A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion.

The homeostasis of most organelles requires membrane fusion mediated by soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis-SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N-ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does N-ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics.

Hyper-activated motility in sperm capacitation is mediated by phospholipase D-dependent actin polymerization.

In order to fertilize the oocyte, sperm must undergo a series of biochemical changes in the female reproductive tract, known as capacitation. Once capacitated, spermatozoon can bind to the zona pellucida of the egg and undergo the acrosome reaction (AR), a process that enables its penetration and fertilization of the oocyte. Important processes that characterize sperm capacitation are actin polymerization and the development of hyper-activated motility (HAM). Previously, we showed that Phospholipase D (PLD)-dependent actin polymerization occurs during sperm capacitation, however the role of this process in sperm capacitation is not yet known. In the present study, we showed for the first time the involvement of PLD-dependent actin polymerization in sperm motility during mouse and human capacitation. Sperm incubated under capacitation conditions revealed a time dependent increase in actin polymerization and HAM. Inhibition of Phosphatidic Acid (PA) formation by PLD using butan-1-ol, inhibited actin polymerization and motility, as well as in vitro fertilization (IVF) and the ability of the sperm to undergo the AR. The inhibition of sperm HAM by low concentration of butan-1-ol is completely restored by adding PA, further indicating the involvement of PLD in these processes. Furthermore, exogenous PA enhanced rapid actin polymerization that was followed by a rise in the HAM, as well as an increased in IVF rate. In conclusion, our results demonstrate that PLD-dependent actin polymerization is a critical step needed for the development of HAM during mouse and human sperm capacitation.

Phospholipase d promotes lipid microdomain-associated signaling events in mast cells.

Initial IgE-dependent signaling events are associated with detergent-resistant membrane microdomains. Following Ag stimulation, the IgE-receptor (Fc(epsilon)RI ) accumulates within these domains. This facilitates the phosphorylation of Fc(epsilon)RI subunits by the Src kinase, Lyn, and the interaction with adaptor proteins, such as the linker for activation of T cells. Among the phospholipases (PL) subsequently activated, PLD is of interest because of its presence in lipid microdomains and the possibility that its product, phosphatidic acid, may regulate signal transduction and membrane trafficking. We find that in Ag-stimulated RBL-2H3 mast cells, the association of Fc(epsilon)RI with detergent-resistant membrane fractions is inhibited by 1-butanol, which subverts production of phosphatidic acid to the biologically inert phosphatidylbutanol. Furthermore, the knockdown of PLD2, and to a lesser extent PLD1 with small inhibitory RNAs, also suppressed the accumulation of Fc(epsilon)RI and Lyn in these fractions as well as the phosphorylation of Src kinases, Fc(epsilon)RI , linker for activation of T cells, and degranulation. These effects were accompanied by changes in distribution of the lipid microdomain component, ganglioside 1, in the plasma membrane as determined by binding of fluorescent-tagged cholera toxin B subunit and confocal microscopy in live cells. Collectively, these findings suggest that PLD activity plays an important role in promoting IgE-dependent signaling events within lipid microdomains in mast cells.

Membrane Rigidity and Phosphatidic Acid (PtdOH) Signal: Two Important Events in Acinetobacter guillouiae SFC 500-1A Exposed to Chromium(VI) and Phenol.

The remodeling of membrane lipids is a mechanism that allows microorganisms to survive in unfavorable environments such as industrial effluents, which often contain inorganic and organic pollutants, like chromium and phenol. In the present work, we evaluated the effect of Cr(VI) and phenol on the membrane of Acinetobacter guillouiae SFC 500-1A, a bacterial strain isolated from tannery sediments where such pollutants can be found. The presence of lipid kinases and phospholipases and the changes in their activities under exposure to these pollutants were determined. Cr(VI) and Cr(VI) + phenol caused the membrane to become more rigid for up to 16 h after exposure. This could be due to an increase in cardiolipin (Ptd2 Gro) and a decrease in phosphatidylethanolamine (PtdEtn), which are indicative of more order and rigidity in the membrane. Increased phospholipase A activity (PLA, EC 3.1.1.4) could be responsible for the decrease in PtdEtn levels. Moreover, our results indicate that Cr(VI) and Cr(VI) + phenol trigger the phosphatidic acid (PtdOH) signal. The finding of significantly increased phosphatidylinositol-4-phosphate (PtdIns-4-P) levels means this is likely achieved via PtdIns-PLC/DGK. This report provides the first evidence that A. guillouiae SFC 500-1A is able to sense Cr(VI) and phenol, transduce this signal through changes in the physical state of the membrane, and trigger lipid-signaling events.

