Corneal endothelial NKCC: molecular identification, location, and contribution to fluid transport.
Although Na(+)-K(+)-2Cl(-) cotransport has been demonstrated in cultured bovine corneal endothelial cells, its presence and role in the native tissue have been disputed. Using RT-PCR we have now identified a partial clone of the cotransporter protein in freshly dissected as well as in cultured corneal endothelial and epithelial cells. The deduced amino acid sequence of this protein segment is 99% identical to that of the bovine isoform (bNKCC1). [(3)H]bumetanide binding shows that the cotransporter sites are located in the basolateral membrane region at a density of 1.6 pmol/mg of protein, close to that in lung epithelium. Immunocytochemistry confirms the basolateral location of the cotransporter. We calculate the turnover rate of the cotransporter to be 83 s(-1). Transendothelial fluid transport, determined from deepithelialized rabbit corneal thickness measurements, is partially inhibited (30%) by bumetanide in a dose-dependent manner. Our results demonstrate that Na(+)-K(+)-2Cl(-) cotransporters are present in the basolateral domain of freshly dissected bovine corneal endothelial cells and contribute to fluid transport across corneal endothelial preparations.
Kuang
K
K
Department of Ophthalmology, Columbia University, New York 10032, USA.
Li
Y
Y
Wen
Q
Q
Wang
Z
Z
Li
J
J
Yang
Y
Y
Iserovich
P
P
Reinach
P S
PS
Sparrow
J
J
Diecke
F P
FP
Fischbarg
J
J
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D001826:Body Fluids / Q000378:metabolism*; D002034:Bumetanide / Q000378:metabolism / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics* / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003315:Cornea / Q000033:anatomy & histology / Q000187:drug effects / Q000378:metabolism; D004728:Endothelium, Corneal / Q000378:metabolism*; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D028021:Sodium-Potassium-Chloride Symporters; D014018:Tissue Distribution
D002352:Carrier Proteins; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide