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

Abstract

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-alpha) 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 x 10(-10) M) had no effect on Ca2+ release due to EGF, TGR-alpha or bradykinin but resulted in a rapid return of [Ca2+]i to baseline levels for EGF or TGF-alpha. Addition of the PKC inhibitor staurosporine (1 and 10 nM) 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 microM TPA produced the converse effect, namely prolonged Ca2+ entry following stimulation with EGF or TGF-alpha. 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-alpha resulted in Ca2+ release and Mn2+ entry. TPA but not the inactive phorbol ester, 4-alpha-phorbol-12,13-didecanoate, inhibited the Mn2+ influx. Thus, PKC is able to regulate Ca2+ entry due to EGF or TGF-alpha in this cell type. A431 cells treated with higher concentrations of TPA (5 x 10(-8) M) inhibited not only Ca2+ entry but also Ca2+ release due to EGF/TGF-alpha 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-alpha gave a single transient of [Ca2+]i, showing a common pool of Ca2+ for these agonists. In contrast, sequential addition of EGF (or TGF-alpha) and bradykinin resulted in two [Ca2+]i transients equal in size to those obtained with a single agonist.(ABSTRACT TRUNCATED AT 400 WORDS)

Aequorin-Based Functional Assays for G-Protein-Coupled Receptors, Ion Channels, and Tyrosine Kinase Receptors

“…As RTKs increase intracellular Ca ++ via the production of inositol phosphates and subsequent release of intracellular Ca ++ stores, the superiority of mitochondrial over cytoplasmic aequorin, as shown with GPCRs, is most probably also true for RTKs. The pharmacology of the three agonists and two antagonists tested was comparable to the one observed for these compounds when analyzed in a Ca ++ assay (Wheeler et al 1990) or in other functional assays (Marquardt et al 1984;Gazit et al 1989;Levitzki et al 1995). EGF's EC 50 varies according to the functional assay used; it is in the rank of order of 10 −10 to 10 −11 M when analyzed in cell proliferation assays (Marquardt et al 1984), and was described to be 1 nM to 3 nM in Fura-2 performed Ca ++ assays in COS cells (Takeuchi et al 2000) or in 3T3 fibroblasts (Pandiella et al 1988), or in aequorin-loaded 3T3 fibroblasts (Olsen et al 1988).…”

The Ras-Related Protein Cdc42Hs and Bradykinin Promote Formation of Peripheral Actin Microspikes and Filopodia in Swiss 3T3 Fibroblasts

“…We found that bradykinin but not PDGF, insulin, PMA, LPA, or bombesin treatment of cells resulted in morphological and cytoskeletal effects similar to that seen upon Cdc42Hs microinjection. Bradykinin is a mediator of inflammation and hypotension and is a mitogen, thought to act through bradykinin receptor-linked G proteins, which leads to activation of phospholipases C and D and release of internal calcium (9,48,50). Bombesin and LPA are, like bradykinin, thought to act through receptors linked to G proteins (7,49) but cause different morphological and cytoskeletal effects.…”

Manipulation of Intracellular Calcium in NCB‐20 Cells

“…In many excitable (Schlegel et al, 1987;Fasolato et al, 1988) and nonexcitable cells (Jackson et al, 1988;Putney, 1990;Wheeler et al, 1990), hormones induce a rapid transient increase in [Ca2+], followed by a sustained elevation. The persistent effect requires extracellular Ca2+, and is presumed to be due to the modulation of ion channel activity and PKC activation in excitable cells and the result ofa yet to be defined mechanism that may involve inositol polyphosphates in nonexcitable cells (Putney, 1990).…”

“…An additional means of determining whether Ca" influx contributes to the rise in [Ca2+], induced by BK is to assess the effect of BK on the rate of influx of Mn2+ into the cell (Wheeler et al, 1990). MnZ+, which quenches fura-2 fluorescence, enters certain cells, e.g., ' Cells were pretreated with PTX in six different experiments and BK was subsequently added in concentrations varying between 1 &and 1 pM, to accommodate the possibility that PTX would be effective only at submaximal or maximal concentrations of BK.…”