ETOH inhibits embryonic neural stem/precursor cell proliferation via PLD signaling.

While a mother's excessive alcohol consumption during pregnancy is known to have adverse effects on fetal neural development, little is known about the underlying mechanism of these effects. In order to investigate these mechanisms, we investigated the toxic effect of ethanol (ETOH) on neural stem/precursor cell (NSC) proliferation. In cultures of NSCs, phospholipase D (PLD) is activated following stimulation with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Exposure of NSCs to ETOH suppresses cell proliferation, while it has no effect on cell death. Phosphatidic acid (PA), which is a signaling messenger produced by PLD, reverses ETOH inhibition of NSC proliferation. Blocking the PLD signal by 1-butanol suppresses the proliferation. ETOH-induced suppression of NSC proliferation and the protective effect of PA for ETOH-induced suppression are mediated through extracellular signal-regulated kinase signaling. These results indicate that exposure to ETOH impairs NSC proliferation by altering the PLD signaling pathway.

Diethylstilbestrol inhibits phospholipase D activity and degranulation by stimulated human neutrophils.

In the present study the effects of diethylstilbestrol on phospholipase D activity and degranulation by human neutrophils were examined. Diethylstilbestrol is a synthetic estrogen and has structural similarity to resveratrol. Resveratrol is a natural polyphenolic antioxidant and has been shown to inhibit the activity of phospholipase D in stimulated neutrophils. Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid and choline. It also catalyzes the transfer of the phosphatidyl group to ethanol forming phosphatidylethanol at the expense of phosphatidic acid. Phospholipase D activation is associated with degranulation by neutrophils stimulated with chemotactic peptide, formyl-methionyl-leucyl-phenylalanine. The results show that diethylstilbestrol at 100 microM induced a complete inhibition of phosphatidic acid formation in neutrophils, the latter activated by chemotactic peptide. In the presence of ethanol, diethylstilbestrol dose dependently reduced phosphatidylethanol formation induced by chemotactic peptide or by phorbol 12-myristate 13-acetate, indicative of diethylstilbestyrol inhibition of phospholipase D activity. The results also demonstrate that diethylstilbestrol inhibited degranulation by chemotactic peptide-stimulated neutrophils. In comparison to resveratrol, diethylstilbestrol exhibits a stronger inhibition on PA formation, phospholipase D activity and degranulation. These findings suggest that diethylstilbestrol-like resveratrol, may have anti-inflammatory effect in vitro.

Role of RhoA and Rho kinase in lysophosphatidic acid-induced endothelial barrier dysfunction.

In the present study, the roles of the small GTPase RhoA and its target Rho kinase in endothelial permeability were investigated in vitro. We have shown previously that, in addition to a rise in the intracellular Ca(2+) concentration ([Ca(2+)](i)), RhoA is involved in the prolonged thrombin-induced barrier dysfunction. To study the role of RhoA and Rho kinase more specifically, endothelial cells were stimulated with lysophosphatidic acid (LPA), a commonly used RhoA activator. LPA induced a 2- to 3-fold increase in the passage of horseradish peroxidase (HRP) across endothelial monolayers that lasted for several hours, whereas thrombin induced a 5- to 10-fold increase. Comparable to the thrombin-induced barrier dysfunction, the LPA-induced barrier dysfunction was accompanied by a reorganization of the F-actin cytoskeleton and the formation of focal attachment sites. LPA induced only a transient increase in myosin light-chain (MLC) phosphorylation, which returned to basal level within 10 minutes. In endothelial cells, [Ca(2+)](i) was not elevated by LPA. Chelation of Ca(2+)(i) ions by 1, 2-bis(2-aminophenoxy)ethane-N:,N:,N:',N:'-tetraacetic acid did not prevent the LPA-induced passage of HRP. Apparently, a low degree of MLC kinase activation occurred, because the MLC kinase inhibitor KT5926 reduced the levels of both basal and LPA-stimulated HRP passage. Inhibition of RhoA by the C3 transferase from Clostridium botulinum inhibited the LPA-induced cytoskeletal changes and prevented the LPA-induced HRP passage. Inhibition of Rho kinase by Y-27632 completely prevented the LPA-induced increase in HRP passage without affecting basal permeability. These data indicate that LPA-induced endothelial hyperpermeability occurs without a change in [Ca(2+)](i) and requires activation of RhoA and Rho kinase.

