Disturbed phospholipid homeostasis in endoplasmic reticulum initiates tri-o-cresyl phosphate-induced delayed neurotoxicity.

Tri-o-cresyl phosphate (TOCP) is a widely used organophosphorus compound, which can cause a neurodegenerative disorder, i.e., organophosphate-induced delayed neurotoxicity (OPIDN). The biochemical events in the initiation of OPIDN were not fully understood except for the essential inhibition of neuropathy target esterase (NTE). NTE, located in endoplasmic reticulum (ER), catalyzes the deacylation of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) to glycerophosphocholine (GPC). The present study aims to study the changes of ER phospholipids profile as well as levels of important intermediates of phospholipid synthesis such as diacylglycerol (DAG) and phosphatidic acid (PA) at the initiation stage of OPIDN. Hens are the most commonly used animal models of OPIDN. The spinal cord phospholipidomic profiles of hens treated by TOCP were studied by using HPLC-MS-MS. The results revealed that TOCP induced an increase of PC, LPC, and sphingomyelin (SM) levels and a decrease of GPC, phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), phosphatidylglycerol (PG), and phosphatidylinositol (PI) levels., Levels of DAG and PA were also decreased. Pretreatment with phenylmethylsulfonyl fluoride (PMSF) 24 h before TOCP administration prevented OPIDN and restored the TOCP-induced changes of phospholipids except GPC. Thus, the disruption of ER phospholipid homeostasis may contribute to the initiation of organophosphate-induced delayed neurotoxicity.

Tuberous sclerosis complex 2 loss increases lysophosphatidylcholine synthesis in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a destructive lung disease affecting women. LAM is caused by mutations in the tuberous sclerosis complex (TSC) genes. The TSC protein complex inhibits the mechanistic/mammalian target of rapamycin complex 1 (mTORC1), which is a master regulator of cellular metabolism. Using mass spectrometry-based lipid profiling, we analyzed plasma from patients with LAM and discovered elevated levels of four lysophosphatidylcholine (LPC) species (C16:0, C18:0, C18:1, and C20:4) compared with those in healthy control women. To investigate whether these lipids are generated in a TSC2-dependent manner, we profiled in vitro preclinical models of TSC/LAM and found significant LPC accumulation in TSC2-deficient cells relative to TSC2-expressing control cells. These lysoglycerophospholipid changes occurred alongside changes in other phospholipid and neutral lipid species. Treatment with rapamycin or torin1 or down-regulation of sterol regulatory element-binding protein (SREBP), a lipogenic transcription factor, did not suppress LPC in TSC2-deficient cells. Inhibition of distinct isoforms of phospholipase A2 decreased the proliferation of TSC2-deficient cells. Collectively, these results demonstrate that TSC2-deficient cells have enhanced choline phospholipid metabolism and reveal a novel function of the TSC proteins in choline lysoglycerophospholipid metabolism, with implications for disease pathogenesis and targeted therapeutic strategies.

Targeted lipid analysis of haemolytic mycelial extracts of Aspergillus niger.

Ethanolic extracts of mycelia from Aspergillus niger (strain N402) grown in liquid media were observed to have haemolytic activity on bovine erythrocytes. This haemolytic activity decreased significantly during the time of growth (1-3 days). Moreover, when A. niger was grown on carbon-deprived medium, the efficiency of this haemolytic activity in the ethanolic extracts was much lower than when grown in carbon-enriched medium, and became almost undetectable after 3 days of growth in carbon-deprived medium. The lipid composition of these ethanolic extracts was analysed by liquid chromatography-electrospray ionisation tandem mass spectrometry. This haemolytic activity can be mainly linked to the relative levels of the molar ratios of the unsaturated fatty acids and lysophosphatidylcholines.

Pre-emptive morphine treatment abolishes nerve injury-induced lysophospholipid synthesis in mass spectrometrical analysis.

