Ethanol inhibits astroglial cell proliferation by disruption of phospholipase D-mediated signaling

The activation of phospholipase D (PLD) is a common response to mitogenic stimuli in various cell types. As PLD-mediated signaling is known to be disrupted in the presence of ethanol, we tested whether PLD is involved in the ethanol-induced inhibition of cell proliferation in rat cortical primary astrocytes. Readdition of fetal calf serum (FCS) to serum-deprived astroglial cultures caused a rapid, threefold increase of PLD activity and a strong mitogenic response; both effects were dependent on tyrosine kinases but not on protein kinase C. Ethanol (0.1-2%) suppressed the FCS-induced, PLD-mediated formation of phosphatidic acid (PA) as well as astroglial cell proliferation in a concentration-dependent manner. Moreover, exogenous bacterial PLD increased astroglial proliferation in an ethanol-sensitive manner, whereas exogenous PA or lysophosphatidic acid was less effective. Formation of PA and astroglial proliferation were strongly inhibited by 1-butanol (0.1-1%), a substrate of PLD, but were unaffected by t-butanol, a non-substrate; 2-butanol had intermediate effects. Platelet-derived growth factor and endothelin-1 mimicked the mitogenic effect of FCS; their effects were also inhibited by the butanols in the potency order 1-butanol > 2-butanol > tert-butanol. Our results, in particular, the differential effects of 1-, 2-, and tert-butanol with respect to PA formation and astroglial proliferation, strongly suggest that the antiproliferative effects of ethanol in glial cells are due to the disruption of the PLD signaling pathway. This mechanism may also contribute to the inhibition of astroglial growth and brain development observed in alcoholic embryopathy.     

Role of phospholipase D signaling in ethanol-induced inhibition of carbachol-stimulated DNA synthesis of 1321N1 astrocytoma cells

Inhibition of astrocyte proliferation has been suggested to be an important event in the developmental neurotoxicity associated with ethanol. We have previously shown that the acetylcholine analog carbachol induces astroglial cell proliferation through activation of muscarinic M3 receptors, and that ethanol strongly inhibits this effect by inhibiting activation of protein kinase C (PKC) zeta and its down-stream effector 70-kDa ribosomal S6 kinase (p70S6K). In this study, we investigated whether inhibition by ethanol of this signal transduction pathway in 1321N1 human astrocytoma cells may be due, at least in part, to inhibition of the formation of the PKC zeta activator phosphatidic acid (PA), which is formed by hydrolysis of phosphatidylcholine by phospholipase D (PLD). 1-Butanol, which is a substrate for PLD and inhibits PA formation, inhibited carbachol-induced cell proliferation and the underlying intracellular signaling, whereas its analog tert-butanol, which is a poor substrate for PLD, was much less effective. In addition, exogenous PAs were able to increase DNA synthesis and to activate PKC zeta and p70S6K. Furthermore, in carbachol-stimulated cells, ethanol increased the formation of phosphatidylethanol and inhibited the formation of PA. Taken together, these results indicate that PLD activation plays an important role in carbachol-induced astroglial cell proliferation by generating the second messenger PA, which activates PKC zeta. Moreover, the effect of ethanol on carbachol-induced proliferation appears to be mediated, at least in part, by its ability to interact with PLD leading to a decreased synthesis of PA.     

Stability of phospholipase D in primary astrocytes

Induction of expression and proteolytic breakdown of phospholipase D (PLD) isoforms in primary astrocyte cultures have been investigated. Astrocytes express both PLD1 and 2 and are dependent on PLD activity for cell proliferation [K. K?tter, J. Klein, J. Neurochem. 73 (1999) 2517]. Competitive RT-PCR analysis demonstrated a higher level of PLD1 mRNA than PLD2 mRNA (8.9 vs. 0.9amol/microg RNA, respectively). Treatment of astroglial cultures with the phorbol ester, 4beta-phorbol-12beta,13alpha-dibutyrate (0.1 microM), for 24-48h selectively induced PLD1b but not PLD1a or 2 expression as shown by PCR and Western blot; the effect was sensitive to G? 6976. In cells transiently permeabilized with streptolysin-O, antisense oligonucleotides directed against PLD1 or 2 entered the cytoplasm as shown by immunofluorescence experiments but did not affect astroglial proliferation within 2-6 days. Treatment of the cultures with cycloheximide revealed that PLD1 and 2 proteins had biological half-lives of 2-3 days (PLD2) and 4-6 days (PLD1), respectively. It has been concluded that astroglial PLD1b is up-regulated by phorbol esters via protein kinase C activation. Down-regulation of PLD isoforms is prevented by extended biological half-lives of the PLD proteins.     

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.     

Differentiation of U937 cells enables a phospholipase D-dependent pathway of cytosolic phospholipase A2 activation.

Treatment with dibutyryl cyclic AMP (dBcAMP) of the human, premonocytic U937 cell line results in differentiation toward a monocyte/granulocyte-like cell. This differentiation enables the cell to activate cytosolic phospholipase A2 (cPLA2) to release arachidonate upon stimulation. In contrast, undifferentiated cells are unable to release arachidonate even when stimulated with calcium ionophores. In the present research, a role for phospholipase D (PLD) in the regulation of cPLA2 was shown based on a number of observations. First, the ionomycin- and fMLP-stimulated production of arachidonate in differentiated cells was sensitive to ethanol (2% (v/v)). Ethanol acts as an alternate substrate in place of water for PLD producing phosphatidylethanol (PEt) instead of phosphatidic acid. Indeed, ionomycin stimulation of differentiated cells produced a 14-fold increase in PEt levels. Further evidence for the involvement of PLD in the regulation of cPLA2 came from the observation that the stimulated production of diacylglycerol (for which phosphatidic acid is a major source) was greatly diminished in undifferentiated cells as compared to differentiated cells. Moreover, the normally deficient activation of cPLA2 in undifferentiated cells could be stimulated to release arachidonate if the cells were electroporated in the presence of GTP[gamma]S and MgATP. This treatment stimulates phosphatidylinositol-4,5-bisphosphate (PIP2) production which appears to activate PLD and cPLA2 in subsequent steps. The phosphatidic acid (and diacylglycerol derived from phosphatidic acid) appears to greatly regulate the action of cPLA2 by an unknown mechanism, and undifferentiated cells lack the ability to stimulate PLD activity due to a dysfunction of PIP2 production.     

Functions and pathophysiological roles of phospholipase D in the brain

Ten years after the isoforms of mammalian phospholipase D (PLD), PLD1 and 2, were cloned, their roles in the brain remain speculative but several lines of evidence now implicate these enzymes in basic cell functions such as vesicular trafficking as well as in brain development. Many mitogenic factors, including neurotransmitters and growth factors, activate PLD in neurons and astrocytes. Activation of PLD downstream of protein kinase C seems to be a required step for astroglial proliferation. The characteristic disruption of the PLD signaling pathway by ethanol probably contributes to the delay of brain growth in fetal alcohol syndrome. The post-natal increase of PLD activities concurs with synapto- and myelinogenesis in the brain and PLD is apparently involved in neurite formation. In the adult and aging brain, PLD activity has antiapoptotic properties suppressing ceramide formation. Increased PLD activities in acute and chronic neurodegeneration as well as in inflammatory processes are evidently due to astrogliosis and may be associated with protective responses of tissue repair and remodeling. ARF-regulated PLD participates in receptor endocytosis as well as in exocytosis of neurotransmitters where PLD seems to favor vesicle fusion by modifications of the shape and charge of lipid membranes. Finally, PLD activities contribute free choline for the synthesis of acetylcholine in the brain. Novel tools such as RNA interference should help to further elucidate the roles of PLD isoforms in brain physiology and pathology.     

Phosphatidic acid produced by phospholipase D is required for tobacco pollen tube growth

Phospholipase D (PLD) and its product phosphatidic acid (PA) are involved in a number of signalling pathways regulating cell proliferation, membrane vesicle trafficking and defence responses in eukaryotic cells. Here we report that PLD and PA have a role in the process of polarised plant cell expansion as represented by pollen tube growth. Both phosphatidylinositol-4,5-bisphosphate-dependent and independent PLD activities were identified in pollen tube extracts, and activity levels during pollen tube germination and growth were measured. PLD-mediated PA production in vivo can be blocked by primary alcohols, which serve as a substrate for the transphosphatidylation reaction. Both pollen germination and tube growth are stopped in the presence 0.5% 1-butanol, whereas secondary and tertiary isomers do not show any effect. This inhibition could be overcome by addition of exogenous PA-containing liposomes. In the absence of n-butanol, addition of a micromolar concentration of PA specifically stimulates pollen germination and tube elongation. Furthermore, a recently established link between PLD and microtubule dynamics was supported by taxol-mediated partial rescue of the 1-butanol-inhibited pollen tubes. The potential signalling role for PLD-derived PA in plant cell expansion is discussed.     

