Stimulated Phospholipase D Activity Research Articles

Wortmannin has opposite effects on phorbol ester-induced DNA synthesis and phosphatidylcholine hydrolysis

The tumor promoter phorbol 12-myristate 13-acetate (PMA) and hormonal activators of protein kinase C (PKC) commonly stimulate phospholipase D (PLD)-mediated formation of phosphatidic acid from phosphatidylcholine (PtdCho) in fibroblasts and other cell types. On the basis that phosphatidic acid is a mitogen, PLD is often considered to have a major role in the regulation of cell growth by PKC activators. However, we found that in NIH 3T3 fibroblasts wortmannin, an inhibitor of phosphatidylinositol 3-kinase (P13K), strongly inhibited DNA synthesis induced by 100 nM PMA, while it actually enhanced PMA-stimulated PtdCho hydrolysis. These results indicate that stimulation of PLD activity is either not required or not sufficient for the mitogenic action of PMA.

Effect of prostaglandin E2 on phospholipase D activity in osteoblast-like MC3T3-E1 cells.

Recent evidence indicates that phosphatidylcholine breakdown by phospholipase D (PLD) is an important cellular control mechanism. We investigated the signaling pathway participating in prostaglandin E2 (PGE2)-induced PLD activation in osteoblast-like MC3T3-E1 cells. PGE2 stimulated PLD activity, as measured by choline generated from phosphatidylcholine, just after the stimulation. The reaction reached a plateau 15 minutes later. PGE2 stimulated PLD activity in a dose-related manner and also increased inositol phosphate (IP) formation. However, the EC50 value for PGE2-induced IP formation is lower than that for PLD activation. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, stimulated PLD activity, and a combination of PGE2 and TPA potentiated it in an additive manner. Although NaF, a heterotrimeric GTP-binding protein activator, significantly stimulated PLD activity, this effect was not augmented by combination with PGE2. PGE2-induced PLD activity was markedly suppressed by either chelating extracellular Ca2+ by EGTA or pertussis toxin. These findings suggest that osteoblasts might have at least two PLD activation mechanisms which involve PKC-dependent or -independent pathways. However, present results indicate that PKC is unlikely to be essential to PGE2-induced PLD activation. On the contrary, pertussis toxin-sensitive GTP-binding protein and extracellular Ca2+ might play important roles in the pathway of PGE2-induced PLD activation.

Purinergic agonist and G protein stimulation of phospholipase D in rat liver plasma membranes. Independence from phospholipase C activation

Hormonal regulation of phospholipase D (PLD) was studied in isolated rat liver plasma membranes. Purinergic agents and a submaximal concentration of guanosine 5′-0-(3-thiotriphosphate) (GTPγS), a non-hydrolyzable analog of GTP, synergistically stimulate phosphatidylethanol formation, a measure of PLD activity. The rank order of efficacy for stimulation of PLD activity in the presence of 0.2 μM GTPγS was β, γ-methylene-ATP > adenosine 5′-0-(3-thiotriphosphate) = ATP = ADP = 2-methylthio-ATP > α, β-methylene-ATP = UTP. This pattern of activation does not conform to the series at known P 2 receptors. GTPγS stimulated PLD activity in a dose-dependent manner, and the GTPyS dose-response curve for phosphatidylethanol formation was shifted to the left by an analog of ATP. Activation of PLD by purinergic agents in the presence of GTPγS supports the involvement of a purinergic receptor of the P 2 class and a GTP-binding protein. Purinergic agents competitively inhibited [ 35S]adenosine 5′-0-(3-thiotriphosphate) binding to plasma membranes in the rank order adenosine 5′-0′(3-thiotriphosphate) > ATP > α, β-methylene-ATP = UTP ⪢ β γ-methylene-ATP = ADP. Stimulation of phosphoinositide phospholipase C (PI-PLC) by purinergic agents, as measured by release of radioactivity from endogenously myo[ 3H] inositol-labeled plasma membranes, occurred in the order α, β- methylene- ATP ⪢ 2- methylthio- ATP. β, γ-meth-ylene-ATP had little effect on PI-PLC activity. Different dose-response relationships for agonist-stimulation of PI-PLC and PLD indicate that activation of PI-PLC is not involved in stimulation of PLD in rat liver plasma membranes, and suggest that purinergic activation of PLD occurs via a pathway involving a G protein and a heretofore uncharacterized P 2 receptor.

Partial Purification and Characterization of Arf-sensitive Phospholipase D from Porcine Brain

Phospholipase D (PLD) activity from membranes of cultured cells can be activated by guanosine 5'-O-(3-thiotriphosphate) and the small GTP-dependent protein, Arf. While this activity was readily apparent in membranes from HL60 cells, it was much lower or not observable in membranes from various mammalian tissues. However, extraction of porcine brain membranes with detergent and subsequent chromatography with SP-Sepharose revealed a large peak of Arf-sensitive PLD activity. This activity has been enriched through several steps of chromatography and characterized with respect to size, nucleotide specificity, and sensitivity to different Arf and Arf-like proteins. Hydrodynamic analysis indicated that the enriched PLD had an s20,w of 5.1 and a Stokes radius of 4.3 nm. These parameters indicate that the enzyme has an apparent molecular mass of 95,000 Da. Effective stimulation of the enriched enzyme was achieved with GTP as well as nonhydrolyzable analogs. All of the Arf subtypes tested were effective activators of PLD activity. Arf derived from yeast could activate mammalian PLD but with lower potency. The Arf-related Arl proteins were ineffective. PLD that has been highly enriched retained a requirement for phosphatidylinositol 4,5-bisphosphate for efficient expression of activity. Additionally, the ability of recombinant or purified porcine brain Arf to stimulate PLD activity was reduced relative to impure fractions of Arf activity. Thus, porcine PLD that has been purified about 5,000-10,000-fold is synergistically activated by Arf in combination with other cytosolic components that are described in the accompanying paper (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950). Taken together, these data suggest that physiological regulation of Arf-sensitive PLD may involve the coordinate assembly of several interacting regulatory subunits.

Characterization of a regulated form of phospholipase D in the yeast Saccharomyces cerevisiae.

Phospholipase D (PLD), which is present in bacterial, plant and animal cells, can serve as an important element of signal-transduction pathways. This study examined the potential role of this enzyme in the regulation of Saccharomyces cerevisiae. An assay in vitro using a fluorescent 1-acyl-2-alkyl glycerophosphocholine as substrate was used to assess PLD activity in yeast cell extracts. A neutral PLD activity is present in membranes prepared from both haploid and diploid yeast cells, as evidenced by the production of phosphatidic acid and phosphatidylbutanol in the presence of butanol. Alcohols, in addition to serving as substrates for transphosphatidylation, stimulate PLD activity. Increased PLD activity is detected in membranes when either haploid or diploid cells are incubated in the presence of a non-fermentable carbon source. Membrane PLD activity increases within 10 min after diploid cells are placed in a sporulation-inducing medium lacking nitrogen and containing a non-fermentable carbon source. The increased activity persists for 2-3 h, and then declines to control values. This response occurs in the presence of cycloheximide, an inhibitor of protein synthesis. These data indicate that PLD activity is present in yeast, and that activation of PLD is an early event in sporulation in this organism.

Oestradiol-17β modulates PAF-evoked phospholipase D activity but not inositide-lipid hydrolysis in human endometrial cell line, HEC-1B

Platelet-activating factor (PAF) has been shown to stimulate phospholipase D (PLD) activity in human endometrium. The effect of 17β-oestradiol on PAF- and 12-O-tetradecanoylphorbol 13-acetate (TPA)-evoked PLD activity assayed as an accumulation of [ 3H]phosphatidylbutanol was examined in [ 3H]myristic acid labelled in a human endometrial epithelial cell line HEC-1B. TPA stimulated PLD activity in a dose-dependent manner whereas PAF had no significant effect on PLD activity. Following 48 h pretreatment with 100 nM 17β-oestradiol, PAF evoked PLD activity while leaving inositol trisphosphate accumulation in myo-[2- 3H] inositol-labelled HEC-1B cells unaffected. In the 17β-oestradiol-treated cells, TPA-stimulated PLD activity was significantly elevated at 100 nM TPA ( P < 0.05) and 1 μM TPA ( P < 0.05) compared to responses in the untreated cells, suggesting that 17β-oestradiol may upregulate PKC activity. Interestingly, following a 30 min pretreatment of HEC-1B cells with a range of 17β-oestradiol concentrations, TPA (10 nM) and PAF (100 nM) stimulated PLD activity. However, TPA-stimulated PLD activity levels fell 10-fold while PAF-mediated PLD activity remained elevated at 10 nM and 100 nM concentrations of 17β-oestradiol suggesting a different mechanism of activation. These results indicate that 17β-oestradiol can upregulate PAF-induced PLD activity in HEC-1B cells.

