PLD1 promotes reactive oxygen species production in vascular smooth muscle cells and injury-induced neointima formation

Phospholipase D (PLD) generates the signaling lipid phosphatidic acid (PA) and has been known to mediate proliferation signal in vascular smooth muscle cells (VSMCs). However, it remains unclear how PLD contributes to vascular diseases. VSMC proliferation directly contributes to the development and progression of cardiovascular disease, such as atherosclerosis and restenosis after angioplasty. Using the mouse carotid artery ligation model, we find that deletion of Pld1 gene inhibits neointima formation of the injuried blood vessels. PLD1 deficiency reduces the proliferation of VSMCs in both injured artery and primary cultures through the inhibition of ERK1/2 and AKT signals. Immunohistochemical staining of injured artery and flow cytometry analysis of VSMCs shows a reduction of the levels of reactive oxygen species (ROS) in Pld1^?/? VSMCs. An increase of intracellular ROS by hydrogen peroxide stimulation restored the reduced activities of ERK and AKT in Pld1^?/? VSMCs, whereas a reduction of ROS by N-acetyl-l-cysteine (NAC) scavenger lowered their activity in wild-type VSMCs. These results indicate that PLD1 plays a critical role in neointima, and that PLD1 mediates VSMC proliferation signal through promoting the production of ROS. Therefore, inhibition of PLD1 may be used as a therapeutic approach to suppress neointimal formation in atherosclerosis and restenosis after angioplasty.     

Implication of phospholipase D2 in oxidant-induced phosphoinositide 3-kinase signaling via Pyk2 activation in PC12 cells

The role of phospholipase D (PLD) activation in hydrogen peroxide (H(2)O(2))-induced signal transduction and cellular responses is not completely understood. Here we present evidence that Ca(2+)-dependent tyrosine kinase, Pyk2, requires PLD activation to mediate survival pathways in rat pheochromocytoma PC12 cells under oxidative stress. The H(2)O(2)-induced phosphorylation of two Pyk2 sites (Tyr(580), and Tyr(881)) was suppressed by 1-butanol, an inhibitor of transphosphatidylation by PLD, and also by transfection of catalytically negative mouse PLD2K758R (PLD2KR). Furthermore, we found that PLD2 was associated with Pyk2 and Src, and that activation of PLD2 was required for H(2)O(2)-enhanced association of Src with Pyk2 leading to full activation of Pyk2. H(2)O(2)-induced phosphorylation of Akt and p70S6K was dependent on phosphatidylinositol 3-kinase (PI3K) activity and was abolished by 1-butanol but not t-butanol. Furthermore, the PI3K/Akt activation in response to H(2)O(2) was reduced by transfection of either PLD2KR or the dominant negative Pyk2DN. This study is the first demonstration that PLD2 activation is implicated in Src-dependent phosphorylation of Pyk2 (Tyr(580) and Tyr(881)) by promoting the complex formation between Pyk2 and activated Src in PC12 cells exposed to H(2)O(2), thereby resulting in activation of the survival signaling pathway PI3K/Akt/p70S6K.     

Mechanotransduction through fibronectin-integrin focal adhesion in microvascular smooth muscle cells: is calcium essential?

