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.

Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation

1997 ◽  
Vol 273 (4) ◽  
pp. C1378-C1385 ◽  
Author(s):  
Jerrold R. Turner ◽  
Brian K. Rill ◽  
Susan L. Carlson ◽  
Denise Carnes ◽  
Rachel Kerner ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Inhibitors ◽  
Tight Junction Regulation ◽  
Tight Junction Permeability ◽  
Mlc Phosphorylation

Tight junctions serve as the rate-limiting barrier to passive movement of hydrophilic solutes across intestinal epithelia. After activation of Na+-glucose cotransport, the permeability of intestinal tight junctions is increased. Because previous analyses of this physiological tight junction regulation have been restricted to intact mucosae, dissection of the mechanisms underlying this process has been limited. To characterize this process, we have developed a reductionist model consisting of Caco-2 intestinal epithelial cells transfected with the intestinal Na+-glucose cotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstrate physiological Na+-glucose cotransport. Activation of SGLT1 results in a 22 ± 5% fall in transepithelial resistance (TER) ( P< 0.001). Similarly, inactivation of SGLT1 by addition of phloridzin increases TER by 24 ± 2% ( P < 0.001). The increased tight junction permeability is size selective, with increased flux of small nutrient-sized molecules, e.g., mannitol, but not of larger molecules, e.g., inulin. SGLT1-dependent increases in tight junction permeability are inhibited by myosin light-chain kinase inhibitors (20 μM ML-7 or 40 μM ML-9), suggesting that myosin regulatory light-chain (MLC) phosphorylation is involved in tight junction regulation. Analysis of MLC phosphorylation showed a 2.08-fold increase after activation of SGLT1 ( P< 0.01), which was inhibited by ML-9 ( P < 0.01). Thus monolayers incubated with glucose and myosin light-chain kinase inhibitors are comparable to monolayers incubated with phloridzin. ML-9 also inhibits SGLT1-mediated tight junction regulation in small intestinal mucosa ( P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junction regulation subsequent to SGLT1 activation. The intimate relationship between tight junction regulation and MLC phosphorylation suggests that a critical step in regulation of epithelial tight junction permeability may be myosin ATPase-mediated contraction of the perijunctional actomyosin ring and subsequent physical tension on the tight junction.

FK506 BINDING PROTEIN 8 (FKBP8) INTERACTIONS WITH MYOSIN LIGHT CHAIN KINASE 1 ARE REQUIRED FOR TNF-INDUCED TIGHT JUNCTION BARRIER LOSS

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S25-S25
Author(s):  
Li Zuo ◽  
Feng Cao ◽  
Wei-Ting Kuo ◽  
Jerrold Turner
Keyword(s):  
Tight Junction ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Intestinal Barrier ◽  
Intestinal Epithelial ◽  
Binding Interaction ◽  
Chaperone Protein ◽  
Tight Junction Permeability ◽  
Mlc Phosphorylation

