Calcium-switch technique and junctional permeability in native rabbit esophageal epithelium

N. A. Tobey, C. M. Argote, S. S. Hosseini, and R. C. Orlando

Department of Medicine, Tulane University Health Sciences Center and the Veterans Administration Hospital, New Orleans, Louisiana 70112

Submitted 5 September 2003 ; accepted in final form 15 January 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The Ca2+-switch technique was used to investigate the nature of the barrier governing (paracellular) permeability across the junctions of "native" rabbit esophageal epithelium. This was done by mounting esophageal epithelium in Ussing chambers to monitor transepithelial electrical resistance (RT), a marker of junctional permeability. When exposed to Ca2+-free Ringer solutions containing EDTA, RT declined ~35% below baseline over 2 h, and this decline reversed within 2 h by restoration of (1.2 mM) Ca2+-containing, normal Ringer solution ("Ca2+-switch technique"). Junctional resealing, i.e., increased RT on Ca2+ replacement, was assessed by the Ca2+-switch technique and shown to be 1) specific for Ca2+, with only Mn2+ among substituted divalent cations yielding partial resealing; 2) a function of extracellular Ca2+ levels because maneuvers (BAPTA/AM or A23187 [GenBank] exposure) to alter intracellular Ca2+ had no effect; 3) dose dependent, requiring as a minimum >=0.5 mM Ca2+ and 1.2 mM Ca2+ for optimization; and 4) independent of protein synthesis because it was not inhibited by cycloheximide. Resealing was also inhibited by luminal antibodies or synthetic peptides to the extracellular domain of E-cadherin. Immunohistochemistry revealed E-cadherin within all layers of stratum corneum in Ca2+-free but not Ca2+-containing solution. The present investigation documents, using the Ca2+-switch technique, that esophageal epithelial junctions contain a major Ca2+-dependent component and that this component reflects adhesion between the extracellular domains of E-cadherin containing a histidine-alanine-valine recognition sequence.

E-cadherin; paracellullar permeability; epithelial barrier; ussing chamber


THE ESOPHAGEAL EPITHELIUM (EE) acts as a barrier between luminal content-containing meal components and digestive juices and interstitital fluid and blood. Its major components include multiple layers of squamous cells and their intercellular junctional complexes (19). There is limited knowledge about the nature of the junctional complexes that govern paracellular permeability in mammalian EE, and much of what is known is derived from studies in rabbit EE. In rabbit EE, the junctional complex is confined to seven or eight of the most lumen-oriented cell layers of stratum corneum (20). This was established by exposing tissues to lanthanum and demonstrating by transmission electron microscopy (TEM) that the lanthanum failed to penetrate the cells or intercellular spaces of stratum corneum from either luminal or serosal sides. In contrast, serosal exposure to lanthanum enabled it to freely diffuse throughout the intercellular spaces of all the lower cell strata, i.e., stratum germinativum and stratum spinosum. Although these observations localized the junctional barrier to the cell layers comprising the stratum corneum, the nature of the structures preventing lanthanum permeation into the intercellular spaces was less obvious.

The major junctional components in most epithelia consist of "tight junctions" (zonula occludens), zonula adherens (belt desmosomes), and spot desmosomes (macula adherens), with barrier function attributed both to tight junctions and zonula adherens (22). Close examination of rabbit EE on TEM revealed tight junctions as short sections of membrane fusions or kisses between adjacent cells of stratum corneum (20), and structures consistent with tight junctions were again recently verified within the upper living cell layers of esophagus (16). Freeze-fracture replicas of rabbit EE, however, showed that the tight junctions only consisted of a few (1–2) strands per cell layer (20). Although for all cell layers of stratum corneum, this cumulatively adds up to 8–16 tight junctional strands in series, the possibility was considered that other structures within the intercellular spaces of EE may contribute to the tissue's reported high transepithelial electrical resistance (RT; average range 1,000–3,000 {Omega}·cm2) (16, 22, 32). In the above study, an amorphous glycoconjugate matrix was identified that was also felt to contribute to the junctional barrier in rabbit EE. This was based on the inability of lanthanum to penetrate the intercellular spaces of stratum corneum even under conditions (immersion) where it had access to the tissue's lateral cut edges. Additionally, it appeared that the glycoconjugate material was glycoprotein rather than glycolipid and that these glycoproteins were a complex mixture of both neutral and acidic side chains (20). An intercellular matrix with similar staining characteristics has also been identified in human EE, although this material was considered to be lipid rather than protein based on its appearance on TEM (1, 9). Another established junctional structure that was prominent throughout the rabbit EE on TEM was the spot desmosome (20). These desmosomes appear on TEM as "spot" welds between adjacent cells in association with thick dark plaques within the membrane, being particularly prominent within the layers of stratum corneum. However, unlike tight junctions, spot desmosomes do not encircle the cell membrane and consequently provide structural integrity but not barrier function (22). Notably lacking on TEM of rabbit EE were structures typical of zonula adherens; these were of importance because of their reported role, along with tight junctions, in establishing the junctional barrier in most epithelia. However, failure to identify zonula adherens on TEM does not exclude their presence, because difficulty distinguishing desmosomes from zonula adherens by TEM has been reported in other stratified squamous epithelium (human epidermis) (14).

