Cytoplasmic regulation of tight-junction permeability: effect of plant cytokinins

1980 ◽  
Vol 239 (3) ◽  
pp. C75-C89 ◽  
Author(s):  
C. J. Bentzel ◽  
B. Hainau ◽  
S. Ho ◽  
S. W. Hui ◽  
A. Edelman ◽  
...  
Keyword(s):  
Tight Junction ◽  
Cytochalasin B ◽  
De Novo ◽  
Morphological Changes ◽  
Freeze Fracture ◽  
Transepithelial Resistance ◽  
Molecular Flow ◽  
Necturus Gallbladder ◽  
Tight Junction Permeability ◽  
Flow Through

The significance of the "leaky" tight junction might be understood better if cells of the epithelial monolayer possessed mechanisms to regulate molecular flow through the junction. To test this possibility, Necturus gallbladder, a representative leaky epithelium, was studied before, during, and after mucosal exposure to plant cytokinins and two other microfilament-active drugs, cytochalasin B and phalloidin. Concomitant with morphological changes in microfilaments, cytokinins induced rapid reversible increases in transepithelial resistance and potential difference (PD) and decreases in NaCl dilution potentials, with no change in the ratio of relative cell membrane resistances. Cytochalasin B (0.2-1.2 microM) and phalloidin (0.6-12.7 microM) caused similar changes in transepithelial resistance and PD. When the intramembranous structure of tight junctions was studied by freeze fracture, peak cytokinin-induced increments in transepithelial resistance were associated with more disorder in the strand meshwork resulting in a small increase in tight junction depth, but there was no evidence of de novo strand assembly. These studies suggest that permeability of the tight junction of Necturus gallbladder is subject to rapid reversible modulation, possibly under cytoskeletal control.

Ca2+ regulation of tight-junction permeability and structure in Necturus gallbladder

1983 ◽  
Vol 245 (3) ◽  
pp. C203-C212 ◽  
Author(s):  
C. E. Palant ◽  
M. E. Duffey ◽  
B. K. Mookerjee ◽  
S. Ho ◽  
C. J. Bentzel
Keyword(s):  
Cell Membrane ◽  
Tight Junction ◽  
Morphological Changes ◽  
Freeze Fracture ◽  
Transepithelial Resistance ◽  
Mucosal Cell ◽  
Base Line ◽  
Ionophore A23187 ◽  
Necturus Gallbladder ◽  
Tight Junction Permeability

To explore the role of Ca2+ in tight-junction permeability, the Necturus gallbladder was exposed to varying Ca2+ concentrations and to the Ca2+ ionophore A23187 added to the mucosal side (1.9 X 10(-6) to 6.8 X 10(-5) M). Electrophysiological parameters measured in an Ussing-type chamber were correlated with tight-junction morphology revealed by freeze-fracture electron microscopy. In Ca2+-free bathing media, transepithelial resistance decreases and tight-junctional ultrastructure is fragmented. In 1.8 mM Ca2+ media, A23187 induces an initial drop in transepithelial resistance, followed by an increase in transepithelial resistance to a value 20% above base line. At peak response to A23187, NaCl diffusion potentials decrease. Freeze-fracture replicas reveal that the number of junctional strands increase pari passu with junctional depth. Both physiological and morphological changes were partially reversible. The initial decrease in transepithelial resistance coincided with a persistent hyperpolarization of the mucosal cell membrane potential difference and a decrease in the mucosal-to-serosal cell membrane resistance ratio. Thus A23187 alters both the transcellular and paracellular pathway, resulting in opposing effects on transepithelial resistance.

scholarly journals Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance.

1988 ◽  
Vol 107 (6) ◽  
pp. 2401-2408 ◽  
Author(s):  
B R Stevenson ◽  
J M Anderson ◽  
D A Goodenough ◽  
M S Mooseker
Keyword(s):  
Tight Junction ◽  
Mdck Cells ◽  
Freeze Fracture ◽  
Transepithelial Resistance ◽  
Madin Darby Canine Kidney ◽  
Junctional Permeability ◽  
Tight Junction Permeability ◽  
Junction Structure ◽  
Darby Canine Kidney ◽  
Canine Kidney

