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 RPMCs 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 [3H]5-hydroxytryptamine ([3H]5-HT) from [3H]5-HT-loaded RPMCs served as measure of secretion. U73122 potently inhibited the exocytotic response induced by 10 microM GTPgammaS (Ca2+: 10(-6) M) in permeabilised cells (IC50: 0.6 microM, n=5) in an insurmountable manner. In intact RPMCs, with a nearly equal potency (IC50: 4 microM, 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).


CONCLUSIONS
U73122 exerts potent PI-PLC-independent secretostatic effects, limiting its use to define PI-PLC function within exocytotic processes.
Gloyna
Wiebke
W
St. Josef Hospital, Ruhr-University of Bochum, Gudrunstr. 56, D-44791 Bochum, Germany.
Schmitz
Frank
F
Seebeck
Jörg
J
D000818:Animals; D001426:Bacterial Proteins / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004963:Estrenes / Q000494:pharmacology*; D005089:Exocytosis; D005260:Female; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020128:Inhibitory Concentration 50; D036341:Intercellular Signaling Peptides and Proteins; D008297:Male; D008407:Mast Cells / Q000166:cytology; D010455:Peptides; D010537:Peritoneum / Q000166:cytology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction; D013301:Streptolysins / Q000494:pharmacology; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism*; D014862:Wasp Venoms / Q000494:pharmacology
D001426:Bacterial Proteins; D004963:Estrenes; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D047428:Protein Kinase Inhibitors; D011760:Pyrrolidinones; D013301:Streptolysins; D014862:Wasp Venoms; C025964:streptolysin O; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C074854:U 73343; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C037500:mastoparan; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium