[Choline requirement of laying hens. 3. The effect of a varying choline supply on the choline content of the liver, body and eggs and on the brain and gallbladder weights].
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Animal Feed↗
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[Potentiating effect of choline on the action of phenylacetate. 3. Effect of choline analogues and choline esters on the isolated guinea pig ileum].
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Amino Acids↗
Lipid monolayers: action of phospholipase A of Crotalus atrox and Naja naja venoms on phosphatidyl choline and phosphatidal choline.
The activity of phospholipase A on phosphatidyl choline and phosphatidal choline spread as monolayers on phosphate buffers containing snake venom (Crotalus atrox or Naja naja) was studied by measuring the fall of surface potential as a function of time, pH, film pressure, temperature, and concentrations of phosphate and venom. At 25 degrees C, pH 7.0, and 0.2 micrograms of venom per ml, optimal activity was observed with both venoms on both substrates at 12 dynes/cm film pressure on 0.04 m phosphate. Under these conditions, the pH optimum for C. atrox was broad (6.6-7.4) and that for N. naja was sharp (8.0) for the action on phosphatidyl choline, whereas both venoms had a sharp optimum at pH 8.0 in their action on phosphatidal choline. The optimal temperature with phosphatidyl choline was 27.5 degrees C for N. naja and 40 degrees C for C. atrox. In line with studies of phospholipase A activity in bulk phase in ether, phosphatidal choline was attacked much more slowly than phosphatidyl choline by C. atrox. Under conditions where both venoms had equal activity on phosphatidyl choline, C. atrox was only half as active as N. naja on phosphatidal choline. The studies suggest that the linkage of the hydrophobic chains in glycerophosphatides may affect their interaction with proteins.
Animals↗
The effect of cytidine-diphosphate choline (CDP-choline) on brain lipid changes during aging.
Lipid synthesis has been tested in vivo in different brain areas of 12-month-old male rats. Cortex, striatum, brainstem, and subcortex of brain have been examined. The cerebellum was discarded. Mixtures of (2-3H)glycerol and (Me-14C)choline were injected into the lateral ventricle of the brain as lipid precursors, and their incorporation into total lipid, water-soluble intermediates and choline-containing phospholipids was examined 1 hr after isotope injection. In another series of experiments cytidine-5'-diphosphate choline (CDP-choline) was injected intraventricularly to the aged rats 10 min before sacrifice with a simultaneous injection, and radioactivity assays were performed as above. Distribution of radioactivity content of CDP-choline among brain areas 10 min after its administration showed a noticeable enrichment of the nucleotide and water-soluble-related compounds in the examined areas, but to a lesser degree in the cerebral cortex. The incorporation of labelled glycerol, which is severely depressed in aged rats in all four areas [Gaiti et al, 1982, 1983], was increased only in the cortex, and apparently decreased in the other areas. This last result is probably due to a dilution effect brought about by the administered cold CDP-choline upon the (14C)-containing water-soluble metabolites. As a consequence, the (3H)/(14C) ratio in total lipid and in isolated phosphatidylcholine and choline plasmalogen increased after CDP-choline treatment.
Aging↗
Evidence for the extreme overestimation of choline acetyltransferase in human sperm, human seminal plasma and rat heart: a case of mistaking carnitine acetyltransferase for choline acetyltransferase.
Detection of choline acetyltransferase (ChAc) in a number of non-neuronal tissues has been extremely overestimated. There are two major types of errors encountered. Type 1 error occurs when endogenous substrates (e.g. L-carnitine) are acetylated by acetyltransferase enzymes (e.g. carnitine acetyltransferase ( CarAc ) ) yielding an acetylated product mistaken for acetylcholine (AcCh). In the past, human sperm and human seminal plasma putative ChAc activity has been extremely overestimated due to Type 1 error. This study demonstrates (1) an endogenous acetyltransferase and substrate activity in human sperm and human seminal plasma forming an acetylated product that is not AcCh but probably acetylcarnitine ( AcCar ); (2) that the addition of 5 mM choline substrate does not significantly increase acetyltransferase activity; (3) that boiled seminal plasma contains an endogenous acetyltransferase substrate which is not choline, but probably L-carnitine. Type 2 error occurs when endogenous carnitine acetyltransferase synthesizes true AcCh, resulting in mistaken evidence for ChAc. This is demonstrated by the fact that the choline substrate Km-value for the neuronal or true ChAc from mouse brain is 0.73 +/- 0.06 mM while the Km-value of choline substrate for purified CarAc from pigeon breast muscle is 108 +/- 4 mM. Type 2 error has occurred for the estimation of putative ChAc in rat heart. The rat heart ChAc was measured in previous studies utilizing a concentration of 30 mM choline substrate. While saturation of neuronal ChAc is observed at 2-5 mM choline, saturation of the rat heart CarAc enzyme is not reached until over 800 mM. Purified CarAc significantly synthesizes AcCh at 30 mM choline. Thus, putative ChAc has been greatly overestimated in the scientific literature for mammalian sperm, human seminal plasma and rat heart.
