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Activation of the type 1 histamine (Hl) or the type 2 protease-activated (PAR-2) G protein-coupled receptors interrupts E-cadherin adhesion and decreases the transepithelial resistance (TER) of epithelium. Several reports suggest that cadherin adhesive function depends on the association of cadherin with [beta]-catenin and that this association is regulated by phosphorylation of tyrosines in [beta]-catenin. We tested the hypothesis that loss of cadherin adhesion and compromise of TER on activation of the H I or PAR-2 receptor is due to phosphorylation of tyrosines in [beta]-catenin. L cells were stably transfected to express E-cadherin (L-E-cad cells) and HI (L-H1-E-cad cells). L cells and Madin-Darby canine kidney (MDCK) cells constitutively express PAR-2. Stably transfected L-E-cad, L-H1-E-cad. and MDCK cells were also stably transfected with FLAG-tagged wild-type (WT) or mutant [beta]-catenin. converting tyrosine 142, 489, or 654 to the nonphosphorylatable mimetic, phenylahmine (WT. Y142F, Y489F. or Y654F). Activation of H1 or PAR-2 interrupted adhesion to an immobilized E-cadherin-Fc fusion protein of L-H1-E-cad, L-E-cad. and MDCK cells expressing WT or Y142F [beta]-catenin but did not interrupt adhesion of L-H1-E-cad, L-E-cad, and MDCK cells expressing the Y489F or Y654F mutant [beta]-catenins. PAR-2 activation decreased the TER of monolayers of MDCK cells expressing WT or Y142F [beta]-catenin 40-45%. However, PAR-2 activation did not decrease the TER of monolayers of MDCK cells expressing Y489F or Y654F [beta]-catenin. The protein tyrosine phosphatase PTP1B binds to the cadherin cytoplasmic domain and dephosphorylates [beta]-catenin. Inhibition of PTP1B interrupted adhesion to E-cadherin-Fc of MDCK cells expressing WT [beta]-catenin but did not affect the adhesion of MDCK cells expressing Y489F or Y654F [beta]-catenin. Similarly, inhibition of PTP1B compromised the TER of MDCK cells expressing WT [beta]-catenin but did not affect the TER of MDCK cells expressing Y489F or Y654F [beta]-catenin. We conclude that phosphorylation of tyrosines 489 and 654 in [beta]-catenin is a necessary step in the process by which G protein-coupled HI and PAR-2 receptors interrupt E-cadherin adhesion. We also conclude that activation of PAR-2 has no effect on the TER without first interrupting E-cadherin adhesion.

The airway epithelium is an important barrier between the environment and subepithelial tissues. The epithelium is also divided into functionally restricted apical and basolateral domains, and this restriction is dependent on the elements of the barrier. The protease-activated receptor-2 (PAR2) receptor is expressed in airway epithelium, and its activation initiates multiple effects including enhanced airway inflammation and reactivity. We hypothesized that activation of PAR2 would interrupt E-cadherin adhesion and compromise the airway epithelial barrier. The PAR2-activating peptide (PAR2-AP, SLIGRL) caused an immediate ~50% decrease in the transepithelial resistance of primary human airway epithelium that persisted for 6-10 rain. The decrease in resistance was accompanied by an increase in mannitol flux across the epithelium and occurred in cystic fibrosis transmembrane conductance receptor (CFTR) epithelium pretreated with amiloride to block Na and Cl conductances, confirming that the decrease in resistance represented an increase in paracellular conductance. In parallel experiments, activation of PAR2 interrupted the adhesion of E-cadherin-expressing L cells and of primary airway epithelial cells to an immobilized E-cadherin extracellular domain, confirming the hypothesis that activation of PAR2 interrupts E-cadherin adhesion. Selective interruption of E-cadherin adhesion with antibody to E-cadherin decreased the transepithelial resistance of primary airway epithelium by >80%. Pretreatment of airway epithelium or the E-cadherin-expressing L cells with the long-acting [beta]-agonist salmeterol prevented PAR2 activation from interrupting Ecadherin adhesion and compromising the airway epithelial barrier. Activation of PAR2 interrupts E-cadherin adhesion and compromises the airway epithelial barrier. protease-activated receptor; histamine; harrier function

