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• W2106180589 abstract "This study presents evidence that phosphoinositide 3-kinase (PI3K) plays a concerted role with phospholipase Cγ in initiating antigen-mediated Ca2+ signaling in mast cells via a phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3)-sensitive Ca2+ entry pathway. Exogenous PI(3,4,5)P3 at concentrations close to its physiological level induces instantaneous Ca2+ influx into RBL-2H3 cells. This PI(3,4,5)P3-induced intracellular Ca2+ increase is independent of phospholipase C activity or the depletion of internal stores. Moreover, inhibition of PI3K by LY294002 or by overexpression of the dominant negative inhibitor Δp85 suppresses the Ca2+ response to the cross-linking of the high affinity receptor for IgE (FcεRI). Concomitant treatment of RBL-2H3 cells with LY294002 or Δp85 and 2-aminoethyl diphenylborate, a cell-permeant antagonist of d-myo-inositol 1,4,5-trisphosphate receptors, abrogates antigen-induced Ca2+ signals, whereas either treatment alone gives rise to partial inhibition. Conceivably, PI(3,4,5)P3-sensitive Ca2+ entry and capacitative Ca2+ entry represent major Ca2+ influx pathways that sustain elevated [Ca2+]i to achieve optimal physiological responses. This study also refutes the second messenger role ofd-myo-inositol 1,3,4,5-tetrakisphosphate in regulating FcεRI-mediated Ca2+ response. Considering the underlying mechanism, our data suggest that PI(3,4,5)P3directly stimulates a Ca2+ transport system in plasma membranes. Together, these data provide a molecular basis to account for the role of PI3K in the regulation of FcεRI-mediated degranulation in mast cells. This study presents evidence that phosphoinositide 3-kinase (PI3K) plays a concerted role with phospholipase Cγ in initiating antigen-mediated Ca2+ signaling in mast cells via a phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3)-sensitive Ca2+ entry pathway. Exogenous PI(3,4,5)P3 at concentrations close to its physiological level induces instantaneous Ca2+ influx into RBL-2H3 cells. This PI(3,4,5)P3-induced intracellular Ca2+ increase is independent of phospholipase C activity or the depletion of internal stores. Moreover, inhibition of PI3K by LY294002 or by overexpression of the dominant negative inhibitor Δp85 suppresses the Ca2+ response to the cross-linking of the high affinity receptor for IgE (FcεRI). Concomitant treatment of RBL-2H3 cells with LY294002 or Δp85 and 2-aminoethyl diphenylborate, a cell-permeant antagonist of d-myo-inositol 1,4,5-trisphosphate receptors, abrogates antigen-induced Ca2+ signals, whereas either treatment alone gives rise to partial inhibition. Conceivably, PI(3,4,5)P3-sensitive Ca2+ entry and capacitative Ca2+ entry represent major Ca2+ influx pathways that sustain elevated [Ca2+]i to achieve optimal physiological responses. This study also refutes the second messenger role ofd-myo-inositol 1,3,4,5-tetrakisphosphate in regulating FcεRI-mediated Ca2+ response. Considering the underlying mechanism, our data suggest that PI(3,4,5)P3directly stimulates a Ca2+ transport system in plasma membranes. Together, these data provide a molecular basis to account for the role of PI3K in the regulation of FcεRI-mediated degranulation in mast cells. the high affinity receptor for IgE phosphoinositide 3-kinase phospholipase C 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate 4)P2, phosphatidylinositol 3,4-bisphosphate 5)P2, phosphatidylinositol 4,5-bisphosphate phosphatidylinositol 3-monophosphate 4,5)P3, d-myo-inositol 1,4,5-trisphosphate 3,4,5)P4,d- myo-inositol 1,3,4,5-tetrakisphosphate immunoreceptor tyrosine-based activation motif protein-tyrosine kinase Src homology-2 Bruton's tyrosine