The role of protein phosphatases in the regulation of Na-K-Cl cotransport was 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 immunologically detected. The protein phosphatase inhibitor calyculin A stimulated Na-K-Cl cotransport by 89 +/- 12% at 10 n M, whereas okadaic acid had no effect at concentrations less than 100 n M. Calyculin A had no significant effect on either Na-K ATPase or ouabain-insensitive, bumetanide-insensitive 86Rb+uptake. These data suggest that PP1 plays a role in the inhibition of Na-K-Cl cotransport in PE cells. Treatment of cells with phorbol 12-myristate, 13-acetate (PMA), a protein kinase C (PKC) activator caused an 82% inhibition of Na-K-Cl cotransport. When cells were first treated for 5 min with PMA, 10 n M calyculin A stimulated Na-K-Cl cotransport by 53% compared to 101% 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 cotransport activity. These data suggest that maximal inhibition of Na-K-Cl cotransport by PKC requires PP1 activity, but that a part of PKCs inhibitory effect is independent of PP1. The effect of PKC activation on PP1 was further examined by determining PP1 activity in cells pretreated with PMA. PP1 activity increased 38+/-8% in cells exposed to 1 microM PMA for 5 min. This stimulation was blocked by 100 n M staurosporine or 1 microM bisindolylmaleimide, two PKC inhibitors. An isomer which does not activate PKC (4 alpha phorbol 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 PP1 and PKA function in a kinase-phosphatase regulatory loop. To determine whether other protein kinases might also be involved, several protein kinase inhibitors were tested, including KT5823 (protein kinase G, type II-specific), KN62 (calmodulin activated kinase-specific) and ML7 (myosin light chain kinase-specific). None prevented activation of Na-K-Cl cotransport by calyculin A, suggesting that these kinases are not involved in the activation of Na-K-Cl cotransport.