Basolateral Na(+)-K(+)-2Cl(-) cotransport in cultured and fresh bovine corneal endothelium

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 cells. 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 microM) 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 apparent.