The relationship of tight junction permeability to junction structure and composition was examined using two strains of Madin-Darby canine kidney (MDCK) cells (I and II) which differ greater than 30-fold in transepithelial resistance. This parameter is largely determined by paracellular, and hence junctional, permeability under most conditions. When these two strains of cells were grown on permeable filter supports, they formed monolayers with equivalent linear amounts of junction/area of monolayer. Ultrastructural analysis of these monolayers by thin section EM revealed no differences in overall cellular morphology or in tight junction organization. Morphometric analysis of freeze-fractured preparations indicated that the tight junctions of these two cell strains were similar in both number and density of junctional fibrils. Prediction of transepithelial resistance for the two strains from this freeze-fracture data and a published structure-function formulation (Claude, P. 1978, J. Memb. Biol. 39:219-232) yielded values (I = 26.5 omega/cm2, II = 35.7 omega/cm2) that were significantly lower than those observed (I = 2,500-5,000 omega/cm2, II = 50-70 omega/cm2). Consistent with these structural studies, a comparison of the distribution and cellular content of ZO-1, a polypeptide localized exclusively to the tight junction, revealed no significant differences in either the localization of ZO-1 or the amount of ZO-1 per micron of junction (I = 1,415 +/- 101 molecules/micron, II = 1,514 +/- 215 molecules/micron).

Transepithelial resistance can be regulated by the intestinal brush-border Na+/H+ exchanger NHE3

2000 ◽  
Vol 279 (6) ◽  
pp. C1918-C1924 ◽  
Author(s):  
Jerrold R. Turner ◽  
Eric D. Black ◽  
Jeff Ward ◽  
Chung-Ming Tse ◽  
Frederick A. Uchwat ◽  
...  
Keyword(s):  
Tight Junction ◽  
Myosin Ii ◽  
Transepithelial Resistance ◽  
Cell Swelling ◽  
Intestinal Brush Border ◽  
Tight Junction Regulation ◽  
Cytoplasmic Acidification ◽  
Tight Junction Permeability ◽  
Dimethyl Amiloride ◽  
Mlc Phosphorylation

Initiation of intestinal Na+-glucose cotransport results in transient cell swelling and sustained increases in tight junction permeability. Since Na+/H+ exchange has been implicated in volume regulation after physiological cell swelling, we hypothesized that Na+/H+ exchange might also be required for Na+-glucose cotransport-dependent tight junction regulation. In Caco-2 monolayers with active Na+-glucose cotransport, inhibition of Na+/H+ exchange with 200 μM 5-( N, N-dimethyl)- amiloride induced 36 ± 2% increases in transepithelial resistance (TER). Evaluation using multiple Na+/H+ exchange inhibitors showed that inhibition of the Na+/H+ exchanger 3 (NHE3) isoform was most closely related to TER increases. TER increases due to NHE3 inhibition were related to cytoplasmic acidification because cytoplasmic alkalinization with 5 mM NH4Cl prevented both cytoplasmic acidification and TER increases. However, NHE3 inhibition did not affect TER when Na+-glucose cotransport was inhibited. Myosin II regulatory light chain (MLC) phosphorylation decreased up to 43 ± 5% after inhibition of Na+/H+ exchange, similar to previous studies that associate decreased MLC phosphorylation with increased TER after inhibition of Na+-glucose cotransport. However, NHE3 inhibitors did not diminish Na+-glucose cotransport. These data demonstrate that inhibition of NHE3 results in decreased MLC phosphorylation and increased TER and suggest that NHE3 may participate in the signaling pathway of Na+-glucose cotransport-dependent tight junction regulation.

scholarly journals Circulating Ouabain Modulates Expression of Claudins in Rat Intestine and Cerebral Blood Vessels

2020 ◽  
Vol 21 (14) ◽  
pp. 5067
Author(s):  
Alexander G. Markov ◽  
Arina A. Fedorova ◽  
Violetta V. Kravtsova ◽  
Anastasia E. Bikmurzina ◽  
Larisa S. Okorokova ◽  
...  
Keyword(s):  
Tight Junction ◽  
Blood Vessels ◽  
Barrier Properties ◽  
Transepithelial Resistance ◽  
Epithelial Barrier ◽  
Cerebral Blood Vessels ◽  
Down Regulation ◽  
Barrier Formation ◽  
Tight Junction Permeability

