Characterization of anoctamin in porcine ciliary epithelium

Abstract

Purpose : It has been shown that swelling-activated Cl- channels play an important role in regulating Cl- and aqueous humor secretion across the ciliary epithelium. In particular, Cl- efflux by non-pigmented ciliary epithelial (NPE) cells likely limits the rate of aqueous humor secretion, although the identities of Cl- channels have not been fully understood. Recently, it has been demonstrated that anoctamin-6 (ANO-6) is expressed in bovine trabecular meshwork cells, contributing to the regulation of swelling-activated Cl- current and cell volume. In this study, we aim to 1) characterize the expression of ANO isoforms in porcine ciliary epithelium; and 2) determine the functional significance of the most abundant ANO isoform in modulating transepithelial electrical and fluid measurements across excised porcine ciliary epithelium Methods : The ciliary epithelium of the porcine eye was studied since the porcine eye is an excellent model of the human eye. Gene and protein expressions of anoctamin isoforms in porcine ciliary epithelium were characterized by real-time PCR (qPCR) and Western blots, respectively. Transepithelial electrical parameters and fluid movement across excised ciliary body/epithelium were determined using custom-made fluid flow chambers. Results : Among all 10 ANO isoforms tested, ANO-6 was found to be the most abundant ANO isoform expressed in porcine ciliary epithelium. The presence of ANO-6 protein was confirmed by Western blot. Using intact porcine ciliary body/epithelium, administration of hypotonic bathing solution to the NPE surface elicited a significant increase in transepithelial fluid movement (from 2.79±0.31μL/h/preparation to 4.79±0.36μL/h/preparation) into the eye (n=6; p<0.01). Subsequent addition of 100μM tannic acid, a specific ANO-6 blocker, significantly inhibited hypotonicity-induced fluid flow by 85±9% (p<0.001), strongly supporting the involvement of ANO-6 in regulating aqueous humor secretion. Conclusions : Our results show that ANO-6 is the most abundant ANO isoform expressed in the porcine ciliary epithelium. The inhibition exerted by tannic acid on hypotonicity-induced fluid flow strongly suggests that ANO-6 may be a plausible candidate for mediating Cl- and aqueous humor secretion into the human eye.