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PubMed1999

Opposite regulation of transepithelial electrical resistance and paracellular permeability by Rho in Madin-Darby canine kidney cells.

Small GTPase Rho has been thought to be important for the formation and the maintenance of tight junction in epithelial cells, but the role of Rho in the regulation of barrier function of tight junction is not well understood. We here examined whether Rho was involved in the barrier function of tight junction in Madin-Darby canine kidney (MDCK) cells. The activation of prostaglandin EP3beta receptor, coupled to a Rho activation pathway, induced the increase in transepithelial electrical resistance (TER) but the increase in paracellular flux of mannitol in the preformed monolayer of the MDCK cells expressing the EP3beta receptor. This effect of the EP3 receptor was mimicked by the expression of constitutively active RhoA but not by active Rac1 in MDCK cells, using an isopropyl-beta-D-thiogalactoside-inducible expression system. On the other hand, the activation of EP3beta receptor suppressed the elevation of TER and the decrease in paracellular mannitol flux during Ca(2+) switch-induced tight junction formation, whereas the expression of active RhoA or Rac1 did not apparently affect the TER development in the Ca(2+) switch. These results demonstrate that the EP3 receptor and active RhoA regulate permeabilities of ionic and nonionic molecules in opposite directions in the preformed monolayer, and the EP3 receptor suppresses the elevation of TER during the tight junction formation.

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PubMed2000

Urothelial pathophysiological changes in feline interstitial cystitis: a human model.

Unique barrier properties of the urothelial surface membrane permit urine storage. Interstitial cystitis causes disabling dysuria, and frequency. Similarly, feline interstitial cystitis (FIC) occurs in cats. These studies define the permeability and structural properties of normal and FIC urothelium. To determine the effects of bladder filling, groups were studied before and after hydrodistention. Normal urothelium with or without hydrodistention exhibited high transepithelial resistances (TER) and low water and urea permeabilities, resembling other species. Fluorescence confocal microscopy revealed localization of the marker AE-31 to the apical surface of all umbrella cells in normal urothelium, with the tight junction protein ZO-1 localized to tight junctions. Scanning and transmission electron microscopy revealed uniform distribution of luminal cells with characteristic apical membrane and tight junction morphology. Urothelium in FIC animals displayed reduced TER and increased water and urea permeability following hydrodistention. Structural studies in FIC revealed denuded urothelium, with appearance of AE-31 in underlying epithelial cells. The results demonstrate severe epithelial damage and dysfunction in FIC and suggest novel approaches toward examining the etiology and therapy of IC.

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PubMed2005

Cerebral ischemia enhances tyrosine phosphorylation of occludin in brain capillaries.

Cerebral ischemia induces disruption of the blood-brain barrier (BBB), and this disruption can initiate the development of brain injuries. Although the molecular structure of tight junctional complexes in the BBB has been identified, little is known about alterations of tight junctional proteins after cerebral ischemia. Therefore, we investigated alterations of tight junctional proteins, i.e., occludin and zonula occludens (ZO)-1, in isolated rat brain capillaries after microsphere-induced cerebral embolism. We demonstrated that the levels of occludin and ZO-1 had decreased after the embolism. The embolism also resulted in a marked increase in tyrosine phosphorylation of occludin, which was coincident with an increase in the activity of c-Src. These results suggest that a decrease in the levels of occludin and ZO-1, and an increase in tyrosine phosphorylation of occludin may play an important role in the disruption of tight junctions, which may lead to dysfunction of the BBB after cerebral ischemia.

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PubMed2004

sPAR-3, a splicing variant of PAR-3, shows cellular localization and an expression pattern different from that of PAR-3 during enterocyte polarization.

PAR-3 (partitioning-defective) is a scaffold-like PDZ (postsynaptic density-95/discs large/zonula occludens-1) domain-containing protein that forms a complex with PAR-6 and atypical PKC, localizes to tight junctions, and contributes to the formation of functional tight junctions. There are several alternatively spliced isoforms of PAR-3, although their physiological significance remains unknown. In this study, we show that one of the major isoforms of PAR-3, sPAR-3, is predominantly expressed in the Caco-2 cells derived from colon carcinoma and is used as a model to investigate the events involved in the epithelial cell differentiation and cell polarity development. During the polarization of Caco-2 cells, the expression of PAR-3 increases as do those of other cell-cell junction proteins, whereas the expression of sPAR-3 decreases. Biochemical characterization revealed that sPAR-3 associates with atypical PKC, as does PAR-3. On the other hand, immunofluorescence microscopy revealed that sPAR-3 does not concentrate at the cell-cell contact region in fully polarized cells, whereas it concentrates at premature cell-cell junctions. This makes a contrast to PAR-3, which concentrates at tight junctions in fully polarized cells. These results provide evidence suggesting the difference in the role between sPAR-3 and PAR-3 in epithelial cells.

