Changes in Lipid Composition During Manganese-Induced Apoptosis in PC12 Cells.

Lipid composition of membranes is fundamental to modulate signaling pathways relying on lipid metabolites and/or membrane proteins, thus resulting in the regulation of important cell processes such as apoptosis. In this case, membrane remodeling is an early event important for the activation of signaling leading to cell death and removal of apoptotic cells. In the present study, we analyzed phospholipid, cholesterol and fatty acid content during apoptosis induced by manganese in PC12 cells. Lipid analysis of whole cells and detergent-resistant membranes was carried out by HPLC/GC. Results showed that apoptosis is associated with changes in lipid composition detectable in whole cell extracts, namely cholesterol, phosphatidylserine and phosphatidylethanolamine decreases. Noteworthy, phosphatidylserine level reduction was detectable before to the detection of apoptosis, in correlation with our previous study carried out by radioactive labelling. By contrast, phosphatidylserine and phosphatidylethanolamine changes were not detected in detergent resistant membranes, which instead showed an altered composition in phosphatidylinositol, phosphatidylcholine and sphingomyelin in apoptotic cells.

Phosphatidylserine metabolism modification precedes manganese-induced apoptosis and phosphatidylserine exposure in PC12 cells.

Long-term exposure to high manganese (Mn) levels can lead to Parkinson-like neurological disorders. Molecular mechanisms underlying Mn cytotoxicity have been not defined. It is known that Mn induces apoptosis in PC12 cells and that this involves the activation of some signal transduction pathways. Although the role of phospholipids in apoptosis and signal transduction is well-known, the membrane phospholipid component in Mn-related damage has not yet been investigated. Phosphatidylserine (PS) facilitates protein translocation from cytosol to plasma membrane and PS exposure on the cell surface allows macrophage recognition of apoptotic cells. This study investigates the effects of MnCl2 on PS metabolism in PC12 cells, relating them to those on cell apoptosis. Apoptosis induction decreased PS radioactivity of PC12 cells incubated with radioactive serine. MnCl2 reduced PS radioactivity even under conditions that did not affect cell viability or PS exposure, suggesting that the effects on PS metabolism may represent an early event in cell apoptosis. Thus the latter conditions that also induced a greater PS decarboxylation were utilized for further investigating on the effects on PS synthesis, by measuring the activity and expression of PS-synthesizing enzymes, in cell lysates and in total cellular membranes (TM). Compared with corresponding controls, enzyme activity of MnCl2-treated cells was lower in cell lysates and greater in TM. Evaluating the expression of two isoforms of PS-synthesizing enzyme (PSS), PSSII was increased both in cell lysate and TM, while PSSI was unchanged. MnCl2 addition to control cell lysate reduced enzyme activity. These results suggest Mn plays a dual role on PS synthesis. Once inside the cell, Mn inhibits the enzyme/s, thus accounting for reduced PS synthesis in lysates and intact cells. On the other hand, it increases PSSII expression in cell membranes. The possibility that this occurs to counteract the direct effects of Mn ions on enzyme activity cannot be excluded. The effects on membrane enzyme activity and expression may also participate to PS exposure, observed at longer periods of treatment, by increasing membrane PS content.

Dexamethasone increases the incorporation of [3H]serine into phosphatidylserine and the activity of serine base exchange enzyme in mouse thymocytes: a possible relation between serine base exchange enzyme and apoptosis.

The exposure of phosphatidylserine toward the external surface of the membrane is a well-established event of programmed cell death. The possibility that an apoptotic stimulus influences the metabolism of this phospholipid could be relevant not only in relation to the previously mentioned event but also in relation to the capability of membrane phosphatidylserine to influence PKC activity. The present investigation demonstrates that treatment of mouse thymocytes with the apoptotic stimulus dexamethasone, enhances the incorporation of [3H]serine into phosphatidylserine. Cell treatment with dexamethasone also enhanced the activity of serine base exchange enzyme, assayed in thymocyte lysate. Both the effects were observed at periods of treatment preceding DNA fragmentation. The addition of unlabelled ethanolamine, together with [3H]serine to the medium containing dexamethasone-treated thymocytes lowered the radioactivity into phosphatidylserine. Serine base exchange enzyme activity was influenced by the procedure used to prepare thymocyte lysate and was lowered by the addition of fluoroaluminate, that is widely used as a G-protein activator. The increase of serine base exchange enzyme activity induced by dexamethasone treatment was observed independently by the procedure used to prepare cell lysate and by the presence or absence of fluoroaluminate.

