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PubMed1984

Determination of choline in erythrocytes using high-resolution proton nuclear magnetic resonance spectroscopy: comparison with a choline oxidase method.

Detailed operating conditions are reported for the determination of choline in human erythrocytes using proton nuclear magnetic resonance spectroscopy in conjunction with the inversion-recovery spin-echo pulse sequence. The results of the NMR method were in excellent agreement with those obtained using an enzymatic (choline oxidase) assay; however, they were approximately three times higher than those reported using gas chromatography/mass spectrometry techniques. The differences may be partly due to the method of preparing or sampling cells since there is a distribution of choline in cells of different ages. However, choline levels were not affected by the methods used in the present study for storing or preparing cells.

Alcohol Oxidoreductases↗
PubMed1994

Choline acetyltransferase and acetylcholinesterase in the hippocampus of aged rats: sensitivity to choline alphoscerate treatment.

The influence of aging on the acetylcholine synthesising and the degrading enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) was studied in the hippocampus of male Wstar rats at 2 months (young), 12 months (adult) and 27 months (old) of age using biochemical, immunocytochemical and histochemical techniques. The influence of treatment for 6 months with a daily dose of 100 mg/kg of choline alphoscerate (L-alpha-glycerylphosphorylcholine) on the parameters examined was also investigated in old rats. Biochemical analysis of ChAT and AChE revealed the highest of the enzymatic activities in the hippocampus of adult rats and no significant differences between young and old animals. Immunocytochemical analysis of ChAT immunoreactivity revealed the highest immunostaining in adult rats followed in descending order by young then old animals. Histochemical evaluation of AChE reactivity revealed the highest expression in adult rats followed in descending order by old then young animals. Biochemical analysis of the effects of choline alphoscerate did not reveal any effect on ChAT activity and in increased expression of AChE activity. Moreover, the compound restored, in part, ChAT immunoreactivity in the hippocampus of old rats and increased the expression of AChE reactivity primarily in the CA3 sub field in old rats. The above results suggest that appropriate quantitative immunocytochemical and histochemical techniques may represent a useful tool for assessing age-dependent changes in cholinergic neurotransmission markers. The functional and pharmacological significance of the effects of choline alphoscerate on the expression of ChAT and AChE in the hippocampus of aged rats should be clarified in future studies.

Acetylcholinesterase↗
PubMed1993

Liquid chromatography with electrochemical detection for quantitation of bound choline liberated by phospholipase D hydrolysis from phospholipids containing choline in rat plasma.

This report describes the optimal conditions for the determination of bound choline in rat plasma. The method used was based on the liberation of choline by phospholipase D from phospholipids containing choline in plasma, followed by high-performance liquid chromatographic analysis. Normal concentrations of total, free and bound choline in rat plasma were found to be 1278.7 +/- 132.5, 11.5 +/- 2.2 and 1267.2 +/- 125.5 nmol/ml, respectively. The described procedure has the advantages of rapidity, specificity, excellent precision and the need for only a small amount of the sample.

Animals↗
PubMed1972

Choline metabolism in the cerebral cortex of guinea pigs. Phosphorylcholine and lipid choline.

1. The labelling of phosphorylcholine and choline-containing phospholipids in the subcellular fractions of guinea-pig cerebral cortex after the intraventricular injection of [N-Me-(3)H]choline into conscious animals has been studied. Special emphasis was placed upon the synaptosome fraction and early time-periods after administration. 2. The labelling of phosphorylcholine was rapid compared with that of phospholipid and was confined to two distinct subcellular fractions: the soluble cytoplasmic fraction and the synaptosome fraction. Most of the labelled phosphorylcholine of the synaptosome fraction was readily released by osmotic rupture indicating location in the nerve-ending cytoplasm. The two pools of phosphorylcholine had similar specific radioactivities at all observed times. 3. (3)H-labelled phospholipid was found in all membranous fractions. The labelling was confined to choline-containing phospholipids, notably phosphatidylcholine. 4. The labelling of the different membranous fractions was similar. 5. The half-life of the choline-containing phospholipids in the synaptic vesicle fraction was very much greater than the acetylcholine in this fraction. 6. Evidence is presented that synthesis de novo of phosphatidylcholine at nerve terminals occurs in vivo.

Acetylcholine↗
PubMed1988

Affinity labelling and identification of the high-affinity choline carrier from synaptic membranes of Torpedo electromotor nerve terminals with [3H]choline mustard.

The physiological mechanisms regulating activity of the sodium-dependent, high-affinity choline transporter and the molecular events in the translocation process remain unclear; the protein has not been purified or characterized biochemically. In the present study, [3H]choline mustard aziridinium ion [( 3H]ChM Az), a nitrogen mustard analogue of choline, bound irreversibly to presynaptic plasma membranes from Torpedo electric organ in a hemicholinium-sensitive, and sodium-, time-, and temperature-dependent manner. Specific binding of this ligand was greatest when it was incubated with membranes in the presence of sodium at 30 degrees C. Separation of the 3H-labelled membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the radiolabel was associated with a polypeptide of apparent molecular mass of approximately 42,000 daltons; labelling of this species was abolished in membranes incubated with ligand in the presence of HC-3. Two other 3H-labelled polypeptides were detected, with apparent molecular masses of approximately 58,000 and 90,000 daltons; radiolabelling of the former was also HC-3 sensitive. [3H]ChM Az may be a useful affinity ligand in the purification of the choline carrier from cholinergic neurons.

Affinity Labels↗
PubMed1957

Some actions of substituted choline phenyl ethers, particularly of choline 2:6-xylyl ether.

Marked nicotine-like stimulant properties are possessed by choline phenyl ether and choline o-tolyl ether, and to a decreasing extent by choline 2:6-xylyl ether and choline 2:4:6-mesityl ether. The compounds all show neuromuscular blocking properties, which are of short duration and pass from mainly decamethonium-like to mainly curare-like as more methyl groups are added to the phenyl nucleus. This series of compounds also possesses muscarinic, weak anti-adrenaline and vasodilator properties, as well as long-lasting local anaesthetic effects in the two compounds tested by intradermal injection.

Choline↗
PubMed1998

Uptake and incorporation of choline and ethanolamine into lipoteichoic acid and teichoic acid by the choline-independent mutant JY2190 of Streptococcus pneumoniae.

Lipoteichoic acid- and teichoic acid-containing muropeptides were isolated from choline- or ethanolamine-grown cells of the choline-independent mutant JY2190 of Streptococcus pneumoniae. Choline was taken up and incorporated into lipoteichoic acid and teichoic acid with 81% efficiency, compared with the parent strain Rx1. With similar efficiency, ethanolamine was incorporated. Accordingly, the mutant is a valuable tool for identifying the individual genes encoding the enzymes of choline utilisation, because any of these genes can be deleted without affecting viability and growth rate.

Choline↗
PubMed1988

Long-term effects on biotransformation of labelled choline in different parts of the rat brain induced by single choline injections.

The long-term effects of single choline (Ch) injections on the uptake and metabolism of a tracer dose of 3H-Ch were studied in male rats. Choline was administered as a threshold infusion to obtain convulsions 10 and 4 weeks before sacrifice (group 1). At a single threshold infusion of choline 4 weeks before sacrifice no convulsions were induced in 50% of the animals in a second group (group 2--) whereas convulsions were induced in the remainder of the animals in this group (group 2+). Group 3 contained control animals. One min. after administration of a tracer dose of 3H-Ch the animals were sacrificed and examined for 3H-total activity, 3H-Ch, 3H-acetylcholine (3H-ACh) and 3H-phosphorylcholine (3H-PhCh). These activities were determined in three parts of the brain (cortex, striatum, midbrain + medulla oblongata). In the cortex a significant negative correlation between brain weight and 3H-ACh synthesis was seen in group 1. A comparison between group 2+ and group 2- indicated that induced convulsions were not critical for this effect. In the striatum there was a significant reduction in the total uptake of radioactivity in group 1 and group 2- when values were compared to the control group. Furthermore a significant positive correlation was found between the concentration of radiolabel and 3H-ACh synthesis and a negative relationship with the level of 3H-Ch. In the midbrain preparation the synthesis of 3H-ACh was reduced in group 1 where a significant negative correlation was found between the average threshold dose of choline and both 3H-ACh and 3H-PhCh synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

Distribution of choline acetyltransferase, acetylcholinesterase, muscarinic receptor binding, and choline uptake in discrete areas of the rat medulla oblongata.

Quantitative measurements were made of choline acetyltransferase (CAT) activity, acetylcholinesterase (AChE) activity and cholinergic muscarinic receptor binding ([3H]QNB) in eight areas of a cross-section of the rat medulla oblongata. A fourth cholinergic parameter, high-affinity choline uptake, was measured in three groups of these areas. CAT, AChE and [3H]QNB binding were found to be highest in the hypoglossal nucleus and the dorsal motor nucleus of the vagus; the lowest value was in the area which contains the inferior olive and the corticospinal tract. The distribution of AChE and CAT activities varied approximately 7- to 10-fold among the eight regions examined, whereas that of the muscarinic receptor varied only about 4-fold. The Na+-dependent high-affinity choline uptake varied approximately 20-fold from the region with the lowest activity (inferior olivary nucleus and corticospinal tract) to that with the highest activity (tissue areas containing the dorsal motor nucleus, hypoglossal nucleus of the solitary tract and nucleus cuneatus). The four cholinergic parameters are statistically correlated throughout all the areas of the medulla which were studied.

