Incorporation of [3H]ethanolamine into acetylcholine by a human cholinergic neuroblastoma clone

Human neuroblastoma cholinergic LA-N-2 cells were used as an experimental model to test the possibility that the methylation of phosphoethanolamine (PEtn) to phosphocholine (PCho) and free choline (Cho) (Andriamampandry et al. 1989) could contribute to acetylcholine (AcCho) synthesis. LA-N-2 cells were incubated with [³H]Cho for 90 min and 22.7% of the radioactivity was present in PCho, 18.5% in free Cho and 4.8% as AcCho. The ratio of Cho/AcCho, however, was of about 1 after 16 hours of incubation. The incorporation of 10M [³H]ethanolamine (Etn) into MeEtn, PMeEtn, PMe₂Etn and their corresponding phospholipids was reduced in cells incubated in medium containing 7.2M choline as compared to cells incubated in medium devoid of choline indicating that the lack of Cho from the incubation medium stimulated the conversion of PEtn to Cho water soluble derivatives. Incubation of LA-N-2 cells with [³H]Etn led to the labelling of [³H]AcCho. Cultures incubated in parallel with [³H]Cho showed that roughly 10% of [³H]AcCho obtained after 16 hrs of incubation with the Cho label derived from [³H]Etn. The synthesis of Cho and AcCho from Etn may be enhanced after cellular differentiation induced by the growth of the cells in the presence of retinoic acid (RA). The results indicate that the methylation of [³H]Etn and/or of [³H]PEtn may be used by cholinergic neurons as precursor for AcCho.Abbreviations Etn ethanolamine - MeEtn monomethylethanolamine - Me₂Etn dimethylethanolamine - P- phosphoryl - AcCho acetylcholine - Ptd phosphatidyl - LPtd lysophosphatidyl - RA retinoic acid     

Partial purification of two forms of Choline kinase and separation of Choline kinase from sphingosine kinase of rat brain

Choline kinase of rat brain was purified approximately 200,000 fold using acid precipitation, ammonium sulphate fractionation, Q-Sepharose, Octyl-Sepharose and AH-Sepharose chromatography. The ability of this enzyme to catalyze the phosphorylation of choline, ethanolamine (Etn), monomethylethanolamine (MeEtn), dimethylethanolamine (Me₂Etn) and sphingosine was investigated. Choline kinase was separated from sphingosine kinase. The fraction with highly purified choline kinase had four major polypeptides with different molecular masses and possessed activities towards choline, Etn, MeEtn and Me₂Etn. Two forms of choline kinase were obtained when the enzymatically active fractions eluted from the Q-Sepharose column were subjected to a horizontal isoelectrofocusing electrophoresis. One form focused around pH 4.7 and is able to phosphorylate choline, Etn, MeEtn and Me₂Etn. The other form focused around pH 10 and possessed only choline kinase activity. The latter form of choline kinase did not display classical Michaelis-Menten's mechanism but revealed a positive co-operative pattern for two choline binding sites. This form was purified to apparent homogeneity with a approximate molecular mass of 14.4 kDa.Abbreviations Etn ethanolamine - MeEtn N-monomethylethanolamine - Me₂Etn N, N-dimethylethanolamine     

