Phosphoinositide 3-Kinase Facilitates Antigen-stimulated Ca 2 1 Influx in RBL-2H3 Mast Cells via a Phosphatidylinositol 3,4,5-Trisphosphate-sensitive Ca 2 1 Entry Mechanism*
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This study presents evidence that phosphoinositide 3-kinase (PI3K) plays a concerted role with phospholipase C g in initiating antigen-mediated Ca 2 1 signaling in mast cells via a phosphatidylinositol 3,4,5-trisphos-phate (PI(3,4,5)P 3 )-sensitive Ca 2 1 entry pathway. Exogenous PI(3,4,5)P 3 at concentrations close to its physio- logical level induces instantaneous Ca 2 1 influx into RBL-2H3 cells. This PI(3,4,5)P 3 -induced intracellular Ca 2 1 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 D p85 suppresses the Ca 2 1 response to the cross-linking of the high affinity receptor for IgE (Fc e RI). Concomitant treatment of RBL-2H3 cells with LY294002 or D p85 and 2-aminoethyl diphenylborate, a cell-permeant antagonist of D - myo -inositol 1,4,5-trisphosphate receptors, abrogates antigen-in-duced Ca 2 1 signals, whereas either treatment alone gives rise to partial inhibition. Conceivably, PI(3,4,5)P 3 D p85; PH, pleckstrin homology; fura-2 AM, fura-2 acetoxymethyl ester; fluo-3 AM, fluo-3 acetoxymethyl ester; CMV, cytomegalovirus; di-C8-PI(3,4,5)P 3 , 1- O -(1,2-di- O -octanoyl- sn -glycero-3- O -phosphoryl)- D - myo -inositol 3,4,5- trisphosphate. O O sn D - -inositol form (PI(3)P) synthe-sized as previously reported (21, 22). The identity and purity of all inositol phosphates and inositol lipids were verified by 1 H and 31 P NMR and high resolution mass spectrometry. Published data from this and other laboratories have shown that PI(3,4,5)P 3 and other inositol lipids cell-permeant can readily fuse with cell membranes to exert cellular responses in different types of cells, platelets adipocytes Jurkat Fura-2