Acetylation↗
Global in vivo replacement of choline by N-aminodeanol. Testing a hypothesis about progressive degenerative dementia: I. Dynamics of choline replacement.
Severe disruption of certain cholinergic pathways is a characteristic feature of Alzheimer's disease. Attempts to establish animal models by interfering with cholinergic function have not been very successful. We now present data which show a substantial and progressive replacement of free and phospholipid-bound choline by the novel choline isostere N-amino-N,N-dimethylaminoethanol during its dietary administration in place of choline. Free choline in blood fell to approximately 20% of controls after 10 to 30 days on diet. Phospholipid-bound choline in plasma was reduced to less than 15%, and in erythrocytes to about 22%. After 120 days of diet free and bound choline were reduced in most tissues to approximately 30% of controls. Only liver retained more than 80% of free choline. Acetylcholine was decreased to 33 to 50% of control. Total true and false transmitter in experimental animals was in all tissues less that acetylcholine in controls, suggesting that muscarinic transmission would be impaired. Moderate reduction of choline acetyltransferase activity was seen in striatum and myenteric plexus, and of QNB-binding in hippocampus, striatum and myenteric plexus.
Acetylation↗
AF64A-induced cytotoxicity and changes in choline acetyltransferase activity in the LA-N-2 neuroblastoma cell line are modulated by choline and hemicholinium-3.
The cholinergic neurotoxin AF64A (ethylcholine aziridinium) has been used to selectively destroy the cholinergic system. Due to its structural similarity to choline, this compound may be selectively taken up by the cholinergic terminal via the high-affinity choline transport (HAChT) system to produce persistent and selective cholinergic deficits. The mechanism by which it exerts its cholinotoxicity remains to be elucidated. We have examined the effects of AF64A in the human neuroblastoma cell line, LA-N-2, which has an intact sodium-coupled choline uptake system, and is capable of synthesizing acetylcholine (ACh). AF64A (25, 50 and 100 microM) produced dose-dependent increases in cell kill as measured by colony formation assay. The addition of increasing concentrations (10(-5), 10(-4) and 10(-3) M) of choline and hemicholinium-3 (HC-3) protected the cells from the cytotoxic effects of AF64A. At the same doses, AF64A also decreased choline acetyltransferase (ChAT) activity. In the presence of the highest concentration of choline or HC-3 (10(-3) M) which produced complete protection against AF64A's cytotoxicity in the colony formation assay, ChAT activity was restored to control values. These results demonstrate that agents that utilize (i.e., choline) or inhibit (i.e., HC-3) the choline uptake system prevented AF64A-induced cytotoxicity and decreases in ChAT activity, in a manner similar to that which has been observed in chick and rat primary cholinergic cultures in vitro. The LA-N-2 neuroblastoma cell line thus serves well as an in vitro model of the cholinergic neuron and provides a useful system to study the mode of cholinotoxicity induced by AF64A.
Animals↗
Choline derived from the phosphatidylcholine specific phospholipase D is not directly available for the CDP choline pathway in phorbol ester-treated C3H10T1/2 Cl 8 fibroblasts.
We have shown that 12-O-tetradecanoylphorbol 13-acetate (TPA) increases protein kinase C (PKC)-mediated choline transport, incorporation of choline into phosphatidylcholine (PtdCho) and PtdCho degradation by phospholipase D (PLD) in C3H10T1/2 Cl 8 cells. Dual prelabeling experiment using [3H]/[14C]choline indicated that intracellular choline generated from the PLD reaction was not directly recycled to PtdCho synthesis within the cell, and that a large fraction of the choline was transported out of the TPA-treated cells. In contrast, medium derived choline was preferably channeled to PtdCho synthesis. These results indicate that in TPA-treated cells, the choline derived from the PKC-mediated increased PLD activity and the choline newly taken up by the cell behave as two distinctly different metabolic pools.
