Activation of a CFTR-mediated chloride current in a rabbit corneal epithelial cell line.
OBJECTIVE
To determine whether there is gene expression and functional activity of cystic fibrosis transmembrane conductance regulator protein (CFTR) in an SV40-immortalized rabbit corneal epithelial cell line, tRCE.


METHODS
Both whole-cell and cell-attached patch-clamp techniques were used to examine the biophysical characteristics of the cAMP-dependent chloride current. The molecular identity of this conductance was evaluated using RT-PCR analysis.


RESULTS
In whole-cell patch-clamp studies, a cAMP-dependent chloride conductance was further facilitated by the known CFTR activator genistein (20 microM). Kinetic analysis of cell-attached patches containing few channels ascertained that genistein increased the chloride channel activity by increasing channel open probability (via an increased channel open time and a decreased channel closed time). In addition, in the presence of a reduced forskolin concentration (i.e., 100 nM), the chloride conductance generated could be augmented by the nonspecific phosphodiesterase enzyme inhibitor, IBMX (100 microM), implicating the importance of intracellular cAMP in the regulation of this conductance. Furthermore, this conductance exhibited voltage-dependent inhibition in the presence of the CFTR chloride channel blocker glibenclamide (250 microM), but was DIDS insensitive (500 microM). Consistent with the presence of a CFTR-mediated chloride conductance, the expression of CFTR-mRNA was detected using RT-PCR. Sequence analysis of the product revealed 99.4% homology to that described for rabbit CFTR.


CONCLUSIONS
In tRCE cells, there is gene expression and functional CFTR activity. Its presence may have important therapeutic implications in corneal epithelial diseases resulting from declines in transepithelial secretory and fluid transport activity.
Al-Nakkash
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Department of Physiology, School of Medicine, University of Missouri-Columbia, 65212, USA. al-nakkashl@missouri.edu
Reinach
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D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D003315:Cornea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression; D019833:Genistein / Q000494:pharmacology; D005905:Glyburide / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002712:Chlorides; D010726:Phosphodiesterase Inhibitors; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D019833:Genistein; D000242:Cyclic AMP; D005905:Glyburide; D015056:1-Methyl-3-isobutylxanthine