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PubMedDate not supplied

Effect of chloroquine on the formation of tight junctions in cultured immature rat Sertoli cells.

Adjoining immature Sertoli cells in the seminiferous epithelium form a tight junctional complex leading to the development of the blood-testis barrier. Protease and antiprotease activities have been implicated in the process of formation of tight junctions. Here, we report the effect of chloroquine, an antimalarial drug with antiprotease activity, on the development of intercellular tight junctions in cultured immature rat Sertoli cells. For positive control, the classical lysosomotropic agent ammonium chloride was used. Sertoli cells were seeded in serum-free defined medium at a density of 3 x 10(6) cells/0.64-cm2 well on Matrigel-covered Millicell-HA filters. Chloroquine at concentrations ranging from 25 to 100 microM was added to the outer chamber of the bicameral system on either day 1 or 7 of the culture. The formation of the tight junction was monitored by the measurement of the transepithelial resistance (TER) at 24-hour intervals using an impedance meter. TER in untreated controls was 50 ohms/cm2 on day 1; it increased progressively to 80 ohms/cm2 by day 7 and plateaued until day 12. The cells treated from day 1 with chloroquine also showed a dose-dependent progressive increase in TER until day 9, reaching 225 ohms/cm2 in cells treated with the 100 microM concentration. In comparison to controls, the increase in TER was significantly higher. In cells treated with chloroquine starting from day 7 of culture onwards, there was no observable difference in TER from the untreated control. These observations demonstrate that chloroquine and ammonium chloride increase the TER of immature Sertoli cells in the bicameral chamber.

Amines↗
PubMed1994

A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells.

Small rab/Ypt1/Sec4 GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways in eucaryotic cells. Polarized epithelial cells have morphologically and functionally distinct apical and basolateral surfaces separated by tight junctions. The establishment and maintenance of these structures require delivery of membrane proteins and lipids to these domains. In this work, we have isolated a cDNA clone from a human intestinal cDNA library encoding a small GTPase, rab13, closely related to the yeast Sec4 protein. Confocal microscopy analysis on polarized Caco-2 cells shows that rab13 protein colocalized with the tight junction marker ZO-1. Cryostat sections of tissues confirm that rab13 localized to the junctional complex region of a variety of epithelia, including intestine, kidney, liver, and of endothelial cells. This localization requires assembly and integrity of the tight junctions. Disruption of tight junctions by incubation in low Ca2+ media induces the redistribution of rab13. In cells devoid of tight junctions, rab13 was found associated with vesicles dispersed throughout the cytoplasm. Cell-cell contacts initiated by E-cadherin in transfected L cells do not recruit rab13 to the resulting adherens-like junction complexes. The participation of rab13 in polarized transport, in the assembly and/or the activity of tight junctions is discussed.

Amino Acid Sequence↗
PubMed2000

Organization of choroid plexus epithelial and endothelial cell tight junctions and regulation of claudin-1, -2 and -5 expression by protein kinase C.

Claudins are components of the tight junctional complex in epithelial and endothelial cells. We characterized the composition of tight junctions in the choroid plexus of the lateral ventricle in the rat brain and tested whether protein kinase C induced changes in their composition. Claudin-1, -2 and -5 were present in the epithelial cells at and near the tight junctions, respectively. In the endothelial cells, claudin-5 was stronger expressed than claudin-1 and -2. Twenty-four hours after the phorbolester injection into the ventricle, claudin-1 immunoreactivity of the epithelial cells was increased and spread to the cytoplasm. The claudin-2 and -5 immunoreactivities were reduced. These findings are consistent with an influence of protein kinase C on the composition of the tight junctions in the choroid plexus.

Animals↗
PubMed1999

Airway epithelial tight junctions and binding and cytotoxicity of Pseudomonas aeruginosa.

