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PubMed2001

Phospholipase D and phosphatidic acid-mediated generation of superoxide in Arabidopsis.

Phospholipase D (PLD), which hydrolyzes phospholipids into free head groups and phosphatidic acid (PA), may regulate cellular processes through the production of lipid and lipid-derived messengers. We have genetically abrogated PLD alpha, the most prevalent isoform of PLD in plants, and the depletion of PLD alpha in Arabidopsis decreased the levels of PA and superoxide production in Arabidopsis leaf extracts. Addition of PA promoted the synthesis of superoxide in the PLD alpha-depleted plants, as measured by chemiluminescence and superoxide dismutase-inhibitable, NADPH-dependent reduction of cytochrome c and nitroblue tetrazolium. The PA-enhanced generation of superoxide was associated mainly with microsomal membranes. Among various lipids tested, PA was the most effective stimulator with the optimal concentrations between 100 and 200 microM. The PA-promoted production of superoxide was observed also in leaves directly infiltrated with PA. The added PA was more effective in stimulating superoxide generation in the PLD alpha-depleted leaves than in the PLD alpha-containing, wild-type leaves, suggesting that PA produced in the cell was more effective than added PA in promoting superoxide production. These data indicate that PLD plays a role in mediating superoxide production in plants through the generation of PA as a lipid messenger.

Antisense Elements (Genetics)↗
PubMed2003

The oleate-stimulated phospholipase D, PLDdelta, and phosphatidic acid decrease H2O2-induced cell death in Arabidopsis.

Hydrolysis of common membrane phospholipids occurs in response to various environmental stresses, but the control and cellular function of this hydrolysis are not fully understood. Hydrogen peroxide (H2O2) is a pivotal signaling molecule involved in various stress responses. Here, we show that the plasma membrane-bound phospholipase D, PLDdelta, is activated in response to H2O2 and that the resulting phosphatidic acid (PA) functions to decrease H2O2-promoted programmed cell death. The Arabidopsis genome has 12 PLD genes, and knockout of PLDdelta abolishes specifically the oleate-stimulated PLD activity. H2O2 treatment of Arabidopsis cells activates PLD enzyme activity, and ablation of PLDdelta abolishes that activation. PLDdelta-null cells display increased sensitivity to H2O2-induced cell death. The addition of PA to PLDdelta-null cells mitigates the H2O2 effect, whereas suppression of the H2O2-induced PA formation in wild-type cells increases the effect. PLDdelta-ablated plants exhibit increased susceptibility to stress. These results demonstrate that activation of oleate-stimulated PLDdelta constitutes an important step in the plant response to H2O2 and increasing plant stress tolerance.

Apoptosis↗
PubMed2004

Phosphatidic acid accumulation is an early response in the Cf-4/Avr4 interaction.

The Cladosporium fulvum (Cf)-4 gene of tomato confers resistance to the fungus C. fulvum, expressing the corresponding avirulence (Avr)4 gene, which codes for an elicitor protein. Little is known about how such mechanisms work, but previous studies have shown that elicitor recognition activates Ca(2+) signalling and protein kinases, such as mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase (CDPK). Here, we provide evidence that a new signalling component, the lipid second messenger phosphatidic acid (PA), is produced within a few minutes of AVR4/Cf-4 interaction. Using transgenic tobacco cells expressing the tomato Cf-4-resistance gene as a model system, phospholipid signalling pathways were studied by pre-labelling the cells with (32)P(i) and assaying for the formation of lipid signals after challenge with the fungal elicitor AVR4. A dramatic rapid response was an increase in (32)P-PA, together with its metabolic product diacylglycerol pyrophosphate (DGPP). AVR4 increased the levels of PA and DGPP in a Cf-4(+)-, time- and dose-dependent manner, while the non-matching elicitor AVR9 did not trigger any response. In general, PA signalling can be triggered by two different pathways: via phospholipase D (PLD), which generates PA directly by hydrolysing structural phospholipids like phosphatidylcholine (PC), or via PLC, which generates diacylglycerol (DAG) that is subsequently phosphorylated to PA by DAG kinase (DGK). To determine the origin of the AVR4-induced PA formation, a PLD-specific transphosphatidylation assay and a differential (32)P-labelling protocol were used. The results clearly demonstrated that most PA was produced via the phosphorylation of DAG. Neomycin and U73122, inhibitors of PLC activity, inhibited AVR4-induced PA accumulation, suggesting that the increase in DGK activity was because of increased PLC activity producing DAG. Lastly, evidence is provided that PLC signalling and, in particular, PA production could play a role in triggering responses, such as the AVR4-induced oxidative burst. For example, PLC inhibitors inhibited the oxidative burst, and when PA was added to cells, an oxidative burst was induced.

