Differential Effects of the Protein Kinase C Activator Phorbol 12-Myristate 13-Acetate on Calcium Responses and Secretion in Adherent and Suspended RBL-2H3 Mucosal Mast Cells
1996; Elsevier BV; Volume: 271; Issue: 12 Linguagem: Inglês
10.1074/jbc.271.12.6658
ISSN1083-351X
AutoresPatricia C. Wolfe, En‐Yuh Chang, Juan Rivera, Clare Fewtrell,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoAdhesion of RBL-2H3 mucosal mast cells to fibronectin-coated surfaces has been linked to changes in secretion and tyrosine kinase activity. We now show that adhesion affects the sensitivity of RBL cells to the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). In suspended cells, PMA inhibited antigen-induced calcium influx (as measured by manganese influx) and changes in intracellular free calcium and had complex effects on antigen-stimulated secretion. However, in adherent cells PMA had little effect on these responses. Suspended cells only secreted in response to thapsigargin if they were co-treated with PMA, while adherent cells secreted in response to thapsigargin alone. The thapsigargin-induced secretion in adherent cells was inhibited by protein kinase C down-regulation and by the protein kinase C inhibitor GF 109203X, but not by calphostin C. We suggest that protein kinase C is constitutively activated in adherent cells, possibly due to modification of the regulatory domain of the enzyme. Adhesion of RBL-2H3 mucosal mast cells to fibronectin-coated surfaces has been linked to changes in secretion and tyrosine kinase activity. We now show that adhesion affects the sensitivity of RBL cells to the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). In suspended cells, PMA inhibited antigen-induced calcium influx (as measured by manganese influx) and changes in intracellular free calcium and had complex effects on antigen-stimulated secretion. However, in adherent cells PMA had little effect on these responses. Suspended cells only secreted in response to thapsigargin if they were co-treated with PMA, while adherent cells secreted in response to thapsigargin alone. The thapsigargin-induced secretion in adherent cells was inhibited by protein kinase C down-regulation and by the protein kinase C inhibitor GF 109203X, but not by calphostin C. We suggest that protein kinase C is constitutively activated in adherent cells, possibly due to modification of the regulatory domain of the enzyme. INTRODUCTIONThe RBL-2H3 mucosal mast cell line has been used extensively as a model of stimulus secretion coupling(1.Fewtrell C. Metzger H. Becker E.L. Simon A.S. Austen K.F. Biochemistry of the Acute Allergic Reactions. Alan R. Liss, Inc., New York1981: 295-314Google Scholar). Activation of these cells by antigen leads to a complex series of events including tyrosine phosphorylation of various proteins(2.Benhamou M. Stephan V. Robbins K.C. Siraganian R.P. J. Biol. Chem. 1992; 267: 7310-7314Abstract Full Text PDF PubMed Google Scholar, 3.Hamawy M.M. Swaim W.D. Minoguchi K. de Feijter A.W. Mergenhagen S.E. Siraganian R.P. J. Immunol. 1994; 153: 4655-4662PubMed Google Scholar), including the receptor for immunoglobulin E (IgE) 1The abbreviations used are: IgEimmunoglobulin Efura-2/AMfura-2 acetoxymethyl esterIP3inositol-1,4,5-trisphosphatePMAphorbol 12-myristate 13-acetateRBLrat basophilic leukemiamIgEαDNPmouse monoclonal IgE anti-dinitrophenyl. (4.Paolini R. Jouvin M.-H. Kinet J.-P. Nature. 1991; 353: 855-858Crossref PubMed Scopus (219) Google Scholar), phosphoinositide breakdown (5.Beaven M.A. Moore J.P. Smith G.A. Hesketh T.R. Metcalfe J.C. J. Biol. Chem. 1984; 259: 7137-7142Abstract Full Text PDF PubMed Google Scholar) leading to activation of protein kinase C(6.White K.N. Metzger H. J. Immunol. 