The junctional complex consisting of tight junctions (zonula occludens) and zonula adherens is known to be Ca2+ dependent, with various epithelial models showing that exposure to Ca2+-free solution can disaggregate adhesed cells, prevent aggregation of suspended cells, and lower RT in polarized cell monlayers mounted in Ussing chambers (2, 7, 11, 12, 18). As examples of the latter, when monolayers of Madin-Darby canine kidney (MDCK) cells or monolayers of CaSki cells, a human cervix squamous cell line, are mounted in Ussing chambers and exposed to Ca2+-free bathing solutions, RT, a marker of junctional permeability, falls to low levels, and on Ca2+ restoration, RT returns to baseline. Because RT reflects junctional permeability, the rise in RT toward baseline on restoration of Ca2+ has been referred to as junctional "resealing," and the monitoring of these biological changes when changing from Ca2+-containing to Ca2+-free and back to Ca2+-containing solutions is referred to as the "Ca2+-switch technique" (2, 7, 11, 12, 18).

In the last 40 years, the Ca2+-switch technique with modifications has been used successfully to characterize the nature of the junctional barrier in epithelial models using both cultured cells and native epithelia (2, 7, 10, 11, 12, 18, 27). In the present investigation, we mounted native rabbit EE in Ussing chambers and performed the standard Ca2+-switch technique while monitoring RT as a measure of junctional permeability. The ability to use RT in this way relies on the fact that stratified squamous epithelia behave as a syncytium, and so their electrical properties as measured in Ussing chambers parallel those generated by simpler, single cell-layered epithelia (22). This concept is supported by performance of circuit analysis and by correlation of mannitol and dextran fluxes with RT in multilayered epithelia (5, 17, 23, 24, 2933). Notably, EE responded predictably with a significant decline in RT in Ca2+-free solution and restoration of RT to baseline on Ca2+ restoration. For this reason, we used this technique to further explore the nature of the junctional barrier in this tissue. The results of our investigation indicate that the junctional barrier in native EE has a significant Ca2+-dependent component and that this component reflects the presence and adhesion between the extracellular domains of E-cadherin molecules within adjacent cells. Furthermore, for adhesion, the extracellular domains of E-cadherin contain a three amino acid histidine-alanine-valine (HAV) sequence recognition site.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
New Zealand white male rabbits weighing 8–9 lbs were killed by intravenous overdose of pentobarbital sodium (60 mg/ml). The animal protocol was approved by the institutional Animal Welfare Committee. The esophagus was excised, opened, and stripped of its muscle layers in a paraffin tray containing ice-cold oxygenated normal Ringer solution so that a sheet of tissue was obtained consisting of stratified squamous epithelium and a small amount of underlying connective tissue. From this tissue, four sections were cut and mounted as flat sheets between Lucite half-chambers with an aperture of 1.13 cm2 for measurements of potential difference (PD), short-circuit current (Isc), and calculation of total RT. Tissues were bathed in normal Ringer solution of (in mM): 140 Na+, 119.8 Cl, 5.2 K+, 25 HCO3, 1.2 Ca2+, 1.2 Mg2+, 2.4 HPO42–, and 0.4 H2PO4, 268 mosmol/kgH2O, pH 7.5 when gassed with 95% O2-5% CO2 at 37°C. Mucosal and serosal solutions were connected to calomel and Ag/AgCl electrodes with Ringer-agar bridges for measurements of PD and automatic short circuiting except for 5–10 s when the open-circuit PD was read. RT was calculated using Ohm's Law by dividing the open-circuit PD by the Isc (or from the current deflection to imposed voltage).