Muscarinic binding sites in a catecholaminergic human neuroblastoma cell line

Tyrosine hydroxylase (TH) a characteristic enzyme activity for the catecholaminergic clonal cell line LA-N-1 and choline acetyltransferase (ChAT) a characteristic enzyme activity for the cholinergic clonal cell line LA-N-2 were previously shown to be increased in these cells exposed to 10^–5 M retinoic acid (RA) as differentiating agent. An investigation of the receptor characteristics suggests a complementarity between the two cell lines. The binding of QNB, a muscarinic ligand, was undetectable with the LA-N-2 cells but was present in the LA-N-1 cells and possessed a kD of 1.8 nM and 2.2 nM and a B_max of 0.56 and 0.68 for control and RA grown cells respectively. There was a gradual increase in QNB binding to LA-N-1 cells from 2 days in vitro (DIV) until 6 DIV in both control and RA grown cells. An IC₅₀ of 2.5×10^–8 M and 0.9×10^–8 M for atropine inhibition was obtained for the control and RA grown cells respectively. The corresponding values for carbachol inhibition were 7×10^–2 M and 3×10^–2 M respectively. The inhibition by the agonist oxotremorine is comparable to that of carbachol and 1 mM pilocarpine inhibited the binding by 21%. QNB binding showed a low affinity for pirenzepine and for AF-DX-116 but was inhibited with a rather high affinity by 4-DAMP (IC₅₀:110 M) thus suggesting the presence of an M₃ receptor. Acetylcholine (100 M) plus eserine (50 M) and BW284c55 (1 M), an acetylcholinesterase inhibitor, reduced the binding of QNB by approximately 25%. Nicotine (1 mM) caused a 36% reduction of binding and hemicholinium-3 (HC-3) (1 M), an inhibitor of choline uptake, inhibited the binding by 53%. There was a down regulation of QNB binding observed with cells grown for 24 hours with either the antagonist atropine or the agonists carbachol or oxotremorine. Low amounts of -bungarotoxin (-BgTx) binding sites were barely detectable in both LA-N-1 and LA-N-2 cells. The LA-N-1 muscarinic receptor is coupled to polyphosphoinositide hydrolysis without increased cyclic AMP formation further suggesting its being an M₃ receptor.     

Partial purification of a phosphoethanolamine methyltransferase from rat brain cytosol

The conversion of phosphoethanolamine to phosphocholine requires 3 separate N-methyltransferases. We had previously purified the enzyme catalyzing the last methylation, phosphodimethylethanolamine N-methyltransferase. We have successfully purified the enzyme catalyzing the initial methylation of phosphoethanolamine. A 434 fold purified enzyme from rat brain was obtained by the sequential use of ammonium sulfate fractionation, Q-Sepharose fast flow column chromatography and a -aminoethyl agarose column chromatography. The pH optimum was 11 or greater, the Km value for phosphoethanolamine was 167.8±41.7 M and the Vmax was 487.3±85 mmoles/mg/hr. The kinetics for S-adenosyl-methionine, the methyldonor, has characteristics of cooperative binding with a Km of 1.805±0.59 mM and a Vmax of 16.9±3.6 moles/mg/hr. The activity was stimulated 6 fold by 2.5 mM MnCl₂ and inhibited by DZA and S-adenosylhomocysteine. These results reinforce the early in vivo observations which had provided suggestive evidence for the existence of a pathway for the methylation of phosphoethanolamine to phosphocholine in rat brain.Abbreviations used Adomet S-adenosylmethionine - AdoHcy S-adenosyl-homocysteine - CAPS 3-(cyclohexyl)amino-1-propanesulphonic acid - Cho choline - 3-DZA 3-deazaadenosine - Etn ethanolamine - N-MT N-methyltransferase - PEG polyethyleneglycol - PMSF phenylmethanesulphonyl fluoride - PEtn phosphoethanolamine - PCho phosphocholine - PMe₂Etn phosphodimethylethanolamine - PtdCho phosphatidylcholine - PtdEtn phosphatidylethanolamine     

Effect of Retinoic Acid on the Ca2+-Independent Phospholipase A2 in Nuclei of LA-N-1 Neuroblastoma Cells

LA-N-1 neuroblastoma cell cultures contain Ca²⁺-independent phospholipases A₂ hydrolyzing phosphatidylethanolamine and ethanolamine plasmalogens. These enzymes differ from each other in their molecular mass, substrate specificity, and kinetic properties. Subcellular distribution studies have indicated that the activity of these phospholipases is not only localized in the cytosol but also in non-nuclear membranes and in nuclei. The treatment of LA-N-1 neuroblastoma cell cultures with retinoic acid results in a marked stimulation of Ca²⁺-independent phospholipases A₂ hydrolyzing phosphatidylethanolamine and plasmenylethanolamine. The increase of the activities of both enzymes was first observed in nuclei followed by those present in the cytosol. No effect of retinoic acid on either phospholipase activity could be observed in non-nuclear membranes. The stimulation of these enzymes may be involved in the generation and regulation of arachidonic acid and its metabolites during differentiation.     

