Characterization of cyclic AMP-regulated chloride conductance in the pigmented rabbit conjunctival epithelial cells

Abstract

We have previously reported that the pigmented rabbit conjunctiva is a Cl–secreting tissue, subject to cAMP, Ca2+, and PKC modulation. The present study was conducted to characterize, at the cellular and molecular levels, cAMP-regulated Cl–channels in rabbit conjunctival epithelial cells. cAMP-inducible Cl–channel properties were evaluated by monitoring the whole-cell currents using patch clamp techniques. Results showed that 10 μM forskolin significantly stimulated a glibenclamide-inhibitable whole-cell conductance by approximately five-fold. Furthermore, reduction of the Cl–concentration in the bathing solution through partial substitution of NaCl with Na-isethionate resulted in a rightward shift of the reversal potential for both baseline and forskolin-stimulated whole-cell currents from 0 to values close to the theoretical Cl–reversal potential predicted by the Nernst equation. Western blot analysis with a monoclonal antibody recognizing the epitope in the C-terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) showed a positive band at its molecular weight, approximately 170 kD. Immunostaining under confocal microscopy revealed a CFTR specific signal in the apical sections of primary conjunctival epithelial cells. In addition, RT-PCR detection amplified a cDNA fragment 100% identical to the predicted portion of the cloned rabbit CFTR message. The stage is thus set for determining the extent of CFTR contribution to cAMP-regulated Cl–conductance in pigmented rabbit conjunctival epithelial cells.Key words: conjunctiva, chloride current, CFTR, chloride channel, patch clamp, cyclic AMP, whole-cell.