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PubMed1994

Lidocaine inhibits choline uptake and phosphatidylcholine biosynthesis in human leukemic monocyte-like U937 cells.

The effect of lidocaine on [3H]choline uptake and the incorporation of label into phosphatidylcholine (PC) in human monocyte-like U937 cells was investigated. Lidocaine inhibited the rate of choline uptake in a dose-dependent manner; at 3.2 mM it resulted in a drastic reduction, by as much as 65 per cent (n = 10; p < 0.0005) or 55 per cent (n = 10; p < 0.0006) in a 3- or 6-h incubation, respectively. Lidocaine also decreased the rate of choline incorporation into PC in a dose-dependent manner. At the highest dose, nearly 70 per cent or 45 per cent reduction was seen in a 3- or 6-h incubation, respectively. Analysis of choline-containing metabolites showed that the major label association with phosphocholine and PC was reduced to a similar extent which was also parallel to the inhibition of choline uptake. At 3.2 mM lidocaine, the reduction of choline uptake was shown to follow a competitive inhibition. In the case of [3H] choline incorporation into PC, the inhibitory pattern was shown to be of a mixed type. The pulse-chase study dissecting the effect on choline metabolism from that on total choline uptake indicated that lidocaine exerted an additionally inhibitory effect on intracellular choline metabolism into PC. In a separate protocol in which the labelled cells were first allowed to be chased until 3H-incorporation into PC reached a steady state, lidocaine no longer showed any effect. These results seem to exclude the possibility of enhanced PC breakdown and further suggest that the main inhibitory effect is on the CDP-choline pathway for PC biosynthesis. After a 3-h treatment, CTP: cholinephosphate cytidylyltransferase (CYT) in both the cytosolic and microsomal fractions was inhibited by approximately 20 per cent, while choline kinase (CK) and choline phosphotransferase (CPT) remain relatively unchanged. There was no evidence for translocation of CYT between cytosol and microsomes. Taken together, we have demonstrated a dual inhibitory function of lidocaine which inhibits PC biosynthesis in addition to its ability to block choline uptake profoundly in U937 cells.

Biological Transport↗
PubMed1984

Mechanisms of aldehyde-induced adenosinetriphosphatase activities of kinases.

Aldehyde analogues of the normal alcohol substrates induce ATPase activities by glycerokinase (D-glyceraldehyde), fructose-6-phosphate kinase (2,5-anhydromannose 6-phosphate), fructokinase (2,5-anhydromannose or 2,5-anhydrotalose), hexokinase (D-gluco-hexodialdose), choline kinase (betaine aldehyde), and pyruvate kinase (glyoxylate). Since purified deuterated aldehydes give V and V/K isotope effects near 1.0 for glycerokinase, fructokinase with 2,5-anhydro[1-2H]talose, hexokinase, choline kinase, and pyruvate kinase, the hydrates of these almost fully hydrated aldehydes are the activators of the ATPase reactions. Fructose-6-phosphate kinase and fructokinase with 2,5-anhydro[1-2H]mannose show V/K deuterium isotope effects of 1.10 and 1.22, respectively, suggesting either that both hydrate and free aldehyde may be activators (predicted values are 1.37 if only the free aldehyde activates the ATPase) or, more likely, that the phosphorylated hydrate breaks down in a rate-limiting step on the enzyme while MgADP is still present and the back-reaction to yield free hydrate in solution is still possible. 18O was transferred from the aldehyde hydrate to phosphate during the ATPase reactions of glycerokinase, fructose-6-phosphate kinase, fructokinase, and hexokinase but not with choline kinase or pyruvate kinase. Thus, direct phosphorylation of the hydrates by the first four enzymes gives the phosphate adduct of the aldehyde, which decomposes nonenzymatically, while with choline kinase and pyruvate kinase the hydrates induce transfer to water (metal-bound hydroxide or water with pyruvate kinase on the basis of pH profiles). Observation of a lag in the release of phosphate from the glycerokinase ATPase reaction at 15 degrees C supports the existence of a phosphorylated hydrate intermediate with a rate constant for breakdown of 0.035-0.043 s-1 at this temperature. Kinases that phosphorylate creatine, 3-phosphoglycerate, and acetate did not exhibit ATPase activities in the presence of keto or aldehyde analogues (N-methylhydantoic acid, D-glyceraldehyde 3-phosphate, and acetaldehyde, respectively), possibly because of the absence of an acid-base catalytic group in the latter two cases. These analogues were competitive inhibitors vs. the normal substrates, and in the latter case, the hydrate of acetaldehyde was shown to be the inhibitory species on the basis of the deuterium isotope effect on the inhibition constant.

