POTASSIUM-TRANSPORT IN NONPIGMENTED EPITHELIAL-CELLS OF OCULAR CILIARY BODY - INHIBITION OF A NA+, K+, CL- COTRANSPORTER BY PROTEIN-KINASE-C

The mechanisms by which Rb-86+ (used as a tracer for K+) enters human nonpigmented ciliary epithelial cells were investigated. Ouabain-inhibitable bumetanide-insensitive Rb-86+ transport accounted for approximately 70-80% of total, whereas bumetanide-inhibitable ouabain-insensitive uptake accounted for 15-25% of total. K+ channel blockers such as BaCl2 reduced uptake by approximately 5%. Bumetanide inhibited Rb-86+ uptake with an IC50 of 0.5-mu-M, while furosemide inhibited with an of about 20-mu-M. Bumetanide-inhibitable Rb-86+ uptake was reduced in Na+-free or Cl--free media, suggesting that Na+ and Cl- were required for optimal uptake via this mechanism. These characteristics are consistent with a Na+, K+, Cl- cotransporter in NPE cells. Treatment of NPE cells for 15 min with phorbol 12-myristate, 13-acetate (PMA), an activator of protein kinase C, caused a 50-70% decrease in Rb-86+ uptake via the Na+, K+, Cl- cotransporter. Other Rb-86+ uptake mechanisms were not affected. Rb-86+ uptake via the Na+, K+, Cl- cotransporter could be inhibited by other phorbol esters and by dioctanoylglycerol, an annlog of diacylglycerol, but not by 4-alpha-phorbol didecanoate, an ineffective activator of protein kinase C. Staurosporine, a protein kinase C inhibitor, blocked phorbol ester inhibition of Rb-86+ uptake. These data suggest that a Na+ K+, Cl- cotransporter in NPE cells is inhibited by activation of protein kinase C.