Abstract Background Tumor necrosis factor (TNF) regulates intestinal epithelial tight junction permeability by activating myosin light chain kinase 1 (MLCK1) expression and enzymatic activity. MLCK1 recruitment to the apical perijunctional actomyosin ring (PAMR) is, however, required for barrier regulation; Divertin, a small molecule drug that blocks this recruitment, prevents barrier loss and attenuates both acute and chronic experimental diarrheal disease. We therefore hypothesized that MLCK1 recruitment to the PAMR requires interactions with as yet unidentified chaperone protein(s). Objective To identify binding partners and define the mechanisms by which they activate MLCK1 recruitment to the PAMR. Results We performed a yeast-2-hybrid (Y2H) screen using the MLCK1 domains required for PAMR recruitment as bait. FKBP8, which encodes a peptidyl-prolyl cis-trans isomerase (PPI), was recovered, and direct binding to the MLCK1 domains (Kd=~5mM) was confirmed using microscale thermophoresis (MST). This binding interaction required the FK506-binding PPI domain and was specifically inhibited by FK506 (tacrolimus). Immunofluorescent microscopy demonstrated partial colocalization of MLCK1 and FKBP8 within intestinal epithelial monolayers; TNF caused both to concentrate around the PAMR. To further characterize this interaction, we performed proximity ligation assays (PLA) and found that TNF increased interaction between MLCK1 and FKBP8 &gt; 2-fold. FK506 prevented TNF-induced increases in PLA signal. FK506 was also able to reverse TNF-induced increases in myosin light chain (MLC) phosphorylation and tight junction permeability. In Caco-2 monolayers, FKBP8 knockout blocked TNF-induced MLCK1 recruitment, MLC phosphorylation, and tight junction barrier loss; all of which were restored by FKBP8 re-expression. In mice, MLC phosphorylation and intestinal barrier loss triggered by acute, anti-CD3-induced, T cell activation were blocked by luminal FK506. Importantly, this local FK506 treatment did not prevent anti-CD3-induced increases in mucosal TNF, IL-1b, and IFNg. Immunostains of biopsies from IBD patients documented increased PAMR MLC phosphorylation, MLCK1 recruitment, FKBP8 recruitment, and MLCK1-FKBP8 PLA signal relative to control subjects. Conclusions FKBP8 is a chaperone protein required for TNF-induced MLCK1 recruitment and barrier loss. This requires direct interaction between MLCK1 and the PPI domain of FKBP8. FK506 binding to the PPI domain displaces MLCK1 thereby preventing recruitment to the PAMR and barrier loss. These activities are separate from the immunosuppressive effects of FK506. We speculate that molecular blockade of the FKBP8-MLCK1 interaction may be a novel approach to barrier restoration and therapy of diseases associated with intestinal barrier dysfunction. Support NIH (DK068271, DK061931) and the NNSF of China (81800464, 82070548).

PKC-dependent regulation of transepithelial resistance: roles of MLC and MLC kinase

1999 ◽  
Vol 277 (3) ◽  
pp. C554-C562 ◽  
Author(s):  
J. R. Turner ◽  
J. M. Angle ◽  
E. D. Black ◽  
J. L. Joyal ◽  
D. B. Sacks ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Myosin Ii ◽  
Transepithelial Resistance ◽  
Pkc Inhibitor ◽  
Kinase C ◽  
H 51 ◽  
Mlc Phosphorylation ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Pkc Activation

The mechanisms by which protein kinase C (PKC) activation results in increased transepithelial resistance (TER) are unknown [G. Hecht, B. Robinson, and A. Koutsouris. Am. J. Physiol. 266 ( Gastrointest. Liver Physiol. 29): G214–G221, 1994]. We have previously shown that phosphorylation of the regulatory light chain of myosin II (MLC) is associated with decreases in TER and have suggested that contraction of the perijunctional actomyosin ring (PAMR) increases tight junction (TJ) permeability [J. R. Turner, B. K. Rill, S. L. Carlson, D. Carnes, R. Kerner, R. J. Mrsny, and J. L. Madara. Am. J. Physiol. 273 ( Cell Physiol. 42): C1378–C1385, 1997]. We therefore hypothesized that PKC activation alters TER via relaxation of the PAMR. Activation of PKC by the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a progressive dose-dependent increase in TER that was apparent within 15 min (111% of controls) and maximal within 2 h (142% of controls). Similar increases were induced by a diacylglycerol analog, and the effects of both PMA and the diacylglycerol analog were prevented by the PKC inhibitor bisindolylmaleimide I. PMA treatment caused progressive decreases in MLC phosphorylation, by 12% at 15 min and 41% at 2 h. Phosphorylation of MLC kinase (MLCK) increased by 64% within 15 min of PMA treatment and was stable over 2 h (51% greater than that of controls). Thus increases in MLCK phosphorylation preceded decreases in MLC phosphorylation. These data suggest that PKC regulates TER via decreased phosphorylation of MLC, possibly due to inhibitory phosphorylation of MLCK. The decreased phosphorylation of MLC likely reduces PAMR tension, leading to decreased TJ permeability.