kinase dinitrophenol-conjugated human serum albumin 2-aminoethoxy diphenylborate hemagglutinin epitope-tagged Δp85 pleckstrin homology fura-2 acetoxymethyl ester fluo-3 acetoxymethyl ester cytomegalovirus 4,5)P3, 1-O-(1,2-di-O-octanoyl-sn-glycero-3-O-phosphoryl)-d-myo-inositol 3,4,5- trisphosphate Activation of mast cells by the cross-linking of the high affinity receptor for IgE (FcεRI)1leads to the secretion and generation of an array of mediators that induce immediate allergic inflammation (for review, see Ref. 1Metcalfe D.D. Baram D. Mekori Y.A. Physiol. Rev. 1997; 77: 1033-1079Crossref PubMed Scopus (1779) Google Scholar). Although the FcεRI-mediated signaling cascade has been characterized, the regulatory mechanism governing mast cell degranulation is only partially understood. FcεRI is a heterotrimeric protein complex (αβγ2) that contains immunoreceptor tyrosine-based activation motifs (ITAMs) in both the β and γ subunit cytoplasmic domains (2Metzger H. Immunol. Rev. 1992; 125: 37-48Crossref PubMed Scopus (222) Google Scholar). The protein-tyrosine kinase (PTK) Lyn is associated with the β subunit at the resting state (3Vonakis B.M. Chen H. Haleem-Smith H. Metzger H. J. Biol. Chem. 1997; 272: 24072-24080Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), and its action is promoted by FcεRI cross-linking (4Pribluda V.S. Pribluda C. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11246-11250Crossref PubMed Scopus (173) Google Scholar). Lyn phosphorylates ITAMs of the β and γ subunits, resulting in the recruitment of Lyn and Syk, respectively, through Src homology-2 (SH2) domain-mediated interactions with phosphotyrosine residues (5Jouvin M.H. Adamczewski M. Numerof R. Letourneur O. Valle A. Kinet J.P. J. Biol. Chem. 1994; 269: 5918-5925Abstract Full Text PDF PubMed Google Scholar, 6Kihara H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Abstract Full Text PDF PubMed Google Scholar). Activation of these newly recruited PTKs, in turn, facilitates the translocation and phosphorylation of multiple substrates, including phospholipase Cγ (PLCγ) isozymes and phosphoinositide 3-kinase (PI3K) (ref review, see Ref. 1Metcalfe D.D. Baram D. Mekori Y.A. Physiol. Rev. 1997; 77: 1033-1079Crossref PubMed Scopus (1779) Google Scholar). The activated PLCγ hydrolyzes phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) tod-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol, which induce the release of Ca2+ from intracellular stores and the activation of protein kinase C, respectively. On the other hand, stimulation of PI3K results in transient accumulation of micromolar levels of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Although the involvement of PI3K in antigen-induced degranulation is established (7Yano H. Nakanishi S. Kimura K. Hanai N. Saitoh Y. Fukui Y. Nonomura Y. Matsuda Y. J. Biol. Chem. 1993; 268: 25846-25856Abstract Full Text PDF PubMed Google Scholar, 8Yano H. Agatsuma T. Nakanishi S. Saitoh Y. Fukui Y. Nonomura Y. Matsuda Y. Biochem. J. 1995; 312: 145-150Crossref PubMed Scopus (60) Google Scholar, 9Hirasawa N. Sato Y. Yomogida S. Mue S. Ohuchi K. Cell Signal. 1997; 9: 305-310Crossref PubMed Scopus (21) Google Scholar, 10Pelletier C. Guerin-Marchand C. Iannascoli B. Marchand F. David B. Weyer A. Blank U. Inflamm. Res. 1998; 47: 493-500Crossref PubMed Scopus (31) Google Scholar, 11Barker S.A. Lujan D. Wilson B.S. J. Leukoc. Biol. 1999; 65: 321-329Crossref PubMed Scopus (71) Google Scholar), a clear consensus regarding how lipid products derived from the action of PI3K mediate the cellular responses has yet to emerge. Putative downstream targets for PI(3,4,5)P3 and PI(3,4)P2 include SH2- or pleckstrin homology (PH) domain-containing signaling enzymes such as PLC-γ, Akt, and Bruton's tyrosine kinase (Btk) (12Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1221) Google Scholar, 13Rameh L.E. Cantley L.C. J. Biol. Chem. 1999; 274: 8347-8350Abstract Full Text Full Text PDF PubMed Scopus (849) Google Scholar), which transduce the signal to the respective signaling pathways. In addition, data from several laboratories suggest a role of PI3K in the regulation of intracellular Ca2+ ([Ca2+]i) increase in mast cells (9Hirasawa N. Sato Y. Yomogida S. Mue S. Ohuchi K. Cell Signal. 