Ras Mediates the Activation of Phospholipase D by v-Src

We demonstrated previously that v-Src activates a phospholipase D (PLD) activity (Song, J., Pfeffer, L.M., and Foster, D.A. (1991) Mol. Cell. Biol. 11, 4903-4908) and that this activation is dependent upon a G protein(s) (Jiang H., Alexandropoulos, K., Song, J., and Foster, D.A. (1994) Mol. Cell. Biol. 14, 3676-3682). An in vitro PLD assay was developed to study G protein involvement in v-Src-induced PLD activity. Maximal PLD activity in membranes isolated from v-Src-transformed cells was dependent upon both GTP and cytosol. In this report, we present three lines of evidence demonstrating that v-Src-induced PLD activity is mediated by Ras. First, a neutralizing Ras monoclonal antibody (Y13-259) inhibits PLD activity in membranes isolated from v-Src-transformed Balb/c 3T3 cells. Second, immobilized Ras protein depleted cytosol of the ability to stimulate PLD activity. This effect was dependent upon preloading immobilized Ras with GTP. Last, expression of a dominant negative Ras mutant in v-Src-transformed cells reduced PLD activity to the level observed in the nontransformed parental cells. These data establish a novel role for Ras in the regulation of PLD activity.

Tamoxifen stimulates phospholipase D activity by an estrogen receptor-independent mechanism

The effects of tamoxifen (TAM), a widely used agent in the treatment of breast cancer, were examined on phospholipase D (PLD)-mediated phospholipid hydrolysis. In drug-sensitive MCF-7 human breast carcinoma cells TAM, similar to several well-established activators of PLD, had no effect on phospholipid hydrolysis. In an estrogen receptor-deficient multidrug-resistant subline of MCF-7 cells, TAM preferentially stimulated the hydrolysis of phosphatidylethanolamine; two-fold stimulation required 2.5 or 5 μM TAM in the absence or presence of serum, respectively. In NIH 3T3 fibroblasts significant (4- to 4.8-fold) stimulation of phosphatidylethanolamine and phosphatidylcholine hydrolysis in the presence of serum required 10 μM TAM. These data establish that TAM can stimulate PLD activity by an estrogen receptor-independent mechanism.

Stimulation of protein synthesis and phospholipase D activity by vasopressin and phorbol ester in L6 myoblasts

The effects of 12- O-tetradecanoylphorbol-13-acetate (TPA) and vasopressin on protein synthesis and phospholipase D (PLD) activity were investigated in L6 myoblasts. TPA stimulated a concentration-dependent increase in protein synthesis (EC 50 approx. 10 nM) during a 90 min incubation, but had no effect after 6 h. The maximum increase was about 15% and was mediated through changes in translation, as TPA had no effect on RNA accretion and the response was not prevented by actinomycin D. TPA also stimulated PLD activity as measured by an 8-fold increase in the formation of phosphatidylbutanol (PtdBuOH) and the release of choline (EC 50 5–10 nM). In contrast to TPA, vasopressin stimulated protein synthesis (maximum increase 30%, EC 50 approx. 10 nM) and RNA accretion after 6 h, but had no effect after 90 min. Vasopressin also increased PtdBuOH production 4–5-fold (EC 50 approx. 0.5 nM) and choline release (EC 50 approx. 1 nM). The addition of a highly purified preparation of PLD (2–10 units/ml) from Streptomyces sp. to L6 cells stimulated a concentration-dependent increase in choline release and protein synthesis after both 90 min (maximum stimulation 13%) and 6 h (maximum stimulation 12%). PLD also stimulated RNA accretion after 6 h but not 90 min. The data support a role for PLD in the regulation of protein synthesis in L6 cells.