It is believed that increased transmural pressure exerts force on vascular smooth muscle cells (VSMCs) and triggers Ca(2+) signaling as an initiating event responsible for the arteriolar myogenic response. However, the mechanisms linking the pressure increase to Ca(2+) signaling are unclear. We have shown previously using atomic force microscopy (AFM) that mechanical force induces a VSMC contractile response when applied to single fibronectin (FN; Sun Z, Martinez-Lemus LA, Hill MA, Meininger GA. Am J Physiol Cell Physiol 295; C268-C278, 2008) focal adhesion sites. This current study seeks to determine whether application of force to single focal adhesions can cause a change in VSMC Ca(2+). Experiments were performed in low passage (p3～10) as well as in freshly isolated skeletal muscle arteriole VSMCs. AFM-attached microbeads (5 μm) were coated with FN or collagen type I (CN-I) or type IV (CN-IV) and placed on a VSMC for 20 min, resulting in formation of a focal adhesion between the cell and the microbead. In low passage VSMCs, mechanically pulling on the FN-coated beads (800～3000 pN) did not induce a Ca(2+) increase but did cause a contractile response. In freshly isolated VSMCs, application of an FN or CN-I-coated bead onto the cell surface induced global Ca(2+) increases. However, these Ca(2+) increases were not correlated with the application of AFM pulling force to the bead or with the VSMC contractile responses to FN-coupled pulling. Chelating cytosolic Ca(2+) using BAPTA loading had no negative effect on the focal adhesion-related contractile response in both freshly isolated and low passage VSMCs, while the Rho-kinase inhibitor Y27632 abolished the micromyogenic response in both cases. These observations suggest that, in freshly isolated and cultured VSMCs, application of mechanical force to a focal adhesion does not invoke an acute global Ca(2+) increase. On the other hand, our data support a role for Rho-linked signaling mechanism involved in mechanotransduction leading to focal contraction that is independent of the need for a global increase in VSMC Ca(2+).     

Evidence for and characterization of Ca2+ binding to the catalytic region of Arabidopsis thaliana phospholipase Dbeta

Most types of plant phospholipase D (PLD) require Ca(2+) for activity, but how Ca(2+) affects PLD activity is not well understood. We reported previously that Ca(2+) binds to the regulatory C2 domain that occurs in the N terminus of the Ca(2+)-requiring PLDs. Using Arabidopsis thaliana PLDbeta and C2-deleted PLDbeta (PLDbetacat), we now show that Ca(2+) also interacts with the catalytic regions of PLD. PLDbetacat exhibited Ca(2+)-dependent activity, was much less active, and required a higher level of Ca(2+) than the full-length PLDbeta. Ca(2+) binding of the proteins was stimulated by phospholipids; phosphatidylserine was the most effective among those tested. Scatchard plot analysis of Ca(2+) binding data yielded an estimate of 3.6 high affinity (K(d) = 29 mum) binding sites on PLDbeta. The Ca(2+)-PLDbetacat interaction increased the affinity of the protein for the activator, phosphatidylinositol 4,5-bisphosphate, but not for the substrate, phosphatidylcholine. This is in contrast to the effect of Ca(2+) binding to the C2 domain, which stimulates phosphatidylcholine binding but inhibits phosphatidylinositol 4,5-bisphosphate binding of the domain. These results demonstrate the contrasting and complementary effects of the Ca(2+)- and lipid-binding properties of the C2 and catalytic domains of plant PLD and provide insight into the mechanism by which Ca(2+) regulates PLD activity.     

Phosphohydrolase and transphosphatidylation reactions of two Streptomyces phospholipase D enzymes: covalent versus noncovalent catalysis

A kinetic comparison of the hydrolase and transferase activities of two bacterial phospholipase D (PLD) enzymes with little sequence homology provides insights into mechanistic differences and also the more general role of Ca(2+) in modulating PLD reactions. Although the two PLDs exhibit similar substrate specificity (phosphatidylcholine preferred), sensitivity to substrate aggregation or Ca(2+), and pH optima are quite distinct. Streptomyces sp. PMF PLD, a member of the PLD superfamily, generates both hydrolase and transferase products in parallel, consistent with a mechanism that proceeds through a covalent phosphatidylhistidyl intermediate where the rate-limiting step is formation of the covalent intermediate. For Streptomyces chromofuscus PLD, the two reactions exhibit different pH profiles, a result consistent with a mechanism likely to involve direct attack of water or an alcohol on the phosphorus. Ca(2+), not required for monomer or micelle hydrolysis, can activate both PLDs for hydrolysis of PC unilamellar vesicles. In the case of Streptomyces sp. PMF PLD, Ca(2+) relieves product inhibition by interactions with the phosphatidic acid (PA). A similar rate enhancement could occur with other HxKx(4)D-motif PLDs as well. For S. chromofuscus PLD, Ca(2+) is absolutely critical for binding of the enzyme to PC vesicles and for PA activation. That the Ca(2+)-PA activation involves a discreet site on the protein is suggested by the observation that the identity of the C-terminal residue in S. chromofuscus PLD can modulate the extent of product activation.     