1988; 141: 942-947PubMed Google Scholar), emptying of intracellular calcium stores by inositol 1,4,5-trisphosphate (IP3)(7.Mohr F.C. Fewtrell C. J. Cell Biol. 1987; 104: 783-792Crossref PubMed Scopus (100) Google Scholar, 8.Beaven M.A. Cunha-Melo J.R. Prog. Allergy. 1988; 42: 123-184PubMed Google Scholar), and influx of calcium across the plasma membrane(9.Kanner B.I. Metzger H. J. Biol. Chem. 1984; 259: 10188-10193Abstract Full Text PDF PubMed Google Scholar, 10.Crews F.T. Morita Y. McGivney A. Hirata F. Siraganian R.P. Axelrod J. Arch. Biochem. Biophys. 1981; 212: 561-571Crossref PubMed Scopus (67) Google Scholar, 11.Fewtrell C. Sherman E. Biochemistry. 1987; 26: 6995-7003Crossref PubMed Scopus (30) Google Scholar). These events culminate in the secretion of various mediators of the inflammatory response(1.Fewtrell C. Metzger H. Becker E.L. Simon A.S. Austen K.F. Biochemistry of the Acute Allergic Reactions. Alan R. Liss, Inc., New York1981: 295-314Google Scholar, 8.Beaven M.A. Cunha-Melo J.R. Prog. Allergy. 1988; 42: 123-184PubMed Google Scholar). It is clear that both the increase in intracellular calcium and protein kinase C activation are important steps in the signaling pathway and that these two signals act synergistically to promote secretion(12.Sagi-Eisenberg R. Pecht I. Immunol. Lett. 1984; 8: 237-241Crossref PubMed Scopus (35) Google Scholar, 13.Beaven M.A. Guthrie D.F. Moore J.P. Smith G.A. Hesketh T.R. Metcalfe J.C. J. Cell Biol. 1987; 105: 1129-1136Crossref PubMed Scopus (69) Google Scholar).Activation of protein kinase C with the phorbol ester phorbol 12-myristate 13-acetate (PMA), alone, does not induce secretion in rat basophilic leukemia (RBL) cells(12.Sagi-Eisenberg R. Pecht I. Immunol. Lett. 1984; 8: 237-241Crossref PubMed Scopus (35) Google Scholar, 13.Beaven M.A. Guthrie D.F. Moore J.P. Smith G.A. Hesketh T.R. Metcalfe J.C. J. Cell Biol. 1987; 105: 1129-1136Crossref PubMed Scopus (69) Google Scholar, 14.Cunha-Melo J.R. Gonzaga H.M.S. Ali H. Huang F.L. Huang K.-P. Beaven M.A. J. Immunol. 1989; 143: 2617-2625PubMed Google Scholar). Some laboratories have reported that PMA potentiates antigen-induced secretion at concentrations below 15 nM(12.Sagi-Eisenberg R. Pecht I. Immunol. Lett. 1984; 8: 237-241Crossref PubMed Scopus (35) Google Scholar, 15.Sagi-Eisenberg R. Lieman H. Pecht I. Nature. 1985; 313: 59-60Crossref PubMed Scopus (116) Google Scholar), but other reports do not support this finding(13.Beaven M.A. Guthrie D.F. Moore J.P. Smith G.A. Hesketh T.R. Metcalfe J.C. J. Cell Biol. 1987; 105: 1129-1136Crossref PubMed Scopus (69) Google Scholar, 14.Cunha-Melo J.R. Gonzaga H.M.S. Ali H. Huang F.L. Huang K.-P. Beaven M.A. J. Immunol. 1989; 143: 2617-2625PubMed Google Scholar). Nevertheless, there is general agreement that PMA markedly potentiates secretion in response to calcium ionophore(12.Sagi-Eisenberg R. Pecht I. Immunol. Lett. 1984; 8: 237-241Crossref PubMed Scopus (35) Google Scholar, 13.Beaven M.A. Guthrie D.F. Moore J.P. Smith G.A. Hesketh T.R. Metcalfe J.C. J. Cell Biol. 1987; 105: 1129-1136Crossref PubMed Scopus (69) Google Scholar, 14.Cunha-Melo J.R. Gonzaga H.M.S. Ali H. Huang F.L. Huang K.-P. Beaven M.A. J. Immunol. 1989; 143: 2617-2625PubMed Google Scholar). A similar synergism has been seen when protein kinase C is activated by PMA while intracellular calcium is increased by treatment with the endoplasmic reticulum Ca2⁺-ATPase inhibitors thapsigargin 2F. C. Mohr, personal communication. or cyclopiazonic acid(16.Falcone D. Fewtrell C. J. Cell. Physiol. 1995; 164: 205-213Crossref PubMed Scopus (15) Google Scholar). Additionally, the protein kinase C inhibitors staurosporine, Ro31-7549, and calphostin C have been shown to inhibit antigen-stimulated secretion(17.Yamada K. Jelsema C.L. Beaven M.A. J. Immunol. 