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PubMed2005

Changes of occludin expression in intestinal mucosa after burn in rats.

In severely burned rats, hyperemia, edema and other pathological injuries occur in the intestinal mucosa. Ultramicroscopically, the microvilli, tight junction and organelles are disrupted. Occludin is a functional component of tight junctions. The purpose of the present study is to investigate changes of occludin expression, and to further elucidate the relationship between occludin expression and ultrastructure damage. The fluorescence intensity of occludin was detected in intestinal wall by the method of immunofluorescence histochemistry and confocal laser scanning microscopy (CLSM). Expression of occludin and its mRNA were determined by western blotting and RT-PCR, respectively. Changes of intestinal mucosa ultrastructure were observed by TEM. The results showed that fluorescence intensity of occludin at 3PBH was enhanced, higher than that of the control group, being 80.77+/-8.38 and 72.86+/-4.74, respectively, and reached a peak at 12PBH (116.14+/-6.89). The expression levels of occludin at 3PBH and 6PBH were 1.21+/-0.02 and 1.53+/-0.14 times that of the control group, respectively, and there were significant differences (P<0.01) between 3PBH group and 6PBH group and control group. The levels of occludin mRNA were also enhanced. At 12PBH, the level reached a peak (P<0.01), being 2.00+/-0.24 times that of the control group. Coincidently, the structure of the tight junction between epithelial cells was disrupted on a large scale under TEM. We speculate that up-regulation of epithelial occludin may play a role in enhancing paracellular permeability and be related to the damage to the tight junction.

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PubMed1996

Dependence of epithelial intercellular junction biogenesis on thapsigargin-sensitive intracellular calcium stores.

Perturbation of potentially regulatable endoplasmic reticulum (ER) calcium stores with the Ca-ATPase inhibitor, thapsigargin (TG), perturbs the formation of desmosomes and tight junctions during polarized epithelial cell biogenesis, despite the development of cell contact. In a Madin-Darby canine kidney cell model for intercellular junction assembly, TG treatment inhibited the development of transepithelial electrical resistance (TER), a measure of tight junction assembly, in a dose-dependent manner. The TG-induced inhibition of tight junction assembly was paralleled by a defect in the sorting of the tight junction protein, ZO-1. An even more dramatic delay in sorting of the desmosomal protein, desmoplakin, was observed in the presence of TG. In addition, while both ZO-1 and desmoplakin-I in control cells were shown to become associated with the Triton X-100 insoluble cytoskeleton during intercellular junction assembly, prior treatment with 100 nM TG diminished this biochemical stabilization into the detergent-insoluble fraction, particularly in the case of ZO-1. Although spectrofluorimetric measurements in fura-2 loaded Madin-Darby canine kidney cells confirmed the occurrence of TG-mediated release of calcium from internal stores, total cytosolic calcium during junction assembly remained similar to untreated cells. Therefore, the presence of cytosolic calcium alone is not sufficient for normal intercellular junction biogenesis if intracellular stores are perturbed by TG. The results indicate the presence of calcium-sensitive intracellular mechanisms involved in the sorting and cytoskeletal stabilization of both tight junction and desmosomes and suggest a role for calcium-dependent signaling pathways at an early (possibly common) step in polarized epithelial biogenesis.

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PubMed1995

Cholestasis-induced alterations of the trans- and paracellular pathways in rat hepatocytes.

