Phospholipase D signaling pathway
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Publication
Journal: Nature Immunology
November/17/2008
Abstract
Mast cells can function as effector and immunoregulatory cells in immunoglobulin E-associated allergic disorders, as well as in certain innate and adaptive immune responses. This review focuses on exciting new developments in the field of mast cell biology published in the past year. We highlight advances in the understanding of FcvarepsilonRI-mediated signaling and mast cell-activation events, as well as in the use of genetic models to study mast cell function in vivo. Finally, we discuss newly identified functions for mast cells or individual mast cell products, such as proteases and interleukin 10, in host defense, cardiovascular disease and tumor biology and in settings in which mast cells have anti-inflammatory or immunosuppressive functions.
Publication
Journal: Blood
June/12/2000
Abstract
Chemokines are small peptides that are potent activators and chemoattractants for leukocyte subpopulations and some nonhemopoietic cells. Their actions are mediated by a family of 7-transmembrane G-protein-coupled receptors, the size of which has grown considerably in recent years and now includes 18 members. Chemokine receptor expression on different cell types and their binding and response to specific chemokines are highly variable. Significant advances have been made in understanding the regulation of chemokine receptor expression and the intracellular signaling mechanisms used in bringing about cell activation. Chemokine receptors have also recently been implicated in several disease states including allergy, psoriasis, atherosclerosis, and malaria. However, most fascinating has been the observation that some of these receptors are used by human immunodeficiency virus type 1 in gaining entry into permissive cells. This review will discuss structural and functional aspects of chemokine receptor biology and will consider the roles these receptors play in inflammation and in infectious diseases.
Publication
Journal: Cellular and Molecular Life Sciences
December/13/2005
Abstract
Phospholipase D (PLD) hydrolyzes the phosphodiester bond of the glycerolipid phosphatidylcholine, resulting in the production of phosphatidic acid and free choline. Phosphatidic acid is widely considered to be the intracellular lipid mediator of many of the biological functions attributed to PLD. However, phosphatidic acid is a tightly regulated lipid in cells and can be converted to other potentially bioactive lipids, including diacylglycerol and lysophosphatidic acid. PLD activities have been described in multiple organisms, including plants, mammals, bacteria and yeast. In mammalian systems, PLD activity regulates the actin cytoskeleton, vesicle trafficking for secretion and endocytosis, and receptor signaling. PLD is in turn regulated by phosphatidylinositol-4,5-bisphosphate, protein kinase C and ADP Ribosylation Factor and Rho family GTPases. This review focuses on the lipid precursors and products of mammalian PLD metabolism, especially phosphatidic acid and the roles this lipid performs in the mediation of the functions of PLD.
Publication
Journal: Biochimica et Biophysica Acta - General Subjects
October/6/2009
Abstract
During the past decade elevated phospholipase D (PLD) activity has been reported in virtually all cancers where it has been examined. PLD catalyzes the hydrolysis of phosphatidylcholine to generate the lipid second messenger phosphatidic acid (PA). While many targets of PA signaling have been identified, the most critical target of PA in cancer cells is likely to be mTOR - the mammalian target of rapamycin. mTOR has been widely implicated in signals that suppress apoptotic programs in cancer cells - frequently referred to as survival signals. mTOR exists as two multi-component complexes known as mTORC1 and mTORC2. Recent data has revealed that PA is required for the stability of both mTORC1 and mTORC2 complexes - and therefore also required for the kinase activity of both mTORC1 and mTORC2. PA interacts with mTOR in a manner that is competitive with rapamycin, and as a consequence, elevated PLD activity confers rapamycin resistance - a point that has been largely overlooked in clinical trials involving rapamycin-based strategies. The earliest genetic changes occurring in an emerging tumor are generally ones that suppress default apoptotic programs that likely represent the first line of defense of cancer. Targeting survival signals in human cancers represents a rational anti-cancer therapeutic strategy. Therefore, understanding the signals that regulate PA levels and how PA impacts upon mTOR could be important for developing strategies to de-repress the survival signals that suppress apoptosis. This review summarizes the role of PA in regulating the mTOR-mediated signals that promote cancer cell survival.
Publication
Journal: Cancer Research
January/29/2007
Abstract
Interest in the regulation of the mammalian target of rapamycin (mTOR) has increased substantially in recent years largely because of an apparent link between mTOR and survival signals in human cancer cells. Much has been learned about the regulation of mTOR in response to survival signals generated by phosphatidylinositol 3-kinase (PI3K). However, another mechanism for regulating mTOR has been proposed involving the generation of phosphatidic acid (PA). PA is the metabolic product of phospholipase D (PLD), whose activity is elevated in a large number of human cancers, and, like PI3K, has been implicated in the survival of human cancer cells. Although the regulation of mTOR by the PI3K signaling pathway is well established, a role for PLD and PA in regulating mTOR has been controversial. In this review, the evidence implicating PLD and PA in the regulation of mTOR is summarized, and the implications of this novel and potentially important mechanism for regulating mTOR are discussed.
Publication
Journal: Chemical Reviews
January/29/2012
Publication
Journal: Journal of Allergy and Clinical Immunology
November/12/2009
Abstract
The recent development of a consensus definition and proposed diagnostic criteria for anaphylaxis offers promise for research efforts and a better understanding of the epidemiology and pathogenesis of this enigmatic and life-threatening disease. This review examines basic principles and recent research advances in the mechanisms of mast cell signaling believed to underlie anaphylaxis. The unfolding complexity of mast cell signaling suggests that the system is sensitive to regulation by any of several individual signaling pathways and intermediates and that complementary pathways regulate mast cell activation by amplified signals. The signaling events underlying anaphylactic reactions have largely been identified through experiments in genetically modified mice and supported by biochemical studies of mast cells derived from these mice. These studies have revealed that signaling pathways exist to both upregulate and downregulate mast cell responses. In this review we will thus describe the key molecular players in these pathways in the context of anaphylaxis.
Publication
Journal: Journal of Biological Chemistry
December/13/2004
Abstract
Sphingosine kinase 1 (SK1) phosphorylates sphingosine to generate sphingosine 1-phosphate (S1P). Because both substrate and product of the enzyme are potentially important signaling molecules, the regulation of SK1 is of considerable interest. We report that SK1, which is ordinarily a cytosolic enzyme, translocates in vivo and in vitro to membrane compartments enriched in phosphatidic acid (PA), the lipid product of phospholipase D. This translocation depends on direct interaction of SK1 with PA, because recombinant purified enzyme shows strong affinity for pure PA coupled to Affi-Gel. The SK1-PA interaction maps to the C terminus of SK1 and is independent of catalytic activity or of the diacylglycerol kinase-like domain of the enzyme. Thus SK1 constitutes a novel, physiologically relevant PA effector.
Publication
Journal: Nature Cell Biology
June/8/2006
Abstract
Dynamin is a large GTP-binding protein that mediates endocytosis by hydrolyzing GTP. Previously, we reported that phospholipase D2 (PLD2) interacts with dynamin in a GTP-dependent manner. This implies that PLD may regulate the GTPase cycle of dynamin. Here, we show that PLD functions as a GTPase activating protein (GAP) through its phox homology domain (PX), which directly activates the GTPase domain of dynamin, and that the arginine residues in the PLD-PX are vital for this GAP function. Moreover, wild-type PLD-PX, but not mutated PLD-PXs defective for GAP function in vitro, increased epidermal growth factor receptor (EGFR) endocytosis at physiological EGF concentrations. In addition, the silencing of PLDs was shown to retard EGFR endocytosis and the addition of wild-type PLDs or lipase-inactive PLDs, but not PLD1 mutants with defective GAP activity for dynamin in vitro, resulted in the recovery of EGFR endocytosis. These findings suggest that PLD, functioning as an intermolecular GAP for dynamin, accelerates EGFR endocytosis. Moreover, we determined that the phox homology domain itself had GAP activity - a novel function in addition to its role as a binding motif for proteins or lipids.
Publication
Journal: Nature Reviews Cancer
February/3/2013
Abstract
Phospholipases (PLC, PLD and PLA) are essential mediators of intracellular and intercellular signalling. They can function as phospholipid-hydrolysing enzymes that can generate many bioactive lipid mediators, such as diacylglycerol, phosphatidic acid, lysophosphatidic acid and arachidonic acid. Lipid mediators generated by phospholipases regulate multiple cellular processes that can promote tumorigenesis, including proliferation, migration, invasion and angiogenesis. Although many individual phospholipases have been extensively studied, how phospholipases regulate diverse cancer-associated cellular processes and the interplay between different phospholipases have yet to be fully elucidated. A thorough understanding of the cancer-associated signalling networks of phospholipases is necessary to determine whether these enzymes can be targeted therapeutically.
