THE METABOLISM OF LABELLED ETHANOLAMINE IN NEURONAL AND GLIAL CELLS OF THE RABBIT IN VIVO1

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.     

Quantitative evaluation of two pathways for phosphatidylcholine biosynthesis in rat brain in vivo

[Me-³H] choline and [³²P] orthophosphate were injected intraventricularly into adult female rats. After variable intervals from injection (1–10 min) the animals were sacrificed by means of a microwave apparatus, and phosphorylcholine and choline phosphoglycerides extracted from brain and counted for radioactivity content after separation. The kinetic constants (K) for phosphorylcholine incorporation into lipids were determined both for [³²P] and [³H] labeling. From the data obtained by these procedures it is concluded that base-exchange reactions for choline incorporation into lipids are operating in rat brain in vivo and that they represent a rapidly equilibrating system.     

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.     

Axonal transport of phospholipids in rat visual system.

Abstract— Seventeen day old rats were injected intraocularly with a phospholipid precursor, [³²P]phosphate, and a glycoprotein precursor, [³H]fucose. Animals were killed between 1 h and 21 days later, and structures of the visual pathway (retina, optic nerve, optic tract, lateral geniculate body, and superior colliculus) were dissected. Radioactivity in phospholipids ([³²P] in solvent-extracted material) and in glycoproteins ([³H] in solvent-extracted residue) was determined. Incorporation of [³H]fucose into retinal glycoproteins peaked at 6–8 h. Labelled glycoproteins were present in superior colliculus by 2h after injection, indicating a rapid rate of transport; maximal labelling was at 8–10 h after injection. Incorporation of [³²P]phosphate into retinal phospholipids peaked at 1 day after injection. Phospholipids were also rapidly transported since label was present in the superior colliculus by 3 h after injection: however, maximal labelling did not occur until 5–6 days. These results indicate that newly synthesized phospholipids enter a preexisting pool, part of which is later committed to transport at a rapid rate. Transported phospholipids were catabolized at the nerve endings with a maximum half-life of several days; there was minimal recycling of precursor label. Lipids were fractionated by thin-layer chromatography, and radioactivity in individual phospholipid classes determined. Choline and ethanolamine phosphoglycerides were the major transported phospholipids, together accounting for approx 85% of the total transported lipid radioactivity. At early time points, the ratio of radioactivity in choline phosphoglycerides to that in ethanolamine phosphoglycerides increased in structures progressively removed from the site of synthesis (retina) but by 2 days approached a constant value. In each structure, choline phosphoglyceride-ethanolamine phosphoglyceride radioactivity ratios decreased with time, rapidly at first, but plateaued by 2 days. These results indicate that choline phosphoglycerides are committed to transport sooner than ethanolamine phosphoglycerides. Some experiments were also conducted using [2-³H]glycerol as a phospholipid precursor. Results concerning incorporation of this precursor into individual phospholipid classes and their subsequent axonal transport were comparable to those obtained using [³²P]phosphate, with the following exceptions: (a) incorporation of [2-³H]glycerol into retinal phospholipids was relatively rapid (near-maximal levels at 1 h after injection) although transport to the superior colliculus showed an extended time course very similar to [³²P]-labelled lipids; (b) [2-³H]glycerol was somewhat less efficient than [³²P]phosphate in labelling lipids committed to transport relative to labelling those which remained in the retina; and (c) [2-³H]glycerol did not label plasmalogens.     

The calcium-stimulated incorporation of ethanolamine and serine into the phospholipids of the housefly Musca domestica

1. The calcium-stimulated incorporation of [2-¹⁴C]ethanolamine and l-[3-¹⁴C]-serine into the phospholipids of homogenates of the fat bodies from larval houseflies (Musca domestica) was studied. 2. Ethanolamine and serine acted as competitive inhibitors with one another. N-Methylethanolamine was not distinguished from ethanolamine by the system. Tris buffer was also a competitor with these compounds, and a number of other amino alcohols were inhibitory, probably competitively. 3. The incorporation of [³²P]phosphorylethanolamine into phospholipids was observed in suspensions of whole fat bodies. This incorporation was stimulated by magnesium. 4. During the incubation of the homogenates, a calcium-stimulated breakdown of phospholipids by a phospholipase A occurred. 5. These results are compared with results published for similar mammalian systems, and their possible physiological significance is discussed.     

Formation of ceramide phosphorylethanolamine from phosphatidylethanolamine in the rumen protozoon Entodinium caudatum (Short Communication)

From `pulse'-labelling experiments of Entodinium caudatum with [¹⁴C]ethanolamine and by incubating the organism with [³²P]phosphatidylethanolamine it is concluded that phosphatidylethanolamine can act as a direct precursor of the phosphorylethanolamine moiety of ceramide phosphorylethanolamine. The phosphorylethanolamine is probably never liberated in the free form but is transferred directly to a ceramide or ceramide-containing acceptor. The results are also in agreement with previous conclusions that phosphatidylethanolamine is the direct lipid precursor of N-(1-carboxyethyl)phosphatidylethanolamine.     

Lipid synthesis by oligodendrocytes from adult pig brain maintained in long-term culture

Oligodendrocytes were isolated from adult pig brain and cultivated for 18–24 days. [¹⁴C]acetate, [³H]galactose or [³⁵S]sulfate were added to the medium for an additional 24 h. Lipids were extracted and separated by high-performance thin-layer chromatography. The labeled lipids were studied by fluorography and scintillation counting. [¹⁴C]acetate was incorporated in decreasing order into neutral lipids, phosphatidylcholine, ethanolamine phosphatides, galactocerebrosides, phosphatidylinositol, phosphatidylserine, sulfatides and sphingomyelin. From the [¹⁴C]acetate incorporated into ethanolamine and choline phosphatides, 71.6 and 14.8%, respectively, were found in plasmalogens. Among neutral lipids, [¹⁴C]acetate labeled not only cholesterol but also large amounts of triglycerides. No cholesterol esters were synthesized. [³H]galactose primarily labeled galactocerebrosides, sulfatides, and monogalactosyl diglyceride. [³⁵S]sulfate incorporation was restricted to sulfatides. Together with our previous results concerning proteins, these data show that: (1) oligodendrocytes remain highly differentiated in long-term cultures; (2) they are able to synthesize the major components of myelin; (3) they synthesize surprisingly high amounts of triglycerides and of monogalactosyl diglyceride, a marker for myelination.     

Ethanol inhibits phosphatidylcholine and phosphatidylethanolamine biosynthesis in human leukemic monocyte-like U937 cells

The effect of ethanol (ETOH) on the incorporation of [¹⁴C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [¹⁴C] 18 : 1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (⩽ 1·0 per cent; v/v) had no effect. At concentrations greater than 1·5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased. The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [³H]choline or [¹⁴C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (≥ 1·5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40–50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [¹⁴C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18 : 1 and PL-base, the ratios of incorporation (base/18 : 1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [¹⁴C] ethanolamine into [¹⁴C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway.     

Inositol-containing lipids in suspension-cultured plant cells: an isotopic study

Polar lipids were extracted from suspension-cultured tomato (Lycopersicon esculentum Mill.) cells and analyzed by thin layer chromatography. Four major inositol-containing compounds were found, and incorporation of [³²P]orthosphosphate, [2-³H]glycerol, and myo-[2-³H]inositol was studied. Results showed that phosphatidylinositol-monophosphate is the phospholipid in these cells displaying the most rapid incorporation of [³²P]orthophosphate. We suggest that the tracer is incorporated primarily into the phosphomonoester group. Two inositol-containing lipids showed chromatographic behavior similar to phosphatidylinositol-4,5-bisphosphate when using standard thin layer chromatography techniques. The labeling pattern of these compounds, however, reveals that it is unlikely that either of these is identical to phosphatidylinositol-4,5-bisphosphate. Should phosphatidylinositol-bisphosphate be present in suspension cultured plant cells, our data indicate chemical abundancies substantially lower than previously reported.     

Axonal transport of glycerophospholipids following intracerebral injection of glycerol into substantia nigra or lateral geniculate body