Lisofylline, an inhibitor of unsaturated phosphatidic acid generation, ameliorates interleukin-1 beta-induced dysfunction in cultured rat islets.

Interleukin-1 beta (IL-1 beta) causes rat islet cell dysfunction through mechanisms that involve inducible nitric oxide synthase (iNOS). However, IL-1 beta also activates several lipid pathways, including those generating phosphatidic acid (PA). Lisofylline (LSF), a water-soluble, nontoxic, selective inhibitor of the PA-1 alpha subspecies, which is stimulated by IL-1 beta and tumor necrosis factor-alpha, has been shown to prevent cytokine-induced cytotoxicity in in vivo animal models. To evaluate the effect of LSF on acute IL-1 beta-induced islet dysfunction, rat islets were exposed to IL-1 beta (0.1 ng/ml) with or without LSF (100 microM) for 24 h, followed by 25 mM glucose (G) stimulation, measurement of rat insulin by RIA, and calculation of the insulin secretion rate. In other experiments, rat islets were precultured for 48 h, then treated for 48 h in 25 mM G with or without IL-1 beta (0.1 ng/ml) and LSF (400 microM), and aliquots of medium were removed at 0, 24, and 48 h for measurement of rat insulin. In addition, islets were exposed to 25 mM G with or without IL-1 beta and LSF, lipids were then extracted, and PA subspecies were identified by TLC and mass spectroscopy, and quantitated using normal phase HPLC. Islets were also exposed to IL-1 beta with or without LSF, and Western immunoblots were performed to evaluate the effect of LSF on iNOS protein expression. IL-1 beta caused a 44% decrease in islet G-stimulated insulin secretion compared to that in untreated islets (P < 0.0005), which was totally reversed by LSF. In addition, IL-1 beta decreased the G-stimulated medium insulin content by 75% at 24 h (P = 0.0004) and 86% at 48 h compared to that in control islets (P < 0.0001). LSF-treated islets maintained 70% of medium insulin content at 24 h (P = 0.11) and 50% at 48 h (P < 0.0001) compared to control islets. HPLC quantitation of PA-1 alpha extracted from islets treated with IL-1 beta alone showed an approximately 15-fold increase over the PA-1 alpha content of islets treated with IL-1 beta and LSF. IL-1 beta-induced expression of iNOS was unchanged with the addition of LSF. These results suggest that LSF is effective in reducing IL-1 beta-induced islet dysfunction, thus supporting the role of lipid mediators such as PA in cytokine-induced islet toxicity.

The activation of phospholipase D participates in the mitogenic action of arginine vasopressin in cultured rat glomerular mesangial cells.

The present study was undertaken to determine whether phospholipase D participates in the mitogenic action of arginine vasopressin (AVP) in cultured rat glomerular mesangial cells. AVP promptly increased the phosphatidylethanol formation in a concentration-dependent manner, which indicates the activation of phospholipase D. When cells were preincubated with 2,3-diphosphoglycerate or carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK), inhibitors of phospholipase D, the 1 x 10(-7) M AVP-produced phosphatidylethanol was significantly attenuated. Also, inhibitors of protein kinase C, staurosporine and calphostin C, reduced the AVP-induced increase in phosphatidylethanol. AVP activated mitogen-activated protein (MAP) kinase in a concentration-dependent manner. Such an activation was significantly reduced by 2,3-diphosphoglycerate, zLYCK, or staurosporine. Also, AVP stimulated [3H]thymidine incorporation, an effect significantly less in the presence of 2,3-diphosphoglycerate or zLYCK. Similar results were obtained with exogenous bacterial phospholipase D. Both MAP kinase and [3H]thymidine incorporation were not altered by 2,3-diphosphoglycerate or zLYCK per se. These results indicate that AVP activates phospholipase D and promotes cellular growth mediated through phospholipase D, in addition to a phospholipase C-dependent signal transduction, in glomerular mesangial cells.

Phosphatidic acid induces the respiratory burst of electropermeabilized human neutrophils by acting on a downstream step of protein kinase C.