Animals↗
PubMed2004

Isolation and identification of phosphatidic acid targets from plants.

Phosphatidic acid (PA) is emerging as an important lipid signalling molecule. In plants, it is implicated in various stress-signalling pathways and is formed in response to wounding, osmotic stress, cold stress, pathogen elicitors, Nod factors, ethylene and abscisic acid. How PA exerts its effects is still unknown, mainly because of the lack of characterized PA targets. In an approach to isolate such targets we have used PA-affinity chromatography. Several PA-binding proteins were present in the soluble fraction of tomato and Arabidopsis cells. Using mass spectrometric analysis, several of these proteins, including Hsp90, 14-3-3 proteins, an SnRK2 serine/threonine protein kinase and the PP2A regulatory subunit RCN1 could be identified. As an example, the binding of one major PA-binding protein, phosphoenolpyruvate carboxylase (PEPC), was characterized further. Competition experiments with different phospholipids confirmed specificity for PA. Hypo-osmotic treatment of the cells increased the amount of PEPC that bound the PA beads without increasing the absolute amount of PEPC. This suggests that PEPC's affinity for PA had increased. The work shows that PA-affinity chromatography/mass spectrometry is an effective way to isolate and identify PA-binding proteins from plants.

Arabidopsis↗
PubMed2006

Metabolomic approaches reveal that phosphatidic and phosphatidyl glycerol phospholipids are major discriminatory non-polar metabolites in responses by Brachypodium distachyon to challenge by Magnaporthe grisea.

Metabolomic approaches were used to elucidate some key metabolite changes occurring during interactions of Magnaporthe grisea--the cause of rice blast disease--with an alternate host, Brachypodium distachyon. Fourier-transform infrared (FT-IR) spectroscopy provided a high-throughput metabolic fingerprint of M. grisea interacting with the B. distachyon accessions ABR1 (susceptible) and ABR5 (resistant). Principal component-discriminant function analysis (PC-DFA) allowed the differentiation between developing disease symptoms and host resistance. Alignment of projected 'test-set' on to 'training-set' data indicated that our experimental approach produced highly reproducible data. Examination of PC-DFA loading plots indicated that fatty acids were one chemical group that discriminated between responses by ABR1 and ABR5 to M. grisea. To identify these, non-polar extracts of M. grisea-challenged B. distachyon were directly infused into an electrospray ionization mass spectrometer (ESI-MS). PC-DFA indicated that M. grisea-challenged ABR1 and ABR5 were differentially clustered away from healthy material. Subtraction spectra and PC-DFA loadings plots revealed discriminatory analytes (m/z) between each interaction and seven metabolites were subsequently identified as phospholipids (PLs) by ESI-MS-MS. Phosphatidyl glycerol (PG) PLs were suppressed during both resistant and susceptible responses. By contrast, different phosphatidic acid PLs either increased or were reduced during resistance or during disease development. This suggests considerable and differential PL processing of membrane lipids during each interaction which may be associated with the elaboration/suppression of defence mechanisms or developing disease symptoms.

Gas Chromatography-Mass Spectrometry↗
PubMed1988

Rates of production and consumption of phosphatidic acid upon thrombin stimulation of human platelets.

