Stimulation of phosphatidylcholine biosynthesis by hemicholinium-3, a potent inhibitor of choline uptake in human leukemic monocyte-like U937 cells

The effect of hemicholinium-3 (HC-3) on choline uptake and phosphatidylcholine (PC) biosynthesis was examined in human leukemic monocyte-like U937 cells. HC-3 inhibited [³H]choline uptake in a dose- and time-dependent manner. After a 3 h treatment, HC-3 (100 μM) decreased choline uptake by as much as 80 per cent (p < 0·0001; n = 4). Reduction of incorporation of label into PC was also detected in a dose-dependent manner; the extent of inhibition, however, was always 10–20 per cent less than that observed in the total uptake. At 3 h HC-3 decreased the incorporation into PC by 65 per cent (p < 0·0001; n = 5). Kinetic studies in vivo showed that HC-3 inhibited total uptake and incorporation into PC differently, suggesting that the labelling of PC is not simply dictated by [³H]choline uptake. In separate experiments, cells were pretreated with 100 μM HC-3 for 3 h. After washing, the inhibitory effect on total uptake was no longer observed, while a 20 per cent stimulation of the incorporation into PC was obtained in these pretreated cells. In pulse-chase studies, the cells were prelabelled with [³H]choline for 30 min and chased with HC-3 for up to 3 h; the results showed a significant stimulation of incorporation into PC in a longer chase with 100 μM HC-3. After a 3 h treatment, the cytosolic CTP:cholinephosphate cytidylytransferase (CT) was activated by 56 per cent, while choline kinase (CK) was inhibited slightly. The stimulation of CT was not simply due to the intact HC-3 molecule, and there was no redistribution of CT between cytosol and microsomes. Taken together, the results suggest that HC-3 activates PC biosynthesis apart from the inhibitory effect on choline uptake.     

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.     

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.     

The effects of inhibitors of protein synthesis on incorporation of lipids into myelin

Abstract— Following intracranial injections of puromycin, the incorporation of [³H]leucine into brain protein was inhibited by 80 per cent. Conversely, incorporation of [³⁵S]sulphate into sulphatide or [2-³H]glycerol into phosphatidyl choline was not inhibited. Under these conditions, appearance of labelled protein in myelin was inhibited by 90 per cent, while the appearance of newly labelled sulphatide and phosphatidyl choline in myelin membrane was not greatly affected. Experiments with cycloheximide gave similar results with phosphatidyl choline, but incorporation of [³⁵S]sulphate into total sulphatide was decreased by about 30 per cent in animals given cycloheximide. Neither puromycin nor cycloheximide had any inhibitory effect on galactocerebroside sulphotransferase.     

Mechanism by which ethanol inhibits phosphatidylcholine biosynthesis in human leukemic monocyte-like U937 cells

A previous study showing that ethanol (ETOH) blocked [³H]choline incorporation into phosphatidylcholine (PC) suggested an inhibition of PC biosynthesis in human leukemic monocyte-like U937 cells. The mechanism of the inhibitory action of ETOH was investigated. Cells were pulsed with [³H]choline for 30 min and chased in the presence or absence of ETOH for up to 6 h. PC biosynthesis was inhibited drastically within 1 h after exposure to ETOH which increased intracellular cAMP appreciably. After a 3-h treatment, ETOH significantly inhibited both choline kinase (CK) and the cytosolic CTP: cholinephosphate cytidylyltransferase (CT). The inactivated CT was no longer stimulated by exogenous phosphatidylglycerol (PG). There was no evidence for redistribution of CT activity between cytosol and microsomes. When cells were exposed to 8-Bromo-cAMP ranging from 100 to 300 μM, PC biosynthesis remained unaffected despite the drastically elevated cAMP. These results seem to suggest that the raised cAMP is not a prerequisite for the inhibition of PC biosynthesis in U937 cells. Following pretreatment with protein kinase inhibitors (H-89 and K-252a), PC biosynthesis was decreased significantly and the inhibitory effect of ETOH was potentiated. Taken together, our results suggest that the inhibition of PC biosynthesis and the inhibitory effect of ETOH are independent of the activation of cAMP-dependent protein kinase. Unlike protein kinase inhibitors, pretreatment with tyrosine kinase inhibitors (erbstatin, genistein and tyrphostin 25) resulted in differential effects on PC biosynthesis and on the inhibitory action of ETOH. Genistein stimulated PC biosynthesis by 30 per cent as well as partially preventing /reversing the ETOH action, while tyrphostin 25 produced a synergistic inhibition. The relevance of tyrosine phosphorylation/dephosphorylation to the regulation of PC biosynthesis and ETOH action remains to be established.     

UPTAKE OF [3H-METHYL]CHOLINE BY MICROSOMAL, SYNAPTOSOMAL, MITOCHONDRIAL AND SYNAPTIC VESICLE FRACTIONS OF RAT BRAIN

Abstract— Microsomal, mitochondrial, synaptosomal and synaptic vesicle fractions of rat brain took up [³H-methyl]choline by a similar carrier-mediated transport system. The apparent K_m for the uptake of [³H-methyl]choline in these subcellular fractions was about 5 × 10^?5 M. Choline uptake was also observed in microsomal fractions prepared from liver and skeletal muscle. Virtually identical kinetic properties for [³H-methyl]choline transport were found in the synaptosomal fractions prepared from the whole brain, cerebellum or basal ganglia. Countertransport of [³H-methyl]choline from the synaptosomal fraction was demonstrated against a concentration gradient. HC-3 was a competitive inhibitor of the uptake of [³H-methyl]choline in brain microsomal, synaptosomal and mitochondria] fractions with respective values for K_i of 4.0, 2.1 and 2.3 × 10^?5 M. HC-15 was a competitive inhibitor of the transport of [³H-methyl]choline in the synaptosomal fraction, with a K_i of 1.7 × 10^?4 M. Upon entry into the microsomal fraction, 74 per cent of the radioactivity could be recovered as unaltered choline, 10 per cent as phosphorylcholine, 1.5 per cent as acetylcholine and 2.5 per cent as phospholipid. Choline acetyltransferase (EC 2.3.1.6) was assayed with [¹⁴C]acetylCoA in synaptosomal fractions prepared from basal ganglia and cerebellum, and in the 31,000 g supernatant fraction of a rat brain homogenate. Enzyme activity was 11-fold greater in the synaptosomal fraction from the basal ganglia than in that from the cerebellum. HC-3 did not inhibit choline acetyltransferase and there was no evidence for acetylation of HC-3. Our findings suggest that choline uptake is a ubiquitous property of membranes in the CNS and cannot serve to distinguish cholinergic nerve endings and their synaptic vesicles.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

Metabolism of phosphatidylcholine, phosphatidylinositol and palmityl carnitine in synaptosomes from rat brain

Abstract— Synthesis of phosphatidylcholine, phosphatidylinositol and palmityl carnitine in synaptosomes isolated from rat brain was investigated and compared with the synthesis of these compounds in microsomes and mitochondria. Electron microscopic and marker enzyme studies showed the contaminants in the synaptosomal preparation to consist of a few microsomes and almost no free mitochondria. In synaptosomes, addition of 1,2-diglyceride exerted no effect on the incorporation of [¹⁴C]choline into phosphatidylcholine or on the incorporation of [³H]myo-inositol into phosphatidylinositol, but it stimulated the incorporation of CDP[1,2-¹⁴C]choline into phosphatidylcholine by more than 50 per cent. The incorporation of the latter in intact synaptosomes, lysed synaptosomes and purified mitochondria was 15-6, 27 and 9-9 per cent, respectively, of that in the microsomes. The incorporation of [³H]myo-inositol into the phosphatidylinositol of synaptosomes and purified mitochondria was 15-8 and 11-1 per cent, respectively, of that in the microsomes. Maximal incorporation of [³H]myo-inositol occurred at pH 7–5 in a medium containing Mg²⁺ and CTP; it was linear with time and protein concentration and was inhibited by 1 mM Ca^{2 +} but unaffected by the presence of ATP. This incorporation of myo-inositol appeared to occur through the reversal of the CDP-diglyceride: inositol transferase reaction. The demonstration of carnitine palmityl transferase in synaptosomes indicated that, as in mitochondrial and erythrocyte membranes, fatty acids can be transported across the synaptosomal membrane. In contrast to mitochondria where maximal incorporation of [¹⁴C]carnitine into palmityl carnitine was observed after 20 min of incubation, the incorporation in synaptosomes increased as a function of time up to 60 min of incubation. We conclude that synaptosomes can carry on de novo synthesis of lipids, although at a limited rate. From the present data we cannot state with certainty how much of this synthesis is attributable to membranes originating from the endoplasmic reticulum.     