Phosphatidic acid (PA) dose-dependently induced superoxide (O2-) production of electropermeabilized human neutrophils but not of intact neutrophils, indicating that PA induces the activation of NADPH oxidase by acting on an intracellular target. The O2- production by PA was not inhibited by protein kinase C (PKC) inhibitors, such as staurosporine and calphostin C, and an inhibitor of PA phosphohydrolase, propranolol. These observations suggest that the activation of the oxidase by PA is independent of the activity of PKC and may dominate the activation by diacylglycerol which is formed from PA via the action of PA phosphohydrolase. Furthermore, the production by PA, as well as that by phorbol myristate acetate, was inhibited by cyclic AMP and GDP beta S. Therefore, PA seems to act at a site downstream of PKC.

Effect in supralethally irradiated rats of granulocyte colony-stimulating factor and lisofylline on hematopoietic reconstitution by syngeneic bone marrow or whole organ passenger leukocytes.

We have previously shown the existence of migratory hematopoietic stem cells in adult solid organs. This study demonstrates that granulocyte colony-stimulating factor (G-CSF) and lisofylline, a phosphatidic acid inhibitor that suppresses hematopoiesis-inhibiting cytokines, can enhance the engraftment of organ-based hematopoietic stem cells. When syngeneic heart grafts or liver nonparenchymal cells were transplanted into lethally irradiated (9.5 Gy) Lewis rats, complete hematopoietic reconstitution and animal survival were significantly improved by treating the recipient with G-CSF or, to a lesser extent, with lisofylline. Pretreatment of hepatic nonparenchymal cell donors with G-CSF, but not lisofylline, also resulted in striking improvement of recipient survival which was associated with an augmented subpopulation of donor stem cells. The results suggest that these drugs can be used to enhance the chimerism that we postulate to be the basis of organ allograft acceptance.

Staurosporine and its derivatives enhance f-Met-Leu-Phe-induced superoxide production via phospholipase D activation in human polymorphonuclear leukocytes.

Staurosporine (STAR) is one of the most potent inhibitors of protein kinase C (PKC). It is known that in human polymorphonuclear leukocytes (PMNs), the phorbol ester-induced generation of superoxide anion (respiratory burst) is effectively inhibited by STAR in a dose-dependent manner, whereas superoxide generation induced by chemoattractants, e.g. n-formyl-methionyl-leucyl-phenylalanine (FMLP) or PAF, is regulated biphasically by STAR. We compared the effects of STAR and K252a on FMLP-induced superoxide production from PMNs and examined the effects of propranolol, a inhibitor of phosphatidic acid (PA) phosphohydrolase, on the potentiation of the production by STAR. We also examined the effects of some derivatives of STAR and K252a on the production and the alteration of the effects induced by propranolol pretreatment. When PMNs were stimulated with FMLP, STAR potentiated superoxide production by 240.5 +/- 30.9% at a low concentration (100 nmol/l). Propranolol pretreatment specifically inhibited the potentiation. When phorbol-12-myristate-13-acetate (PMA) was used as a stimulant, STAR inhibited superoxide production dose-dependently and did not enhance the production. K252a inhibited PMA or FMLP-induced superoxide production dose-dependently and did not enhance FMLP-induced superoxide production. STAR derivatives showed potentiation of FMLP-induced superoxide production similar to that of STAR at concentrations ranging from 10-100 nmol/l, and propranolol (200 mumol/l) effectively inhibited it. K252a derivative NA332 did not show any potentiative effect on the production. PMA-induced superoxide production was inhibited by all compounds dose-dependently.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in cell phospholipid metabolism in vitro in the presence of HEMA and its degradation products.