Effect of Monomethylethanolamine, Dimethylethanolamine, Gangliosides, Isoproterenol, and 2-Hydroxyethylhydrazine on the Conversion of Ethanolamine to Methylated Products by Cultured Chick Brain Neurons

The sequential methylation of ethanolamine and its phosphorylated derivatives has been studied with chick neurons in culture in the presence of several pharmacological agents. Incubation with [3H]ethanolamine in the presence of monomethylethanolamine and dimethylethanolamine indicated that in these neurons the preferential conversion to choline-containing compounds is via the methylation of phosphorylethanolamine. The possibility that there are two separate enzymes, i.e., one responsible for the methylation of water-soluble ethanolamine-containing compounds and another for the ethanolamine phospholipids, was examined with agents believed to influence these conversions. Incubation of neurons in the presence of a mixture of exogenous gangliosides at 10(-8) M and 10(-5) M concentrations showed that these neuritogenic compounds stimulate the methylation of phosphatidylethanolamine and decrease that of phosphorylethanolamine. The inhibitor of phosphatidylethanolamine methyltransferase (EC 2.1.1.17), 2-hydroxyethylhydrazine, decreased the conversion of phosphatidylethanolamine to phosphatidylcholine and increased that of phosphorylethanolamine to phosphorylcholine. The possible effects of adrenergic stimulation were studied by the incubation of neurons with isoproterenol at 10(-6) M and 10(-5) M concentrations. There was a reduction of phosphorylethanolamine methylation and a stimulation of that of phosphatidylethanolamine, and these effects were counteracted by the presence of 5 x 10(-5) M propranolol.     

Phosphatidylcholine Metabolism in Nuclei of Phorbol Ester-Activated LA-N-1 Neuroblastoma Cells

The agonist stimulation of a variety of cells results in the induction of specific lipid metabolism in nuclear membranes, supporting the hypothesis of an important role of the lipids in nuclear signal transduction. While the existence of a phosphatidylinositol cycle has been reported in cellular nuclei, little attention has been given to the metabolism of phosphatidylcholine in nuclear signaling. In the present study the metabolism of phosphatidylcholine in the nuclei of neuro-blastoma cells LA-N-1 was investigated. The incubation of LA-N-1 nuclei with radioactive choline, phosphocholine or CDP-choline led to the production of labelled phosphatidylcholine. The incorporation of choline and phosphocholine but not CDP-choline was enhanced in nuclei of TPA treated cells. Moreover the presence of choline kinase, phosphocholine cytidylyltransferase and phosphocholine transferase activities were detected in the nuclei and the TPA treatment of the cells stimulated the activity of the phosphocholine cytidylyltransferase. When cells prelabelled with [³H]palmitic acid were stimulated with TPA in the presence of ethanol, an increase of labelled diacylglycerol and phosphatidylethanol in the nuclei was observed. Similarly, an increase of labelled diacylglycerol and phosphatidic acid but not of phosphatidylethanol occurred in [³H]palmitic acid prelabelled nuclei stimulated with TPA in the presence of ethanol. However the production of phosphatidylethanol was observed when the nuclei were treated with TPA in the presence of ATP and GTPS. The stimulation of [³H]choline prelabelled nuclei with TPA also generated the release of free choline and phosphocholine. The results indicate the presence of PLD and probably PLC activities in LA-N-1 nuclei and the involvement of phosphatidylcholine in the production of nuclear lipid second messengers upon TPA stimulation of LA-N-1 cells. The correlation of the disappearance of phosphatidylcholine, the production of diacylglycerol and phosphatidic acid with the stimulation of phosphatidylcholine synthesis in nuclei of TPA treated LA-N-1 suggests the existence of a phosphatidylcholine cycle in these nuclei.     

Phospholipid biosynthesis in the anaerobic protozoon Entodinium caudatum.

