title: Antisense oligonucleotides targeting human protein kinase C-alpha inhibit phorbol ester-induced reduction of bradykinin-evoked calcium mobilization in A549 cells.
abstract: Regulation of the bradykinin-evoked increase in intracellular Ca2+ concentration by protein kinase C (PKC)-alpha was investigated in A549 human lung carcinoma cells. Bradykinin, a potent and selective kinin B2 receptor agonist, induces calcium mobilization in a concentration-dependent fashion in this cell line. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, is known to reduce the amplitude of agonist-induced calcium mobilization in various cell lines. Because PKC-alpha is a major PKC isozyme in A549 cells, we investigated whether this isozyme plays a role in this process. A 20-mer phosphorothioate oligonucleotide targeting the 3'-untranslated region of the human PKC-alpha mRNA, which contains 2'-methoxyethyl modifications incorporated into the 5' and 3' segments of the oligonucleotide, was used to assess the putative role of PKC-alpha in the receptor regulation. ISIS 9606 reduced PKC-alpha mRNA for > or = 72 hr after the initial treatment and the reduction was concentration dependent, whereas the mismatch control, ISIS 13009, had no effect. Concentrations of ISIS 9606 of 150 nM specifically reduced the level of immunoreactive PKC-alpha protein by 66.3 +/- 2.5% at 72 hr after treatment, without an effect on immunoreactive PKC-delta protein. This reduction in PKC-alpha was sufficient to inhibit the reduction of bradykinin-induced calcium mobilization by TPA. This finding is corroborated by the use of staurosporine, a nonselective PKC inhibitor, that prevented the effect of TPA. These results suggest that PKC-alpha is involved in kinin B2 receptor regulation by phorbol esters in A549 cells.
authors.affiliation: Isis Pharmaceuticals, Department of Molecular Pharmacology, Carlsbad, California 92008, USA.
authors.forename: L
authors.initials: L
authors.lastname: Levesque
authors.forename: N M
authors.initials: NM
authors.lastname: Dean
authors.forename: H
authors.initials: H
authors.lastname: Sasmor
authors.forename: S T
authors.initials: ST
authors.lastname: Crooke
mesh_terms: D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002277:Carcinoma / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008168:Lung / Q000187:drug effects*; D008175:Lung Neoplasms / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010703:Phorbol Esters / Q000494:pharmacology*; D011493:Protein Kinase C / Q000187:drug effects*; D014407:Tumor Cells, Cultured
chemical_list: D016376:Oligonucleotides, Antisense; D010703:Phorbol Esters; D011493:Protein Kinase C; D001920:Bradykinin; D002118:Calcium