Publication
Journal: Journal of Biological Chemistry
December/15/2014
Abstract
Phosphatidic acid (PA) is a critical metabolite at the heart of membrane phospholipid biosynthesis. However, PA also serves as a critical lipid second messenger that regulates several proteins implicated in the control of cell cycle progression and cell growth. Three major metabolic pathways generate PA: phospholipase D (PLD), diacylglycerol kinase (DGK), and lysophosphatidic acid acyltransferase (LPAAT). The LPAAT pathway is integral to de novo membrane phospholipid biosynthesis, whereas the PLD and DGK pathways are activated in response to growth factors and stress. The PLD pathway is also responsive to nutrients. A key target for the lipid second messenger function of PA is mTOR, the mammalian/mechanistic target of rapamycin, which integrates both nutrient and growth factor signals to control cell growth and proliferation. Although PLD has been widely implicated in the generation of PA needed for mTOR activation, it is becoming clear that PA generated via the LPAAT and DGK pathways is also involved in the regulation of mTOR. In this minireview, we highlight the coordinated maintenance of intracellular PA levels that regulate mTOR signals stimulated by growth factors and nutrients, including amino acids, lipids, glucose, and Gln. Emerging evidence indicates compensatory increases in one source of PA when another source is compromised, highlighting the importance of being able to adapt to stressful conditions that interfere with PA production. The regulation of PA levels has important implications for cancer cells that depend on PA and mTOR activity for survival.
Publication
Journal: Naunyn-Schmiedeberg's Archives of Pharmacology
June/17/2007
Abstract
Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of the versatile lipid second messenger, phosphatidic acid (PA), which is involved in fundamental cellular processes, including membrane trafficking, actin cytoskeleton remodeling, cell proliferation and cell survival. PLD activity can be dramatically stimulated by a large number of cell surface receptors and is elaborately regulated by intracellular factors, including protein kinase C isoforms, small GTPases of the ARF, Rho and Ras families and, particularly, by the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP(2)). PIP(2) is well known as substrate for the generation of second messengers by phospholipase C, but is now also understood to recruit and/or activate a variety of actin regulatory proteins, ion channels and other signaling proteins, including PLD, by direct interaction. The synthesis of PIP(2) by phosphoinositide 5-kinase (PIP5K) isoforms is tightly regulated by small GTPases and, interestingly, by PA as well, and the concerted formation of PIP(2) and PA has been shown to mediate receptor-regulated cellular events. This review highlights the regulation of PLD by membrane receptors, and describes how the close encounter of PLD and PIP5K isoforms with small GTPases permits the execution of specific cellular functions.
Publication
Journal: Biochimica et Biophysica Acta - General Subjects
May/29/2007
Abstract
G protein-coupled receptors (GPCRs) control a variety of fundamental cellular processes by regulating phospholipid signaling pathways. Essential for signaling by a large number of receptors is the hydrolysis of the membrane phosphoinositide PIP(2) by phospholipase C (PLC) into the second messengers IP(3) and DAG. Many receptors also stimulate phospholipase D (PLD), leading to the generation of the versatile lipid, phosphatidic acid. Particular PLC and PLD isoforms take differential positions in receptor signaling and are additionally regulated by small GTPases of the Ras, Rho and ARF families. It is now recognized that the PLC substrate, PIP(2), has signaling capacity by itself and can, by direct interaction, affect the activity and subcellular localization of PLD and several other proteins. As expected, the synthesis of PIP(2) by phosphoinositide 5-kinases is tightly regulated as well. In this review, we present an overview of how these signaling pathways are governed by GPCRs, explain the molecular basis for the spatially and temporally organized, highly dynamic quality of phospholipid signaling, and point to the functional connection of the pathways.
Publication
Journal: Biochimica et Biophysica Acta - General Subjects
October/6/2009
Abstract
Epidermal growth factor receptor (EGFR) is a representative model of receptor tyrosine kinases (RTKs), and offers a means of understanding their common principles and fundamental mechanisms. Furthermore, EGFR plays an essential role in cell proliferation and migration, and the disruption of EGFR signaling has been implicated in the development and growth of cancer. Phospholipase D (PLD) is a key mediator of EGFR function, and can be directly regulated by upstream binding partners in an EGF-dependent manner. PLD regulates downstream molecules by generating phosphatidic acid (PA), but it also dynamically interacts with a variety of intracellular molecules and these interactions spatiotemporally regulate EGFR function and serve as a hub that orchestrates signaling flow. This review summarizes the interrelationship between PLD and its binding molecules in the context of EGFR signaling, and addresses the roles of PLD in the mediation and coordination of this signaling.
