Ceramide induces serotonin release from RBL-2H3 mast cells through calcium mediated activation of phospholipase A2

Ceramide has been suggested to function as a mediator of exocytosis in response to the addition of a calcium ionophore from PC12 cells. Here, we show that although cell-permeable C6-ceramide or a calcium ionophore alone did not increase either the degranulation of serotonin or the release of arachidonic acid (AA) from RBL-2H3 cells, their combined effect significantly stimulated these processes in a time- and dose-dependent manner. This effect was inhibited by the presence of an exogenous calcium chelator and significantly suppressed by the CERK inhibitor (K1) and phospholipase A2 (PLA2) inhibitors. Moreover, cytosolic PLA2 GIVA (cPLA2 GIVA) siRNA-transfected RBL-2H3 cells showed a lower level of serotonin release than scramble siRNA-transfected cells.Little is known about the regulation of degranulation proximal to the activation of cytosolic phospholipase A2 GIVA, the initial rate-limiting step in RBL-2H3 cells. In this study, we suggest that CERK, ceramide-1-phosphate, and PLA2 are involved in degranulation in a calcium-dependent manner. Inhibition of p44/p42 mitogen-activated protein kinase partially decreased the AA release, but did not affect degranulation. Furthermore, treatment of the cells with AA (ω-6, C20:4), not linoleic acid (ω-6, C18:2) or α-linolenic acid (ω-6, C18:3), induced degranulation. Taken together, these results suggest that ceramide is involved in mast cell degranulation via the calcium-mediated activation of PLA2.

► C6-ceramide or A23187 alone did not induce the release of either AA or serotonin.
► Degranulation significantly increased when Cer and A23187 were added in combination.
► AA and serotonin release were significantly inhibited by CERK and PLA2 inhibitors.
► Transfection of cPLA2siRNA resulted in decreased serotonin release.
► Treatment of the cells with AA effectively induced serotonin release.