The Ca2+-switch technique has been previously employed to study junctional barrier function in cultured epithelial cell monolayers mounted in Ussing chambers (2, 7, 8, 1113, 18). In the present experiments, the Ca2+-switch technique was adopted to study the nature of the junctional barrier in native EE. This was done by mounting intact sections of rabbit EE in Ussing chambers and exposing them, both luminally and serosally, to Ca2+-free bathing solutions containing 3 mM EDTA. After this exposure, RT was continuously monitored and declined progressively over time until a plateau was reached, usually within 2 h. Subsequently, bathing solutions were replaced with Ca2+-containing Ringer solution, and this resulted in progressive restoration of RT to baseline, a process known as "resealing." The sequence of exposure to Ca2+-free solution and restoration of Ca2+-containing solution constitutes the Ca2+-switch technique. Because the Ca2+-switch technique, herein employed, showed readily reversible changes in RT, the technique was further employed to investigate the nature and characteristics of the junctional barrier in native EE.

To assess the nature of the proteins involved with junctional resealing, the Ca2+-switch experiment was modified in the following way. First, electrically paired (by RT) sections of rabbit EE in Ussing chambers (holding 10 ml of bathing solution on each side) were exposed to Ca2+-free bathing solutions with EDTA to lower RT for 2 h. After 2 h (for cost savings on antibody and peptide purchases), the luminal solution only was replaced with 1 ml of Ca2+-free bathing solution (without EDTA) containing 10 ug/ml of monoclonal antibody or nonspecific IgG as control or 25 ug/ml synthetic peptide or no treatment as control. The serosal solutions retained the 10 ml of Ca2+-free, EDTA-containing bathing solution. After 20 min of exposure to antibody, IgG, or peptide in Ca2+-free solution, both luminal and serosal solutions were replaced with 10 ml of Ca2+-containing solutions (normal Ringer) in both baths so that resealing could be monitored by recovery of RT toward baseline. The ability of an antibody or peptide to reduce the rate and/or degree of resealing compared with simultaneously studied controls was taken as evidence in support of involvement of a particular protein or epitope in junctional resealing. Tested in this system were monoclonal antibodies to E-cadherin obtained from Zymed Laboratories (San Francisco, CA) and peptides custom synthesized by Multiple Peptide Systems (San Diego, CA). BAPTA was from Molecular Probes (Eugene, Oregon), and all other chemicals from Sigma (St. Louis, MO).

Immunohistochemistry. In some experiments, tissues were fixed in 4% buffered paraformaldehyde for 20 min and then stored in PBS before dehydration and embedding in paraffin. Five-micrometer sections were mounted on Superfrost plus microscope slides (Fisher Scientific, Pittsburgh, PA), blocked with 20% normal horse serum (Vector Laboratories, Burlingame, CA), incubated with optimized concentrations of E-cadherin monoclonal antibody (HECD-1; Zymed Laboratories), stained with Vectastatin ABC-elite reagents, and detected with 3,3'-diaminobenzidine substrate (Vector Laboratories) according to the manufacturer's instructions.

Statistics. Statistical significance was determined using Student's t-test for parametric data. All data were reported as the means ± SE.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ca2+-switch technique. Sections of rabbit EE were mounted in Ussing chambers and equilibrated for 45 min in physiological Ringer solution (containing 1.2 mM Ca2+) to obtain stable basal readings of RT. Both luminal and serosal bathing solutions of normal Ringer solution were then replaced with Ca2+-free Ringer solution containing 3 mM EDTA to ensure adequate depletion of bathing solution Ca2+. The effect of this Ca2+-free environment on paracellular (junctional) permeability was monitored for 2 h by recording the changes in RT. After 2 h of exposure to Ca2+-free environment, the luminal and serosal bathing solutions were removed and replaced with normal (Ca2+ containing) Ringer solution, and RT was monitored again for an additional 2 h. As shown in Fig. 1, exposure to Ca2+-free environment produced a progressive decline in RT, indicative of "junctional opening" and restoration of Ca2+ in the form of normal Ringer solution, resulted in a progressive increase in RT toward baseline indicative of "junctional resealing" (note, EDTA alone has no effect on RT; data not shown). In Ca2+-free solution, RT declined ~35% over the 2-h period, and on Ca2+ restoration, RT returned to within 5% of baseline over 2 h. If tissues were kept in Ca2+-free solution for up to 4 h, after 2 h, RT declined at a much slower rate, reaching a nadir of ~45% below baseline by 4 h, a value that was only 10% below that observed at 2 h. Moreover, if Ca2+ restoration was delayed beyond 2 h (data not shown), restoration of RT toward baseline was both slower and less complete over the ensuing 2 h than that shown in Fig. 1.



