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C.W. Do, W.N. Lu, C.H. Mitchell, M.M. Civan; Functional and Molecular Characterization of Swelling–Activated Cl– Currents in Native Bovine Non–Pigmented Ciliary Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3323.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To examine the functional significance of swelling–activated Cl– current (ICl,swell) in native bovine non–pigmented ciliary epithelial (NPE) cells and to determine the potential role of ClC–3 Cl– channel in mediating the ICl,swell. Methods: Fresh bovine ciliary epithelium was excised and mounted in an Ussing chamber to monitor transepithelial currents. In addition, whole–cell currents were recorded in freshly harvested native bovine NPE cells by patch–clamp measurements either in the presence or absence of a functional anti–ClC–3 antibody. Results: Osmotic swelling stimulated short–circuit current, which was completely blocked by the addition of 100 µM 5–nitro–2–(phenylpropylamino)–benzoate (NPPB) to the aqueous surface. In parallel with the transepithelial measurements, hypotonic cell swelling stimulated whole–cell, outwardly rectifying ICl,swell, which was reversibly inhibited by the Cl– channel blockers, phloretin (300 µM) or NPPB (100 µM). Intracellular dialysis with carboxy–terminal anti–ClC–3 C670–687 antibody did not affect baseline currents under isotonic conditions, but delayed and inhibited hypotonic stimulation of ICl,swell. Preabsorption of the antibody with its antigen prevented the inhibition of ICl,swell by antibody. Western immunoblots confirmed that the antibody recognized ClC–3, and that the ClC–3 band was not detected with preabsorbed antibody. Additionally, intracellular dialysis with control Ex133–148 antibody to an extracellular ClC–3 epitope did not affect the ICl,swell. Conclusions: Our results suggest that hypotonic swelling stimulates whole–cell ICl,swell, leading to an increase in transepithelial Cl– secretion. Endogenous ClC–3 is involved in mediating ICl,swell of native bovine NPE cells. This information will be useful for targeting ClC–3 in the development of selective therapies to control aqueous humor formation and thereby intraocular pressure.
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