1997; 9: 305-310Crossref PubMed Scopus (21) Google Scholar, 11Barker S.A. Lujan D. Wilson B.S. J. Leukoc. Biol. 1999; 65: 321-329Crossref PubMed Scopus (71) Google Scholar, 14Miura K. MacGlashan D.W. Blood. 2000; 96: 2199-2205Crossref PubMed Google Scholar). Evidence suggests that PI3K may mediate [Ca2+]i elevation in mast cells via two distinct mechanisms. First, in vitro data indicate that PI(3,4,5)P3 stimulates Ins(1,4,5)P3 production by activating PLCγ isozymes (15Bae Y.S. Cantley L.G. Chen C.S. Kim S.R. Kwon K.S. Rhee S.G. J. Biol. Chem. 1998; 273: 4465-4469Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 16Barker S.A. Caldwell K.K. Pfeiffer J.R. Wilson B.S. Mol. Biol. Cell. 1998; 9: 483-496Crossref PubMed Scopus (94) Google Scholar). This PLCγ activation may be attained directly by facilitating membrane translocation (15Bae Y.S. Cantley L.G. Chen C.S. Kim S.R. Kwon K.S. Rhee S.G. J. Biol. Chem. 1998; 273: 4465-4469Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 16Barker S.A. Caldwell K.K. Pfeiffer J.R. Wilson B.S. Mol. Biol. Cell. 1998; 9: 483-496Crossref PubMed Scopus (94) Google Scholar) or indirectly via Btk (14Miura K. MacGlashan D.W. Blood. 2000; 96: 2199-2205Crossref PubMed Google Scholar, 17Kawakami Y. Kitaura J. Satterthwaite A.B. Kato R.M. Asai K. Hartman S.E. Maeda-Yamamoto M. Lowell C.A. Rawlings D.J. Witte O.N. Kawakami T. J. Immunol. 2000; 165: 1210-1219Crossref PubMed Scopus (151) Google Scholar). Second, PI3K may increase [Ca2+]i by facilitating Ca2+mobilization across plasma membranes (11Barker S.A. Lujan D. Wilson B.S. J. Leukoc. Biol. 1999; 65: 321-329Crossref PubMed Scopus (71) Google Scholar). This premise was prompted by data showing the inhibitory effect of PI3K inhibitors on antigen-induced Ca2+ response (9Hirasawa N. Sato Y. Yomogida S. Mue S. Ohuchi K. Cell Signal. 1997; 9: 305-310Crossref PubMed Scopus (21) Google Scholar, 11Barker S.A. Lujan D. Wilson B.S. J. Leukoc. Biol. 1999; 65: 321-329Crossref PubMed Scopus (71) Google Scholar, 14Miura K. MacGlashan D.W. Blood. 2000; 96: 2199-2205Crossref PubMed Google Scholar), and is in line with the notion that two distinct Ca2+ influx pathways (capacitative versus non-capacitative) are operative in antigen-stimulated RBL-2H3 cells (18Lee R.J. Oliver J.M. Mol. Biol. Cell. 1995; 6: 825-839Crossref PubMed Scopus (40) Google Scholar). It is noteworthy that the proposed involvement of PI3K in the regulation of a non-capacitative Ca2+ influx pathway is reminiscent of our finding of a PI(3,4,5)P3-sensitive Ca2+ entry mechanism in platelets (19Lu P.J. Hsu A.L. Wang D.S. Chen C.S. Biochemistry. 1998; 37: 9776-9783Crossref PubMed Scopus (32) Google Scholar) and Jurkat T cells (20Hsu A.L. Ching T.T. Sen G. Wang D.S. Bondada S. Authi K.S. Chen C.S. J. Biol. Chem. 2000; 275: 16242-16250Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Consequently, we hypothesize that this PI(3,4,5)P3-sensitive Ca2+ entry is a conserved mechanism that plays a crucial role in the regulation of receptor-mediated Ca2+ signaling among these hematopoietic cells. This hypothesis is corroborated by the present data showing the involvement of this PI(3,4,5)P3-mediated Ca2+influx in FcεRI-mediated