Basic FGF activates phospholipase D in endothelial cells in the absence of inositol-lipid hydrolysis.

In the absence of inositol-lipid hydrolysis, mitogenic concentrations of basic fibroblast growth factor (bFGF) stimulated phosphatidylbutanol formation in the presence of butan-1-ol in [3H]myristate-labeled human umbilical vascular endothelial (HUVE) cells, indicating that the fibroblast growth factor receptor was able to couple to the activation of phospholipase D (PLD). The ability of bFGF and 12-O-tetradecanoylphorbol-13-acetate (TPA) to stimulate PLD activity was completely abolished in cells pretreated with 400 nM TPA for 48 h to downregulate protein kinase C (PKC). bFGF-stimulated PLD activity was inhibited by genistein (5 microM; P < 0.02) and the PKC inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7, 5 microM; P < 0.001) as well as by the removal of calcium from extracellular environment. bFGF induced DNA synthesis in a dose-dependent manner, and pretreatment of cells with H-7 inhibited the mitogenic activity of bFGF. These results indicate that activation of PKC is responsible for bFGF-induced PLD activation and the mitogenic activity of bFGF in HUVE cells. A coupled PLD/3-sn-phosphatidate phosphohydrolase pathway may play a role in the regulation of endothelial cell proliferation.

The Regulation of Agonist-Stimulated Phospholipase D Activity in Swiss 3T3 Fibroblasts

The work in this thesis aimed to characterise the regulation of phospholipase D (PLD) activity by receptor occupancy in Swiss 3T3 fibroblasts. Bombesin-stimulated PLD activity had previously been found to be partially dependent on protein kinase C (PKC) suggesting that activation of PLD was to some extent down stream of the G-protein coupled receptor activated phosphatidylinositol 4,5, bisphosphate (PtdIns(4,5)P2) hydrolysis (Cook et al., 1992). Using a permeabilised cell system to introduce non-hydrolysable analogues of guanine-nucleotides, bombesin-stimulated PLD activity was found to be indirectly regulated by a G-protein. The phosphorylation of proteins on tyrosine was determined using immunoblotting with a monoclonal anti-phosphotyrosine antibody. The elevation of tyrosine phosphorylation using pervanadate, an inhibitor of tyrosine phosphatases, was found to activate PLD activity. PLD activity was found to be reduced by pretreatment with the tyrosine kinase inhibitor Genistein. Pretreatment with both Genistein and the selective PKC inhibitor Ro-31-8220 reduced bombesin-stimulated PLD activity to basal. Elevation of Ca2+ from intracellular pools using thapsigargin, an inhibitor of the Ca2+-ATPase on the intracellular stores was found to be insufficient to activate PLD alone nor did it affect bombesin-stimulated PLD activity. Elevation of cyclic AMP using forskolin and dibutyrylcyclicAMP did not affect bombesin-stimulated PLD activity nor did it alone affect the enzyme. A common event in cells exposed to agonist is a loss of responsiveness to the stimulant, referred to as desensitisation. Bombesin-stimulated PLD activity was found to desensitise rapidly with a complete loss in the response after a 40 second agonist exposure. Desensitisation was reversible and occurred within 4.5 minutes of agonist removal, however was never complete. In the continuous presence of agonist resensitisation of PLD activity occurred. The resensitised rate was reduced from that generated in response to the initial stimulus but activity continued over a period of an hour. The recovery of bombesin-stimulated total inositol phosphate generation commenced later than that of bombesin-stimulated PLD activation and reached completion within 11.5 minutes after removal of the desensitising stimulus. Radio-receptor binding studies showed that there was a transient loss of binding sites from the cell surface which was due to internalisation and recycling of the receptor. Although the rate of receptor loss and recovery paralleled that of the desensitisation and resensitisation of PLD activity, differences were observed in the extent of the two processes. Readdition of bombesin was essential for a stimulation of PLD activity to be observed on resensitisation of the enzyme. Hence degradation of the ligand was occurring before the receptor was recycled. Desensitisation and resensitisation was independent of PKC activation and a desensitising pretreatment with bombesin did not affect the PMA- stimulated PLD activity. A23187-stimulated PLD activity also underwent homologous desensitisation and could not stimulate enzyme activity after prior desensitisation by bombesin pretreatment. Thus desensitisation was not due to a decrease in intracellular Ca2+ levels. Bombesin also induced heterologous desensitisation. Pretreatment with bombesin or vasopressin reduced a subsequent stimulation with the other agonist by approximately 70%. A desensitising pretreatment with either agonist attenuated the GTP?S-stimulated PLD activity in permeabilised cells by approximately 50%. It was thus proposed that desensitisation not only affected the cell surface receptors but also occurred at or distal to the receptor-coupled G-protein. These findings support a role for PLD-derived PtdOH in mitogenic signalling, in view of the resensitisation and subsequent maintenance of PLD activity in an early mitogenic situation when the agonist is continually present. The kinetics of the agonist-stimulated PLD activity are discussed with relation to the regulatory pathways of PLD activation and mitogenic signalling.