[2-³H]Glycerol was injected into one substantia nigra of adult rats. Incorporation of radioactivity into lipids at the injection site was maximal by 2 hr, after which it declined. Rapidly transported³H-labeled lipids were just beginning to accumulate in the primary projection site, the ipsilateral corpus striatum by 2 hr, as evidenced by 20-fold higher levels of lipid radioactivity in the projection site relative to control regions. However, the bulk of labeled lipid arrived between 6 hr and 3 days postinjection, suggesting either a prolonged period of release of rapidly transported lipids from the nerve cell bodies or a slow rate of transport for the later arriving lipids. Colchicine applied locally to the fibers of this tract blocked the axonal transport of lipids to the striatum almost completely. Choline and ethanolamine phosphoglycerides were the major transported lipids, accounting for approximately 60% and 25%, respectively, of the total. Similar results were obtained in studies of [2-³H]glycerol-labeled lipids synthesized in the lateral geniculate body and transported to the visual cortex. The rapid axonal transport of lipids labeled with [³²P]phosphate (injected simultaneously with [2-³H]glycerol) could also be demonstrated in both tracts. However, in contrast to [2-³H]glycerol, considerable amounts of³²P soluble label were present in the projection sites, and colchicine only partially blocked the accumulation of³²P-labeled lipid. These results demonstrate the relative utility of [2-³H]glycerol as a lipid precursor for examination of axonal transport in intrabrain tracts. Characteristics of lipid axonal transport in these two intrabrain tracts are similar to each other and are also similar to those previously described for retinal ganglion cells, indicating a common requirement for the axonal transport of these membrane constituents to axons and nerve endings in widely divergent CNS tracts.Presented in part at the 11th meeting of the American Society for Neurochemistry, Houston, Texas, March 1980.     

Studies on the preferential incorporation of [3H]glycerol over [32P]phosphate into major phospholipids of human platelets

It is well known that platelets readily incorporate radioactive glycerol, but not radioactive phosphate into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in vitro, thus not in accordance with de novo synthesis according to the Kennedy pathway. In attempts to understand the reason for the discrepancy, gel-filtered platelets were incubated simultaneously with [32P]Pi and [3H]glycerol, and the specific and relative radioactivities of products and intermediates were determined. Both precursors were incorporated into phosphatidylinositol (PI) with a 32P/3H ratio similar to that in glycerol 3-phosphate (in accordance with the Kennedy pathway). However, PC and PE obtained a much lower ratio. The specific 32P radioactivity in phosphorylcholine was similar to that of the gamma-phosphoryl of ATP and 650-times higher than that of PC. The specific 32P radioactivity of phosphorylethanolamine was 20-times less than that of phosphorylcholine. Both mass and 32P labelling of CDP-choline were below the detection limits. It is concluded that the incorporation of [32P]Pi into PC via phosphorylcholine is insignificant while the preferential incorporation of [3H]glycerol could be explained by exchange of diacyl[3H]glycerol in the reversible choline phosphotransferase (CDP-choline: 1,2-diacylglycerol cholinephosphotransferase) reaction. The same mechanism would explain the preferential incorporation of 3H over 32P into PE, although dilution of 32P at the phosphorylethanolamine stage would account for part of the feeble 32P incorporation. Although other mechanisms are also possible, our results clearly show that the appearance of [3H]glycerol in PC and PE is not a reliable method of monitoring de novo synthesis of these phospholipids.     

Differential patterns of mitochondrial,chloroplastic and nuclear DNA synthesis in the synchronous cell cycle of Chlamydomonas reinhardtii

Nuclear DNA (ncDNA) synthesis in Chlamydomonas reinhardtii was measured by both ³²P[or-thophosphoric acid] (³²P) and [¹⁴C]adenine incorporation and found to be highly synchronous. Ca. 85% of incorporation was confined to the first 6 h of the dark period of a synchronized regime consisting of an alternating light-dark period of 12 h each. In contrast, no such synchronous incorporation pattern was found for chloroplast (cp) and mitochondrial (mt) DNAs in the same cell population. These two organellar DNAs also exhibited different ³²P-incorporation patterns in the cell cycle. Considerable amounts of ³²P were incorporated into cpDNA throughout the light-dark synchronous cycle under both mixo- and phototrophic growth conditions, although the second 6-h light period under phototrophy showed an increase not apparent under mixotrophy. This change in growth conditions did not affect ³²P incorporation into mtDNA, which was found throughout the cell cycle, with a modest peak in the first 6-h of the dark period. The pattern of [³H]thymidine incorporation into cpDNA was also determined. Under synchronous phototrophic conditions, this pattern was quite different from that obtained with ³²P. Most [³H]thymidine incorporation occurred during the light period of the synchronous cycle; this period had been shown previously by density transfer experiments to be the time of cpDNA duplication. Such preferential [³H]thymidine incorporation into cpDNA in the light period was not observed under mixotrophic synchronous growth conditions; in these, [³H]thymidine incorporation was detected throughout the cell cycle. This lack of coincidence between the patterns of ³²P- and of [³H]thymidine incorporation into cpDNA during the synchronous cell cycle indicates that in addition to replication, the considerably reiterated organelle-DNA molecules may also regularly undergo an extensive repair process during each cell cycle.     

