Abstract
Purpose :
The role of ion and aquaporin water channels allowing tear film homeostasis remains poorly understood, in particular due to the lack of valuable human cell models. Here, we developed various preparations of human conjunctival (HC) epithelial cells to study ion and water fluxes and to identify key ion channels and aquaporins.
Methods :
We obtained, isolated and cultured Human conjunctival epithelial cells from 3 donors and used immortalized Human conjunctival epithelial cells line (Innoprot). Transmembrane ion fluxes characterization was performed by Ussing Chamber to record short circuit current (Isc) on conjunctival human reconstructed epithelia. To study water transport regulation, we recorded the optical path difference (OPD) in HC epithelial cells by the quadriwave lateral shearing interferometry (QWLSI) method. This quantitative phase imaging technique is based on the measurement of the light wave shift when passing through a living sample. We completed our study by RT-PCR and Western Blotting.
Results :
Ussing chambers experiments revealed the function of ENaC, CFTR and CaCC. The basal ENaC-dependent short circuit Isc was blocked by amiloride (100µM). The CFTR-dependent Isc was stimulated by forskolin (10µM) and by the CFTR potentiator Vx770 (10µM) and inhibited by the selective CFTR inhibitor CFTRinh172 (10µM). Finally UTP stimulated a calcium-dependent chloride Isc, showing expression of CaCC, probably TMEM16A. We identified the water flows by recording OPD variations stimulated by forskolin and inhibited by HgCl2 consistent with the function of aquaporin water channel. We confirmed by RT-PCR and Western blot expression of ENaC, CFTR and Aquaporins.
Conclusions :
Using our HC cell models we were able to identify ENaC, CFTR and CaCC and characterized a mercury-sensitive, cAMP-dependent water transport. Our study thus established our HC epithelial cells as a valuable model to dissect the molecular regulation and pharmacology of ions and water flows in human conjunctival epithelial cells.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.