ATP-induced potentiation of G-protein-dependent phospholipase D activity in a cell-free system from U937 promonocytic leukocytes.

Although G-protein- and protein kinase-mediated pathways have been reported to activate phospholipase D (PLD) following cell stimulation, the relation between these activation pathways and the mechanistic details of lipase stimulation remain unknown. We have studied activation of PLD by GTP gamma S (guanosine 5'-O-(thiotriphosphate)), and its potentiation by ATP, in a cell-free system derived from U937 human promonocytic leukocytes. ATP, in the micromolar to millimolar range, significantly augmented GTP gamma S-stimulated PLD activity (2.6-fold) and the combination resulted in a 15-fold increase in PLD activity compared to control. ATP alone did not stimulate PLD activity. Measurement of endogenous cytosolic ATP levels and nucleotide depletion with activated charcoal demonstrated that stimulation of PLD by GTP gamma S proceeds by both ATP-dependent and -independent pathways. Nucleotide specificity data suggested that the ATP-dependent pathway involves kinase activity. The tyrosine phosphatase inhibitor vanadate augmented PLD activity stimulated by GTP gamma S/ATP by 41% (p < 0.01). Conversely, the tyrosine kinase inhibitors genistein and herbimycin A decreased PLD activity stimulated by GTP gamma S/ATP by 58 and 35%, respectively (p < 0.001 for each). Mixing experiments utilizing subcellular fractions from herbimycin A-treated cells suggested that the relevant tyrosine kinase activity is membrane-associated. Despite its role in ATP-induced potentiation, tyrosine kinase activity is neither necessary nor sufficient for activation of PLD in this system. Protein kinase C (PKC) is unlikely to play a role in potentiation by ATP as PKC activity is not stimulated under conditions of maximal PLD activation.

Rat brain cytosol contains a factor which reconstitutes guanine-nucleotide-binding-protein-regulated phospholipase-D activation in HL60 cells previously permeabilized with streptolysin O.

We report that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) can stimulate phospholipase D (PLD) in HL60 cells acutely permeabilized with streptolysin O. The ability of GTP[S] to stimulate PLD is impaired if the cells are previously permeabilized such that the majority of the cytosol has leaked out. Rat brain and HL60 cytosols were both found to restore GTP[S]-stimulated PLD activity in a reconstitution assay consisting of previously permeabilized HL60 cells. Rat brain cytosol was fractionated on heparin agarose and assayed for reconstitution of GTP[S]-stimulated PLD activity. The active fractions were pooled, concentrated and chromatographed on gel filtration to assess its molecular mass. The molecular mass of the reconstituting factor was found to be 16 kDa. Reconstitution by the cytosolic factor was dependent on GTP[S]. Ca2+ (pCa 5), MgATP and MgCl2 enhanced GTP[S]-dependent reconstitution of PLD activity in the previously permeabilized HL60 cells. These results demonstrate the presence in rat brain cytosol of a factor which is an activator of GTP[S]-stimulated PLD.