UPTAKE OF 3-O-METHYL-d-GLUCOSE INTO CULTURED HUMAN GLIOMA CELLS

Human glioma cells (138 MG) were found to take up 3-O-methyl-d -glucose (3-OMG) by a saturable low affinity transport system with a K_m of 20 mm and a V_max of 500 nmol/mg protein/min. About 20 per cent of the total uptake was due to passive diffusion. d -Glucose was a competitive inhibitor with a K_i of 10 mm . Follow-up experiments indicated that the same transport mechanism is involved in the uptake of n-glucose and 3-OMG. Phloretin (0·02 mm ) and cytochalasin B (0·002 mm ) strongly inhibited the uptake of 3-OMG, whereas phlorizin (0·02 mm ), ouabain (0·1 mm ), NaCN (0·5 mm ) and iodoacetic acid (1·0 mm ) had no effect. The data suggest that 3-OMG and d -glucose enter 138 MG cells mainly by a Na⁺-independent passive carrier-mediated transport system. Serum-deprivation doubled the population doubling time (T_d) without affecting the total uptake of 3-OMG. An increase in the non-specific (diffusional) uptake was balanced by a decrease in the specific (carrier-medíated) uptake. After addition of dibutyryl cyclic AMP (dbcAMP, 0·25 mm ) the cells attained a morphology characteristic of differentiated glia cells. T_d was maintained unchanged. The non-specific uptake of 3-OMG was not affected in cells grown in serum-containing medium plus dbcAMP, whereas the specific uptake increased by 40 per cent and there-fore also the total uptake. Similar, but more pronounced, changes were observed if serum-deprived cells were treated with dbcAMP.     

Inhibition by 1-beta-D-arabinofuranosylcytosine of the incorporation of N-acetylneuraminic acid into glycolipids and glycoproteins of hamster embryo cells

1-β-D-Arabinofuranosylcytosine which interferes with DNA synthesis in bacteria and mammalian cells and brings about transformation of hamster embryo fibroblasts, has been found to inhibit the incorporation of N-Acetylneuraminic acid into glycolipids and glycoproteins of both normal and transformed hamster embryo cells in tissue culture. Three hours after commencement of treatment (10^?3M ara-C), incorporation of [¹⁴C] thymidine into DNA was inhibited by 95 per cent, while incorporation of [³H] D-glycosamine (precursor of sialic acid) into glycolipids and glycoproteins was inhibited by 85 per cent. At 24 hours, the inhibition of incorporation of the two labelled components was 83 and 80 per cent respectively. In homogenates of both cell types, incorporation of [¹⁴C] N-acetylneuraminic acid was competitively inhibited by ara-CMP. Ara-C was found to have no effect on the incorporation of [¹⁴C] choline into phospholipids of cells grown in tissue culture. These results suggest that interference with DNA synthesis by ara-C may not be the only factor involved in cell transformation by this substance.     

Utilization of fatty acids by Pieris brassicae reared on artificial and natural diets

The fatty acid composition of Pieris brassicae was measured from larvae reared on four different diets. Pieris can alter the composition of fatty acids in the diet through selective incorporation and synthesis. Oleate is preferentially accumulated on artificial diets (15·9 per cent in diet, 43·8 per cent in neutral lipid (NL) of fifth instar larvae), but not equally on natural diets (18·1 per cent in Brassica napus, 25·6 per cent in the NL of fifth instar larvae). Incorporation of linolenate appears to depend on the concentration of both linolenate and linoleate in the diet. With dietary levels of 35·7% linolenate and 32·2% linoleate, fifth instar larvae contain 12·2 and 16·0 per cent, respectively, of these acids. With 45·8% linolenate and 12·5% linoleate in the diet, fifth instar larvae contain 44·1 and 11·6 per cent of these acids, respectively, in the NL. Palmitoleate is actively synthetized on the artificial diets; with trace amounts of dietary palmitoleate, fifth instar larvae have 9·3 per cent of this acid in the NL. Pieris regulates the uptake of linoleate from the diet at the intestinal wall as was shown by linoleic acid-1-¹⁴C, and is unable to convert dietary linoleate to any of the 18-carbon analogues. The female apparently accumulates linolenate into egg phospholipids on the artificial diet, but in general the fatty acid composition of the eggs resembles that of the fat body.     

