Studies on the expression of mRNA for anion transport related proteins in corneal endothelial cells.
OBJECTIVE
Chloride and bicarbonate are necessary for maintenance of fluid transport by the corneal endothelium, however there is little information on the identity of anion transport proteins that could serve as anion efflux mechanisms in endothelial cells. Therefore, we ask whether mRNA for the anion transport related proteins, CFTR, CLC-2, ClC-3, ClC-5 and AE2, are expressed in human, bovine or rabbit corneal endothelium.


METHODS
RT-PCR was performed for CFTR, CLC-2, ClC-3, ClC-5 and AE2 using total RNA from fresh human, bovine and rabbit corneal endothelium as well as cultured bovine corneal endothelial cells (CBCEC). Specificity of PCR products was confirmed by sequencing.


RESULTS
RT-PCR analysis gave positive bands at the predicted size for CLC-3 and CLC-5 from fresh human, rabbit and bovine as well as CBCEC. However, for CLC-2, no band was apparent around the predicted size from fresh and cultured corneal endothelium. A band at the predicted size was obtained for CFTR from fresh human, rabbit and bovine endothelium, as well as from CBCEC. RT-PCR analysis for AE2 produced specific bands from fresh human, rabbit and bovine corneal endothelium, but no positive band was obtained from CBCEC. Sequencing analysis further confirmed the identities of CLC-3, CLC-5, CFTR and AE2 in corneal endothelium.


CONCLUSIONS
CFTR, CLC-3 and ClC-5 are expressed in fresh and cultured corneal endothelial cells. However, consistent with previous immunoblots studies, AE2 is only expressed in fresh corneal endothelium. These results have implications for modeling possible apical anion efflux mechanisms in corneal endothelium.
Sun
X C
XC
School of Optometry, Indiana University, Bloomington, IN 47401, USA.
McCutheon
C
C
Bertram
P
P
Xie
Q
Q
Bonanno
J A
JA
D000818:Animals; D027321:Anion Transport Proteins; D017920:Antiporters; D001639:Bicarbonates / Q000378:metabolism; D001692:Biological Transport; D002417:Cattle; D002478:Cells, Cultured; D018118:Chloride Channels / Q000096:biosynthesis / Q000235:genetics*; D002712:Chlorides / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004728:Endothelium, Corneal / Q000378:metabolism*; D005136:Eye Proteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis*; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D064606:SLC4A Proteins
D027321:Anion Transport Proteins; D017920:Antiporters; D001639:Bicarbonates; C505032:CFTR protein, human; C097503:CLC-5 chloride channel; D018118:Chloride Channels; D002712:Chlorides; C115376:ClC-3 channel; D017931:DNA Primers; D005136:Eye Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D064606:SLC4A Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator