Regulation of Na–K–2Cl cotransport in cultured bovine corneal endothelial cells

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We have previously demonstrated the presence of a Na+–K+–2Cl cotransporter in cultured bovine corneal endothelial cells (CBCEC) and determined that this cotransporter is located in the basolateral membrane. This transporter may contribute to volume regulation and transendothelial fluid transport. We have now investigated factors regulating the activity of the cotransporter. This activity was assessed by measuring the bumetanide-sensitive 86Rubidium (86Rb) uptake in 86Rb-containing solutions. Data were normalized to protein content determined with a Lowry protein assay. We investigated the regulation by extracellular and intracellular ion concentrations, by osmotic gradients, and by second messengers. Our results indicate that extracellular Na+ and K+ each are required for activation of the cotransporter and activate with first-order kinetics at half-maximally effective concentrations (k1/2) of 21·1 and 1·33 mm, respectively. Extracellular Cl is also required for cotransport activation, but shows higher order kinetics; the k1/2 for Cl is 28·1 mm and the Hill coefficient 2·1. Symbol exerts a modulating effect on cotransporter activity; at 0 Symbol the bumetanide-sensitive K+ uptake is reduced by 30% compared to that at 26 mm Symbol. Manipulations of the intracellular [Cl] by preincubation in Cl-free solution or inhibition of Cl efflux resulted in increased uptake at low [Cl]i and decreased uptake at high [Cl]i. To assess the role of protein kinases in the regulation of cotransport, we have determined the effect of protein kinase inhibitors. H-89 and KT5270, inhibitors of PKA, inhibit cotransport almost completely, while calphostin C, an inhibitor of PKC, produces a small activation of cotransport. The tyrosine kinase inhibitor genistein reduced K+ uptake while its inactive analog daidzein was without effect. The calmodulin kinase inhibitor KN-93 was without effect. We also investigated the effects of phosphatase inhibitors. Calyculin A (k1/2=21 nm) and okadaic acid (k1/2=915 nm) produced approximate doubling of K+ uptake, suggesting that phosphatase 1 is dominant. We also investigated the role of the cytoskeleton and its activation. Reduction of Symbol by preincubation in Ca2+-free medium as well as by exposure to W-7, an inhibitor of the binding of Ca2+ to calmodulin, reduced K+ uptake. Consistent with this, ML-7, a relatively specific inhibitor of the Ca2+–calmodulin activated myosin light chain kinase, inhibited cotransport by 40%. The Ca2+–calmodulin activated myosin light chain kinase contributes to the modulation of the cytoskeleton by regulating the actin–myosin interaction. Consistent with the above, disruption of the actin polymerization by cytochalasin D led to a decrease in K+ uptake. We conclude that extracellular Na+, K+ and Cl are requirements for the function of the CBCEC Na+–K+–2Cl cotransporter, while intracellular Cl and extracellular Symbol modulate its activity. Several protein kinases, including PKA, PKC, tyrosine kinase, and myosin light chain kinase, modulate the K+ uptake. Another modulating pathway for cotransport involves the state of the cytoskeleton.

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