The ability of exogenous low ouabain concentrations to affect claudin expression and therefore epithelial barrier properties was demonstrated previously in cultured cell studies. We hypothesized that chronic elevation of circulating ouabain in vivo can affect the expression of claudins and tight junction permeability in different tissues. We tested this hypothesis in rats intraperitoneally injected with ouabain (1 μg/kg) for 4 days. Rat jejunum, colon and brain frontal lobes, which are variable in the expressed claudins and tight junction permeability, were examined. Moreover, the porcine jejunum cell line IPEC-J2 was studied. In IPEC-J2-cells, ouabain (10 nM, 19 days of incubation) stimulated epithelial barrier formation, increased transepithelial resistance and the level of cSrc-kinase activation by phosphorylation, accompanied with an increased expression of claudin-1, -5 and down-regulation of claudin-12; the expression of claudin-3, -4, -8 and tricellulin was not changed. In the jejunum, chronic ouabain increased the expression of claudin-1, -3 and -5 without an effect on claudin-2 and -4 expression. In the colon, only down-regulation of claudin-3 was observed. Chronic ouabain protected the intestine transepithelial resistance against functional injury induced by lipopolysaccharide treatment or by modeled acute microgravity; this regulation was most pronounced in the jejunum. Claudin-1 was also up-regulated in cerebral blood vessels. This was associated with reduction of claudin-3 expression while the expression of claudin-5 and occludin was not affected. Altogether, our results confirm that circulating ouabain can functionally and tissue-specifically affect barrier properties of epithelial and endothelial tissues via Na,K-ATPase-mediated modulation of claudins expression.

Osmotic water flow pathways across Necturus gallbladder: role of the tight junction

1994 ◽  
Vol 266 (4) ◽  
pp. G722-G730 ◽  
Author(s):  
K. Loeschke ◽  
C. J. Bentzel
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Water Flow ◽  
Freeze Fracture ◽  
Electrical Conductance ◽  
Leaky Epithelia ◽  
Necturus Gallbladder ◽  
Osmotic Water ◽  
Flow Pathways ◽  
Osmotic Water Flow

To explore the quantitative significance of passive water flow through tight junctions of leaky epithelia, transepithelial water flow rates were measured in Necturus gallbladder mounted in chambers. Osmotic flows generated by raffinose gradients were asymmetrical with the greater flow in the mucosal-to-serosal direction. In tissue fixed in situ, intercellular spaces were dilated during mucosal-to-serosal flow and closed during serosal-to-mucosal flow. Tight junctions were focally separated (blistered), which correlated with the magnitude of mucosal-to-serosal flow. Blisters were not observed during serosal-to-mucosal flow or in nontransporting gallbladders. In freeze-fracture replicas, blisters appeared as pockets between intramembranous strands. Protamine, which decreases electrical conductance and increases depth and complexity of the tight junction, reduced osmotic water flow by approximately 30% in the mucosal-to-serosal direction (100 mosmol/kg gradient) without altering serosal-to-mucosal flow. We suggest that in the steady state, at least 30% of osmotically driven water passes transjunctionally in the mucosal-to-serosal direction, but flow is transcellular in the serosal-to-mucosal direction. Directionally divergent pathways may account for flow asymmetry.

HOCl effects on the tight junctions of rabbit tracheal epithelium

1996 ◽  
Vol 270 (2) ◽  
pp. L224-L231 ◽  
Author(s):  
Y. Guo ◽  
M. Krumwiede ◽  
J. G. White ◽  
O. D. Wangensteen
Keyword(s):  
Tight Junction ◽  
Morphological Changes ◽  
Cell Damage ◽  
Freeze Fracture ◽  
Tracheal Epithelium ◽  
Epithelial Tissue ◽  
Laser Confocal Microscopy ◽  
Transmission Electron ◽  
Junction Protein ◽  
Before And After

We previously found that HOCl, produced from neutrophil products in infected airways, decreases electrical resistance (R) of rabbit tracheal epithelium. Interestingly, HOCl at 6 mM, a reasonable concentration in diseased airways, decreased R without apparent cell damage. This study sought to determine whether this noncytotoxic dose of HOCl causes morphological changes that correlate with the decrease in R. Excised rabbit tracheas were treated with 6 mM HOCl for 15 min, before and after which epithelial R was determined. Epithelial tissue was then fixed and prepared for transmission electron microscopy, immunofluorescent labeling of F-actin or the tight junction protein ZO-1, or freeze fracture to examine tight junction strands. HOCl treatment caused a 50% decrease in R. Electron micrographs showed no cell, cell membrane, or tight junction changes. By laser confocal microscopy, 6 mM HOCl did not affect the distribution of F-actin or ZO-1. However, morphometric analysis of freeze-fracture replicas showed that tight junction strand number was significantly decreased from 7.06 +/- 0.09 to 4.79 +/- 0.11 and junctional width was significantly decreased from 0.306 +/- 0.007 to 0.214 +/- 0.006 microns. These latter changes may have contributed to the observed decrease in epithelial R.

scholarly journals Structural integrity of hepatocyte tight junctions.