Human platelets were labelled with [32P]Pi and [3H]glycerol before gel filtration. In unstimulated cells, the specific 32P radioactivity in phosphatidic acid (PtdOH) was similar to that of phosphatidylinositol (PtdIns) but only 4% of that of the gamma-phosphate of ATP. Upon 3 min of stimulation with 0.5 U/ml of thrombin, there was a 20-fold increase in specific 32P radioactivity of PtdOH which approached that of the ATP gamma-phosphate. Based on constant rates of synthesis and removal, this thrombin-induced increase in specific 32P radioactivity in PtdOH allowed us to calculate the flux of phosphate through PtdOH upon stimulation. Synthesis and removal occurred at rates of 107 and 52 nmol min-1/10(11) cells, respectively. The specific [3H]glycerol radioactivity was similar in PtdIns, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in unstimulated platelets. In PtdOH, it was 50% of that of the inositol phospholipids. Thrombin stimulation induced no changes in the specific 3H radioactivity of the inositol phospholipids whereas specific [3H]PtdOH increased to the level of these lipids. It is concluded that PtdIns, PtdInsP and PtdInsP2 exist in a metabolic homogenous pool in human platelets.

Adenosine Triphosphate↗
PubMed1988

Phosphatidic acid and phosphoinositide turnover in myelin and its stimulation by acetylcholine.

Brain slices obtained from the forebrains of adult female rats were incubated with [32P]phosphate and [3H]glycerol for 60 min, and lipids extracted and analyzed by TLC. The 32P in brain slice lipids was primarily in polyphosphoinositides, phosphatidylinositol (PI), and phosphatidate (PA). Distribution of the 32P-labeled lipids in isolated myelin was biased toward PA, 38%, relative to 16% in whole tissue slice lipids. About 33% of the total labeled PA in brain slices was accounted for by that in myelin. On a per milligram protein basis, PA labeling in myelin is about 2.5-fold greater than that of whole brain slice. Since incorporation of [3H]glycerol (indicative of synthesis by the de novo synthetic pathway) was at very low levels, we conclude that [32P]phosphate entered into myelin PA primarily through a pathway involving phospholipase C activity. Much of the production of PA relates to hydrolysis of phosphoinositides, yielding diacylglycerol which is then phosphorylated within myelin. The distribution of label among the inositol-containing lipids suggests that only a fraction of the myelin polyphosphoinositides serve as substrate for rapid diglyceride production. In the presence of 10 mM acetylcholine (ACh) there was a 20-60% stimulation of [32P]phosphate incorporation into PA and PI of brain slice lipids and purified myelin. Stimulation by ACh was blocked by atropine. The observed increase in the 32P/3H ratio, relative to controls, indicated that for both total lipids and myelin lipids there was selective stimulation of a phospholipase C-dependent cycle relative to de novo biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1991

Influence of bradykinin on diacylglycerol and phosphatidic acid accumulation in cultured bovine adrenal chromaffin cells.

Earlier studies have shown that bradykinin stimulated release of catecholamines from chromaffin cells by an influx of calcium through dihydropyridine-insensitive channels, and also that bradykinin stimulated (poly)phosphoinositide hydrolysis. To investigate membrane-bound second messengers in chromaffin cells, and to elucidate any role these may play in stimulus-secretion coupling, we have studied the influence of bradykinin on diacylglycerol and phosphatidic acid (PA). Using equilibrium labelling of primary cultures of chromaffin cells with [3H]arachidonic acid or [3H]glycerol, we found no influence of bradykinin (10 nM) on labelled diacylglycerol formation, either in the presence or absence of inhibitors of diacylglycerol lipase or kinase. However, when we used cells prelabelled with 32Pi for 2.5 h, we found that bradykinin produced a substantial stimulation of label found in PA, with an EC50 value of about 1 nM. This bradykinin stimulation of [32P]PA formation was only partially dependent on extracellular calcium, in contrast to the smaller response to nicotine, which was completely dependent on extracellular calcium. Short (10 min) pretreatment with tetradecanoylphorbol acetate (TPA) almost completely eliminated the bradykinin-stimulated formation of inositol phosphates, but failed to affect bradykinin stimulation of label in PA, suggesting that PA production in response to bradykinin is not downstream of phospholipase C activation. TPA alone failed to stimulate [32P]PA substantially, whereas long-term (24 or 48 h) treatment with TPA failed to attenuate the response to bradykinin. Diacylglycerol kinase inhibitors were also without effect on the bradykinin stimulation of [32P]PA. These results suggest that bradykinin stimulates PA production by a mechanism independent of the activation of protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Lack of phospholipase D activity in chromaffin cells: bradykinin-stimulated phosphatidic acid formation involves phospholipase C in chromaffin cells but phospholipase D in PC12 cells.