Adenosine Triphosphatases↗
PubMed1988

Effect of dibutyryl cyclic AMP on lipid synthesis in Microsporum gypseum.

The influence of intracellular levels of cAMP on the lipid synthesis of Microsporum gypseum has been examined by exogenous supplementation of dibutyryl cAMP and its activators/inhibitors. Incorporation of [14C]acetate into various lipid fractions of M. gypseum was markedly enhanced in the presence of dibutyryl cAMP and its modulators, probably as a consequence of increased intracellular cAMP levels, which, in turn, affected the lipid biosynthesis. Increased activities of phosphatidic acid phosphatase, glycerol kinase, ethanolamine kinase and choline kinase in the presence of these additives supports the enhanced synthesis of phospholipids and suggests that lipid biosynthesis is being controlled by cAMP in M. gypseum.

Alprostadil↗
PubMed1991

Modulation of CTP:phosphocholine cytidylyltransferase translocation by oleic acid and the antitumoral alkylphospholipid in HL-60 cells.

Short time effect of oleate and 1-O-alkyl-2-O-methyl-rac-glycero-3-phosphocholine (AMGPC) on choline incorporation into phosphatidylcholines were studied in HL-60 cells. The non lytic concentration of 50 microM oleate induced a three-fold increase in [3H]choline incorporation into phosphatidylcholine. This stimulation was accompanied by a translocation of the CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) from cytosol to membranes. By contrast, the ether-lipid AMGPC inhibited [3H]choline incorporation into phosphatidylcholine by 60% at 10 microM. AMGPC had no effect on choline kinase or choline phosphotransferase activities. When AMGPC was added separately to an homogenate, a particulate or a cytosolic fraction, cytidylyltransferase inhibition was observed only in the homogenate. However on particulates recovered from homogenates treated with increasing concentrations of AMGPC, membranous cytidylyltransferase activity decreased dose-dependently. Thus AMGPC had no effect on cytidylyltransferase activity itself but inhibited its translocation from cytosol to membrane. At variance with the well-established positive effect on cytidylyltransferase translocation induced by fatty acids, this is the first demonstration that AMGPC can inhibit cytidylyltransferase translocation in cell-free system.

Antineoplastic Agents↗
PubMed1983

Effect of renal transplantation on the levels of choline in the plasma of uremic humans.

Plasma choline levels were measured in patients who received a kidney transplant, in donors who underwent nephrectomy and in nonrenal surgical patients. Choline was measured using a choline kinase assay. Choline levels in patients receiving a kidney fell from 29.8 +/- 1.86 microM before transplantation to 15.7 +/- 2.32 1 day later; this normal level was maintained for at least 7 months and in a single case for 2 years. Kidney donors and nonrenal surgery patients showed a significant decrease in plasma choline on the day following surgery but choline levels returned to normal by 3 days after surgery. Thus a transplanted functional kidney reduced the high plasma choline levels, associated with uremia, to normal and maintained these normal levels throughout the period of observation.

Choline↗
PubMed1984

Inhibition of ornithine decarboxylase and glutamic acid decarboxylase activities by phosphorylethanolamine and phosphorylcholine.

Ornithine decarboxylase, which catalyzes the first step in polyamine biosynthesis, is rapidly and transiently increased in various tissues during growth and after various hormonal or noxious stimuli, prior to an elevation in choline kinase activity. Polyamines themselves have been demonstrated to activate choline kinase. The present study sought to determine the effect of phosphorylcholine, the product of the reaction catalyzed by choline kinase, on ornithine decarboxylase activity. The data demonstrate that ornithine decarboxylase activity. The data demonstrate that ornithine decarboxylase activity is inhibited by phosphorylcholine and more potently by the related compound phosphorylethanolamine. The inhibition by both compounds led to decreased affinity of partially purified ornithine decarboxylase for ornithine. The inhibition is not time dependent and reversible. Both compounds also inhibit glutamic acid decarboxylase activity. The results suggest that high intracellular levels of phosphorylethanolamine and phosphorylcholine can serve as natural inhibitors of decarboxylases.

Animals↗
PubMed1988

Synthesis of phosphatidylcholines in ozone-exposed alveolar type II cells isolated from adult rat lung: is glycerolphosphate acyltransferase a rate-limiting enzyme?

Type II cells were exposed to ozone by gas diffusion through the thin Teflon bottom of culture dishes. The rate of phosphatidylcholine synthesis by type II cells, monitored by the incorporation of [Me-14C]choline, was impaired by ozone at concentrations that did not affect other cellular parameters. The enzymes choline kinase and cholinephosphate cytidylyltransferase were not susceptible to inactivation by ozone at concentrations at which the activity of glycerolphosphate acyltransferase was decreased. The enzyme activity of lactate dehydrogenase increased after ozone exposure. The specific activity of choline kinase in the cytosolic fraction of type II cells was fivefold that in whole lung. The metabolism of [Me-14C]choline was studied as a function of the choline concentration. Maximal rates of phosphatidylcholine synthesis were already attained at a concentration of 20 microM choline. Exposure of type II cells to ozone did not affect the recovery of label from [Me-14C]choline in choline phosphate and CDP choline. However, the maximal rate of phosphatidylcholine synthesis decreased after ozone exposure, which indicates that the decreased apparent activity of glycerolphosphate acyltransferase limits the supply of diacylglycerols and thereby the rate of phosphatidylcholine synthesis. If the flux through the diacylglycerol pathway was stimulated by the addition of palmitic acid, a higher maximal rate of phosphatidylcholine synthesis was observed. The uptake of [Me-14C]choline and the recovery of label in CDPcholine were not altered by the addition of different concentrations of palmitate. It is concluded that type II cells take up choline very efficiently, probably due to the high specific activity of choline kinase. At low choline concentrations the rate of phosphatidylcholine synthesis is determined by the supply of CDPcholine. At concentrations of choline in the upper physiological range, the rate of phosphatidylcholine synthesis is determined by the availability of diacylglycerols, which in turn is limited by the apparent activity of glycerolphosphate acyltransferase.

Acyltransferases↗
PubMed1999

Isolation and characterization of the Saccharomyces cerevisiae EKI1 gene encoding ethanolamine kinase.

Ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1. 82) catalyzes the committed step of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. The gene encoding ethanolamine kinase (EKI1) was identified from the Saccharomyces Genome Data Base (locus YDR147W) based on its homology to the Saccharomyces cerevisiae CKI1-encoded choline kinase, which also exhibits ethanolamine kinase activity. The EKI1 gene was isolated and used to construct eki1Delta and eki1Delta cki1Delta mutants. A multicopy plasmid containing the EKI1 gene directed the overexpression of ethanolamine kinase activity in wild-type, eki1Delta mutant, cki1Delta mutant, and eki1Delta cki1Delta double mutant cells. The heterologous expression of the S. cerevisiae EKI1 gene in Sf-9 insect cells resulted in a 165,500-fold overexpression of ethanolamine kinase activity relative to control insect cells. The EKI1 gene product also exhibited choline kinase activity. Biochemical analyses of the enzyme expressed in insect cells, in eki1Delta mutants, and in cki1Delta mutants indicated that ethanolamine was the preferred substrate. The eki1Delta mutant did not exhibit a growth phenotype. Biochemical analyses of eki1Delta, cki1Delta, and eki1Delta cki1Delta mutants showed that the EKI1 and CKI1 gene products encoded all of the ethanolamine kinase and choline kinase activities in S. cerevisiae. In vivo labeling experiments showed that the EKI1 and CKI1 gene products had overlapping functions with respect to phospholipid synthesis. Whereas the EKI1 gene product was primarily responsible for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway, the CKI1 gene product was primarily responsible for phosphatidylcholine synthesis via the CDP-choline pathway. Unlike cki1Delta mutants, eki1Delta mutants did not suppress the essential function of Sec14p.