We have previously demonstrated that lysophosphatidic acid (LPA) production in the spinal cord following partial sciatic nerve injury (SCNI) and its signaling initiate neuropathic pain. In order to examine whether LPA production depends on the intense nociceptive signal, we have attempted to see suppression by pre-emptive treatment with centrally administered morphine, which mainly inhibits nociceptive signal at the level of spinal cord. In the present study, we developed a quantitative mass spectrometry assay to simultaneously analyze several species of lysophosphatidyl choline (LPC). The levels of 16:0-, 18:0- and 18:1-LPC in the spinal cord and dorsal root were maximally increased at 75 min after SCNI and then declined, as LPC is converted to LPA by autotaxin (ATX). In atx(+/-)-mice, on the other hand, these levels were similar to wild-type mice at 75 min, but maximal at 120 min, suggesting that this difference is partly due to the low conversion of LPC to LPA in atx(+/-)-mice. When morphine was centrally administered before SCNI, the injury-induced increase of LPC was completely abolished. These results suggest that LPC (or LPA) is produced by injury-induced nociceptive signal, which is effectively and pre-emptively suppressed by central morphine, possibly through known descending anti-nociceptive pathways.

Imaging mass spectrometry revealed the production of lyso-phosphatidylcholine in the injured ischemic rat brain.

To develop an effective neuroprotective strategy against ischemic injury, it is important to identify the key molecules involved in the progression of injury. Direct molecular analysis of tissue using mass spectrometry (MS) is a subject of much interest in the field of metabolomics. Most notably, imaging mass spectrometry (IMS) allows visualization of molecular distributions on the tissue surface. To understand lipid dynamics during ischemic injury, we performed IMS analysis on rat brain tissue sections with focal cerebral ischemia. Sprague-Dawley rats were sacrificed at 24 h after middle cerebral artery occlusion, and brain sections were prepared. IMS analyses were conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) in positive ion mode. To determine the molecular structures, the detected ions were subjected to tandem MS. The intensity counts of the ion signals of m/z 798.5 and m/z 760.5 that are revealed to be a phosphatidylcholine, PC (16:0/18:1) are reduced in the area of focal cerebral ischemia as compared to the normal cerebral area. In contrast, the signal of m/z 496.3, identified as a lyso-phosphatidylcholine, LPC (16:0), was clearly increased in the area of focal cerebral ischemia. In IMS analyses, changes of PC (16:0/18:1) and LPC (16:0) are observed beyond the border of the injured area. Together with previous reports--that PCs are hydrolyzed by phospholipase A(2) (PLA(2)) and produce LPCs,--our present results suggest that LPC (16:0) is generated during the injury process after cerebral ischemia, presumably via PLA(2) activation, and that PC (16:0/18:1) is one of its precursor molecules.

Phospholipid biosynthesis in the gut of Spodoptera litura larvae and effects of tannic acid ingestion.

Tannins are polyphenols and defensive molecules occurring widely in plants. Dietary tannin may bind digestive enzymes of herbivores and affect them by reducing digestibility. In lepidopteran caterpillars, lysophosphatidylcholine (lysoPC), known as surfactants present in the guts, are thought to interfere with tannin-protein interactions. However, details of biosynthetic pathways of lysoPC in caterpillars have not been paid attention. Here, we investigated biosyntheses of lysoPC in Spodoptera litura larvae. S. litura larvae were fed on artificial diets enriched with [U-(13)C]linolenic acid for 3h, and then phospholipids in the gut contents were analyzed by LCMS-IT-TOF. Labeled linolenic acid was incorporated into lysoPC as well as diacylPC, diacylphosphatidylethanolamine (diacylPE) and diacylphosphatidylinositol (diacylPI). Detailed investigation on the biosynthesis revealed that [U-(13)C]linolenic acid was incorporated into phospholipids in gut tissues and then the biosynthesized phospholipids were released to the gut lumen, and then some of diacylPC were hydrolyzed to lysoPC in the gut lumen. When S. litura larvae were fed on artificial diets enriched with tannic acid, a significant increase in the activity to produce diacylPC in the gut tissues was observed. The activated diacylPC production leading to an increase of lysoPC in the larvae might be the adaptively-tuned mechanisms against plant tannins.

Lysophosphatidylcholine metabolism in Saccharomyces cerevisiae: the role of P-type ATPases in transport and a broad specificity acyltransferase in acylation.

We recently described a new route for the synthesis of phosphatidylethanolamine (PtdEtn) from exogenous lyso-PtdEtn, which we have termed the exogenous lysolipid metabolism (ELM) pathway. The ELM pathway for lyso-PtdEtn requires the action of plasma membrane P-type ATPases Dnf1p and Dnf2p and their requisite beta-subunit, Lem3p, for the active uptake of lyso-PtdEtn. In addition, the acyl-CoA-dependent acyltransferase, Ale1p, mediates the acylation of the imported lysolipid to form PtdEtn. We now report that these components of the lyso-PtdEtn ELM pathway are also active with lyso-1-acyl-2-hydroxyl-sn-glycero-3-phosphocholine (PtdCho) as a substrate. Lyso-PtdCho supports the growth of a choline auxotrophic pem1Delta pem2Delta strain. Uptake of radiolabeled lyso-PtdCho was impaired by the dnf2Delta and lem3Delta mutations. Introduction of a lem3Delta mutation into a pem1Delta pem2Delta background impaired the ability of the resulting strain to grow with lyso-PtdCho as the sole precursor of PtdCho. After import of lyso-PtdCho, the recently characterized lyso-PtdEtn acyltransferase, Ale1p, functioned as the sole lyso-PtdCho acyltransferase in yeast. A pem1Delta pem2Delta ale1Delta strain grew with lyso-PtdCho as a substrate but showed a profound reduction in PtdCho content when lyso-PtdCho was the only precursor of PtdCho. Ale1p acylates lyso-PtdCho with a preference for monounsaturated acyl-CoA species, and the specific LPCAT activity of Ale1p in yeast membranes is >50-fold higher than the basal rate of de novo aminoglycerophospholipid biosynthesis from phosphatidylserine synthase activity. In addition to lyso-PtdCho, lyso-PtdEtn, and lyso-phosphatidic acid, Ale1p was also active with lysophosphatidylserine, lysophosphatidylglycerol, and lysophosphatidylinositol as substrates. These results establish a new pathway for the net synthesis of PtdCho in yeast and provide new tools for the study of PtdCho synthesis, transport, and remodeling.

Identification of a novel lysophospholipid acyltransferase in Saccharomyces cerevisiae.

The incorporation of unsaturated acyl chains into phospholipids during de novo synthesis is primarily mediated by the 1-acyl-sn-glycerol-3-phosphate acyltransferase reaction. In Saccharomyces cerevisiae, Slc1 has been shown to mediate this reaction, but distinct activity remains after its removal from the genome. To identify the enzyme that mediates the remaining activity, we performed synthetic genetic array analysis using a slc1Delta strain. One of the genes identified by the screen, LPT1, was found to encode for an acyltransferase that uses a variety of lysophospholipid species, including 1-acyl-sn-glycerol-3-phosphate. Deletion of LPT1 had a minimal effect on 1-acyl-sn-glycerol-3-phosphate acyltransferase activity, but overexpression increased activity 7-fold. Deletion of LPT1 abrogated the esterification of other lysophospholipids, and overexpression increased lysophosphatidylcholine acyltransferase activity 7-fold. The majority of this activity co-purified with microsomes. To test the putative role for this enzyme in selectively incorporating unsaturated acyl chains into phospholipids in vitro, substrate concentration series experiments were performed with the four acyl-CoA species commonly found in yeast. Although the saturated palmitoyl-CoA and stearoyl-CoA showed a lower apparent Km, the monounsaturated palmitoleoyl-CoA and oleoyl-CoA showed a higher apparent Vmax. Arachidonyl-CoA, although not abundant in yeast, also had a high apparent Vmax. Pulse-labeling of lpt1Delta strains showed a 30% reduction in [3H]oleate incorporation into phosphatidylcholine only. Therefore, Lpt1p, a member of the membrane-bound o-acyltransferase gene family, seems to work in conjunction with Slc1 to mediate the incorporation of unsaturated acyl chains into the sn-2 position of phospholipids.

Local production of lipoprotein-associated phospholipase A2 and lysophosphatidylcholine in the coronary circulation: association with early coronary atherosclerosis and endothelial dysfunction in humans.

BACKGROUND: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a novel marker and participant in vascular inflammation. Inflammation also is associated with coronary atherosclerosis. We tested the hypothesis that local coronary production of Lp-PLA2 is enhanced in patients with early coronary atherosclerosis and associated with local endothelial function. METHODS AND RESULTS: Coronary angiography, blood flow, flow reserve, endothelial function assessment, and intravascular ultrasound with volumetric analysis were performed in 15 patients with mild coronary atherosclerosis and in 15 control subjects. Plasma samples were collected simultaneously from the left main coronary artery and coronary sinus for measurement of Lp-PLA2, lysophosphatidylcholine (a product of Lp-PLA2), and C-reactive protein. Hemodynamic parameters and cholesterol were similar in both groups. Arterial Lp-PLA2 levels were similar in patients and control subjects: 225 ng/mL (interquartile range [IQR], 196 to 273 ng/mL) versus 221 ng/mL (IQR, 177 to 294 ng/mL). Lp-PLA2 net production in the coronary circulation was higher in patients compared with control subjects: 519 ng/min (IQR, 198 to 1276 ng/min) versus -529 ng/min (IQR, -872 to -79 ng/min; P=0.001) and correlated with percent atheroma volume (r(s)=0.37, P=0.04). Net production of lysophosphatidylcholine was higher in patients compared with control subjects: 199 ng/min (IQR, -592 to 470 ng/min) versus -505 ng/min (IQR, -1119 to 0 ng/min; P=0.03) and correlated with coronary endothelial dysfunction (r(s)=0.5, P=0.005). C-reactive protein was not significantly different between the groups. CONCLUSIONS: Early coronary atherosclerosis in humans is characterized by local production of Lp-PLA2. Local coronary production of lysophosphatidylcholine, the active product of Lp-PLA2, is associated with endothelial dysfunction. These results support the role for Lp-PLA2 in the mechanism of regional vascular inflammation and atherosclerosis in humans.

The role and characterization of phospholipase A1 in mediating lysophosphatidylcholine synthesis in Trypanosoma brucei.

Lysophospholipids are ubiquitous intermediates in a variety of metabolic and signalling pathways in eukaryotic cells. We have reported recently that lysoglycerophosphatidylcholine (lyso-GPCho) synthesis in the insect form of the ancient eukaryote Trypanosoma brucei is mediated by a novel phospholipase A1 (TbPLA1). In the present study, we show that despite equal levels of TbPLA1 gene expression in wild-type insect and bloodstream trypomastigotes, both TbPLA1 enzyme levels and lysoGPCho metabolites are approx. 3-fold higher in the bloodstream form. Both of these parasite stages synthesize identical molecular species of lysoGPCho. TbPLA1 null mutants in the bloodstream form of the parasite are viable, but are deficient in lysoGPCho synthesis, a defect that can be overcome by the expression of an ectopic copy of TbPLA1. The biochemical attributes of TbPLA1-mediated lysoGPCho synthesis were examined in vitro using recombinant TbPLA1. Although TbPLA1 possesses an active-site serine residue, it is insensitive to serine-modifying reagents, such as di-isopropyl fluorophosphate and PMSF, a characteristic shared by lipases that possess lid-sheltered catalytic triads. TbPLA1 does not require metal co-factors for activity, but it does require interfacial activation prior to catalysis. Results from size-exclusion chromatography and binding kinetics analysis revealed that TbPLA1 activation by Triton X-100/GPCho mixed micelle surfaces was not specific and did not require the pre-formation of a specific enzyme-substrate complex to achieve surface binding.

Crystal structure analysis of phosphatidylcholine-GM2-activator product complexes: evidence for hydrolase activity.

GM2-activator protein (GM2AP) is a lysosomal lipid transfer protein with important biological roles in ganglioside catabolism, phospholipid metabolism, and T-cell activation. Previous studies of crystal structures of GM2AP complexed with the physiological ligand GM2 and platelet activating factor (PAF) have shown binding at two specific locations within the spacious apolar pocket and an ordering effect of endogenous resident lipids. To investigate the structural basis of phospholipid binding further, GM2AP was cocrystallized with phosphatidylcholine (PC), known to interact with GM2AP. Analysis of three crystal forms revealed binding of single chain lipids and fatty acids only and surprisingly not intact PC. The regions of best defined electron density are consistent with the presence of lyso-PC and oleic acid, which constitute deacylation products of PC. Their acyl tails are in stacking contact with shorter, less well-defined stretches of electron density that may represent resident fatty acids. The GM2AP associated hydrolytic activity that generates lyso-PC was further confirmed by mass spectrometry and enzymatic assays. In addition, we report the structures of (i) mutant Y137S, assessing the role of Tyr137 in lipid transfer via the hydrophobic cleft, and (ii) apo-mouse GM2AP, revealing a hydrophobic pocket with a constricted opening. Our structural results provide new insights into the biological functions of GM2AP. The combined effect of hydrolytic and lipid transfer properties has profound implications in cellular signaling.

The role of phospholipid methylation in 1-methyl-4-phenyl-pyridinium ion (MPP+)-induced neurotoxicity in PC12 cells.

Excessive methylation has been proposed to be involved in the pathogenesis of Parkinson's disease (PD), via mechanisms that involve phospholipid methylation. Meanwhile, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was found to stimulate phospholipid methylation via the oxidized metabolite, 1-methyl-4-phenyl-pyridinium (MPP+), in the rat brain and liver tissues. In the present study, we investigated the effect of MPP+ on phosphatidylethanolamine N-methyltransferases (PENMT) and the potential role of this pathway in MPP(+)-induced neurotoxicity using PC12 cells. The results obtained indicate that MPP+ stimulated phosphatidylethanolamine (PTE) methylation to phosphatidylcholine (PTC) and correspondingly increased the formation of lysophosphatidylcholine (lyso-PTC). Moreover, the addition of S-adenosylmethionine (SAM) to the cell culture medium increases MPP(+)-induced cytotoxicity. The incubation of 1mM MPP+ and various concentrations of SAM (0-4 mM) decreased the viability of PC12 cells from 80% with MPP+ alone to 38% viability with 4 mM SAM for 4 days incubation. The data also revealed that the addition of S-adenosylhomocysteine (SAH), a methylation inhibitor, offered significant protection against MPP(+)-induced cytotoxicity, indicating that methylation plays a role in MPP(+)-induced cytotoxicity. Interestingly, lyso-PTC showed similar actions to MPP+ in causing many cytotoxic changes with at least 10 times higher potency. Lyso-PTC induced dopamine release and inhibited dopamine uptake in PC12 cells. Lyso-PTC also caused the inhibition of mitochondrial potential and increased the formation of reactive oxygen species in PC12 cells. These results indicate that phospholipid methylation pathway might be involved in MPP+ neurotoxicity and lyso-PTC might play a role in MPP(+)-induced neurotoxicity.

The highly selective production of 2-arachidonoyl lysophosphatidylcholine catalyzed by purified calcium-independent phospholipase A2gamma: identification of a novel enzymatic mediator for the generation of a key branch point intermediate in eicosanoid signaling.