Integrin-mediated mechanotransduction in renal vascular smooth muscle cells: activation of calcium sparks

Integrins are transmembrane heterodimeric proteins that link extracellular matrix (ECM) to cytoskeleton and have been shown to function as mechanotransducers in nonmuscle cells. Synthetic integrin-binding peptide triggers Ca(2+) mobilization and contraction in vascular smooth muscle cells (VSMCs) of rat afferent arteriole, indicating that interactions between the ECM and integrins modulate vascular tone. To examine whether integrins transduce extracellular mechanical stress into intracellular Ca(2+) signaling events in VSMCs, unidirectional mechanical force was applied to freshly isolated renal VSMCs through paramagnetic beads coated with fibronectin (natural ligand of alpha(5)beta(1)-integrin in VSMCs). Pulling of fibronectin-coated beads with an electromagnet triggered Ca(2+) sparks, followed by global Ca(2+) mobilization. Paramagnetic beads coated with low-density lipoprotein, whose receptors are not linked to cytoskeleton, were minimally effective in triggering Ca(2+) sparks and global Ca(2+) mobilization. Preincubation with ryanodine, cytochalasin-D, or colchicine substantially reduced the occurrence of Ca(2+) sparks triggered by fibronectin-coated beads. Binding of VSMCs with antibodies specific to the extracellular domains of alpha(5-) and beta(1)-integrins triggered Ca(2+) sparks simulating the effects of fibronectin-coated beads. Preincubation of microperfused afferent arterioles with ryanodine or integrin-specific binding peptide inhibited pressure-induced myogenic constriction. In conclusion, integrins transduce mechanical force into intracellular Ca(2+) signaling events in renal VSMCs. Integrin-mediated mechanotransduction is probably involved in myogenic response of afferent arterioles.     

Effect of nitric oxides and hydrogen peroxide on Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase activity in myometrium sarcolemma

The action of sodium nitroprusside, nitrite-anions and hydrogen peroxide on Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase (Ca(2+)-independent) enzymatic activity in myometrium sarcolemma fraction is investigated. It is established, that 0.1 mM sodium nitroprusside and 10(-8)-10(-5) M nitrite-anions essentially reduce Ca2+, Mg(2+)-ATPase activity whereas Mg(2+)-ATPase proved to be absolutely resistant to them. At rather high concentration of nitrite-anions (0.1 mM) appreciable stimulation of Ca2+, Mg(2+)-ATPase was observed. Hydrogen peroxide (10(-8)-10(-4)), depending on the concentration suppressed both enzymes activity. However, Ca2+, Mg(2+)-ATPase proved to be more sensitive to the action of H2O2 (seeming K(i) = 0.42 +/- 0.1 microM), than Mg(2+)-ATPase (seeming K(i) = 3.1 +/- 0.9 microM). At presence of 1 mM ditiothreitole (a reducer of SH groups of the membrane surface) action of investigated substances considerably decreased. Reagents on carboxic- (dicyclogexilcarbodiimid) and amino- groups of the membrane (trinitrobenzolsulfonic acid) inhibited both Ca2+, Mg(2+)-ATPase, and Mg(2+)-ATPase activity in membrane fractions. In the presence of noted reagents sodium nitroprusside and nitrite-anions action was not almost shown. Hence, nitrogen oxide, nitrite-anions and hydrogen peroxide suppress Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase (only hydrogen peroxide) activity in the plasmatic membrane of myometrium cells, and this action can be connected with direct updating of superficial chemical groups of the membrane.     

