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PubMed2003

Regulation of the blood-biliary barrier: interaction between gap and tight junctions in hepatocytes.

Hepatocytes tightly connect with each other by intercellular junctions to form liver cell plates. The junctions composed of gap, tight, and adherens junctions and desmosomes concentrate around bile canaliculi. In particular, tight junctions serve as a barrier to keep bile in bile canaliculi away from the blood circulation. Thus, it is very reasonable to call tight junctions of hepatocytes the blood-biliary barrier. On the other hand, gap junctions of hepatocytes are considered to enable ordered contraction of bile canaculi from centrizonal to periportal hepatocytes by their function of intercellular communication. Gap and tight junctions may thus play a crucial role in bile secretion, one of the most differentiated functions of the liver. In intrahepatic cholestasis, a common pathological condition of the liver, downregulation of gap and tight junctional functions is seen, which results in impaired intercellular communication and in leaky tight junctions. Although the changes in gap and tight junctions had been considered to be independent of each other, recent findings that the tight junction-associated proteins ZO-1 and occludin bind to connexins indicate the possibility of either coordinate or reciprocal regulation of macromolecular complexes containing gap- and tight-junction proteins. In this review, we introduce the interaction and regulation between gap and tight junctions of hepatocytes in vitro and discuss the regulatory mechanisms of the "blood-biliary barrier" to study the molecular pathogenesis of cholestasis.

Actins↗

PubMed1995

Regulation of tight-junction permeability during nutrient absorption across the intestinal epithelium.

Tight junctions are located at the luminal aspect of adjacent epithelial cells and form a barrier that limits the paracellular diffusion of hydrophilic solutes. In recent years, evidence has accumulated to indicate that tight-junction permeability is regulated by the absorption of various nutrients. In this review, we present the physiological basis and importance of tight-junction regulation in intestinal epithelium. The molecular structure of tight junctions and their interactions with the cell cytoskeleton as well as the physical and chemical forces that influence tight junction permeability are described. Much of this review addresses the controversial Pappenheimer hypothesis, which states that a major portion of intestinal glucose absorption occurs through tight junctions and not by saturable transcellular active transport. The absorption of a significant portion of glucose through tight junctions requires increased junctional permeability, a very high intralumenal glucose concentration, and a sufficient osmotic gradient to promote volume flow.

Calcium↗

PubMed1985

The structure of tight junctions in the ciliary epithelium.

The tight junctions of the ciliary epithelium act as a barrier preventing the passage of blood borne macromolecules into the posterior chamber. The use of the freeze-fracture technique has led to a good knowledge of their morphological pattern in various species. However, in order to attempt a correlation of the morphology of the tight junctions with their physiological properties, their intimate substructure must be considered. As in other glutaraldehyde-fixed epithelia, the tight junctions appear as networks (variable in their apico-basal thickness) of more or less discontinuous P-face ridges and as complementary E-face furrows in which some particles or short bars are found. The significance of the discontinuities of ridges has been analysed. The continuity of the junctional fibrils was evident as assessed both by quantitative measurements as well as morphological examination of complementary fracture faces. In addition, the absence of loss of junctional material showed that the integrity of the junction was preserved during the freeze-fracture process, even in conditions where an increase in "transfer" of junctional elements was experimentally induced. Most of all, a "pore system" due to visualizable gaps in the fibrils is not tenable for the ciliary epithelium. Furthermore, the analysis of transition steps at the level of membrane "fusion" showed that the tight junctions of the ciliary epithelium must now be considered as formed by two slightly offset fibrils, one per adjacent plasma membrane.

Animals↗

PubMed2006

Absence of tight junctions between microvascular endothelial cells in human cerebellar hemangioblastomas.

