Labeling Of Phosphatidylcholine Research Articles

Organelle-selective click labeling coupled with flow cytometry allows pooled CRISPR screening of genes involved in phosphatidylcholine metabolism

Cellular lipid synthesis and transport are governed by intricate protein networks. Although genetic screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain-especially for high-throughput readouts of lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine (PC) with flow cytometry-based CRISPR screening technologies to convert organellar PC phenotypes into a simple fluorescence readout for genome-wide screening. This technique, named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screens to identify previously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, we revealed previously uncharacterized roles of FLVCR1 as a choline uptake facilitator, CHEK1 as a post-translational regulator of the PC-synthetic pathway, and CDC50A as responsible for the translocation of PC to the outside of the plasma membrane bilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedented platform for genetic dissection of cellular lipid metabolism.

Kinetic complexities of triacylglycerol accumulation in developing embryos from Camelina sativa provide evidence for multiple biosynthetic systems

Quantitative flux maps describing glycerolipid synthesis can be important tools for rational engineering of lipid content and composition in oilseeds. Lipid accumulation in cultured embryos of Camelina sativa is known to mimic that of seeds in terms of rate of lipid synthesis and composition. To assess the kinetic complexity of the glycerolipid flux network, cultured embryos were incubated with [14C/13C]glycerol, and initial and steady state rates of [14C/13Cglyceryl] lipid accumulation were measured. At steady state, the linear accumulations of labeled lipid classes matched those expected from mass compositions. The system showed an apparently simple kinetic precursor–product relationship between the intermediate pool, dominated by diacylglycerol (DAG) and phosphatidylcholine (PC), and the triacylglycerol (TAG) product. We also conducted isotopomer analyses on hydrogenated lipid class species. [13C3glyceryl] labeling of DAG and PC, together with estimates of endogenous [12C3glyceryl] dilution, showed that each biosynthetically active lipid pool is ∼30% of the total by moles. This validates the concept that lipid sub-pools can describe lipid biosynthetic networks. By tracking the kinetics of [13C3glyceryl] and [13C2acyl] labeling, we observed two distinct TAG synthesis components. The major TAG synthesis flux (∼75%) was associated with >95% of the DAG/PC intermediate pool, with little glycerol being metabolized to fatty acids, and with little dilution from endogenous glycerol; a smaller flux exhibited converse characteristics. This kinetic heterogeneity was further explored using postlabeling embryo dissection and differential lipid extractions. The minor flux was tentatively localized to surface cells across the whole embryo. Such heterogeneity must be recognized in order to construct accurate gene expression patterns and metabolic networks describing lipid biosynthesis in developing embryos.

Organelle membrane-specific chemical labeling and dynamic imaging in living cells.

Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking.

Diverse pathways of phosphatidylcholine biosynthesis in algae as estimated by labeling studies and genomic sequence analysis.

Phosphatidylcholine (PC) is an almost ubiquitous phospholipid in eukaryotic algae and plants but is not found in a few species, for example Chlamydomonas reinhardtii. We recently found that some species of the genus Chlamydomonas possess PC. In the universal pathway, PC is synthesized de novo by methylation of phosphatidylethanolamine (PE) or transfer of phosphocholine from cytidine diphosphate (CDP)-choline to diacylglycerol. Phosphocholine, the direct precursor to CDP-choline, is synthesized either by methylation of phosphoethanolamine or phosphorylation of choline. Here we analyzed the mechanism of PC biosynthesis in two species of Chlamydomonas (asymmetrica and sphaeroides) as well as in a red alga, Cyanidioschyzon merolae. Comparative genomic analysis of enzymes involved in PC biosynthesis indicated that C.merolae possesses only the PE methylation pathway. Radioactive tracer experiments using [(32) P]phosphate showed delayed labeling of PC with respect to PE, which was consistent with the PE methylation pathway. In Chlamydomonas asymmetrica, labeling of PC was detected from the early time of incubation with [(32) P]phosphate, suggesting the operation of phosphoethanolamine methylation pathway. Genomic analysis indeed detected the genes for the phosphoethanolamine methylation pathway. In contrast, the labeling of PC in C.sphaeroides was slow, suggesting that the PE methylation pathway was at work. These results as well as biochemical and computational results uncover an unexpected diversity of the mechanisms for PC biosynthesis in algae. Based on these results, we will discuss plausible mechanisms for the scattered distribution of the ability to biosynthesize PC in the genus Chlamydomonas.

Altered molecular specificity of surfactant phosphatidycholine synthesis in patients with acute respiratory distress syndrome.

BackgroundAcute respiratory distress syndrome (ARDS) is a life-threatening critical illness, characterised by qualitative and quantitative surfactant compositional changes associated with premature airway collapse, gas-exchange abnormalities and acute hypoxic respiratory failure. The underlying mechanisms for this dysregulation in surfactant metabolisms are not fully explored. Lack of therapeutic benefits from clinical trials, highlight the importance of detailed in-vivo analysis and characterisation of ARDS patients according to patterns of surfactant synthesis and metabolism.MethodsTen patients with moderate to severe ARDS were recruited. Most (90%) suffered from pneumonia. They had an infusion of methyl-D9-choline chloride and small volume bronchoalveolar lavage fluid (BALF) was obtained at 0,6,12,24,48,72 and 96 hours. Controls were healthy volunteers, who had BALF at 24 and 48 hours after methyl-D9-choline infusion. Compositional analysis and enrichment patterns of stable isotope labelling of surfactant phosphatidylcholine (PC) was determined by electrospray ionisation mass spectrometry.ResultsBALF of patients with ARDS consisted of diminished total PC and fractional PC16:0/16:0 concentrations compared to healthy controls. Compositional analysis revealed, reductions in fractional compositions of saturated PC species with elevated levels of longer acyl chain unsaturated PC species. Molecular specificity of newly synthesised PC fraction showed time course variation, with lower PC16:0/16:0 composition at earlier time points, but achieved near equilibrium with endogenous composition at 48 hours after methyl-D9-choline infusion. The enrichment of methyl-D9-choline into surfactant total PC is nearly doubled in patients, with considerable variation between individuals.ConclusionsThis study demonstrate significant alterations in composition and kinetics of surfactant PC extracted from ARDS patients. This novel approach may facilitate biochemical phenotyping of ARDS patients according to surfactant synthesis and metabolism, enabling individualised treatment approaches for the management of ARDS patients in the future.

Daily rhythms of glycerophospholipid synthesis in fibroblast cultures involve differential enzyme contributions

Circadian clocks regulate the temporal organization of several biochemical processes, including lipid metabolism, and their disruption leads to severe metabolic disorders. Immortalized cell lines acting as circadian clocks display daily variations in [(32)P]phospholipid labeling; however, the regulation of glycerophospholipid (GPL) synthesis by internal clocks remains unknown. Here we found that arrested NIH 3T3 cells synchronized with a 2 h-serum shock exhibited temporal oscillations in a) the labeling of total [(3)H] GPLs, with lowest levels around 28 and 56 h, and b) the activity of GPL-synthesizing and GPL-remodeling enzymes, such as phosphatidate phosphohydrolase 1 (PAP-1) and lysophospholipid acyltransferases (LPLAT), respectively, with antiphase profiles. In addition, we investigated the temporal regulation of phosphatidylcholine (PC) biosynthesis. PC is mainly synthesized through the Kennedy pathway with choline kinase (ChoK) and CTP:phosphocholine cytidylyltranferase (CCT) as key regulatory enzymes. We observed that the PC labeling exhibited daily changes, with the lowest levels every ~28 h, that were accompanied by brief increases in CCT activity and the oscillation in ChoK mRNA expression and activity. Results demonstrate that the metabolisms of GPLs and particularly of PC in synchronized fibroblasts are subject to a complex temporal control involving concerted changes in the expression and/or activities of specific synthesizing enzymes.

Rapid Kinetic Labeling of Arabidopsis Cell Suspension Cultures: Implications for Models of Lipid Export from Plastids

Cell cultures allow rapid kinetic labeling experiments that can provide information on precursor-product relationships and intermediate pools. T-87 suspension cells are increasingly used in Arabidopsis (Arabidopsis thaliana) research, but there are no reports describing their lipid composition or biosynthesis. To facilitate application of T-87 cells for analysis of glycerolipid metabolism, including tests of gene functions, we determined composition and accumulation of lipids of light- and dark-grown cultures. Fatty acid synthesis in T-87 cells was 7- to 8-fold higher than in leaves. Similar to other plant tissues, phosphatidylcholine (PC) and phosphatidylethanolamine were major phospholipids, but galactolipid levels were 3- to 4-fold lower than Arabidopsis leaves. Triacylglycerol represented 10% of total acyl chains, a greater percentage than in most nonseed tissues. The initial steps in T-87 cell lipid assembly were evaluated by pulse labeling cultures with [(14)C]acetate and [(14)C]glycerol. [(14)C]acetate was very rapidly incorporated into PC, preferentially at sn-2 and without an apparent precursor-product relationship to diacylglycerol (DAG). By contrast, [(14)C]glycerol most rapidly labeled DAG. These results indicate that acyl editing of PC is the major pathway for initial incorporation of fatty acids into glycerolipids of cells derived from a 16:3 plant. A very short lag time (5.4 s) for [(14)C]acetate labeling of PC implied channeled incorporation of acyl chains without mixing with the bulk acyl-CoA pool. Subcellular fractionation of pea (Pisum sativum) leaf protoplasts indicated that 30% of lysophosphatidylcholine acyltransferase activity colocalized with chloroplasts. Together, these data support a model in which PC participates in trafficking of newly synthesized acyl chains from plastids to the endoplasmic reticulum.

Salinity regulates N-methylation of phosphatidylethanolamine in euryhaline crustaceans hepatopancreas and exchange of newly-formed phosphatidylcholine with hemolymph

Phosphatidylcholine (PC), the main phospholipid in eukaryotes, is synthesized via two different routes, the phosphatidylethanolamine N-methyl transferase (PEMT) and the CDP-choline pathways. We previously showed in euryhaline fish that salinity impacts the relative contribution of the two pathways for PC biosynthesis, with PEMT pathway being activated in the liver of sea water (SW)-adapted animals. To address the occurrence of such phenomenon in other animals we performed in vivo metabolic studies in two crustacean species: the Chinese crab (Eriocheir sinensis) and the green crab (Carcinus maenas). In both species, the levels of PC and phosphatidylethanolamine in hepatopancreas and hemolymph were not modified by SW-adaptation. In E. sinensis, SW-adaptation activated PC labeling from L-(U-(14)C)-serine in the hepatopancreas and resulted in an increased ratio of PC specific activities between hemolymph and hepatopancreas. In C. maenas, incorporation of L-(3-(3)H)-serine and L-(2-(14)C)-ethanolamine into PC of hepatopancreas was strongly inhibited after acclimation to fresh water (FW). The results show that PC synthesis via the PEMT pathway and its subsequent release into hemolymph are both activated in SW- compared to FW-adapted animals. SW-adaptation also resulted in increased tissue concentrations of betaine and labeling from L-(U-(14)C)-serine, suggesting that the PEMT-derived PC is used for the synthesis of organic osmolytes. The physiological relevance of these observations is discussed.

Conversion of Low Density Lipoprotein-associated Phosphatidylcholine to Triacylglycerol by Primary Hepatocytes

