Inhibits Phosphatidylcholine Synthesis Research Articles

A coumarin derivative C7 exhibits antiviral activity against WSSV by reducing phosphatidylcholine content in shrimp

The white spot syndrome virus (WSSV) causes white spot disease (WSD), a severe condition in crustacean aquaculture, leading to significant economic losses. Our previous study demonstrated that C7 is an effective therapeutic agent against WSSV infection in aquaculture. It specifically blocked viral horizontal transmission and reduced shrimp mortality in a dose- and time-dependent manner. Here, we report the potential antiviral mechanism of C7 in shrimp. C7 regulated abnormal glycerophospholipid metabolism caused by WSSV and inhibited phosphatidylcholine (PC) synthesis by more than twofold, potentially enhancing shrimp resistance to viral infection. As the primary phospholipid in the cell membrane, PC is one of the main reactants in lipid peroxidation. Our results indicated that C7 significantly reduced the levels of lipid peroxidation products 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) induced by WSSV, whereas PC had the opposite effect. Accumulation of lipid peroxidation products inhibits stimulator of interferon genes (STING) signaling. Further evidence showed that C7 promoted STING transport from the endoplasmic reticulum to the Golgi apparatus, significantly activating the expression of the shrimp interferon analogue Vago4 gene. In contrast, PC suppressed Vago4 expression. Our results demonstrated that C7 inhibited PC synthesis, reduced the degree of lipid peroxidation, promoted STING translocation, activated Vago4 expression, and ultimately exerted antiviral effects. Therefore, C7 exhibits immunoregulatory activity as a preventative agent for enhancing the innate immunity of shrimp, making it potentially useful for future immunomodulatory approaches.

Alteration of Plasma Membrane Organization by an Anticancer Lysophosphatidylcholine Analogue Induces Intracellular Acidification and Internalization of Plasma Membrane Transporters in Yeast

The lysophosphatidylcholine analogue edelfosine is a potent antitumor lipid that targets cellular membranes. The underlying mechanisms leading to cell death remain controversial, although two cellular membranes have emerged as primary targets of edelfosine, the plasma membrane (PM) and the endoplasmic reticulum. In an effort to identify conditions that enhance or prevent the cytotoxic effect of edelfosine, we have conducted genome-wide surveys of edelfosine sensitivity and resistance in Saccharomyces cerevisiae presented in this work and the accompanying paper (Cuesta-Marbán, Á., Botet, J., Czyz, O., Cacharro, L. M., Gajate, C., Hornillos, V., Delgado, J., Zhang, H., Amat-Guerri, F., Acuña, A. U., McMaster, C. R., Revuelta, J. L., Zaremberg, V., and Mollinedo, F. (January 23, 2013) J. Biol. Chem. 288,), respectively. Our results point to maintenance of pH homeostasis as a major player in modulating susceptibility to edelfosine with the PM proton pump Pma1p playing a main role. We demonstrate that edelfosine alters PM organization and induces intracellular acidification. Significantly, we show that edelfosine selectively reduces lateral segregation of PM proteins like Pma1p and nutrient H(+)-symporters inducing their ubiquitination and internalization. The biology associated to the mode of action of edelfosine we have unveiled includes selective modification of lipid raft integrity altering pH homeostasis, which in turn regulates cell growth.

Membrane Lipid Biosynthesis As a Potential Therapeutic Target in Multiple Myeloma

AbstractAbstract 1836Phosphatidylcholine (PC) is the most prominent phospholipid in mammalian endoplasmic reticulum (ER) membranes. The rate-limiting step in PC synthesis through the Kennedy pathway is the conversion of phosphocholine + cytidine triphosphate (CTP) to cytidine diphosphocholine, (CDP)-choline, via the enzyme CTP:phosphocholine cytidylyltransferase (CCT) (see figure). [Display omitted] Multiple myeloma (MM) cells may be particularly dependent on this biosynthetic reaction because of their high consistent level of ER stress and requirement to continuously replenish ER membranes. Indeed, CCT-null mice have a defect in differentiation of B lymphocytes to plasma cells and deficiencies in Ig synthesis. To test whether this pathway remains critical in survival of malignant MM cells, we exposed MM cell lines to an inhibitor shown to inhibit CCT activity, HexPC. HexPC induced apoptosis in all MM cell lines in a concentration- and time-dependent manner. The addition of lysophosphatidylcholine (LPC), presumably converted to PC independently of the Kennedy pathway, completely rescued MM cell apoptosis. In contrast, similar concentrations of LPC in the same cell lines could not rescue apoptosis induced by bortezomib. An additional intervention to inhibit phosphatidylcholine synthesis, namely inducing pyrimidine starvation, also resulted in MM cell apoptosis and down-regulation of CDP-choline levels. Apoptosis of MM cells induced by HexPC was associated with induction of ER stress as shown by enhanced phosphorylation of IRE1 and eIF-2alpha. This ER stress was also prevented when LPC was added to HexPC although LPC could not prevent similar ER stress induced by bortezomib. These results underscore the importance of this phosphatidylcholine synthesis pathway in MM cells and provide new targets for future therapy. Disclosures:No relevant conflicts of interest to declare.