1992; 149: 1031-1037PubMed Google Scholar). In general, it appears that the combination of protein kinase C activation and increases in intracellular calcium are sufficient to induce secretion.In addition to promoting secretion, activation protein kinase C by PMA has a second, inhibitory effect on RBL cells in suspension(12.Sagi-Eisenberg R. Pecht I. Immunol. Lett. 1984; 8: 237-241Crossref PubMed Scopus (35) Google Scholar, 13.Beaven M.A. Guthrie D.F. Moore J.P. Smith G.A. Hesketh T.R. Metcalfe J.C. J. Cell Biol. 1987; 105: 1129-1136Crossref PubMed Scopus (69) Google Scholar, 15.Sagi-Eisenberg R. Lieman H. Pecht I. Nature. 1985; 313: 59-60Crossref PubMed Scopus (116) Google Scholar). Increases in intracellular Ca2⁺ are inhibited at concentrations above 10 nM(12.Sagi-Eisenberg R. Pecht I. Immunol. Lett. 1984; 8: 237-241Crossref PubMed Scopus (35) Google Scholar, 13.Beaven M.A. Guthrie D.F. Moore J.P. Smith G.A. Hesketh T.R. Metcalfe J.C. J. Cell Biol. 1987; 105: 1129-1136Crossref PubMed Scopus (69) Google Scholar, 15.Sagi-Eisenberg R. Lieman H. Pecht I. Nature. 1985; 313: 59-60Crossref PubMed Scopus (116) Google Scholar), possibly by the inhibition of phospholipase C-γ(13.Beaven M.A. Guthrie D.F. Moore J.P. Smith G.A. Hesketh T.R. Metcalfe J.C. J. Cell Biol. 1987; 105: 1129-1136Crossref PubMed Scopus (69) Google Scholar, 18.Ozawa K. Yamada K. Kazanietz M.G. Blumberg P.M. Beaven M.A. J. Biol. Chem. 1993; 268: 2280-2283Abstract Full Text PDF PubMed Google Scholar), thus preventing phosphoinositide breakdown. Some groups have also shown that antigen-stimulated secretion is inhibited by high concentrations of PMA (12.Sagi-Eisenberg R. Pecht I. Immunol. Lett. 1984; 8: 237-241Crossref PubMed Scopus (35) Google Scholar, 15.Sagi-Eisenberg R. Lieman H. Pecht I. Nature. 1985; 313: 59-60Crossref PubMed Scopus (116) Google Scholar), presumably due to the inhibition of the Ca2⁺ response.In the past, experiments on RBL cells have been performed interchangeably with cells in suspension or with adherent cells. However, recent experiments have shown that adhesion itself affects RBL cell responses. Adhesion of RBL cells results in the tyrosine phosphorylation of several proteins including pp125FAK(19.Hamawy M.M. Mergenhagen S.E. Siraganian R.P. J. Biol. Chem. 1993; 268: 6851-6854Abstract Full Text PDF PubMed Google Scholar). In addition, antigen-stimulated secretion is enhanced in adherent RBL cells(20.Hamawy M.M. Oliver C. Mergenhagen S.E. Siraganian R.P. J. Immunol. 1992; 149: 615-621PubMed Google Scholar). In studying the effects of protein kinase C activation on secretion and calcium handling, we have discovered another effect of adhesion on RBL cell responses, namely a loss of sensitivity to the effects of the protein kinase C activator, PMA.EXPERIMENTAL PROCEDURESSensitized RBL CellsAll experiments were performed with the secreting subline 2H3 of rat basophilic leukemia cells (21.Barsumian E.L. Isersky C. Petrino M.G. Siraganian R.P. Eur. J. Immunol. 1981; 11: 317-323Crossref PubMed Scopus (481) Google Scholar) maintained in monolayer culture in Eagle's minimum essential medium containing 10% fetal bovine serum, 8% newborn bovine serum, and antibiotics as described(22.Taurog J.D. Fewtrell C. Becker E. J. Immunol. 