Figure 4.

Catestatins mediate intracellular Ca²⁺ mobilization and mast cell chemotaxis. (a) LAD2 cells (2 × 10⁵ cells) were incubated for 1 hr at 37° in Hanks' balanced salt solution containing HEPES, probenecid and Calcium 3 Reagent, and then stimulated with 1·25–5 μm wild-type catestatin (WT), Gly364Ser (G364S), Pro370Leu (P370L) or Arg374Gln (R374Q), or diluent 0·01% acetic acid (Ctrl, control). The data presented are representative of four separate experiments and are shown as the changes in fluorescence. (b) LAD2 cells (50 μl from 3 × 10⁶ cells/ml) were placed in the upper wells of a chemotaxis micro-chamber and allowed to migrate towards 0·02–1·25 μm WT catestatin, G364S, P370L or R374Q, 0·32 μm scrambled catestatin (sCst), or diluent 0·01% acetic acid (Ctrl, control) for 3 hr at 37°. Chemotaxis was assessed by counting under a light microscope the number of cells that migrated through the polycarbonate membrane with 8-μm diameter pores, in three randomly chosen high-power fields. Values were compared between stimulated and non-stimulated cells (Ctrl, control). *P < 0·05, **P < 0·01, #P < 0·001. Each bar represents the mean ± SD of nine separate experiments. (c) Peripheral blood-derived mast cells (50 μl from 3 × 10⁶ cells/ml) were allowed to migrate towards 0·32 μm WT catestatin, G364S, P370L or R374Q, or diluent 0·01% acetic acid (Ctrl, control), and chemotaxis assay was performed as above. Values were compared between stimulated and untreated cells (Ctrl, control). *P < 0·001. Each bar represents the mean ± SD of three separate experiments.