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Fig. 1. Effects of bathing solution Ca2+ removal on junctional permeability as reflected by changes in transepithelial electrical resistance (R) in Ussing-chambered rabbit esophageal epithelium. Replacement with Ca2+-free solutions is shown to produce a progressive decline in R with time, a process that is reversible (resealing) as shown by a return in R toward basal levels following replacement of Ca2+-free solutions with normal Ringer solution (1.2 mM Ca2+). R = %initial R before Ca2+ removal. Note, EDTA is also added to the bathing solutions to ensure Ca2+ depletion but addition of EDTA alone has no effect on R: data not shown. Also the initial absolute values for R = 1,659 ± 110 for Ca2+ restored and 1,404 ± 213 for Ca2+-free/EDTA throughout.

 
Junctional resealing. The reproducibility of the changes in RT during the Ca2+-switch technique prompted its further use to characterize the nature of the junctional barrier. This was done initially by monitoring the process of junctional resealing during Ca2+ restoration under a variety of conditions. First, resealing was monitored under conditions of varying the Ca2+ concentration of the restored Ringer bathing solution. As shown in Fig. 2, Ca2+-induced resealing, as indicated by the 2-h recovery of RT from nadir in Ca2+-free solution, was dose dependent. Significant resealing required a concentration of at least 0.5 mM Ca2+, whereas optimum resealing required 1.2 mM Ca2+. Second, resealing was monitored under conditions of varying divalent cation substitutions for Ca2+ in the replacement Ringer solution. As shown in Fig. 3, neither 1.2 mM Mn2+, Mg2+, Ba2+, nor Sr2+ was as effective as Ca2+ in the resealing process, although Mn2+ showed evidence of partial resealing. Third, resealing was examined under conditions in which tissues were bathed luminally and serosally with either 50 µM BAPTA/AM, an intracellular chelator of Ca2+, or 10–5 M A23187 [GenBank] , an ionophore for Ca2+, maneuvers designed to change intracellular Ca2+. Because the results showed that neither BAPTA/AM, which lowers intracellular Ca2+, nor A23187 [GenBank] , which raises intracellular Ca2+, had any effect on resealing (or on basal RT, data not shown; Fig. 4), the findings suggest that Ca2+ manipulation alters RT via an extracellular effect. Fourth, resealing was examined under conditions in which tissues were bathed luminally and serosally with the protein synthesis inhibitor cycloheximide (7 µg/ml). Because, as shown in Fig. 5, cycloheximide had no effect on resealing, the findings suggest that the changes in RT reflect changes in the conformation of junctional proteins rather than new protein synthesis.



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Fig. 2. Effect of bathing solution Ca2+ concentration on junctional resealing in Ussing-chambered rabbit esophageal epithelium. After reduction in R by exposure to Ca2+-free solutions for 2 h, solutions were replaced with Ringer containing varying concentrations of Ca2+. The %recovery of R toward baseline levels in Ca2+-containing Ringer solutions at 2 h is shown. %Recovery is calculated by dividing the maximum increase in R in Ca2+-containing solution at 2 h from the nadir in Ca2+-free solution by the maximum decline in R in Ca2+-free solution from baseline in normal (1.2 mM Ca2+) Ringer solution x 100. The results indicate that resealing requires a minimum Ca2+ concentration of 0.5 mM with physiological Ca2+ concentrations of 1.2 mM providing the maximum rate of resealing.

 


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Fig. 3. Effect of substituting various divalent cations for bathing solution Ca2+ on junctional resealing in Ussing-chambered rabbit esophageal epithelium. After reduction in R by exposure to Ca2+-free solutions for 2 h, bathing solutions were replaced with Ringer containing, instead of 1.2 mM Ca2+, either 1.2 mM Mn2+, Sr2+, Ba2+, or Mg2+. The %recovery of R toward baseline levels for the divalent cations is compared with that of Ca2+. %Recovery is calculated by dividing the maximum increase in R in cation-containing solutions at 2 h from the nadir in Ca2+-free solution by the maximum decline in R in Ca2+-free solution from baseline in normal (1.2 mM Ca2+) Ringer solution x 100. The results show the dependency of resealing on Ca2+ because there is limited or no capacity for other divalent cations to serve as an effective substitute for the process.