Ca2+ response in RBL-2H3 mast cells. Considering the crucial role of Ca2+ influx in the secretion of inflammatory mediators, this mechanism provides a molecular basis whereby PI3K regulates mast cell function. Moreover, given that PI3K and PLCγ are the downstream effectors of the FcεRI-mediated tyrosine kinase cascade, we propose that these two enzymes act in a concerted manner to initiate Ca2+ response to antigen stimulation. In stimulated cells, PI3K and PLCγ act on the mutual substrate PI(4,5)P2 to generate PI(3,4,5)P3 and Ins(1,4,5)P3, respectively, which activate Ca2+ channels at different cellular compartments to provide the elevated [Ca2+]irequired for optimal physiological responses. d-myo-Inositol 1,3,4,5-tetrakisphosphate, potassium salt (Ins(1,3,4,5)P4) 1-O-(1,2-di-O-palmitoyl-sn-glycero-3-O-phosphoryl)-d-myo-inositol 3,4,5-trisphosphate, protonated form (PI(3,4,5)P3), 1-O-(1,2-di-O-octanoyl-sn-glycero-3-O-phosphoryl)-d-myo-inositol 3,4,5-trisphosphate, protonated form (di-C8-PI(3,4,5)P3), 1-O-(1,2-di-O-palmitoyl-sn-glycero-3-O-phosphoryl)-d-myo-inositol 3,4-bisphosphate, protonated form (PI(3,4)P2), 1-O-(1,2-di-O-palmitoyl-sn-glycero- 3-O-phosphoryl)-d-myo-inositol 4,5-bisphosphate, protonated form (PI(4,5)P2), and 1-O-(1,2-di-O-palmitoyl-sn-glycero-3-O-phosphoryl)-d-myo-inositol 3-monophosphate, protonated form (PI(3)P) were synthesized as previously reported (21Lu P.J. Gou D.M. Shieh W.R. Chen C.S. Biochemistry. 1994; 33: 11586-11597Crossref PubMed Scopus (48) Google Scholar, 22Wang D.-S. Chen C.-S. J. Org. Chem. 1996; 61: 5905-5910Crossref Scopus (56) Google Scholar). The identity and purity of all inositol phosphates and inositol lipids were verified by 1H and31P NMR and high resolution mass spectrometry. Published data from this and other laboratories have shown that PI(3,4,5)P3 and other inositol lipids are cell-permeant and can readily fuse with cell membranes to exert cellular responses in different types of cells, including platelets (19Lu P.J. Hsu A.L. Wang D.S. Chen C.S. Biochemistry. 1998; 37: 9776-9783Crossref PubMed Scopus (32) Google Scholar), NIH3T3 cells (23Derman M.P. Toker A. Hartwig J.H. Spokes K. Falck J.R. Chen C.S. Cantley L.C. Cantley L.G. J. Biol. Chem. 1997; 272: 6465-6470Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), adipocytes (24Gagnon A. Chen C.S. Sorisky A. Diabetes. 1999; 48: 691-698Crossref PubMed Scopus (58) Google Scholar), and Jurkat T cells (20Hsu A.L. Ching T.T. Sen G. Wang D.S. Bondada S. Authi K.S. Chen C.S. J. Biol. Chem. 2000; 275: 16242-16250Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Fura-2 acetoxymethyl ester (fura-2 AM), fluo-3 acetoxymethyl ester (fluo-3 AM), and 2-aminoethyoxy diphenylborate (2-APB) were purchased from Calbiochem. Leupeptin, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), and the antigen dinitrophenol-conjugated human serum albumin (DNP-HSA) were products from Sigma. [3H]Inositol was purchased from PerkinElmer Life Sciences. DNP-specific monoclonal IgE was a kind gift from Dr. Henry Metzger (Chemical Immunology Section, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health). The construct expressing hemagglutinin (HA)-tagged Δp85 (HA·Δp85) was provided by Professor Alex Toker (Harvard Medical School). Monoclonal anti-HA and rabbit anti-Btk antibodies were purchased from Roche Molecular Biochemicals and BD Biosciences, respectively. RBL-2H3 cells (from ATCC) were maintained in monolayer culture in 75-cm3 plastic tissue culture flasks containing 15 ml of Eagle's minimum essential medium with 10% fetal bovine serum and 0.1% gentamicin at 37 °C in the presence of 5% CO2. [Ca2+]i was monitored by change in the fluorescence intensity of fura-2-loaded cells. RBL-2H3 cells (1 × 107 cells/ml) were suspended in 1 ml of assay buffer consisting of 4.3 mmNa2HPO4, 24.3 mmNaH2PO4, 4.3 mmK2HPO4, 113 mm NaCl, 5 mm glucose, pH 7.4. fura-2 loading was achieved by exposing the cells to 10 μm fura-2 AM in the presence of 0.5% bovine serum albumin and 2 mm probenacid in the dark for 1 h at 37 °C. The cells were then pelleted by centrifugation at 1000 × g for 10 min, washed with assay buffer twice, and resuspended at 8 × 105 cells/ml in the same buffer containing 1 mm Ca2+. The effect of various inositol lipids on [Ca2+]i was examined by fura-2 fluorescence in a Hitachi F-2000 spectrofluorimeter at 37 °C with excitation and emission wavelengths at 340 and 510 nm, respectively, as described in the literature (25da Silva C.P. Emmrich F. Guse A.H. J. Biol. Chem. 1994; 269: 12521-12526Abstract Full Text PDF PubMed Google Scholar, 26Wolfe P.C. Chang E.Y. Rivera J. Fewtrell C. J. Biol. Chem. 1996; 271: 6658-6665Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The maximum fura-2 fluorescence intensity (Fmax) in RBL-2H3 cells was determined by adding 4-bromo-A23187 (1 μm), and the minimum fluorescence (Fmin) was determined following depletion of external Ca2+ by 5 mmEGTA. The [Ca2+]i was calculated according to the equation [Ca2+]i =Kd(F −Fmin)/(Fmax −F), where Kd denotes the apparent dissociation constant (Kd = 224 nm) of the fluorescence dye-Ca2+ complex (27Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). RBL-2H3 cells were sensitized with 1 μg/ml mouse DNP-specific IgE overnight. Cells were collected, suspended in assay buffer, loaded with fura-2, and activated by the antigen DNP-HSA (1 μg/ml). Changes in [Ca2+]i were monitored by fura-2 fluorimetry as described above. All the above fura-2 experiments were also repeated with fluo-3-loaded cells in a similar manner. fluo-3 displays a lowerKd (864 nm at 37 °C) and a longer excitation wavelength (505 nm) of the dye-Ca2+ complexvis à vis fura-2 (28Merritt J.E. McCarthy S.A. Davies M.P. Moores K.E. Biochem. J. 1990; 269: 513-519Crossref PubMed Scopus (278) Google Scholar). Consistent results were obtained with both fluorescence dyes. Δp85 is a deletion mutant that lacks a region required for tight association with p110 but is still able to bind to appropriate phosphotyrosine targets. Thus, Δp85 can compete with native p85 for binding to essential signaling proteins and behaves as a dominant negative mutant. Transient transfection of HA·Δp85 was carried out using the LipofectAMINE Plus reagent according to the protocol supplied by the manufacturer. In brief, Opti-MEM medium (1.5 ml) containing the indicated amount of the HA·Δp85 expression vector (3–12 μg) was incubated with 120 μl of the Plus reagent at 25 °C for 15 min, and 12 μl of the LipofectAMINE reagent in 1.5 ml of Opti-MEM was added. In parallel, an empty pCMV/blue plasmid (12 μg) was subjected to the same treatment as a negative control. The mixture was incubated at 25 °C for 15 min and added to 9 ml of serum-free Opti-MEM (Life Technologies). RBL-2H3 cells in a T-75 flask were washed with serum-free Opti-MEM and were added to the aforementioned transfection medium. After 3 h at 37 °C, the transfection media were replaced with 15 ml of Eagle's minimum essential medium containing 10% fetal bovine serum. The transfected cells were allowed to grow for 2 days to express foreign DNA. The collected cells were analyzed for antigen-induced Ca2+ response by fluorescence spectrometry and for HA·Δp85 expression by Western blot analysis using anti-HA antibody. The release of mast cell mediators by exocytosis was monitored by the β-hexosaminidase assay. RBL-2H3 cells were grown in 12-well plates and passively sensitized with DNP-specific IgE. The IgE-sensitized