The role of tight junctions in the binding and cytoxicity of Pseudomonas aeruginosa to apical or basolateral membranes of lung airway epithelial cells was tested with fluorescence microscopy on living cells. Binding of noncytotoxic P. aeruginosa strain O1 was assessed with P. aeruginosa that expressed green fluorescent protein. Binding of cytotoxic P. aeruginosa strain 6206 was assessed with FITC-labeled P. aeruginosa; cytotoxicity was determined from nuclear uptake of the impermeant dye propidium iodide. The role of direct contact of P. aeruginosa to epithelial cells was tested with filters with small (0.45-micrometer) or large (2.0-micrometer) pores. High transepithelial resistance (R(t)) Calu-3 and cultured bovine tracheal monolayers (R(t) > 1,000 Omega. cm(2)) bound P. aeruginosa very infrequently (<1 P. aeruginosa/100 cells) at the apical membrane, but P. aeruginosa bound frequently to cells near "free edges" at holes, wounds, islands, and perimeters; cytotoxicity required direct interaction with basolateral membranes. Wounded high R(t) epithelia showed increased P. aeruginosa binding and cytotoxicity at the free edges because basolateral membranes were accessible to P. aeruginosa, and dead and living cells near the wound bound P. aeruginosa similarly. Compared with high R(t) epithelia, low R(t) CFT1 (R(t) = 100-200 Omega. cm(2)) and EGTA-treated Calu-3 monolayers were 25 times more susceptible to P. aeruginosa binding throughout the monolayer. Cytotoxicity to CFT1 cells (throughout the confluent monolayer, not only at the free edge) occurred after a shorter delay (0.25 vs. 2.0 h) and then five times faster than to Calu-3 cells, indicating that the time course of P. aeruginosa cytotoxicity may be limited by the rate of gaining access through tight junctions and that this occurred faster in low R(t) than in high R(t) airway epithelia. Cytotoxicity appeared to occur in a sequential process that led first to a loss of fura 2 and a later uptake of propidium iodide. P. aeruginosa bound three times more frequently to regions between cells (tight junctions?) than to cell membranes of low R(t) CFT1 cells.

Animals↗
PubMed2000

Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells.

In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell-cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin-mediated cell-cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell-cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell-cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO-1, but not claudin-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.

Animals↗
PubMed1994

Mammary epithelial cell tight junction integrity and mammary blood flow during an extended milking interval in goats.

The timing and relation of changes in mammary epithelial cell tight junction integrity and mammary blood flow during a 36-h milking interval were studied in six lactating Saanen goats. An increase in lactose concentration in plasma, a decrease in transepithelial potential difference, and changes in ionic milk composition were used to indicate tight junction patency. After 36 h of milk accumulation, mammary tight junctions had become disrupted. Further analyses indicated that this disruption began after 21 h of milk accumulation and that mammary blood flow also started to decline after 21 h. The time when both events occurred was not significantly different from the time when milk secretion began to decline (19 h). Moreover, positive but nonsignificant correlations existed between these events. Mammary tight junctions became disrupted when milk secretion declined, suggesting that impairment of mammary tight junction integrity is associated with decreased milk secretion during an extended milking interval. The decline in mammary blood flow may be the result of a negative feedback response to a reduced demand for metabolites, which is due to a reduced rate of milk secretion.

Animals↗
PubMed2004

The cytoplasmic tails of claudins can influence tight junction barrier properties through effects on protein stability.

The tight junction seal formed between epithelial cells varies among tissues in both tightness and ionic charge selectivity. We recently demonstrated that the extracellular domains of the claudin family of proteins are determinants of both characteristics, but in that study other unidentified domains in the claudins clearly contributed to their physiological potency. To investigate the importance of the cytoplasmic carboxyl-terminal domains in determining the degree to which a claudin can influence barrier properties, we constructed chimeras by exchanging the tails of claudin-2 and -4 and expressing them in MDCK II cells. Although swapping these domains had little effect on claudin localization, we found that the tail of claudin-2 could stabilize claudin-4, with a concomitant increase in both protein level and physiologic influence. This difference in stability was not an artifact of their chimeric structure, since metabolic radio-labeling experiments revealed that the half-life of endogenous claudin-2 is more than three times longer than claudin-4 (>12 h and approximately 4 h respectively). Further, half-life was not affected by removing the carboxyl-terminal three amino acids, which form a PDZ-binding motif. The finding that cytoplasmic tails of claudins strongly influence stability reveals a potential mechanism by which cells can establish their tight junction protein composition and thus function.

Animals↗
PubMed1992

Influence of metabolic inhibitors on the degradation of tight junctions in HT29 cells.

The human colon adenocarcinoma cell line HT 29 grows in culture without tight junctions (TJ). Tight junction strands of the fascia occludens type can be induced by treatment with proteases and are subsequently degraded during a period of about 3 h. Experiments using a variety of metabolic inhibitors such as 2-deoxyglucose, 2,4-dinitrophenol, and CCCP show that the degradation of TJ is retarded under conditions of ATP depletion. Thus it appears that the removal of TJ from the cell surface is an energy-dependent process. Moreover, DNP can specifically inhibit the degradation of TJ even in the absence of ATP depletion. The possible involvement of a proton gradient in the mechanism of TJ degradation is discussed.