The role of lipid-bound second messengers in the regulation of neurotransmitter secretion is an important but poorly understood subject. Both bovine adrenal chromaffin cells and rat phoeochromocytoma (PC12) cells, two widely studied models of neuronal function, respond to bradykinin by generating phosphatidic acid (PA). This putative second messenger may be produced by two receptor-linked pathways: sequential action of phospholipase C (PLC) and diacylglycerol kinase (DAG kinase), or directly by phospholipase D (PLD). Here we show that bradykinin stimulation of chromaffin cells prelabelled (24 h) with 32Pi leads to production of [32P]PA which is not affected by 50 mM butanol. However, bradykinin stimulation of PC12 cells leads to [32P]PA formation, all of which is converted to phosphatidylbutanol in the presence of butanol. When chromaffin cells prelabelled with [3H]choline were stimulated with bradykinin there was no enhancement of formation of water soluble products of phosphatidylcholine hydrolysis. When chromaffin cells were permeabilised with pneumolysin and incubated in the presence of [gamma-32P]ATP, the formation of [32P]PA was still stimulated by bradykinin. These results show that, although both neuronal models synthesize PA in response to bradykinin, they do so by quite different routes: PLC/DAG kinase for chromaffin cells and PLD for PC12 cells. The observation that neither bradykinin nor tetradecanoyl phorbol acetate stimulate PLD in chromaffin cells suggests that these cells lack PLD activity. The conservation of PA formation, albeit by different routes, may indicate an essential role of PA in the regulation of cellular events by bradykinin.

Adrenal Gland Neoplasms↗
PubMed2006

Dynamics and function of phospholipase D and phosphatidic acid during phagocytosis.

Phospholipase D (PLD) produces phosphatidic acid (PA), an established intracellular signalling lipid that has been also implicated in vesicular trafficking, and as such, PLD could play multiple roles during phagocytosis. Using an RNA interference strategy, we show that endogenous PLD1 and PLD2 are necessary for efficient phagocytosis in murine macrophages, in line with results obtained with wild-type constructs and catalytically inactive PLD mutants which, respectively, enhance and inhibit phagocytosis. Furthermore, we found that PA is transiently produced at sites of phagosome formation. Macrophage PLD1 and PLD2 differ in their subcellular distributions. PLD1 is associated with cytoplasmic vesicles, identified as a late endosomal/lysosomal compartment, whereas PLD2 localizes at the plasma membrane. In living cells undergoing phagocytosis, PLD1 vesicles are recruited to nascent and internalized phagosomes, whereas PLD2 is only observed on nascent phagosomes. These results provide evidence that both PLD isoforms are required for phagosome formation, but only PLD1 seems to be implicated in later stages of phagocytosis occurring after phagosomal internalization.

Animals↗
PubMed2005

Janus kinase-signal transducer and activator of transcription mediates phosphatidic acid-induced interleukin (IL)-1beta and IL-6 production.