Serine base exchange enzyme in porcine lyophilised platelets: enzyme properties and modulation by AlF4- and different types of heparin.

Phosphatidylserine is one of the PKC modulators and thus it may play an important role in signal transduction. Regulation of the synthesis of this phospholipid is not yet clarified. The contrasting reports are possibly related to the existence of different enzymes which, in mammalian tissues, catalyse the exchange between free serine and the nitrogen base of a membrane phospholipid. This study demonstrates that serine base exchange reactions of commercially available lyophilised porcine platelets exhibit similar pH optima, temperature and Ca2+ dependence as observed in fresh tissues. Analysis of fatty acids composition of the three phospholipid classes involved in base exchange reactions also demonstrated a similarity with fresh platelets. Serine and ethanolamine base exchange enzyme activities were assayed in parallel in platelet lysate subjected to preincubation at various temperatures (30-60 degrees C). When dithioerithrol was omitted from the incubation medium, the two base exchange reactions were inhibited with a similar temperature-dependent pattern. Addition of the reducing agent enhanced the sensitivity to preincubation only for the serine base exchange reaction which was inhibited by 80% after preincubation at 45 degrees C. With respect to its regulation, porcine platelet serine base exchange enzyme(s) was inhibited by fluoroalluminate, a widely used G-protein activator, and stimulated by unfractionated heparin. Low mol. wt. heparin did not influence enzyme activity. Unfractionated heparin greatly stimulated SBEE activity assayed at pH 7.4, a pH value far from the optimal pH.

Effect of serine and ethanolamine administration on phospholipid-related compounds and neurotransmitter amino acids in the rabbit hippocampus.

The report concerns mechanisms for the increase of extracellular levels of ethanolamine and phosphoethanolamine in CNS regions, such as the hippocampus, in transient brain ischemia, hypoglycemia, seizures, etc. L-Serine (2.5-10 mM), D-serine (10 mM), or ethanolamine (10 mM) was administered for 20 min via a microdialysis tubing to the hippocampus of unanesthetized rabbits. The concentrations of primary amines were determined in the dialysates. When levels were elevated 10-100 times in the extracellular fluid, L-serine caused a dose-dependent increase of the concentration of extracellular ethanolamine. Ethanolamine caused a corresponding, although somewhat smaller, increase in serine levels. Furthermore, L-serine also induced an increased concentration of phosphoethanolamine that was delayed in time relative to the peak of ethanolamine. D-Serine was as effective as L-serine in raising ethanolamine levels but had no effect on phosphoethanolamine. Ethanolamine, but not L-serine, also increased extracellular glutamate/aspartate levels in an MK-801-dependent fashion. A similar effect, but delayed in time, was observed with D-serine. These effects were inhibited by MK-801. The concentrations of other amino acids were not significantly affected. The characteristics of the effects are suggestive of base exchange reactions between serine and ethanolamine and between ethanolamine and serine glycerophospholipids, respectively, in neuronal plasma membranes.

Synthesis of ethanolamine phosphoglycerides in rat cerebral cortex subjected in vitro to experimental hypoxia with and without hypocapnia.