Resistance to alkyl-lysophospholipid-induced apoptosis due to downregulated sphingomyelin synthase 1 expression with consequent sphingomyelin- and cholesterol-deficiency in lipid rafts

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; Et-18-OCH3) induces apoptosis in S49 mouse lymphoma cells. To this end, ALP is internalized by lipid raft-dependent endocytosis and inhibits phosphatidylcholine synthesis. A variant cell-line, S49AR, which is resistant to ALP, was shown previously to be unable to internalize ALP via this lipid raft pathway. The reason for this uptake failure is not understood. In the present study, we show that S49AR cells are unable to synthesize SM (sphingomyelin) due to down-regulated SMS1 (SM synthase 1) expression. In parental S49 cells, resistance to ALP could be mimicked by small interfering RNA-induced SMS1 suppression, resulting in SM deficiency and blockage of raft-dependent internalization of ALP and induction of apoptosis. Similar results were obtained by treatment of the cells with myriocin/ISP-1, an inhibitor of general sphingolipid synthesis, or with U18666A, a cholesterol homoeostasis perturbing agent. U18666A is known to inhibit Niemann-Pick C1 protein-dependent vesicular transport of cholesterol from endosomal compartments to the trans-Golgi network and the plasma membrane. U18666A reduced cholesterol partitioning in detergent-resistant lipid rafts and inhibited SM synthesis in S49 cells, causing ALP resistance similar to that observed in S49AR cells. The results are explained by the strong physical interaction between (newly synthesized) SM and available cholesterol at the Golgi, where they facilitate lipid raft formation. We propose that ALP internalization by lipid-raft-dependent endocytosis represents the retrograde route of a constitutive SMS1- and lipid-raft-dependent membrane vesicular recycling process.

Oxysterols Inhibit Phosphatidylcholine Synthesis via ERK Docking and Phosphorylation of CTP:Phosphocholine Cytidylyltransferase

Surfactant deficiency contributes to acute lung injury and may result from the elaboration of bioactive lipids such as oxysterols. We observed that the oxysterol 22-hydroxycholesterol (22-HC) in combination with its obligate partner, 9-cis-retinoic acid (9-cis-RA), decreased surfactant phosphatidylcholine (PtdCho) synthesis by increasing phosphorylation of the regulatory enzyme CTP:phosphocholine cytidylyltransferase-alpha (CCTalpha). Phosphorylation of CCTalpha decreased its activity. 22-HC/9-cis-RA inhibition of PtdCho synthesis was blocked by PD98059 or dominant-negative ERK (p42 kinase). Overexpression of constitutively active MEK1, the kinase upstream of p42 kinase, increased CCTalpha phosphorylation. Expression of truncated CCTalpha mutants lacking proline-directed sites within the C-terminal phosphorylation domain partially blocked oxysterol-mediated inhibition of PtdCho synthesis. Mutagenesis of Ser315 within CCTalpha was both required and sufficient to confer significant resistance to 22-HC/9-cis-RA inhibition of PtdCho synthesis. A novel putative ERK-docking domain N-terminal to this phosphoacceptor site was mapped within the CCTalpha membrane-binding domain (residues 287-300). The results are the first demonstration of a physiologically relevant phosphorylation site and docking domain within CCTalpha that serve as targets for ERKs, resulting in inhibition of surfactant synthesis.

