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

PURPOSE. To examine whether Na+-K+-2Cl(-) cotransport has the potential to contribute to corneal endothelial ion and fluid transport in cultured and f resh bovine corneal endothelial cells. METHODS. Cl- and Na+ sensitive fluorescent dyes were used to measure furose mide-dependent ion fluxes in cultured and fresh endothelial cells. Immunobl ot analysis and immunofluorescence were used to determine expression and lo cation of the Na+-K+-2Cl(-) cotransporter (NKCC1). RESULTS. Application of furosemide (50-100 mu M) 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 slowe d Cl- efflux when cells were bathed in Cl--free Ringer's. Double-sided perf usion of cultured cells indicated that furosemide-sensitive Cl- fluxes were located on the basolateral side. Immunoblot analysis revealed 174-kDa band s in both fresh and cultured cells, but the bands were denser in fresh endo thelial 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 c ultured bovine corneal endothelium, and the expression is apparently higher in the fresh cells. The cotransporter is present on the lateral membrane c onsistent with a rule in loading endothelial cells with Cl-, thereby possib ly contributing to a transendothelial Cl- flux. However, in the resting cel l, net flux through the transporter is often not apparent.