An indirect pathway of receptor-mediated 1,2-diacylglycerol formation in mast cells. I. IgE receptor-mediated activation of phospholipase D

The current studies explore the role of phospholipase D (PLD) in mast cell activation. Although most investigators believe that receptor-mediated accumulation of 1,2-diacylglycerol (DAG) occurs by phospholipase C hydrolysis of phosphoinositides, our previous work indicated a modest role for these substrates and suggested that phosphatidylcholine (PC) is the more likely substrate. PLD cleaves the terminal phosphodiester bond of phospholipids to yield phosphatidic acid (PA), but in the presence of ethanol, it transfers the phosphatidyl moiety of the phospholipid substrate to ethanol producing phosphatidylethanol (PEt); a reaction termed transphosphatidylation. In purified rat mast cells prelabeled with [3H]arachidonic acid, [3H]palmitic acid, or 1-O-[3H]alkyl-lysoPC, a receptor-associated increase in PLD activity was initially suggested by the rapid accumulation of labeled PA, although other mechanisms might be involved. PLD activity was assessed more directly by the production of labeled PEt by PLD-mediated transphosphatidylation in the presence of ethanol. IgE receptor cross-linking resulted in a 3- to 10-fold increase in PLD activity during the 10 min after stimulation, approximately 50% of which occurred during the first two min. PEt formation was dependent on the concentration of ethanol and was maximal at 0.5%. At concentrations of ethanol greater than or equal to 0.2%, receptor-dependent formation of PA was reduced suggesting that the ethanol promoted transphosphatidylation at the expense of hydrolysis. The dose-related decline in PA accumulation seen in the presence of ethanol was similar to ethanol-mediated inhibition of exocytosis suggesting that receptor-mediated PA formation may be of regulatory importance. These observations indicate that PLD-mediated formation of PA occurs in stimulated mast cells and, in conjunction with separate findings of PA phosphohydrolase conversion of PA to DAG in mast cells, suggest that a major mechanism of DAG formation during mast cell activation is PC----PA----DAG.     

Ceramide pathways modulate ethanol-induced cell death in astrocytes

We showed previously that alcohol exposure during in vivo brain development induced astroglial damage and caused cell death. Because ceramide modulates a number of biochemical and cellular responses to stress, including apoptosis, we now investigate whether ethanol-induced cell death in astrocytes is mediated by ceramide signalling pathways triggering apoptosis. Here we show that both ethanol and ceramide are able to induce apoptotic death in cultured astrocytes, in a dose-dependent manner, and that C2-ceramide addition potentiates the apoptotic effects of ethanol. Cell death induced by ethanol is associated with stimulation of neutral and acidic sphingomyelinase (SMase) and ceramide generation, as well as with activation of stress-related kinases, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38) and extracellular signal-regulated kinase (ERK) pathways. We also provide evidence for the participation of JNK and p38 in ethanol-induced cell death, because pharmacological inhibitors of these kinases largely prevent the apoptosis induced by ethanol or by ethanol and C2-ceramide. Furthermore, we show that ethanol-induced ERK activation triggers the stimulation of cyclo-oxygenase-2 (COX-2) and the release of prostaglandin E2, and that blockade of the mitogen-activated protein kinase kinase (MEK)/ERK pathway by PD98059 abolishes the up-regulation of COX-2 induced by ethanol plus ceramide, and decreases the ethanol-induced apoptosis. These results strongly suggest that ethanol is able to stimulate the SMase-ceramide pathway, leading to the activation of signalling pathways implicated in cell death. These findings provide an insight into the mechanisms involved in ethanol-induced astroglial cell death during brain development.     