Amino Acid Sequence↗
PubMed1987

Choline glycerophospholipid biosynthesis in the guinea pig heart.

The aims of this study were to (i) elucidate the biosynthetic pathways for the formation of plasmenylcholine in the mammalian heart and (ii) investigate whether the control of choline glycerophospholipid production is different in hearts with high plasmenylcholine content. Guinea pig hearts were used throughout this study, since 34% of the cardiac choline glycerophospholipids in this species is present in the plasmenylcholine form. By perfusion of the guinea pig heart in the Langendorff mode with labeled choline, we demonstrated that the majority of plasmenylcholine in the heart was synthesized via the CDP-choline pathway. The ability of the heart to form plasmenylcholine from CDP-choline and 1-alkenyl-2-acylglycerol was also shown. We postulate that 1-alkenyl-2-acylglycerol in the guinea pig heart might originate from the hydrolysis of plasmenylethanolamine. In mammalian liver and other tissues, the CDP-choline pathway is the major pathway for phosphatidylcholine biosynthesis and the rate-limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase. The results obtained from the present study support this supposition. In addition, evidence was obtained indicating that phosphorylation of choline by choline kinase in the CDP-choline pathway may also be rate limiting. Although the involvement of choline kinase as a rate-limiting enzyme in the CDP-choline pathway has been shown in a number of cell cultures, the rate-limiting role of this enzyme in intact mammalian organs has not been previously reported. The rationale for the presence of more than one rate-limiting step in the CDP-choline pathway in the guinea pig heart remains undefined.

Animals↗
PubMed1985

Action of hemicholinium-3 on phospholipid metabolism in Krebs II ascites cells.

Incorporation of [Me-14C]choline or/and [2-14C]ethanolamine into phospholipids of Krebs II ascites cells in toto have been tested in the presence of hemicholinium-3. With [Me-14C]choline, labelling of cell pellet, intracellular choline, phosphocholine and total lipid extract is inhibited by hemicholinium-3 in a dose-dependent way between 6.25 X 10(-6) M and 10(-3) M. These effects are caused by a diminution of the choline or/and ethanolamine transport across the cell membrane and by a choline-kinase inhibition. In Krebs cells, choline is taken up by a low affinity Na+ sensitive uptake system (KT = 46 X 10(-6) M) which is competitively inhibited by hemicholinium-3 (KTi = 161 X 10(-6) M). Krebs cells exert a counter-transport (i.e. an exchange of choline across the membrane) against a concentration gradient of 10 mM choline whereas 10 mM hemicholinium-3 has no effect. Choline-kinase is also inhibited (I50 = 57 X 10(-6) M) in Krebs cells in toto and time-course data suggest that choline transport and phosphorylation might be tightly coupled. Specific radioactivities of phosphocholine and choline-glycerophospholipids decrease owing to the effect of the drug on the uptake and phosphorylation system. With 4 X 10(-5) M hemicholinium-3 and [Me-14C]choline as a marker, labelled choline-glycerophospholipids are decreased by 22%. With [2-14C]ethanolamine, labelled ethanolamine-phospholipids are decreased by 26% and choline-glycerophospholipids remain unlabelled. With the two markers, the additional effect produces a 35% decrease. It is concluded that hemicholinium-3 might be able to induce a depression of the intracellular choline and phosphocholine pool which could provoke a serious quantitative deficiency of major phospholipids in Krebs cells.

Animals↗
PubMedDate not supplied

Effect of potassium loading on choline pathways in renal cells.