2,4-Dinitrophenol↗
PubMed1998

An anti-inflammatory component derived from milk of hyperimmunised cows reduces tight junction permeability in vitro.

OBJECTIVE AND DESIGN: Effects of hyperimmune milk factor (HIMF), an anti-inflammatory factor from milk of hyperimmunised cows, on tight junction permeability and cell growth were studied in vitro. MATERIAL OR SUBJECTS: Mammary (HC11) and kidney (MDCK) epithelial cell lines were used. TREATMENT: HIMF was used at a final concentration of 2 mg/ml. METHODS: Tight junction permeability was assessed by measuring transepithelial electrical resistance (TER) across confluent monolayers, following the addition of HIMF with or without an inflammatory challenge. Cell growth was assessed by measuring total DNA of cultures with and without HIMF. Data were analysed by analyses of variance. RESULTS: HIMF promoted tight junction formation and prevented loss of TER following a challenge in both epithelia. Post-challenge recovery of TER was also faster with HIMF. HIMF inhibited cell growth. CONCLUSIONS: HIMF stimulates tight junction maintenance and formation, and its previously reported anti-inflammatory properties may be mediated by restricting the extravasation of white blood cells through tight junctions.

Animals↗
PubMed2001

Claudin-1, claudin-2 and claudin-11 are present in tight junctions of choroid plexus epithelium of the mouse.

The choroid plexus epithelium forms the blood-cerebrospinal fluid (CSF) barrier and is responsible for the secretion of the CSF from the blood. The morphological correlate of the blood-CSF barrier are the tight junctions of choroid plexus epithelium. By freeze-fracture electron microscopy it has been demonstrated that choroid plexus epithelial tight junctions form parallel strands resembling those of Sertoli cells building the blood-testis barrier and those of the myelin sheaths of oligodendrocytes. As the oligodendrocyte specific protein/claudin-11 has been shown to be the central mediator of parallel-array tight junctions in Sertoli cells and myelin sheaths in mice, we asked whether claudin-11 is present in the tight junctions of choroid plexus epithelial cells of the mouse. Here, we present the first direct evidence that claudin-11 besides claudin-1 and -2, occludin and the zonula occludens protein ZO-1 is present in choroid plexus epithelial tight junctions. During inflammation in the central nervous system such as experimental autoimmune encephalomyelitis, the molecular composition of choroid plexus epithelial tight junctions does not change considerably. Their unique molecular composition, with claudin-11 accompanied by claudin-1 and claudin-2 points to a unique regulatory mechanism of the blood-CSF-barrier function.

Animals↗
PubMed1992

The altered glomerular filtration slits seen in puromycin aminonucleoside nephrosis and protamine sulfate-treated rats contain the tight junction protein ZO-1.

Nephrosis induced in rats by puromycin aminonucleoside treatment (PAN) results in the apical displacement of the glomerular filtration slit membrane by newly formed, intercellular occluding-type junctions. Similar changes can also be induced by acute kidney perfusion with protamine sulfate (PS). We have investigated the molecular nature of these altered junctions using an antibody to ZO-1, a protein found exclusively in tight junctions. Immunoblotting demonstrates ZO-1, a 225-kd band, in glomerular extracts of normal, PAN-, and PS-treated rats. By immunofluorescence, ZO-1 was localized at the base of podocytes outlining the capillary loops of glomeruli from all three experimental groups. At the electron microscope level, using immunoperoxidase or immunogold labeling, ZO-1 was concentrated along the cytoplasmic surfaces of the slit diaphragms of normal rats. In PAN or PS rats, it was concentrated along both the newly formed occluding-type junctions and the remaining slit diaphragms. When podocalyxin (the major membrane sialoprotein of the podocyte) was similarly localized, it was found exclusively apical to the displaced slit membrane. Based on morphology and the presence of ZO-1, the altered junctions seen in PAN and PS rats appear to represent bona fide tight junctions. Their rapid (15-minute) induction in PS-treated rats suggests that on neutralization of the cell surface charge by polycation perfusion, discontinuous tight junctions form from a preexisting pool of junctional proteins. These findings raise the possibility that glomerular hydraulic conductivity may be regulated in part by regulating the relative patency and width of the filtration slits through focal tight junction assembly.

