function they also may have functions independent of each other. Both aPKCzeta and aPKCiota/lambda, are expressed in the epidermis but only aPKCiota/lambda showed a strong enrichment in the junctions, suggesting that this aPKC isoform is important for epidermal tight junction function. Indeed, inhibition of aPKC function showed that endogenous aPKC is crucial for in vitro barrier function and this required the presence of both the Par3 and Par6 binding sites.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Ultrastructure of the blood-brain barrier in the rabbit.

The morphology of the normal blood-brain barrier in the rabbit by thin section and freeze-fracture electron microscopy is reported. Exogenous tracer horseradish peroxidase was injected to visualize the integrity of the blood-brain barrier in New Zealand White rabbits. Freeze-fracture was used to determine the intramembrane architecture of the tight junctions. Thin sections (60-100 nm) of brain capillaries from animals injected with horseradish peroxidase (HRP) possessed few pinocytotic vesicles in the cytoplasm. Junctional profiles between adjoining plasma membranes were present. Thin sections of capillaries containing electron dense HRP reaction product (HRP-RP) in the lumen revealed focal fusions of apposing plasma membranes that occluded reaction product from entering the junctional clefts. Some cytoplasmic vesicles were filled with HRP-RP; however, basal laminae and brain interstitium were free of HRP-RP in all vessel profiles examined. Freeze-fracture electron microscopy revealed tight junctions as an elaborate network of interconnecting strands of intramembrane particles appearing as ridges on the EF face and corresponding grooves on the PF face on platinum replicas. Results of this study demonstrate the architecture of rabbit brain microvessel endothelial junctions (blood-brain barrier) and provide evidence that the tight junctions prevent HRP extravasation. It is concluded that rabbit brain endothelial tight junctions (zonulae occludentes), as in other mammals, form the anatomical basis of the blood-brain barrier. Consequently, the rabbit brain microvasculature can be a useful model for establishing stereotactic radiosurgical procedures to treat brain astrocytomas (tumours).

Animals↗
PubMed2006

Intercellular junctions in rabbit eye ora serrata.

Summary The aim of this study was to describe and localize the intercellular junctions in the ora serrata region of albino and pigmented rabbit eyes. Eyes of albino and pigmented rabbits were fixed and processed for transmission electron microscopy. Light and electron microscope examination was carried out on semithin and ultrathin sections. The ora serrata region showed adherens, gap and tight junctions in the retinal and ciliary margins of albino and pigmented rabbit eyes. In the retinal margin, zonulae adherens between Müller cells and photoreceptors are associated with tight junctions. In the ciliary margin, epithelial cells are joined by adherens, gap and tight junctions localized between apical and apicolateral cell membranes. Tight junctions appear as zonulae occludens in the non-pigmented apicolateral cell membranes and as tight focal junctions between pigmented and non-pigmented apical cell membranes. Between the ciliary and retinal margins there are adherens and tight focal junctions which attach pigmented apical cell membranes to adjacent cells. There were no differences in the distribution of intercellular junctions between albino and pigmented rabbits.

Animals↗
PubMed2000

Claudins regulate the intestinal barrier in response to immune mediators.

BACKGROUND & AIMS: To determine the functional role of immune mediators in the formation of the intestinal barrier, we have examined the regulation of claudin expression by interleukin (IL)-17 in human intestinal epithelial cells. METHODS: Expression of claudins, extracellular signal-related (ERK) mitogen-activated protein kinases (MAPKs), and activated ERK MAPKs was determined by immunoblotting. Claudin membrane association was assessed by immunohistochemistry and claudin messenger RNA expression by Northern blot analysis. Intestinal epithelial barrier function was characterized through transepithelial electrical resistance and mannitol tracer flux. RESULTS: IL-17 induced the development of a paracellular barrier of T84 cell monolayers. Inhibition of ERK activation with the MEK inhibitor PD98059 blocked IL-17 as well as basal development of tight junctions in T84 cells. IL-17 induced formation of tight junctions correlated with up-regulation of claudin-1 and claudin-2 gene transcription. Inhibition of MEK reduced the activated and basal expression of claudin-2 messenger RNA and protein expression. Functional MEK was required for the expression and membrane association of claudin-2 but not claudin-1 in T84 cells. CONCLUSIONS: MEK activity is required for claudin-mediated formation of tight junctions. IL-17 is able to regulate the intestinal barrier through the ERK MAPK pathway.

Blotting, Northern↗
PubMed1985

Freeze-fracture quantitative comparison of rabbit corneal epithelial and endothelial membranes.

The intramembrane structure of epithelial apical, lateral and basal membranes was compared with that of the endothelium in normal rabbit corneas. The cytoplasmic membrane leaflet (P-face) of apical epithelial membrane displays randomly scattered intramembrane particles over the apical microvilli. Apical endothelial P-face membranes, by comparison, are covered by densely-packed particles. The lateral membranes of superficial epithelial cells display delicate tight junctions, punctate particle aggregates characteristic of desmosomes, and small gap junctions. Background intramembrane particles are randomly distributed and not densely packed. Large gap junctions and fewer desmosomes are present on deeper epithelial cells, including basal cells. By comparison, the endothelial lateral membranes display densely-packed, background particles and numerous triangular-shaped, gap junctional particle aggregates bordered by strands of particles resembling tight junctions. At the apical border, long anastomosing tight junctional strands are present. The basal E-face membranes of both cell layers adjacent to the basement membrane are similar in that they have sparse particles and vesicle fusion sites. Small particle aggregates are present on basal epithelial membranes at sites of hemidesmosomes. Quantitation of particle size and density on P-face apical and lateral membranes of each cell layer reveals that particle density on endothelial membranes is more than twice that on epithelial membranes. Particle sizes are similar on both cell layers and mean diameter ranges from 8.7 to 9.2 nm. The differences in intramembrane particle density and junctional aggregates on epithelial and endothelial membranes reflect membrane constituents associated with functional differences of these two cell layers.

Animals↗