1979; 122: 2150-2153PubMed Google Scholar). For secretion experiments in adherent cells, 0.4 × 106 cells in 0.3 ml of culture medium containing 0.18 μg of mouse monoclonal IgE anti-dinitrophenyl (mIgEαDNP) for sensitization were plated into each well of 24-well multiwell plates (Falcon, Oxnard, CA) and incubated overnight at 37°C in a humid atmosphere containing 5% CO2. For fura-2 experiments with adherent cells, aliquots of 5 × 106 cells in 2.5 ml of culture medium containing 1 μg/ml mIgEαDNP were added to 35-mm plastic culture dishes, each of which contained a 22 × 22-mm glass coverslip that had been scored down the middle. The cells were incubated overnight at 37°C in a humid atmosphere containing 5% CO2. For secretion and fura-2 experiments with cell suspensions, cells grown to confluence in a 75-cm2 tissue culture flask were incubated overnight at 37°C in 10 ml of culture medium containing 6 μg of mIgEαDNP.SolutionsThe standard saline solution used was a modified Tyrode's solution composed of 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 0.05% gelatin, and 10 mM HEPES adjusted to pH 7.4 with NaOH. Ice-cold quenching solution for secretion experiments contained 135 mM NaCl, 5 mM KCl, and 10 mM Na-HEPES (pH 7.4). For loading cells with fura-2, the saline solution contained 250 μM sulfinpyrazone (Sigma) and 0.1% bovine serum albumin instead of gelatin to maximize uptake and retention of the dye(23.Beaven M.A. Rogers J. Moore J.P. Hesketh T.R. Smith G.A. Metcalfe J.C. J. Biol. Chem. 1984; 259: 7129-7136Abstract Full Text PDF PubMed Google Scholar, 24.Millard P.J. Ryan T.A. Webb W.W. Fewtrell C. J. Biol. Chem. 1989; 264: 19730-19739Abstract Full Text PDF PubMed Google Scholar).ReagentsFura-2 acetoxymethyl ester (fura-2/AM) was purchased from Molecular Probes (Junction City, OR). Calphostin C and GF 109203X were purchased from Calbiochem-Novabiochem International (San Diego, CA). PMA, thapsigargin, and 4-methylumbelliferyl-N-acetyl β-D-glucosaminide were purchased from Sigma. Stock solutions of fura-2/AM, PMA, thapsigargin, 4-methylumbelliferyl-N-acetyl β-D-glucosaminide, calphostin C, and GF 109203X were prepared in dry dimethyl sulfoxide. Cells were never exposed to >0.2% dimethyl sulfoxide, and at this or lower concentrations the solvent did not affect the responses of RBL cells. Purified mIgEαDNP (25.Liu F.-T. Bohn J.W. Ferry E.L. Yamamoto H. Molinaro C.A. Sherman L.A. Klinman N.R. Katz D.H. J. Immunol. 1980; 124: 2728-2737PubMed Google Scholar) was a gift from Barbara Baird and David Holowka, Department of Chemistry, Cornell University. The antigen used was bovine γ-globulin to which an average of 10 dinitrophenyl groups/molecule had been coupled(26.Eisen H.N. Kern M. Newton W.T. Helmreich E. J. Exp. Med. 1959; 110: 187-206Crossref PubMed Scopus (67) Google Scholar), except for the measurements of translocation and tyrosine phosphorylation of protein kinase C, where the dinitrophenyl groups were coupled to bovine serum albumin.SecretionThis was determined from the release of the granule-associated enzyme, β-hexosaminidase. Secretion was carried out in 24-well plates in which cells had been plated overnight and washed with saline solution, or in polystyrene tubes containing 0.5 × 106 cells in saline solution. Secretion was initiated by antigen (1 μg/ml) added directly to the wells or tubes, and was terminated by adding ice-cold quenching solution to each well after 30 min for adherent cells or 60 min for suspended cells. These incubation times resulted in maximal secretion. An aliquot from each supernatant was assayed fluorimetrically for β-hexosaminidase (excitation 360, emission 450) using 4-methylumbelliferyl-N-acetyl β-D-glucosaminide as the substrate. Secretion is expressed as a percent of the β-hexosaminidase content of the cells prior to stimulation.Fura-2 Measurements in Cell SuspensionsFor measurements of free ionized calcium, sensitized cells (106/ml) were incubated with 0.5 μM fura-2/AM for 1 h at 37°C. In manganese quench experiments, the fura-2/AM concentration was 5 μM. The cells were then washed and resuspended in