Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways
Calcium signaling systems in nonexcitable cells involve activation of Ca2+ entry across the plasma membrane and release from intracellular stores as well as activation of Ca2+ pumps and inhibition of passive Ca2+ pathways to ensure exact regulation of free cytosolic Ca2+ concentration ([Ca2+] i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca2+ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α) both stimulated Ca2+ entry and release while bradykinin appeared only to release Ca2+ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca2+] i response to these agonists was examined by four methods. Low concentrations of TPA (2×10−10m) had no effect on Ca2+ release due to EGF, TGR-α or bradykinin but resulted in a rapid return of [Ca2+] i to baseline levels for EGF or TGF-α. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca2+] i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 μm TPA produced the converse effect, namely prolonged Ca2+ entry following stimulation with EGF or TGF-α. To show that one effect of TPA was on Ca2+ entry, fura-2 loaded cells were suspended in Mn2+ rather than Ca2+ buffers. Addition of EGF or TGF-α resulted in Ca2+ release and Mn2+ entry. TPA but not the inactive phorbol ester, 4-α-phorbol-12,13-didecanoate, inhibited the Mn2+ influx. Thus, PKC is able to regulate Ca2+ entry due to EGF or TGF-α in this cell type. A431 cells treated with higher concentrations of TPA (5×10−8m) inhibited not only Ca2+ entry but also Ca2+ release due to EGF/TGF-α but had no effect on bradykinin-mediated Ca2+ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF-α gave a single transient of [Ca2+] i , showing a common pool of Ca2+ for these agonists. In contrast, sequential addition of EGF (or TGF-α) and bradykinin resulted in two [Ca2+] i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca2+ stores, whereas ionomycin following either EGF (or TGF-α) or bradykinin gave an elevation of the [Ca2+] i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca2+ for EGF-mediated Ca2+ release which also respond differently to TPA.
Key Wordsprotein kinase C cytosolic Ca2+ Ca2+ entry A431 cells
Unable to display preview. Download preview PDF.
- 2.Brown, A., Birnbaumer, L. 1988. Direct G protein gating of ion channels.Am. J. Physiol. 23:H401-H410Google Scholar
- 9.Hepler, J., Nakahata, N., Lovenberg, T., DiGuiseppi, J., Jerman, B., Earp, H., Harden, T. 1987. Epidermal growth factor stimulates the rapid accumulation of inositol (1,4,5)-triphosphate and a rise in cytosolic calcium mobilized from intracellular stores in A431 cells.J. Biol. Chem. 262:2951–2956PubMedGoogle Scholar
- 15.Molski, T.-F.P., Mege, J.-L., Tao, J., Gomez-Cambronero, J., Huang, C.-K., Becker, E.L., Sha'afi, R.I. 1988. Diacylglycerol kinase inhibitor, R59022, and stimulated neutrophil responses.FASEB J. 2:6778 (Abstr.)Google Scholar
- 23.Pike, L.J., Marquardt, H., Todaro, G.J., Gallis, B., Casnellie, J.E., Bornstein, P., Krebs, E.G. 1983. Transforming growth factor and epidermal growth factor stimulate the phosphorylation of a synthetic, tyrosine-containing peptide in a similar manner.J. Biol. Chem. 257:14628–14631Google Scholar
- 27.Sekar, M.C., Hokin, L.E. 1986. The role of phosphoinositides in signal transduction.J. Membrane Biol. 89:193–210Google Scholar
- 29.Smith, K., Losonzcy, I., Sahai, A., Pannerselvam, M., Fehnel, P., Salomon, D. 1983. Effect of 12-0-tetradecanoylphobol-13-acetate (TPA) on the growth inhibitory and increased phosphatidylinositol (PI) responses induced by epidermal growth factor (EGF) in A431 cells.J. Cell Physiol. 117: 91–100CrossRefPubMedGoogle Scholar