cells were washed twice with Tyrode's buffer consisting of 10 mm Hepes, pH 7.4, 130 mm NaCl, 5 mm KCl, 1.4 mmCaCl2, 1 mm MgCl2, 5.6 mm glucose, and 0.1% bovine serum albumin. Secretion was initiated by exposing cells to the antigen DNP-HSA (1 μg/ml). After 1 h, the reaction was terminated by placing the plate on ice. The enzyme activities of β-hexosaminidase in 50 μl of supernatants and attached cells solubilized with 0.5% Triton X-100 were measured with 200 μl of 1 mm p-nitrophenylN-acetyl-β-d-glucosaminide in 0.1m sodium citrate, pH 4.5, at 37 °C for 1 h. The reaction was stopped by the addition of 500 μl of 0.1 mNaHCO3. The release of the product p-nitrophenol was measured by monitoring the absorbance at 400 nm. The percentage of degranulation was calculated by dividing the absorbance of the supernatant over the combined absorbance of the supernatant and cell lysate. The examination of phosphoinositol turnover was carried out according to a modification of the procedure described previously (20Hsu A.L. Ching T.T. Sen G. Wang D.S. Bondada S. Authi K.S. Chen C.S. J. Biol. Chem. 2000; 275: 16242-16250Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In brief, RBL-2H3 cells were incubated withmyo-[2-3H]inositol (10 μCi/106cells/ml) in Eagle's minimum essential medium supplemented with 10% fetal bovine serum. The cells were then washed with the medium, sensitized with DNP-specific IgE, and washed with 20 mmHepes, pH 7.4, containing 285 mm NaCl, 11 mmKCl, 1.3 mm Na2HPO4, 1 mm KH2PO4, 8.3 mmNaHCO3, 1.6 mm MgSO4, 2.2 mm MgCl2, 2.2 mm CaCl2, and 5.6 mm glucose. Aliquots containing 1 × 106 cells were each resuspended in 0.3 ml of the aforementioned assay buffer and transferred to 1.5-ml microcentrifuge tubes. Each sample was incubated with 0.3 μg of DNP-HSA for the indicated times and quenched by adding 0.25 ml of 6% trichloroacetic acid. The tubes were centrifuged for 2 min at 12,000 ×g. The supernatant (200 μl) was analyzed by high pressure liquid chromatography on a 5-μm Adsorbosphere Sax column (4.6 × 200 mm) equilibrated with H2O. The [3H]inositol phosphates were eluted with a linear gradient of 0–0.9 mNH4H2PO4 in 60 min at a flow rate of 1 ml/min. Fractions were collected every 1 ml, and their radioactivity was measured by liquid scintillation. Synthetic [3H]Ins(1,3,4,5)P4, [3H]Ins(1,4,5)P3, [3H]Ins(4,5)P2, and Ins(4)P were used as standards. The respective retention times were 60, 48, 43, and 31 min. For the Ca2+ release assay, the plasma membrane was purified as previously described (20Hsu A.L. Ching T.T. Sen G. Wang D.S. Bondada S. Authi K.S. Chen C.S. J. Biol. Chem. 2000; 275: 16242-16250Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In brief, RBL-2H3 cells (4 × 108 cells) were washed with phosphate-buffered saline and suspended in 5 ml of PM buffer consisting of 20 mm Hepes, pH 7.2, 110 mm KCl, 10 mm NaCl, 2 mm MgCl2, 5 mmKH2PO4, 1 mm dithiothreitol, 1 mm EGTA, 1 mm AEBSF, and 20 μg/ml leupeptin. The cell suspension was homogenized in a Dounce homogenizer using a loose pestle with five strokes up and down. The homogenate was centrifuged at 1500 × g for 10 min. The pellet was suspended in 3.125 ml of PM buffer and mixed with 5.5 ml of 69% (w/w) sucrose to make a final 44% (w/w) sucrose-membrane mixture. The mixture was overlaid with 42.3% (w/w) sucrose, and the two-phase suspension was subjected to centrifugation at 90,000 ×g for 2 h in a swinging bucket rotor. The membrane material at the interface of the phases contained the greatest enrichment in plasma membranes based on the activity of (Na+-K+)-ATPase. This fraction was collected, suspended in 5 ml of 10 mm Hepes, pH 7.5, containing 1 mm dithiothreitol, 1 mm AEBSF, and 20 μg/ml leupeptin, and centrifuged