We have found previously that phosphatidic acid (PA) can induce inflammatory mediators such as cytokines, which implies that PA plays a role in inflammatory response. In the present study, we provide evidence of the PA-mediated activation of the Janus tyrosine kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, which results in the production of interleukin (IL)-1beta and IL-6. PA elicited the rapid phosphorylations of JAK2 and STAT1/3, and the subsequent nuclear translocation. Macrophages that had been transiently transfected with a luciferase reporter construct containing eight consecutive gamma-interferon activating sequence (GAS) elements, a known STAT binding site, exhibited enhanced reporter gene activity in response to PA stimulation, which further supports the involvement of JAK-STAT activation in the PA-induced signaling pathway. Of the inflammatory cytokines, IL-1beta, IL-6, and tumor necrosis factor (TNF)-alpha were detected in media from macrophages stimulated with PA. Moreover, the JAK2 inhibitor alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG-490) abolished PA-induced IL-1beta and IL-6 release but not TNF-alpha production, which is consistent with the notion that IL-1beta and IL-6 but not TNF-alpha contain a STAT binding element in their promoter region. The knockdown of JAK2 in macrophages by small interfering RNA significantly attenuated PA-induced IL-1beta and IL-6 production. In addition, JAK2 inhibitor suppressed PA-induced Akt phosphorylation, and the Akt inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) blocked GAS activation (GAS contains a promoter that responds to PA), suggesting that PA-mediated JAK2 activation leads to phosphatidylinositol 3-kinase/Akt phosphorylation and STAT activation, and the subsequent translocation of STAT to the nucleus. Together, our data demonstrate that PA-activated macrophages produce IL-1beta and IL-6 and that these processes require the activation of the JAK2-STAT1/3 or JAK2-Akt-STAT signaling pathways.

Active Transport, Cell Nucleus↗
PubMed1997

Selective activation of rolipram-sensitive, cAMP-specific phosphodiesterase isoforms by phosphatidic acid.

In rat thymic lymphocytes, accumulation of phosphatidic acid (PA) occurs at the same time as decrease in cAMP levels and activation of a cAMP-specific phosphodiesterase (PDE) [type 4, EC 3.1.4.17 (PDE4)]. We investigated the nature of the PDE activated by PA and the mechanism of activation by using recombinant cAMP-specific PDE4 isoforms derived from three different genes (PDE4A, PDE4B, and PDE4D). The "long" variants expressed from each gene (PDE4A5, PDE4B1, and PDE4D3) were activated by PA, whereas the "short" variants (PDE4A1, PDE4B2, PDE4D1, and PDE4D2) were not. Phosphatidylserine was an activator that was as effective as PA, whereas phosphatidylcholine was ineffective, indicating that activation was restricted to anionic phospholipids. PA caused an increase in the Vmax value of PDE4D3 without affecting the Km value of the enzyme for the cAMP substrate. PA also caused a change in the Mg2+ requirement for hydrolysis. Half-maximal stimulation of the PDE was obtained with approximately 10 microg/ml PA. Although protein kinase A-mediated phosphorylation of PDE4D3 produces effects similar to those elicited by PA, the mechanism of PA-induced activation was not found to involve a phosphorylation. Instead, several observations suggest that PA may directly interact with the enzyme. The stimulation of cAMP PDEs by PA and other acidic phospholipids may be a mechanism by which growth factors and hormones modulate the cAMP-dependent signal transduction pathway during cell stimulation.

Animals↗
PubMed2001

Phosphatidic acid-mediated mitogenic activation of mTOR signaling.

The mammalian target of rapamycin (mTOR) governs cell growth and proliferation by mediating the mitogen- and nutrient-dependent signal transduction that regulates messenger RNA translation. We identified phosphatidic acid (PA) as a critical component of mTOR signaling. In our study, mitogenic stimulation of mammalian cells led to a phospholipase D-dependent accumulation of cellular PA, which was required for activation of mTOR downstream effectors. PA directly interacted with the domain in mTOR that is targeted by rapamycin, and this interaction was positively correlated with mTOR's ability to activate downstream effectors. The involvement of PA in mTOR signaling reveals an important function of this lipid in signal transduction and protein synthesis, as well as a direct link between mTOR and mitogens. Furthermore, these studies suggest a potential mechanism for the in vivo actions of the immunosuppressant rapamycin.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Evidence for coupling of Clostridium perfringens alpha-toxin-induced hemolysis to stimulated phosphatidic acid formation in rabbit erythrocytes.

