Sodium, potassium, two chloride cotransport in corneal endothelium: characterization and possible role in volume regulation and fluid transport.

PURPOSE

To search for membrane transporter proteins that could contribute to volume regulation and fluid transport by corneal endothelium. As an initial step, the authors have focused on Na+-K+-2Cl- cotransporters.

METHODS

Bovine corneal endothelial cells were cultured to confluence. 86Rubidium was used as a tracer for K+ uptake determinations; uptake values were normalized per milligram of cell protein.

RESULTS

Three components of K+ uptake were characterized: ouabain (1 mM) sensitive, bumetanide (0.1 mM) sensitive, and ouabain-bumetanide insensitive. Both the ouabain-sensitive and bumetanide-sensitive components increased in the presence of 26.2 mM HCO3-; 0.5 mM 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid abolished this increase. The bumetanide-sensitive component was completely inhibited in the absence of Na+ or Cl-. This component was increased 33% by a 33% hypertonic solution and was decreased 38% by a 33% hypotonic solution. The protein kinase C activator phorbol 12-myristate 13-acetate decreased the activity of the cotransporter, whereas forskolin, in the presence of isobutylmethylxanthine, decreased it. Calyculin A (100 nM), an inhibitor of phosphatases 1 and 2a, produced a large (97%) activation of this component.

CONCLUSIONS

These results provided for the first time conclusive evidence for the presence of a Na+-K+-2Cl- cotransporter in corneal endothelium and of its possible involvement in volume-regulatory processes in these cells. Given the uptake values reported here, such cotransporter could contribute significantly to electrolyte transport and hence to fluid transport across this preparation.