Herein, we report the heterologous expression of the human peroxisomal 63-kDa calcium-independent phospholipase A2gamma (iPLA2gamma) isoform in Sf9 cells, purification of the N-terminal His-tagged enzyme by affinity chromatography, and the identification of its remarkable substrate selectivity that results in the highly selective generation of 2-arachidonoyl lysophosphatidylcholine. Mass spectrometric analyses demonstrated that purified iPLA2gamma hydrolyzed saturated or monounsaturated aliphatic groups readily from either the sn-1 or sn-2 positions of phospholipids. In addition, purified iPLA2gamma effectively liberated arachidonic acid from the sn-2 position of plasmenylcholine substrates. In contrast, incubation of iPLA2gamma with 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine resulted in the rapid release of palmitic acid and the selective accumulation of 2-arachidonoyl lysophosphatidylcholine (LPC), which was not metabolized further by iPLA2gamma. The putative regiospecificity of the 2-arachidonoyl LPC product was authenticated by its diagnostic fragmentation pattern during tandem mass spectrometric analysis. To identify the physiological relevance of iPLA2gamma-mediated 2-arachidonoyl LPC production utilizing naturally occurring membranes, we incubated purified rat hepatic peroxisomes with iPLA2gamma and similarly identified the selective accumulation of 2-arachidonoyl LPC. Furthermore, tandem mass spectrometric analysis demonstrated that 2-arachidonoyl LPC is a natural product in human myocardium, a tissue in which iPLA2gamma expression is robust. Because 2-arachidonoyl LPC represents a key branch point intermediate that can potentially lead to a variety of bioactive molecules in eicosanoid signaling (e.g. arachidonic acid, 2-arachidonoylglycerol), these results have uncovered a novel eicosanoid selective pathway through iPLA2gamma-mediated 2-arachidonoyl LPC production to amplify and diversify the repertoire of biologic lipid second messengers in response to cellular stimulation.

Lysophosphatidylcholine decreases locomotor activities and dopamine turnover rate in rats.

Lysophosphatidylcholine (lyso-PTC), a secondary product of S-adenosylmethionine (SAM)-dependent phosphatidylethanolamine (PTE) methylation, is a potent cytotoxin and might be involved in the pathogenesis of Parkinson's disease (PD). Our previous studies showed that the injection of SAM into the brain caused PD-like changes in rodents. Moreover, 1-methyl-4-phenylpyridinium (MPP+), a Parkinsonism-inducing agent, increased lyso-PTC formation via the stimulation of PTE methylation pathway. These results indicate a possible role of lyso-PTC in the PD-like changes seen following the injection of SAM or MPP+. In the present study, lyso-PTC was injected into the lateral ventricle of rats and locomotor activities and the biogenic amine levels were measured to evaluate the effects of lyso-PTC on the dopaminergic system. Quinacrine, a phospholipase A2 (PLA2) inhibitor, was employed to determine its protective effect on SAM-induced PD-like changes by the inhibition of lyso-PTC formation. The results showed that 1 h after the injection, 0.4 and 0.8 micromol of lyso-PTC increased striatal dopamine (DA) by 20 and 24%, decreased 3,4-dihydroxyphenylacetic acid (DOPAC) by 37 and 45% and decreased homovanilic acid (HVA) by 24 and 13%, respectively. Consequently, dopamine turnover rate, (DOPAC + HVA)/DA, was significantly reduced by 44 and 48% in the rat striatum. Meanwhile, the administration of 0.4 or 0.8 micromol of lyso-PTC decreased movement time by 52 and 63%, total distance by 44 and 48% and the number of movements by 43 and 64%, respectively. Quinacrine attenuated SAM-induced hypokinesia without affecting SAM metabolism prior to its action on rat brain. The results obtained indicate that the hypokinesia observed following the administration of lyso-PTC might be related to the decline in DA turnover in the striatum in response to lyso-PTC exposure. The present study suggests that inhibitory effects of lyso-PTC on dopaminergic neurotransmission is one of the contributing factors in SAM and MPP+-induced PD-like changes.

Human serum paraoxonase 1 decreases macrophage cholesterol biosynthesis: possible role for its phospholipase-A2-like activity and lysophosphatidylcholine formation.

