Depletion of protein kinase C-alpha by antisense oligonucleotides alters beta-adrenergic function and reverses the phorbol ester-induced reduction of isoproterenol-induced adenosine 3'-5'-cyclic monophosphate accumulation in murine Swiss 3T3 fibroblasts.
Beta-adrenergic agonists are well known to increase the activity of adenylate cyclase, yielding increases of the intracellular concentration of cAMP. It has been reported that activation of protein kinase C (PKC) by phorbol esters reduces the amplitude of isoproterenol-induced cAMP production in a 3T3-L1 cell line. In this study, we investigated whether PKC-alpha is involved in this process in murine Swiss 3T3 fibroblasts. A 20-mer phosphorothioate oligonucleotide designed to hybridize to the AUG initiation codon of the murine PKC-alpha mRNA, which contains 2'-O-methoxyethyl modifications incorporated into the 5' and 3' segments of the oligonucleotide, was used to assess the putative role of PKC-alpha in the beta-adrenergic receptor regulation. ISIS 14012 reduced PKC-alpha mRNA for over 72 hr after the initial treatment and the reduction was concentration dependent, whereas the mismatch control, ISIS 13818, had no effect. This depletion was found to be selective; ISIS 14012 had no effect on the mRNA expression of PKC-delta and PKC-zeta. ISIS 14012 reduced in a time and concentration-dependent fashion the levels of immunoreactive PKC-alpha protein by over 85% at 72 hr after treatment. Depletion of PKC-alpha inhibited the effect of isoproterenol-induced cAMP production by phorbol dibutyrate (PdBu). This finding is corroborated by the use of a nonspecific inhibitor of PKC, GF-109203x, which also prevented the effect of PdBu. Depletion of PKC-alpha by ISIS 14012 potentiated isoproterenol-induced cAMP production in cells untreated with PdBu. However, neither depletion of PKC-alpha nor PKC activation by a phorbol ester altered beta-adrenergic receptor affinity and density. PKC activation by PdBu did not alter forskolin-induced cAMP levels, but enhanced cAMP production by cholera toxin. PKC-alpha inhibition by ISIS 14012 had no effect on either cholera toxin-induced increases in cAMP or the acute effects of phorbol esters on cholera toxin in induction of cAMP. Thus, PKC-alpha appears to be involved in the regulation of beta-adrenergic receptor coupling to adenylate cyclase, possibly by phosphorylating the Gs protein, but other PKC isotypes must be involved in the effects observed when cells are treated with cholera toxin.
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Isis Pharmaceuticals 2292 Faraday Ave., Department of Molecular Pharmacology 2292 Faraday Ave., Carlsbad, California 92008, USA.
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D016475:3T3 Cells; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis*; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000502:physiology*; D007545:Isoproterenol / Q000494:pharmacology*; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D011943:Receptors, Adrenergic, beta / Q000032:analysis / Q000187:drug effects / Q000502:physiology*
D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011943:Receptors, Adrenergic, beta; D005576:Colforsin; D015240:Phorbol 12,13-Dibutyrate; D002772:Cholera Toxin; D000242:Cyclic AMP; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D007545:Isoproterenol; C070515:bisindolylmaleimide I