Publication
Journal: FASEB Journal
February/23/2004
Abstract
Mitogens activate protein translation through phosphorylation of p7S6 kinase (p70(S6K)) and eIF4E binding protein 1 (4E-BP1) mediated by the mammalian target of rapamycin (mTOR) or phosphoinositide 3-kinase (PI3K). A recent report (Science 294, 1942, 2001) has implicated phospholipase D (PLD) in mTOR signaling. We studied the role of PLD in the phosphorylation of p70(S6K) and 4E-BP1 induced by lysophosphatidic acid (LPA) and platelet-derived growth factor (PDGF) using fibroblasts deficient in PLD activity and also 1-butanol, which inhibits phosphatidic acid production by PLD. The reduction in PLD activity in both situations impaired the effect of LPA on mTOR signaling but did not inhibit the effect of PDGF. PDGF induced marked phosphorylation of Akt (a PI3K target) but this was not affected by PLD deficiency. LPA caused much less phosphorylation of Akt and this was dependent on PLD activity. Toxin B, which inactivates Rho GTPases, markedly impaired PLD1 activation and phosphorylation of Akt, p70(S6K), and 4E-BP1 induced by LPA but had a minimal or no effect on the actions of PDGF. These results support the hypothesis that LPA activates protein translation through the action of PLD1-generated PA on mTOR and the PI3K/Akt pathway whereas PDGF acts through P13K/Akt independent of PLD1.
Publication
Journal: Journal of Cellular Physiology
April/21/1997
Abstract
Arg8-vasopressin (AVP) is a potent inducer of myogenic differentiation stimulating the expression of myogenic regulatory factors. To understand the mechanism of its effect on myogenesis, we investigated the early signals induced by AVP in myogenic target cells. In the rat skeletal muscle cell line L6, AVP selectively stimulates phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho) breakdown, through the activation of phospholipases C and D (PLC, PLD), as shown by the generation of Ins(1,4,5)P3 and phosphatidylethanol (PtdEtOH), respectively. AVP induces the biphasic increase of sn-1,2-diacylglycerol (DAG) consisting in a rapid peak followed by a sustained phase, and the monophasic generation of phosphatidic acid (PA). Propranolol (a PA phosphatase inhibitor) and Zn2+ (a PLD inhibitor), abolish the sustained phase of DAG generation. Our data indicate that PtdIns-PLC activity is mainly responsible for the rapid phase of AVP-dependent DAG generation, whereas the sustained phase is dependent upon PtdCho-PLD activity and PA dephosphorylation, ruling out any significant role of DAG kinase. Modifications of PA level correlate with parallel changes of PLC activity, indicating a possible cross-talk between the two signal transduction pathways in the intact cell. PLD activation is elicited at AVP concentrations two orders of magnitude lower than those required for PLC activation. The differentiation of L6 myoblasts into multinucleated fibers is stimulated significantly by AVP at concentrations at which PLD, but not PLC, is activated. These data provide the first evidence for an important role of PLD in the mechanism of AVP-induced muscle differentiation.
Publication
Journal: European journal of biochemistry
October/20/1997
Abstract
We have shown previously that the stem cell factor (SCF) receptor undergoes phosphorylation on serine residues following ligand stimulation, and that this phopshorylation is dependent mainly on the activity of protein kinase C (PKC). In the present study, we have further investigated the molecular mechanisms behind SCF-stimulated activation of PKC, and found that SCF does not activate phosphatidylinositol-specific phospholipase C. In contrast, phospholipase D (PLD) is activated in response to SCF in a dose-dependent manner. Activation of PLD was not inhibited by calphostin C, an inhibitor of PKC. On the other hand, inhibitors of phosphatidylinositol PtdIns 3'-kinase (PtdIns 3'-kinase), i.e. wortmannin and LY294002, inhibited SCF-induced PLD activation. Moreover, a mutant SCF receptor in which Tyr721, which is responsible for activation of PtdIns 3'-kinase, is mutated to a phenylalanine residue was unable to mediate activation of PLD. Thus, PtdIns 3'-kinase appears to be essential for SCF-induced PLD activation. Furthermore, we demonstrate that phosphatidic acid (PtdH), generated through the action of PLD in response to SCF, is metabolized to diacylglycerol by dephosphorylation. Diacylglycerol can then activate PKC, and, moreover, after deacylation by a diacylglycerol lipase, yield arachidonic acid, an important second messenger in cell signaling.
