E. Francescangeli

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.

Properties of PAF-synthesizing phosphocholinetransferase and evidence for lysoPAF acetyltransferase activity in rat brain

Several reports have indicated that platelet-activating factor (PAF) may play a role in the physiopathology of nervous tissue. We previously have demonstrated the presence, in the microsomal fractions of rat brain, of a phosphocholinetransferase which is able to synthesize PAF by thede novo pathway. The presence of dithiothreitol in the medium increases the rate of PAF biosynthesis, whereas it inhibits the synthesis of long-chain alkylacyl- and diacyl-glycerophosphocholines (GPC), including dioctanoyl-GPC. This and other properties, such as pH dependence and thermal stability, indicate that rat brain may have two distinct enzymes for the synthesis of PAF and other choline phospholipids. The affinity of these enzymes for CDPcholine is similar to that reported for other tissues, the Km being 42 μm and 55 μm with alkylacetylglycerol and dioctanoylglycerol as lipid substrates, respectively. The Vmax values were 3.0 and 2.2 nmol/mg prot/min for PAF and dioctanoyl-GPC, respectively. In addition, it was shown that the microsomal fraction of rat brain contains an acetyltransferase which can convert lysoPAF to PAF. Since it has been reported previously that brain tissue possesses phospholipase A2 activity that can hydrolyze alkylacyl-GPC to lysoPAF, we conclude that brain tissue has all enzymic activities for the synthesis of PAF by the “remodeling pathway”. The role of the two routes of PAF biosynthesis in nervous tissue remains to be established.

Enzymic synthesis of ethanolamine plasmalogens through ethanolaminephosphotransferase activity in neurons and glial cells of rabbit in vitro

The de novo synthesis of ethanolamine plasmalogen in isolated neuronal and glial cells from adult rabbit brain cortex was investigated in vitro, using labeled cytidine-5′-diphosphate ethanolamine as lipid precursor. The neuronal cell enriched fraction was found to possess a twofold ethanolaminephosphotransferase activity (EC 2.7.8.1), as compared to the glial fraction. The neuronal/glial ratio was similar both in the absence and in the presence of saturating alkenylacyl glycerol. Under the most favorable conditions, rates of 31 nmoles and 16 nmoles ethanolamine plasmalogen/mg protein/30 min were obtained for neurons and glia, respectively. Several kinetic properties of the phosphotransferase were found to be similar both in neurons and glia, e.g., Km of cytidine-5′-diphosphate ethanolamine, pH optimum, need for divalent cations; the Km value for alkenylacyl glycerol was twofold higher in glia (4 mM) than in neurons (2 mM). The neuronal/glial ratio for the phosphatidylethanolamine synthesizing activity was 2, 4.5, and 6 on using diacyl glycerols prepared from ox heart, ox brain, and soybean, respectively. It is concluded that the cytidine-dependent system for ethanolamine plasmalogen and phosphatidylethanolamine synthesis is concentrated prevalently in the neuronal cells, as compared to glia.

Relative contribution of the de novo and remodelling pathways to the synthesis of platelet-activating factor in brain areas and during ischemia

Two distinct pathways for the synthesis of platelet-activating factor (PAF) have been demonstrated in the nervous tissue. This potent lipid mediator is involved in physiological and pathological processes. The relative contribution of the two pathways to its synthesis during various conditions needs to be defined, thus the activities of the enzymes directly responsible for PAF synthesis, PAF-synthesizing phosphocholinetransferase (PAF-PCT) and lyso-PAF acetlytransferase (lyso-PAF AcT), have been assayed in rat brain areas. The former catalyses the last reaction of the de novo pathway and the latter that of the remodelling one. PAF-PCT activity was always more elevated than that of lyso PAF AcT. No differences were observed among different brain areas when enzyme activities were assayed in their homogenates. In microsomes, the highest PAF-PCT activity was found in cerebellum whereas lyso-PAF AcT activity was greater in cerebellum and in hippocampus than in the other brain areas. The activity of PAF-synthesizing enzymes was also studied in the gerbil during ischemia and reperfusion. After 6 min from bilateral occlusion of the carotid arteries, a significant increase of lyso-PAF AcT activity was observed in the hippocampus. This enzyme activity remained relatively high up to 3 days after reperfusion whereas, in other brain areas it reached basal levels much earlier. Since it has been shown that the PAF levels increase in the brain of animals during ischemia, these results suggest that the remodelling pathway may provide an important contribution to its synthesis particularly in the hippocampus, where a selective neuronal death is observed. In this area during reperfusion, a further contribution to PAF synthesis might be also provided by the de novo pathway.

The metabolism of labelled choline in neuronal and glial cells of the rabbit in vivo.

Abstract— Adult rabbits were injected intraventricularly with [14C]ethanolamine and the incorporation of the base into the phosphatidylethanolamine and ethanolamine plasmalogen (and their water‐soluble precursors) of isolated neuronal and glial cells was investigated. All the radioactivity was incorporated into the base moiety of the ethanolamine lipids for the time intervals examined in both types of cells. In neurons, maximum labelling of the two ethanolamine lipids occurred at 7 h after administration, whereas the highest specific radioactivity for glial phosphatidylethanolamine and ethanolamine plasmalogen was reached at 20 and 36 h, respectively. The two lipids had a faster turnover in neurons than in glia, and in both populations incorporated the base at a faster rate than did whole brain tissue. The maximum incorporation rates for phosphorylethanolamine and CDP‐ethanolamine were reached in both types of cell at about 6 h after administration but the content of radioactivity per unit protein for phosphorylethanolamine was much higher in glial than in neuronal cells. It is concluded that the site of most active synthesis of ethanolamine phospholipids in vivo is the neuronal cell, with a possible transfer of intact lipid molecule to the glial compartment.