Plant phospholipase Dalpha is an acidic phospholipase active at near-physiological Ca(2+) concentrations.

The conventional plant phospholipase D (PLD) requires Ca(2+) for activity; however, the most distinct and puzzling feature of this PLD is its in vitro need for 20 to 100 mM Ca(2+). This noncytoplasmic Ca(2+) requirement has raised doubt about the role of Ca(2+) in regulating its function in vivo. Using the cloned conventional castor bean PLD, PLDalpha, expressed in Escherichia coli, this study demonstrates that this PLD is active at micromolar, near-physiological concentrations of Ca(2+), and this activity at low Ca(2+) requires an acidic pH (4.5-5.5). By comparison, the newly cloned PLDbeta and -gamma were active only at neutral pH under the same Ca(2+) concentrations. This study also shows that PLDalpha activity at low Ca(2+) needs substrates presented as a mixture of membrane lipids. Phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate are equally effective in stimulating the acidic PLDalpha activity, whereas phophatidylinositol is inactive. These results suggest that the conventional plant PLD in vivo is an acidic phospholipase that is active at near-physiological Ca(2+) concentrations. The possible physiological significance of these findings will be discussed.     

Potential effect of resistin on the ET-1-increased reactions of blood pressure in rats and Ca2+ signaling in vascular smooth muscle cells

Resistin and endothelin-1 (ET-1) are upregulated in people with type II diabetes mellitus, central obesity, and hypertension. ET-1 signaling is involved in Ca(2+)-contraction coupling and related to blood pressure regulation. The aim of this study is to investigate the role of resistin on ET-1-increased blood pressure and Ca(2+) signaling. The blood pressure and cytosolic Ca(2+) of vascular smooth muscle cells (VSMCs) of Sprague-Dawley rats were detected. The data demonstrated that resistin accelerated and prolonged ET-1-induced increases in blood pressure and had significant effects on ET-1-increased Ca(2+) reactions. Resistin-enhanced ET-1-increased Ca(2+) reactions were reversed by blockers of store-operated Ca(2+) entry (SOCE) and extracellular-signal-regulated kinase (ERK). The endogenous expression of Orai and stromal interaction molecular (STIM) were characterized in the VSMCs. Furthermore, resistin-enhanced ET-1 Ca(2+) reactions and the resistin-dependent activation of SOCE were abolished under STIM1-siRNA treatment, indicating that STIM1 plays an important role in resistin-enhanced ET-1 Ca(2+) reactions in VSMCs. Resistin appears to exert effects on ET-1-induced Ca(2+) increases by enhancing the activity of ERK-dependent SOCE (STIM1-partcipated), and may accelerate and prolong ET-1-increased blood pressure via the same pathway.     

Essential role of protein kinase G and decreased cytoplasmic Ca2+ levels in NO-induced inhibition of rat aortic smooth muscle cell motility

Hyperinsulinemia is a major risk factor for the development of vascular disease. We have reported that insulin increases the motility of vascular smooth muscle cells via a hydrogen peroxide-mediated mechanism and that nitric oxide (NO) attenuates insulin-induced motility via a cGMP-mediated mechanism. Events downstream of cGMP elevation have not yet been investigated. The aim of our study was to test the hypothesis that antimotogenic effects of NO and cGMP in cultured rat aortic smooth muscle cells are mediated via PKG, followed by reduction of cytoplasmic Ca(2+) levels and increased protein tyrosine phosphatase-proline, glutamate, serine, and threonine activity, leading to suppression of agonist-induced elevation of hydrogen peroxide levels and cell motility. Treatment of primary cultures with adenovirus expressing PKG-1alpha mimicked NO-induced inhibition of insulin-elicited hydrogen peroxide elevation and cell motility, whereas treatment with the pharmacological PKG inhibitor Rp-8-bromo-3',5'-cyclic monophosphorothioate (Rp-8-Br-cGMPS) rescued the stimulatory effects of insulin that were suppressed by NO donor. Treatment of cells with insulin failed to increase cytoplasmic Ca(2+) levels, whereas NO donor decreased cytoplasmic Ca(2+) levels in the presence or absence of insulin. Treatment of cells with the Ca(2+) chelator BAPTA mimicked the effects of PKG and the NO donor and increased the activity of PTP-PEST. Finally, treatment with a dominant negative allele of PTP-PEST reversed the inhibitory effect of BAPTA on cell motility and hydrogen peroxide elevation. We conclude that NO-induced inhibition of cell motility occurs via PKG-mediated reduction of basal cytoplasmic Ca(2+) levels, followed by increased PTP-PEST activity, leading to decreased hydrogen peroxide levels and reduced cell motility.     

