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

Abstract

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, RTdeclined ∼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 RTon 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.