Incorporation of [14C] ethanolamine and [3H] Methionine into phospholipids of rat brain and liver in vivo and in vitro

Comparative studies were undertaken on the in vivo and in vitro incorporation of [¹⁴C] ethanolamine, [³H] methionine and [¹⁴C] S-adenosyl-methionine into phosphatidylethanolamine (PhE) and phosphatidylcholine (PhC) of rat liver and brain. It was observed that brain can synthesize de novo PhC from PhE via the transmethylation pathway, however synthesis rates were (1) markedly lower than those of liver and (2) decreased significantly with age. In the choline-containing lipids more than 95% of the radioactivity was found in PhC. Studies on the localization of the radioactivity in PhC following the intracranial injection of [³H] methionine or [¹⁴C] ethanolamine revealed that both precursors are incorporated almost exclusively into the choline moiety of this phospholipid. There was significant labeling of PhC only when the precursors were administered intracranially and much less incorporation was observed with the systemic routes. Thus following the intravenous administration of [¹⁴C] ethanolamine, the specific radioactivities of liver PhE and PhC were up to 75 times as high as those of brain and 4 to 5 times as high in the organs of the 20-day old as those of the adult. In contrast, when this precursor was administered intracranially the specific radioactivities of both phospholipids in liver were only twice as high as those of brain. Although the short-and long-term time-course studies on the in vivo incorporation of [¹⁴C] ethanolamine and [³H] methionine into PhC of both organs could suggest a precursor-product relationship between the biosynthesis of this phospholipid in liver and brain, this apparent relationship could also be due to the high turnover of PhE in liver, with half-life of 2.87 hr, and its low turnover in brain, with half-life of 10.7 days. The present findings on the low rate of formation of PhC from PhE in brain coupled with the fact that this conversion declines sharply with age, especially when the isotopes are administered systemically, could explain the observation of previous investigators that the brain cannot synthesize its own choline and thus it must derive its choline from exogenous sources such as lipid-choline. It was concluded that the brain can synthesize its own choline; however it remains also dependent on liver and dietary choline which are probably transported into the brain as free choline.     

Effect of calmodulin antagonists on lysosomal enzyme secretion and phospholipid metabolism in guinea-pig macrophages

The effects of calmodulin antagonists on the secretion of lysosomal enzyme and lipid metabolism in guinea-pig peritoneal macrophages were studied. Calmodulin antagonists, such as trifluoperazine, dibucaine and quinacrine, inhibited the secretion of N-acetyl-β-d-glucosaminidase from cytochalasin B-treated macrophages when the macrophages were stimulated by the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (f Met-Leu-Phe) or the Ca²⁺ ionophore A23187. The effect of calmodulin antagonists on the incorporation of [³²P]P_i or [³H]glycerol into glycerolipids as well as on the redistribution of [¹⁴C]glycerol or [³H]arachidonic acid in [¹⁴C]glycerol- or [³H]arachidonic acid-prelabelled lipids were examined. Trifluoperazine, dibucaine or quinacrine stimulated [³²P]P_i incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) without significant effect on the labelling of phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), lysophosphatidylcholine (lyso-PtdCho) and lysophosphatidylethanolamine (lyso-PtdEtn). The incorporation of [³²P]P_i into phosphatidylcholine (PtdCho) was, on the contrary, inhibited. When calmodulin antagonists were added to macrophages stimulated by fMet-Leu-Phe, [³²P]P_i incorporation into PtdIns and PtdA was synergistically increased compared with that induced only by calmodulin antagonists. Trifluoperazine inhibited the incorporation of [³H]glycerol into PtdCho, triacylglycerol and PtdEtn. Also in this case, the incorporation of [³H]glycerol into PtdA and PtdIns was greatly enhanced. But [³H]glycerol incorporation into PtdSer, lyso-PtdEtn and lyso-PtdCho was not affected by the drug. On the other hand, diacylglycerol labelling with [³H]glycerol was maximally activated by 10μm-trifluoperazine and levelled off with the increasing concentration. When the effect of calmodulin antagonists on the redistribution of [¹⁴C]glycerol among lipids was examined in pulse-chase experiments, no significant effect on [¹⁴C]glycerol redistribution in PtdEtn, PtdCho, PtdIns, PtdSer, PtdA and tri- and di-acylglycerol could be detected. When macrophages prelabelled with [³H]arachidonic acid were treated with trifluoperazine, dibucaine or quinacrine, the [³H]arachidonic acid moiety in PtdEtn and PtdCho was decreased and that in PtdA was increased. The formation of [arachidonate-³H]diacylglycerol and non-esterified [³H]-arachidonic acid was also enhanced, but the increase in [³H]arachidonic acid was only observed at concentrations between 1 and 50μm. [Arachidonate-³H]PtdIns was not significantly affected. The activated formation of [arachidonate-³H]PtdA, diacylglycerol and non-esterified arachidonic acid by these drugs was synergistically enhanced in the presence of fMet-Leu-Phe.     