When rabbit erythrocytes were exposed to low concentrations of Clostridium perfringens alpha-toxin, hot-cold hemolysis was observed. The toxin induced production of phosphatidic acid (PA) in a dose-dependent manner when incubated with erythrocytes at 37 degrees C. When erythrocyte membranes were incubated with the toxin and [gamma-32P]ATP in the presence or absence of ethanol, [32P]PA formation was maximal within 30 s, then sharply decreased, and began again after 5 min of incubation. Ethanol had no effect on the early appearance (at approximately 5 min) of PA formation induced by the toxin but significantly inhibited formation of PA over 10 min of incubation. Treatment of erythrocyte membranes with alpha-toxin resulted in the biphasic formation of 1,2-diacylglycerol and PA as well as an increase of inositol-1,4,5-trisphosphate (IP3) and decrease of phosphatidylinositol-4,5-bisphosphate (PIP2) within 30 s. Neomycin inhibited the toxin-induced increase in turbidity of egg yolk suspensions but did not inhibit the toxin-induced hemolysis of intact erythrocytes. On the other hand, neomycin inhibited the toxin-induced hemolysis of saponin-treated erythrocytes. In addition, neomycin inhibited PA formation induced by the toxin in erythrocyte membranes. IP3 was released by incubation of PIP2 with erythrocyte membranes but not by incubation of PIP2 with the toxin. The toxin stimulated the membrane-induced release of IP3 from PIP2. These data suggest that the toxin-induced hemolysis is dependent on the action of phospholipase C in erythrocyte membranes.

Animals↗
PubMed1977

Phosphatidic acid synthesis in the heart. 1. Effect of age and species difference in the mitochondrial and microsomal synthesis.

Rates of syntheses of monoacyl- and diacyl-glycerol 3-P (phosphate) were determined in the mitochondrial and microsomal fractions prepared from hearts of rats and rabbits, to compare characteristics of the acylation reactions by the two subcellular fractions. The assays were carried out with the subcellular fractions prepared from (i) hearts of hyperthyroid animals, and (ii) hearts of newborn and weanling rats. In addition, the effect of an addition of bovine serum albumin in the assay system was examined. (1) Administration of thyroid hormones increased the acyltransferase activity in rabbit hearts but not that in rat hearts. (2) Mitochondrial and microsomal fractions of hearts of newborn rats acylated glycerol 3-P at a rate 1.3-4 times greater than those of adult rats. The rate of acylation by the mitochondrial fraction of weanling rats was also high, but the rate of microsomal acylation was slightly lower than that of adult rats. By contrast, in newborn rats, diacylglycerol 3-P formation by the liver microsomes was not greater than that of the adults, although its formation by the newborn liver mitochondria was greater. (3) The accumulation of monoacylglycerol 3-P during the assay was accelerated by the addition of increasing amounts of bovine serum albumin. Therefore, the monoacylglycerol 3-P formation was less than 10% of the diacylglycerol 3-P formation in the assay containing no albumin and with the rat subcellular fractions, whereas nearly four times more monoacyl- than diacyl-glycerol 3-P was synthesized in the presence of 20 mg albumin. (4) The ratio of monoacyl- to diacyl-glycerol 3-P formation by the mitochondrial fraction was greater than that of microsomal fraction at any concentration of albumin in both rats and rabbits. At an equal albumin concentration in the assay, relatively more diacyl- than monoacyl-glycerol 3-P was formed in the mitochondrial fraction of newborn rat hearts as compared with adult hearts; (5) In conclusion, our data concerning the age and species differences in acyltransferase activities support a view that the mitochondrial fraction of both rat and rabbit hearts, in addition to the microsomal enzymes, is capable of catalyzing the de novo synthesis of phosphatidic acid.

Acyltransferases↗
PubMed1985

Effects of prostaglandins and luteinizing hormone-releasing hormone on phosphatidic acid-phosphatidylinositol labeling in rat granulosa cells.