Animals↗
PubMed2000

Tight junction proteins ZO-1, ZO-2, and occludin along isolated renal tubules.

BACKGROUND: Tight junctions play a critical role in tubular function. In mammalian kidney, the transepithelial electrical resistance and the complexity of the tight junction increase from the proximal to the collecting tubule. The differential expression of three tight junction proteins, ZO-1, ZO-2, and occludin, along isolated rabbit renal tubules is examined in this article. METHODS: Microdissected rabbit renal tubules were processed for immunofluorescence detection of ZO-1, ZO-2, and occludin. The quantitation of these proteins was done by Western blot determinations in Percoll isolated tubules. RESULTS: ZO-1 stained cell boundaries independently of the identity of the tubule. However, the amount found in distal segments was significantly higher than that expressed in proximal regions. ZO-2 in the proximal region was found diffusely distributed in the cytoplasm, with faint staining at cell borders, while a clear signal at cell perimeters was detectable from the Henle's loop to collecting tubules. Nuclear staining of ZO-2 was found along the whole nephron. The presence of occludin at the proximal region was faint and discontinuous, while its expression in the more distant portions was conspicuous. The quantity of ZO-2 and occludin present at the distal region was significantly higher compared with the proximal segment. CONCLUSIONS: The distribution of ZO-1, ZO-2, and occludin follows the increase in junction complexity encountered in renal tubules. The amount of the three proteins found in proximal and distal segments is significantly higher in the latter.

Animals↗
PubMed1979

Degradation of tight junctions in HT29, a human colon adenocarcinoma cell line.

The process of tight junction degradation was followed in a cell line of human colon adenocarcinoma. Tight junctions are degraded by 2 mechanisms: (1) breakdown of junctional elements to intramembrane particles; (2) bleb formation by which tight-junctional elements are internalized into the cytoplasm or excluded into the medium. It is suggested that the first mechanism allows preservation of membrane particles for re-use, whereas the second is a mechanism by which the cells eliminate unneeded junctional elements.

Adenocarcinoma↗
PubMed1995

Determination of the Na permeability of the tight junctions of MDCK cells by fluorescence microscopy.

The kinetics of Na movement across the tight junctions of MDCK cells, grown on coverslips and perfused with HEPES or bicarbonate Ringer at 37 degrees C, were investigated after filling the lateral intercellular spaces (LIS) of the epithelium with SBFO, an Na-sensitive fluorescent dye. Dilution and bi-ionic potential measurements showed that MDCK cell tight junctions, although cation-selective, were poorly permeable to N-methyl-D-glucamine Cl (NMDG) but freely permeable to Li. In previous experiments in which Na was replaced by NMDG, a very slow decrease in LIS Na concentration (time constant = 4.8 min) resulted. In the present study, reduction of perfusate Na from 142 to 14 or 24 mM with Na replaced by Li caused LIS Na concentration to decrease with a time constant of 0.43 min. The time constant for Na increase of the LIS was 0.28 min, significantly shorter than that for Na decrease because of the additional component of transcellular Na influx. Ouabain eliminated the transcellular component and equalized the time constants for Na influx and efflux. These results were incorporated into a mathematical model which enabled calculation of the transcellular and paracellular Na fluxes during fluid reabsorption. Regulation of the Na permeability of individual tight junctions by protein kinase A (PKA) was evaluated by treating the monolayers with the Sp-cAMPS, a cAMP substitute, or Rp-cAMPS, a specific inhibitor of PKA. Stimulation of PKA strikingly increased tight junctional permeability while PKA inhibition diminished junctional Na permeability.

Animals↗
PubMed2004

Transport of octreotide and evaluation of mechanism of opening the paracellular tight junctions using superporous hydrogel polymers in Caco-2 cell monolayers.

The purpose of this study was to investigate the mechanism of opening of tight junctions in Caco-2 cell monolayers using superporous hydrogel (SPH) and SPH composite (SPHC) polymers as permeation enhancers for peptide drug delivery. Moreover, the transport of octreotide across Caco-2 cell monolayers was assessed by application of SPH and SPHC polymers on Caco-2 cell monolayers. In these experiments, N,N,N-trimethyl chitosan chloride with 60% quaternization (TMC60) was used as a positive control for opening of tight junctions. Transepithelial electrical resistance (TEER) studies showed that all three polymers (TMC60, SPH, and SPHC) were able to decrease TEER values to approximately 30% of the initial values, indicating the ability of these polymers to open the tight junctions. Recovery TEER studies showed that the effects of the polymers on Caco-2 cell monolayers were reversible, indicating viability of the cells after incubation with polymers. Both SPH and SPHC (compared with TMC60) were able to increase the paracellular transport of octreotide by their mechanical pressures on tight junctions. The mechanistic studies showed that junctional proteins, including actin, occludin, and claudin-1, were influenced by application of SPH and SPHC polymers to the Caco-2 cell monolayers. SPH and SPHC induced clear changes in the staining pattern of all three proteins compared with the control, indicating that the expression of these proteins in the tight junctions was increased, most likely due to the mechanical pressure of the polymers on the junctional proteins.