Hexadecylphosphocholine inhibits phosphatidylcholine synthesis via both the methylation of phosphatidylethanolamine and CDP-choline pathways in HepG2 cells

We reported in a recent publication that hexadecylphosphocholine (HePC), a lysophospholipid analogue, reduces cell proliferation in HepG2 cells and at the same time inhibits the biosynthesis of phosphatidylcholine (PC) via CDP-choline by acting upon CTP:phosphocholine cytidylyltransferase (CT). We describe here the results of our study into the influence of HePC on other biosynthetic pathways of glycerolipids. HePC clearly decreased the incorporation of the exogenous precursor [1,2,3- 3 H ]glycerol into PC and phosphatidylserine (PS) whilst increasing that of the neutral lipids diacylglycerol (DAG) and triacylglycerol (TAG). Interestingly, the uptake of l-[3- 3 H ]serine into PS and other phospholipids remained unchanged by HePC and neither was the activity of either PS synthase or PS decarboxylase altered, demonstrating that the biosynthesis of PS is unaffected by HePC. We also analyzed the water-soluble intermediates and final product of the CDP-ethanolamine pathway and found that HePC caused an increase in the incorporation of [1,2- 14 C ]ethanolamine into CDP-ethanolamine and phosphatidylethanolamine (PE) and a decrease in ethanolamine phosphate, which might be interpreted in terms of a stimulation of CTP:phosphoethanolamine cytidylyltransferase activity. Since PE can be methylated to give PC, we studied this process further and observed that HePC decreased the synthesis of PC from PE by inhibiting the PE N-methyltransferase activity. These results constitute the first experimental evidence that the inhibition of the synthesis of PC via CDP-choline by HePC is not counterbalanced by any increase in its formation via methylation. On the contrary, in the presence of HePC both pathways seem to contribute jointly to a decrease in the overall synthesis of PC in HepG2 cells.

Alkyl-lysophospholipid Accumulates in Lipid Rafts and Induces Apoptosis via Raft-dependent Endocytosis and Inhibition of Phosphatidylcholine Synthesis

The synthetic alkyl-lysophospholipid (ALP), 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, is an antitumor agent that acts on cell membranes and can induce apoptosis. We investigated how ALP is taken up by cells, how it affects de novo biosynthesis of phosphatidylcholine (PC), and how critical this is to initiate apoptosis. We compared an ALP-sensitive mouse lymphoma cell line, S49, with an ALP-resistant variant, S49(AR). ALP inhibited PC synthesis at the CTP:phosphocholine cytidylyltransferase (CT) step in S49 cells, but not in S49(AR) cells. Exogenous lysophosphatidylcholine, providing cells with an alternative way (acylation) to generate PC, rescued cells from ALP-induced apoptosis, indicating that continuous rapid PC turnover is essential for cell survival. Apoptosis induced by other stimuli that do not target PC synthesis remained unaffected by lysophosphatidylcholine. Using monensin, low temperature and albumin back-extraction, we demonstrated that ALP is internalized by endocytosis, a process defective in S49(AR) cells. This defect neither involved clathrin-coated pit- nor fluid-phase endocytosis, but depended on lipid rafts, because disruption of these microdomains with methyl-beta-cyclodextrin or filipin (sequestering cholesterol) or bacterial sphingomyelinase reduced uptake of ALP. Furthermore, ALP was found accumulated in isolated rafts and disruption of rafts also prevented the inhibition of PC synthesis and apoptosis induction in S49 cells. In summary, ALP is internalized by raft-dependent endocytosis to inhibit PC synthesis, which triggers apoptosis.

Inhibition of phosphatidylcholine synthesis is associated with excitotoxic cell death in cerebellar granule cell cultures.

Glucose deprivation (GD) enhances the sensitivity of cerebellar granule cells to die by excitotoxicity. Neither 70 min of GD, a treatment that depletes cell energy resources, nor exposure to 20 microM glutamate (GLU) for 30 min, induce significant cell death in cultures of cerebellar granule cells. However, the combined treatment with GLU and GD induces choline (Cho) release before excitotoxic cell death. We investigated whether the neurotoxic effect of this treatment is related with inhibition of phosphatidylcholine (PC) synthesis. We found that exposure to GLU for 30 min, to GD for 70 min, and to the combination of both, inhibited PC synthesis at the end of treatment by 71%, 92% and 91%, respectively. The inhibition of PC synthesis was accompanied by a decrease in the incorporation of [(3)H]Cho into phosphocholine and by an increase of the intracellular content of free [(3)H]Cho, indicating that these treatments inhibit the synthesis of PC by inhibiting choline kinase activity. However, only the combined treatment with GLU and GD induced a prolonged inhibition of PC synthesis that extended after the end of treatment. These results show that excitotoxic death is associated with sustained inhibition of PC synthesis and suggest that this effect of the combined treatment with GLU and GD on PC synthesis is produced by an action on an enzymatic step downstream of choline kinase activity.