OBJECTIVES: Diacylglycerol-kinase (DAG-kinase) is an enzyme that phosphorylates diacylglycerol (DAG) to phosphatidic acid (PA), which serves as a precursor to phosphoglycerides involved in cell signaling or as cell membrane structural components. DAG-kinase can be inhibited by diacylethylene glycols (DAEG). We hypothesize that 2-hydroxyethyl methacrylate (HEMA) may alter phosphorylation of DAG to PA following intracellular formation of DAEG. METHODS: Cultured rabbit kidney (RK13) epithelial cells were treated with HEMA, EG, or known inhibitors of DAG-kinase for 24 h, then exposed to [32P]O4- in the presence of a synthetic diacylglycerol for 2 h. Other cultures were radiolabeled with [3H]-oleic acid for 24 h, then exposed to HEMA for an additional 24 h. The cells were harvested and the lipids extracted. Radioactive lipids were separated by thin layer chromatography, located by autoradiography, and quantitated as cpm/ug protein. Cell cultures treated with HEMA were homogenized and the DAG-kinase activity was assayed and expressed as cpm/ug protein. Data were analyzed by one-way ANOVA and Newman-Keuls Multiple Comparison Test. RESULTS: Cultures exposed to HEMA or known DAG-kinase inhibitors exhibited reduced incorporation of radioactivity in the PA fraction compared to control cultures. Direct assays of DAG-kinase activity from cells exposed to HEMA demonstrated decreased enzyme activity. Evaluation of cell phospholipid synthesis showed altered formation of phosphatidylethanolamine and phosphatidylcholine. SIGNIFICANCE: Results suggest that HEMA impairs formation of PA, possibly by acylation of EG released by hydrolysis of the HEMA and resultant production of the inhibitor DAEG. The decreased availability of PA may alter PA-dependent cell structural lipid pathways and lipid-dependent signaling pathways, altering cell growth.

Topical combinations aimed at treating microvascular dysfunction reduce allodynia in rat models of CRPS-I and neuropathic pain.

UNLABELLED: Growing evidence indicates that various chronic pain syndromes exhibit tissue abnormalities caused by microvasculature dysfunction in the blood vessels of skin, muscle, or nerve. We tested whether topical combinations aimed at improving microvascular function would relieve allodynia in animal models of complex regional pain syndrome type I (CRPS-I) and neuropathic pain. We hypothesized that topical administration of either α(2)-adrenergic (α(2)A) receptor agonists or nitric oxide (NO) donors combined with either phosphodiesterase (PDE) or phosphatidic acid (PA) inhibitors would effectively reduce allodynia in these animal models of chronic pain. Single topical agents produced significant dose-dependent antiallodynic effects in rats with chronic postischemia pain, and the antiallodynic dose-response curves of PDE and PA inhibitors were shifted 2.5- to 10-fold leftward when combined with nonanalgesic doses of α(2)A receptor agonists or NO donors. Topical combinations also produced significant antiallodynic effects in rats with sciatic nerve injury, painful diabetic neuropathy, and chemotherapy-induced painful neuropathy. These effects were shown to be produced by a local action, lasted up to 6 hours after acute treatment, and did not produce tolerance over 15 days of chronic daily dosing. The present results support the hypothesis that allodynia in animal models of CRPS-I and neuropathic pain is effectively relieved by topical combinations of α(2)A or NO donors with PDE or PA inhibitors. This suggests that topical treatments aimed at improving microvascular function may reduce allodynia in patients with CRPS-I and neuropathic pain. PERSPECTIVE: This article presents the synergistic antiallodynic effects of combinations of α(2)A or NO donors with PDE or PA inhibitors in animal models of CRPS-I and neuropathic pain. The data suggest that effective clinical treatment of chronic neuropathic pain may be achieved by therapies that alleviate microvascular dysfunction in affected areas.

Effect of phosphate on aluminium-inhibited growth and signal transduction pathways in Coffea arabica suspension cells.

In acid soils, aluminium (Al) toxicity and phosphate (Pi) deficiency are the most significant constraints on plant growth. Al inhibits cell growth and disrupts signal transduction processes, thus interfering with metabolism of phospholipase C (PLC), an enzyme involved in second messenger production in the cell. Using a Coffea arabica suspension cell model, we demonstrate that cell growth inhibition by Al toxicity is mitigated at a high Pi concentration. Aluminium-induced cell growth inhibition may be due to culture medium Pi deficiency, since Pi forms complexes with Al, reducing Pi availability to cells. Phosphate does not mitigate inhibition of PLC activity by Al toxicity. Other enzymes of the phosphoinositide signal transduction pathway were also evaluated. Aluminium disrupts production of second messengers such as inositol 1,4,5-trisphosphate (IP(3)) and phosphatidic acid (PA) by blocking PLC activity; however, phospholipase D (PLD) and diacylglycerol kinase (DGK) activities are stimulated by Al, a response probably aimed at counteracting Al effects on PA formation. Phosphate deprivation also induces PLC and DGK activity. These results suggest that Al-induced cell growth inhibition is not linked to PLC activity inhibition.