Bile secretion depends on the vectorial transport of solutes from blood to bile and involves three different pathways: transcellular pathways mediated by transport proteins distributed asymmetrically in the basolateral and canalicular plasma membrane and by transcytotic vesicles, and a paracellular pathway allowing selective diffusion through tight junctions. All three pathways are impaired differentially by extrahepatic (bile duct ligation) or intrahepatic (ethinyloestradiol) cholestasis. Ethinyloestradiol treatment leads to tight junctional defects that are less severe than those induced by bile duct ligation. Junctional impairment is reflected functionally in increased permeability for horseradish peroxidase and structurally by decreased strand numbers and increased junctional length, but not by alterations at the level of the individual strands. The parallelism of physiological and morphological perturbations indicates a structure-function relationship in hepatocellular tight junctions. In addition, impaired functional integrity of tight junctions following bile duct ligation is reflected in a partial loss of hepatocellular surface polarity owing to redistribution of some, but not all, domain-specific plasma membrane antigens, which might mimic the behaviour of transport systems. After ethinyloestradiol treatment no alterations of surface polarity were observed. Thus, immunohistochemistry supports the view that ethinyloestradiol results in less severe impairment of the tight junctions than bile duct ligation. Finally, bile duct ligation, but not ethinyloestradiol, affects the transcytotic vesicular pathway; severe impairment of this is reflected in the absence of a late horseradish peroxidase peak in bile and also in the accumulation of pericanalicular vesicles that are immunopositive for canalicular membrane proteins and accessible for bulk phase endocytic markers.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed2003

Apical and basal regulation of the permeability of the retinal pigment epithelium.

PURPOSE: The functional characteristics of tight junctions in the outer blood-retinal barrier change during embryonic development and in the presence of disease. A culture model of developing retinal pigment epithelium (RPE) was used to examine the regulation of the tight junctions. METHODS: RPE from chick embryos was cultured on filters that separated the apical and basal medium compartments. Cultures were maintained in various combinations of serum-free medium, serum-free medium that was conditioned by neural retinas, or serum-free medium that was supplemented with bovine pituitary extract, serum, or various hormones. Function was monitored by the transepithelial electrical resistance (TER) or the permeation of small organic tracers. Structure was monitored by immunofluorescence and freeze-fracture electron microscopy. RESULTS: Functional analysis indicated differences in permeability among RPE of different embryonic age and culture conditions. In serum-free medium, the tight junctions were leaky or failed to form. Barrier properties increased if pituitary extract was added to the basal medium chamber or retina-conditioned medium was added to the apical chamber. Retina-conditioned medium was more effective at organizing tight junctional strands into a continuous network, but bovine pituitary extract appeared to modulate the permeability of that network. In combination, they synergistically elevated the TER to physiological levels. Although the thyroid hormone T3 had no effect, serum in the apical medium chamber inhibited the ability of RPE cells to respond to retina-conditioned medium. CONCLUSIONS: Diffusible factors secreted by the neural retina acted synergistically with basolateral stimulation to regulate the structure and function of RPE tight junctions. Serum on the apical side of the RPE monolayer inhibited the ability of retinal factors to upregulate the tight junction barrier.

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PubMed1998

Differentiation of blood-brain barrier endothelial cells.

The vascular system of the central nervous system is derived from capillary endothelial cells, which have invaded the early embryonic neuroectoderm from the perineural vascular plexus. This process is called angiogenesis and is probably regulated by brain-derived factors. Vascular endothelial cell growth factor (VEGF) is an angiogenic growth factor whose expression correlated with embryonic brain angiogenesis, i.e. expression is high in the embryonic brain when angiogenesis occurs and low in the adult brain when angiogenesis is shut off under normal physiological conditions. VEGF receptors 1 and 2 (flt-1 and flk-1) as well as the recently identified angiopoietin receptors (tie-1 and tie-2) are receptor tyrosine kinases specifically expressed in endothelial cells. Expression of these receptors is high during brain angiogenesis but low in adult blood-brain barrier endothelium. They are required for the proper development of a vascular system, and particularly tie-2 is necessary for brain angiogenesis. Signal transduction by these receptors regulates endothelial cell growth, permeability and differentiation. Blood-brain barrier endothelial cell characteristics (complex tight junctions, low number of vesicles, specialized transport systems) are induced by the local brain environment, e.g. neurons and astrocytes. Tight junctions between brain endothelial cells are the structural basis for the paracellular impermeability and high electrical resistance of blood-brain barrier endothelium. Association of tight junction particles with the P-face rather than the number or branching frequency of tight junction stands correlated with blood-brain barrier development and function suggesting that the cytoplasmic anchoring of the tight junctions plays an important role. During inflammation, leukocytes migrate through blood-brain barrier endothelium. ICAM-1 and VCAM-1 on blood-brain barrier endothelial cells appear to be the major mediators of these processes while the selectins are absent from brain endothelium in vivo.

