title: Ceramide inhibition of phospholipase D and its relationship to RhoA and ARF1 translocation in GTP gamma S-stimulated polymorphonuclear leukocytes.
abstract: Phospholipase D (PLD) regulates the polymorphonuclear leukocyte (PMN) functions of phagocytosis, degranulation, and oxidant production. Ceramide inhibition of PLD suppresses PMN function. In streptolysin O-permeabilized PMNs, PLD was directly activated by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) stimulation of adenosine diphosphate (ADP)-ribosylation factor (ARF) and Rho, stimulating release of lactoferrin from specific granules of permeabilized PMNs; PLD activation and degranulation were inhibited by C2-ceramide but not dihydro-C2-ceramide. To investigate the mechanism of ceramide's inhibitory effect on PLD, we used a cell-free system to examine PLD activity and translocation from cytosol to plasma membrane of ARF, protein kinase C (PKC)alpha and beta, and RhoA, all of which can activate PLD. GTP gamma S-activated cytosol stimulated PLD activity and translocation of ARF, PKC alpha and beta, and RhoA when recombined with cell membranes. Prior incubation of PMNs with 10 microM C2-ceramide inhibited PLD activity and RhoA translocation, but not ARF1, ARF6, PKC alpha, or PKC beta translocation. However, in intact PMNs stimulated with N-formyl-1-methionyl-1-leucyl-1-phenylalamine (FMLP) or permeabilized PMNs stimulated with GTP gamma S, C2-ceramide did not inhibit RhoA translocation. Exogenous RhoA did not restore ceramide-inhibited PLD activity but bound to membranes despite ceramide treatment. These observations suggest that, although ceramide may affect RhoA in some systems, ceramide inhibits PLD through another mechanism, perhaps related to the ability of ceramide to inhibit phosphatidylinositol-bisphosphate (PIP2) interaction with PLD.
authors: Department of Pediatrics, Division of Hematology/Oncology, University of Michigan, Ann Arbor, 48109, USA.
authors: Pamela J
authors: PJ
authors: Mansfield
authors: Shannon S
authors: SS
authors: Carey
authors: Vania
authors: V
authors: Hinkovska-Galcheva
authors: James A
authors: JA
authors: Shayman
authors: Laurence A
authors: LA
authors: Boxer
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism*; D001426:Bacterial Proteins; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007781:Lactoferrin / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects* / Q000201:enzymology; D010739:Phospholipase D / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010766:Phosphorylation; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D013301:Streptolysins / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; C064769:N-acetylsphingosine; D013301:Streptolysins; C025964:streptolysin O; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010739:Phospholipase D; D007781:Lactoferrin; D020823:ADP-Ribosylation Factor 1; D020742:rhoA GTP-Binding Protein; D013110:Sphingosine