Protein Kinase C Regulation of a Na +, K +, Cl - Cotransporter in Fetal Human Pigmented Ciliary Epithelial Cells

Abstract

The epithelium of the ciliary body is the site of aqueous humor secretion in the eye. We have begun to study ion transport in this tissue by investigating the mechanisms of K +-transport in fetal human pigmented ciliary epithelial (PE) cells using 86Rb + as a tracer for K +. In PE three mechanisms were found: (1) ouabain-sensitive, bumetanide-insensitive 86Rb + uptake (∼ 70% of total): (2) bumetanide-sensitive, ouabain-insensitive 86Rb + uptake (∼ 15% of total): and (3) K + channel blocker-sensitive 86 Rb + uptake (∼ 15% of total). Evidence that the ouabain-insensitive component of 86Rb + uptake includes a Na +, K +, Cl - cotransporter is the following: (1) bumetanide inhibited 86Rb+ uptake with an IC 50 of 0·6 μM and (2) bumetanide-sensitive 86Rb + uptake was substantially reduced in media lacking Na + or Cl -, suggesting that both extracellular Na + and Cl - are required for optimal 86Rb + uptake via this mechanism. Treatment of cells for 15 min with phorbol 12-myristate, 13-acetate (PMA) caused a dose-dependent inhibition of bumetanide-sensitive 86 Rb + uptake. No other 86Rb + uptake mechanism was affected. Efflux studies revealed that efflux via the bumetanide-sensitive mechanism was likewise inhibited by PMA. Treatment with other phorbol esters or an analog of diacylglycerol inhibited bumetanide-sensitive 86Rb + uptake, while the protein kinase C inhibitor staurosporine blocked inhibition by phorbol esters. These data suggest that a Na +, K +, Cl - cotransporter in human PE cells is inhibited by activation of protein kinase C.