SODIUM, POTASSIUM, 2 CHLORIDE COTRANSPORT IN CORNEAL ENDOTHELIUM - CHARACTERIZATION AND POSSIBLE ROLE IN VOLUME REGULATION AND FLUID TRANSPORT

PURPOSE. To search for membrane transporter proteins that could contri bute to volume regulation and fluid transport by corneal endothelium. As an initial step, the authors have focused an Na+-K+-2Cl(-) cotransp orters. METHODS. Bovine corneal endothelial cells were cultured to con fluence. (86)Rubidium was used as a tracer for K+ uptake determination s; uptake values were normalized per milligram of cell protein. RESULT S. Three components of K+ uptake were characterized: ouabain (1 mM) se nsitive, bumetanide (0.1 mM) sensitive, and ouabain-bumetanide insensi tive. Both the ouabain-scnsitive and bumetanide-sensitive components i ncreased in the presence of 26.2 mM HCO3-; 0.5 mM 4,4'-diisothiocyanat o-stilbene-2,2'-disulfonic acid abolished this increase, The bumetanid e-sensitive component was completely inhibited in the absence of Na+ o r Cl-. This component was increased 33% by a 33% hypertonic solution a nd was decreased 38% by a 33% hypotonic solution. The protein kinase C activator phorbol 12-myristate 13-acetate decreased the activity of t he cotransporter, whereas forskolin, in the presence of isobutylmethyl xanthine, decreased it. Calyculin A (100 nM), an inhibitor of phosphat ases 1 and 2a, produced a large (97%) activation of this component. CO NCLUSIONS. These results provided for the first time conclusive eviden ce for the presence of a Na+-K+-2Cl(-) cotransporter in corneal endoth elium and of its possible involvement in volume-regulatory processes i n these cells. Given the uptake values reported here, such cotransport er could contribute significantly to electrolyte transport and hence t o fluid transport across this preparation.