1. The anaerobic rumen protozoon Entodinium caudatum was incubated either intact or with various radioactive precursors of phospholipids after ultrasonication. 2. Pulse-chase experiments showed a rapid turnover of phosphatidylinositol and much slower turnovers of phosphatidylethanolamine and phosphatidylcholine. 3. E. caudatum imbibed choline very rapidly; this was immediately and exclusively converted into phosphatidylcholine which was shown by radioautography after 10 min to be distributed throughout the cell membranes. 4. Phosphatidylcholine was synthesized through a phosphorylcholine-CDP-choline pathway, the methylation or base-exchange pathways not being present. 5. Under suitable conditions [Me-14C]choline can be substantially (50-60%) converted into CDP-choline by sonicated E. caudatum and this provides an excellent method of preparing this biosynthetic intermediary. 6. [2-14C]Ethanolamine was taken up much less readily than choline. The former was incorporated into phosphatidylethanolamine by the CDP-ethanolamine pathway. 7. Doubly labelled [32P]phosphatidyl[2-3H]ethanolamine was converted into ceramide phosphorylethanolamine and N-(1-carboxyethyl)phosphatidyl-ethanolamine, without change in the isotopic ratio. Ceramide phosphoryl [2-14C]-ethanolamine was converted into phsophatidylethanolamine. 8. Palmitic acid, oleic acid and linoleic acid were taken by E. caudatum cells and incorporated into phospholipids. By contrast, although stearic acid was taken up it was hardly incorporated into phospholipids.     

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. K_m of cytidine-5′-phosphate ethanolamine, K_m of diacyl glycerol, pH optimum, need for divalent cations), but the K_m value for cytidine-5′-phosphate choline in glial cells was much lower (2.3 × 10^?4m ) than in neurons (1 × 10^?3m ). The K_mfor 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.     

Plants synthesize ethanolamine by direct decarboxylation of serine using a pyridoxal phosphate enzyme.

The established pathways from serine to ethanolamine are indirect and involve decarboxylation of phosphatidylserine. Here we show that plants can decarboxylate serine directly. Using a radioassay based on ethanolamine (Etn) formation, pyridoxal 5'-phosphate-dependent l-serine decarboxylase (SDC) activity was readily detected in soluble extracts from leaves of diverse species, including spinach, Arabidopsis, and rapeseed. A putative Arabidopsis SDC cDNA was identified by searching GenBank for sequences homologous to other amino acid decarboxylases and shown by expression in Escherichia coli to encode a soluble protein with SDC activity. This cDNA was further authenticated by complementing the Etn requirement of a yeast psd1 psd2 mutant. In a parallel approach, a cDNA was isolated from a rapeseed library by its ability to complement the Etn requirement of a yeast cho1 mutant and shown by expression in E. coli to specify SDC. The deduced Arabidopsis and rapeseed SDC polypeptides are 90% identical, lack obvious targeting signals, and belong to amino acid decarboxylase group II. Recombinant Arabidopsis SDC was shown to exist as a tetramer and to contain pyridoxal 5'-phosphate. It does not attack d-serine, l-phosphoserine, other l-amino acids, or phosphatidylserine and is not inhibited by Etn, choline, or their phosphoesters. As a soluble, pyridoxal 5'-phosphate enzyme, SDC contrasts sharply with phosphatidylserine decarboxylases, which are membrane proteins that have a pyruvoyl cofactor.     

Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemispheres in culture. I. Uptake and phosphorylation of [U-14C]ethanoloamine and the effect of various inhibitors.

Cultured dissociated cells from rat embryo cerebral hemispheres were incubated with [U-14C]ethanolamine and the resulting cellular labeled products were identified. A highly efficient uptake for ethanolamine with a Km of approximately 8.3 muM was calculated. A rapid labeling of phosphorylethanolamine was observed prior to the appearance of label in lipids. A lag period of 2.5 min for the phosphorylation reaction was observed, followed by an almost linear rate for up to 40 min. After a 1-min incubation, a plateau for free ethanolamine taken up by the cells was established. Respiratory inhibitiors such as cyanide, 2,4-dinitrophenol, and N-ethylmaleimide decreased by the formation of phosphorylethanolamine. However, the amount offree ethanolamine present in the cells increased 1.6-fold after 10 min of incubation with N-ethylmaleimide. 2-Chloroethylamine, a structural analog of ethanolamine, and choline were both competitive inhibitiors with an apparent Ki of 0.1 mM and 0.36 mM, respectively. Incubations of short duration suggest that both compounds affect ethanolamine transport into the cells. Based on these studies it is suggested that ethanolamine transport and the phosphorylation reaction are independent events. Evidence based on studies with hemicholinium-3 and chloroethylamine suggest that ethanolamine uptake may proceed by a pathway independent of either choline or serine uptake.     

Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line

The ability of retinoic acid (RA) to modulate acetylcholinesterase (AChE) activity in a human neuroblastoma cell line (LN-N-5) was examined. The specific activity of AChE was significantly increased 3 days after exposure of LA-N-5 to RA and reached its maximum values after 9 or more days of culturing. Dose-response experiments demonstrated that large increases of AChE occurred at RA concentrations between 10(-7) and 10(-6) M with maximum AChE values detected at 10(-6)-10(-5) M. Increased AChE activity paralleled neurite outgrowth in LA-N-5 cultures. These findings demonstrate that RA can regulate specific AChE activity in human neuroblastoma cells in a manner consistent with neuronal maturation.     

Localization of neuropeptides in efferent terminals of the eye in the marine snail,Bulla gouldiana

Summary Retinoic acid (RA), a naturally occurring metabolite of vitamin A, increased the number of receptors for nerve growth factor (NGF) in cultured human neuroblastoma cells (LA-N-1), as indicated by an immunofluorescence assay of cell surface receptors and by specific binding of ¹²⁵I-NGF to solubilized receptors. Analysis of ¹²⁵I-NGF binding showed that RA increased the number of both high affinity and low affinity receptors for NGF without affecting the equilibrium dissociation constants. Neurite outgrowth similar to that produced by NGF occurred following RA-treatment in LA-N-1 cells, in the SY5Y subclone of SK-N-SH human neuroblastoma cells and in explanted chick dorsal root ganglia (DRG). Whether morphological changes following RA treatment are directly related to the increase in NGF receptors is unknown. Data presented here are consistent with literature reports that RA modifies cell surface glycoproteins, including those that act as cell surface receptors for epidermal growth factor and insulin.Abbreviations DRG dorsal root ganglia - NGF nerve growth factor - RA retinoic acid     

Evidence from engineering that decarboxylation of free serine is the major source of ethanolamine moieties in plants

Plants form ethanolamine (Etn) moieties by decarboxylating serine or phosphatidylserine (PtdSer), and use them to make phosphatidylethanolamine, phosphatidylcholine, choline, and glycine betaine. Serine decarboxylation is mediated by a serine decarboxylase (SDC) that is unique to plants and has a characteristic N-terminal extension. This extension was shown to have little influence on function of the enzyme in vitro. To explore the importance of SDC and its extension in vivo, native or truncated versions of the Arabidopsis enzyme were expressed in tobacco. Transgene expression increased SDC activity by up to 10-fold and free Etn level up to 6-fold, but did not change levels of serine, choline, phosphocholine, or phosphatidyl bases. The truncated enzyme gave significantly higher Etn levels. These results show that SDC activity exerts substantial control over flux to Etn, and suggest that the enzyme's N-terminus may have a regulatory role. In complementary studies with Arabidopsis, we showed that a mutant with 9-fold elevated mitochondrial PtdSer decarboxylase activity had normal pools of serine, Etn, and Etn metabolites. Taken together, these data indicate that serine decarboxylation is the main source of Etn moieties in plants. The ability to enhance serine --> Etn flux should advance engineering of choline and glycine betaine accumulation.     

Tetracycline resistance element of pBR322 mediates potassium transport

High concentrations of choline and phosphorylcholine blocked the adsorption of pneumococcal autolytic enzyme to homologous cell walls and inhibited enzymatic cell wall hydrolysis in a noncompetitive manner. Enzyme adsorption had an absolute requirement for the presence of choline residues in the wall teichoic acid. Other amino alcohols and derivatives such as ethanolamine, monomethylaminoethanolamine , and phosphorylethanolamine had no effect on enzyme adsorption or hydrolytic activity. It is proposed that enzymatic hydrolysis of cell walls requires prior adsorption of enzyme molecules to the insoluble wall substrate and that cholin residues of the wall teichoic acid have the role of adsorption ligands in this process.     