植物信号传导中的磷脂酶

20世纪 80年代早期人们意识到构成细胞膜的磷脂不只是一道将细胞物质与外界隔开的屏障,而且是细胞对外界环境刺激作出应答的物质基础。磷脂酰肌醇（ｐｈｏｓｐｈｏｔｉｄｙｌｉｎｏｓｉｔｏｌ,ＰＩ）不但是构成细胞膜的重要组分（约占细胞膜组分的 10 %）,在细胞内外环境信号的传递方面也起着重要的作用［1］。磷脂酶（ｐｈｏｓｐｈｏｌｉｐａｓｅ）水解磷脂后产生的三磷酸肌醇 （ｉｎｏｓ ｉｔｏｌｔｒｉｓｐｈｏｓｐｈａｔｅ,ＩＰ3 ）／二酰基甘油（ｄｉａｃｙｌｇｌｙｃｅｒｏｌ,ＤＡＧ）、磷脂酸（ｐｈｏｓｐｈａｔｉｄｉｃａｃｉ…     

Phospholipase D regulation and localisation is dependent upon a phosphatidylinositol 4,5-biphosphate-specific PH domain

The signalling pathway leading, for example, to actin cytoskeletal reorganisation, secretion or superoxide generation involves phospholipase D (PLD)-catalysed hydrolysis of phosphatidylcholine to generate phosphatidic acid, which appears to mediate the messenger functions of this pathway. Two PLD genes (PLD1 and PLD2) with similar domain structures have been doned and progress has been made in identifying the protein regulators of PLD1 activation, for example Arf and Rho family members. The activities of both PLD isoforms are dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and our sequence analysis suggested the presence of a pleckstrin homology (PH) domain in PLD1, although its absence has also been daimed. Investigation of the inositide dependence showed that a bis-phosphorylated lipid with a vicinal pair of phosphates was required for PLD1 activity. Furthermore, PLD1 bound specifically and with high affinity to lipid surfaces containing PI(4,5)P2 independently of the substrate phosphatidylcholine, suggesting a key role for the PH domain in PLD function. Importantly, a glutathione-S-transferase (GST) fusion protein comprising GST and the PH domain of PLD1 (GST-PLD1-PH) also bound specifically to supported lipid monolayers containing PI(4,5)P2. Point mutations within the PLD1 PH domain inhibited enzyme activity, whereas deletion of the domain both inhibited enzyme activity and disrupted normal PLD1 localisation. Thus, the functional PH domain regulates PLD by mediating its interaction with polyphosphoinositide-containing membranes; this might also induce a conformational change, thereby regulating catalytic activity.     

The oligodendrocyte precursor mitogen PDGF stimulates proliferation by activation of alpha(v)beta3 integrins

Central nervous system development requires precise and localized regulation of neural precursor behaviour. Here we show how the interaction between growth factor and integrin signalling pathways provides a mechanism for such precision in oligodendrocyte progenitor (OP) proliferation. While physiological concentrations of platelet-derived growth factor (PDGF) were not in themselves sufficient to promote OP proliferation, they did so on extracellular matrix (ECM) substrates that bind alpha(v)beta3 integrin. Upon PDGF-AA exposure and alpha(v)beta3 engagement, a physical co-association between both receptors was demonstrated, confirming the interaction between these signalling pathways. Furthermore, we found that PDGFalphaR stimulated a protein kinase C-dependent activation of integrin alpha(v)beta3, which in turn induced OP proliferation via a phosphatidylinositol 3-kinase-dependent signalling pathway. These studies establish a mechanism by which OP proliferation is dependent on the availability of both an ECM ligand and a mitogenic growth factor. Growth factor- mediated integrin activation is the critical integrative step in proliferation signalling, and ensures that the response of neural precursor cells to long-range cues can be regulated by their cellular neighbours, allowing precise control of cell behaviour during development.     

Phospholipase D Activity in the Tetrahymena pyriformis GL

Phospholipase D (PLD) is an enzyme which participates in the signalling mechanism cleaving phosphatidylcholine (PC) to choline and phosphatidic acid (PA). In Tetrahymena pyriformis GL this enzyme activity is enhanced by different kinds of agonists (sodium orthovanadate, sodium fluoride and phorbol 12-myristate 13-acetate), and its activity can be inhibited by inhibitors such as pertussis toxin, calphostin C, genistein, trifluoperazine. These results suggest that the PLD signalling pathway is connected with the tyrosine kinase, phospholipase C, phosphatidylinositol and G-protein coupled signalling pathways. By demonstrating the PLD activity in Tetrahymena our knowledge on the signalling mechanisms at a unicellular level has been extended. The results support our view that most transducing mechanisms that are characteristic of mammalian cells are also in the protozoan Tetrahymena. © 1997 John Wiley & Sons, Ltd.     