at 25,000 × g for 10 min. The pellet was suspended in 1 ml of the same buffer. To investigate the involvement of PI3K in antigen-stimulated Ca2+ response in mast cells, we first examined the effect of exogenous PI(3,4,5)P3 on [Ca2+]i in RBL-2H3 cells. Exposure of fura-2-loaded RBL-2H3 cells to PI(3,4,5)P3, ranging from 1 to 20 μm, elicited an instantaneous [Ca2+]i increase in a dose-dependent manner (Fig. 1 A, upper panel). This PI(3,4,5)P3 effect became saturated at 20 μm, beyond which no significant enhancement in the amplitude of Ca2+ response was noted (data not shown). The PI(3,4,5)P3-induced [Ca2+]i rise was largely attributable to Ca2+ influx, because the increase could be abolished by Ca2+ depletion with 5 mmEGTA (Fig. 1 A, lower panel). With regard to other phosphoinositides examined, PI(3,4)P2 at high doses (20 μm) could also elicit [Ca2+]iincrease but to a much lesser extent than PI(3,4,5)P3 (Fig.1 B, upper panel), however. The potency was ∼10% of that of PI(3,4,5)P3. On the other hand, PI(4,5)P2 and PI(3)P did not show any appreciable effect on [Ca2+]i (lower panel). These data suggest the presence of a PI(3,4,5)P3-sensitive Ca2+ influx mechanism in RBL-2H3 mast cells. Data from several groups suggest that PI(3,4,5)P3 might stimulate Ins(1,4,5)P3production by activating PLCγ isozymes via discrete mechanisms (9Hirasawa N. Sato Y. Yomogida S. Mue S. Ohuchi K. Cell Signal. 1997; 9: 305-310Crossref PubMed Scopus (21) Google Scholar,11Barker S.A. Lujan D. Wilson B.S. J. Leukoc. Biol. 1999; 65: 321-329Crossref PubMed Scopus (71) Google Scholar, 14Miura K. MacGlashan D.W. Blood. 2000; 96: 2199-2205Crossref PubMed Google Scholar). This raised a concern that PI(3,4,5)P3-induced [Ca2+]i increase might, in part, be due to Ca2+ release from endoplasmic reticulum stores. To refute this possibility, we first examined the effect of two distinct pharmacological inhibitors that blocked Ins(1,4,5)P3-induced Ca2+ mobilization. These included the PLC inhibitor U73122 (10 μm) and the cell-permeant antagonist of Ins(1,4,5)P3 receptors 2-APB (40 μm) (29Maruyama T. Kanaji T. Nakade S. Kanno T. Mikoshiba K. J. Biochem. ( Tokyo ). 1997; 122: 498-505Crossref PubMed Scopus (770) Google Scholar, 30Ma H.T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (531) Google Scholar). Although these inhibitors were effective in attenuating FcεRI-mediated Ca2+ response in RBL-2H3 cells (Figs. 3 and 4), they did not display significant effect on PI(3,4,5)P3-induced [Ca2+]iresponse. The extents of the [Ca2+]i increase elicited by 20 μm PI(3,4,5)P3 in RBL-2H3 cells pretreated with U73122 and 2-APB were 95 ± 2% (n = 3) and 98 ± 2% (n = 3), respectively, of that of the untreated control.Figure 4Overexpression of Δp85 inhibits FcεRI-mediated Ca2+ response and secretion in RBL-2H3 cells. A, expression levels of HA·Δp85 in RBL-2H3 cells that had been transfected with 1 μg/ml of a control pCMV/blue plasmid (a) or with increasing amounts of the HA·Δp85-expressing plasmid (b, 0.25 μg/ml; c, 0.5 μg/ml;d, 0.75 μg/ml; e, 1 μg/ml) for 2 days. The Western analysis was carried out using antibodies against the HA tag. The loading amounts of individual samples were calibrated in reference to actin as internal standard (data not shown). Data are representative of three independent experiments. B, Δp85 inhibited FcεRI-mediated Ca2+ response in a dose-dependent manner. C, Δp85 inhibited FcεRI-mediated secretion of β-hexosaminidase in a dose-dependent manner. Cells expressing different levels of Δp85 (a–e, as indicated above) were collected after overnight sensitization with DNP-specific IgE. The collected cells were loaded with fura-2 and stimulated with DNP-HSA (Ag) to test for Ca2+ response and β-hexosaminidase