 


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Fig. 4. Effect of exposure to 50 µM BAPTA/AM or 10–5 M A23187 [GenBank] on junctional resealing in Ussing-chambered rabbit esophageal epithelium. Exposure to the agents both luminally and serosally had no effect on basal R, magnitude of the decline in R on exposure to Ca2+-free solutions for 2 h (data not shown), nor on the magnitude of the recovery (resealing) of R following restoration of normal Ringer solution. The %recovery of R toward baseline levels in BAPTA/AM or A23187 [GenBank] is shown compared with normal Ringer controls. %Recovery is calculated by dividing the maximum increase in R in Ca2+-containing solution at 2 h from the nadir in Ca2+-free solution by the maximum decline in R in Ca2+-free solution from baseline in normal (1.2 mM Ca2+) Ringer solution x 100. The results indicate that manipulation of intracellular Ca2+ concentrations have little effect on junctional permeability or resealing in this tissue.

 


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Fig. 5. Effect of exposure to 7.5 µg/ml cycloheximide on junctional resealing in Ussing-chambered rabbit esophageal epithelium. Exposure to the agent, both luminally and serosally, had no effect on basal R, magnitude of the decline in R on exposure to calcium (Ca2+)-free solutions for 2 h (data not shown) nor on the magnitude of the recovery (resealing) of R following restoration of normal Ringer solution. The %recovery of R toward baseline levels in cycloheximide is shown compared with normal Ringer controls. %Recovery is calculated by dividing the maximum increase in R in Ca2+-containing solution at 2 h from the nadir in Ca2+-free solution by the maximum decline in R in Ca2+-free solution from baseline in normal (1.2 mM Ca2+) Ringer solution x 100. The results indicate that inhibition of protein synthesis has little effect on junctional permeability or resealing in this tissue.

 
E-cadherin. The monitoring of junctional resealing during the Ca2+-switch technique was also used to establish whether E-cadherin, a Ca2+-dependent protein known to be present in EE (28), was involved in the regulation of paracellular permeability in native EE. This was done by luminal exposure of EE to monoclonal antibodies to E-cadherin, two of which (DECMA-1, HECD-1) were known to bind to the extracellular domain of E-cadherin and one of which (4A2C7) was known to bind to the intracellular domain of E-cadherin. Exposure to a similar amount of luminal IgG served as control (note, Ca2+-switch experiments comparing IgG to no treatment showed a similar rate or degree of resealing over 2 h; data not shown). As shown in Fig. 6, A and B, in the presence of 10 µg/ml of monoclonal antibody to DECMA-1, junctional resealing was significantly impaired and a similar inhibitory effect on resealing was observed with exposure to HECD-1. In contrast, no inhibition of junctional resealing was observed with 4A2C7, a monoclonal antibody directed against the intracellular (COOH terminus) domain of E-cadherin (Fig. 6B).



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Fig. 6. A: effect of rat monoclonal antibodies (DECMA-1) to the extracellular portion of mouse E-cadherin on junctional resealing in Ussing-chambered rabbit esophageal epithelium. After reduction in R by exposure to calcium (Ca2+)-free solutions for 2 h, tissues were exposed luminally for 20 min to 10 µg DECMA-1 or, as control, 10 µg of rat IgG, and this was followed by restoration of normal (Ca2+ containing) Ringer solution. The results, depicted as the change in R as %initial R in normal Ringer solution, show that DECMA-1 inhibits junctional resealing following Ca2+ restoration. Initial absolute value for R = 1,561 ± 114 for DECMA-1 and 1,407 ± 64 for IgG control. B: comparison of rat or mouse monoclonal antibodies to either the extracellular portion of mouse E-cadherin (DECMA-1; data from Fig. 6), human E-cadherin (HECD-1), or the intracellular portion of human E-cadherin (4A2C7) on junctional resealing in Ussing-chambered rabbit esophageal epithelium. After reduction in R by exposure to Ca2+-free solutions for 2 h, tissues were exposed luminally for 20 min to 10 µg of DECMA-1, HECD-1, 4A2C7, or, as control, 10 µg of rat or mouse IgG, and this was followed by restoration of normal (Ca2+ containing) Ringer solution. The %recovery of R toward baseline levels in Ca2+-containing Ringer solutions at 2 h is shown. %Recovery is calculated by dividing the maximum increase in R in Ca2+-containing solution at 2 h from the nadir in Ca2+-free solution by the maximum decline in R in Ca2+-free solution from baseline in normal (1.2 mM Ca2+) Ringer solution x 100. The results indicate that antibodies to the extracellular, but not intracellular, domain of E-cadherin can effectively inhibit junctional resealing following Ca2+ restoration.

 
The concept that Ca2+-free solution exposes the extracellular domains of E-cadherin to reactive molecules in the bathing solution is also supported by our investigation using immunohistochemistry to localize the sites of E-cadherin within native EE. As shown in Fig. 7, when immunohistochemical staining to localize the HECD-1 monoclonal antibody to the extracellular domain of E-cadherin was done in the presence of physiological levels (1.2 mM) of Ca2+, E-cadherin was identified on the luminal surface of stratum corneum (some cells in process of sloughing into the lumen) but not within the remaining cell layers of stratum corneum. E-cadherin was also noted to be present to a much lesser extent within the membranes of cells within the stratum spinosum. Notably, tissues exposed to HECD-1 in Ca2+-free solution showed dense staining of the membranes for all cells throughout the stratum corneum. There was also an increase in the level of staining for cells within the stratum spinosum and stratum basalis. In addition, E-cadherin distribution was investigated in tissues from the same animal that had initially been opened by Ca2+-free solution and then exposed to either a subphysiological concentration of bathing solution Ca2+ (0.5 mM) or physiological (1.2 mM) levels of bathing solution Ca2+. The results showed that whether RT recovered 56% (1.2 mM Ca2+) or only 10% (0.5 mM Ca2+), immunohistochemistry revealed staining that differed from those in Ca2+-free solution (see Fig. 7B). In fact, they closely resembled those tissues unexposed to calcium-free solution, having minimal staining of the membranes of cells within the stratum corneum (see Fig. 7A). This suggests that transassociation of E-cadherin molecules within the junctions of the stratum corneum can occur in Ca2+-containing solutions, even when Ca2+ is present in suboptimal concentrations, and this well before the restoration of RT. The latter observation may reflect the requirement for transassociation of E-cadherin before the adhesion of other (tight junction) proteins that are necessary for the complete junctional sealing reflected in values for RT.



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Fig. 7. Immunohistochemistry using monoclonal antibodies (HECD-1) to the extracellular domain of human E-cadherin in the presence (A) or absence (B) of bathing solution Ca2+. In Ca2+-containing solution, the E-cadherin staining is principally localized to the surface cell layer of stratum corneum and, to a much lesser extent, to the membranes of cells within the layer of stratum spinosum. There is no staining of the stratum basalis. In contrast, tissues exposed to Ca2+-free solution show marked staining for E-cadherin in the membranes of all cells within the multilayered stratum corneum as well as increased staining of the membranes for all cells within the stratum spinosum. Note that control tissues unexposed to antibody have no staining (data not shown). Magnification, x40.

 
Adhesion recognition sequence. Because the above experiments indicated that the extracellular domain of E-cadherin was needed for junctional resealing in EE, we sought the nature of the amino acid adhesion recognition sequence by having synthesized three short peptides to the extracellular domain of E-cadherin. Two of the peptides (H-LRAHAVDVNG-NH2 or H-LGHHAVPSNG-NH2) contained an HAV amino acid sequence previously shown by Blaschuk et al. (3, 4) to be important for E-cadherin adhesion and compared their effect on resealing to another peptide (H-VCDCEGAAGVCR-NH2) devoid of the HAV sequence. However, when the Ca2+-switch technique was employed using luminal exposure to 25 ug/ml of each synthetic peptide, there was no inhibition of resealing on Ca2+ replacement for any of the peptides. Nonetheless, because these experiments were carried out with the peptides effectively "washed out," i.e., no peptide was present in the Ca2+-containing solution during the monitoring of resealing, we repeated the experiments in a similar manner (see MATERIALS AND METHODS) but monitored resealing with 25 ug/ml of each peptide present (or absent in controls) in 1 ml of replacement Ca2+-containing Ringer solution. As shown in Fig. 8A and supporting the importance of the HAV adhesion recognition sequence, resealing was dramatically inhibited during exposure to the two synthetic peptides containing HAV (peptide 1: H-LRAHAVDVNG-NH2 or peptide 2: H-LGHHAVPSNG-NH2), whereas that without the HAV sequence (peptide 3) had essentially no effect (note, resealing is similar in rate and degree irrespective of whether the replacement luminal baths contained 1 or 10 ml of Ca2+-containing Ringer solution; data not shown).