Acetylcholinesterase↗
PubMed1994

Striatal extracellular choline and acetylcholine in choline-free plasma rats.

Choline (Ch)-free plasma rats were induced successfully by intravenous (IV) injection of choline oxidase (ChO) (16). However, brain acetylcholine (ACh) levels were not affected by ChO treatment, maintaining the same levels as those in controls, although brain Ch levels were significantly decreased. To clarify the reasons for this, in vivo microdialysis was carried out in the treated rats' striata. The ChO treatment induced not only a 70% decrease of extracellular Ch levels but also a 40% decrease of extracellular ACh levels, reflecting the amount of ACh released from cholinergic terminals. In addition, plasma-bound Ch levels and choline acetyltransferase (CAT) and acetylcholinesterase (AChE) activities in the brain were examined in the rats receiving ChO treatment. No significant differences from controls were observed in these levels. The results suggest that: approximately 70% of striatal extracellular Ch is physiologically supplied from circulating plasma-free Ch; the inhibition of ACh release is related to the maintenance of tissue (intraneuronal) ACh levels under the condition of halting of the supply of free Ch from blood to the brain; if there is a compensative supply of free Ch from de novo synthesis, autocannibalism, or plasma-bound Ch, this may be supplied within neuronal cells, because the level of extracellular-free Ch maintained its depressed level even 11-14 h after ChO treatment.

Acetylcholine↗
PubMed2005

Differential expression and regulation of the high-affinity choline transporter CHT1 and choline acetyltransferase in neurons of superior cervical ganglia.

Previous studies revealed that leukemia inhibitory factor (LIF) and retinoic acid (RA) induce a noradrenergic to cholinergic switch in cultured sympathetic neurons of superior cervical ganglia (SCG) by up-regulating the coordinate expression of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter. Here, we examined the effect of both factors on high-affinity choline uptake (HACU) and on expression of the high-affinity choline transporter CHT1. We found that HACU and CHT1-mRNA levels are up-regulated by LIF and down-regulated by RA in these neurons. Thus, in contrast to LIF, RA differentially regulates the expression of the presynaptic cholinergic proteins. Moreover, we showed that untreated SCG neurons express HACU and CHT1-mRNAs at much higher levels than ChAT activity and transcripts. In intact SCG, CHT1-mRNAs are abundant and synthesized by the noradrenergic neurons themselves. This study provides the first example of CHT1 expression in neurons which do not use acetylcholine as neurotransmitter.

Acetylcholine↗
PubMed1978

Different time course of development for high-affinity choline uptake and choline acetyltransferase in the chick retina.

Synthesis and storage of [3H]acetylcholine in isolated pieces of chick retina increased in two stages during embryogenesis. The first increase coincided with a 100-fold rise in the activity of choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6), but during the second increase the activity of this enzyme remained essentially constant. The second increase instead was linked to an approximately 6-fold increase in the Vmax for high-affinity uptake of choline.

Animals↗
PubMed1989

Synaptosomal "membrane-bound" choline acetyltransferase is most sensitive to inhibition by choline mustard.

The objectives of the present study were to validate the presence of cytoplasmic and membrane-associated pools of choline acetyltransferase (ChAT) in rat brain synaptosomes, and to evaluate inhibition of these different forms of the enzyme by the nitrogen mustard analogue of choline, choline mustard aziridinium ion (ChM Az). The relative distribution of ChAT and lactate dehydrogenase (LDH) was followed in subfractions of synaptosomes to establish whether ChAT activity associated with salt-washed presynaptic membranes represents membrane-bound protein rather than cytosolic enzyme trapped within undisrupted synaptosomes or revesiculated membrane fragments. The percentage of total synaptosomal ChAT activity (14%) recovered in the final membrane pellet always exceeded that of LDH (6%), lending support to the hypothesis that much of the ChAT associated with the membranes was a membrane bound form of the enzyme. Incubation of purified synaptosomes with ChM Az led to irreversible inhibition of ChAT activity; this loss of enzyme activity could not be accounted for by lysis of nerve terminals during incubation in the presence of the mustard analogue. Subfractionation of the ChM Az-treated nerve terminals revealed that the membrane-bound form of ChAT was inhibited to the greatest extent, followed by the ionically membrane-associated enzyme, with the activity of the water-solubilized enzyme not differing significantly from control. Preparation of the synaptosomal ChAT subfractions from untreated nerve terminals prior to incubation with varying concentrations of ChM Az or naphthylvinylpyridine revealed that under these conditions water-solubilized, ionically membrane-associated, and detergent-solubilized membrane-bound pools of ChAT were not differentially inhibited by either compound.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Preneoplastic and neoplastic lesions in the pancreas of rats fed choline-devoid or choline-supplemented diets.

