The Journal of Membrane Biology

, Volume 118, Issue 1, pp 77–91 | Cite as

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways

  • Larry A. Wheeler
  • Danon D. Goodrum
  • George Sachs
Articles

Summary

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 Words

protein kinase C cytosolic Ca2+ Ca2+ entry A431 cells 

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Copyright information

© Springer-Verlag New York Inc 1990

Authors and Affiliations

  • Larry A. Wheeler
    • 1
  • Danon D. Goodrum
    • 1
  • George Sachs
    • 2
  1. 1.Department of Biological SciencesDiscovery Research Allergan, Inc./Herbert LabsIrvine
  2. 2.CUREWadsworth VA HospitalLos Angeles

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