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Am J Physiol Cell Physiol 280: C491-C499, 2001;
0363-6143/01 $5.00
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Vol. 280, Issue 3, C491-C499, March 2001

Corneal endothelial NKCC: molecular identification, location, and contribution to fluid transport

Kunyan Kuang1, Yansui Li1, Quan Wen1, Zheng Wang2, Jun Li1, Yingqing Yang1, Pavel Iserovich1, Peter S. Reinach2, Janet Sparrow3, Friedrich P. J. Diecke4, and Jorge Fischbarg1,5

1 Departments of Ophthalmology, 5 Physiology and Cellular Biophysics, and 3 Anatomy and Cell Biology, Columbia University, New York 10032; 4 Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07103; and 2 Department of Biological Sciences, State University of New York-Optometry, New York, New York 10010

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). [3H]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.

bumetanide binding; epithelial polarization; stromal thickness; specular microscopy; turnover rate; sodium-potassium-two chloride cotransporter


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