Lipid metabolism in T47D human breast cancer cells: 31P and 13C-NMR studies of choline and ethanolamine uptake.

31P and 13C-NMR were used to determine the kinetics of choline and ethanolamine incorporation in T47D clone 11 human breast cancer cells grown as small (150 microns) spheroids. Spheroids were perfused inside the spectrometer with 1,2-13C-labeled choline or 1,2-13C-labeled ethanolamine (0.028 mM) and the buildup of labeled phosphoryl-choline (PC) or phosphorylethanolamine (PE) was monitored. Alternatively the PC and GPC pools were prelabeled with 13C and the reduction of label was monitored. 31P spectra were recorded from which the overall energetic status as well as total pool sizes could be determined. The ATP content was 8 +/- 1 fmol/cell, and the total PC and PE pool sizes were 16 and 14 fmol/cell, respectively. PC either increased by 50% over 24 h or remained constant, while PE remained constant in medium without added ethanolamine but increased 2-fold within 30 h in medium containing ethanolamine, indicating a dependence on precursor concentration in the medium. The 31P and 13C data yielded similar kinetic results: the rate of the enzymes phosphocholine kinase and phosphoethanolamine kinase were both on the order of 1.0 fmol/cell per h, and the rate constants for CTP:phosphocholine cytidyltransferase and CTP:phosphoethanolamine kinase were 0.06 h-1 for both enzymes. The kinetics of choline incorporation did not alter in the presence of 0.028 mM ethanolamine indicating that they have non-competing pathways.     

Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemipheres in culture. II. Incorporation of [U-14C]ethanolamine into 1-alkenyl,2-acyl-and 1,2 diacyl-ethanolamine phosphoglycerides.

Cultured dissociated cells from rat embryo cerebral hemisphere incorporate [3H]-and [U-14C]ethanolamine into cellular lipids. Nearly all radioactivity in the lipid fractions is incorporated into 1,2-diacylethanolamine phosphoglycerides and 1-alkenyl,2-acylethanolamine phosphoglycerides (plasmalogen). Kinetic data suggest that the rate of labeling of both ethanolamine phospholipids from the phosphorylethanolamine is similar. A relative increase of the plasmalogen labeling is observed when free ethanolamine is continually present in the medium. The rate of incorporation of label from ethanolamine and phosphorylethanolamine into lipids was measured using a double label technique. Based upon these studies, an independent labeling pattern of the ethanolamine moiety of plasmalogens is suggested. A relative delay for the incorporation of label in plasmalogens could be explained by the presence of a variety of cell types which may differ in their capacity for phospholipid biosynthesis. The rate of incorporation of phosphorylethanolamine into the phosphatidylethanolamine was not affected by the presence of high concentrations of either choline or serine.     

Studies of Myo-inositol and Plasmalogen Metabolism in Rat Brain

Plasmalogens are ether-linked phospholipids that are abundant in nervous tissues. Their biological role is unclear, but may involve membrane structure/function and antioxidant activities. This study further investigates a recent report that chronic administration of myo-inositol in rats increased brain phosphatidylethanolamine plasmalogen (PlsEtn). We examined the effects of myo-inositol administration on the incorporation of [2-(13)C]ethanolamine ([2-(13)C]Etn) into rat brain phospholipids using NMR spectroscopy. Rats received either acute myo-inositol (single dose) +/- [2-(13)C]Etn, or chronic myo-inositol (10-day treatment) + [2-(13)C]Etn. Controls received saline rather than myo-inositol. Acute myo-inositol produced a 68% increase in brain [myo-inositol] and an increase in the incorporation of [2-(13)C]Etn into phospholipids (P < .05). The PlsEtn/phosphatidylethanolamine ratio and the [PlsEtn] were increased by 27% and 30%, respectively. The PlsEtn content as a mole percentage of total phospholipids was elevated (P < or = .05). Acute administration of myo-inositol + ethanolamine illustrates a positive correlation between the brain [myo-inositol] and the biosynthesis of ethanolamine phospholipids, with preferential synthesis of PlsEtn.