Animals↗
Differential effects of nerve growth factor on expression of choline acetyltransferase and sodium-coupled choline transport in basal forebrain cholinergic neurons in culture.
Nerve growth factor (NGF) treatment of primary cultures of embryonic day 17 rat basal forebrain differentially altered activity of choline acetyltransferase (ChAT) and high-affinity choline transport; ChAT specific activity was increased by threefold in neurons grown in the presence of NGF for between 4 and 8 days, whereas high-affinity choline transport activity was not changed relative to control. Dose-response studies revealed that enhancement of neuronal ChAT activity occurred at low concentrations of NGF with an EC50 of 7 ng/ml, with no enhancement of high-affinity choline transport observed at NGF concentrations up to 100 ng/ml. In addition, synthesis of acetylcholine (ACh) and ACh content in neurons grown in the presence of NGF for up to 6 days was increased significantly compared with controls. These results suggest that regulation of ACh synthesis in primary cultures of basal forebrain neurons is not limited by provision of choline by the high-affinity choline transport system and that increased ChAT activity in the presence of NGF without a concomitant increase in high-affinity choline transport is sufficient to increase ACh synthesis. This further suggests that intracellular pools of choline, which do not normally serve as substrate for ACh synthesis, may be made available for ACh synthesis in the presence of NGF.
Animals↗
Effects of prolonged (1 year) choline deficiency and subsequent re-feeding of choline on 1,2-sn-diradylglycerol, fatty acids and protein kinase C in rat liver.
Rats fed a choline-deficient diet develop foci of enzyme-altered hepatocytes with subsequent formation of hepatic tumors. They also develop fatty livers, because choline is needed for hepatic secretion of lipoproteins. We have previously reported that 1,2-sn-diradylglycerol accumulates in the livers of rats fed a choline-deficient diet for 1-27 weeks, and that protein kinase C activity in the hepatic plasma membrane is elevated during that time (da Costa et al., J. Biol. Chem., 268, 2100-2105, 1993). In the present study, we examined the changes that occur in rat liver at 52 weeks of choline deficiency and determined whether these changes were reversible when choline was returned to the diet of the deficient animals for 1 or 16 weeks. At 52 weeks, non-tumor liver samples from the experimental animals had increased 1,2-sn-diradylglycerol concentrations in the lipid droplets compared with control animals. Plasma membrane 1,2-sn-diradylglycerol levels in the liver did not differ between the two groups, but an age-related increase in membrane 1,2-sn-diradylglycerol concentrations was observed. Unsaturated free fatty acids, another activator of protein kinase C, accumulated in the deficient livers. Protein kinase C activity associated with the plasma membrane remained significantly elevated at 52 weeks in deficient livers. Hepatic foci expressing gamma-glutamyltranspeptidase were detected only in the deficient rats (0.83% of liver volume) and 15% of these rats had hepatocellular carcinoma at 1 year on the diet. At 53 weeks (1 week after choline was returned to the deficient group), 1,2-sn-diradylglycerol concentrations in the lipid droplets and hepatic free fatty acids had dropped to control levels. By 68 weeks (16 weeks of re-feeding choline), the membrane protein kinase C activity had returned to normal. At this time, 14% of the experimental animals had hepatocellular carcinoma. We suggest that choline deficiency altered the protein kinase C-mediated signal transduction within liver and this contributed to hepatic carcinogenesis in these animals.
Aging↗
Acetylation of choline and homocholine by membrane-bound choline-O-acetyltransferase in mouse forebrain nerve endings.
The choline analog homocholine is not acetylated in vitro by choline-O-acetyltransferase (ChAT, EC 2.3.1.6), which is solubilized by 100 mM-sodium phosphate buffer washes of a crude vesicular fraction of mouse forebrain. However, both homocholine and choline are acetylated by a form of ChAT which is nonionically associated with a subcellular fraction of mouse forebrain containing membrane-associated organelles and occluded acetylcholine (P4). Acetylation of homocholine by membrane-associated ChAT is saturable. 4-(1-Naphthylvinyl)pyridine (NVP) inhibits the acetylation of both choline (60%) and homocholine (40%) by membrane-associated ChAT but reduces the acetylation of choline alone by soluble ChAT (76%). Choline and homocholine serve as competitive alternative substrates for the same membrane-associated ChAT, whereas homocholine acts only as a competitive inhibitor of choline acetylation by soluble ChAT. Acetylhomocholine competitively inhibits the acetylation of choline by both soluble and membrane-associated ChAT more dramatically than does the natural end product, acetylcholine.