Publication
Journal: Molecular Immunology
July/21/2003
Abstract
Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidate (PA). Two mammalian phospholipase Ds (PLD1 and PLD2) have been cloned and both are present in RBL-2H3 mast cells. PLD1 is localised to secretory granules whilst PLD2 is localised to the plasma membrane, and the activity of both enzymes is increased upon antigen stimulation. Primary alcohols specifically interfere with the production of PLD-derived PA and are found to be potent inhibitors of antigen-stimulated exocytosis. One major intracellular regulator for PLD activity and exocytosis is ARF proteins, as depletion by permeabilisation leads to loss of both antigen-mediated PLD activation and exocytosis. Both responses can be restored in depleted cells by re-addition of ARF1 or ARF6. ARF proteins and PLD-derived PA synergistically regulate the activity of a Type I PIP 5-kinasealpha. It is suggested that ARF, by activating PLD and PIP 5-kinase activities regulate PA and PI(4,5)P(2) levels, and both are critical components of the exocytosis machinery in mast cells.
Publication
Journal: Biochemical Journal
January/17/2001
Abstract
Phospholipase D (PLD)1 is quiescent in vitro and in vivo until stimulated by classical protein kinase C (PKC) isoforms, ADP-ribosylation factor or Rho family members. By contrast, PLD2 has high basal activity, and the mechanisms involved in agonist-induced activation of PLD2 are poorly understood. Using transiently transfected human embryonic kidney (HEK)-293 cells as a model system, we report in the present study that PLD2 overexpressed in HEK-293 cells exhibits regulatory properties similar to PLD1 when stimulated in response to insulin and phorbol ester. Co-expression of PLD1 or PLD2 with PKC alpha results in constitutive activation of both PLD isoforms, which cannot be further stimulated by insulin. Co-expression of PLD1 with phospholipase C (PLC)gamma has the same effect, while co-expression of PLD2 with PLC gamma allows PLD2 activity to be stimulated in an insulin-dependent manner. The PKC-specific inhibitors bisindolylmaleimide and Gö 6976 abolish insulin-induced PLD2 activation in HEK-293 cells co-expressing the insulin receptor, PLC gamma and PLD2, confirming that not only PLD1, but PLD2 as well, is regulated in a PKC-dependent manner. Finally, we provide evidence that PKC alpha is constitutively associated with PLD2. In summary, we demonstrate that insulin treatment results in activation of both PLD1 and PLD2 in appropriate cell types when the appropriate upstream intermediate signalling components, i.e. PKC alpha and PLC gamma, are expressed at sufficient levels.
Publication
Journal: Biochemical Journal
April/14/1994
Abstract
In [3H]myristic acid-labelled osteoblast-like MC3T3-E1 cells, prostaglandin F2 alpha (PGF2 alpha)-induced PLD activity was assessed by measuring the [3H]phosphatidylethanol (PEt) formation in the presence of ethanol. Inhibition of the increase in intracellular Ca2+ concentration ([Ca2+]i) by U73122, an inhibitor of phosphoinositide-specific phospholipase C (PI-PLC), or chelation of extracellular Ca2+ with EGTA or of intracellular Ca2+ with BAPTA, suppressed PGF2 alpha-induced phospholipase D (PLD) activation. Neither protein kinase C (PKC) inhibitors nor PKC down-regulation with phorbol 12-myristate 13-acetate affected PGF2 alpha-induced [3H]PEt formation. In permeabilized cells, guanosine 5'-[gamma-thio]triphosphate enhanced PGF2 alpha 's potency in [3H]PEt formation in the presence of Ca2+. The pretreatment of intact cells with pertussis toxin failed to inhibit PGF2 alpha-induced [3H]PEt formation. PGF2 alpha caused a biphasic production of [3H]1,2-diacylglycerol ([3H]1,2-DAG) in [3H]glycerol-labelled cells. The initial transient phase was decreased by U73122, whereas the late sustained phase was decreased by ethanol and the phosphatidic acid phosphohydrolase inhibitor, propranolol. From these results, it was suggested that PGF2 alpha-induced PLD activation was mediated by the dual control of the [Ca2+]i increase due to PI-PLC activation and activation of pertussis-toxin-insensitive G-protein, but not mediated by PKC, and also that PLD activation was involved in the late sustained 1,2-DAG generation in MC3T3-E1 cells.