The effect of potassium loading on 14C-choline incorporation into phospholipids, via choline kinase, was measured in young rats after 10 days on a high potassium (1.34 mEq/g) diet, when urinary potassium excretion rose sixfold. Renal choline kinase activity fell by 20%, from 47.3 +/- SEM 2.2 to 37.8 +/- 1.4 pmol 14C-choline incorporated into phosphorylcholine/min/mg protein in the potassium-loaded rats (p less than 0.005). This inhibition was overcome during 48 h of renal compensatory growth following uninephrectomy. There was a 25% increase in the activity of the enzyme, which was associated with a 24% increase in renal weight in potassium-loaded rats following uninephrectomy. Uninephrectomized rats on a normal diet also showed a 20% increase in both renal choline kinase activity and renal weight compared to sham-operated rats. The effect of potassium loading on 14C-choline incorporation into phospholipids was also studied on renal-derived cells having some of the characteristics of either porcine proximal or canine distal tubule cells. Incubation of either cell type in media with 40 mM potassium for 30 min resulted in a 20-30% decrease in the incorporation of 14C-choline into phospholipids (p less than 0.05). Thus, renal utilization of choline for the incorporation of membrane phospholipids for growth can be partially inhibited by chronic potassium loading in young rats and in cells incubated in vitro with high potassium concentrations. The inhibitory effect of potassium loading on renal phospholipid incorporation can be overcome by the stimulating effect of renal compensatory growth on phospholipid incorporation, resulting in increased growth in the young rat kidney.

Aldosterone↗
PubMed1980

Alteration in enzyme activities of de novo phosphatidylcholine biosynthesis in rat liver by treatment with typical inducers of microsomal drug-metabolizing system.

1. The activities of choline kinase, ethanolamine kinase, cholinephosphate cytidylytransferase(s) and cholinephosphotransferase were compared in the liver subcellular fractions after the treatment of rats for two successive days with phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls. 2. The administration of phenobarbital resulted in a significant decrease in choline kinase activity while not affecting ethanolamine kinase activity. Both 3-methylcholanthrene and polychlorinated biphenyls caused considerable enhancement of choline kinase activity concomitantly with ethanolamine kinase activity. 3. The activity of cytosolic cytidylytransferase was not affected by any of the inducers while the microsomal activity was significantly depressed by the administration of either phenobarbital or polychlorinated biphenyls. 4. The activity of microsomal cholinephosphotransferase decreased significantly after the treatment with both 3-methylcholanthrene and polychlorinated biphenyls and increased slightly after phenobarbital administration. 5. The observed opposite effects of phenobarbital and 3-methylcholanthrene on the enzymes in de novo phosphatidylcholine synthesis indicate that there exist a possible relation between induction of microsomal drug-metabolizing system and modulation of phosphatidylcholine biosynthesis in animal liver.

Animals↗
PubMed1989

Induction of phosphatidylcholine biosynthesis via CDPcholine pathway in lung and liver of rats following intratracheal administration of DDT and endosulfan.

The induction of phosphatidylcholine (PC) biosynthesis via the CDPcholine pathway in lung and liver of rats has been shown following the intratracheal administration of 1,1,1-trichloro-2m2-bis(p-chlorophenyl) ethane (DDT) (5 mg/100 g body weight) and endosulfan (1 mg/100 g body weight) for 3 days. Controls received only the vehicle solution (groundnut oil, 0.1 m1/100 g body weight). The treatment of DDT and endosulfan significantly increased the PC contents and the incorporation of radioactive [methyl-3H]choline into PC of lung and liver microsomes. The incorporation of radioactive [methyl-14C]methionine into microsomal PC of lung and liver was not affected significantly by treatment with either of the insecticides. 1,4,5,6,7-hexachloro-5-norbornene-2,3-dimethano cyclic sulfite (endosulfan) administration significantly increased the activity of choline kinase and phosphocholine cytidylyltransferase (both cytosolic and microsomal) of lung, whereas DDT increased the activity of only latter. In liver, both DDT and endosulfan administration significantly increased the activity of choline kinase and phosphocholine cytidylyltransferase (both cytosolic and microsomal). However, the activity of phosphocholinetransferase was not affected in both lung and liver microsomes of rats treated with these insecticides. The PC precursor pool sizes, choline and phosphorylcholine, of lung and liver tissues were not altered by DDT and endosulfan treatments. The present results suggest that the increased level of PC and incorporation of radioactive [methyl-3H]choline into microsomal PC could be the result of increased activity of choline kinase and phosphocholine cytidylyltransferase of lung and liver of rats following intratracheal administration of DDT and endosulfan.