Epithelial cell motility is triggered by activation of the EGF receptor through phosphatidic acid signaling.

Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid, and there is currently much interest in elucidating messenger functions for this molecule. We report here that wounding sheets of corneal epithelial and Madin Darby canine kidney cells induces strong activation of phospholipase D, and we provide evidence that activation is amplified through a positive feed-back loop. Short-chain analogues of phosphatidic acid induce motility robustly in corneal and other epithelial cell types. The effects of these analogues were not the result of their conversion to the corresponding diacylglycerol or lysophosphatidic acid, implying that phosphatidic acid acts directly on one or more cellular targets. Strikingly, phosphatidic acid signaling was found to stimulate the epidermal growth factor receptor (EGFR) through a transactivation process. Healing of wounds in sheets of corneal epithelial cells is absolutely dependent on epidermal growth factor receptor signaling, and the present data suggest that its activation is a result of wound-induced phospholipase D activation.

An essential role for phospholipase D in the activation of protein kinase C and degranulation in mast cells.

Activation of phospholipase D (PLD) and protein kinase C (PKC) as well as calcium mobilization are essential signals for degranulation of mast cells. However, the exact role of PLD in degranulation remains undefined. In this study we have tested the hypothesis that the PLD product, phosphatidic acid, and diacylglycerides generated therefrom might promote activation of PKC. Studies were conducted in two rodent mast cell lines that were stimulated with Ag via FcepsilonRI and a pharmacologic agent, thapsigargin. Diversion of production of phosphatidic acid to phosphatidylbutanol (the transphosphatidylation reaction) by addition of l-butanol suppressed both the translocation of diacylglyceride-dependent isoforms of PKC to the membrane and degranulation. Tertiary-butanol, which is not a substrate for the transphosphatidylation, had a minimal effect on PKC translocation and degranulation, and 1-butanol itself had no effect on PKC translocation when PKC was stimulated directly with phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. Also, in cells transfected with small inhibitory RNAs directed against PLD1 and PLD2, activation of PLD, generation of diacylglycerides, translocation of PKC, and degranulation were all suppressed. Phorbol ester, which did not stimulate degranulation by itself, restored degranulation when used in combination with thapsigargin whether PLD function was disrupted with 1-butanol or the small inhibitory RNAs. However, degranulation was not restored when cells were costimulated with Ag and phorbol ester. These results suggested that the production of phosphatidic acid by PLD facilitates activation of PKC and, in turn, degranulation, although additional PLD-dependent processes appear to be critical for Ag-mediated degranulation.

Stimulation of phospholipase D activity in human neuroblastoma (LA-N-2) cells by activation of muscarinic acetylcholine receptors or by phorbol esters: relationship to phosphoinositide turnover.

We have investigated the coupling of muscarinic acetylcholine receptors (mAChR) to phospholipid hydrolysis in a human neuroblastoma cell line, LA-N-2, by measuring the formation of 3H-inositol phosphates (3H-IP) and of [3H]phosphatidylethanol ([3H]PEt) in cells prelabeled with [3H]inositol and [3H]oleic acid. The muscarinic agonist carbachol (CCh) stimulated the phospholipase C (PLC)-mediated formation of 3H-IP in a time- and dose-dependent manner (EC50 = 40-55 microM). In addition, in the presence of ethanol (170-300 mM), CCh elevated levels of [3H]PEt [which is regarded as a specific indicator of phospholipase D (PLD) activity] by three- to sixfold. The effect of CCh on PEt formation also was dose dependent (EC50 = 50 microM). Both effects of CCh were antagonized by atropine, indicating that they were mediated by mAChR. Incubation of LA-N-2 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA, 0.1 microM; 10 min) increased [3H]PEt levels by up to 10-fold. This effect was inhibited by the protein kinase C (PKC) inhibitor staurosporine (1 microM) or by pretreatment for 24 h with 0.1 microM PMA, by 74% and 65%, respectively. In contrast, the effect of CCh on PEt accumulation was attenuated by only 28% in the presence of staurosporine (1 microM). In summary, these results suggest that, in LA-N-2 neuroblastoma cells, mAChR are coupled both to phosphoinositide-specific PLC and to PLD. PKC is capable of stimulating PLD activity in these cells; however, it is not required for stimulation of the enzyme by mAChR activation.