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PubMed1981

The development of cell junction during nephrogenesis.

The differentiation and distribution of intercellular junctions especially during the early development stages of the rabbit nephron was studied by freeze-fracture electron microscopy. Metanephrogenic cells were found to be connected by sporadic focal tight junctions. During the formation of the renal vesicle similar tight junctions occurred on the periphery as well as near the developing lumen. These focal tight junction increased in size and coalesced to broad zonulae occludentes lining the vesicular lumen at a later stage. Broad occluding junctions were also observed in the different nephron segments of the S-shaped stage. Ultrastructurally, these early maculae and zonulae occludentes consisted of beaded rows of particles. As development progressed, continuous tight junctions formed, whereas the number of strands decreased with the exception of the distal tubule. In contrast to the parietal glomerular epithelium, the initial occluding zonules of the visceral glomerular cells were gradually reduced to maculae occludentes, and finally disappeared. These results suggest that zonulae occludentes appear synchronously with the establishing lumen; the ultrastructural differentiation of tight junction strands seems to be completed with the onset of glomerular filtration.

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PubMed1988

Freeze-fracture study of the perineurium around frog dorsal root ganglia.

The perineurium around frog dorsal root ganglia consists of layers of flattened cells separated by extracellular connective tissue elements. The number of layers is smaller than that in the perineurium around adjacent peripheral nerves, and some of the layers are discontinuous, but in both cases, cells in the same layer overlap and form tight junctions with each other, sometimes accompanied by desmosomes or gap junctions. In freeze-fracture replicas the tight junctions between perineurial cells around peripheral nerves consist of 13-91 strands (mean: approximately 38). Some of these are parallel to the cell borders and some are oblique, forming elaborate meshworks. The overall width of each junction averages approximately 12 microns. In contrast, the tight junctions between perineurial cells around ganglia are much narrower, averaging approximately 2 microns in width, and they consist of only 1-14 strands (mean: approximately 6) with few anastomoses and many free ends. These structural differences provide a morphological basis for a less complete diffusion barrier around dorsal root ganglia.

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PubMed2005

Matrix metalloproteinases in early diabetic retinopathy and their role in alteration of the blood-retinal barrier.

One of the early features of diabetic retinopathy is the alteration of the blood-retinal barrier (BRB), which may involve the breakdown of endothelial cell tight junctions. The aim of this study was to examine the expression of extracellular proteinases in an animal model of early diabetic retinopathy and to determine their role in the alteration of the BRB. Matrix metalloproteinase (MMP) expression was studied in the retinas of rats with 12 weeks of diabetes. The role of MMPs in regulating tight junction function was investigated in retinal endothelial and pigment epithelial cells by measuring transepithelial electrical resistance (TER). The retinas of diabetic animals demonstrated elevated levels of MMP-2, MMP-9 and MMP-14 messenger RNA. A significant increase in the production of MMP-9 was seen when cells were exposed to high glucose conditions. Both cell types treated with purified MMP-2 or MMP-9 were found to have alterations of tight junction function as shown by decreased TER. Western blot analysis of cell extracts treated with MMP-2 or MMP-9, revealed specific degradation of the tight junction protein, occludin. Results suggest that elevated expression of MMPs in the retina may facilitate an increase in vascular permeability by a mechanism involving proteolytic degradation of the tight junction protein occludin followed by disruption of the overall tight junction complex.

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PubMed1996

Localization and expression of zonula occludens-1 tight junction-associated protein in baboon (Papio anubis) corpora lutea.