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Fig. 8. A–C: effect of 3 small synthetic peptides to the extracellular adhesive domain of E-cadherin (peptide 1: H-LRAHAVDVNG-NH2; peptide 2: H-LGHHAVPSNG-NH2, peptide 3: H-VCDCEGAAGVCR-NH2) on junctional resealing in Ussing-chambered rabbit EE. After reduction in R by exposure to Ca2+-free solution for 2 h, tissues were exposed luminally for 20 min to 25 µg, and this was followed by restoration of normal (Ca2+ containing) Ringer solution. The results, depicted as the change in R as %initial R in normal Ringer solution, show that the 2 synthetic peptides containing the histidine-alanine-valine (HAV) recognition sequence of the extracellular domain of E-cadherin but not the peptide that did not have the HAV recognition sequence inhibit junctional resealing following Ca2+ restoration. Note, absolute initial R = 2,013 ± 311 for peptide 1 and 1,882 ± 195 for control; 1,644 ± 181 for peptide 2 and 1,327 ± 153 for control; and 1,302 ± 180 for peptide 3 and 1,376 ± 228 for control.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, we applied the Ca2+-switch technique to the investigation of the junctional barrier in native rabbit EE. When mounted in Ussing chambers and exposed to Ca2+-free solution (with EDTA), the EE exhibited a progressive decline in RT over 2 h to ~35% below basal levels. Beyond 2 h, RT declined further but at a much slower rate so that by 3–4 h, RT had plateaued at ~45% below basal levels. Because declines in RT of Ussing-chambered EE, when unassociated with cell necrosis, are closely paralleled by increases in mannitol and dextran fluxes (5, 17, 2933), these observations establish the junctional barrier in EE as being comprised of significant Ca2+-dependent and -independent components. Furthermore, the Ca2+-dependent component could be readily reversed (within 2 h) on Ca2+ restoration to the bathing solution as a reflection of junctional resealing. This reversibility, and the lack of cell necrosis morphologically, support the absence of material damage to the tissue during the period of transient Ca2+ removal.

An important role for Ca2+ in cell adhesion has long been established in single-layered epithelia, and this through a major interaction with the Ca2+-dependent membrane protein E-cadherin. Furthermore, research has demonstrated that at very low bathing solution concentrations of Ca2+ (~0.05 mM), E-cadherin's rodlike structure is stabilized, whereas concentrations in the 0.5 mM range are required for its dimerization. Finally, it takes concentrations of Ca2+ in the physiological range, i.e., >1 mM, for the transassociation of E-cadherin dimers, which are necessary for junction formation and cell-cell adhesion. Notably, this critical role for Ca2+ in cell adhesion is mediated extracellularly and not through alteration of intracellular Ca2+ levels, and furthermore, E-cadherin's role in junctional resealing does not require new protein synthesis (2, 7, 8, 1113, 21). Although these concepts are well established in single-layered epithelia, documentation of similar action has not been established in a multilayered epithelium. For this reason and given the reproducibility of the Ca2+-switch technique for opening and closing the junctions in EE, we used this tool to reevaluate these same concepts in rabbit EE. This was done by 1) performing a dose response to Ca2+, 2) substituting various divalent cations for Ca2+, 3) exposing the tissue to two chemicals, BAPTA/AM or A23187 [GenBank] , known to lower or raise intracellular Ca2+, and 4) assessing the effect on the process of cycloheximide, a protein synthesis inhibitor. The results of these experiments showed that resealing was relatively rapid (recovery mostly complete by 2 h), dependent on Ca2+, with only Mn2+ among the divalent cations tested being able to partially substitute for Ca2+, and requiring as a minimum 0.5 mM Ca2+ and for optimization, 1.2 mM Ca2+. Notably, neither BAPTA/AM nor A23187 [GenBank] had an effect on RT, either basally or on resealing, supporting the concept that the effects of Ca2+ were mediated extracellularly and not intracellularly. Furthermore, both the speed and the lack of effect of cycloheximide on the process indicated that junctional resealing occurred by Ca2+-induced changes in protein trafficking or conformation rather than through the new synthesis of junctional protein. These observations are all in general agreement with those of Gumbiner and Simons (12) and Gorodeski et al. (11) in Ussing-chambered monolayers of MDCK cells or CaSki cells, the latter being a human cervix-derived squamous cell line.