The effect of methionine on the regional and intracellular disposition of [3H]-methionine sulphoximine in rat brain

—The intracellular disposition of the convulsant agent, methionine sulphoximine (MSO), administered as methyl-labelled [³H]MSO, was examined in rat brain. Intraperitoneal (i.p.) and intrathecal (i.th.) routes were compared. The effect of simultaneous administration of methionine on the uptake, the regional distribution and the intracellular disposition of [³H]MSO was also assessed: (1) The peak uptake of i.p. [³H]MSO was at 2 h and amounted to about 1 per cent of the dose; the peak uptake of i.th. [³H]MSO was at 30 min post-injection and amounted to 40 per cent of the administered dose. The uptake was effectively reduced when methionine was simultaneously administered. (2) The regional distribution of [³H]MSO as a function of time after injection revealed a rather uniform penetration of the entire brain by the drug. A maximum of 43 per cent of the tissue radioactivity was found in the cerebellum 2 h after i.p. injection, while 49 per cent accumulated in the extracortical portion of the brain 3·5 h after i.th. administration. Methionine did not affect the regional distribution of [³H]MSO. (3) Differential centrifugation of samples of cortex and cerebellum revealed an association of [³H]MSO with intracellular particulate fractions. Since closely similar proportions of MSO occurred in the crude mitochondrial and the microsomal fractions, these fractions were analysed further: (a) [³H]MSO was bound to nerve endings sedimenting at the 1·0 m–1·2 m-sucrose interface; this binding was not abolished by prior increase of the endogenous cerebral methionine pool; and (b) [³H]MSO was released by subjecting the nerve endings to osmotic shock. However, the striking finding was that [³H]MSO could not be released from the nerve endings of the cerebellum from animals pre-treated with methionine. (4) An association of [³H]MSO was observed with the membranes of the endoplasmic reticulum and specifically with its agranular component. (5)The results implicate the cerebellum as the primary target for MSO, in confirmation of the original observations of Lodin (1958).     

Effect of neurotransmitters on phospholipid metabolism in rat cerebral-cortex slices: cellular and subcellular distribution

Abstract— Young rat cerebral-cortex slices were incubated with ³²Pi in the absence and presence of ACh plus eserine, norepinephrine, dopamine or serotonin for 1 h. their cellular and subcellular fractions were isolated, and the specific radioactivities of the various phospholipids determined. In the neuronal- and astroglial-enriched fractions ACh plus eserine increased the ³²P-labelling of phosphatidyl inositol (PhI) phosphatidic acid (PhA) and phosphatidylcholine (PhC) by increments which ranged from 108 per cent for PhI to 30 per cent for PhC and in the presence of norepinephrine or dopamine these increments ranged from 180 per cent for PhI to 29 per cent for PhC. In the subcellular fractions ACh plus eserine exerted maximal stimulatory effect on the labelling of the synaptosomal phospholipids, which was 88 per cent for PhI and 79 per cent for PhA, followed by those of microsomes, mitochondria and nuclei. ACh plus eserine exerted no effect on [^l4C]glucose incorporation, but inhibited the incorporation of [¹⁴C]glycerol into phospholipids by amounts which ranged from 30 per cent for PhI to 3 per cent for PhE. Although the rate of incorporation of ³²Pi into phospholipids of 0.2 mm slices was higher than that of the 0.5 mm slices the stimulatory effect of ACh plus eserine on the ³²Pi incorporation into the lipids of the latter was higher. When neuronal- and astroglial enriched fractions were first isolated from the cerebra then incubated with ³²Pi or [¹⁴C]choline, labelling of phospholipids in the neuronal fraction was higher than that of the astroglial fraction; however, ACh plus eserine had no effect on the incorporation of ³²Pi into the lipids of either fraction. ACh plus eserine stimulated the activity of phosphatidic acid phosphatase in the various subcellular fractions by increments which ranged from 13 per cent in nuclei to 37 per cent in microsomes. It was concluded that the nonspecific localization of the neurotransmitter effect could be due to the widespread distribution of the enzymes which appear to be responsive to cholinergic and adrenergic neurotransmitters.     

