Evidence for G-Protein-Dependent and G-Protein-Independent Activation of Phospholipase D in Lymphocytes

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    Evidence for G-Protein-Dependent and G-Protein-IndependentActivation of Phospholipase D in Lymphocytes

    Yu-Zhang Cao, Padala V. Reddy, Lorraine M. Sordillo,George R. Hildenbrandt, and C. Channa Reddy1

    Environmental Resources Research Institute and Department of Veterinary Science, The Pennsylvania StateUniversity, University Park, Pennsylvania 16802

    Received October 24, 1996

    Previously we reported that tumor-promoting phorbol esters stimulate phospholipase D (PLD) independentof protein kinase C (PKC) activation in bovine lymph node lymphocytes. (Cao et al., Biochem. Biophys.Res. Commun. 171, 955962, 1990; 217, 908915, 1995). In the present study, we examined the effectsof prostagladins (PGs), E2 , F2a , D2, and H2 on PLD activity as measured by conversion of [1-14C] arachidonicacid-labeled phospholipids into phosphatidylethanol (PEt) in bovine lymph node lymphocytes. Prostaglan-dins stimulated the formation of PEt at an optimal concentration of 10 mM with relative stimulatory effecton the order of PGE2 PGF2a PGH2 PGD2. The PGE2-stimulated formation of PEt was dose-dependent in the range of 0.1 to 10 mM and was not inhibited by PKC inhibitors staurosporine and K252a.When both PGE2 and 12-0-tetradecanoylphorbol-13-acetate (TPA) were included, their effect on the PLDactivation was additive. Furthermore, NaF, a G-protein activator, stimulated the PEt formation. Interestingly,the stimulatory effects of PGE2 and NaF were not additive; however, the formation of PEt by NaF andTPA was additive. These results suggest that similar to TPA, PGs increase PLD activity independent ofPKC and the stimulation by PGs and TPA in lymphocytes may involve both G-protein-dependent and G-protein-independent signaling pathways. q 1996 Academic Press, Inc.

    Phospholipase D (EC (PLD) activation has been recognized as an important routeof signal transduction in a number of cell types. It has been implicated in the regulation ofDNA synthesis, cell proliferation and many other cellular functions (1). Several factors, likeGTP-binding protein (G-protein), protein kinase C (PKC), protein-tyrosine kinase and calciumions have been reported to regulate PLD activation. Phospholipase D activity was shown tobe enhanced by the non-hydrolyzable GTP analogue GTP-g-S in a number of systems, includ-ing HL-60 cells (1), human neutrophils (2), dog brain microsomes (3), and rat liver plasmamembranes (4). Recently, it was reported that prostaglandin (PG) F2a , an arachidonic acidmetabolite, stimulates the activation of PLD in osteoblast-like cells(5) and in chinese hamsterovary (CHO) cells (6).

    In the previous studies (7,8), we demonstrated that the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) activates PLD via a PKC-independent mechanism in bovine lymphnode lymphocytes. However, the precise mechanism(s) of PLD activation by TPA in immunecells have not been established. The present study was undertaken to delineate the molecularmechanisms of PLD activation by TPA as well as the role of PGs and NaF, a G-proteinactivator, in PLD activation in bovine lymphocytes.

    MATERIALS AND METHODSMaterials. [1-14C] arachidonic acid ( specific activity 51.7 mCi/mmol) was purchased from Dupont NEN, Boston,

    MA. DMSO, TPA, PGE2, PGD2, PGF2a , staurosporine, NaF, and calcium ionophore A23187 were purchased from

    1 Corresponding author. Fax: (814) 863-1696. E-mail: ccr1@psu.edu.

    0006-291X/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.


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    FIG. 1. Relative effects of various agonists on PLD activity. Autoradiography of TLC separation of lipids extractedfrom cell cultures prelabeled with 1-14C arachidonic acid and incubated with various test compounds: (1) 0.1% DMSO;(2) 10 mM PGE2; (3) 100 nM TPA; (4) 0.2 mM 15-HPETE; (5) 40 mM NaF; (6) 1 mM Calcium ionophore A23187;(7) 3 mM Staurosporine / 100 nM TPA; (8) 1 mM K252a / 100 nM TPA. Incubation was for 3 h in the presenceof 1% ethanol or 0.1% DMSO and indicated test compounds.

    Sigma Chemical Co., St. Louis, MO. PGH2 was prepared in our laboratory as previously described (9). K252a wasobtained from Kamiya Biochemical Co., Thousand Oaks, CA. RPMI-1640 with L-glutamine medium was purchasedfrom Mediatech Co., Washington, DC. Precoated silica gel 150A plates were from Whatman Laboratory, Clifton, NJ.

    Preparation of bovine lymph node lymphocytes. Lymphocytes were prepared from bovine retropharyngeal lymphnodes and cultured as described previously (10). Cell cultures (108 cells/5 ml) were prelabeled with [1-14C] arachidonicacid (0.1 mCi/ml) at 377C for 1 h, washed once with RPMI-1640 medium and resuspended in fresh medium.