Acetylation↗
Influence of choline and ethanolamine administration on choline and ethanolamine phosphorylating activities of mouse liver and kidney.
The administration of ethanolamine to adult male mice resulted in a significant increase in ethanolamine kinase activity in liver and kidney. Similarly, choline administration resulted in a significant increase in choline kinase activity in liver and kidney. The administration of ethanolamine resulted in enhancement of choline kinase activity concomitantly with ethanolamine kinase activity in liver and kidney. The administration of choline, however, did not result in any significant increase in ethanolamine kinase activity in liver or kidney. Cycloheximide administration along with choline-ethanolamine prevented the increase in kinase activity in liver and kidney. The results obtained have been discussed in relation to the regulatory role of choline kinase and ethanolamine kinase by de novo synthesis in response to enhanced substrate concentration, the secondary nature of choline kinase induction on ethanolamine administration, and possible distinction between choline kinase and ethanolamine kinase.
Animals↗
Hepatic free choline and betaine and the utilization of dietary protein in the choline-deficient rat.
In the rat, exclusion of choline from a carbohydrate-rich, 10% casein diet, resulted in a drastic reduction of betaine in the liver. The amount of unesterified choline was not lowered to the same extent. Both unesterified choline and total lipids in the liver were increased by rats fed a choline-deficient diet supplemented with 0.5% L-methionine compared to rats given an unsupplemented choline-deficient diet. The amount of betaine was low in both groups compared to choline-supplemented rats. Dietary choline deficiency lowered the biological value of casein only slightly. Statistically significant effects were seen only in some of the experiments when potato starch was used as the carbohydrate source but not when maize starch was used. The choline-deficient potato starch diet gave rise to a more severe liver fattening that the corresponding maize starch diet.
Adipose Tissue↗
Choline incorporation by Schistosoma mansoni: distribution of choline metabolites during development and after sexual differentiation.
Choline metabolism was investigated in Schistosoma mansoni during the main phases of its development, namely, schistosomula, 11- and 15-day-old worms, and adults. At the physiological choline concentration used in the assay (20 microM), betaine was, along with phosphatidylcholine, one of the most abundant choline metabolites, revealing considerable choline oxidation activity. Very little radioactivity was associated with CDP-choline, whereas a sustained incorporation into phosphocholine occurred. These results provide good evidence that CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) plays a regulatory role in the de novo pathway of phosphatidylcholine biosynthesis. During development, the incorporation of choline into its various metabolites was maximal in 11-day-old worms. At this stage, the oxidative pathway predominated over the Kennedy pathway, whereas at all other stages the de novo phosphatidylcholine biosynthesis was predominant. Furthermore, choline incorporation into betaine was much more important in the adult female worm than in the male, indicating a major difference in choline incorporation and distribution between the 2 sexes of the adult worms.
Animals↗
Environmental temperature and choline requirements in rats. II. Choline and methionine requirements for lipotropic activity.
Young rats were fed choline-deficient diets and maintained at different environmental temperatures. The hepatic lipid level remained normal in rats at 2 degrees when 25 mg of choline per 100 g of food was fed; 50 mg of choline per 100 g food was required at 21 degrees and 100 mg of choline per 100 g food at 33 degrees to prevent excessive lipid accumulation. These values were equivalent to a mean daily intake per rat of 3 mg of choline at 2 degrees, 5.5 mg at 21 degrees, and 7 mg at 33 degrees respectively. When the growth rate was slower owing to a slight inadequacy of histidine in the basal choline-deficient diet, normal hepatic lipid was maintained by supplements of 50 mg of choline per 100 g food at 21 degrees and 33 degrees. Increasing the methionine content of the diet two- or three-fold from a basal value of 340 mg per 100 g food was as effective as 200 mg of choline per 100 g of food in lowering hepatic lipids at 2 degrees, 21 degrees, and 33 degrees.
Animals↗
Reciprocal regulation of choline acetyltransferase and choline kinase in sympathetic neurons during cholinergic differentiation.