Sphingomyelin metabolites inhibit sphingomyelin synthase and CTP:phosphocholine cytidylyltransferase.

Tissue injury in inflammation involves the release of several cytokines that activate sphingomyelinases and generate ceramide. In the lung, the impaired metabolism of surfactant phosphatidylcholine (PC) accompanies this acute and chronic injury. These effects are long-lived and extend beyond the time frame over which tumor necrosis factor (TNF)-alpha and interleukin-1beta are elevated. In this paper, we demonstrate that in H441 lung cells these two processes, cytokine-induced metabolism of sphingomyelin and the inhibition of PC metabolism, are directly interrelated. First, metabolites of sphingomyelin hydrolysis themselves inhibit key enzymes necessary for restoring homeostasis between sphingomyelin and its metabolites. Ceramide stimulates sphingomyelinases as effectively as TNF-alpha, thereby amplifying the sphingomyelinase activation, and TNF-alpha, ceramide, and sphingosine all inhibit PC:ceramide phosphocholine transferase (sphingomyelin synthase), the enzyme that restores homeostasis between sphingomyelin and ceramide pools. Second, ceramide inhibits PC synthesis, probably because of its effects on CTP:phosphocholine cytidylyltransferase, the rate-limiting enzymatic step in de novo PC synthesis. The data presented here suggest that TNF-alpha may be an inhibitor of phospholipid metabolism in inflammatory tissue injury. These actions may be amplified because of the ability of metabolites of sphingomyelin to inhibit the pathways that should restore the normal ceramide-sphingomyelin homeostasis.

Overactivation of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate and N‐methyl‐d‐aspartate but not kainate receptors inhibits phosphatidylcholine synthesis before excitotoxic neuronal death

Glutamate receptor overactivation induces excitotoxic neuronal death, but the contribution of glutamate receptor subtypes to this excitotoxicity is unclear. We have previously shown that excitotoxicity by NMDA receptor overactivation is associated with choline release and inhibition of phosphatidylcholine synthesis. We have now investigated whether the ability of non‐NMDA ionotropic glutamate receptor subtypes to induce excitotoxicity is related to the ability to inhibit phosphatidylcholine synthesis. α‐Amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)‐induced a concentration‐dependent increase in extracellular choline and inhibited phosphatidylcholine synthesis when receptor desensitization was prevented. Kainate released choline and inhibited phosphatidylcholine synthesis by an action at AMPA receptors, because these effects of kainate were blocked by the AMPA receptor antagonist LY300164. Selective activation of kainate receptors failed to release choline, even when kainate receptor desensitization was prevented. The inhibition of phosphatidylcholine synthesis evoked by activation of non‐desensitizing AMPA receptors was followed by neuronal death. In contrast, specific kainate receptor activation, which did not inhibit phosphatidylcholine synthesis, did not produce neuronal death. Choline release and inhibition of phosphatidylcholine synthesis were induced by AMPA at non‐desensitizing AMPA receptors well before excitotoxicity. Furthermore, choline release by AMPA required the entry of Ca2+ through the receptor channel. Our results show that AMPA, but not kainate, receptor overactivation induces excitotoxic cell death, and that this effect is directly related to the ability to inhibit phosphatidylcholine synthesis. Moreover, these results indicate that inhibition of phosphatidylcholine synthesis is an early event of the excitotoxic process, downstream of glutamate receptor‐mediated Ca2+ overload.

Overactivation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate but not kainate receptors inhibits phosphatidylcholine synthesis before excitotoxic neuronal death.