    Measurement of phospholipase D activity. Quantitation of radioactive phosphatidylethanol (PEt) formed from theendogenous prelabeled phospholipids in the presence of ethanol was used to measure PLD activity. All incubationson prelabeled cultures were conducted in presence of test compounds and 1.0 % ethanol for 3 h or as indicated inthe legends of Figure and Tables. Test compounds were included in the 1.0 % ethanol additions except for TPAwhich was added in DMSO (0.1 % final concentration). In the experiments containing PKC inhibitor, the inhibitorwas added 30 min prior to the addition of test compound and 1.0 % ethanol. The cells were centrifuged at 300 1 gfor 10 min and washed once with phosphate-buffered saline (PBS) and the lipids were extracted. The lipid extractswere evaporated and applied to precoated silica gel 150A plates and the chromatograms developed with the organicphase of ethyl acetate-2,2,4-trimethylpentane-acetic acid-water (11:5:2:10, v/v/v/v). The lipids were visualized byiodine vapors and the radioactivity in PEt was determined by scraping the PEt band and subjecting it to liquidscintillation spectrometry as described before (7).

    Preparation of PEt standard. A PEt standard was synthesized using cabbage PLD and egg lecithin as describedby Kobayashi and Kanfer (11).

    Data analysis. Data were analyzed by the Students t test and the statistical significance was assigned at p 0.05.


    Effect of various agonists on PEt formation catalyzed by PLD. It is well recognized thatthe assay of PEt formation has many advantages over the assay of phosphatidic acid (PA) inthe measurement of PLD activity. Double-labeling method demonstrated that PEt was formedfrom phospholipids exclusively by the action of PLD and it was metabolically stable, lastingup to 12 h after agonist treatment (12,13). Thus, in the present study, the PEt formation wasmeasured to evaluate the activity of PLD in intact cells. As shown in Figure 1, TPA greatlystimulated PEt formation in lymphocytes. PGE2 and NaF also stimulated the PEt formation. Aspreviously reported by us, the time course of PEt accumulation in TPA stimulated lymphocytesindicates that significant PEt accumulated within 15 min. The rate of increase remained rela-tively linear for 3 h. The increase in PEt with TPA was approximately 10 times greater thanthat with 0.1% DMSO alone with the highest stimulation of PEt synthesis seen at 1 1 1007M TPA (7).

    The relative effects of different PGs on PLD-mediated PEt formation. As shown in Table631

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    TABLE 1Relative Effects of PGs on PLD-Mediated

    PEt Formation

    Test compounds PEt formation (CPM)

    Control 320 { 35PGE2a 1123b { 115PGF2aa 729b { 103PGD2a 469b { 37PGH2a 561b { 45

    a All incubations on prelabeled cultures wereconducted in the presence of test compoundsat 10 mM concentration and 1% ethanol for3 h.

    b The values with a superscript are signifi-cantly different from the control value (p 0.05). Data are expressed as mean { S.D. forthree individual experiments.

    1, at 10 mM concentration, PGE2, PGF2a , PGD2, and PGH2 caused significant increase (p0.05)in PLD activity in bovine lymph node lymphocytes with PGE2 showing the highest stimulatoryeffect followed by PGF2a PGH2 PGD2. Time course of PGE2-stimulated PEt formationin bovine lymphocytes revealed that significant PEt accumulated within 1 h and the linearincrease continued up to 3 h. A dose response study (not shown) revealed that PGE2 stimulatedthe formation of PEt in a concentration dependent manner in the range between 0.1-10 mMwith the maximum stimulation of PEt formation achieved at 10 mM. At this concentration thePEt formation by PGE2 was increased approximately to 3 fold over the control (Table 1).

    The effect of PKC inhibitors on the activation of PLD by TPA and PGE2. We examinedthe effects of two potent PKC inhibitors K252a and staurosporine on TPA- and PGE2-stimulatedPEt formation in intact lymphocytes. These two compounds have been shown to be veryselective in inhibiting PKC activity in different cells (14). Both K252a and staurosporine failedto affect PEt formation stimulated by TPA and PGE2 (Figure 1 and Table 2). These resultsstrongly suggest that PKC is not involved in the activation of PLD by TPA and PGE2 inbovine lymph node lymphocytes.

    The comparative effects of PGE2, NaF, and TPA on PEt formation. Sodium fluoride (NaF),a nonspecific activator of G-protein which has been widely used to activate G-protein (4,15),was employed to check its effect on activation of PLD. It was found that NaF at the concentra-tion of 10-40 mM stimulates the formation of PEt in bovine lymphocytes with the maximumstimulation observed at 40 mM. At the maximum stimulatory concentration , PGE2 (10 mM)and NaF (40 mM) together did not result in additiv


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