saline solution containing 250 μM sulfinpyrazone and 0.05% gelatin. Three-ml aliquots of cell suspension (106 cells/ml) were added to acrylic cuvettes maintained at 37°C and constantly stirred. Fura-2 fluorescence at 510 nm was monitored with a Perkin-Elmer LS-5 fluorescence spectrophotometer. Fura-2 was excited at 334 nm for measurements of free ionized calcium, or at 360 nm for manganese quench experiments.Fura-2 Measurements in Adherent CellsSensitized cells on coverslips were washed twice and incubated for 45 min with 1 μM fura-2/AM for measurements of free ionized calcium, or 5 μM fura-2/AM for manganese quench experiments. After loading, the cells were washed twice and each coverslip half was placed in a holder made from a 1.5-ml centrifuge tube, which was then inserted into a 3-ml acrylic cuvette containing 2.5 ml of saline solution containing 250 μM sulfinpyrazone and 0.05% gelatin. The temperature was maintained at 37°C, and fluorescence was monitored as described above for suspended cells.Measurement of Translocation and Tyrosine Phosphorylation of Protein Kinase C Isozymes Derived from Suspended and Adherent CellsSuspended or adherent cells were sensitized and activated with antigen essentially as described above, with activation times of 1 min. In some experiments cells were treated with either 50 or 100 nM PMA for 2 min, followed by the addition, or not, of antigen for 1 min. In experiments using thapsigargin, cells were incubated with 500 nM thapsigargin for 5 min in order to achieve a maximal increase in intracellular calcium. All incubations were in 0.9 ml of saline solution. Following activation, 0.1 ml of a 10 × sonication buffer (27.Haleem-Smith H. Chang E.-Y. Szallasi Z. Blumberg P.M. Rivera J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9112-9116Crossref PubMed Scopus (69) Google Scholar) was added and samples were immediately sonicated at 4°C. Suspended cells were treated as described(27.Haleem-Smith H. Chang E.-Y. Szallasi Z. Blumberg P.M. Rivera J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9112-9116Crossref PubMed Scopus (69) Google Scholar), while adherent cells were activated and sonicated directly in the 25-cm2 flasks in which the cells were cultured. A 0.1-ml sample of the nuclei-free sonicate was mixed with an equal volume of 2 × Tris-glycine SDS sample buffer for determination of the amount of protein kinase C isozymes present in the cells. The soluble and pelleted fractions were then recovered from the remaining nuclei-free sonicate as described(27.Haleem-Smith H. Chang E.-Y. Szallasi Z. Blumberg P.M. Rivera J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9112-9116Crossref PubMed Scopus (69) Google Scholar). Pelleted fractions were resuspended to 0.9 ml, and 0.1-ml aliquots were removed from both soluble and pelleted fractions and mixed with the 2 × Tris-glycine SDS sample buffer as above. Proteins derived from the soluble and particulate fractions were resolved by SDS-PAGE (8%) and transferred to nitrocellulose for analysis of the relative amounts of protein kinase C isozymes present in each fraction. Analysis was by Western blots using the polyclonal or monoclonal antibodies described previously(28.Szallasi Z. Smith C.B. Pettit G.B. Blumberg P.M. J. Biol. Chem. 1994; 269: 2118-2124Abstract Full Text PDF PubMed Google Scholar), except for the antibody to protein kinase C-ζ, which was obtained from Transduction Laboratories, Lexington, KY.Immunoprecipitation of protein kinase C isozymes for analysis of tyrosine phosphorylation was done as described previously(27.Haleem-Smith H. Chang E.