OBJECTIVE: Human serum paraoxonase 1 (PON1) activity is inversely related to the risk of developing an atherosclerotic lesion, which contains cholesterol-loaded macrophage foam cells. To assess a possible mechanism for this relationship, we analyzed the effect of PON1 on cellular cholesterol biosynthesis. METHODS AND RESULTS: Mouse peritoneal macrophages (MPMs) were harvested from PON1-deficient mice (PON1o and PON1o/Eo mice on the genetic background of C57BL/6J and Eo mice, respectively). PON1o/Eo mice exhibited a significantly 51% increased atherosclerotic lesion area and 35% increased macrophage cholesterol content compared with control E degrees mice. In parallel, macrophage cholesterol biosynthesis rates were increased in PON1-deficient mice MPMs by 50% compared with their controls. Incubation of macrophages with human PON1 revealed a dose-dependent inhibitory effect (up to 84%) on macrophage cholesterol biosynthesis. We demonstrated a PON1 phospholipase-A2-like activity on MPMs, evidenced by release of polyunsaturated fatty acids and formation of lysophosphatidylcholine. On incubation of macrophages with lysophosphatidylcholine, a dose-dependent inhibition (up to 40%) of cellular cholesterol biosynthesis was noted. The inhibitory effect of PON1 on macrophage cholesterol biosynthesis was shown to be downstream to mevalonate, probably at the lanosterol metabolic point. CONCLUSIONS: PON1 inhibits macrophage cholesterol biosynthesis and atherogenesis probably through its phospholipase-A2-like activity.

Elicitor-activated phospholipase A(2) generates lysophosphatidylcholines that mobilize the vacuolar H(+) pool for pH signaling via the activation of Na(+)-dependent proton fluxes.

The elicitation of phytoalexin biosynthesis in cultured cells of California poppy involves a shift of cytoplasmic pH via the transient efflux of vacuolar protons. Intracellular effectors of vacuolar proton transport were identified by a novel in situ approach based on the selective permeabilization of the plasma membrane for molecules of < or = 10 kD. Subsequent fluorescence imaging of the vacuolar pH correctly reported experimental changes of activity of the tonoplast proton transporters. Lysophosphatidylcholine (LPC) caused a transient increase of the vacuolar pH by increasing the Na(+) sensitivity of a Na(+)-dependent proton efflux that was inhibited by amiloride. In intact cells, yeast elicitor activated phospholipase A(2), as demonstrated by the formation of LPC from fluorescent substrate analogs, and caused a transient increase of endogenous LPC, as determined by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. It is suggested that LPC generated by phospholipase A(2) at the plasma membrane transduces the elicitor-triggered signal into the activation of a tonoplast H(+)/Na(+) antiporter.

Is a role of phospholipase A(2) in cholesterol gallstone formation phospholipid species-dependent?

Phospholipase A(2) plays a role in cholesterol gallstone formation by hydrolyzing bile phospholipids into lysolecithin and free fatty acids. This study investigated its effects on cholesterol crystallization in model bile systems. Supersaturated model bile solutions with different cholesterol saturation indexes (1.2, 1.4, and 1.6) were prepared using cholesterol, taurocholate, and egg yolk phosphatidylcholine, soybean phosphatidylcholine, palmitoyl-oleoyl phosphatidylcholine, or palmitoyl-linoleoyl phosphatidylcholine. Then the effect of digestion of phosphatidylcholine by phospholipase A(2) on bile metastability was assessed by spectrophotometry and video-enhanced differential contrast microscopy. Addition of phospholipase A(2) caused the release of free fatty acids in a time-dependent manner. Cholesterol crystallization was enhanced by an increased crystal growth rate in model bile containing hydrophilic species such as soybean or palmitoyl-linoleoyl phosphatidylcholine, consisting predominantly of polyunsaturated fatty acids. Because phospholipase A(2) enhanced cholesterol crystallization in bile containing hydrophilic phosphatidylcholine species, but not hydrophobic phosphatidylcholine species, release of polyunsaturated fatty acids by hydrolysis may be responsible for such enhancement. Therefore, the role of phospholipase A(2) in cholesterol gallstone formation depends on the phospholipid species present in bile, so that phospholipid species selection during hepatic excretion is, in part, crucial to the cholesterol stone formation.

