Abstract
Regulation of the bradykinin-evoked increase in intracellular Ca2+ concentration by protein kinase C (PKC)-α 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-α 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-α 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-α in the receptor regulation. ISIS 9606 reduced PKC-α mRNA for ≥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 nmspecifically reduced the level of immunoreactive PKC-α protein by 66.3 ± 2.5% at 72 hr after treatment, without an effect on immunoreactive PKC-δ protein. This reduction in PKC-α 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-α is involved in kinin B2 receptor regulation by phorbol esters in A549 cells.
Footnotes
- Received July 31, 1996.
- Accepted October 30, 1996.
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Send reprint requests to: Dr. Stanley T. Crooke, Isis Pharmaceuticals, Department of Molecular Pharmacology, 2292 Faraday Avenue, Carlsbad, CA 92008. E-mail:dmusacchia{at}isisph.com
- The American Society for Pharmacology and Experimental Therapeutics
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