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Sergey Jelamskii, Xing Cai Sun, Peter Herse, Joseph A. Bonanno; Basolateral Na+-K+-2Cl− Cotransport in Cultured and Fresh Bovine Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2000;41(2):488-495.
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purpose. To examine whether Na+-K+-2Cl− cotransport has the potential to contribute to corneal endothelial ion
and fluid transport in cultured and fresh bovine corneal endothelial
methods. Cl− and Na+ sensitive fluorescent dyes were
used to measure furosemide-dependent ion fluxes in cultured and fresh
endothelial cells. Immunoblot analysis and immunofluorescence were used
to determine expression and location of the
Na+-K+-2Cl− cotransporter (NKCC1).
results. Application of furosemide (50–100 μM) reduced Cl− and
Na+ influx in approximately 50% of trials using cultured
cells and only 10% of trials with fresh cells; however, in all cases
pretreatment with furosemide slowed Cl− efflux when cells
were bathed in Cl−-free Ringer’s. Double-sided perfusion
of cultured cells indicated that furosemide-sensitive Cl− fluxes were located on the basolateral side. Immunoblot analysis
revealed 174-kDa bands in both fresh and cultured cells, but the bands
were denser in fresh endothelial cells. Immunofluorescence showed
distinct lateral membrane staining in addition to significant amounts
of perinuclear staining.
conclusions. The Na+-K+-2Cl− cotransporter is
present in both fresh and cultured bovine corneal endothelium, and the
expression is apparently higher in the fresh cells. The cotransporter
is present on the lateral membrane consistent with a role in loading
endothelial cells with Cl−, thereby possibly contributing
to a transendothelial Cl− flux. However, in the resting
cell, net flux through the transporter is often not
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