secretion(a–e in accordance with the above designations). In addition, cells expressing the highest level of Δp85 were treated with 2-APB (40 μm) for 10 min before antigen stimulation. As shown, the concerted action of Δp85 and 2-APB abrogated the Ca2+ signal (trace f in B) and β-hexosaminidase secretion (vertical bar f inC). Traces are representative of three independent experiments. Vertical bars shown are means ± S.D. (n = 3). Consistent results were obtained when another fluorescence dye fluo-3 was used in the above experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Furthermore, we obtained evidence that thapsigargin-sensitive Ca2+ pools were not disturbed by PI(3,4,5)P3. RBL-2H3 cells were exposed to 20 μmPI(3,4,5)P3 in a Ca2+-depleted medium, followed by 1 μm thapsigargin. As shown in Fig.2, PI(3,4,5)P3 did not affect the extent of thapsigargin-induced Ca2+ response visà vis that without PI(3,4,5)P3 pretreatment. Together, these results bore out the premise that PI(3,4,5)P3-induced Ca2+ response was independent of PLC activity and was solely attributable to Ca2+ influx from the medium. The existence of a PI(3,4,5)P3-sensitive Ca2+ entry mechanism suggests a link between PI3K and receptor-activated Ca2+signaling in RBL-2H3 cells. Because both PI3K and PLCγ are downstream effectors in FcεRI-mediated tyrosine kinase cascades, we hypothesized that PI3K acted in concert with PLCγ in initiating the Ca2+ response to FcεRI cross-linking. To test this hypothesis, a combination of pharmacological and molecular genetic approaches was employed to characterize the role of PI3K in FcεRI-induced Ca2+ response. First, the effect of the PI3K inhibitor LY294002 on antigen-stimulated Ca2+ response in fura-2-loaded RBL-2H3 cells was assessed. In this study, LY294002 was used in lieu of wortmannin to inhibit PI3K in cells in light of the nonspecific effect of wortmannin on myosin light-chain kinase, one of the key regulatory enzymes in mast cells (8Yano H. Agatsuma T. Nakanishi S. Saitoh Y. Fukui Y. Nonomura Y. Matsuda Y. Biochem. J. 1995; 312: 145-150Crossref PubMed Scopus (60) Google Scholar). RBL-2H3 cells were sensitized with DNP-specific IgE overnight, loaded with the fluorescence indicator, and pretreated with varying concentrations of LY294002. After stimulation with the antigen DNP-HSA (1 μg/ml), changes in [Ca2+]i were monitored by fluorimetry. In line with the earlier reports that the antigen-stimulated Ca2+ response was largely attributable to Ca2+ influx (11Barker S.A. Lujan D. Wilson B.S. J. Leukoc. Biol. 1999; 65: 321-329Crossref PubMed Scopus (71) Google Scholar, 31Kim T.D. Eddlestone G.T. Mahmoud S.F. Kuchtey J. Fewtrell C. J. Biol. Chem. 1997; 272: 31225-31229Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), depletion of external Ca2+ by EGTA substantially diminished the Ca2+signal following antigen stimulation (Fig.3 A, inset). Fig. 3 indicates that LY294002 pretreatment attenuated the amplitude of antigen-induced Ca2+ response in a dose-dependent manner. The effect of LY294002 on the Ca2+ response attained maximum at 200 μm, beyond which no further decrease was noticed (data not shown). Presumably, the residual Ca2+ response following LY294002 treatment was attributed to Ins(1,4,5)P3-induced Ca2+ release and the consequent c" @default.
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• W2106180589 title "Phosphoinositide 3-Kinase Facilitates Antigen-stimulated Ca2+ Influx in RBL-2H3 Mast Cells via a Phosphatidylinositol 3,4,5-Trisphosphate-sensitive Ca2+Entry Mechanism" @default.
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• W2106180589 doi "https://doi.org/10.1074/jbc.m009851200" @default.
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