We have studied the uptake and metabolism of phosphatidylcholine (PC), the major phospholipid of low density lipoproteins (LDL), by cultures of primary hepatocytes. Strikingly, in the absence of the LDL receptor, PC incorporation into hepatocytes was inhibited by only 30%, whereas cholesteryl ether uptake was inhibited by 60-70%. On the other hand, scavenger receptor class B, type I, the other important receptor for LDL in the liver, was found to be responsible for the uptake of the remaining 30-40% of LDL-cholesteryl ether. PC uptake was, however, only partially inhibited (30%) in scavenger receptor class B, type I, knock-out hepatocytes. Once LDL-PC was taken up by hepatocytes, approximately 50% of LDL-[(3)H]oleate-PC was converted to triacylglycerol rather than degraded in lysosomes as occurs for LDL-derived cholesteryl esters. The remainder of the LDL-derived PC was not significantly metabolized to other products. Triacylglycerol synthesis from LDL-PC requires a PC-phospholipase C activity as demonstrated by inhibition with the phospholipase C inhibitor D609 or activation with rattlesnake venom. Small interfering RNA-mediated suppression of acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2), but not DGAT1, decreased the acylation of the LDL-derived diacylglycerol. These findings show that PC in LDL particles is taken up not only by the classical receptors but also by additional mechanism(s) followed by metabolism that is completely different from the cholesteryl esters or apoB100, the other main components of LDL.

Lipid Homeostasis and Lipoprotein Secretion in Niemann-Pick C1-deficient Hepatocytes

Niemann-Pick C (NPC) disease is a fatal inherited disorder characterized by an accumulation of cholesterol and other lipids in late endosomes/lysosomes. Although this disease is considered to be primarily a neurodegenerative disorder, many NPC patients suffer from liver disease. We have investigated alterations that occur in hepatic lipid homeostasis using primary hepatocytes isolated from NPC1-deficient mice. The cholesterol content of Npc1(-/-) hepatocytes was 5-fold higher than that of Npc1(+/+) hepatocytes; phospholipids and cholesteryl esters also accumulated. In contrast, the triacylglycerol content of Npc1(-/-) hepatocytes was 50% lower than of Npc1(+/+) hepatocytes. We hypothesized that the cholesterol sequestration induced by NPC1 deficiency might inhibit very low density lipoprotein secretion. However, this process was enhanced by NPC1 deficiency and the secreted particles were enriched in cholesteryl esters. We investigated the mechanisms responsible for these changes. The synthesis of phosphatidylcholine, cholesteryl esters, and cholesterol in hepatocytes was increased by NPC1 deficiency and the amount of the mature form of sterol response element-binding protein-1 was also increased. These observations indicate that the enhanced secretion of lipoproteins from NPC1-deficient hepatocytes is due, at least in part, to increased lipid synthesis.

Evidence for a different metabolism of PC and PE in shoots and roots

We investigated phosphatidylcholine (PC) and phosphatidylethanolamine (PE) labelling in shoots and roots from leek plantlets, maize seedlings and Arabidopsis thaliana through the incorporation of radiolabelled acetate. Regardless of the pathway followed in shoots, PC labelling was always higher than PE labelling. However, we obtained an opposite situation in leek and A. thaliana roots since PC labelling was much lower than PE labelling. Several hypotheses to explain the origin(s) of these discrepancies between roots and shoots were tested. Among them, neither the level of the respective AAPT activities, nor specific regulations of PC biosynthesis through the mRNA levels of several enzymes (choline citidylyltransferase ( CCT), ethanolamine citidylyltransferase ( ECT), phosphoethanolamine methyltransferase ( PEAMT)), nor the fatty acyl chain composition of PC, PE, and diacylglycerol, were responsible for the differences observed between PC and PE metabolism in roots and shoots. Finally, we investigated the acylation of PC and PE in vitro in both shoots and roots of A. thaliana seedlings, and demonstrated that some specific remodelling of PC and PE by acylation was responsible for the differences in labelling observed in vivo.

2n-fatty acids from phosphatidylcholine label sphingolipids—A novel role of phospholipase A 2?