Association between leukotriene B 4-induced phospholipase D activation and degranulation of human neutrophils

We have explored the role of phospholipase D (PLD) activation in leukotriene B 4 (LTB 4)-induced Ca 2+ mobilization and degranulation of human neutrophils. Stimulation of [ 3H]alkyl-acylphosphatidylcholine-labeled neutrophils with LTB 4 resulted in a rapid accumulation of [ 3H]alkylphosphatidic acid (PA) as well as a somewhat slower accumulation of [ 3H]alkyl-diglyceride (DG). In the presence of ethanol, PLD catalyzed a transphosphatidylation reaction in which LTB 4 increased [ 3H]alkyl-phosphatidylethanol formation and simultaneously decreased LTB 4-induced PA and DG accumulation. This pattern of lipid metabolism is consistent with the conclusion that LTB 4 stimulates PLD activity in human neutrophils. Additional studies in which the extracellular and intracellular concentrations of Ca 2+ were varied indicated that maximal LTB 4-induced PLD activation was dependent upon Ca 2+ and potentiated by inhibitors of protein kinase C. The time-course and concentration-response curves for LTB 4-induced PLD activation were different from those for LTB 4-induced Ca 2+ mobilization, as measured by fura-2 fluorescence. On the other hand, the concentration-response curve for LTB 4-induced PLD activation was similar to that for LTB 4-induced degranulation. Preincubation of the cells with ethanol inhibited LTB 4-induced PA and DG accumulation, as well as degranulation, suggesting that one or both of these metabolites were important for this response. In contrast, ethanol had no effect on LTB 4-induced Ca 2+ mobilization. Propranolol, an inhibitor of phosphatidate phosphohydrolase, abolished DG accumulation in response to LTB 4 but had no effect on degranulation, suggesting that PA is more important than DG as a mediator of degranulation. Taken collectively, these data indicate that LTB 4-induced activation of PLD in human neutrophils is mediated by a Ca 2+-dependent mechanism, but not by protein kinase C. In addition, PLD activation in these cells may induce degranulation, but not Ca 2+ mobilization.

Distinct mechanisms of phospholipase D activation and attenuation utilized by different mitogens in NIH-3T3 fibroblasts.

The activation of phospholipase D (PLD) by platelet-derived growth factor (PDGF), prostaglandin F2 alpha and 12-O-tetradecanoylphorbol 13-acetate (TPA) was studied in NIH-3T3 fibroblasts. PLD activation was determined by measuring the production of both [3H]phosphatidic acid and [3H]phosphatidylpropanol (products of the PLD-catalyzed hydrolysis and transphosphatidylation reactions, respectively), in cells that were metabolically pre-labeled with [3H]oleic acid. All mitogens caused a rapid (within 2 min) activation of PLD. Activation of PLD by prostaglandin F2 alpha and PDGF was transient and declined to near basal levels by 15 min and 55 min, respectively. In contrast, TPA-induced activation of PLD was sustained for at least 60 min of incubation. A combination of maximally effective concentrations of PDGF and TPA stimulated PLD activity in a non-additive manner, while the effect of prostaglandin F2 alpha was additional to that of either PDGF or TPA. The protein kinase inhibitor staurosporine inhibited PLD activation by PDGF or TPA with almost identical dose/response curves. In contrast, staurosporine potentiated prostaglandin-F2 alpha-induced PLD activation. The specific protein kinase C inhibitor GF109203X (a bisindolylmaleimide) inhibited PLD activation by prostaglandin F2 alpha and PDGF at concentrations higher than those required for inhibition of PLD activation induced by TPA. Depletion of cellular protein kinase C abolished PLD activation by all three mitogens without affecting in vitro activity of membrane-bound PLD. The distinct kinetics of PLD activation and its differential susceptibility to protein kinase inhibitors suggest the existence of agonist-specific activation and/or inactivation mechanisms. The results indicate also that protein kinase C participates in the mechanism of PLD activation via PDGF, while the effect of prostaglandin F2 alpha involves a pathway independent of protein kinase C.

