Ontogeny of corneal epithelial tight junctions: stratal locale of biosynthetic activities.

PURPOSE On reaching the surface position, corneal epithelial cells assemble a tight paracellular barrier rapidly. The purpose of this study was to identify at what maturation stage the cells acquire biochemical elements underpinning this ability. METHODS Rabbit corneas were subjected to a digitonin-exposure protocol that results in the devitalization and desquamation of the three outermost cell layers of the 5-6-layer thick epithelium and thus, exposes winglike cells to the tear surface. Corneas were then mounted in Ussing-type chambers to measure transepithelial resistance and solute fluxes. RESULTS After the devitalization treatment transepithelial resistance remained near baseline for 2 hours and then rose to 8.24 +/- 3.12 K omega.cm2 (+/- SD, n = 15) during the next 8 hours. This increase was matched by increases in the resistance to the paracellular flow of mannitol. Transmission electron microscopy confirmed de novo formation of tight junctions over this time span in the new surface. The addition of transcription (actinomycin D; alpha-amanitin), translation (cycloheximide), core glycosylation (tunicamycin), and endoplasmic reticulum-to-Golgi traffic (brefeldin A) inhibitors immediately after the devitalization protocol prevented the transepithelial resistance raise. Introducing time delays between the cell devitalization and the addition of these biosynthesis inhibitors reduced their effect; a time delay of 90 minutes abolished the inhibition by alpha-amanitin. CONCLUSION Devitalization of overlying cells induces the assembly of a tight paracellular barrier in the wing cells. The process requires and includes the synthesis of essential transcripts and proteins that were initially absent in these cells. In vivo, the synthesis of these elements must occur as cells reach the squamous stage of maturation.