-Y. Szallasi Z. Blumberg P.M. Rivera J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9112-9116Crossref PubMed Scopus (69) Google Scholar). Triton X-100 (final concentration 0.5%) was added to the remaining volume of the particulate fraction (see above), and the detergent lysates were used for immunoprecipitation of the individual protein kinase C isozymes. Antibodies for immunoprecipitations have been described(28.Szallasi Z. Smith C.B. Pettit G.B. Blumberg P.M. J. Biol. Chem. 1994; 269: 2118-2124Abstract Full Text PDF PubMed Google Scholar). Proteins were resolved and transferred to nitrocellulose as above. The tyrosine phosphorylation of protein kinase C-α, -δ, and -∈ derived from suspended or adherent cells was analyzed by immunoblotting with a mouse monoclonal antibody to phosphotyrosine (4G10, Upstate Biotechnology, Inc., Lake Placid, NY). Tyrosine phosphorylation of the β isozyme was not assessed due to the unavailability of an immunoprecipitating antibody. Detection was by enhanced chemiluminescence, and relative quantitation of immunoblots was performed by densitometry as described(29.Germano P. Gomez J. Kazanietz M.G. Blumberg P.M. Rivera J. J. Biol. Chem. 1994; 269: 23102-23107Abstract Full Text PDF PubMed Google Scholar).RESULTSAntigen-stimulated SecretionWe have examined the effect of the protein kinase C activator PMA on adherent and suspended RBL cells to determine whether cell adherence can explain the conflicting reports in the literature on the effects of PMA on antigen-stimulated secretion (12.Sagi-Eisenberg R. Pecht I. Immunol. Lett. 1984; 8: 237-241Crossref PubMed Scopus (35) Google Scholar, 13.Beaven M.A. Guthrie D.F. Moore J.P. Smith G.A. Hesketh T.R. Metcalfe J.C. J. Cell Biol. 1987; 105: 1129-1136Crossref PubMed Scopus (69) Google Scholar, 14.Cunha-Melo J.R. Gonzaga H.M.S. Ali H. Huang F.L. Huang K.-P. Beaven M.A. J. Immunol. 1989; 143: 2617-2625PubMed Google Scholar, 15.Sagi-Eisenberg R. Lieman H. Pecht I. Nature. 1985; 313: 59-60Crossref PubMed Scopus (116) Google Scholar). In cell suspensions, concentrations of PMA higher than about 15 nM inhibited antigen-stimulated secretion, while lower concentrations of PMA potentiated secretion somewhat (Fig. 1A). The potentiation of secretion by low concentrations of PMA varied between experiments; Fig. 1A (inset) shows an experiment in which this potentiation was especially striking. In adherent RBL cells, however, PMA had only a small effect on secretion (Fig. 1B).Fig. 2 shows that the protein kinase C inhibitor GF 109203X (30.Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charon D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar) inhibits antigen-stimulated secretion in both suspended and adherent RBL cells, thus confirming the central role of protein kinase C in secretion from RBL cells. Although high concentrations of PMA can abolish antigen-stimulated secretion from cells in suspension (Fig. 1A), while PMA has little effect on adherent cells (Fig. 1B), the results in Fig. 2 clearly demonstrate that protein kinase C activity is necessary for secretion in both adherent and suspended cells. This result supports previous studies showing that secretion can be reconstituted in protein kinase C-depleted cells by the protein kinase C isozymes β and δ(31.Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar).Figure 2:The protein kinase C inhibitor GF 109203X inhibits antigen-stimulated secretion in suspended and adherent cells. Antigen-stimulated β-hexosaminidase secretion was measured in suspended (A) and adherent (B) cells in the presence of the indicated concentrations of GF 109203X. Spontaneous secretion was subtracted from stimulated secretion at each inhibitor concentration. Data are expressed as the fraction of control (antigen-stimulated secretion without inhibitor) and represent the mean and standard