OBJECTIVE: Endothelial tight junctions form the main barrier of the blood-brain barrier (BBB). In human hemangioblastomas, cyst formation is a common and important clinical manifestation. Although most researchers consider that the cyst formation in hemangioblastomas may be caused by the breakdown of the BBB, the underlying molecular mechanisms for cyst formation remain unknown. At present, there are few reports about the change of tight junctions in microvessel endothelium of human hemangioblastomas. The purpose of this research is to investigate the change of tight junction and its major molecular components in microvessel endothelium of human hemangioblastomas. METHODS: Twenty-four consecutive patients with cerebellar hemangioblastomas were studied. Tight junctions in the microvessels of hemangioblastomas and the control brain were examined by electron microscopy. Immunohistochemistry and double immunofluorescent microscopy were used to analyze the expression of CLN5 and its relationship with astrocytic endfeet in the control brain and hemangioblastomas. Quantitative real-time reverse-transcriptase polymerase chain reaction and Western blots were used to investigate the expression level of CLN5 in hemangioblastomas. Triple immunofluorescent microscopy was used to analyze the coexpression of vascular endothelial growth factor, vascular endothelial growth factor-R1, and placenta growth factor on microvessels of hemangioblastomas. Clinical and experimental data were correlated and analyzed by the one-way analysis of variance, Kruskal-Wallis test, and Spearman rank correlation test. RESULTS: In the control brain, the paracellular cleft between adjacent endothelial cells is sealed by continuous strands of tight junctions. In cystic hemangioblastomas, a significant paracellular cleft could be found between adjacent endothelial cells. Some endothelial cells were connected with adherens junction and no tight junction was found between them. Compared with the control brain, expression of CLN5 was decreased in cystic hemangioblastomas (P < 0.05). Phosphorylated CLN5 was detected in most hemangioblastomas, but not in the control brain. Microvessels in hemangioblastomas showed a significant absence of astrocytic endfeet. Coexpression of vascular endothelial growth factor, vascular endothelial growth factor-R1, and placenta growth factor was detected in the endothelial cells. The Spearman rank correlation test showed a significant correlation between a greater degree of CLN5 expression and less morphological cystic formation in these patients studied (correlation coefficient = -0.520; P = 0.009). CONCLUSION: The continuity of tight junctions of the BBB is interrupted in human cerebellar hemangioblastomas. Significant absence of astrocytic endfeet and tight junctions can be found in microvessels of hemangioblastomas, which may lead to the breakdown of the BBB in these tumors. These findings suggest that the absence of tight junctions might play a role in cyst formation of hemangioblastomas.

Adult↗

PubMed1997

Development of tight junctions between odontoblasts in early dentinogenesis as revealed by freeze-fracture.

BACKGROUND: Mature odontoblasts possess junctional structures constituted by adherens, gap, and tight junctions. Although adherens and gap junctions appear early between odontoblasts, there is no information on the appearance and development of tight junctions between odontoblasts. In this study, we have examined freeze-fracture replicas of early dentinogenesis to study the development of tight junctions between odontoblasts and to determine whether these junctions are of zonular or macular type. METHODS: Upper first molar tooth germs of Wistar rats between 1 and 3 days old were fixed in buffered 4% glutaraldehyde/4% formaldehyde and subsequently cryoprotected with cacodylate-buffered glycerol. Freeze-fracture replicas were obtained in a Balzers 301 apparatus, and early stages of dentinogenesis were examined in a Jeol 100 CX II electron microscope. RESULTS: In the stage of early dentine matrix prior to mineralization, odontoblasts exhibit only gap junctions. With the progression of development, the distal plasma membranes of odontoblasts show numerous short tight junctions formed by fused particles and grooves. In the stage of advanced mineralization, branched and continuous rows of fused particles or grooves constitute tight junctions of the focal or macular type. CONCLUSIONS: The present study shows that tight junctions of focal or macular type appear on distal plasma membrane of early odontoblasts during differentiation. Formation of tight junctions indicates the establishment of a distal membrane domain and maturation of odontoblasts. These events occur as mantle dentine formation ceases and circumpulpar dentine formation begins.

Age Factors↗

PubMed2006

An ultrastructural study of microvascular inter-endothelial tight junctions in normal endometrium.

OBJECTIVE: To investigate changes in tight junctions between microvascular endothelial cells in normal endometrium. STUDY DESIGN: A quantitative transmission electron microscopy study of changes in the number of tight junctions per microvessel, and widths and lengths of individual junctions at different phases of the menstrual cycle. RESULTS: Two types of tight junctions were defined ("open" and "closed") which varied in the degree of separation between apposed plasma membranes of endometrial endothelial cells. Tight junction density was greater in proliferative than secretory phases (p<0.02). The width and length measurements of individual tight junctions changed during menstruation. CONCLUSIONS: The two types of tight junctional contacts and the changes in width and density may reflect variation in structure and function between adjacent endothelial cells. These junctions may have importance in the processes of implantation and menstruation.

Adolescent↗

PubMed2001

Dynamic changes in protein components of the tight junction during liver regeneration.