Potential role of phospholipase D2 in increasing interleukin-2 production by T-lymphocytes through activation of mitogen-activated protein kinases ERK1/ERK2

Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of phosphatidic acid (PA), which is itself a source of diacylglycerol (DAG). These two versatile lipid second messengers are at the centre of a phospholipid signalling network and as such are involved in several cellular functions. However, their role in T-cell activation and functions are still enigmatic. In order to elucidate this role, we generated a human and a murine T-cell line that stably overexpressed the PLD2 isoform. Analysis of the Ras-MAPK pathway upon phorbol myristate acetate (PMA) and ionomycin stimulation revealed that PLD2 promoted an early and sustained increase in ERK1/2 phosphorylation in both cell lines. This response was inhibited by 1-butanol, a well known distracter of PLD activity, or upon overexpression of a dominant negative PLD2, and it was concomitant with a boost of PA/DAG production. As a functional consequence of this PLD2-dependent MAPK activation, interleukin-2 production evoked by PMA/ionomycin stimulation or CD3/CD28 engagement was enhanced in the two T-cell lines overexpressing PLD2. Thus, PLD2 emerged as an early player upstream of the Ras-MAPK-IL-2 pathway in T-cells via PA and DAG production, raising new possibilities of pharmacological manipulation in immune disorders.     

Rapid attenuation of receptor-induced diacylglycerol and phosphatidic acid by phospholipase D-mediated transphosphatidylation: formation of bisphosphatidic acid.

Generation and attenuation of lipid second messengers are key processes in cellular signalling. Receptor-mediated increase in 1,2-diacylglycerol (DG) levels is attenuated by DG kinase and DG lipase. We here report a novel mechanism of DG attenuation by phospholipase D (PLD), which also precludes the production of another (putative) second messenger, phosphatidic acid (PA). In the presence of an alcohol, PLD converts phosphatidylcholine (PC) into a phosphatidylalcohol (by transphosphatidylation) rather than into PA. We found in bradykinin-stimulated human fibroblasts that PLD mediates transphosphatidylation from PC (donor) to the endogenous 'alcohol' DG (acceptor), yielding bis(1,2-diacylglycero)-3-sn-phosphate (bisphosphatidic acid; bisPA). This uncommon phospholipid is thus a condensation product of the phospholipase C (PLC) and PLD signalling pathways, where PLC produces DG and PLD couples this DG to a phosphatidyl moiety. Long-term phorbol ester treatment blocks bradykinin-induced activation of PLD and consequent bisPA formation, thereby unveiling rapid formation of DG. BisPA formation is rapid (15 s) and transient (peaks at 2-10 min) and is also induced by other stimuli capable of raising DG and activating PLD simultaneously, e.g. endothelin, lysophosphatidic acid, fetal calf serum, phorbol ester, dioctanoylglycerol or bacterial PLC. This novel metabolic route counteracts rapid accumulation of receptor-induced DG and PA, and assigns for the first time a physiological role to the transphosphatidylation activity of PLD, that is signal attenuation.     

The Role of Phospholipase D in Modulating the MTOR Signaling Pathway in Polycystic Kidney Disease

The mammalian target of rapamycin (mTOR) signaling pathway is aberrantly activated in polycystic kidney disease (PKD). Emerging evidence suggests that phospholipase D (PLD) and its product phosphatidic acid (PA) regulate mTOR activity. In this study, we assessed in vitro the regulatory function of PLD and PA on the mTOR signaling pathway in PKD. We found that the basal level of PLD activity was elevated in PKD cells. Targeting PLD by small molecule inhibitors reduced cell proliferation and blocked mTOR signaling, whereas exogenous PA stimulated mTOR signaling and abolished the inhibitory effect of PLD on PKD cell proliferation. We also show that blocking PLD activity enhanced the sensitivity of PKD cells to rapamycin and that combining PLD inhibitors and rapamycin synergistically inhibited PKD cell proliferation. Furthermore, we demonstrate that targeting mTOR did not induce autophagy, whereas targeting PLD induced autophagosome formation. Taken together, our findings suggest that deregulated mTOR pathway activation is mediated partly by increased PLD signaling in PKD cells. Targeting PLD isoforms with pharmacological inhibitors may represent a new therapeutic strategy in PKD.     

Atrial natriuretic peptide effects on intracellular pH changes and ROS production in HEPG2 cells: role of p38 MAPK and phospholipase D.

