Characterization of cyclic AMP-regulated chloride conductance in the pigmented rabbit conjunctival epithelial cells.
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 microM 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.
Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles 90089-9121, USA.
Michael H I
MH
Shiue
Hovhannes J
HJ
Gukasyan
Kwang-Jin
KJ
Kim
Donald D F
DD
Loo
Vincent H L
VH
Lee
D000818:Animals; D015153:Blotting, Western; D002469:Cell Separation; D018118:Chloride Channels / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology; D003228:Conjunctiva / Q000166:cytology / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008297:Male; D018613:Microscopy, Confocal; D018408:Patch-Clamp Techniques; D010858:Pigmentation / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018118:Chloride Channels; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D000242:Cyclic AMP