The identification of the cell junction-forming proteins connexin-43, a gap junction protein and E-cadherin, which is a component of adherent junctions, in the corpus luteum of both humans and baboons suggests that cell-cell interactions and metabolic cooperation must occur in this tissue. Occluding junctions are a third type of junction which form a physical barrier between cells. Thus, our aims in this study were firstly to examine the presence of the tight junction-associated protein zonula occludens-1 (ZO-1) by immunohistochemistry, and secondly to determine the concentrations of this protein in the early-, mid- and late luteal phase baboon corpora lutea of the menstrual cycle by a Western analysis. ZO-1 was localized mainly at the periphery of the luteal cells, and the intensity of immunoreactivity varied through the luteal phase, with comparatively stronger immunoreactivity in the mid-luteal phase than the early and late luteal phases. Atretic corpora lutea were devoid of activity. By Western analysis, bands of immunoreactivity were observed at 225 kDa, further confirming the presence of the protein. Maximum activity, as determined by densitometry, was observed in the mid-luteal phase. These data infer the presence of tight junctions in the corpus luteum and suggest that expression of the ZO-1 protein forming these junctions may be hormonally regulated within this tissue.

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PubMed1984

Plasma membrane organization of astrocytes in elasmobranchs with special reference to the brain barrier system.

The structural machinery contributing to the blood-brain barrier in elasmobranchs has been examined mainly using freeze-fracture techniques. Capillary endothelial cells, which show local aggregations of pinocytotic vesicles and infrequent fenestrations, are connected by poorly developed tight junctions. Astrocytic processes investing the capillary are linked by well-developed tight junctions between lateral membranes immediately beneath the perivascular space. The tight junctions consist of continuous strands of multiple layers coursing circumferentially around the astrocytic processes parallel to one another as well as to the perivascular space. The presence of intramembrane particles (IMPs) within E-face grooves may result in discontinuities in IMP rows on the P-face. Thus, in compensation for the capillary endothelium, perivascular astrocytes constitute the morphological site of the blood-brain barrier in elasmobranchs. Continuous strands of tight junctions are also detected between astrocytic processes forming the glia limitans at the brain surface. These may act as a barrier between meningeal connective tissue and brain parenchyma. Astrocytic membranes have numerous IMPs of 8-9 nm in diameter on their P-faces. These IMPs are uniformly distributed so that astrocytic membranes are easily distinguished from neuronal membranes even in the neuropil. Ependymal cells also have numerous IMPs in all their membrane domains. Orthogonal arrays are not detected in either astrocytic or ependymal plasma membranes.

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PubMed1992

A permeability barrier to lanthanum and the presence of collagen between odontoblasts in pig molars.

Previous experiments in rat incisors indicate that the odontoblasts form an impermeable barrier which prevents fluid movement between pulp and dentine. The permeability of the odontoblast layer has now been investigated in pig molars which are more analogous to human teeth. The heads and necks of anesthetised piglets were perfused intra-arterially with lanthanum nitrate in Ringer's solution or with Ringer's solution alone. Molar tooth germs were removed, sliced, fixed by immersion and embedded in resin. Ultrathin sections including pulp and dentine were examined by transmission electron microscopy. Fenestrated capillaries were permeable to the electron dense lanthanum which thus entered the extracellular space between the odontoblast cell bodies. The lanthanum was excluded from predentine indicating that a barrier to permeability is present. In the above specimens and in others from 2 animals which were fixed by perfusion fixation, longitudinally oriented bundles of collagen fibrils were found passing from dentine through predentine into the odontoblast layer. Longitudinal collagen was also present between odontoblast cell bodies and entering the pulp at their basal ends. This suggests that classical von Korff fibres are present during primary circumpulpal dentinogenesis. In some sections longitudinally oriented collagen was absent. The junctions showed features of classical tight junctions but open tight junctions containing longitudinal collagen were also observed, suggesting that the junctions may modulate. Despite a trace of evidence that lanthanum can leak through adjacent to longitudinally penetrating collagen we concluded that the biological permeability barrier is maintained. The presence of the barrier indicates that other than the longitudinal collagen fibrils of which the source is unknown, all molecules incorporated into dentine are deposited there by the odontoblasts. An advantage of the barrier may be that it provides a closed environment for the orderly process of matrix deposition and mineralisation of dentine.

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PubMed1984

[Ultrastructural changes in female germ cells during the meiotic prophase in the rat: a special study of membranes after cryofracture].