Caco-2 Cells↗
PubMed2004

Tight junction protein MAGI-1 is up-regulated by transfection with connexin 32 in an immortalized mouse hepatic cell line: cDNA microarray analysis.

Gap junctions are considered to play a crucial role in differentiation of epithelial cells, including hepatocytes. Recently, we found that Cx32 but not Cx26 was closely related to tight junctional proteins in primary cultured rat hepatocytes (Kojima et al., Exp Cell Res 263:193-201, 2001) and that Cx32 formation and/or Cx32-mediated intercellular communication could induce expression and function of tight junctions in a mouse hepatic cell line (Kojima et al., Exp Cell Res 276:40-51, 2002). In this study, to investigate the mechanisms of induction of tight junctions by transfection with Cx32, we performed cDNA microarray analysis of Cx32 transfectants, compared with parental cells derived from Cx32-deficient hepatocytes. In cDNA microarray analysis, a 2.5-fold increase in expression of membrane-associated guanylate kinase with inverted orientation-1 (MAGI-1), which is known to be localized at adherens and tight junction regions, was observed. High expression of MAGI-1 in Cx32 transfectants was confirmed by Western blotting and RT-PCR. MAGI-1 was colocalized with occludin, claudin-2, ZO-1, and F-actin, but not with E-cadherin in the apical-most regions at cell borders of Cx32 transfectants, similar to junctional adhesion molecule-1 (JAM-1), which may play a crucial role in formation and assembly of tight junctions. Treatment with the gap junction blocker 18beta-glycyrrhetinic acid did not affect expression of MAGI-1 and JAM-1 in Cx32 transfectants. These results suggest that Cx32 expression is in part related to induction of tight junctions through modulation of MAGI-1 expression in an immortalized mouse hepatic cell line.

Actins↗
PubMed1977

The effect of HCG on pinocytosis iwthin the canine inter-sertoli cell tight junction. A preliminary report.

The role of tight junctions in permeability barriers is now well established. Other roles for this junction have been postulated but have not been demonstrated. In both adult and prepubertal dogs, stimulation with human chorionic gonadotropin (hCG) results in an increased number of pinocytotic vesicles on the membrane faces enclosed by tight junctions within the inter-Sertoli cell junctions of the testis. It is suggested that these membranous regions, protected from the normal flow of intercellular fluids, may act as regions of selective intercellular exchange.

Animals↗
PubMed1990

Autonomic regulation of tight junctional permeability in the rat submandibular gland.

The permeability of tight junctions to tracers having different molecular weights was investigated in the submandibular gland of rats stimulated parasympathetically, sympathetically, or both. Lactoperoxidase (82,000 daltons), horseradish peroxidase (40,000 daltons), and microperoxidase (1,630 daltons) were used as the tracers. The tracers were administered by close arterial infusion via the glandular artery, and their secretion into the saliva was quantified biochemically, and their secretory routes within the gland were determined histochemically at the electron microscopic level. Microperoxidase and horseradish peroxidase passed into the saliva by electrical stimulation of either the chorda or superior cervical ganglion, and the combined stimulation of both caused a larger output of both tracers. No output of lactoperoxidase into the saliva occurred with any type of nerve stimulation. Electron microscopic histochemical observations showed that molecules of molecular weight equal to or lower than that of horseradish peroxidase entered the lumen through the tight junctions between adjacent acinar cells following combined stimulations of chorda and superior cervical ganglion. These findings indicate that electrical stimulation of parasympathetic and sympathetic nerves causes an increase in tight junctional permeability of acinar cells to microperoxidase and horseradish peroxidase. Although the combined stimulation of parasympathetic and sympathetic nerves resulted in increased junctional permeability to these tracers, the junctions remained impermeable to larger molecules, i.e., lactoperoxidase.

Animals↗