Increase In Tight Junction Permeability Research Articles

Relationship of serine/threonine phosphorylation/dephosphorylation signaling to glucocorticoid regulation of tight junction permeability and ZO-1 distribution in nontransformed mammary epithelial cells.

The synthetic glucocorticoid dexamethasone regulates tight junction permeability resulting in an increased transepithelial electrical resistance (TER) of cultured 31EG4 mammary epithelial cells. Inhibition of cellular type 1 and type 2A protein phosphatase activity by okadaic acid reduced the TER of dexamethasone-treated monolayers of 31EG4 cells to basal levels within 24 h. Coincident with the increase in tight junction permeability, immunofluorescence revealed that okadaic acid caused a partial cellular redistribution of the ZO-1 tight junction-associated protein. The potent glucocorticoid antagonist RU486 had no effect on TER or ZO-1 distribution, indicating that the effects of okadaic acid are not a result of disrupting glucocorticoid receptor function. Immunoprecipitation of 32P-labeled cells and V8 protease peptide mapping demonstrated that dexamethasone did not alter ZO-1 phosphorylation. However, consistent with the changes in TER, dexamethasone induced a 2.3-fold stimulation in ZO-1 protein levels which was reduced to 73% of basal levels by okadaic acid. No effects on ZO-1 transcript levels were observed. Monolayers grown in the presence of glucocorticoids had only 28% less junction density and 16.5% more linear junction/cell, which cannot account for the observed increases of TER and ZO-1 protein levels. Taken together, our results have shown that a disruption of phosphorylation/dephosphorylation activity overrides the glucocorticoid regulation of tight junction permeability in 31EG4 mammary cells.

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

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 tumor necrosis factor-induced increase in renal (LLC-PK1) transepithelial permeability.

Tumor necrosis factor-alpha (TNF) causes a spontaneously reversible increase in tight junction permeability. TNF was the only cytokine tested that produced this effect. The effect on transepithelial permeability proceeds in four distinct phases: 1) a 60- to 90-min delay from time of application of TNF, 2) a rapid decrease in transepithelial resistance, 3) a recovery of transepithelial resistance to control level within 1 h, and 4) a further increase of transepithelial resistance above control levels. The recovery of transepithelial resistance occurs with or without TNF in the culture medium. Different protein kinase inhibitors affected different phases of this overall process. The tyrosine kinase inhibitor genistein significantly blocked the TNF effect. Neither transcription nor protein synthesis was required for transepithelial permeability to increase, but were required for the recovery. After the tight junctions have opened at 2 h in response to TNF, a second application of TNF will not produce the effect again for at least 12 h. The tight junctions will, however, open in response to phorbol esters during this time frame. Electron microscopy studies using apically applied ruthenium red suggest that TNF action results in < 10% of the junctions having increased permeability at any given time during the resistance decrease. The role of epithelial barrier permeability changes in TNF action in vivo is discussed.

Tight junction permeability and liver plasma membrane fluidity in lithocholate-induced cholestasis

The present study correlated the reversibility of bile flow (BF) impairment with biochemical and morphological changes in the liver after injection of a cholestatic dose (12 μmole/100 g body weight) of lithocholic acid (LCA). BF declined maximally at 60 min but recovered totally at 210 min after LCA treatment. During the cholestatic period, there was an increase in tight junction permeability as measured by the bile to plasma (B/P) ratio of inulin and using lanthanum as a tracer. Cholesterol content and the cholesterol/phospholvpid ratio in liver plasma membranes (LPM) were augmented while the fluidity of bile canalicular membranes (BCM) was decreased at 30 and 60 min after LCA injection. These changes in cholesterol content and membrane fluidity seemed to be correlated with LCA incorporation in LPM; their reversal at 120 min preceded the recovery of BF (210 min). Some biochemical disorders were evident after LCA injection, but they did not correlate with the variation in BF. These data suggest that increased tight junction permeability and decreased BCM fluidity are important pathogenic steps in LCA-induced cholestasis.

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

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.

Permeability changes in Necturus proximal tubule during volume expansion.

The permeability of Necturus proximal tubule to hydrophilic nonelectrolytes of varying molecular size was studied under control conditions and during isotonic expansion of the animal's extracellular volume. Transepithelial permeability was measured in perfused tubular segments under conditions of zero net water flux. During volume expansion, tubular permeability to urea increased slightly, whereas mannitol decreased slightly and permeability to sucrose was significantly decreased. Volume expansion had a greater effect on osmotic flow parameters; the NaCl reflection coefficient decreased from 0.64 to 0.47 (summer animals) and from 0.41 to 0.27 (winter animals). Osmotic water flux and hydraulic conductivity increased but only in the lumen-to-capillary direction. Reflection coefficients of nonelectrolytes measured at the apical surface were reduced during volume expansion for probing molecules greater than 3 A in radius and were unchanged for smaller molecules, less than 3 A, suggesting two pore populations. We propose that an increase in tight-junction permeability can account for modification of osmotic flow parameters, whereas the whole thickness of the epithelium, particularly the intercellular space, plays the dominant role in regulation of diffusional permeability.