Ultrastructural changes in the nuclear and cytoplasmic elements in the germ cells of female rats were followed before meiotic prophase (15.50 days post-coïtum and 17.25 days post-coïtum) and during it (17.75 days post-coïtum to birth). We observed: modifications in the nuclear envelope which was thick during the oogonial stage, becoming thinner when the chromosomes entered preleptotene stage. The thinning of the envelope was due to the disappearance of the chromatin material lining it; variations in the number and distribution of germ cell nuclear pores according to stage; the pores were first scattered in small clusters of 6 to 8 over the entire nuclear membrane. From the preleptotene to zygotene stage, these clusters enriched in pores to form large areas. Finally, in the pachytene and diplotene stages, clusters of more than 100 pores were seen; nucleolar fragmentation from the preleptotene stage, followed by the formation of a new active nucleole in the diplotene; polarization of the mitochondria in the oldest oogonia just before the beginning of meiotic prophase. This polarization disappeared after the onset of the meiotic processes, then appeared again near the developing Golgi apparatus at the end of the pachytene stage; the formation of large gap junctions and numerous bands of tight junctions between the somatic cells; these formations contrasted with small gap junctions, and the tight junctions became scarce just before the meiotic process began. These observations, as well as those concerning nuclear pore distribution were made using the cryofracture technique.

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PubMed1980

A freeze-fracture study of intercellular junctions between various kinds of epithelial cells surrounding common endolymphatic space in the hearing organ of the chick.

Intercellular junctions between various epithelial cells in the hearing organ (basilar papilla) of the chick were studied with the freeze-fracture technique. The effects of hypertonic solutions on the intercellular junctions were also examined. The basilar papilla of the chick is primarily composed of hair cells and supporting cells in the neuroepithelium, specialized columnar cells (TMC) which attach to the tectorial membrane, and light cells (LC) and dark cells (DC) in the tegmentum vasculosum. All of these epithelial cells surround a common endolymphatic space. The tight junctions between hair and supporting cells, and those between adjacent supporting cells in the neuroepithelium are 0.1--0.3 micrometer in depth and display the usual network of branching and anastomosing strands of shared intramembrane proteins. The tight junctions in the tegmentum vasculosum have the same structure as in the neuroepithelium. In contrast, the tight junctions between the TMCs are extremely well developed. They are 1--2 micrometer in depth. In freeze-fracture replicas, they appear as a fingerprint pattern of unbranched parallel particulate strands, running both parallel and perpendicular to the cell surface. After exposure to hypertonic solutions, all the epithelial cells are shrunken and intercellular spaces are expanded; all tight junctions, however, are intact. Thus, tight junctions in the basilar papillae are resistant to dissociation by hypertonic solutions. The usual zonulae occuludentes in the neuroepithelium and tegmentum vasculosum are thought to prevent diffusion of endolymph through the intercellular spaces of epithelial cells. However, the tight junctions on the TMCs may function not only as a diffusion barrier, but also provide structural support to the cells anchoring the tectorial membrane which receives mechanical forces induced by the vibration of the basilar membrane. Extensive gap junctions are found between all the supporting cells (supporting cells in the neuroepithelium, TMCs, and LCs in the tegmentum vasculosum) surrounding the endolymphatic space.

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PubMed1975

Freeze-fracture replication of junctionsl complexes in unincubated and incubated chick embryos.

Junctional complexes have been investigated in the epiblast of young chick embryos by examination of freeze-fracture replicas and of sections of comparable specimens stained with lanthanum nitrate. By means of freeze-fracture, tight junctions were shown to be present in the unincubated embryo (stage 1 of Hamburger and Hamilton). The number of ridges or grooves was found to vary between 2 and 10 near the dorsal border, whereas isolated ridges were found more ventrally. Lanthanum was unable to penetrate between the cells in the region of the dorsally situated tight junctions. Similar tight junctions were found in incubated embryos (stage 3) examined by both techniques. Tight junctions were also seen in cleavage (pre-laying) embryos examined in section. Gap junctions were extremely uncommon in unincubated embryos, though occasional aggregates of gap junction particles were seen on the lateral cell membranes close to the dorsal surface. In only one instance were associated pits visible. By contrast, gap junctions were more frequently encountered by stage 3, and these junctions possessed both pits and particles. Desmosomes were never seen in the freeze-fracture replicas at either stages 1 or 3, though structures which might be developing desmosomes were visible in sections. The functions of both the tight and gap junctions in the young chick embryo are discussed. The results are also considered in relation to recent theories about the way in which gap junctions are formed.

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