Antisense oligonucleotide to PKC-epsilon alters cAMP-dependent stimulation of CFTR in Calu-3 cells.
Protein kinase C (PKC) regulates cystic fibrosis transmembrane conductance regulator (CFTR) channel activity but the PKC signaling mechanism is not yet known. The goal of these studies was to identify PKC isotype(s) required for control of CFTR function. CFTR activity was measured as 36Cl efflux in a Chinese hamster ovary cell line stably expressing wild-type CFTR (CHO-wtCFTR) and in a Calu-3 cell line. Chelerythrine, a PKC inhibitor, delayed increased CFTR activity induced with phorbol 12-myristate 13-acetate or with the cAMP-generating agents (-)-epinephrine or forskolin plus 8-(4-chlorophenylthio)adenosine 3',5'- cyclic monophosphate. Immunoblot analysis of Calu-3 cells revealed that PKC-alpha, -betaII, -delta, -epsilon, and -zeta were expressed in confluent cell cultures. Pretreatment of cell monolayers with Lipofectin plus antisense oligonucleotide to PKC-epsilon for 48 h prevented stimulation of CFTR with (-)-epinephrine, reduced PKC-epsilon activity in unstimulated cells by 52.1%, and decreased PKC-epsilon mass by 76.1% but did not affect hormone-activated protein kinase A activity. Sense oligonucleotide to PKC-epsilon and antisense oligonucleotide to PKC-delta and -zeta did not alter (-)-epinephrine-stimulated CFTR activity. These results demonstrate the selective regulation of CFTR function by constitutively active PKC-epsilon.
Liedtke
C M
CM
Cystic Fibrosis Center and Departments of Pediatrics and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Cole
T S
TS
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects / Q000502:physiology*; D004837:Epinephrine / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D011493:Protein Kinase C / Q000096:biosynthesis / Q000235:genetics*; D051744:Protein Kinase C-epsilon; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection
D002712:Chlorides; D007527:Isoenzymes; D020319:Oligodeoxyribonucleotides, Antisense; D011994:Recombinant Proteins; D013873:Thionucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; C497256:Prkce protein, mouse; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate; D004837:Epinephrine