Sequence of prolactin effects on phospholipid synthesis in mouse mammary gland explants

In cultured mouse mammary gland explants derived from 12-14 day pregnant mice, the effect of prolactin (PRL) on the rate of incorporation of several precursors into neutral lipids and phospholipids was determined. Employing [14C]-acetate as a substrate, PRL stimulates its incorporation into a) neutral lipids by 4-6 hours, b) phosphatidyl choline (PC) and phosphatidyl inositol-phosphatidyl serine (PI-PS) by 1-2 hours, and c) phosphatidyl ethanolamine (PE) by 2-4 hours. Using [3H]-glycerol as a substrate, the temporal response to PRL for its incorporation into the neutral lipids was the same as that for [14C]-acetate, however, PRL did not enhance the rate of [3H]-glycerol incorporation into the phospholipids at any time through 16 hours. PRL similarly had no effect on the rates of [3H]-choline, [3H]-serine, [3H]-ethanolamine, or [32P]O4 incorporation into the phospholipids at hormone exposure periods of 8 hours or more. And finally, PRL had no effect on the rates of [3H]-arachidonate or [14C]-linoleate incorporation into neutral lipids or phospholipids at culture periods up to 18 hours. These data suggest that the early effect of PRL on [14C]-acetate incorporation into the phospholipids is due to either the insertion of newly synthesized fatty acids and/or the extension of fatty acids contained in the phospholipids.     

Biosynthesis of Choline and Ethanolamine Phospholipids in Crithidia fasciculata*

The incorporation of radioactivity from [1,2-³⁴C]choline, [1,2-³⁴C]ethanolamine, [3-¹⁴C]serine and [methyl-¹⁴C]methionine into lipids was studied in growing cultures of Crithidia fasciculata. Lecithin was formed both from choline and by the methylation of phosphatidylethanolamine. Mono- and dimethylphosphatidylethanolamines were present in no more than trace amounts. Growth of the protozoa in media containing choline (1 mM) did not decrease synthesis by the methylation pathway. Phosphatidylethanolamine was formed from ethanolamine. Radioactivity from serine also was present in both phosphatidylethanolamine and lecithin; however, the presumed intermediate, phosphatidylserine, could not be detected.     

Effect of neomycin on phosphoinositide labelling and calcium binding in guinea-pig inner ear tissues in vivo and in vitro.

—Phospholipids of guinea-pig inner ear tissues were labelled in vivo by perilymphatic perfusion of the cochlea with [³²P]orthophosphate or myo-[³H]inositol. After 20-40 min the most highly labelled ³²P-lipids were phosphatidylinositol phosphate and diphosphate. Incorporation of [³H]inositol proceeded in the order phosphatidylinositol > phosphatidylinositol phosphate > phosphatidylinositol diphosphate. After treatment of animals with neomycin for 3 weeks, ³²P-incorporation into phosphatidylinositol diphosphate, but not into other lipids, was significantly decreased in the preparations of the organ of Corti and stria vascularis. In homogenates of inner ear tissues, the labelling of the polyphosphoinositides by [γ-³²P]ATP was increased and the hydrolysis of these lipids was blocked in the presence of 10^?4m -neomycin. Neomycin also competitively inhibited the binding of ⁴⁵Ca²⁺ to homogenates of these tissues.     