Glutamate receptor overactivation induces excitotoxic neuronal death, but the contribution of glutamate receptor subtypes to this excitotoxicity is unclear. We have previously shown that excitotoxicity by NMDA receptor overactivation is associated with choline release and inhibition of phosphatidylcholine synthesis. We have now investigated whether the ability of non-NMDA ionotropic glutamate receptor subtypes to induce excitotoxicity is related to the ability to inhibit phosphatidylcholine synthesis. alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-induced a concentration-dependent increase in extracellular choline and inhibited phosphatidylcholine synthesis when receptor desensitization was prevented. Kainate released choline and inhibited phosphatidylcholine synthesis by an action at AMPA receptors, because these effects of kainate were blocked by the AMPA receptor antagonist LY300164. Selective activation of kainate receptors failed to release choline, even when kainate receptor desensitization was prevented. The inhibition of phosphatidylcholine synthesis evoked by activation of non-desensitizing AMPA receptors was followed by neuronal death. In contrast, specific kainate receptor activation, which did not inhibit phosphatidylcholine synthesis, did not produce neuronal death. Choline release and inhibition of phosphatidylcholine synthesis were induced by AMPA at non-desensitizing AMPA receptors well before excitotoxicity. Furthermore, choline release by AMPA required the entry of Ca(2+) through the receptor channel. Our results show that AMPA, but not kainate, receptor overactivation induces excitotoxic cell death, and that this effect is directly related to the ability to inhibit phosphatidylcholine synthesis. Moreover, these results indicate that inhibition of phosphatidylcholine synthesis is an early event of the excitotoxic process, downstream of glutamate receptor-mediated Ca(2+) overload.

Diethanolamine Inhibits Choline Uptake and Phosphatidylcholine Synthesis in Chinese Hamster Ovary Cells

Diethanolamine (DEA), an alkanolamine used widely in industry, is hepatocarcinogenic in mice. The goal of this work was to determine whether DEA altered choline homeostasis in cultured cells, so as to ascertain whether the liver tumor response may be related to choline deficiency. CHO cells were cultured in Ham's F-12 medium containing DEA (0-1000 μg/ml) and [33P]-phosphorus was used to label phospholipid pools. After 48 hours incubation, lipids were extracted and [33P]-labeled phospholipids were quantified by autoradiography after thin layer chromatographic separation. In control cells, phosphatidylcholine (PC) accounted for 51 ± 0.7% of the total lipid 33P incorporation. DEA had no effect on cell number or total phospholipid biosynthesis, but it significantly decreased the incorporation of 33P into PC at concentrations ≥50 μg/ml. DEA (≥20 μg/ml) also inhibited the uptake of [3H]-choline into CHO cells, with 95% inhibition observed at 250 μg/ml. To determine whether supplemental choline prevented PC synthesis inhibition by DEA, CHO cells were cultured with or without excess choline (30 mM) and DEA (500 μg/ml). DEA reduced PC synthesis to 27 ± 3% of total phospholipids, but had no effect on PC synthesis in choline-supplemented cells. When [14C]-DEA was incubated with CHO cells, it was also incorporated into the phospholipid fraction. Collectively, these results indicate that DEA reversibly inhibits PC synthesis by blocking choline uptake and competing for utilization in the CDP-choline pathway in CHO cells.

Competitive Inhibition of Choline Phosphotransferase by Geranylgeraniol and Farnesol Inhibits Phosphatidylcholine Synthesis and Induces Apoptosis in Human Lung Adenocarcinoma A549 Cells

We have previously shown that, among various isoprenoids, farnesol and geranylgeraniol specifically induced actin fiber disorganization, growth inhibition, and apoptosis in human lung adenocarcinoma A549 cells (Miquel, K., Pradines, A., and Favre, G. (1996) Biochem. Biophys. Res. Commun. 225, 869-876). Here we demonstrate that isoprenoid-induced apoptosis was preceded by an arrest in G0/G1 phase. The isoprenoid effects were independent of protein prenylation and of mitogen-activated protein kinase activity. Moreover, geranylgeraniol and farnesol induced a rapid inhibition of phosphatidylcholine biosynthesis at the last step of the CDP-choline pathway controlled by choline phosphotransferase and not at the level of CTP:phosphocholine cytidylyltransferase, the key enzyme of the pathway. Inhibition of choline phosphotransferase was confirmed by in vitro assays on microsomal fractions, which clearly showed that the isoprenoids acted by competitive inhibition with the diacylglycerol binding. Exogenous phosphatidylcholine addition prevented all the biological effects of the isoprenoids, including actin fiber disorganization and apoptosis, suggesting that inhibition of phosphatidylcholine biosynthesis might be the primary event of the isoprenoid action. These data demonstrate the molecular mechanism of geranylgeraniol and farnesol effects and suggest that the mevalonate pathway, leading notably to prenylated proteins, might be linked to the control of cell proliferation through the regulation of phosphatidylcholine biosynthesis.