A role for calcium in sphingosine 1-phosphate-induced phospholipase D activity in C2C12 myoblasts

Receptor-regulated phospholipase D (PLD) is a key signaling pathway implicated in the control of fundamental biological processes. Here evidence is presented that in addition to protein kinase C (PKC) and Rho GTPases, Ca(2+) response evoked by sphingosine 1-phosphate (S1P) also participates to the enzyme regulation. Ca(2+) was found critical for PKC(alpha)-mediated PLD activation. Moreover, S1P-induced PLD activity resulted diminished by calmodulin inhibitors such as W-7 and CGS9343B implicating its involvement in the process. A plausible candidate for Ca(2+)-dependent PLD regulation by S1P was represented by calcineurin, in view of the observed reduction of the stimulatory effect by cyclosporin A. In contrast, monomeric GTP-binding protein Ral was translocated to membranes by S1P in a Ca(2+)-independent manner, ruling out its possible role in agonist-mediated regulation of PLD.     

Reactive oxygen species produced up- or downstream of calcium influx regulate proinflammatory mediator release from mast cells: role of NADPH oxidase and mitochondria

Earlier studies have demonstrated that mast cells produce reactive oxygen species (ROS), which play a role in regulating Ca(2+) influx, while in other cell types ROS are produced in a Ca(2+)-dependent manner. We sought to determine whether ROS are produced downstream of the extracellular Ca(2+) entry in mast cells. Thapsigargin (TG), a receptor-independent agonist, could evoke a robust burst of intracellular ROS. However, this response was distinct from the antigen-induced burst of ROS with respect to time course and dependence on Ca(2+) and phosphatidylinositol-3-kinase (PI3K). The antigen-induced ROS generation occurred immediately, while the TG-induced ROS generation occurred with a significant lag time (~2 min). Antigen but not TG caused extracellular release of superoxide (O(2)(*-))/hydrogen peroxide (H(2)O(2)), which was blocked by diphenyleneiodonium, apocynin, and wortmannin. A capacitative Ca(2+) entry resulted in the generation of O(2)(*-) in the mitochondria in a PI3K-independent manner. Blockade of ROS generation inhibited TG-induced mediator release. Finally, when used together, antigen and TG evoked the release of leukotriene C(4), tumor necrosis factor-alpha, and interleukin-13 as well as ROS generation synergistically. These results suggest that ROS produced upstream of Ca(2+) influx by NADPH oxidase and downstream of Ca(2+) influx by the mitochondria regulate the proinflammatory response of mast cells.     

Amlodipine inhibits thapsigargin-sensitive CA(2+) stores in thrombin-stimulated vascular smooth muscle cells