Differential effects of unsaturated fatty acids on phospholipid synthesis in human leukemia monocytic U937 cells

The biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in monocyte-like leukemia U937 cells was monitored by adding [³H]choline, [¹⁴C]ethanolamine or [¹⁴C]glycerol to the culture media; incorporation into phospholipid (PL) increased with time. The effect of unsaturated fatty acids (UFA) on PC and PE synthesis was investigated by pretreating U937 cells for 72h with 10 μM 18:1 (n –9), 18:2 (n –6), 18:3 (n –3), 20:4 (n –6) and 20:5 (n –3). The UFA caused no alteration in cell growth, as evidenced by light microscopy and the incorporation of [³H]thymidine and [³H]leucine. Total cellular uptake of radioactive precursors remained unaffected by all the treatments. Pretreatment with 20:5 resulted in approximately 25 per cent reduction in the incorporation of [³H]choline into PL, while no significant effect was detected with the other UFAs. 18:3, 20:4 and 20:5 depressed the incorporation of [¹⁴C]ethanolamine into PL by 34 per cent, 28 per cent and 49 per cent respectively. However, there was no redistribution of label with any of the treatments. 18:3, 20:4 and 20:5 also antagonized the stimulatory effect of endotoxin (LPS) on PC and PE synthesis. In addition, the incorporation from [¹⁴C]glycerol into PC and PE was reduced by 18:3, 20:4 and 20:5. Although the PL composition of the cells remained essentially unaffected, our study shows that chronic treatment of U937 cells with n –3 PUFA (20:5) depressed PC and PE synthesis, and 18:3 and 20:4 also caused inhibition of PE synthesis.     

Isolation and characterization of a Chinese hamster ovary cell mutant with altered regulation of phosphatidylserine biosynthesis

We have screened approximately 10,000 colonies of Chinese hamster ovary (CHO) cells immobilized on polyester cloth for mutants defective in [14C]ethanolamine incorporation into trichloroacetic acid-precipitable phospholipids. In mutant 29, discovered in this way, the activities of enzymes involved in the CDP-ethanolamine pathway were normal; however, the intracellular pool of phosphorylethanolamine was elevated, being more than 10-fold that in the parental CHO-K1 cells. These results suggested that the reduced incorporation of [14C]ethanolamine into phosphatidylethanolamine in mutant 29 was due to dilution of phosphoryl-[14C]ethanolamine with the increased amount of cellular phosphorylethanolamine. Interestingly, the rate of incorporation of serine into phosphatidylserine and the content of phosphatidylserine in mutant 29 cells were increased 3-fold and 1.5-fold, respectively, compared with the parent cells. The overproduction of phosphorylethanolamine in mutant 29 cells was ascribed to the elevated level of phosphatidylserine biosynthesis, because ethanolamine is produced as a reaction product on the conversion of phosphatidylethanolamine to phosphatidylserine, which is catalyzed by phospholipid-serine base-exchange enzymes. Using both intact cells and the particulate fraction of a cell extract, phosphatidylserine biosynthesis in CHO-K1 cells was shown to be inhibited by phosphatidylserine itself, whereas that in mutant 29 cells was greatly resistant to the inhibition, compared with the parental cells. As a conclusion, it may be assumed that mutant 29 cells have a lesion in the regulation of phosphatidylserine biosynthesis by serine-exchange enzyme activity, which results in the overproduction of phosphatidylserine and phosphorylethanolamine as well.     

Exogenous gangliosides may affect methylation mechanisms in neuronal cell cultures

Primary neurons in culture from chick embryo cerebral hemispheres were treated with a mixture of gangliosides added to the growth medium (final concentration: 10^–5M and 10^–8M) from the 3rd to the 6th day in vitro. Under these conditions methylation processes measured with [³H] and [³⁵S] methionine and [³H]ethanolamine as precursors showed an increased methylation of [³H]ethanolamine containing phospholipids, a correspondent increased conversion of these compounds to [³H]choline containing phospholipids, and a general increased methylation of trichloroacetic acid precipitable macromolecules containing labeled methionine. A small increase in protein synthesis was observed after incubation of neurons with [³H]- and [³⁵S]methionine. This was confirmed after electrophoretic separation of a protein extract with increased³H-and³⁵S-labeling in protein bands with moecular weights between 50 and 60 KDaltons. A protein band of about 55 KDaltons appeared to be preferentially labelled when [³H] methionine was the precursor. The treatment with gangliosides increased the incorporation of [methyl-³H] label after incubation of neurons with [³H] methionine, into total DNA and decreased that of total RNA. The treatment of neurons in culture with exogenous gangliosides hence affects differently methylation processes, a finding which may confirm the involvement of gangliosides on the intracellular mediation of neuronal information mechanisms.