PKC signaling in CF/T43 cell line: regulation of NKCC1 by PKC-delta isotype.
Cystic fibrosis (CF) airway epithelial cells have a reduced mass of ether-linked diacylglycerols which might alter protein kinase C (PKC)-regulated Cl secretion. PKC regulation of basolateral Na-K-2Cl cotransport (NKCC1) was investigated in CF nasal polyp epithelial cells and a CF/T43 cell line to ascertain whether PKC signaling was altered in CF. NKCC1 was detected as bumetanide-sensitive (86)Rb influx. Methoxamine, a alpha(1)-adrenergic agonist, increased PKC activity in cytosol and a particulate fraction for a prolonged time period, as predicted from previous studies on the generation of diglycerides induced with methoxamine. Short-term stimulation of CF/T43 cells for 40 s promoted a shift in PKC-delta and -zeta to a particulate fraction, increased activity of immune complexes of cytosolic PKC-delta and of particulate PKC-zeta and increased activity of NKCC1. Pretreatment with antisense oligonucleotide to PKC-delta blocked methoxamine-stimulated PKC-delta activity, reduced PKC-delta mass by 61.4%, and prevented methoxamine-stimulated activity of NKCC1. Sense and missense oligonucleotide to PKC-delta and antisense oligonucleotide to PKC-zeta did not alter expression of PKC-delta or the effects of methoxamine. These results demonstrate that PKC-delta-dependent activation of NKCC1 is preserved in CF cells and suggest that regulation of NKCC1 is independent of low ether-linked diglyceride mass.
Liedtke
C M
CM
The Cystic Fibrosis Center, Departments of Pediatrics and Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106-4948, USA. cx17@po.cwru.edu
Cole
T S
TS
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D003550:Cystic Fibrosis / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008729:Methoxamine / Q000494:pharmacology; D009298:Nasal Polyps; D009841:Oligonucleotides / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D015398:Signal Transduction; D028021:Sodium-Potassium-Chloride Symporters; D014132:Trachea
D000316:Adrenergic alpha-Agonists; D002352:Carrier Proteins; D007527:Isoenzymes; D009841:Oligonucleotides; D028021:Sodium-Potassium-Chloride Symporters; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D008729:Methoxamine