Inhibition of phospholipase C-independent exocytotic responses in rat peritoneal mast cells by U73122

The aminosteroid U73122 has been established as potent, selective, and cell-permeable inhibitor C-type phosphatidylinositol-specific phospholipases (PI-PLCs), and has been used to define a contribution of PI-PLCs as part of exocytotic signalling pathways in rat peritoneal mast cells (RPMCs). However, doubts have been raised regarding its PI-PLC selectivity of action. Therefore, in the present study, U73122 was tested in PPMCs under experimental conditions allowing to elicit exocytosis PI-PLC independently (streptolysin O [SLO]-permeabilised cells; stimulated by GTPgammaS; in the presence of low concentrations of free Ca2+). The release of [H-3]5-hydroxytryptamine ([H-3]5-HT) from [H-3]5-HT-loaded RPMCs served as measure of secretion. U73122 potently inhibited the exocytotic response induced by 10 muM GTPgammaS (Ca2+: 10(-6) M) in permeabilised cells (IC50: 0.6 muM, n=5) in an insurmountable manner. In intact RPMCs, with a nearly equal potency (IC50: 4 muM, n=4), U73122 also inhibited the PI-PLC-dependent exocytotic response induced by concomitant application of nerve growth factor and lyso-phosphatidylserine (NGF/lyso-PS). Conclusion: U73122 exerts potent PI-PLC-independent secretostatic effects, limiting its use to define PI-PLC function within exocytotic processes. (C) 2004 Elsevier B.V. All rights reserved.