In order to find out whether there is a phospholipase A 2 (PLA 2)-mediated link between glycerophospholipids and sphingolipids, L929 cells were labeled with 1n-palmitoyl-2n-[1- 14C]palmitoyl phosphatidylcholine for 16–18 h or 90 min. After labeling for 16–18 h, 14C-sphingomyelin (SM), 14C-ceramide and 14C-sphingosine were demonstrated on autoradiograms of thin layer chromatograms of untreated or mildly hydrolyzed lipid extracts in different chromatographic systems. Strong hydrolysis of labeled SM proved that both possible moieties of SM, sphingosine and acyl moiety, had been labeled. The identity of SM and its enzymatic degradation product, ceramide, was verified by mass spectrometry. The label in SM-derived ceramide was demonstrated on an autoradiogram after thin layer chromatography. The inhibitor of (dihydro)ceramide synthase fumonisin B1 suppressed the label in sphingolipids significantly during 16–18 h (ceramide and SM), as well as during 90-min labeling (SM). The presence of inhibitors of PLA 2 (bromoenol lactone, aristolochic acid and quinacrine dihydrochloride) diminished the label in SM significantly during the 90-min labeling. These results demonstrate a close metabolic relationship between glycerophospholipids and sphingolipids and give evidence for a novel role of PLA 2.

Phosphatidylcholine metabolism is altered in a monocyte-derived macrophage model of Gaucher disease but not in lymphocytes

Gaucher disease is caused by defective activity of acid-β-glucosidase (GlcCerase), resulting in accumulation of glucosylceramide (GlcCer) mainly in macrophages. We now demonstrate that secondary biochemical pathways regulating levels of phospholipid metabolism are altered in a Gaucher disease macrophage model. Upon treatment of macrophages with the GlcCerase inhibitor, conduritol-B-epoxide, phosphatidylcholine (PC) labeling with the metabolic precursor, [ methyl- 14C]choline, was elevated after 6 or 12 days in macrophages but not in lymphocytes. These changes correlated with increases in the cytoplasmic/nuclear ratio and with levels of [ 3H]GlcCer accumulation. Moreover, metabolic labeling with l-[3- 3H]serine and l-[ methyl- 3H]methionine demonstrated that PC synthesis via the methylation of phosphatidylethanolamine is also increased in CBE-treated macrophages. Since PC is a major structural component of biological membranes and the source of various second messengers, we suggest that changes in its metabolism in macrophages may be relevant for understanding Gaucher disease pathology.

A mandelamide pesticide alters lipid metabolism in Phytophthora infestans

Summary Several pesticide classes have been used for control of the notorious plant pathogen, Phytophthora infestans. Some of these alter lipids, suggesting that lipid metabolism may be a target site. Here, we investigate the action of a new, active, mandelamide compound, SX 623509 (N‐2′‐(4″‐ethoxy‐3″‐methoxy) phenylethyl‐3, 4‐dichloromandelamide), on lipid metabolism. Phytophthora infestans cultured in pea‐broth or minimal media was exposed to different concentrations of SX 623509. Lipid metabolism was followed with radiolabelled acetate, choline or ethanolamine. Products were analysed following separation by thin‐layer chromatography and gas–liquid chromatography. SX 623509 reduced growth and lipid labelling from [14C]acetate in both media. The inhibition in lipid labelling was not caused merely by a reduction in uptake of the radiolabelled precursor. There were changes in labelling patterns, particularly reductions in phosphatidylcholine and triacylglycerol and increases in phosphatidate and diacylglycerol. The effect of SX 623509 on phosphatidylcholine labelling was followed in more detail. We conclude that the mandelamide pesticide SX 623509 reduces lipid synthesis at concentrations similar to those inhibiting growth. Specific effects on lipid labelling patterns are probably caused by inhibition of cholinephosphotransferase, which may be a future target site for pesticide development.

Investigation of the nature of potential phosphorylcholine donors for filarial nematode glycoconjugates