Effect of Cytidine on Membrane Phospholipid Synthesis in Rat Striatal Slices

Abstract: Using rat striatal slices, we examined the effect of cytidine on the conversion of [³H]choline to [³H]-phosphatidylcholine ([³H]PC), and on net syntheses of PC, phosphatidylethanolamine (PE), and phosphatidylserine, when media did or did not also contain choline, ethanolamine, or serine. Incubation of striatal slices with cytidine (50–500 µM) caused dose-dependent increases in intracellular cytidine and cytidine triphosphate (CTP) levels and in the rate of incorporation of [³H]choline into membrane [³H]PC. In pulse-chase experiments, cytidine (200 µM) also increased significantly the conversion of [³H]choline to [³H]PC during the chase period. When slices were incubated with this concentration of cytidine for 1 h, small (7%) but significant elevations were observed in the absolute contents (nmol/mg of protein) of membrane PC and PE (p < 0.05), but not phosphatidylserine, the synthesis of which is independent of cytidine-containing CTP. Concurrent exposure to cytidine (200 µM) and choline (10 µM) caused an additional significant increase (p < 0.05) in tissue PC levels beyond that produced by cytidine alone. Exposure to choline alone at a higher concentration (40 µM) increased the levels of all three membrane phospholipids (p < 0.01); the addition of cytidine, however, did not cause further increases. Concurrent exposure to cytidine (200 µM) and ethanolamine (20 µM) also caused significantly greater elevations (p < 0.05) in tissue PE levels than those caused by cytidine alone. In contrast, the addition of serine (500 µM) did not enhance cytidine's effects on any membrane phospholipid. Exposure to serine alone, however, like exposure to sufficient choline, increased levels of all three membrane phospholipids significantly (p < 0.01). These data show that exogenous cytidine, probably acting via CTP and the Kennedy cycle, can increase the synthesis and levels of membrane PC and PE in brain cells.     

CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN CULTURED BRAIN CELLS FROM CHICK EMBRYOS

—Dissociated cells from brains of 7-day chick embryos were grown in primary culture for as long as 20 days. Many of the plated cells grew out long processes. Others, which proliferated rapidly, formed a confluent layer of flat cells after 4-6 days. Total DNA and protein increased five-fold, and activity of choline acetyltransferase (EC2.3.1.6) increased about 40-fold in 20 days. Acetylcholinesterase (EC3.1.1.7) increased three-fold by the fourth day of culture and then declined. The pattern of increase for choline acetyltransferase was similar to that for the in vivo development of the enzyme. l -Thyroxine, cyclic AMP (adenosine-3′,5′-monophosphate) or theophylline promoted increased levels of both enzymes by 30-200 per cent. l -Thyroxine also increased the activity of acetylcholinesterase in vivo by 40 per cent. When overgrowth by flat cells was prevented by the addition of 10^-3m -5-flourouracil, there was a decrease in the activity of choline acetyltransferase and an increase in the activity of acetylcholinesterase in comparison to control activities. The addition of 10^-3m -morphine or cocaine produced a 30 per cent elevation in the activity of choline acetyltransferase, but this effect could be mimicked with equimolar concentrations of ammonium ion.     

Carrier-mediated choline uptake by Krens II ascites cells

Krebs II ascites cells have a low affinity uptake system for choline (K_m = 36 μM, V_m = 76 nmol/min per 2·10⁸ cells). Choline entered the cells and was rapidly phosphorylated (95% of total intracellular soluble label). Trans acceleration of labeled choline from cells preloaded with radiolabeled choline and postincubated in the presence of unlabeled choline indicates that choline transport in Krebs II ascites cells is carrier mediated. Ethanolamine competed for the choline carrier. The uptake was reduced by hemicholinium-3, iodoacetamide and ouabain. The mechanism of choline transport in Krebs Ii ascites cells is in agreement with a linear transport model.     