Ca(2+) channel blockers, such as amlodipine, inhibit vascular smooth muscle cell (VSMC) growth through interactions with targets other than L-type Ca(2+) channels. The effects of amlodipine on Ca(2+) movements in thrombin- and thapsigargin-stimulated VSMCs were therefore investigated by determining the variations of intracellular free Ca(2+) concentration in fura 2-loaded cultured VSMCs. Results indicated that 10-1,000 nM amlodipine inhibited 1) thrombin-induced Ca(2+) mobilization from a thapsigargin-sensitive pool and 2) thapsigargin-induced Ca(2+) responses, including Ca(2+) mobilization from internal stores and store-operated Ca(2+) entry. These effects of amlodipine do not involve L-type Ca(2+) channels and could not be reproduced with 100 nM isradipine, diltiazem, or verapamil. The inhibition by amlodipine of Ca(2+) mobilization appears therefore to be a specific property of the drug, in addition to its Ca(2+) channel-blocking property. It is suggested that amlodipine acts in this capacity by interacting with Ca(2+)-ATPases of the sarcoplasmic reticulum, thus modulating the enzyme activity. This mechanism might participate in the inhibitory effect of amlodipine on VSMC growth.     

In dialyzed squid axons oxidative stress inhibits the Na+/Ca2+ exchanger by impairing the Cai2+-regulatory site

The Na(+)/Ca(2+) exchanger, a major mechanism by which cells extrude calcium, is involved in several physiological and physiopathological interactions. In this work we have used the dialyzed squid giant axon to study the effects of two oxidants, SIN-1-buffered peroxynitrite and hydrogen peroxide (H(2)O(2)), on the Na(+)/Ca(2+) exchanger in the absence and presence of MgATP upregulation. The results show that oxidative stress induced by peroxynitrite and hydrogen peroxide inhibits the Na(+)/Ca(2+) exchanger by impairing the intracellular Ca(2+) (Ca(i)(2+))-regulatory sites, leaving unharmed the intracellular Na(+)- and Ca(2+)-transporting sites. This effect is efficiently counteracted by the presence of MgATP and by intracellular alkalinization, conditions that also protect H(i)(+) and (H(i)(+) + Na(i)(+)) inhibition of Ca(i)(2+)-regulatory sites. In addition, 1 mM intracellular EGTA reduces oxidant inhibition. However, once the effects of oxidants are installed they cannot be reversed by either MgATP or EGTA. These results have significant implications regarding the role of the Na(+)/Ca(2+) exchanger in response to pathological conditions leading to tissue ischemia-reperfusion and anoxia/reoxygenation; they concur with a marked reduction in ATP concentration, an increase in oxidant production, and a rise in intracellular Ca(2+) concentration that seems to be the main factor responsible for cell damage.     

The Arabidopsis phospholipase D family. Characterization of a calcium-independent and phosphatidylcholine-selective PLD zeta 1 with distinct regulatory domains

Four types of phospholipase D (PLD), PLD alpha, beta, gamma, and delta, have been characterized in Arabidopsis, and they display different requirements for Ca(2+), phosphatidylinositol 4,5-bisphosphate (PIP(2)), substrate vesicle composition, and/or free fatty acids. However, all previously cloned plant PLDs contain a Ca(2+)-dependent phospholipid-binding C2 domain and require Ca(2+) for activity. This study documents a new type of PLD, PLD zeta 1, which is distinctively different from previously characterized PLDs. It contains at the N terminus a Phox homology domain and a pleckstrin homology domain, but not the C2 domain. A full-length cDNA for Arabidopsis PLD zeta 1 has been identified and used to express catalytically active PLD in Escherichia coli. PLD zeta 1 does not require Ca(2+) or any other divalent cation for activity. In addition, it selectively hydrolyzes phosphatidylcholine, whereas the other Arabidopsis PLDs use several phospholipids as substrates. PLD zeta 1 requires PIP(2) for activity, but unlike the PIP(2)-requiring PLD beta or gamma, phosphatidylethanolamine is not needed in substrate vesicles. These differences are described, together with a genomic analysis of 12 putative Arabidopsis PLD genes that are grouped into alpha, beta, delta, gamma, and zeta based on their gene architectures, sequence similarities, domain structures, and biochemical properties.     