The construction of the hepatocyte tight junction is one of the most important events during liver regeneration leading to the reorganization of the bile canaliculi and the repolarization of hepatocytes after cell division. To understand this event at the molecular level, we examined the expression of tight junction proteins by Western blot analysis and their cellular localization by immunofluorescence microscopy in regenerating rat liver after two-thirds hepatectomy. The levels of tight junction components such as claudin-3, ZO-1 and atypical protein kinase C (PKC)-specific interacting protein (ASIP) increased two- to three-fold over control levels in coordination with a peak 2-3 days after partial hepatectomy, whereas occludin levels remained unchanged. The bile canaliculi outlined by tight junction components and actin filaments reveal significant morphological changes from 2-3 days after partial hepatectomy. During this period, claudin-3/ZO-1 and ASIP/ZO-1 were nearly co-localized, whereas occludin was locally reduced or almost absent on the bile canaliculi outlined by ZO-1 staining. The uncoupled localization of F-actin and tight junction components was often observed. The function of hepatocytes, as revealed by the serum bile acids level, was distorted temporally at an early stage of regeneration but mostly restored 3 days after partial hepatectomy. These observations suggest that the de novo construction of tight junctions proceeds mainly 2-3 days after partial hepatectomy in parallel with the cell polarization required for hepatocyte function. However, the complete normalization of the composition of the tight junction components, such as occludin and the association with F-actin, requires additional time, which may support the regeneration of fully polarized normal hepatocytes.

Adaptor Proteins, Signal Transducing↗

PubMed1984

Structural correlates of intestinal tight-junction permeability.

A variety of evidence suggests that specific parameters of tight-junction structure correlate with tight-junction function. Thus, one potential indirect means of probing the functional characteristics of paracellular pathways within epithelia at the cellular level is detailed analysis of tight-junction structure and distribution. In the small intestine such structural studies are confounded by the complex geometry and cellular heterogeneity of the epithelium, but, to date, these studies suggest this epithelium may actually consist of a moderately "tight" (villus) and a "leaky" (crypt) epithelium arranged in parallel. Moreover, it is becoming clear that both the structure and function of tight junctions, including those of the small intestine, are not static. Evidence from several epithelia indicate that a variety of factors (transepithelial osmotic gradients, cyclic nucleotides, intra- and extracellular Ca+2 concentrations) are capable of modulating tight-junction structure and function. Additionally, a variety of indirect evidence suggests that the cytoskeleton of epithelial cells may interact with the tight junction and that perhaps the alterations in junctional structure and function elicited by some of the above factors may be mediated via effects of these agents on the cytoskeleton. It may now be viewed as probable that epithelial cells have the capacity to fine-tune parameters of paracellular transport just as they do transcellular transport. We speculate that, in many instances, they may accomplish this feat by directly manipulating structural units of the tight junction via components of the cytoskeleton.

Animals↗

PubMed1995

Incomplete belts of tight junctions in cultured non-pigmented human ciliary epithelial cells.

Tight junctions of cultured human non-pigmented ciliary epithelial cells were studied with the freeze-fracture technique and related to the transepithelial electrical resistance of these monolayers. Isolated tight junctional fibrils or small groups and networks of tight junctions sometimes associated with gap junctions were revealed in freeze-fracture images of the lateral plasma membrane. The tight junctions always formed incomplete belts, so that the apical and basolateral plasma membrane domains often were in continuity without morphological evidence of interposed intercellular junctions. The monolayers revealed a transepithelial resistance of 19.7 +/- 2.1 omega.cm2. Protamine induced a reversible increase of the transepithelial resistance of the cultures by 91 +/- 12%, but still the tight junctions formed incomplete belts. We conclude that contrary to complete networks of tight junctions in native non-pigmented ciliary epithelium, cultured monolayers only express incomplete belts of tight junctions which may be the morphological correlate of the relatively low transepithelial resistance of these monolayers. Interpretations on transepithelial transport and permeability characteristics of these cultures have to take into account the differences in junctional morphology from their native epithelium.

Cells, Cultured↗

PubMed1985

Development of an apical plasma membrane domain and tight junctions during histogenesis of the mammalian pancreas.

