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Am J Physiol Lung Cell Mol Physiol 282: L1151-L1159, 2002. First published December 14, 2001; doi:10.1152/ajplung.00143.2001
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Vol. 282, Issue 5, L1151-L1159, May 2002

Modulation of Na-K-2Cl cotransport by intracellular Clminus and protein kinase C-delta in Calu-3 cells

Carole M. Liedtke, Robert Papay, and Thomas S. Cole

W. A. Bernbaum Center for Cystic Fibrosis Research, Departments of Pediatrics and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4948

In this study, we tested the hypothesis that intracellular Cl- (Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>) regulates the activity of protein kinase C (PKC)-delta and thus the activation of Na-K-Cl cotransport (NKCC1) in a Calu-3 cell line. The alpha 1-adrenergic agonist methoxamine (MOX) and hypertonic sucrose increased Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and increased or decreased intracellular volume, respectively, without changing Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> concentration ([Cl-]i). Titration of [Cl-]i from 20-140 mM in nystatin-permeabilized cell monolayers did not affect the baseline activity of PKC-delta , PKC-zeta , or rottlerin-sensitive NKCC1. At 200 mM Cl-, rottlerin-sensitive NKCC1 was activated, and PKC isotypes were localized predominantly to a particulate fraction. MOX induced a biphasic increase in NKCC1 activity and PKC-delta in activity and particulate localization of PKC-delta and -zeta . Activity of NKCC1 and PKC-delta decreased with increasing Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> from 20 to 80 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> then increased at 140-200 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> apparently as an additive effect to high [Cl-]i levels. Rottlerin inhibited the effects of MOX, which indicates that PKC-delta was required for activation of NKCC1. The results indicate that, in airway epithelial cells, a Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> electrochemical gradient alone is not sufficient to stimulate NKCC1 activity; rather, elevated activity of PKC-delta is necessary. Further, high Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> levels induce a subcellular redistribution of PKC-delta , which results in increased enzyme activity.

alpha -adrenergic; methoxamine; volume; hyperosmotic stress; bumetanide; shrinkage; cystic fibrosis; rottlerin


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