The role of calcium and protein kinase C in the IgE-dependent activation of phosphatidylcholine-specific phospholipase D in a rat mast (RBL 2H3) cell line.
Our previous studies have suggested that phosphatidylcholine-specific phospholipase D (PtdCho-PLD) plays a role in IgE-dependent diacylglycerol production, protein kinase C activation and mediator release in the RBL 2H3 mast cell line. We have extended these studies to examine the mechanisms by which PtdCho-PLD may be regulated in these cells. RBL 2H3 cellular lipids were labeled with [14C]arachidonic acid or [3H]myristic acid, then PtdCho-PLD activity was monitored by the formation of radiolabeled phosphatidylethanol when ethanol was included in the incubation medium. Trinitrophenol-ovalbumin conjugate (10 ng/ml), when added to cells previously sensitized with anti-(trinitrophenelated mouse IgE) (0.5 microgram/ml), ionomycin (1 microM) and thapsigargin (0.1 microM), stimulated PtdCho-PLD activation and mediator release in cells incubated in buffer containing 1.8 mM calcium, but not in cells incubated in calcium-free, buffer. Phorbol 12-myristate 13-acetate (0.1 microM) activated PtdCho-PLD in both buffers, but on its own did not trigger mediator release. When intracellular calcium was chelated with 5,5'-dimethyl-1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, trinitrophenol-ovalbumin conjugate failed to activate PtdCho-PLD and histamine release. Similarly, down-regulation of protein kinase C activity by long-term exposure to the phorbol ester (0.1 microM) and preincubation of the cells with protein kinase inhibitors resulted in the loss of the trinitrophenol-ovalbumin response on PtdCho-PLD activity and histamine release. Taken together, the above results suggest that IgE-dependent PtdCho-PLD activation is dependent on both activation of protein kinase C and a rise in the intracellular free calcium concentration.
Lin
P
P
Department of Pharmacology, Hoffmann-La Roche, Nutley, NJ 07110.
Gilfillan
A M
AM
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D006636:Histamine Release / Q000187:drug effects; D007073:Immunoglobulin E / Q000502:physiology*; D007211:Indoles / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D008407:Mast Cells; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D019311:Staurosporine; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin
D000470:Alkaloids; D007211:Indoles; D008301:Maleimides; D013729:Terpenes; D007073:Immunoglobulin E; D015759:Ionomycin; D019284:Thapsigargin; D011493:Protein Kinase C; D010739:Phospholipase D; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; C064758:Ro 31-8220; C064755:Ro 31-7549