Down-regulation of Na-K-Cl cotransport by protein kinase C is mediated by protein phosphatase 1 in pigmented ciliary epithelial cells

The role of protein phosphatases in the regulation of Na-K-Cl cotransport w as examined in human pigmented ciliary epithelial (PE) cells. Both a 37 kDa Form and a 72 kDa form of protein phosphatase 1 (PP1) could be immunologic ally detected. The protein phosphatase inhibitor calyculin A stimulated Na- K-Cl cotransport by 89 +/- 12 % at 10 nM, whereas okadaic acid had no effec t at concentrations less than 100 nM. Calyculin A had no significant effect on either Na-K ATP ase or ouabain-insensitive, bumetanide-insensitive Rb-8 6(+) uptake. These data suggest that PP1 plays a role in the inhibition of Na-K-Cl cotransport in FE cells. Treatment of cells with phorbol 12-myrista te, 13-acetate (PMA), a protein kinase C (PKC) activator caused an 82% inhi bition of Na-K-Cl cotransport. When cells were first treated for 5 min with PMR, 10 nM calyculin A stimulated Na-K-CI cotransport by 53% compared to 1 01% by calyculin A alone. Treatment of cells with PMA after stimulation of Na-K-Cl cotransport by calyculin A resulted in a prompt 56% drop in cotrans port activity. These data suggest that maximal inhibition of Na-K-CI cotran sport by PKU requires PP 1 activity, but that a part of PKCs inhibitory eff ect is independent of PPI. The effect of PE;C activation on Prl was further examined by determining PP1 activity in cells pretreated with PMA. PP1 act ivity increased 38 +/- 8 %, in cells exposed to 1 ph;I PMA For 5 min. This stimulation was blocked by 100 nM staurosporine or 1 muM bisindolylmaleimid e, two PI(C inhibitors. An isomer which does not activate PKC (4 alpha phor bol didecanoate), did not stimulate PP1 activity, Thus PKC activation leads to an increase in PP1 activity in PE cells. Pretreatment of cells with the protein kinase A (PKA) inhibitor PHI 14-22 resulted in a partial reduction in calyculin A stimulation of cotransport, suggesting that Prl and PMA fun ction in a kinase-phosphatase regulatory loop. To determine whether other p rotein kinases might also be involved, several protein kinase inhibitors we re tested, including KT5823 (protein kinase G, type II-specific), KN62 (cal modulin activated kinase-specific) and ML7 (myosin light chain kinase-speci fic). None prevented activation of Na-K-Cl cotransport by calyculin A, sugg esting that these kinases are not involved in the activation of Na-K-Cl Cl transport. (C) 2001 Academic Press.