The role of tight junctions (zonula occludens) in the formation of apical plasma membrane (PM) domains was investigated in the embryonic rat pancreas. In the present study, lectin-rhodamine (WGA-TRITC and RCAII-TRITC) and lectin-gold (WGA-Au and RCAII-Au) conjugates were used to monitor apical PM domain formation and freeze-fracture analysis was used to monitor tight junction formation in the pancreatic epithelium of embryonic, neonatal, and adult rats. Fluorescent and TEM analysis of WGA and RCAII binding indicated that an apical PM domain is formed as early as Day 13 of gestation in the pancreatic epithelium. While apical WGA binding remained into adult life, RCAII binding was lost by 1 day after birth. In contrast, tight junctions were not observed until Day 14 of gestation. At this time, tight junctions were found to be incomplete in formation and typically consisted of linear arrays of IMPs or discontinuous arrays of sealing strands (focal adherens). Continuous tight junctions were not completely formed until Day 15 of gestation. Continued development of tight junctions during gestation was characterized by (1) an increase in the number of sealing strands and (2) a more parallel arrangement of sealing strands within each junctional complex. By 8 weeks after birth, tight junctions were more loosely organized and contained fewer sealing strands as compared to that observed in the fetus. These results suggest that lateral diffusion of apical PM glycoconjugates may be restricted even in the absence of complete tight junctional complexes during development of the rat pancreas.

Animals↗

PubMed2002

The small GTPase Rab13 regulates assembly of functional tight junctions in epithelial cells.

Junctional complexes such as tight junctions (TJ) and adherens junctions are required for maintaining cell surface asymmetry and polarized transport in epithelial cells. We have shown that Rab13 is recruited to junctional complexes from a cytosolic pool after cell-cell contact formation. In this study, we investigate the role of Rab13 in modulating TJ structure and functions in epithelial MDCK cells. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delayed the formation of electrically tight epithelial monolayers as monitored by transepithelial electrical resistance (TER) and induced the leakage of small nonionic tracers from the apical domain. It also disrupted the TJ fence diffusion barrier. Freeze-fracture EM analysis revealed that tight junctional structures did not form a continuous belt but rather a discontinuous series of stranded clusters. Immunofluorescence studies showed that the expression of Rab13Q67L delayed the localization of the TJ transmembrane protein, claudin1, at the cell surface. In contrast, the inactive Rab13T22N mutant did not disrupt TJ functions, TJ strand architecture nor claudin1 localization. Our data revealed that Rab13 plays an important role in regulating both the structure and function of tight junctions.

Amino Acid Substitution↗

PubMed2006

Proteomic and bioinformatic analysis of epithelial tight junction reveals an unexpected cluster of synaptic molecules.

BACKGROUND: Zonula occludens, also known as the tight junction, is a specialized cell-cell interaction characterized by membrane "kisses" between epithelial cells. A cytoplasmic plaque of approximately 100 nm corresponding to a meshwork of densely packed proteins underlies the tight junction membrane domain. Due to its enormous size and difficulties in obtaining a biochemically pure fraction, the molecular composition of the tight junction remains largely unknown. RESULTS: A novel biochemical purification protocol has been developed to isolate tight junction protein complexes from cultured human epithelial cells. After identification of proteins by mass spectroscopy and fingerprint analysis, candidate proteins are scored and assessed individually. A simple algorithm has been devised to incorporate transmembrane domains and protein modification sites for scoring membrane proteins. Using this new scoring system, a total of 912 proteins have been identified. These 912 hits are analyzed using a bioinformatics approach to bin the hits in 4 categories: configuration, molecular function, cellular function, and specialized process. Prominent clusters of proteins related to the cytoskeleton, cell adhesion, and vesicular traffic have been identified. Weaker clusters of proteins associated with cell growth, cell migration, translation, and transcription are also found. However, the strongest clusters belong to synaptic proteins and signaling molecules. Localization studies of key components of synaptic transmission have confirmed the presence of both presynaptic and postsynaptic proteins at the tight junction domain. To correlate proteomics data with structure, the tight junction has been examined using electron microscopy. This has revealed many novel structures including end-on cytoskeletal attachments, vesicles fusing/budding at the tight junction membrane domain, secreted substances encased between the tight junction kisses, endocytosis of tight junction double membranes, satellite Golgi apparatus and associated vesicular structures. A working model of the tight junction consisting of multiple functions and sub-domains has been generated using the proteomics and structural data. CONCLUSION: This study provides an unbiased proteomics and bioinformatics approach to elucidate novel functions of the tight junction. The approach has revealed an unexpected cluster associating with synaptic function. This surprising finding suggests that the tight junction may be a novel epithelial synapse for cell-cell communication. REVIEWERS: This article was reviewed by Gáspár Jékely, Etienne Joly and Neil Smalheiser.

