G. Porcellati

Effect of cytidine diphosphate choline (CDP-choline) on ischemia-induced alterations of brain lipid in the gerbil

Brain ischemia was produced in gerbils (Meriones unguiculatus) by the bilateral ligation of the carotid arteries. Definite changes in the energy status of brain demonstrated that carotid occlusion was effective. Five minutes before ligation, an intraventricular injection of either saline or cytidine diphosphate choline (CDP-choline, 0.6 μmol/brain, 3μl) was given to groups of animals. Control animals, with and without CDP-choline, together with the ischemic groups, were decapitated directly into liquid nitrogen; 10 min after arterial ligation. Brain free fatty acids, neutral lipids and phospholipids, which were labeled in vivo by the intraventricular injection of [1-14C] arachidonic acid (0.4–0.6 μCi, 6–9 nmol) 2 hr prior to ligation, were extracted, purified, and separated by thin-layer chromatographic procedures. The CDP-choline treatment noticeably corrected the increase of total and individual fatty acids due to ischemia and the increase of their radioactivity content. The changes in neutral lipids, particularly in the diacyl glycerol fraction, were also corrected by the injection of the nucleotide. CDP-choline partially reversed the decrease of brain phosphatidylcholine and of its labeling, which was due to ischemia. All the data indicate that the prior injection of CDP-choline stimulates the choline phosphotransferase reaction of brain towards synthesis of phosphatidylcholine and prevents the release of free fatty acids, particularly of arachidonic acid, associated with ischemia.

The reverse reaction of cholinephosphotransferase in rat brain microsomes a new pathway for degradation of phosphatidylcholine

The synthesis of phosphatidylcholine is catalyzed by cholinephosphotransferase (EC 2.7.8.2) which is known to be reversible in liver. The reversibility of cholinephosphotransferase in rat brain in demonstrated in this paper. Labeled microsomes were prepared from young rats which had been given an intracerebral injection of labeled choline or oleate 2 h before killing. During incubation of choline-labeled microsomes with CMP, label was lost from ;choline glycerophospholipids and labeled CDPcholine was produced. The Km for CMP was 0.35 mM and V was 3.3 nmol/min per mg protein. Neither AMP nor UMP could substitute for CMP. Oleate-labeled microsomes were pretreated with e mM diisopropylfluorophosphate (lipase inhibitor). During incubation with CMP, label was lost from choline, and ethanolamine glycerophospholipid and labeled diacylglycerols were produced. When the lipase was not inhibited, labeled oleate was produced. We propose that a principal pathway for degradation of phosphatidylcholine, particularly during brain ischemia, is by reversal of cholinephosphotransferase, followed by hydrolysis of diacylglycerols by the lipase.

BASE-EXCHANGE ENZYMIC SYSTEM FOR THE SYNTHESIS OF PHOSPHOLIPIDS IN NEURONAL AND GLIAL CELLS AND THEIR SUBFRACTIONS: A POSSIBLE MARKER FOR NEURONAL MEMBRANES

Abstract— The calcium‐dependent incorporation of L‐[3‐14C]serine and [1,2−14C]ethanolamine into the phospholipid of isolated neuronal and glial cells from rabbit brain was studied, and the distribution of the enzymic system among the correspondent subfractions was examined. The neuronal cell‐enriched fraction was found to possess a much higher rate of exchange of both bases than the glial cell‐enriched fraction. Among the sub‐fractions isolated from the neuronal and glial cells, those corresponding to neuronal plasma membranes and microsomes showed a noticeably higher exchange of serine and ethanolamine compared to the corresponding subfractions from glia. Neuronal/glial ratios of about 6–8 were found for the exchange activity in both plasma membrane‐enriched fraction and in microsomes. Synaptosomes and synaptosomal subfractions contained low activities. It is concluded that the calcium‐dependent enzymic system for the exchange of serine, ethanolamine and other nitrogenous bases with endogenous phospholipid is concentrated mostly in the neuronal perikaryal membranes, and could be used as a neuronal marker.

Properties and function of the calcium-dependent incorporation of choline, ethanolamine and serine into the phospholipids of isolated rat brain microsomes.

Abstract— The calcium‐dependent incorporation of choline, ethanolamine and L‐serine into the phospholipids of isolated rat brain microsomes has been studied in vitro, and various properties of the incorporation have have been examined. The optimum pH for the incorporation of each base was found to vary inversely with the Ca2‐ concentration. Conversely, the optimal Ca2 + concentration for the exchange of the bases increased with decreasing pH values. The enzymic system for the incorporation of ethanolamine appeared to be saturated by two substrate concentrations, i.e. 0‐2 and 1‐7‐2‐0 mM. At low ethanolamine concentration (0‐2 mM] much less incorporation of the base occurred into the alkenylacyl‐ and alkylacyl‐derivatives of ethanolamine phosphoglycerides compared to that into the diacyl species, whereas the difference becomes smaller at a high substrate concentration (1‐7 mM). At pH 81 and 2 mM‐Ca2+ the apparent Km of ethanolamine at low substrate concentration was 80 × 10‐5 M, and this value increased to 16‐2 × 10‐4.viat 10mM‐Ca2+ concentration. At similar pH the Km values for choline and L‐serine were 5.88 × 10‐4M and 40 × 10‐4 M at 2 mM‐ and 10mM‐Ca2 + concentrations, respectively. The properties of the enzyme system show differences for the three substrates when various factors are changed during incubation. These and other results indicate that more than one enzyme is probably involved in the Ca2+‐medialed exchange of nitrogenous bases.

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 SYNTHESIS OF CHOLINE AND ETHANOLAMINE PHOSPHOGLYCERIDES IN NEURONAL AND GLIAL CELLS OF RABBIT IN VITRO

Abstract— The de novo synthesis of phosphatidylcholine and phosphatidylethanolamine in isolated neuronal and glial cells from adult rabbit brain cortex was investigated in vitro, using labelled phosphorylcholine (phosphorylethanolamine) or cytidine‐5′‐phosphate choline (cytidine‐5′‐phosphate ethanolamine), as lipid precursors. Synthesis of phospholipid from phosphorylcholine and phosphorylethanolamine in both fractions was extremely low when compared to that derived from the corresponding cytidine nucleotides. The neuronal cell‐enriched fraction was found to possess a much higher rate of synthesis of both lipids from all precursors. Neuronal/glial ratios of about 5–9 were found for the synthesis of phosphatidylcholine and phosphatidylethanolamine from cytidine‐5′‐phosphate choline and cytidine‐5′‐phosphate ethanolamine, respectively. Several kinetic properties of the choline‐phosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) were found to be similar both in neurons and in glia (e.g. Km of cytidine‐5′‐phosphate ethanolamine, Km of diacyl glycerol, pH optimum, need for divalent cations), but the Km value for cytidine‐5′‐phosphate choline in glial cells was much lower (2.3 × 10−4m) than in neurons (1 × 10−3m). The Kmfor cytidine‐5′‐phosphate ethanolamine in both cells was much lower than in whole brain microsomes. It is concluded that the cytidine‐dependent enzymic system for phosphatidylcholine and phosphatidylethanolamine synthesis is concentrated mostly in the neuronal cells, as compared to glia.

The synthesis in vivo of choline and ethanolamine phosphoglycerides in different brain areas during aging.

The biosynthesis of choline and ethanolamine phosphoglycerides was tested in vivo in different brain areas of the rat during aging. Mixtures of [2−3H] glycerol and [Me-14C] choline or [2-3H] glycerol and [2-14H] ethanolamine were injected into lateral ventricle of the brain as lipid precursors and their incorporation into corresponding phospholipid was examined. A significant decrease of synthesis of both phosphoglycerides takes place in cerebral cortex and in the striatum, and is already apparent at 9 months of age with no further decrease or change therafter. No significant change takes place in the cerebellum. The unchanged absorption of injected water-soluble precursors, together with the lack of any significant change of phospholipid/protein ratio in all examined brain areas, suggests that the incorporation of both glycerol and nitrogen bases are affected by aging.

The metabolism of phosphoric esters and of cytidine-diphosphate esters of choline and ethanolamine in the liver

Abstract 1. 1. The labelled phosphate and cyddine-diphosphate esters of choline and ethanol-amine, together with their bases, were incubated with rat liver homogcnate and microsomal membranes, and the transfer of radioactivity into water-soluble and lipid components examined. 2. 2. Very little conversion of choline into betaine and phosphorylcholine, and of ethanol-amine into phosphorylethanolamine, as well as into their intermediates and lipid material, was observed in both preparations. 3. 3. Small transfer of phosphorylcholine to cytidine-diphosphate choline and to lecithin, and of phospnorylcthanolaminc to cytidine-diphosphate cthanolamine and phosphatidylethanolaminc was observed. 4. 4. Despite a considerable breakdown to hydrosoluble products, the two cyddine-diphosphate esters were noticeably converted into corresponding phosphotipid with both homogcnate and microsomes.

Metabolism of Phosphoglycerides

Glycerophospholipids are present in all living organisms and are necessary for membrane assembly and function. Their presence in nervous tissue has been recognized for many years,1 and, with the development of suitable analytical techniques, their metabolism has been amply studied and is being investigated in different aspects in several laboratories.

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.