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MUCOSAL BIOLOGY

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

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

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 (R_T), a marker of junctional permeability. When exposed to Ca²⁺-free Ringer solutions containing EDTA, R_T 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 R_T 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 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

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