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 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.

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)

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.

scholarly journals Neutrophil migration across a cultured epithelial monolayer elicits a biphasic resistance response representing sequential effects on transcellular and paracellular pathways

1992 ◽  
Vol 117 (4) ◽  
pp. 757-764 ◽  
Author(s):  
CA Parkos ◽  
SP Colgan ◽  
C Delp ◽  
MA Arnaout ◽  
JL Madara
Keyword(s):  
Tight Junction ◽  
Time Course ◽  
Short Circuit ◽  
Neutrophil Migration ◽  
Short Circuit Current ◽  
Transepithelial Resistance ◽  
Sequential Effects ◽  
T84 Cells ◽  
Resistance Response ◽  
Tight Junction Permeability

Migration of polymorphonuclear leukocytes across epithelia is a hallmark of many inflammatory disease states. Neutrophils traverse epithelia by migrating through the paracellular space and crossing intercellular tight junctions. We have previously shown (Nash, S., J. Stafford, and J.L. Madara. 1987. J. Clin. Invest. 80:1104-1113), that leukocyte migration across T84 monolayers, a model human intestinal epithelium, results in enhanced tight junction permeability--an effect quantitated by the use of a simple, standard electrical assay of transepithelial resistance. Here we show that detailed time course studies of the transmigration-elicited decline in resistance has two components, one of which is unrelated to junctional permeability. The initial decrease in resistance, maximal 5-13 min after initiation of transmigration, occurs despite inhibition of transmigration by an antibody to the common beta subunit of neutrophil beta 2 integrins, and is paralleled by an increase in transepithelial short-circuit current. Chloride ion substitution and inhibitor studies indicate that the early-phase resistance decline is not attributable to an increase in tight junction permeability but is due to decreased resistance across epithelial cells resulting from chloride secretion. Since T84 cells are accepted models for studies of the regulation of Cl- and water secretion, our results suggest that neutrophil transmigration across mucosal surfaces (for example, respiratory and intestinal tracts) may initially activate flushing of the surface by salt and water. Equally important, these studies, by providing a concrete example of sequential transcellular and paracellular effects on transepithelial resistance, highlight the fact that this widely used assay cannot simply be viewed as a direct functional probe of tight junction permeability.

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).

scholarly journals Caveolin-1–dependent occludin endocytosis is required for TNF-induced tight junction regulation in vivo

2010 ◽  
Vol 189 (1) ◽  
pp. 111-126 ◽  
Author(s):  
Amanda M. Marchiando ◽  
Le Shen ◽  
W. Vallen Graham ◽  
Christopher R. Weber ◽  
Brad T. Schwarz ◽  
...  
Keyword(s):  
Tight Junction ◽  
Myosin Light Chain ◽  
Structural Reorganization ◽  
Caveolin 1 ◽  
Tight Junction Regulation ◽  
Tight Junction Permeability ◽  
Barrier Regulation ◽  
Factor Α ◽  
Necrosis Factor

Epithelial paracellular barrier function, determined primarily by tight junction permeability, is frequently disrupted in disease. In the intestine, barrier loss can be mediated by tumor necrosis factor (α) (TNF) signaling and epithelial myosin light chain kinase (MLCK) activation. However, TNF induces only limited alteration of tight junction morphology, and the events that couple structural reorganization to barrier regulation have not been defined. We have used in vivo imaging and transgenic mice expressing fluorescent-tagged occludin and ZO-1 fusion proteins to link occludin endocytosis to TNF-induced tight junction regulation. This endocytosis requires caveolin-1 and is essential for structural and functional tight junction regulation. These data demonstrate that MLCK activation triggers caveolin-1–dependent endocytosis of occludin to effect structural and functional tight junction regulation.

Sign in / Sign up

Export Citation Format

Share Document