Figure 4.

Relationship between the FcɛRI-mediated inhibition of M₂PK and mast cell degranulation. (a) Effects of exogenously added pyruvate, an allosteric inhibitor of PK, on mast cell degranulation. (a1) RBL-2H3 cells were treated with 1 mM pyruvate for 20 min, followed by 1 μg mL⁻¹ DNP-BSA treatment for 5 min and PK activity was then measured. ^***P<0.001 versus the control group. (a2) RBL-2H3 cells were treated with increasing concentrations of pyruvate and then stimulated with 1 μg mL⁻¹ DNP-BSA for 10 min. The control is the release of hexosaminidase induced by DNP-BSA in the absence of added pyruvate. ^***P<0.001 versus the ‘no pyruvate added' group. (b) Effects of NaF, a moderately selective PK inhibitor, on RBL cell degranulation. The RBL-2H3 cells were treated with 10 mM NaF for 20 min, followed by 100 ng mL⁻¹ DNP-BSA treatment for 5 min; then PK activity was measured (left panel). ^***P<0.001 compared with the control (NaF(−)) group. For degranulation assay, RBL-2H3 cells were treated with 1 μg mL⁻¹ DNP-BSA for 10 min and then mast cell degranulation was measured (right panel). ^***P<0.001 compared with the control (NaF(−)) group. (c) Effects of exogenous M₁PK on mast cell degranulation. (c1) Wild-type RBL-2H3 cells (WT) or RBL-2H3 cells stably expressing the GFP-encoding vector or GFP-M₁PK were used. ^***P<0.001 versus the GFP group. (c2) Cell lysates (20 μg of protein) from each cell type were analysed by dodecyl sulphate-polyacrylamide gel electrophoresis and blotted with antibodies for M-type PK. BSA, bovine serum albumin; GFP, green fluorescence protein; M₁PK, M₁-type pyruvate kinase; M₂PK, M₂-type pyruvate kinase; PK, pyruvate kinase; RBL-2H3, rat basophilic leukaemia cells.