Lysophosphatidylcholine and 1-O-Octadecyl-2-O-Methyl-rac- Glycero-3-Phosphocholine Inhibit the CDP-Choline Pathway of Phosphatidylcholine Synthesis at the CTP:Phosphocholine Cytidylyltransferase Step

The regulation of the CDP-choline pathway of phosphatidylcholine synthesis at the CTP:phosphocholine cytidylyltransferase (CT) step by lysophosphatidylcholine (LPC) and the nonhydrolyzable LPC analog, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3), was investigated in a colony-stimulating factor 1-dependent murine macrophage cell line. LPC inhibited phosphatidylcholine synthesis in vivo and led to the accumulation of choline and phosphocholine coupled to the disappearance of CDP-choline pointing to CT as the intracellular target. LPC neither inhibited cell growth nor decreased the cellular content of CT or altered the distribution of CT between soluble and particulate subcellular fractions. The inhibition of phosphatidylcholine synthesis was specific for LPC since lysophospholipids lacking the choline headgroup were not inhibitors. ET-18-OCH3 was a more potent inhibitor of phosphatidylcholine synthesis than LPC and caused the translocation of CT from the soluble compartment to the particulate compartment. Both LPC and ET-18-OCH3 were inhibitors of CT activity in vitro and kinetic analysis showed competitive inhibition with respect to the lipid activator. These data point to LPC as a negative regulator of de novo phosphatidylcholine synthesis that acts at the CT step and establish the mechanism for the inhibition of phosphatidylcholine biosynthesis by antineoplastic phospholipids.

Cholecystokinin inhibits phosphatidylcholine synthesis via a Ca(2+)-calmodulin-dependent pathway in isolated rat pancreatic acini. A possible mechanism for diacylglycerol accumulation.

The effects of cholecystokinin (CCK) and other pancreatic secretagogues on phosphatidylcholine (PC) synthesis were studied in isolated rat pancreatic acini. When acini were incubated with [3H]choline in the presence of 1 nM CCK-octapeptide (CCK8) for 60 min, the incorporations of [3H]choline into both water-soluble choline metabolites and PC in acini were reduced by CCK8 to 74 and 41% of control, respectively. Pulse-chase study revealed that CCK8 reduced both the disappearance of phosphocholine and the synthesis of PC. Other Ca(2+)-mobilizing secretagogues such as carbamylcholine, bombesin, and Ca2+ ionophore A23187 also reduced PC synthesis to the same extent as did CCK8. When combined with 1 nM CCK8, A23187 or carbamylcholine did not further inhibit PC synthesis. Furthermore, W-7 or W-5, a calmodulin antagonist, reversed the inhibition by CCK8 of PC synthesis, suggesting that a Ca(2+)-calmodulin-dependent pathway may be involved in CCK-induced inhibition of PC synthesis in acini. By contrast, neither cAMP-dependent secretagogues such as secretin and dibutyryl cAMP nor a phorbol ester had any effect on PC synthesis in acini. Staurosporine or H-7, a protein kinase C inhibitor, did not affect the inhibition by CCK of PC synthesis. The analysis of enzyme activity involved in PC synthesis via CDP-choline pathway showed that CCK treatment of acini reduced CTP:phosphocholine cytidylyltransferase activity in both cytosolic and particulate fraction, a finding consistent with the delayed disappearance of phosphocholine induced by CCK in pulse-chase study. By contrast, CCK treatment of acini did not alter the activities of choline kinase and phosphocholine transferase in acini. The extent of inhibition by CCK of cytidylyltransferase activity became much larger when subcellular fractions of acini were prepared in the presence of phosphatase inhibitors. In addition, W-7 reversed the inhibitory effect of CCK treatment on cytidylyltransferase activity in acini. When acini were labeled with [3H]myristic acid and chased, CCK8 (1 nM) reduced the synthesis of [3H]myristic acid-labeled PC to 27% of control after a 60-min chase period. This inhibition of PC synthesis induced by CCK was accompanied by a delayed disappearance of [3H]diacylglycerol, the radioactivity of which was 225% of control at 60 min. These results indicate that CCK inhibits PC synthesis by inducing both the reduction of choline uptake into acini and the inhibition of CTP:phosphocholine cytidylyltransferase activity. Furthermore, the results suggest the possibility that the activation of Ca(2+)-calmodulin-dependent kinase in response to CCK may phosphorylate cytidylyltransferase thereby decreasing this enzyme activity in pancreatic acinar cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Activated polymorphonuclear leukocytes inhibit phosphatidylcholine synthesis in cultured type II alveolar cells