Animals↗
PubMed1987

Phosphatidylcholine metabolism in neonatal mouse calvaria.

Phosphatidylcholine metabolism was examined in neonatal mouse calvaria in vitro. Incorporation of choline into phosphatidylcholine was slow in this tissue. At 2 h after a pulse of [methyl-3H]choline only 30% of the tissue radioactivity was in the organic phase. Chromatography of the aqueous phase of the tissue extract revealed that more than half of the radioactivity was present as choline at this time. There was no accumulation of phosphocholine, which would have been expected if the cytidylyltransferase were the rate-limiting step in the CDP-choline pathway in the tissue. Choline kinase activity in calvarial cytosol was lower than choline kinase activity in liver cytosol of the same animals. No evidence for significant phosphatidylcholine synthesis through the methylation pathway was found in the calvarial tissue. Although rates of choline-phosphatidylcholine base exchange were higher in bone microsomes than in microsomes from liver, the rate of phosphatidylcholine production through this pathway appeared to be too slow to account for the phosphatidylcholine produced by the calvaria. Phosphatidylcholine synthesis in the calvaria was unaffected by 2 h of treatment with 10 nM-parathyroid hormone, 0.1 nM-0.1 microM-1 alpha,25-dihydroxycholecalciferol, 5 microM-prostaglandin E1 or 2.5 nM-salmon calcitonin, or by 17 h of treatment with 10 nM-parathyroid hormone or 0.1 nM-1 alpha,25-dihydroxycholecalciferol.

Animals↗
PubMed1993

Derangements in acetylcholine metabolism in brain synaptosomes in chronic renal failure.

Chronic renal failure causes abnormalities in the central nervous system function and in norepinephrine metabolism of brain synaptosomes. The present study examined the effect of renal failure on the metabolism of another neurotransmitter, acetylcholine, which is involved in the modulation of behavioral and motor function. We measured acetylcholine content and release, choline content, uptake and release and activity of choline kinase in synaptosomes from rats with renal failure with various duration, renal failure-parathyroid-ectomized rats maintained normocalcemic, renal failure and normal rats treated with verapamil. Acetylcholine content increased while choline content decreased proportionally and significantly (P < 0.01) with the duration of renal failure; choline kinase activity was reduced (P < 0.01). These derangements were prevented by parathyroidectomy of renal failure rats or by their treatment with verapamil. Choline uptake and release were elevated in renal failure and these abnormalities were not corrected by parathyroidectomy or verapamil therapy. Acetylcholine release was elevated in renal failure and parathyroidectomy prevented this derangement. Verapamil reduced acetylcholine release in both normal and renal failure rats. The data show that: (a) renal failure causes significant derangements in acetylcholine metabolism leading to its accumulation in and an increase in its release from brain synaptosomes; (b) this is mainly due to reduced activity of choline kinase, most likely, mediated by the state of secondary hyperparathyroidism of renal failure; (c) blocking the parathyroid hormone-induced calcium influx into synaptosomes by verapamil prevented the abnormalities in acetylcholine metabolism; and (d) the derangement in choline uptake and release in CRF is not related to excess parathyroid hormone since parathyroidectomy or verapamil treatment did not correct them.

Acetylcholine↗
PubMed1987

The regulation of phosphatidylcholine biosynthesis in rye (Secale cereale) roots. Stimulation of the nucleotide pathway by low temperature.

The incorporation of [14C]choline chloride and [14C]glycerol into segments taken from rye (Secale cereale L., cv. Rheidal) roots was greater in segments from roots grown at 5 degrees C than in segments taken from roots growing at 20 degrees C. The incorporation was measured at the temperature at which the root had been growing. Measurements in vitro of the enzymes of the nucleotide pathway showed activity of choline kinase (EC 2.7.1.32), choline-phosphate cytidylyltransferase (EC 2.7.7.15) and cholinephosphotransferase (EC 2.7.8.2) to be higher in homogenates from the cooler roots when assayed at 5 degrees C than the activities assayed at 20 degrees C in the 20 degrees C-root homogenates. Changes in vivo in the pool sizes of the CDP-base intermediates with temperature, relative differences in nucleotide-pathway-enzyme activities and a pulse-chase experiment with [14C]choline indicated that the rate-limiting step for phosphatidylcholine biosynthesis in this tissue, at both temperatures, was the reaction catalysed by cytidylyltransferase.

Choline↗
PubMed1984

Effect of ethyl choline mustard on choline dehydrogenase and other enzymes of choline metabolism.

The effect of ethyl choline mustard (ECMA), and effective irreversible inhibitor of choline transport, was investigated on the enzymes of choline metabolism. ECMA at concentrations of 50 microM hardly affected choline acetyltransferase and caused only a 20% inhibition of choline kinase at a concentration of 1 mM. However, the mustard was an extremely effective inhibitor of choline dehydrogenase, producing 50% inhibition at concentrations of 6 microM. The inhibition was prevented by incubation in the presence of choline or by prior reaction of the mustard with thiosulphate. Separation of the components of the ECMA solution on TLC suggested that only the compound with an aziridine ring was an effective inhibitor of choline dehydrogenase. The inhibition was resistant to the washing out of excess unreacted mustard. The rate constant of inhibition was 395 M-1 X S-1. By the use of [3H]ECMA a single polypeptide in the enzyme preparation having a MW of 67,000 was labelled. The labelling was thiosulphate-sensitive and prevented by incubation with choline. It is concluded that ECMA is an irreversible inhibitor of choline dehydrogenase. It is at least as effective an inhibitor of choline dehydrogenase as of the choline transport system, although it does not appreciably inhibit choline acetyltransferase or choline kinase in the micromolar range.

Alcohol Oxidoreductases↗
PubMed2004

Molecular causes of the aberrant choline phospholipid metabolism in breast cancer.

Proton magnetic resonance spectroscopy ((1)H MRS) consistently detects significant differences in choline phospholipid metabolites of malignant versus benign breast lesions. It is critically important to understand the molecular causes underlying these metabolic differences, because this may identify novel targets for attack in cancer cells. In this study, differences in choline membrane metabolism were characterized in breast cancer cells and normal human mammary epithelial cells (HMECs) labeled with [1,2-(13)C]choline, using (1)H and (13)C magnetic resonance spectroscopy. Metabolic fluxes between membrane and water-soluble pool of choline-containing metabolites were assessed by exposing cells to [1,2-(13)C]choline for long and short periods of time to distinguish between catabolic and anabolic pathways in choline metabolism. Gene expression analysis using microarrays was performed to understand the molecular mechanisms underlying these changes. Breast cancer cells exhibited increased phosphocholine (PC; P < 0.001), total choline-containing metabolites (P < 0.01), and significantly decreased glycerophosphocholine (P < 0.05) compared with normal HMECs. Decreased (13)C-enrichment was detected in choline (P < 0.001) and phosphocholine (P < 0.05, P < 0.001) of breast cancer cells compared with HMECs, indicating a higher metabolic flux from membrane phosphatidylcholine to choline and phosphocholine in breast cancer cells. Choline kinase and phospholipase C were significantly overexpressed, and lysophospholipase 1, phospholipase A2, and phospholipase D were significantly underexpressed, in breast cancer cells compared with HMECs. The magnetic resonance spectroscopy data indicated that elevated phosphocholine in breast cancer cells was primarily attributable to increased choline kinase activity and increased catabolism mediated by increased phospholipase C activity. These observations were consistent with the overexpression of choline kinase and phospholipase C detected in the microarray analyses.

Breast Neoplasms↗