1983 ◽  
Vol 96 (3) ◽  
pp. 745-749 ◽  
Author(s):  
D W Easter ◽  
J B Wade ◽  
J L Boyer
Keyword(s):  
Bile Duct ◽  
Tight Junction ◽  
Structural Breaks ◽  
Structural Integrity ◽  
Bile Duct Ligation ◽  
Freeze Fracture ◽  
Paracellular Permeability ◽  
Extensive Analysis ◽  
Duct Ligation ◽  
Tight Junction Permeability

The significance of discontinuities frequently found in freeze-fracture replicas of the tight junction was evaluated using complementary replicas of hepatocyte junctions from control and bile duct-ligated rats. An extensive analysis of complementary replicas using rotary platinum shadowing indicates that discontinuities in the protoplasmic (P) fracture face do not represent structural breaks in the tight-junctional network. In no case did P-face discontinuities correspond with interruptions in the groove network on the complementary extracellular (E) face. Quantitative analysis of replicas shows that P-face discontinuities result in part from "transfer" of material to the complementary E face (approximately 7% of the junctional length). However, many P-face discontinuities (7-30% of the junctional length) are matched only by a groove on the complementary E face. This finding demonstrates that a significant amount of material can be lost during freeze-fracture. An analysis of junctions from bile duct-ligated rats, which are known to have an increased paracellular permeability, shows comparable transfer and loss of material. However, the number of junctional elements and the tight-junction network density was significantly reduced by bile duct ligation. These observations indicate that discontinuities in tight-junctional elements result during the preparation of freeze-fracture replicas and are not physiologically important features of the junctional barrier. Variation in the number of elements provides the best explanation for observed differences in tight-junction permeability.

Concentration-dependent effects of cytochalasin D on tight junctions and actin filaments in MDCK epithelial cells

1994 ◽  
Vol 107 (3) ◽  
pp. 367-375 ◽  
Author(s):  
B.R. Stevenson ◽  
D.A. Begg
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Actin Filaments ◽  
Mdck Cells ◽  
Microscopic Analysis ◽  
Cytochalasin D ◽  
Transepithelial Resistance ◽  
Actin Bundles ◽  
Filament Bundles ◽  
Tight Junction Permeability

The effects of different concentrations of the actin-disrupting drug cytochalasin D on tight junction permeability and distribution of actin filaments in MDCK epithelial cells were examined. Consistent with previous studies, 2 micrograms/ml cytochalasin D caused a significant decrease in transepithelial resistance, indicative of an increase in tight junction permeability. Surprisingly, increasing concentrations of cytochalasin D caused progressively smaller decreases in transepithelial resistance. The effects of cytochalasin D were reversible. Light microscopic analysis utilizing rhodamine-conjugated phalloidin demonstrated two distinct populations of actin filaments in MDCK cells: an apical peripheral ring of actin, presumably associated with the zonula adherens, and larger actin bundles more basally situated. When treated with 2 micrograms/ml cytochalasin D, both actin populations were severely disrupted and cells became flattened. Actin in the apical ring aggregated along cell boundaries, and these aggregates co-localized with similarly disrupted focal accumulations of the tight junction-associated protein ZO-1. The basal actin filament bundles also reorganized into focal aggregates. Increasing concentrations of cytochalasin D caused gradually less perturbation of the apical actin ring, consistent with the transepithelial resistance observations. However, the basal actin bundles were disrupted at all concentrations of cytochalasin D tested, demonstrating that the two actin populations are differentially sensitive to cytochalasin D and that apical actin filaments are more important in the regulation of tight junction permeability. Finally, treatment of cells with cytochalasin D inhibited the decrease in transepithelial resistance induced by the chelation of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the junctional integrity by low or high concentrations of cytochalasin B and dihydrocytochalasin B in associated with distinct changes in F-actin and ZO-1