In cultures of rat granulosa cells, luteinizing hormone-releasing hormone (LHRH) increases 32P incorporation into both phosphatidylinositol (PI) and phosphatidic acid (PA). After 20 min, the level of radioactivity was three- to four-fold (p less than 0.01) above control in the PI and PA fractions, respectively. The stimulatory effect of LHRH on 32P incorporation was limited to PI and PA. Similar to the effects of LHRH, a rapid and marked increase of 32P incorporation into both PI and PA is observed upon addition of prostaglandin F2 alpha (PGF2 alpha) (10(-5)M) to rat granulosa cells. Incorporation of radioactivity into PA was already increased (p less than 0.05) by 2 min following PGF2 alpha addition, while the increase in 32P-labeled PI became significant (p less than 0.01) by 5 min. In contrast to PGF2 alpha, the labeling of PI and PA following the addition of PGE2 (10(-5)M) was not significantly different from control levels during the entire 10 min of incubation. The sensitivity of the increased PA-PI labeling induced by LHRH and PGF2 alpha is compared in another experiment. After 20 min incubation 10(-6)M LHRH increased PI and PA labeling by six- and four-fold, respectively. Although the effect of PGF2 alpha is less than that of LHRH, 10(-5)M PGF2 alpha significantly (p less than 0.01) increased PI and PA labeling by three- and two-fold, respectively. By contrast, 10(-6)M PGE2 failed to affect 32P incorporation into the various phospholipid fractions, but a small enhancement (p less than 0.05) of PI and PA labeling was observed only at 10(-5)M PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Phosphatidic acid stimulates cardiac KATP channels like phosphatidylinositols, but with novel gating kinetics.

Membrane-bound anionic phospholipids such as phosphatidylinositols have the capacity to modulate ATP-sensitive potassium (K(ATP)) channels through a mechanism involving long-range electrostatic interaction between the lipid headgroup and channel. However, it has not yet been determined whether the multiple effects of phosphatidylinositols reported in the literature all result from this general electrostatic interaction or require a specific headgroup structure. The present study investigated whether phosphatidic acid (PA), an anionic phospholipid substantially different in structure from phosphatidylinositols, evokes effects similar to phosphatidylinositols on native K(ATP) channels of rat heart and heterogeneous Kir6.2/SUR2A channels. Channels treated with PA (0.2-1 mg/ml applied to the cytoplasmic side of the membrane) exhibited higher activity, lower sensitivity to ATP inhibition, less Mg(2+)-dependent nucleotide stimulation, and poor sulfonylurea inhibition. These effects match the spectrum of phosphatidylinositols' effects, but, in addition, PA also induced a novel pattern in gating kinetics, represented by a decreased mean open time (from 12.2 +/- 2.0 to 3.3 +/- 0.7 ms). This impact on gating kinetics clearly distinguishes PA's effects from those of phosphatidylinositols. Results indicate that multiple effects of anionic phospholipids on K(ATP) channels are related phenomena and can likely be attributed to a common mechanism, but additional specific effects due to other mechanisms may also coincide.

Adenosine Triphosphate↗
PubMed1992

Lipid A activation of glomerular mesangial cells: mimicry of the bioactive lipid, phosphatidic acid.

Lipid A, the active component of bacterial endotoxin, stimulates multiple cell types, including glomerular mesangial cells (MC), and yet the molecular mechanisms of cell activation remain unclear. Lipid A, in its monosaccharyl form, structurally resembles the biologically active lipid phosphatidic acid (PA). Given this, it was postulated that lipid A activates cells by acting as a structural and functional mimetic of PA. Lipid A was found to specifically stimulate an MC lyso-PA acyl transferase activity, leading to enhanced synthesis of sn-2-unsaturated forms of PA. Sn-2-unsaturated PA itself, in contrast to sn-2-saturated PA, also stimulated the lyso-PA acyl transferase activity, a positive feedback feature previously noted with lyso-lecithin acyl transferase. Structure-function correlations demonstrated that the phosphate moieties in both PA and lipid A were necessary to feedback stimulation of lyso-PA acyl transferase (AT), as dephosphorylated lipid A and 2-unsaturated 1,2-sn-diacylglycerol had no stimulatory effect on lyso-PA AT. The biologic relevance of the lipid A and PA-mediated increases in lyso-PA acyl transferase activity was shown, whereby limited exposure to these lipids rapidly induced identical MC morphologic and functional alterations characteristic of cellular activation. By mimicking the stimulatory action of PA, per se, on lyso-PA acyl transferase activity, lipid A may initiate a positive feedback cycle of acylation, yielding increased amounts of PA enriched in unsaturated fatty acids. This newly synthesized PA may subsequently act as the proximal mediator of cellular activation.

Acyltransferases↗