ANTISENSE OLIGONUCLEOTIDES TARGETING HUMAN PROTEIN-KINASE C-ALPHA INHIBIT PHORBOL ESTER-INDUCED REDUCTION OF BRADYKININ-EVOKED CALCIUM MOBILIZATION IN A549 CELLS

Regulation of the bradykinin-evoked increase in intracellular Ca2+ con centration by protein kinase C (PKC)-alpha was investigated in A549 hu man lung carcinoma cells. Bradykinin, a potent and selective kinin B-2 receptor agonist, induces calcium mobilization in a concentration-dep endent 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 P KC-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 greater than or equal to 72 hr after the in itial treatment and the reduction was concentration dependent, whereas the mismatch control, ISIS 13009, had no effect. Concentrations of IS IS 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 e ffect 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 staur osporine, a nonselective PKC inhibitor, that prevented the effect of T PA. These results suggest that PKC-alpha is involved in kinin B-2 rece ptor regulation by phorbol esters in A549 cells.