The role of interfacial binding in the activation of Streptomyces chromofuscus phospholipase D by phosphatidic acid

The Streptomyces chromofuscus phospholipase D (PLD) cleavage of phosphatidylcholine in bilayers can be enhanced by the addition of the product phosphatidic acid (PA). Other anionic lipids such as phosphatidylinositol, oleic acid, or phosphatidylmethanol do not activate this PLD. This allosteric activation by PA could involve a conformational change in the enzyme that alters PLD binding to phospholipid surfaces. To test this, the binding of intact PLD and proteolytically cleaved isoforms to styrene divinylbenzene beads coated with a phospholipid monolayer and to unilamellar vesicles was examined. The results indicate that intact PLD has a very high affinity for PA bilayers at pH >/= 7 in the presence of EGTA that is weakened as Ca(2+) or Ba(2+) are added to the system. Proteolytically clipped PLD also binds tightly to PA in the absence of metal ions. However, the isolated catalytic fragment has a considerably weaker affinity for PA surfaces. In contrast to PA surfaces, all PLD forms exhibited very low affinity for PC interfaces with an increased binding when Ba(2+) was added. All PLD forms also bound tightly to other anionic phospholipid surfaces (e.g. phosphatidylserine, phosphatidylinositol, and phosphatidylmethanol). However, this binding was not modulated in the same way by divalent cations. Chemical cross-linking studies suggested that a major effect of PLD binding to PA.Ca(2+) surfaces is aggregation of the enzyme. These results indicate that PLD partitioning to phospholipid surfaces and kinetic activation are two separate events and suggest that the Ca(2+) modulation of PA.PLD binding involves protein aggregation that may be the critical interaction for activation.     

Phospholipase D in hormonal and stress signaling

Phospholipase D (PLD) is a family of diverse enzymes that are differentially regulated by Ca(2+), polyphosphoinositides, free fatty acids, G-proteins, N-acylethanolamines, and membrane lipid environments. Two new types of PLDs were identified in the past year: one is activated by oleic acid and the other requires no cation for activity. The oleate-stimulated PLD is associated with the plasma membrane and binds to microtubules. The Ca(2+)-independent PLD contains a PX and a PH domain, but not the Ca(2+)/phospholipid-binding C2 domain found in most plant PLDs. The mechanism by which Ca(2+), phosphoinositides, and G proteins regulate certain PLDs is better understood. PLDs and their product phosphatidic acid are involved in various stress responses, including water deficits, salts, wounding, and elicitation. Increasing evidence supports a role of PLD in the abscisic acid signaling cascades.     

Ca(2+)- and phospholipase D-dependent and -independent pathways activate mTOR signaling

The mammalian target of rapamycin (mTOR) promotes increased protein synthesis required for cell growth. It has been suggested that phosphatidic acid, produced upon activation of phospholipase D (PLD), is a common mediator of growth factor activation of mTOR signaling. We used Rat-1 fibroblasts expressing the alpha(1A) adrenergic receptor to study if this G(q)-coupled receptor uses PLD to regulate mTOR signaling. Phenylephrine (PE) stimulation of the alpha(1A) adrenergic receptor induced mTOR autophosphorylation at Ser2481 and phosphorylation of two mTOR effectors, 4E-BP1 and p70 S6 kinase. These PE-induced phosphorylations were greatly reduced in cells depleted of intracellular Ca(2+). PE activation of PLD was also inhibited in Ca(2+)-depleted cells. Incubation of cells with 1-butanol to inhibit PLD signaling attenuated PE-induced phosphorylation of mTOR, 4E-BP1 and p70 S6 kinase. By contrast, platelet-derived growth factor (PDGF)-induced phosphorylation of these proteins was not blocked by Ca(2+) depletion or 1-butanol treatment. These results suggest that the alpha(1A) adrenergic receptor promotes mTOR signaling via a pathway that requires an increase in intracellular Ca(2+) and activation of PLD. The PDGF receptor, by contrast, appears to activate mTOR by a distinct pathway that does not require Ca(2+) or PLD.