AIMS: The present study was performed to evaluate Atrial Natriuretic Peptide (ANP) effects on intracellular pH, phospholipase D and ROS production and the possible relationship among them in HepG2 cells. Cancer extracellular microenvironment is more acidic than normal tissues and the activation of NHE-1, the only system able to regulate pHi homeostasis in this condition, can represent an important event in cell proliferation and malignant transformation. METHODS: The ANP effects on pHi were evaluated by fluorescence spectrometry. The effects on p38 MAPK and ROS production were evaluated by immunoblots and analysis of DCF-DA fluorescence, respectively. RT-PCR analysis and Western blotting were used to determine the ANP effect on mRNA NHE-1 expression and protein levels. PLD-catalyzed conversion of phosphatidylcholine to phosphatydilethanol (PetOH), in the presence of ethanol, was monitored by thin layer chromatography. RESULTS: A significant pHi decrease was observed in ANP-treated HepG2 cells and this effect was paralleled by the enhancement of PLD activity and ROS production. The ANP effect on pHi was coupled to an increased p38 MAPK phosphorylation and a down-regulation of mRNA NHE-1 expression and protein levels. Moreover, the relationship between PLD and ROS production was demonstrated by calphostin-c, a potent inhibitor of PLD. At the same time, all assessed ANP-effects were mediated by NPR-C receptors. CONCLUSION: Our results indicate that ANP recruits a signal pathway associated with p38 MAPK, NHE-1 and PLD responsible for ROS production, suggesting a possible role for ANP as novel modulator of ROS generation in HepG2 cells.     

Attenuated PLD1 association and signalling at the H452Y polymorphic form of the 5-HT2A receptor

The 5-HT_2A receptor (5-HT_2AR) is implicated in psychotropic changes within the central nervous system (CNS). A number of polymorphisms have been reported in the 5-HT_2AR gene; one of these results in a non-synonymous change, H452Y, in the carboxy-terminal tail of the receptor protein. The minor allele (9% occurrence) has been statistically associated with CNS dysfunction such as impaired memory processing and resistance to neuroleptic treatment in schizophrenic patients. We investigated the impact of H452Y mutation of the 5-HT_2AR expressed in COS7 cells on distinctly coupled intracellular signalling pathways from the receptor, focusing on the heterotrimeric G protein-independent phospholipase D (PLD) pathway, compared to the conventional Gq/11-linked phospholipase C (PLC) pathway. The H452Y mutation selectively attenuated PLD signalling, which as in the wild-type receptor, was mediated by a molecular complex involving PLD1 docked to the receptor's carboxy-terminal tail domain. Co-immunoprecipitation and GST-fusion protein experiments revealed that the H452Y mutation selectively reduced PLD1 binding to the receptor. Experiments with blocking peptides to mimic short sections of the 5-HT_2AR tail sequence revealed that the peptide spanning residue 452 strongly reduced PLD but not PLC responses of the receptor. Similar observations were made when assessing both PLD responses and PLD-dependent cellular proliferation elicited by activation of 5-HT_2ARs natively expressed in MCF-7 cells. Overall these findings indicate that the H452Y polymorphic variant of the 5-HT_2AR displays selective disruption of its PLD signalling pathway. This may potentially play a role in the CNS dysfunction associated with the H452Y allele of the 5-HT_2AR.     

Phospholipase D as a potential target for the antiproliferative effects of polyunsaturated fatty acids in rat thymocytes

The effects of various saturated and unsaturated fatty acids (FAs) on the proliferative response and phospholipase D (PLD) activity of rat thymocytes were investigated. When added to culture medium as complexes with albumin, all the FAs tested, except stearic acid, inhibited the ConA-induced thymocyte proliferation, eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids being the most inhibitory. Apart from 22:6n-3 which slightly increased the percentage of late apoptotic and necrotic thymocytes in the presence of mitogen, none of the FAs induced significant apoptosis or necrosis. A short 2-h preincubation of rat thymocytes in the presence of FA-albumin complexes was sufficient to induce a significant enrichment of cell phospholipids with each FA and to stimulate thymocyte PLD activity. However, 20:5n-3 was inactive despite a large enrichment in phospholipids. Furthermore, the PLD activity of activated thymocytes was negatively correlated to the proliferative response, with the exception of 20:5n-3-supplemented cells. These results support further our current hypothesis that PLD activity conveys antiproliferative signals in lymphoid cells, and suggest that 20:5n-3 inhibits thymocyte proliferation by a particular mechanism unrelated to that of the other FAs.