Journal Article↗

PubMed1983

A freeze fracture study of Crohn's disease of the terminal ileum: changes in epithelial tight junction organization.

Freeze fracture replicas of the plasma membrane and tight junctions of epithelial cells of the terminal ileum from 10 patients with Crohn's disease involving the small bowel and from the terminal ileum of two patients without inflammatory bowel disease were studied to determine if significant morphological variations could be documented. Samples of diseased tissue were taken from: 1) macroscopically normal ileal resection margins, 2) pinpoint aphthoid ulcers, 3) small 5 mm ulcers, 4) cobblestone mucosa. Samples of ileal mucosa from the proximal margin of ileocolic resections for carcinoma of the right colon were identically processed and served as controls. Control specimens showed normal villus structures lined by absorptive and goblet cells linked to adjacent cells by four to seven tight junction strands, arranged as an anastomosing network of fibrils oriented perpendicularly to the long axis of the cell. Crohn's disease specimens taken from the cobblestone area displayed the greatest degree of tight junction disorganization. Tight junctions commonly formed bizarre patterns, were fragmented, and often showed misalignment in a direction parallel to the cell axis. Specimens taken from areas 2 and 3 displayed a less severe form of tight junction rearrangement. Junctional strand fragmentations, as well as areas of plasma membrane lacking strands, were apparent. The resection margins had minor irregularities in junctional structure in some, but not all, cases. The predominant alteration in those cases which showed change was varying degrees of strand fragmentation. We postulate that the tight junction abnormalities of epithelial cells from the terminal ileum of patients with Crohn's disease may contribute to a disturbance in barrier functions.

Adult↗

PubMed2005

Specific modulation of airway epithelial tight junctions by apical application of an occludin peptide.

Tight junctions are directly involved in regulating the passage of ions and macromolecules (gate functions) in epithelial and endothelial cells. The modulation of these gate functions to transiently regulate the paracellular permeability of large solutes and ions could increase the delivery of pharmacological agents or gene transfer vectors. To reduce the inflammatory responses caused by tight junction-regulating agents, alternative strategies directly targeting specific tight junction proteins could prove to be less toxic to airway epithelia. The apical delivery of peptides corresponding to the first extracellular loop of occludin to transiently modulate apical paracellular flux has been demonstrated in intestinal epithelia. We hypothesized that apical application of these occludin peptides could similarly modulate tight junction permeability in airway epithelia. Thus, we investigated the effects of apically applied occludin peptide on the paracellular permeability of molecular tracers and viral vectors in well differentiated human airway epithelial cells. The effects of occludin peptide on cellular toxicity, tight junction protein expression and localization, and membrane integrity were also assessed. Our data showed that apically applied occludin peptide significantly reduced transepithelial resistance in airway epithelia and altered tight junction permeability in a concentration-dependent manner. These alterations enhanced the paracellular flux of dextrans as well as gene transfer vectors. The occludin peptide redistributed occludin but did not alter the expression or distribution of ZO-1, claudin-1, or claudin-4. These data suggest that specific targeting of occludin could be a better-suited alternative strategy for tight junction modulation in airway epithelial cells compared with current agents that modulate tight junctions.

Amino Acid Sequence↗

PubMed1998

Regulation of the MDCK cell tight junction.

The sodium flux across individual tight junctions (TJ) of low-resistance MDCK cell monolayers grown on glass coverslips was determined as a measure of paracellular permeability. Increases in perfusate glucose concentration from 5 to 25 mM decreased tight junction Na permeability. This permeability decrease was not specific as nonmetabolizable analogues of glucose caused similar diminutions in TJ Na permeability. Stimulation of protein kinase A increased TJ Na permeability, and inhibition of protein kinase A decreased TJ Na permeability. Transepithelial electrical resistance of monolayers grown on permeable supports did not change as predicted from the observed alterations in TJ Na permeability of monolayers grown on glass coverslips. Fluorescent labeling of cell F-actin showed that increased F-actin in the perijunctional ring correlated with higher TJ Na permeability. Although a low dose of cytochalasin D did not change TJ Na permeability, it disrupted the cytoskeleton and blocked the decrease in TJ Na permeability caused by glucose. Cytochalasin D failed to block the effects of protein kinase A stimulation or inhibition on TJ Na permeability. We conclude that tight junction sodium permeability is regulated both by protein kinase A activity and by other processes involving the actin cytoskeleton.

Animals↗

PubMed1977

Tracer and freeze fracture observations on developing tight junctions in fetal rat thyroid.

