title: Modulation of Na-K-2Cl cotransport by intracellular Cl(-) and protein kinase C-delta in Calu-3 cells.
abstract: In this study, we tested the hypothesis that intracellular Cl(-) (Cl) 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 and increased or decreased intracellular volume, respectively, without changing Cl 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 from 20 to 80 mM Cl then increased at 140-200 mM Cl 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 electrochemical gradient alone is not sufficient to stimulate NKCC1 activity; rather, elevated activity of PKC-delta is necessary. Further, high Cl levels induce a subcellular redistribution of PKC-delta, which results in increased enzyme activity.
authors.affiliation: W. A. Bernbaum Center for Cystic Fibrosis Research, Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio 44106-4948, USA. cxl7@po.cwru.edu
authors.forename: Carole M
authors.initials: CM
authors.lastname: Liedtke
authors.forename: Robert
authors.initials: R
authors.lastname: Papay
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Cole
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D001578:Benzopyrans / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002034:Bumetanide / Q000494:pharmacology; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000378:metabolism; D004232:Diuretics / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008729:Methoxamine / Q000494:pharmacology; D009997:Osmotic Pressure; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051745:Protein Kinase C-delta; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2
chemical_list: D000098:Acetophenones; D000316:Adrenergic alpha-Agonists; D001578:Benzopyrans; D002712:Chlorides; D004232:Diuretics; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011942:Receptors, Adrenergic, alpha; C578877:SLC12A2 protein, human; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D002034:Bumetanide; C085746:rottlerin; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D008729:Methoxamine