Intraduodenal infusion of lysophosphatidylcholine restores the intestinal absorption of vitamins A and E in rats fed a low-zinc diet.

Our previous work has shown that the lymphatic absorptions of lipids and lipid-soluble vitamins, retinol and alpha-tocopherol (alphaTP), are lowered markedly in rats fed a low-zinc (LZ) diet in parallel with lower lymphatic phospholipid outputs. Phosphatidylcholine (PC), when infused enterally, restored the absorptions of fat and retinol, but further lowered the absorption of alphaTP in rats fed the LZ diet. This study was conducted to determine whether a luminal infusion of lysophosphatidylcholine, a product of PC hydrolysis by pancreatic phospholipase A2 (PLA2), would simultaneously restore the absorptions of retinol and alphaTP in LZ rats. Rats were trained to consume two meals per day and were divided into two groups. One group was fed an AIN-93G diet containing a LZ (3.0 mg Zn/kg), and the other was fed the same diet, but containing adequate zinc (AZ; 30.0 mg Zn/kg) for 6 weeks. Rats with lymph cannula were infused at 3.0 ml/hr for 8 hr with a lipid emulsion containing retinol, alphaTP, and 14C-labeled triolein (14C-oleic acid) with or without 1-oleoyl-2-hydroxy phosphatidylcholine (lysoPC) in 24 ml of PBS (pH 6.5). When the lipid emulsion without lysoPC was infused, the absorptions of retinol and alphaTP were significantly lower in LZ rats (retinol, 13.2+/-1.5 nmol; alphaTP, 430.6+/-66.8 nmol) than in AZ rats (retinol, 18.2+/-1.0 nmol; alphaTP, 543.8+/-58.9 nmol). The lower absorptions of the vitamins in LZ rats occurred in parallel with a significant decrease in 14C-oleic acid absorption. When the emulsion containing lysoPC was infused, however, absorptions of the vitamins (retinol, 18.4+/-3.0 nmol; alphaTP, 777.2+/-92.1 nmol) in LZ rats were restored completely to the control levels (retinol, 20.4+/-2.8 nmol; alphaTP, 756.3+/-136.1 nmol). The results suggest that the luminal hydrolysis of PC to lysoPC by PLA2 may be impaired in LZ rats, resulting in impaired absorption of fat and the fat-soluble vitamins.

Specific antioxidant activity of caffeoyl derivatives and other natural phenolic compounds: LDL protection against oxidation and decrease in the proinflammatory lysophosphatidylcholine production.

Specific antioxidant activity (SAA) (i.e., activity related to the molar or gallic acid equivalent amount of antioxidant) of natural polyphenolic mixtures or pure phenolic compounds was studied using their capacity to delay the conjugated diene production brought about by in vitro LDL copper-mediated or AAPH-mediated oxidation. The cinnamic acid series (caffeic, sinapic, ferulic acids) displayed a constant SAA over a large range of concentrations, whereas the benzoic acid series (gallic and protocatechuic acids) showed much higher SAA at low concentrations. The natural phenolic mixtures had a constant SAA. The highest SAA was obtained with caffeoyl esters (caffeoylquinic, rosmarinic, and caffeoyltartaric acids) and catechin for the copper-oxidation and the AAPH-oxidation system, respectively. Phenolic mixtures and acids delayed vitamin E depletion and decreased proinflammatory lysophosphatidylcholine production. As with polyphenols, probucol delayed lysophosphatidylcholine and conjugated dienes production, at higher concentrations, but was not effective at preventing vitamin E depletion. Polyphenols prevent the oxidation of LDL and its constituents (vitamin E, phosphatidylcholine), which is compatible with an antiinflammatory and antiatherosclerotic role in pathophysiological conditions.