Antisense oligodeoxynucleotide to PKC-delta blocks alpha 1-adrenergic activation of Na-K-2Cl cotransport.
A role for protein kinase C (PKC)-delta and -zeta isotypes in alpha 1-adrenergic regulation of human tracheal epithelial Na-K-2Cl cotransport was studied with the use of isotype-specific PKC inhibitors and antisense oligodeoxy-nucleotides to PKC-delta or -zeta mRNA. Rottlerin, a PKC-delta inhibitor, blocked 72% of basolateral-to-apical, bumetanide-sensitive 36Cl flux in nystatin-permeabilized cell monolayers stimulated with methoxamine, an alpha 1-adrenergic agonist, with a 50% inhibitory concentration of 2.3 microM. Methoxamine increased PKC activity in cytosol and a particulate fraction; the response was insensitive to PKC-alpha and -beta II isotype-specific inhibitors, but was blocked by general PKC inhibitors and rottlerin. Rottlerin also inhibited methoxamine-induced PKC activity in immune complexes of PKC-delta, but not PKC-zeta. At the subcellular level, methoxamine selectively elevated cytosolic PKC-delta activity and particulate PKC-zeta activity. Pretreatment of cell monolayers with antisense oligodeoxynucleotide to PKC-delta for 48 h reduced the amount of whole cell and cytosolic PKC-delta, diminished whole cell and cytosolic PKC-delta activity, and blocked methoxamine-stimulated Na-K-2Cl cotransport. Sense oligodeoxynucleotide to PKC-delta and antisense oligodeoxynucleotide to PKC-zeta did not alter methoxamine-induced cotransport activity. These results demonstrate the selective activation of Na-K-2Cl cotransport by cytosolic PKC-delta.
Liedtke
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CM
Cystic Fibrosis Center, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Cole
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D000098:Acetophenones / Q000494:pharmacology; D001483:Base Sequence; D001578:Benzopyrans / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002034:Bumetanide / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D008729:Methoxamine / Q000494:pharmacology; D009092:Mucous Membrane / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D009281:Naphthalenes / Q000494:pharmacology; D009761:Nystatin / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000235:genetics; D051745:Protein Kinase C-delta; D012333:RNA, Messenger / Q000096:biosynthesis; D018340:Receptors, Adrenergic, alpha-1 / Q000187:drug effects / Q000502:physiology*; D028021:Sodium-Potassium-Chloride Symporters; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D014132:Trachea / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects*; D000099221:Protein Kinase C zeta
D000098:Acetophenones; D001578:Benzopyrans; D002034:Bumetanide; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002352:Carrier Proteins; D002712:Chlorides; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008729:Methoxamine; D009281:Naphthalenes; D009761:Nystatin; D016376:Oligonucleotides, Antisense; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D012333:RNA, Messenger; D018340:Receptors, Adrenergic, alpha-1; D028021:Sodium-Potassium-Chloride Symporters; D019311:Staurosporine; D000099221:Protein Kinase C zeta; C061465:calphostin complex; C085746:rottlerin; C497258:PRKCD protein, human; C059539:midostaurin