Presently, there are two junctional structures that subserve barrier function in other epithelia, the more lumen-oriented zonula occludens (tight junction), and just below this on the basolateral membrane side, the zonula adherens. Each of these structures restrict ion and molecular flow across the paracellular pathway via the creation of protein bridges that bring neighboring cell membranes into close apposition, and these intercellular bridging (linker) proteins are unique to the structure. For instance, occludin and claudins, the latter which has at least 20 isoforms, are the linker proteins for the zonula occludens, whereas E-cadherin (also known as uvomorulin, 120/80 CAM, L-CAM) is the linker protein for the zonula adherens. Importantly, from the standpoint of the present investigation, E-cadherin but neither occludin nor claudins is known to be Ca2+-dependent (12, 15, 34). Ca2+ enables E-cadherin to subserve barrier function by first enabling its molecular dimerization at low Ca2+ levels within the intercellular space and then, at somewhat higher Ca2+ levels, its ability for homophyllic interaction and association with E-cadherin within the membrane of adjacent cells (21). These observations and the fact that E-cadherin has been reported to be present in EE (28) made it a logical candidate for the Ca2+-dependent component of the junctional barrier in the present investigation. To assess this possibility, we once again returned to the Ca2+-switch technique and used the modification employed by Gumbiner and Simons (12) to establish the nature of the junctional adhesion proteins in Ussing-chambered MDCK monolayers. Specifically, similar to Gumbiner and Simons (12), we showed that we could inhibit junctional resealing by exposing native EE to two different monoclonal antibodies to the extracellular domain of E-cadherin, DECMA-1 and HECD-1; and the specificity of this effect was shown by the fact that neither nonspecific IgG nor monoclonal antibodies to the intracellular domain of E-cadherin had an effect on junctional resealing. Furthermore, Blaschuk et al. (3, 4) have shown that E-cadherin-mediated cell adhesion could be blocked in a compaction assay of eight cell-stage mouse embryos and rat neurite outgrowth on astrocytes by exposure to short synthetic peptides to a recognition sequence of three amino acids (HAV) within the extracellular domain. Again, using the Ca2+-switch technique, we were able to demonstrate that two short synthetic peptides containing the HAV recognition sequence could block resealing in native EE; and the specificity of this effect, shown by the fact that a short peptide to the extracellular domain but devoid of the HAV sequence, had no effect on junctional resealing in EE. These data, taken together, provide compelling evidence that junctional resealing and barrier function in native EE are highly dependent on adhesion of the extracellular domains of E-cadherin and that the adhesive domain contains within it the HAV recognition sequence.

Reinforcement of the above concepts was also evident in a set of experiments that relied on a modification of the Ca2+-switch technique. In these experiments, we observed immunohistochemically that the luminal cell layers of stratum corneum, those previously shown to contain the junctional barrier to the paracellular diffusion of lanthanum (20), contain E-cadherin within their cell membranes. This, in effect, supports, by morphological localization, the conclusion that E-cadherin plays an important role in the junctional barrier in native EE. However, noteworthy was the fact that E-cadherin was only apparent within all layers of stratum corneum when EE was exposed to monoclonal antibodies in Ca2+-free but not Ca2+-containing solution. This supports the concept that the E-cadherin molecules within the stratum corneum were initially inaccessible to antibody binding because they were actively involved with junctional adhesion in Ca2+-containing solution; and that their accessibility to antibodies became apparent after exposure to Ca2+-free solution because of the ability of the latter to induce junctional opening and thus reductions in RT by changing junctional protein conformation.

In summary, the present investigation applies the Ca2+-switch technique to the study of junctional barrier formation in native esophageal epithelia and establishes an important role in the barrier for E-cadherin and the HAV sequence within its extracellular domain. The results of these investigations also suggest that, whereas not identified on TEM, structures consistent with zonula adherens are likely to be present within the membranes of cells comprising the stratum corneum. What contribution other tight junctional proteins, such as occludin and claudins, make to the barrier in EE remain unclear and are not excluded based on the results of the present investigation. This is the case because of the known dependence of tight junction formation on adhesion between E-cadherin molecules of adjacent cells. In effect, creation of the zonula adherens by E-cadherin is required to bring the membranes into close apposition before linker bridges can be formed for the tight junction (8, 13, 35). Moreover, E-cadherin, as a junctional protein, does not function in isolation but within a highly complex and dynamic system modulated by interactions with other cellular proteins, among these are the catenins, mitogen-activated protein kinase, protein kinase C, calmodulin, phospholipase C, and the cytoskeleton. Many of these interactions in the multilayered EE may be accessible to study through the individual or combined application of the Ussing chamber and Ca2+-switch technique.


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This project was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-36013.


    ACKNOWLEDGMENTS
 
The authors thank Luisa Brighton and John L. Carson for expertise in performance of immunohistochemistry.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. A. Tobey, Tulane Univ. Health Sciences Center, SL-35, 1430 Tulane Ave., New Orleans, LA 70112 (E-mail: ntobey{at}tulane.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 RESULTS
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