Slices and homogenates from rat cerebral cortex were used to study the effect of hypoxia, with or without hypocapnia, on phosphatidylethanolamine synthesis. The incorporation of [1-3H]ethanolamine into the corresponding phospholipid was greatest in slices treated with pure nitrogen, intermediate when the nitrogen contained 5% CO2, and least in slices treated with 95% O2-5% CO2. The role of hypocapnia in reinforcing the effect due to hypoxia did not require the integrity of the cell because similar results were obtained by treating homogenates with pure nitrogen or nitrogen plus 5% CO2. In both cases the synthesis of phosphatidylethanolamine was abolished by the addition of EGTA and the degradation of newly synthesized phospholipid by phospholipases was similar to that obtained in controls. When the homogenate was not buffered, changes in the pH due to experimental treatment influenced the response to Ca2+ and to hypoxia plus hypocapnia. Intracellular calcium ions are thought to play a role in the response of cerebrocortical slices to N2-treatment. In fact, although the incorporation was greater in complete medium that contains 2 mM Ca2+ than in the same medium prepared without the addition of this ion, the relative increase of incorporation due to N2-treatment was greater in the medium lacking added Ca2+.

Different mechanisms regulate phosphatidylserine synthesis in rat cerebral cortex.

Transduction of extracellular signals through the membrane involves both the lipid and protein moiety. Phosphatidylserine participates to these processes as a cofactor for protein kinase C activity and thus the existence of a regulatory mechanism for its synthesis ought to be expected. In plasma membranes from rat cerebral cortex, the activity of serine base exchange enzyme, that is mainly responsible for phosphatidylserine synthesis in mammalian tissues, was reduced by the addition to the incubation mixture of AlF4- or GTP-gamma-S, known activators of G proteins, whereas ATP was almost uneffective. GTP-gamma-S inhibited the enzyme activity only at relatively high concentration (> 0.5 mM). When the synthesis of phosphatidylserine in the same cerebral area was investigated by measuring the incorporation of labelled serine into the phospholipid in the homogenate buffered at pH 7.6, ATP had an inhibitory effect as GTP-gamma-S and AlF4-. Heparin activated both serine base exchange enzyme in plasma membranes and phosphatidylserine synthesis in the homogenate. The preincubation of plasma membranes in the buffer without any other addition at 37 degrees C for 15 min reduced by 30% serine base exchange enzyme activity. The remaining activity responded to the addition of GTP-gamma-S but was insensitive to 5 mM AlF-4, a concentration that inhibited by 60% the enzyme assayed without preincubation. These results indicate the existence of different regulatory mechanisms, involving ATP and G proteins, possibly acting on different enzymes responsible for the synthesis of phosphatidylserine. Since previous studies have shown that hypoxia increases the synthesis of this phospholipid in brain slices or homogenate (Mozzi et al. Mol Cell Biochem 126: 101-107, 1993), it is possible that hypoxia may interfere with at least one of these mechanisms. This hypothesis is supported by the observation that in hypoxic homogenate 20 mM AlF-4 was not able to reduce the synthesis of phosphatidylserine as in normoxic samples. A similar difference between oxygenated and hypoxic samples, concerning their response to AlF4-, was observed when the incorporation of ethanolamine into phosphatidylethanolamine was studied. The incorporation of choline into phosphatidilcholine was, on the contrary, inhibited at a similar extent in both experimental conditions.

Original Article: Albumin Prevents TxB, Formation from Thrombin-stimulated Human Platelets by Sequestering the Liberated Arachidonic Acid in the Extracellular Space.

Albumin is the major protein in plasma and possesses high affinity binding sites for fatty acids. Previous studies have shown that albumin interferes at various levels with the metabolism of arachidonic acid by stimulated human platelets. The aim of our study was to further characterise the effect of serum albumin on the release of arachidonic acid from platelet phospholipids and on the formation of thromboxane B(2) In washed prelabeled human platelets, stimulated with thrombin (0.5 U/ml), the presence of albumin in the incubation medium leads to an accumulation of [(3)H]AA in the extracellular space and to a reduced formation of thromboxane B(2). In an albumin-free medium, the radioactivity of thromboxane B(2) is markedly greater, while that of arachidonate is much less. The effect of bovine serum albumin is dose-dependent (0.35%, 1.0% and 3.5%). These data suggest that arachidonic acid liberated by PLA(2)-activation is released to the extracellular space where it binds serum albumin and thus is no longer available for its metabolic conversion to thromboxane B(2).