Inhibition of phospholipase C-independent exocytotic responses in rat peritoneal mast cells by U73122.
ABSTRACT 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). CONCLUSION: U73122 exerts potent PI-PLC-independent secretostatic effects, limiting its use to define PI-PLC function within exocytotic processes.
- [Show abstract] [Hide abstract]
ABSTRACT: A variety of agents, including imidazoles, such as miconazole, elicit elevations of calcium in human leukemic HL-60 cells through a stimulation of influx of calcium and/or a release of intracellular calcium. Release of intracellular calcium by such agents may involve stimulation of IP3 formation. The steroidal maleimide U73122, a potent phospholipase C (PLC) inhibitor, at 3–10 µM blocked elevations of calcium elicited in HL-60 cells by ATP. At a 10&!hyphen; µM concentration, U73122 either blocked or altered elevations of calcium elicited by imidazoles, such as miconazole, SKF 96365, and clotrimazole that are often used to block capacitative calcium entry, and by calmidazolium, a quaternary imidazole that can trigger massive influx of calcium in HL-60 cells. Elevations of calcium elicited by thapsigargin were not affected by 3 µM U73122, but were reduced by higher concentrations. Remarkably, U73122 at 10 µM did not block, but instead slowed and potentiated the elevation of calcium elicited by higher concentrations of imidazoles, an effect that may involve inhibition of Ca2+ -ATPases of the endoplasmic reticulum. The steroidal succinimide U73343 had minimal effects on calcium elevations in HL-60 cells. The results indicate that U73122 requires cautious use in assessing the role of PLC in controlling calcium levels in HL-60 cells, in particular when used with imidazoles, such as SKF 96365, which are used to block capacitative calcium entry. The effects of U73122 on cell calcium are reviewed. In toto, the results suggest that many lipophilic agents may affect intracellular calcium by activating PLC through unknown mechanisms. Drug Dev. Res. 67:519–534, 2006. Published 2006 Wiley-Liss, Inc.Drug Development Research 10/2006; 67(6):519 - 534. · 0.87 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The inositol lipid phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] is involved in a myriad of cellular processes, including the regulation of exocytosis and endocytosis. In this paper, we address the role of PtdIns(4,5)P2 in compound exocytosis from rat peritoneal mast cells. This process involves granule-plasma membrane fusion as well as homotypic granule membrane fusion and occurs without any immediate compensatory endocytosis. Using a novel quantitative immunofluorescence technique, we report that plasma membrane PtdIns(4,5)P2 becomes transiently depleted upon activation of exocytosis, and is not detected on the membranes of fusing granules. Depletion is caused by phospholipase C activity, and is mandatory for exocytosis. Although phospholipase C is required for Ca2+ release from internal stores, the majority of the requirement for PtdIns(4,5)P2 hydrolysis occurs downstream of Ca2+ signalling - as shown in permeabilised cells, where the inositol (1,4,5)-trisphosphate-Ca2+ pathway is bypassed. Neither generation of the PtdIns(4,5)P2 metabolite, diacylglycerol (DAG) or simple removal and/or sequestration of PtdIns(4,5)P2 are sufficient for exocytosis to occur. However, treatment of permeabilised cells with DAG induces a small potentiation of exocytosis, indicating that it may be required. We propose that a cycle of PtdIns(4,5)P2 synthesis and breakdown is crucial for exocytosis to occur in mast cells, and may have a more general role in all professional secretory cells.Journal of Cell Science 06/2006; 119(Pt 10):2084-94. · 5.88 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Phospholipase C (PLC) enzymes are an important family of regulatory proteins involved in numerous cellular functions, primarily through hydrolysis of the polar head group from inositol-containing membrane phospholipids. U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione), one of only a few small molecules reported to inhibit the activity of these enzymes, has been broadly applied as a pharmacological tool to implicate PLCs in diverse experimental phenotypes. The purpose of this study was to develop a better understanding of molecular interactions between U73122 and PLCs. Hence, the effects of U73122 on human PLCβ3 (hPLCβ3) were evaluated in a cell-free micellar system. Surprisingly, U73122 increased the activity of hPLCβ3 in a concentration- and time-dependent manner; up to an 8-fold increase in enzyme activity was observed with an EC50=13.6±5 μm. Activation of hPLCβ3 by U73122 required covalent modification of cysteines as evidenced by the observation that enzyme activation was attenuated by thiol-containing nucleophiles, l-cysteine and glutathione. Mass spectrometric analysis confirmed covalent reaction with U73122 at eight cysteines, although maximum activation was achieved without complete alkylation; the modified residues were identified by LC/MS/MS peptide sequencing. Interestingly, U73122 (10 μm) also activated hPLCγ1 (>10-fold) and hPLCβ2 (∼2-fold); PLCδ1 was neither activated nor inhibited. Therefore, in contrast to its reported inhibitory potential, U73122 failed to inhibit several purified PLCs. Most of these PLCs were directly activated by U73122, and a simple mechanism for the activation is proposed. These results strongly suggest a need to re-evaluate the use of U73122 as a general inhibitor of PLC isozymes.Journal of Biological Chemistry 01/2011; 286(14):12407-16. · 4.65 Impact Factor