deviation of three experiments. Control secretion was 30.9 ± 15.7% in suspended cells and 45.1 ± 8.1% in adherent cells. The antigen concentration was 1 μg/ml. Spontaneous secretion was 7.6 ± 5.9% in suspended cells and 5.4 ± 2.4% in adherent cells; it was unaffected by GF 109203X.View Large Image Figure ViewerDownload (PPT)Antigen-stimulated Calcium ResponsesWe also examined the effects of PMA on antigen-induced changes in intracellular Ca2⁺ in both adherent cells and in cell suspensions, using the fluorescent indicator fura-2. PMA completely abolished the antigen-induced increase of intracellular Ca2⁺ in suspended cells (Fig. 3A), as has been shown previously (13.Beaven M.A. Guthrie D.F. Moore J.P. Smith G.A. Hesketh T.R. Metcalfe J.C. J. Cell Biol. 1987; 105: 1129-1136Crossref PubMed Scopus (69) Google Scholar, 15.Sagi-Eisenberg R. Lieman H. Pecht I. Nature. 1985; 313: 59-60Crossref PubMed Scopus (116) Google Scholar). The IC50 for this inhibition was approximately 15 nM (Fig. 4A), in agreement with results from other groups(12.Sagi-Eisenberg R. Pecht I. Immunol. Lett. 1984; 8: 237-241Crossref PubMed Scopus (35) Google Scholar, 15.Sagi-Eisenberg R. Lieman H. Pecht I. Nature. 1985; 313: 59-60Crossref PubMed Scopus (116) Google Scholar). However, in adherent cells, PMA had no significant effect on the antigen-stimulated Ca2⁺ response at any of the concentrations tested (Fig. 3B and 4B). In addition, GF 109203X was able to reverse the inhibition of the Ca2⁺ response by PMA in suspended cells (Fig. 3A), supporting the idea that this effect of PMA is due to activation of protein kinase C.Figure 3:PMA abolishes antigen-induced increases in intracellular Ca2⁺ in cell suspensions, but has little effect on adherent cells. Suspended (A) and adherent (B) cells loaded with the Ca2⁺ indicator fura-2 were stimulated with 1 μg/ml antigen (Ag) 1 min after treatment with 50 nM PMA as indicated. 5 μM GF 109203X (GF) was added 2 min before antigen in the trace indicated, and was able to reverse the effect of PMA. The quench in fluorescence during the addition of GF 109203X was due to the strong absorbance of the compound. Data show fluorescence traces from one of three representative experiments. PMA had no effect on fluorescence measurements in unstimulated cells.View Large Image Figure ViewerDownload (PPT)Figure 4:Dose-response curves for the effect of PMA on the antigen-induced increase in intracellular calcium in suspended and adherent cells. Antigen-induced changes in fura-2 fluorescence were measured in suspended (A) and adherent (B) cells in experiments similar to those shown in Fig. 3. The maximal change in fluorescence from the pre-stimulation baseline was expressed as a percent of total fluorescence, after correcting for non-fura-2 fluorescence and for leakage of fura-2 from the cells during the experiment. The percent maximal change in fluorescence was then plotted as a fraction of control (percent maximal change in fluorescence without PMA). Data represent the mean and standard deviation of four experiments. Control maximal fluorescence changes were 26.0 ± 5.5% in suspended cells and 26.1 ± 3.3% in adherent cells. The antigen concentration was 1 μg/ml.View Large Image Figure ViewerDownload (PPT)Since PMA abolished not only the initial increase but also the prolonged elevation in intracellular Ca2⁺ in suspended cells, it should inhibit both the release of calcium from intracellular stores and calcium influx across the plasma membrane. We therefore examined the effects of PMA on the calcium influx component of the calcium response using the manganese influx technique(32.Merritt J.E. Jacob R. Hallam T.J. J. Biol. Chem. 1989; 264: 1522-1527Abstract Full Text PDF PubMed Google Scholar). In these experiments, decreases in fura-2 fluorescence are due to quenching of the dye by Mn2⁺, which has entered the cell via a calcium influx pathway(33.Fasolato C. Hoth M. Matthews G. Penner R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3068-3072Crossref PubMed Scopus (137) Google Scholar). As expected, antigen-stimulated Mn2⁺ influx in cell suspensions was abolished by 100 nM PMA (Fig. 5A). In adherent cells, however, PMA had no effect on manganese influx in response to antigen (Fig. 5B).Figure 5:PMA inhibits antigen-induced manganese influx in suspended RBL cells, but had little effect in adherent cells. Suspended (A) and adherent (B) cells were treated with 100 nM PMA 1 min before the addition of 100 μM MnCl2 (Mn). The immediate drop in fluorescence is a result of manganese binding to extracellular fura-2. Two minutes later, the cells were stimulated with 1 μg/ml antigen (Ag). Data show fura-2 fluorescence traces from one experiment representative of three. PMA had no effect on fluorescence measurements in unstimulated cells.View Large Image Figure ViewerDownload (PPT)Responses to ThapsigarginThapsigargin and other inhibitors of the endoplasmic reticulum Ca2⁺-ATPase (34.Thastrup O. Dawson A.P. Scharff O. Foder B. Cullen P.J. Dr⊘bak B.K. Bjerrum P.J. Christensen S.B. Hanley M.R. Agents Actions. 1989; 27: 17-23Crossref PubMed Scopus (434) Google Scholar) deplete intracellular stores of calcium and activate Ca2⁺ influx in RBL cells(16.Falcone D. Fewtrell C. J. Cell. Physiol. 1995; 164: 205-213Crossref PubMed Scopus (15) Google Scholar, 35.Mohr F.C. Dunston S.K. FASEB J. 1991; 5: A1044Google Scholar, 36.Cleveland P.L. Millard P.J. Showell H.J. Fewtrell C.M.S. Cell Calcium. 1993; 14: 1-16Crossref PubMed Scopus (30) Google Scholar, 37.Ali H. Maeyama K. Sagi-Eisenberg R. Beaven M.A. Biochem. J. 1994; 304: 431-440Crossref PubMed Scopus (58) Google Scholar), thus bypassing the IP3-dependent pathway activated by antigen. If protein kinase C activation by PMA directly inhibits the Ca2⁺ influx pathway in suspended cells, then PMA should also prevent the thapsigargin-induced Ca2⁺ influx. However, since the Ca2⁺ response to antigen is completely abolished by PMA, a more likely possibility is that PMA is inhibiting the Ca2⁺ responses at, or prior to, the release of Ca2⁺ from stores. If this is the case then PMA should have no effect on the thapsigargin-induced activation of the calcium influx pathway as monitored by manganese influx, and this is shown in Fig. 6. This is in agreement with recent results obtained by Ali et al.(37.Ali H. Maeyama K. Sagi-Eisenberg R. Beaven M.A. Biochem. J. 1994; 304: 431-440Crossref PubMed Scopus (58) Google Scholar), showing that the thapsigargin-induced increase in intracellular calcium is not inhibited by PMA. Since activation of protein kinase C by PMA is known to inhibit phospholipase C-γ, it seems likely that PMA is inhibiting the Ca2⁺ response to antigen by preventing IP3 production(13.Beaven M.A. Guthrie D.F. Moore J.P. Smith G.A. Hesketh T.R. Metcalfe J.C. J. Cell Biol. 1987; 105: 1129-1136Crossref PubMed Scopus (69) Google Scholar, 18.Ozawa K. Yamada K. Kazanietz M.G. Blumberg P.M. Beaven M.A. J. Biol. Chem. 1993; 268: 2280-2283Abstract Full Text PDF PubMed Google Scholar).Figure 6:Thapsigargin-induced manganese influx in suspended cells is not inhibited by PMA. Suspended cells were treated with 0.1% Me2SO (-PMA) or 100 nM PMA (+PMA) 1 min before the addition of 100 μM MnCl2 (Mn). The immediate drop in fluorescence is a result of manganese binding to extracellular fura-2. Two minutes later, the cells were stimulated with 100 nM thapsigargin (Tg). Data show fura-2 f
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