Phospholipase D activity in phagocytic leucocytes is synergistically regulated by G-protein- and tyrosine kinase-based mechanisms.

The regulation of phospholipase D (PLD)-type effector enzymes by G-proteins and protein kinases/phosphatases was characterized in the U937 human promonocytic leucocyte line. PLD activity was assayed by measuring (in the presence of 1% ethanol) the accumulation of phosphatidylethanol in cells permeabilized with beta-escin, a saponin-like detergent. Basal PLD activity was very low when cells were permeabilized and incubated in cytosol-like medium containing micromolar [Ca2+]. When this medium was supplemented with exogenous MgATP or guanosine 5'-[gamma-thio]triphosphate (GTP[S]), PLD activity increased by 9- and 14-fold respectively. Cells permeabilized in the absence of exogenously added MgATP, but in the presence of 1 microM vanadate/100 microM H2O2, also exhibited a modest 12-fold increase in PLD activity. However, the simultaneous presence of either GTP[S] plus exogenous MgATP or GTP[S] plus vanadate/H2O2 (and endogenous MgATP) induced similar 60-75-fold increases in the rate and extent of phosphatidylethanol accumulation. These latter effects of vanadate/H2O2 were strongly correlated with the very rapid accumulation of multiple tyrosine-phosphorylated proteins. Other studies utilized cells which were permeabilized in the presence of GTP[S] and then washed before assay of PLD. These cells retained approximately 60% of the MgATP-regulatable PLD activity (EC50 approximately = to 100 microM MgATP) observed in freshly permeabilized non-washed cells. In the absence of GTP[S] pre-treatment, washed cells retained minimal PLD activity. Genistein, a tyrosine kinase inhibitor, significantly attenuated the ability of MgATP to stimulate PLD activity and accumulation of tyrosine-phosphorylated proteins in the washed GTP[S]-treated cells. These data suggest that PLD activity in myeloid leucocytes involves co-ordinate regulation by both G-protein(s) and tyrosine phosphorylation.

The regulation of phospholipase D activity and its role in sn-1,2-diradylglycerol formation in bombesin- and phorbol 12-myristate 13-acetate-stimulated Swiss 3T3 cells

Addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) to quiescent Swiss 3T3 cells resulted in a sustained increase in sn-1,2-diradylglycerol (DG) mass and [3H]DG in [3H]palmitate-labelled cells where phosphatidylcholine was the major labelled phospholipid. This occurred in the absence of inositol phosphate accumulation. In [3H]palmitate-labelled cells both bombesin and PMA stimulated the formation of phosphatidylbutanol ([3H]PtdBut) in the presence of 0.3% (v/v) butan-1-ol. The kinetics of [3H]PtdBut formation were consistent with phospholipase D (PLD) activation preceding sustained DG formation. The inclusion of butan-1-ol inhibited 70% of PMA-stimulated DG formation but only 30% of the bombesin response. The ability of bombesin and PMA to stimulate the accumulation of [3H]PtdBut was completely abolished in Swiss 3T3 cells which had been pre-treated with 400 nM-PMA for 48 h to down-regulate protein kinase C activity. PMA-stimulated [3H]PtdBut formation was inhibited by 90% by the protein kinase C inhibitor Ro-31-8220 (10 microM), but bombesin-stimulated PtdBut accumulation was inhibited by at most 50% by the same concentration of inhibitor. Cyclic AMP-elevating agents, i.e. forskolin, dibutyryl cyclic AMP and isobutylmethylxanthine, did not inhibit bombesin stimulation of PLD activity. Bombesin-stimulated PLD activity was inhibited by 50% by buffering of the extracellular Ca2+ concentration to 150 nM, but combination of this treatment with Ro-31-8220 addition was less than additive. Ionophore A23187 alone was able to stimulate PLD activity, but this response was inhibited 50% by Ro-31-8220. Thapsigargin was unable to stimulate PLD activity and had no modulatory effect upon bombesin-stimulated PLD activity at any agonist concentration. The results are discussed in terms of the role of PLD in DG generation and the regulation of PLD activity both by bombesin and by PMA.

Phorbol ester treatment of intact rabbit platelets greatly enhances both the basal and guanosine 5′-[γ-thio]triphosphate-stimulated phospholipase D activities of isolated platelet membranes. Physiological activation of phospholipase D may be secondary to activation of phospholipase C

Rabbit platelets were labelled with [3H]glycerol and incubated with or without phorbol 12-myristate 13-acetate (PMA). Membranes were then isolated and assayed for phospholipase D (PLD) activity by monitoring [3H]phosphatidylethanol formation in the presence of 300 mM-ethanol. At a [Ca2+free] of 1 microM, PLD activity was detected in control membranes, but was 5.4 +/- 0.8-fold (mean +/- S.E.M.) greater in membranes from PMA-treated platelets. Under the same conditions, 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated PLD by 18 +/- 3-fold in control membranes, whereas PMA treatment and GTP[S] interacted synergistically to increase PLD activity by 62 +/- 12-fold. GTP[S]-stimulated PLD activity was observed in the absence of Ca2+, but was increased by 1 microM-Ca2+ (3.5 +/- 0.2-fold and 1.8 +/- 0.1-fold in membranes from control and PMA-treated platelets respectively). GTP exerted effects almost as great as those of GTP[S], but 20-30-fold higher concentrations were required. Guanosine 5'-[beta-thio]diphosphate inhibited the effects of GTP[S] or GTP, suggesting a role for a GTP-binding protein in activation of PLD. Thrombin (2 units/ml) stimulated the PLD activity of platelet membranes only very weakly and in a GTP-independent manner. The actions of PMA and analogues on PLD activity correlated with their ability to stimulate protein kinase C in intact platelets. Staurosporine, a potent protein kinase inhibitor, had both inhibitory and, at higher concentrations, stimulatory effects on the activation of PLD by PMA. The results suggest that PMA not only stimulates PLD via activation of protein kinase C but can also activate the enzyme by a phosphorylation-independent mechanism in the presence of staurosporine. However, under physiological conditions, full activation of platelet PLD may require the interplay of protein kinase C, increased Ca2+ and a GTP-binding protein, and may occur as a secondary effect of the activation of phospholipase C.

A Potential Role for Phospholipase-D in the Angiotensin-II-Induced Stimulation of Aldosterone Secretion from Bovine Adrenal Glomerulosa Cells*

The mechanism by which angiotensin-II (Ang II) stimulates aldosterone secretion from adrenal glomerulosa cells involves a phospholipase-C-mediated increase in phosphoinositide turnover and diacylglycerol (DAG) production. Because agonist-induced activation of phospholipase-D (PLD) also contributes to elevations in DAG in other cell types, the ability of Ang II to stimulate PLD activity in cultured bovine adrenal glomerulosa cells was examined. Ang II elicited significant increases in the levels of phosphatidic acid and, in the presence of ethanol, of phosphatidylethanol, a more specific marker for PLD activation. The potential role of this increased PLD activity in the regulation of aldosterone secretion was examined by investigating the ability of exogenous PLD to alter secretory rates. PLD alone dose-dependently increased aldosterone secretion from 5.9 +/- 0.5 to 135 +/- 48 pg/min.mg protein. In the presence of the calcium channel agonist Bay K 8644, which by itself had only a modest effect on aldosterone production, the stimulatory action of PLD was enhanced, yielding a secretory rate (442 +/- 119 pg/min.mg protein) that was approximately 60% of that elicited by 10 nM Ang II (763 +/- 182 pg/min.mg protein). Exogenous PLD also induced a significant increase in DAG levels (from 0.76 +/- 0.03 to 1.10 +/- 0.1 nmol/mg protein), which was not altered by the addition of Bay K 8644. However, PLD did not stimulate inositol phosphate production. These data indicate that 1) Ang II activates PLD; 2) exogenous PLD can elevate aldosterone secretory rates and DAG levels without eliciting phosphoinositide hydrolysis; and 3) the stimulatory action of exogenous PLD on aldosterone secretion is enhanced in the presence of Bay K 8644. Thus, PLD-induced DAG production may play an important role in the Ang II-mediated stimulation of aldosterone secretion from the adrenal zona glomerulosa.