Phosphatidylcholine-specific phospholipase D-derived 1,2-diacylglycerol does not initiate protein kinase C activation in the RBL 2H3 mast-cell line.
We examined the role of phosphatidylcholine-specific phospholipase D (PC-PLD) in the IgE-dependent activation of protein kinase C (PKC) in RBL 2H3 cells (a model for mast-cell function). Cells were sensitized with mouse monoclonal anti-trinitrophenol (TNP) IgE (0.5 micrograms/ml) and were then triggered with an optimal concentration (10 ng/ml) of TNP-ovalbumin conjugate (TNP-OVA). This resulted in an immediate biphasic increase in the production of 1,2-diacylglycerol (DAG) and activation of PKC. The initial increase in DAG production reached a peak within 30 s, and the second phase reached a plateau within 5 min after stimulation. TNP-OVA-induced PC-PLD activation followed the initial increase in DAG formation in response to IgE-receptor cross-bridging, but coincided with the second peak. Phosphatidic acid (PA), derived from the PC-PLD pathway, is metabolized to DAG by the action of PA phosphohydrolase (PAPase). Propranolol (0.3 mM), which inhibits PAPase, blocked the IgE-dependent increase in DAG, activation of PKC, and subsequently degranulation. The PKC inhibitor staurosporine (0.1 microM) inhibited the second, but not first, peak of DAG accumulation, reversed PKC translocation after 10 min and inhibited subsequent mediator release. Taken together, these results demonstrate that PC-PLD does not initiate, but may play a latent role in, IgE-dependent DAG production, PKC activation and mediator release from RBL 2H3 cells.
Lin
P
P
Department of Pharmacology, Hoffmann-La Roche, Nutley, NJ 07110.
Fung
W J
WJ
Gilfillan
A M
AM
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002451:Cell Compartmentation / Q000187:drug effects; D004075:Diglycerides / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology; D006636:Histamine Release / Q000187:drug effects; D066298:In Vitro Techniques; D008407:Mast Cells / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D011433:Propranolol / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017455:Receptors, IgE / Q000502:physiology; D015398:Signal Transduction; D019311:Staurosporine; D014407:Tumor Cells, Cultured
D000470:Alkaloids; D004075:Diglycerides; D010713:Phosphatidylcholines; D017455:Receptors, IgE; D000431:Ethanol; D011433:Propranolol; D011493:Protein Kinase C; D010739:Phospholipase D; D019311:Staurosporine