Histamine selectively interrupts VE-cadherin adhesion independently of capacitive calcium entry. Am J Physiol Lung Cell Mol Physiol 287: L816-L823, 2004. First published June 25, 2004; 10.1152/ajplung.00056.2004.--Histamine is an important agent of innate immunity, transiently increasing the flux of immune-competent molecules from the vascular space to the tissues and then allowing rapid restoration of the integrity of the endothelial barrier. In previous work we found that histamine alters the endothelial barrier by disrupting cell-cell adhesion and identified VE-cadherin as an essential participant in this process. The previous work did not determine whether histamine directly interrupted VE-cadherin adhesion, whether the effects of histamine were selective for cadherin adhesion, or whether capacitive calcium flux across the cell membrane was necessary for the effects of histamine on cell-cell adhesion. In the current work we found that histamine directly interrupts adhesion of L cells expressing the type 1 histamine (H1) receptor and VE-cadherin to a VE-cadherin-Fc fusion protein. In contrast, integrin-mediated adhesion to fibronectin of the same L cells expressing the H1 receptor was not affected by histamine, demonstrating that the effects of histamine are selective for cadherin adhesion. Some of the effects of many edemagenic agonists on endothelium are dependent on the capacitive flux of calcium across the endothelial cell membrane. Blocking capacitive calcium flux with La[Cl.sub.3] did not prevent histamine from interrupting VE-cadherin adhesion of transfected L cells, nor did it prevent histamine from interrupting cell-cell adhesion of human umbilical vein endothelial ceils. These data support the contentions that histamine directly and selectively interrupts cadherin adhesion and this effect on cadherin adhesion is independent of capacitive calcium flux. endothelium; integrin; human histamine receptor H1; vascular endothelial cadherin

Histamine increases microvascular permeability by creating small transitory (100-400 nm) gaps between adjacent endothelial cells at sites of vascular endothelial (VE)-cadherin-based adhesion. We examined the effects of histamine on the proteins within the VE-cadherin-based adherens junction in primary human umbilical vein endothelial cells. VE-cadherin is linked not only by [beta]- and [alpha]-catenin to cortical actin but also by [gamma]-catenin to the intermediate filament vimentin. In mature human umbilical vein cultures, the VE-cadherin immunoprecipitate contained equivalent amounts of [alpha]- and [beta]-catenin, 130% as much [beta]- as [gamma]-catenin, and 50% as much actin as vimentin. Within 60 s, histamine decreased the fraction of VE-cadherin in the insoluble portion of the cell lysate by 35 [+ or -] 1.5%. At the same time, histamine decreased the amount of vimentin that immunoprecipitated with VE-cadherin by 50 [+ or -] 6%. Histamine did not affect the amount of actin or the amount of [alpha]-, [beta]-, or [gamma]-catenin that immunoprecipitated with VE-cadherin. Within 60 s, histamine simulated a doubling in the phosphorylation of VE-cadherin and [beta]- and [gamma]-catenin. The VE-cadherin immunoprecipitate contained kinase activity that phosphorylated VE-cadherin and [gamma]-catenin in vitro. vascular endothelial cadherin; adhesion

Winter, Michael C., Anant M. Kamath, Dana R. Ries, Sandra S. Shasby, Yih-Tai Chen, and D. Michael Shasby. Histamine alters cadherin-mediated sites of endothelial adhesion. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L988-L995, 1999.--We tested the hypothesis that histamine alters the focal apposition of endothelial cells by acting on sites of cadherin-mediated cell-cell adhesion. Focal apposition was measured as the impedance of a cell-covered electrode, which was partitioned into a cell-matrix resistance, a cell-cell resistance, and membrane capacitance. Histamine causes an immediate, short-lived decrease in the impedance of an electrode covered with human umbilical vein endothelial (HUVE) cells. ECV304 cells are a line of spontaneously transformed HUVE cells that do not express the endothelial cadherin, cadherin-5. Histamine increased ECV304 cell calcium to 600 nM. Histamine did not increase myosin light chain phosphorylation of control or transfected ECV304 cells. ECV304 cells transfected with either E-cadherin or cadherin-5 on a dexamethasone-responsive plasmid (pLKneo) increased their cell-cell resistance when stimulated with dexamethasone, whereas ECV304 cells transfected with pLKneo-lacZ did not. Histamine did not affect the impedance of ECV304 cells transfected with pLKneo-lacZ. In contrast, histamine decreased the cell-cell resistance of ECV304 cells transfected with either pLKneo-E-cadherin or pLKneo-cadherin-5. From these data, we conclude that histamine acts on sites of cadherin-mediated cell-cell apposition. edema; permeability; cell adhesion