1996 ◽  
Vol 16 (4) ◽  
pp. 313-326 ◽  
Author(s):  
Pia Nybom ◽  
Karl-Eric Magnusson
Keyword(s):  
Tight Junction ◽  
Cytochalasin B ◽  
Freeze Fracture ◽  
Permeability Barrier ◽  
Electrical Measurements ◽  
Specific Effects ◽  
Actin Structure ◽  
Junctional Permeability ◽  
Freeze Etching ◽  
High Concentrations

In a study of Necturus gallbladder epithelium Benzel et al. (Benzel et al., 1980) found that low (0.2–1.2 μM) and higher concentrations (1.5 μM and more) of cytochalasin B (CB) caused an increase and decrease in the transepithelial electrical resistance (TER), respectively. Moreover, there were slight changes in the height and complexicity of tight junction (TJ) strands, as visualized by freeze-fracture and freeze-etching. To elucidate the mechanisms of these findings, we first demonstrated that the effect is also present in monolayers of Madin-Darby Canine Kidney strain I (MDCK-I) cells. Thus, a low concentration (0.1 ng/ml) cytochalasin B (CB) strengthened the permeability barrier, as evidenced quantitatively by increases in TER on transepithelial electrical measurements. Furthermore, indirect immunofluorescence and confocal microscopy demonstrated that this effect was paralleled with an accumulation of F-actin and the tight junction marker protein, ZO-1, at the level of TJ. Equimolar concentrations of dihydrocytochalasin B (dhCB), on the other hand, did not lead to a tightening of the epithelium. Confirming previous studies, there was a general decrease in epithelial resistance after treatment with high concentrations (1 μg/ml) of CB and dhCB, which was accompanied by distinct changes in the F-actin network and distribution of ZO-1. We speculate that the divergent effects of CB and dhCB on the F-actin and ZO-1 organization might be due to specific effects on the transport of monosaccharides across the plasma membrane, or that CB and dhCB in distinct ways involve the turnover of phosphatidylinositols in the membrane, thereby modulating junctional permeability and F-actin structure.

scholarly journals Increases in guinea pig small intestinal transepithelial resistance induced by osmotic loads are accompanied by rapid alterations in absorptive-cell tight-junction structure.

1983 ◽  
Vol 97 (1) ◽  
pp. 125-136 ◽  
Author(s):  
J L Madara
Keyword(s):  
Guinea Pig ◽  
Tight Junction ◽  
Ussing Chamber ◽  
Freeze Fracture ◽  
Transepithelial Resistance ◽  
Absorptive Cell ◽  
Cation Selectivity ◽  
Lateral Membrane ◽  
Small Intestinal ◽  
Junction Structure

In some epithelia, mucosal exposure to osmotic loads produces an increase in transepithelial resistance that is presumed to relate to the collapse of the paracellular spaces. Since proximal small intestinal epithelium may transiently encounter osmotic loads during normal digestion, we examined the short-term effect of osmotic loads on resistance and on epithelial structure of mucosal sheets prepared from guinea pig jejunum using Ussing-chamber, thin-section electron-microscopic, and freeze-fracture techniques. After equilibration of mucosal sheets in chambers, mucosal buffer tonicity was increased to 600 mosM with mannitol. This resulted in a 64% increase in resistance within 20 min. Concomitantly, 600 mosM produced a decrease in tight-junction cation selectivity as judged from dilution potentials, collapse of paracellular spaces, decreased cytoplasmic electron density in 10-40% of absorptive cells, and focal absorptive-cell subjunctional lateral-membrane evaginations often associated with microfilament arrays. Freeze-fracture replicas of absorptive-cell tight junctions revealed significant increases in both strand count and depth. Preincubation with 5 micrograms/ml cytochalasin D reduced the 600 mosM resistance increase caused by 600 mosM exposure by 48% but did not prevent the collapse of paracellular spaces. Lowered temperatures that produced morphologic evidence consistent with a gel-phase transition of absorptive-cell lateral membranes prevented both the resistance response and the alterations in tight-junction structure. In conclusion, transient osmotic loads produce an increase in resistance in jejunal epithelium and alter both absorptive-cell tight-junction charge selectivity and structure. These responses, which may have physiologic implications, can be reduced by cytoskeletal inhibitors and ablated by conditions that restrict mobility of absorptive-cell lateral-membrane molecules.

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