Groups of male Fischer 344 rats were chronically fed semipurified choline-devoid or choline-supplemented diets, high in fat (15%), and containing or not containing 0.06% phenobarbital. Atypical acinar cell nodules were observed in the pancreas of the rats, irrespective of the diet fed, with incidences varying from 38% to 100% in the various groups. No consistent differential effects of the dietary treatments on the incidence and growth of the nodules were evident, even though the diameter of the nodules tended to be greater in some of the groups fed the basal choline-devoid diet. The vast majority of the nodules were of the acidophilic type. More advanced pancreatic acinar cell lesions were observed in a few of the rats. Since the rats were not exposed to a chemical carcinogen(s), development of the nodules and of the more advanced lesions, even in rats fed the control diets, was most likely due to evolution of endogenous (spontaneous) initiated pancreatic cells, promoted primarily by the feeding of semipurified diets with a high fat content.

Animals↗
PubMed1983

Cerebral subcellular distribution of CDP-choline and/or its metabolites after oral administration of methyl-14C CDP-choline.

Distribution of radioactivity in subcellular components of a cerebral extract after oral administration of methyl-14C labelled cytidine diphosphate choline (CDP-choline, citicoline, Somazina) was analyzed. Animals were decapitated 24 h after the administration and the brain immediately removed and gently homogenized in a buffer solution. The homogenate was fractionated by differential centrifugation and the protein concentration and the radioactivity were measured in each precipitate as well as in the final supernatant. Radioactivity was found to be associated mainly with membranous microsomal precipitate and with cytoplasmic membrane precipitate. Alternatively, materials sedimenting between 500 X g and 20,000 X g were fractionated in a density gradient centrifugation. Fractions were screened for enzymatic activities as indicators of cytoplasmic membrane, mitochondria, lysosomes, microsomes and synaptosomes. Radioactivity was found to be associated with cytoplasmic and microsomal membrane fractions. Identification of some fractions as enriched in synaptosomes also accumulating radioactivity, as well as the possible incorporation of the radioactivity to choline and phosphorylcholine cerebral pools are discussed.

Administration, Oral↗
PubMed1990

Solubilization and partial characterization of [3H]choline mustard-labeled high-affinity choline carrier from presynaptic plasma membrane of Torpedo electric organ.

The objective of the present study was to characterize the hydrodynamic properties of the detergent-solubilized high-affinity choline carrier from presynaptic plasma membranes of electric organ of Torpedo marmorata following radioaffinity labelling with [3H]choline mustard aziridinium ion [( 3H]ChM Az). Membrane proteins were solubilized with 0.8% CHAPS, then analyzed by gel permeation chromatography and equilibrium density sedimentation in sucrose gradients made in H2O and D2O. The radiolabeled protein eluted from the gel filtration column with a distribution coefficient of 0.36 allowing calculation of a Stokes radius of 4.4 nm. Distribution of the [3H]ChM Az-labeled proteins differed in the H2O and D2O sucrose gradients, with apparent sedimentation coefficients of 5.2 and 4.7, respectively, indicating that detergent may be bound to hydrophobic regions of the protein. The calculated partial specific volume for the protein complex was 0.76. The estimate of molecular mass for the complex was 115,000 Da, with the protein portion having a molecular mass of about 83,000 Da. Analysis of the peak fractions from the gel filtration column and sucrose gradients by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a 3H-labeled polypeptide at 0,000-42,000 Da. The choline carrier may exist as a dimer of this polypeptide.

Animals↗
PubMed1983

The effect of choline deficiency on the activity of a phosphatidylcholine-requiring enzyme: activity and properties of UDP-glucuronyltransferase in choline-deficient rats.

The effect of choline deficiency on the kinetic properties of the microsomal enzyme UDP-glucuronyltransferase (EC2.4.1.17) was investigated in rats. Animals fed choline-deficient diets, as compared with animals fed a choline-replete diet or standard laboratory chow, showed almost a three-fold increase in enzyme activity when the enzyme was assayed at physiological concentrations of UDP-glucuronic acid (0.25 mM). The increase in activity appeared to be due to an enhanced affinity of the enzyme for UDP-glucuronic acid rather than to an increase in the amount of enzyme. These data indicate that the kinetic properties of tightly bound membrane enzymes are altered by a dietary change that is known to cause liver disease in the rat.

Animals↗