Phospholipases Stimulate Secretion in RBL Mast Cells

Abstract

Roles for glycerophospholipids in exocytosis have been proposed, but remain controversial. Phospholipases are stimulated following the activation of the high-affinity receptor for immunoglobulin E (IgE) in mast cells. To study the biochemical sequelae that lead to degranulation, broken cell systems were employed. We demonstrate that the addition of three distinct types of exogenous phospholipases (i.e., bcPLC, scPLD, and tfPLA(2)), all of which hydrolyze phosphatidylcholine (PC), trigger degranulation in permeabilized RBL-2H3 cells, a mucosal mast cell line. Production of bioactive lipids by these phospholipases promotes release of granule contents through the plasma membrane and acts downstream of PKC, PIP(2), and Rho subfamily GTPases in regulated secretion. These exogenous phospholipase-induced degranulation pathways circumvent specific factors activated following stimulation of the IgE receptor as well as in ATP- and GTP-dependent intracellular pathways. Taken together, these results suggest that regulated secretion may be achieved in vitro in the absence of cytosolic factors via phospholipase activation and that products of PC hydrolysis can promote exocytosis in mast cells.

Electrospray ionization mass spectrometry analysis of changes in phospholipids in RBL-2H3 mastocytoma cells during degranulation

“…Interestingly, this increase in PA formation was not apparent from the TLC analysis (see ref. 5), probably because the [ 3 H]myristate-labeled pools were not used in the metabolic formation of PA. It is also possible that tfPLA 2 and bcPLC generate novel substrates that generate PA by secondary metabolism even in these broken cell systems.…”

“…RBL-2H3 cells were grown and permeabilized, and degranulation was stimulated as described (5). Bacillus cereus PC-PLC (bcPLC) and Trimeresurus flavoviridis PLA 2 (tfPLA 2 ) were obtained from Calbiochem.…”

“…Despite extensive study, definition of the roles of phospholipids in secretion and membrane transport processes has been elusive. Recent studies in our laboratory (5) show that changes in membrane phospholipid composition as a result of phospholipase activity are associated with degranulation in mast cells. Because several of the activators of phospholipase D (PLD) activity in mammalian cells (e.g., Arf, Rho, protein kinase C) have themselves been implicated in exocytosis, we decided to explore the effects of changes in lipid composition by using phospholipase enzymes from lower organisms that do not require signal transduction proteins for catalytic regulation.…”

“…Surprisingly, we found that three distinct classes of exogenous phospholipases, all of which were reported to hydrolyze PC preferentially, trigger degranulation in permeabilized RBL-2H3 cells, a mucosal mast cell line. As detected by TLC, production of phosphatidic acid (PA) by PLD, DAG by phospholipase C (PLC), and free fatty acids and lysophosphatidylcholine (LPC) by PLA 2 initiate degranulation independent of endogenous cytosolic factors (5). Production of these bioactive lipids promoted release of granule contents through the plasma membrane.…”

Disruption of lipid order by short-chain ceramides correlates with inhibition of phospholipase D and downstream signaling by FcϵRI

“…These lipases generate important lipid second messengers, as well as amphiphilic products that can modulate membrane structure and influence processes such as vesicle budding and exocytosis (Liscovitch et al, 2000;Cohen and Brown, 2001;Ivanova et al, 2001). Ceramides are the products of sphingomyelin hydrolysis by sphingomyelinases, and these endogenous, long-chain neutral lipids have been implicated as signal transduction mediators in certain cell types (Kolesnick et al, 2000).…”