Publication
Journal: Journal of Molecular and Cellular Cardiology
December/9/2002
Abstract
Several G protein-coupled receptors which stimulate phospholipase C (PLC) also activate phospholipase D (PLD) in cardiomyocytes. Here, we characterized PLD activation in neonatal rat cardiomyocytes by the PLC-stimulatory thrombin receptor PAR1, in comparison to the endothelin-1 receptor ET(A)R, which induces PLD stimulation by activation of protein kinase C (PKC) delta and epsilon. Similar to ET(A)R, activation of PAR1 induced PLD stimulation, which, however, was insensitive to PKC inhibition. Furthermore, in contrast to ET(A)R, PLD stimulation by PAR1 was suppressed by overexpression of regulators of G protein signaling specific for G(12)-type G proteins and treatment with brefeldin A, an inhibitor of guanine nucleotide exchange factors for ADP-ribosylation factor (ARF) GTPases. On the other hand, inactivation of Rho GTPases by Clostridium difficile toxin B and treatment with general tyrosine kinase inhibitors suppressed PAR1- and ET(A)R- as well as phorbol ester-induced PLD stimulation and was associated with a fall in the cellular level of phosphatidylinositol 4,5-bisphosphate (PIP(2)). We conclude that, in contrast to ET(A)R-PLD coupling, PAR1-induced cardiomyocyte PLD stimulation is PKC-independent and mediated by G(12)-type G proteins and ARF GTPases, while Rho and tyrosine kinases regulate PLD stimulation by either receptor, apparently by controlling the cellular level of PIP(2), a common regulator of PLD activity.
Publication
Journal: Membranes
July/4/2014
Abstract
Prolonged agonist exposure of many G-protein coupled receptors induces a rapid receptor phosphorylation and uncoupling from G-proteins. Resensitization of these desensitized receptors requires endocytosis and subsequent dephosphorylation. Numerous studies show the involvement of phospholipid-specific phosphodiesterase phospholipase D (PLD) in the receptor endocytosis and recycling of many G-protein coupled receptors e.g., opioid, formyl or dopamine receptors. The PLD hydrolyzes the headgroup of a phospholipid, generally phosphatidylcholine (PC), to phosphatidic acid (PA) and choline and is assumed to play an important function in cell regulation and receptor trafficking. Protein kinases and GTP binding proteins of the ADP-ribosylation and Rho families regulate the two mammalian PLD isoforms 1 and 2. Mammalian and yeast PLD are also potently stimulated by phosphatidylinositol 4,5-bisphosphate. The PA product is an intracellular lipid messenger. PLD and PA activities are implicated in a wide range of physiological processes and diseases including inflammation, diabetes, oncogenesis or neurodegeneration. This review discusses the characterization, structure, and regulation of PLD in the context of membrane located G-protein coupled receptor function.
Publication
Journal: Archives of Biochemistry and Biophysics
March/14/2000
Abstract
Angiotensin (Ang) II acts as a mitogen in vascular smooth muscle cells (VSMC) via the activation of multiple signaling cascades, including phospholipase C, tyrosine kinase, and mitogen-activated protein kinase pathways. However, increasing evidence supports signal-activated phospholipases A(2) and D (PLD) as additional mechanisms. Stimulation of PLD results in phosphatidic acid (PA) formation, and PA has been linked to cell growth. However, the direct involvement of PA or its metabolite diacylglycerol (DAG) in Ang II-induced growth is unclear. PLD activity was measured in cultured rat VSMC prelabeled with [(3)H]oleic acid, while the incorporation of [(3)H]thymidine was used to monitor growth. We have previously reported the Ang II-dependent, AT(1)-coupled stimulation of PLD and growth in VSMC. Here, we show that Ang II (100 nM) and exogenous PLD (0.1-100 units/mL; Streptomyces chromofuscus) stimulated thymidine incorporation (43-208% above control). PA (100 nM-1 microM) also increased thymidine incorporation to 135% of control. Propranolol (100 nM-10 microM), which inhibits PA phosphohydrolase, blocked the growth stimulated by Ang II, PLD, or PA by as much as 95%, an effect not shared by other beta-adrenergic antagonists. Propranolol also increased the production of PA in the presence of Ang II by 320% and reduced DAG and arachidonic acid (AA) accumulation. The DAG lipase inhibitor RHC-80267 (1-10 microM) increased Ang II-induced DAG production, while attenuating thymidine incorporation and release of AA. Thus, it appears that activation of PLD, formation of PA, conversion of PA to DAG, and metabolism of DAG comprise an important signaling cascade in Ang II-induced growth of VSMC.