The development of tight junctions in fetal rat thyroid from the sixteenth to the twentieth days of gestation was examined with conventional ultrastructural methods and freeze-fracture preparations. These results were compared with those obtained using lanthanum hydroxide and horseradish peroxidase (HRP) tracers. Tight junctions appear to arise on the plasma membranes of fetal thyroid cells by the aggregation and fusion of linear particle chains which appear at several discrete sites on the plasma membrane of developing follicular cells. Tracer studies show that they are effective barriers to the passage of HRP from the outset, are freely penetrated by La3+ at the sixteenth and seventeenth days of gestation, but progressively lose permeability to La3+ from the seventeenth to twentieth days of gestation. However, freeze-fracture observations suggest that La3+ must penetrate into the follicular lumen through the tight junction elements, for the follicular lumen, when it appears, is always completely surrounded by a continuous though sometimes rudimentary meshwork of tight junction elements. The results suggest that the tight junction forms an effective barrier to the passage of large macromolecules, e.g. thyroglobulin, from very early stages in its development. The La3+ results suggest that decreased resistance of the intercellular pathway, possibly related to the development of transepithelial potentials, may occur during this period in development.

Animals↗

PubMed2004

Regulation of tight junctions and loss of barrier function in pathophysiology.

The mechanism by which epithelial and endothelial cells interact to form polarized tissue is of fundamental importance to multicellular organisms. Dysregulation of these barriers occurs in a variety of diseases, destroying the normal cellular environments and leading to organ failure. Increased levels of growth factors are a common characteristic of diseases exhibiting tissue permeability, suggesting that growth factors play a direct role in elevating permeability. Of particular concern for this laboratory, increased expression of vascular endothelial growth factor may enhance vascular permeability in diabetic retinopathy, leading to vision impairment and blindness. However, the mechanism by which growth factors increase permeability is unclear. Polarized cells form strong barriers through the development of tight junctions, which are specialized regions of the junctional complex. Tight junctions are composed of three types of transmembrane proteins, a number of peripheral membrane structural proteins, and are associated with a variety of regulatory proteins. Recent data suggest that growth factor-stimulated alterations in tight junctions contribute to permeability in a variety of disease states. The goal of this review was to elucidate potential mechanisms by which elevated growth factors elicit deregulated paracellular permeability via altered regulation of tight junctions, with particular emphasis on the tight junction proteins occludin and ZO-1, protein kinase C signaling, and endocytosis of junctional proteins. Understanding the molecular mechanisms underlying growth factor-mediated regulation of tight junctions will facilitate the development of novel treatments for diseases such as brain tumors, diabetic retinopathy and other diseases with compromised tight junction barriers.

Cell Membrane Permeability↗

PubMed2002

Role of VASP in reestablishment of epithelial tight junction assembly after Ca2+ switch.

Epithelial permeability is tightly regulated by intracellular messengers. Critical to maintaining barrier integrity is the formation of tight junction complexes. A number of signaling pathways have been implicated in tight junction biogenesis; however, the precise molecular mechanisms are not fully understood. A growing body of evidence suggests a role for intracellular cAMP in tight junction assembly. Using an epithelial model, we investigated the role of cAMP signal transduction in barrier recovery after Ca2+ switch. Our data demonstrate that elevation of intracellular cAMP levels significantly enhanced barrier recovery after Ca2+ switch. Parallel experiments revealed that epithelial barrier recovery is diminished by H-89, a specific and potent inhibitor of cAMP-dependent protein kinase (protein kinase A) activity. Of the possible PKA effector proteins, the vasodilator-stimulated phosphoprotein (VASP) is an attractive candidate, since it has been implicated in actin-binding and cross-linking functions. We therefore hypothesized that VASP may play a role in the cAMP-mediated regulation of epithelial junctional reassembly after Ca2+ switch. We demonstrate here that VASP is phosphorylated via a PKA-dependent process under conditions that enhance barrier recovery. Confocal laser scanning microscopy studies revealed that VASP localizes with ZO-1 at the tight junction and at cell-cell borders and that phospho-VASP appears at the junction after Ca2+ switch. Subsequent transfection studies utilizing epithelial cells expressing truncated forms of VASP abnormal in oligomerization or actin-binding activity revealed a functional diminution of barrier recovery after Ca2+ chelation. Our present studies suggest that VASP may provide a link between cAMP signal transduction and epithelial permeability.

Blotting, Western↗