Filarial nematodes produce proteins containing phosphorylcholine (PC) covalently attached to N-linked glycans. Our previous work has suggested that transfer of PC might be dependent on a metabolite of the Kennedy pathway of phospholipid biosynthesis. In this study we have investigated whether the end product of this pathway, phosphatidylcholine, and in addition, sphingomyelin, could act as PC donors. Pulse-chase experiments employing [ 3H]choline as radiolabel, ruled out sphingomyelin, as the Acanthocheilonema viteae PC-containing protein, ES-62, was radiolabelled 20–30 min prior to the lipid. Phosphatidylcholine however was labelled immediately before ES-62 increasing the possibility that it could act as donor. This was further investigated by radiolabelling phosphatidylcholine synthesised via an alternative pathway such that other metabolites in the Kennedy pathway were not labelled. Specifically, we labelled the choline component of phosphatidylcholine using both [ 3H]serine and [ 3H] S-adenosyl methionine (SAM). Incubation of worms with [ 3H]serine failed to result in labelling of the PC component of ES-62 whereas the presence of [ 3H]SAM in the medium led to labelling of ES-62 but only 24 h after labelling of phosphatidylcholine. As ES-62 is labelled within minutes of phosphatidylcholine when employing [ 3H]choline as radiolabel, this suggests that labelling of ES-62 when using SAM is not due to direct transfer of PC from phosphatidylcholine. It is therefore concluded that neither sphingomyelin nor phosphatidylcholine act as PC donors for filarial nematode glycoproteins. The analysis of PC-containing metabolites and products from A. viteae additionally revealed the presence of PC-substituted glycolipids that were also radiolabelled by the use of [ 14C]choline. The kinetics of radiolabelling however differed from that observed in the case of ES-62, sphingomyelin and phosphatidylcholine in so far as labelled glycolipids were first detectable hours rather than minutes after addition of [ 14C]choline.

Lipid metabolism in the moss Rhytidiadelphus squarrosus (Hedw.) Warnst. from lead-contaminated and non-contaminated populations.

Lipid metabolism and the effect of Pb(2+) and Cu(2+) on this process was studied in the moss Rhytidiadelphus squarrosus collected from both a lead-contaminated and a non-contaminated site. Total radiolabelling of lipids from [1-(14)C]acetate was similar in both populations and Cu or Pb (1 microM, 10 microM) did not cause much alteration in acute exposure experiments. However, there were significant qualitative changes. Of the major labelled neutral lipid classes, samples from the lead-polluted site showed a decrease in labelling of triacylglycerols and an increase in wax esters. Acute lead exposure caused similar effects. Cu caused a decrease in the labelling of wax esters and an increase in diacylglycerols. These data suggest that heavy metals cause a change in carbon flux through the acylation reactions associated with the Kennedy pathway. R. squarrosus obtained from the Pb-contaminated site also showed changes in polar lipid labelling compared to the uncontaminated site. The labelling of phosphatidylcholine was more than halved and replaced by increased labelling of other zwitterionic lipids. The chloroplast glycerolipids were also increasingly labelled. Acute exposure to Pb, however, caused little alteration of labelling patterns within 24 h. R. squarrosus contains high levels of polyunsaturated fatty acids (PUFAs), but moss obtained from the Pb-polluted site had significantly less PUFAs containing three or more double bonds. Such samples, when incubated with [1-(14)C]acetate also showed decreased PUFA labelling. By contrast, acute exposure to Pb produced different results. These data provide a foundation for examining lipid metabolism in bryophytes and the effects of pollution in this important class of organism. The results also emphasize that acute and chronic exposure to heavy metals may produce different effects and that caution must be exercised in extrapolating data from one system to another.

Enhancement of phosphatidylcholine biosynthesis by angiotensin-(1–7) in the rat renal cortex

In the present paper, we investigated the effect of angiotensin-(1–7) (Ang-(1–7)) on phospholipid biosynthesis in the rat renal cortex. A significant increase in phosphatidylcholine (PC) labeling was observed when cortical slices, prelabeled with [ 32 P ]orthophosphate, were incubated for 30 min in the presence of Ang-(1–7) (1 pM to 100 nM). Neither the phospholipase C inhibitors, neomycin or db-cAMP nor the protein kinase C inhibitors, chelerythrine or H7, modified the stimulatory effect induced by 0.1 nM Ang-(1–7). The enhancement of PC biosynthesis caused by 0.1 nM Ang-(1–7) was unmodified by either losartan, an AT 1 receptor antagonist, or (1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1 H-imidazol[4,5- c]pyridine-6-carboxylic acid ditrifluoroacetate) (PD 123319), an AT 2 receptor antagonist, but was partially blocked by [ d-Ala 7]Ang-(1–7), an Ang-(1–7) specific antagonist. However, losartan potentiated the effect of 100 nM Ang-(1–7) on PC biosynthesis. Losartan by itself increased the de novo synthesis of PC. These results suggest that the Ang-(1–7)-mediated increase in PC biosynthesis is independent of AT 1 and AT 2 receptor activation but mediated by a specific Ang-(1–7) receptor. This mechanism is independent of phospholipase C and PKC activation.