Platelet-activating factor and group I metabotropic glutamate receptors interact for full development and maintenance of long-term potentiation in the rat medial vestibular nuclei.

In rat brainstem slices, we investigated the interaction between platelet-activating factor and group I metabotropic glutamate receptors in mediating long-term potentiation within the medial vestibular nuclei. We analysed the N1 field potential wave evoked in the ventral portion of the medial vestibular nuclei by primary vestibular afferent stimulation. The group I metabotropic glutamate receptor antagonist, (R,S)-1-aminoindan-1,5-dicarboxylic acid, prevented long-term potentiation induced by a platelet-activating factor analogue [1-O-hexadecyl-2-O-(methylcarbamyl)-sn-glycero-3-phosphocholine], as well as the full development of potentiation, induced by high-frequency stimulation under the blocking agent for synaptosomal platelet-activating factor receptors (ginkolide B), at drug washout. However, potentiation directly induced by the group I glutamate metabotropic receptor agonist, (R,S)-3,5-dihydroxyphenylglycine, was reduced by ginkolide B. These findings suggest that platelet-activating factor, whether exogenous or released following potentiation induction, exerts its effect through presynaptic group I metabotropic glutamate receptors, mediating the increase of glutamate release. In addition, we found that this mechanism, which led to full potentiation through presynaptic group I metabotropic glutamate receptor activation, was inactivated soon after application of potentiation-inducing stimulus. In fact, the long-lasting block of the platelet-activating factor and metabotropic glutamate receptors prevented the full potentiation development and the induced potentiation progressively declined to null. Moreover, ginkolide B, given when high-frequency-dependent potentiation was established, only reduced it within 5 min after potentiation induction. We conclude that to fully develop vestibular long-term potentiation requires presynaptic events. Platelet-activating factor, released after the activation of postsynaptic mechanisms which induce potentiation, is necessary for coupling postsynaptic and presynaptic phenomena, through the activation of group I metabotropic glutamate receptors, and its action lasts only for a short period. If this coupling does not occur, a full and long-lasting potentiation cannot develop.

Enzymic synthesis of 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamine through ethanolaminephosphotransferase activity in the neuronal and glial cells of rabbit in vitro

The transfer of radioactivity from cytidine-5′-diphosphate ethanolamine into 1-alkyl-2-acyl-sn-glycerophosphorylethanolamine of neuronal and glial cells from adult rabbit brain cortex has been investigated in vitro. The synthesis of 1-alkyl-2-acyl-sn-glycerophosphorylethanolamine in both cell populations was stimulated 23–25-fold by the addition of 6 mM alkylacylglycerol. The neuronal cell-enriched fraction was found to possess/unit protein a 1.7–1.8-fold ethanolaminephosphotransferase activity (EC 2.7.8.1), as compared to the glial fraction, when saturating concentrations (6 mM) of alkylacylglycerols were added in the incubation system. The neuronal/glial ratio was 2.6–2.8 in the absence of lipid acceptor or with low concentrations of alkylacylglycerol. Under most favorable conditions, 6.4 and 3.3. nmoles 1-alkyl-2-acyl-sn-glycerophosphorylethanol-amine/mg protein/30 min was obtained for neurons and glia, respectively. Various kinetic properties of the 1-alkyl-2-acyl-sn-glycerophosphorylethanolamine synthesizing phosphotransferase activity were found to be similar both in neurons and glia.

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.

Properties and regulation of microsomal PAF-synthesizing enzymes in rat brain cortex.

Platelet-activating factor (PAF) is a phospholipid mediator of long-term potentiation, synaptic plasticity and memory formation as well as of the development of brain damage. In brain, PAF is synthesized by two distinct pathways but their relative contribution to its productions, in various physiological and pathological conditions, is not established. We have further investigated on the properties of the two enzymes that catalyze the last step of the de novo or remodeling pathways in rat brain microsomes, PAF-synthesizing phosphocholinetransferase (PAF-PCT) and lysoPAF acetyltransferase (lysoPAF-AT), respectively. The latter enzyme is fully active at μM Ca2+ concentration, inhibited by MgATP and activated by phosphorylation. Because the reversibility of the reaction catalyzed by PAF-PCT, its direction depends on the ratio [CDP-choline]/[CMP] which is related to the energy charge of the cell. These and other properties indicate that the de novo pathway should mainly contribute to PAF synthesis for maintaining its basal levels under physiological conditions. The remodeling pathway should be more involved in the production of PAF during ischemia. During reperfusion, the overproduction of PAF should be the result of the concomitant activation of both pathways.

The effect of CMP on the release of free fatty acids of rat brain in vitro.

With CMP, phosphatidylcholine can be converted to diacylglycerols and CDPcholine by reversal of the cholinephosphotransferase that is normally used for synthesis. Incubation of homogenates of rat brains at pH 8 with 20 mM MgCl2 increased the free fatty acid (FFA) levels 30 to 117%. The FFA levels increased 62 to 212% when 4 mM CMP was included. Diacylglycerols were also produced. Hydrolysis of the diacylglycerols to FFA was markedly inhibited by inclusion of 3 mM diisopropylphosphofluoridate in the incubation mixture. The composition of the fatty acids released by CMP resembles that of phosphatidylcholine except for some polyunsaturated fatty acids. These may have been released from the ethanolamine glycerophospholipids. Most of the CMP-stimulated release of FFA was blocked by inclusion of 1 mM CDPcholine in the incubation mixture. Rat brains were labeled by intracerebral injection of [3H]oleic acid. The labeled oleic acid was released primarily from phosphatidylcholine. Thus, measurements of both mass and radioactivity confirm that the reversal of cholinephosphotransferase followed by diacylglycerol lipase can be an important pathway for the liberation of FFA from phosphatidylcholine.