Induction of intracellular acyl-CoA:cholesterol acyltransferase activity in glioblastoma cells by lidocaine

The perturbation of cellular cholesteryl ester biosynthesis in glioblastoma C-6 cells by lidocaine was investigated. Lidocaine specifically inhibited the incorporation of radioactive oleic acid into cellular cholesteryl ester but had no significant effect on the incorporation of oleic acid into phosphatidylcholine. Oxygenated cholesterol-enhanced cholesteryl ester formation was less sensitive to lidocaine inhibition. Several other local anesthetics were compared. Lidocaine altered cholesteryl ester formation in time- and dose-dependent manners. Lidocaine was a powerful inhibitor initially and its potency declined with time. Lidocaine was capable of directly inhibiting acyl-CoA:cholesterol acyltransferase (ACAT) activity in broken cell homogenates. The lidocaine-mediated inhibition of cellular cholesteryl ester formation triggered an enhanced intracellular ACAT activity that was not fully expressed in the presence of lidocaine. The activation of ACAT activity by lidocaine might represent a compensatory mechanism by which the inhibitory effect of lidocaine was partially overcome with time.     

The Effect of Acetylcholine Release on Choline Fluxes in Isolated Synaptic Terminals

Abstract: As in intact tissues, choline influx into synaptosomes is enhanced after a period of depolarization induced release of acetylcholine. The activation of uptake is dependent on the presence of Ca²⁺ and inhibited by high Mg²⁺ concentrations in the medium during depolarization. Choline transport in erythrocytes was not activated by prior treatment with potassium. The permeability constant of the synaptosome membrane to choline was found to be 2.7 × 10^?8 cm·s^?1 and to acetylcholine 1.8 ′ 10^?8 cm·s^?1. Choline influx has been studied after pre-loading synaptosomes with choline. Different radiolabels were used to measure efflux of preloaded choline and influx simultaneously. Isotopic dilution in flux studies was estimated and corrected for. Influx was stimulated by high internal concentrations of choline, and efflux similarly stimulated by high outside concentrations of choline. The maximal influx and efflux at saturating opposite concentrations of choline were equal with a value of about 500 pmol·min^?1 per mg synaptosomal protein. A reciprocating carrier would explain the equality of the maximal influx and efflux. Acetylcholine competes with choline for binding to the carrier but is itself hardly transported. Increased acetylcholine concentrations were shown to inhibit both choline influx and efflux from the trans position. Raising intrasynaptosomal acetylcholine concentrations by pre-loading abolished the stimulation of influx by prior depolarization. It is proposed that high concentrations of acetylcholine immobilize the carrier on the inside of the synaptic membrane. The stimulation of choline influx consequent upon depolarization is caused by release of ACh which results in relief of this immobilisation. The enhanced supply of choline achieved by this mechanism is likely to be important in maintaining stores of the acetylcholine in vivo.     

Lipids and fatty acids of the southwestern corn borer,Diatraea grandiosella

The lipids of the adults and of several immature stages of the southwestern corn borer, Diatraea grandiosella, were studied after they were fed natural corn stalks or artificial diets. Linoleic acid (18:2) was the major fatty acid of the neutral lipids in both the natural and the artificial diets, but aleic acid (18:1) was the principal neutral lipid in all insect stages. Also, linoleic acid and oleic acid were the principal acids in the insect phospholipids of all stages. The content of linoleic acid in the natural diet was also high, but that in the artificial diet appeared to be much too low for insect requirements. Phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) were the major phospholipids in all growth stages. Thus, in larvae diapausing in the field, the unsaturated fatty acid content of PC was 59·3 per cent, primarily 16:1 and 18:1, and PE was 87·4 per cent, primarily 18:1, 18:2, and 18:3, and the fatty acids in the number 1- and 2-positions of PC were 53·6 and 97·2 per cent unsaturated, respectively. The haemolymph of diapausing southwestern corn borer larvae contained primarily glycerides but also had some PC and PE. Fat body from diapausing larvae contained primarily 16:0, 16:1, and 18:1 in a ratio of 1 : 1 : 2. Thus lipids of the southwestern corn borer do not reflect dietary lipids as closely as do other insects studied.