Rita Mozzi

Metabolism and functions of phosphatidylserine in mammalian brain.

Phosphatidylserine (PtdSer) is involved in cell signaling and apoptosis. The mechanisms regulating its synthesis and degradation are still not defined. Thus, its role in these processes cannot be clearly established at molecular level. In higher eukaryotes, PtdSer is synthesized from phosphatidylethanolamine or phosphatidylcholine through the exchange of the nitrogen base with free serine. PtdSer concentration in the nervous tissue membranes varies with age, brain areas, cells, and subcellular components. At least two serine base exchange enzymes isoforms are present in brain, and their biochemical properties and regulation are still largely unknown because their activities vary with cell type and/or subcellular fraction, developmental stage, and differentiation. These peculiarities may explain the apparent contrasting reports. PtdSer cellular levels also depend on its decarboxylation to phosphatidylethanolamine and conversion to lysoPtdSer by phospholipases. Several aspects of brain PtdSer metabolism and functions seem related to the high polyunsaturated fatty acids content, particularly docosahexaenoic acid (DHA).

Conversion of phosphatidylethanolamine to phosphatidylcholine in rat brain by the methylation pathway

Phosphatidylcholine (Ptd-choline) is synthesized in animal tissues chiefly by the cytidine pathway, although the stepwise methylation of endogenous phosphatidylethanolamine (Ptd-ethanolamine) and the base-exchange reaction also contribute to its production [l-7]. The N-methylation pathway was first demonstrated in vivo in the liver [3]. However, in vivo experiments [8] excluded the occurrence in brain of this pathway, although now contrary evidence has been presented ]9,101. Methyltransferase activity for Ptd-choline synthesis has been observed in liver and brain microsomes [ 101, using phosphatidyl-NJ-dimethylethanolamine as substrate. The authors found a very low activity in brain as compared to liver. The presence of two different enzymes has been reported [ 11,121 in adrenal medulla and erythrocyte membrane acting at different pH and involved in the methylation of Ptd-ethanolamine to Ptd-choline. The first enzyme catalyzes the synthesis of phosphatidyl-N-monomethylethanolamine from S-adenosylmethionine (SAM); the second the other two methylations. The aim of this work is to investigate the N-methylation pathway in brain at two different pH values to establish the activity of both enzymes. The transfer of the last methyl group was examined by adding phosphatidyl-NJ-dimethylethanolamine to the incubation medium. All experiments were carried out with rat brain homogenate prepared after prolonged cardiac perfusion to avoid red cell contamination.

The Mechanism of Action of the Epidermal Growth Factor

The effect of the epidermal growth Factor (EGF) on the rate of synthesis of DNA, RNA and proteins in two different kinds of isolated tumor cells have been studied by timing the uptakes of the labeled precursors 3H-thymidine, 3H-uridine and 14C-amino acids. The results obtained from kinetic data have shown that the EGF stimulates the rate of synthesis of DNA, RNA and proteins. However, the activation of the RNA synthesis appears to be an earlier and more specific effect with respect to stimulation of protein and DNA syntheses.

Synthesis of choline phospholipids in neuronal and glial cell cultures by the methylation pathway.

The synthesis of choline in the nervous tissue has been a subject of debate (reviews [1,2]). Since the work of [3,4] it was believed that the stepwise methylation of ethanolamine and/or phosphatidylethanolamine in nervous tissue was non-existant or irrelevant until the suggestion that choline might be produced de novo in the rat brain [5-7] through methylation of phosphatidylethanolamine. Methyltransferase activity has been shown in rat brain synaptosomes [8,9], suggesting that nervous tissue may have the necessary machinery for the de novo synthesis of phosphatidylcholine. The possibility of obtaining cell cultures containing exclusively neurons or glial cells gave us the opportunity to check, in these isolated systems, whether neurons and/or glia can methylate phosphatidylethanolamine to phosphatidylcholine. The results obtained suggest that both cell types have this capacity and that the synthesis of choline phospholipids through the methylation pathway is much higher in glial cells than in neurons.

The synthesis of choline plasmalogen by the methylation pathway in rat brain

The N-methylation of phosphatidylethanolamine (ptd-ethanolamine) to phosphatidylcholine (ptd-choline) has been demonstrated in rat brain in vitro [ I -4] . Experimental evidence has been also obtained that synthesis of choline plasmalogen may take place in brain microsomes by a similar pathway [4]. Labelled lysophosphatidylcholine was in fact isolated after incubation of rat brain microsomal membranes with S-adenosyl-L[methyl s H] methionine with a maximal incorporation at pH 8.2 [4]. Although the action of phospholipases A on the ptd-choline synthesized from ptd-ethanolamine could be responsible for this finding, we have considered the possibility that the lysocompound originates from choline plasmalogen, thus indicating a methylation of ethanolamine plasmalogen. In fact, because of the acidic extraction procedure [4] the cleavage of the vinyl-ether linkage of plasmalogen occurred, giving rise to the formation of the corresponding labelled lysocompound. Experimental data are here reported which indicate, with the aim of a suitable extraction procedure, the direct transfer of methyl groups from labelled S-adenosyl-L-methionine (SAM) into ethanolamine plasmalogen, thus indicating a possible pathway for choline plasmalogen synthesis both in whole brain homogenate and in brain microsomes in vitro.

Receptor-Mediated Degradation of Choline Plasmalogens and Glycerophospholipid Methylation: A New Hypothesis

The stimulation of receptors on the cell surface initiates biochemical and physical changes in membranes that lead to biological responses by the cells. The biochemical and physical changes include changes in levels of cyclic nucleotides, phosphorylation of proteins in membranes, increased membrane disorder (fluidity), and increased fluxes of Ca2+, Na+, and other ions. Changes in lipid metabolism associated with receptor stimulation may include the release of arachidonic acid and formation of metabolites and changes in phospholipid N-methylation and in polyphosphoinositide metabolism. The large number of studies on the association of phospholipid N-methylation with receptor stimulation and adenylate cyclase suggest that an important biological mechanism is involved. An overall hypothesis linking receptor stimulation directly with increased activity of AdoMet: PtdEtn methyltransferase and phospholipase A2 was proposed (Hirata and Axelrod, 1980; Mato and Alemany, 1983). This hypothesis is no longer tenable because of difficulties of others with reproduction of the results, problems inherent in the methodology, and errors in the interpretation of the results. Most previous studies failed to recognize that stopping a reaction with acid causes the hydrolysis of plasmalogens to lysoGpl. The presence of lyso compounds was instead interpreted as evidence for phospholipase A2 activity.

High Levels of Glycine and Serine as a Cause of the Seizure Symptoms of Cavernous Angiomas

Abstract: Cavernous angiomas are vascular malformations that cause neurodegeneration and symptoms including epileptiform seizures, headache, and motor deficits. Following neurosurgical removal of the angiomas, patients mostly recover well and become seizure‐free. This study reports on the levels of certain amino acids in angiomas, obtained from 13 patients. Distinct zones of the angiomas were analyzed, from the thrombotic core, via gliotic, hemosiderin‐infiltrated intermediate zones, to a periphery without macroscopic abnormalities. The neurotransmitter amino acids glutamate, aspartate, and GABA as well as phosphoethanolamine displayed decreasing levels from the periphery to the core, reflecting the gradual neuronal loss. Compared with normal brain tissue, there was a marked increase in the levels of serine (fivefold), glycine (10‐fold), and ethanolamine (20‐fold) in the peripheral zone of the cavernous angiomas. The results are discussed in relation to seizures and NMDA receptor activation, neuron‐glia interactions, membrane phospholipids, and blood‐brain barrier function.

Phosphatidylserine synthesis in rat cerebral cortex: effects of hypoxia, hypocapnia and development

Phosphatidylserine, which is necessary for protein kinase C activity, is synthesized in mammalian tissues by the Ca2+-dependent base exchange enzyme. The synthesis of phosphatidylserine is greater in slices or homogenates of rat cerebral cortex subjected to hypoxia by N2 treatment when compared with O2 plus 5% CO2. An intermediate effect was observed when the treatment was done with N2 plus 5% CO2. Incorporation rates were dependent on Ca2+ in Krebs-Henseleit Ringer bicarbonate medium, being greater with 2 mM Ca2+ than with the same medium prepared without Ca2+. The increase of phosphatidylserine synthesis, due to hypoxia, was, on the contrary, more evident in the medium lacking added Ca2+. Similar results were obtained with the homogenates. This suggests that elevation of intracellular Ca2+, caused by hypocapnia and hypoxia, may be responsible for the greater incorporation of serine into phosphatidylserine. In both cerebrocortical slices and homogenate, [14C]serine incorporation decreased with development both in O2 plus 5% CO2 and N2-treated preparations. However, in younger rats (14–18 days) hypoxia induced a lesser increase of phosphatidylserine than in 40 day old animals. We suggest that a regulatory mechanisms for phosphatidylserine synthesis is established during development and that N2-treatment can increase phosphatidylserine synthesis by interfering with this regulatory mechanism.

Group B streptococcus (GBS) modifies macrophage phosphatidylserine metabolism during induction of apoptosis

Group B streptococcus (GBS) induced macrophage apoptosis by which it could avoid host defence mechanisms. Macrophages, which constitutively express phosphatidylserine (PtdSer) on the outer leaflet of plasma membrane, increased PtdSer exposure during GBS‐induced apoptosis. Induction of apoptosis decreased PtdSer radioactivity of macrophages incubated with [3H]serine. The effect appeared not due to increasing conversion of PtdSer to phosphatidylethanolamine or phosphatidylcholine nor to the release of radioactive membrane vesicles. The radioactivity in lysoPtdSer was also reduced. These results confirm that induction of apoptosis involves a modification of PtdSer metabolism and point out the typical features of the GBS‐induced apoptosis with respect to other models of apoptosis.

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

Abstract: 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.