As expected, wild-type catestatin and its variants induced considerable increases of intracellular Ca2+ mobilization in human mast cells. These Ca2+ increases were dose-dependent, and catestatin concentrations as low as 1·25 μm caused large amounts of Ca2+ influx, reaching a peak at around 50 seconds after the addition of catestatin peptides (Fig. 4a). Because catestatin is a potent

chemoattractant for monocytes,9 we evaluated whether this peptide would also chemoattract human mast cells. selleck compound In support of our hypothesis, wild-type catestatin and its variants induced mast cell chemotaxis, and the dose-dependence of this effect gave a bell-shaped curve. The optimal chemotactic concentration was as low as 0·32 μm, whereas higher concentrations of catestatin peptides resulted in the inhibition of cell migration. Scrambled catestatin had no effect on LAD2 mast cell migration (Fig. 4b). Similar results with 0·32 μm wild-type catestatin and its variants were observed in human peripheral

blood-derived cultured mast cells (Fig. 4c). To evaluate the cellular mechanisms by which catestatins activate human mast cells, we investigated whether the G-protein and PLC pathways were Linsitinib order involved in catestatin-mediated human mast cell activation by using the specific inhibitors, pertussis toxin and U-73122, respectively. Prior treatment of the mast cells with pertussis toxin or U-73122 significantly suppressed the mast cell degranulation and release of LTC4, PGD2 and PGE2 induced by wild-type catestatin and its variants (Fig. 5a–d). In addition, both inhibitors markedly suppressed mast cell chemotaxis, intracellular Ca2+ mobilization, and the production of cytokines and chemokines (Fig. 5e–j). U-73122 was more potent than pertussis toxin, and its inactive control, U-73343, had no effect on mast cell activation. To further understand the signalling pathways of catestatin peptides in human mast cells, we also examined

whether these peptides could activate MAPK pathways. The MAPK pathway was a likely candidate because it has been reported Farnesyltransferase to be responsible for AMP-mediated activation of mast cells,1,15 and because catestatin induces human monocyte migration via MAPK activation.9 As shown in Fig. 6(a), wild-type catestatin and its variants almost identically enhanced phosphorylation of ERK and JNK, but not p38 in mast cells, as observed after 5 min of stimulation with catestatin peptides. Scrambled catestatin had no effect on MAPK phosphorylation. Notably, longer exposure of mast cells to catestatin peptides, up to 60 min, did not lead to enhanced p38 phosphorylation (data not shown). The requirement for MAPK signalling pathways in catestatin-induced mast cell stimulation was evaluated by pre-treating mast cells with specific inhibitors for ERK and JNK: U0126 and SP600125, respectively. As shown in Fig.