Activated human neutrophils (PMNs) were demonstrated to inhibit total de novo phosphatidylcholine (PC) synthesis in monolayered rat alveolar type II cells (T2C). Non-activated PMNs had no effect on PC synthesis in this system. The magnitude of inhibition T2C PC synthesis by phorbol myristate acetate-activated PMNs in six experiments averaged 59.0 +/- 13%. Exogenous chelated iron (ferric pyrophosphate) did not appear to augment the PMN-mediated inhibition of T2C PC production in this model. Alpha-1-antiprotease usually provided no protection relative to the PMN insult towards the T2C. However, superoxide dismutase and catalase alone or in combination generally provided a significant, protective effect. Although activated PMNs consistently decreased T2C PC synthesis, this effect did not appear to involve generalized T2C cytotoxicity, as assessed by lack of release of cytosolic lactate dehydrogenase. These results indicate that PMNs can inhibit T2C PC synthesis in vitro, probably via oxyradical injury. This type of pulmonary host autoinjury may be operative in a variety of acute lung injury syndromes involving pulmonary sequestration of activated PMNs.

Trifluoperazine and chlorpromazine inhibit phosphatidylcholine biosynthesis and CTP: Phosphocholine cytidylyltransferase in HeLa cells

The influence of chlorpromazine and trifluoperazine on phosphatidylcholine biosynthesis in HeLa cells was investigated. HeLa cells were prelabeled with [ Me- 3H]choline for 1 h. The cells were subsequently incubated with various concentrations of drugs. Both compounds were potent inhibitors of phosphatidylcholine biosynthesis, with 50% inhibition by 5 μm of either drug. Analysis of the radioactivity in the soluble precursors indicated a block in the conversion of phosphocholine to CDPcholine catalyzed by CTP: phosphocholine cytidylyltransferase (CTP: cholinephosphate cytidylyltransferase, EC 2.7.7.15). Inhibition by these drugs was slowly reversed after incubation for more than 2 h, or was immediately abolished when 0.4 mM oleate was included in the cell medium or when the drug-containing medium was removed. The subcellular location of the cytidylyltransferase was unaffected by either drug, nor did the drugs alter the rate of release of cytidylyltransferase from HeLa cells by digitonin treatment. The drugs had a direct inhibitory effect on cytidylyltransferase activity in HeLa cell postmitochondrial supernatants. Half-maximal inhibition was achieved with 30 μM trifluoperazine and 50 μM chlorpromazine. These drugs did not change the apparent K m of the cytidylyltransferase for CTP or phosphocholine. Inhibition of cytidylyltransferase by these compounds was reversible with exogenous phospholipid or oleate in the enzyme assay. The data indicate that both drugs inhibit phosphatidylcholine synthesis by an effect on the cytidylyltransferase. The mechanism of action remains unknown at this time.

Inhibitors of endocytosis perturb phospholipid metabolism in rabbit neutrophils and other cells.

Dansylcadaverine, amantadine, and rimantadine, which have been shown to inhibit the endocytosis of alpha 2-macroglobulin, epidermal growth factor, and vesicular stomatitis virus [Schlegel, R., Dickson, R. B., Willingham, M. C. & Pastan, I. (1982) Proc. Natl. Acad. Sci. USA 79, 2291-2295], were found to decrease phosphatidylcholine synthesis, chemotaxis, and internalization of a formylated peptide but to stimulate the incorporation of inositol into phosphatidylinositol in rabbit neutrophils. Dansylcadaverine decreased phosphatidylcholine synthesis by both the CDP-choline and transmethylation pathways and also inhibited the synthesis of phosphatidylethanolamine by the CDP-ethanolamine pathway. Dansylcadaverine had no effect on the phosphocholine, CDP-choline, or S-adenosyl-L-homocysteine pools but increased 2-fold the S-adenosyl-L-methionine pool. These results suggest that dansylcadaverine in some manner inhibited the condensation of CDP-choline with diacylglycerol to form phosphatidylcholine. Dansylcadaverine also inhibited phosphatidylcholine synthesis in human neutrophils, human fibroblasts, chicken embryo fibroblasts, rat hepatocytes, osteosarcoma cells, and neuroblastoma cells. It did not stimulate phosphatidylinositol synthesis in chicken embryo fibroblasts.