The regulation of the synthesis of acetylcholine and phosphatidylcholine in rat sympathetic neurons was examined in the context of cholinergic differentiation. We demonstrate that the activities of choline acetyltransferase (ChAT) and choline kinase (CK) are inversely affected by treatment of sympathetic neurons with retinoic acid, utilized as an agent that induces cholinergic differentiation. Whereas ChAT specific activity increased 2- to 4-fold after 12 days of treatment with 5 microM retinoic acid, CK specific activity decreased by 25-30%. These changes in enzyme activities were essentially reflected in the incorporation of [methyl-3H]choline into ACh and the metabolites of the CDP-choline pathway for phosphatidylcholine synthesis. When sympathetic neurons were treated under high potassium conditions (50 mM) for 12 days, the specific activity of CK increased 1.3-fold whereas the activity of ChAT decreased by up to 90%. Furthermore, experiments in which the incorporation of [methyl-3H]choline into ACh and the metabolites of the CDP-choline pathway was measured in the absence of Na+ or in the presence of hemicholinium-3 (HC-3), demonstrate that CK has access to the same pool of choline utilized by ChAT. These results provide evidence that the activities of ChAT and CK may be inversely regulated during the process of cholinergic differentiation.
Acetylcholine↗
Isolation of choline and choline esters from Krebs-Ringer solution for gas chromatographic determination.
A simple and efficient novel method for isolating picomole amounts of choline and choline esters in milliliter volumes of Krebs-Ringer solution has been developed. The procedure is based on the observation that the solubility of choline esters in acetonitrile is 10(4)-10(5) times higher than that of the inorganic salt constituents of Krebs-Ringer solution. The glucose content of the medium, which prevented the one-step isolation of choline esters based on acetonitrile extraction from its lyophilizate, was removed using Amberlite CG-50 column chromatography. Bound compounds to the column were eluted in 0.25 N HCl and lyophilized. The lyophilizate was extracted with acetonitrile, which was then decanted and eliminated by evaporation to dryness. The resultant glucose and salt-free residue can be assayed by gas chromatography. Total recoveries of added choline and choline esters over the entire isolation procedure, measured isotopically and/or gas chromatographically, were 93 and 97%, respectively. Due to the high and close-to-equal recoveries of choline esters, and the high purity of the final product, this procedure is suitable for estimating acetylcholine and choline in neural tissue perfusates by gas chromatography, as was demonstrated by this method using hippocampal slices.
Acetonitriles↗
Measurement of choline and choline metabolite concentrations using high-pressure liquid chromatography and gas chromatography-mass spectrometry.
We have developed a reproducible and sensitive procedure for the isolation and measurement of choline, phosphocholine, glycerophosphocholine, phosphatidylcholine, lysophosphatidylcholine and acetylcholine in a single 100-mg sample of biological tissue. Tissues were spiked with 14C-methyl- and 2H-methyl- or 15N-choline labeled internal standards for each compound. They were extracted with chloroform/methanol/water and the aqueous and organic phases were dried. The organic phase was resuspended in chloroform/methanol (1/1, v/v) and an aliquot was applied to a silica-gel thin-layer chromatography plate. The plate was developed in chloroform/methanol/water (65/30/4, v/v). Segments which cochromatographed with external standards of phosphatidylcholine and lysophosphatidylcholine were stained, scraped, and hydrolyzed in 6 M methanolic-HCl at 80 degrees C for 60 min, liberating free choline. The aqueous phase was resuspended in methanol/water and injected onto a silica HPLC column. Choline and its metabolites were eluted using a binary nonlinear gradient of acetonitrile/ethanol/acetic acid/1 M ammonium acetate/water/0.1 M sodium phosphate (800/68/2/3/127/10, v/v changing to 400/68/44/88/400/10, v/v). Peaks were detected with an on-line radiometric detector, collected, and dried under vacuum. Each choline ester was digested in 6 M HCl at 80 degrees C to form choline. Choline was then converted to the propionyl ester and demethylated with sodium benzenethiolate. This volatile derivative was then isolated using gas chromatography and measured with a mass selective detector. Deuterated internal standards were used to correct for variations in recovery. Choline, glycerophosphocholine, phosphocholine, phosphatidylcholine, lysophosphatidylcholine, and acetylcholine were measured in rat liver, heart, muscle, kidney, plasma, red blood cells, and brain and in human plasma.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