Activation of the type 1 histamine (H1) or the type 2 protease-activated (PAR-2) G protein-coupled receptors interrupts E-cadherin adhesion and decreases the transepithelial resistance (TER) of epithelium. Several reports suggest that cadherin adhesive function depends on the association of cadherin with β-catenin and that this association is regulated by phosphorylation of tyrosines in β-catenin. We tested the hypothesis that loss of cadherin adhesion and compromise of TER on activation of the H1 or PAR-2 receptor is due to phosphorylation of tyrosines in β-catenin. L cells were stably transfected to express E-cadherin (L-E-cad cells) and H1 (L-H1-E-cad cells). L cells and Madin-Darby canine kidney (MDCK) cells constitutively express PAR-2. Stably transfected L-E-cad, L-H1-E-cad, and MDCK cells were also stably transfected with FLAG-tagged wild-type (WT) or mutant β-catenin, converting tyrosine 142, 489, or 654 to the non-phosphorylatable mimetic, phenylalanine (WT, Y142F, Y489F, or Y654F). Activation of H1 or PAR-2 interrupted adhesion to an immobilized E-cadherin-Fc fusion protein of L-H1-E-cad, L-E-cad, and MDCK cells expressing WT or Y142F β-catenin but did not interrupt adhesion of L-H1-E-cad, L-E-cad, and MDCK cells expressing the Y489F or Y654F mutant β-catenins. PAR-2 activation decreased the TER of monolayers of MDCK cells expressing WT or Y142F β-catenin 40-45%. However, PAR-2 activation did not decrease the TER of monolayers of MDCK cells expressing Y489F or Y654F β-catenin. The protein tyrosine phosphatase PTP1B binds to the cadherin cytoplasmic domain and dephosphorylates β-catenin. Inhibition of PTP1B interrupted adhesion to E-cadherin-Fc of MDCK cells expressing WT β-catenin but did not affect the adhesion of MDCK cells expressing Y489F or Y654F β-catenin. Similarly, inhibition of PTP1B compromised the TER of MDCK cells expressing WT β-catenin but did not affect the TER of MDCK cells expressing Y489F or Y654F β-catenin. We conclude that phosphorylation of tyrosines 489 and 654 in β-catenin is a necessary step in the process by which G protein-coupled H1 and PAR-2 receptors interrupt E-cadherin adhesion. We also conclude that activation of PAR-2 has no effect on the TER without first interrupting E-cadherin adhesion. [ABSTRACT FROM AUTHOR]

The lung's epithelial surface is at the same time vitally exchanging gas with the environment and acting as a barrier that protects the organism from the environment. We hypothesized that activation of epithelial-cell G-protein-coupled receptors for immune-defense molecules would temporarily interrupt cadherin-dependent cell-cell adhesion of epithelial cells and thereby focally and temporarily compromise the epithelial barrier to facilitate delivery of other immune molecules and cells to challenged sites. Activation of type 1 histamine or type 2 PAR receptors on the basolateral surface of primary airway epithelial cells or L-cells expressing E-cadherin interrupted cadherin adhesion and caused approximately a 50% decrease in the epithelial barrier for 2-3 minutes. Given basic biochemical observations of others, we further hypothesized that activation of the receptors altered the barrier by phosphorylating tyrosines on an essential cadherin-complex component, beta-catenin. Y-F mutations in beta-catenin completely blocked the effects of activating the same receptors on cadherin-dependent adhesion and on the epithelial barrier. Hence, G-protein-coupled receptors responding to immune-defense molecules temporarily and focally interrupt the lung epithelial barrier by compromising cadherin-based adhesion.