Age-dependent variation in membrane lipid synthesis in leaves of garden pea (Pisum sativum L.).

To study membrane lipid synthesis during the life-span of a dicotyledon leaf, the second oldest leaf of 10-40-d-old plants of garden pea (Pisum sativum L.) was labelled with [1-(14)C]acetate and the distribution of radioactivity between the major membrane lipids was followed for 3 d. In the expanding second oldest leaf of 10-d-old plants, acetate was primarily allocated into phosphatidylcholine (PC) during the first 4 h of labelling. During the following 3 d, labelling of PC decreased and monogalactosyldiacylglycerol (MGDG) became the most radioactive lipid. In the fully expanded second oldest leaf of older plants, acetate was predominantly allocated into phosphatidylglycerol (PG), which remained the major radiolabelled lipid during the 3 d studied. The proportion of radioactivity recovered in MGDG decreased with increasing plant age up to 20 d, suggesting that, in expanded leaves, MGDG is more stable and requires renewal to a lower extent than PG. When the second oldest leaf approached senescence, labelling of MGDG again increased, indicating an increased need for thylakoid repair. The proportion of acetate allocated into phosphatidylethanolamine and free sterols was largest in leaves of 18-26-d-old plants and in the youngest leaves, respectively. Thus, these results demonstrate that the distribution of newly synthesized fatty acids between acyl lipid synthesis in the chloroplast and extraplastidial membranes strongly varies with leaf age, as do the proportion utilized for sterol synthesis. The findings emphasize the importance of defining the developmental stage of the leaf material used when performing studies on leaf lipid metabolism.

Colecystokinin octapeptide CCK-8 and carbachol reduce [ 32P]orthophosphate labeling of phosphatidylcholine without modifying phospholipase D activity in rat pancreatic acini

We have studied phospholipase D activation in [ 32P]orthophosphoric acid-prelabeled rat pancreatic acini by measuring the formation of 32P-phosphatidylalcohols as stimulated in the presence of ethanol or butanol. A small but significant and time-dependent basal accumulation of [ 32P]phosphatidylethanol and [ 32P]phosphatidylbutanol was detected, which was further stimulated by phorbol myristate acetate, orthovanadate and pervanadate. However, the secretagogues cholecystokinin octapeptide and carbachol did not enhance basal accumulation of 32P-phosphatidylalcohol, yet they decreased [ 32P]phosphatidylcholine content and stimulated the generation of [ 32P]phosphatidic acid. Our results stress the need to examine the transphosphatidylation reaction as well as agonist effects on the synthesis of phosphatidylcholine in order to assess unambiguously phospholipase D activity.

Surfactant lipid uptake and metabolism by neonatal and adult type II pneumocytes.

Animal experiments suggest developmental changes in surfactant homeostasis. The uptake and metabolism of [(3)H]dipalmitoylphosphatidylcholine-labeled liposomes with a surfactant-like composition were evaluated in type II cells isolated from rats of different postnatal ages. The early part of the uptake process (0-60 min) was more rapid and reached higher levels in cells from 2-day-old rats than in those from 7-day-old, 14-day-old, or adult rats. Temperature independence of this initial phase, differences in response to trypsin-EDTA or neuraminidase treatment, and the dependency of increased neonatal uptake on the presence of phosphatidylglycerol in liposomes suggested binding as a major mechanism of cell-lipid interaction. Although a two to three times larger amount of lipid was associated with neonatal cells, the metabolism of phosphatidylcholine, indicated by a decrease in label in phosphatidylcholine and an accompanying increase in sphingomyelin, was significantly smaller in 2-day-old than in adult cells. These studies support the hypothesis that neonatal and adult cells may have differences in the interaction with alveolar phospholipids and in the metabolism of phosphatidylcholine.