PURPOSE. To determine the potential role of ClC-3, a PKC-inhibitable Cl-channel, in mediating the swelling-activated Cl-current (ICl,swell) of native bovine nonpigmented ciliary epithelial (NPE) cells. METHODS. Native bovine NPE cells were freshly harvested by enzymatic digestion. Whole-cell currents were recorded by patch- clamp measurements either in the presence or absence of a functional anti-ClC-3 antibody. RESULTS. Baseline whole-cell currents were small under isotonic conditions. Hypotonic cell swelling stimulated outwardly rectifying ICl,swell, which was reversibly inhibited by the Cl-channel blockers, phloretin (300 μM) or 5-nitro-2-(phenylpropylamino)- benzoate (NPPB, 100 μM). Intracellular dialysis with anti-ClC-3 C670-687antibody did not affect baseline currents, but significantly delayed and inhibited hypotonic stimulation of ICl,swell. Preabsorption of the antibody with its antigen prevented the inhibition of ICl,swellby antibody. In addition, intracellular dialysis with control Ex133-148antibody did not affect the ICl,swell. Moreover, activation of PKC by pretreatment with 100 nM phorbol 12,13-dibutyrate (PDBu) significantly inhibited the initial stimulation of ICl,swell, but had no effects on the steady state currents. CONCLUSIONS. The results suggest that endogenous ClC-3 is involved in mediating ICl,swellof native bovine NPE cells. The delayed stimulation of ICl,swellby PDBu may reflect upregulation of swelling-activated Cl-channels of different subtypes, especially when the function of ClC-3 is blocked. This information will be useful in understanding the mechanisms controlling aqueous humor formation and thereby intraocular pressure.
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience