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PubMed2002

Cardiovascular effects of intracerebroventricularly injected CDP-choline in normotensive and hypotensive animals: the involvement of cholinergic system.

Intracerebroventricular (i.c.v.) administration of CDP-choline (0.25, 0.5, 1 and 2 micromol) induced prompt, dose- and time-dependent increase in blood pressure in normotensive rats. Equimolar dose of CDP-choline (1 micromol; i.c.v.) and choline (1 micromol; i.c.v.) caused similar increases in blood pressure while cytidine (1 micromol; i.c.v.) failed to produce any pressor effect. In haemorrhagic shock, CDP-choline (0.1, 0.25, 0.5 and 1 micromol; i.c.v.) increased blood pressure dose- and time-dependently. The complete reversal of hypotension was observed with the i.c.v. injection of CDP-choline (1 micromol) and choline (1 micromol). Cytidine (1 micromol; i.c.v.) produced small, but significant ( P<0.05) increase in blood pressure in haemorrhaged rats. Dose-related bradycardia was observed with the injection of CDP-choline in normotensive rats, but the changes in heart rate were not significantly different ( P>0.05) in hypotensive conditions. Choline levels in lateral cerebral ventricle and hypothalamus increased about nine- and fivefold, respectively, after CDP-choline (1 micromol) administration in normotensive rats. In haemorrhagic shock extracellular choline levels in hypothalamus increased sevenfold after an i.c.v. administration of CDP-choline (1 micromol). Hemicholinium-3 (20 microg; i.c.v.), a neuronal high affinity choline uptake blocker, and mecamylamine (50 microg; i.c.v.), nicotinic receptor antagonist, pretreatment abolished the pressor effect of CDP-choline in normal rats. The increase in blood pressure was also attenuated by atropine (10 microg; i.c.v.) pretreatment. Atropine blocked the bradycardic response observed after CDP-choline. In haemorrhaged rats, the pressor effect of CDP-choline was attenuated by hemicholinium-3 and mecamylamine while atropine failed to alter the pressor response to CDP-choline. I.c.v. CDP-choline increased plasma adrenaline and vasopressin levels in normal rats. Haemorrhage, itself, increased plasma catecholamines and vasopressin levels. CDP-choline (1 micromol) produced additional increases in the elevated plasma levels of these hormones. An alpha(1)-adrenoceptor blocker, prazosin (0.5 mg/kg; i.v.), or vasopressin V(1) receptor antagonist, [beta-mercapto, beta,beta-cyclopenta-methylenepropionyl(1), O-Me-Tyr(2)-Arg(8)]-vasopressin (10 micro/kg; i.v.), pretreatments partially blocked the pressor response to CDP-choline (1 micromol; i.c.v.). Simultaneous administration of these two antagonists completely blocked the pressor effect of CDP-choline in haemorrhagic shock. These results show that the exogenous administration of CDP-choline increases blood pressure and reverses hypotension in haemorrhagic shock. In normotensive conditions, increase in blood pressure appears to be due to the activation of both nicotinic and muscarinic central cholinergic receptors through the activation of presynaptic cholinergic mechanisms. In hypotensive rats, activation of nicotinic cholinergic receptors is solely involved in the pressor effect. Increase in plasma vasopressin and adrenaline mediates the pressor response of CDP-choline in both normotensive and hypotensive conditions.

Animals↗
PubMed1974

The distribution in the rabbit of choline administered by injection or infusion.

1. The concentration of choline in plasma, erythrocytes, skeletal muscle, heart, lung, liver, small intestine and kidneys and the changes that follow the injection or infusion of choline have been measured in rabbits anaesthetized with pentobarbitone.2. The concentration of choline in the plasma of arterial blood was 11.8 +/- 0.6 n-mole/ml. and in the erythrocytes 28.4 +/- 1.3 n-mole/ml. blood.3. All tissues contained a higher concentration of free choline than did plasma. The values range from 19.1 +/- 2.2 n-mole/g in skeletal muscle to 500 +/- 25 n-mole/g in the kidney.4. In order of their choline concentrations the tissues were intestine (duodenal end) > kidney > intestine (caecal end) > liver > lung > brain > heart > erythrocytes > (blood) > skeletal muscle > plasma, while in order of the contribution to the total body choline they were liver > intestine > skeletal muscle > (blood) > kidney > erythrocytes > lung > brain > plasma > heart. The total free choline determined by these analyses was between 30-40 mumole/kg body weight, about one third being present in the liver.5. The choline content of the small intestine varied along its length. The lowest amount being present in the portion adjoining the caecum.6. Within 1 min of the injection of choline 100 or 300 mumole/kg, 70-90% had left the circulation. The proportionate loss was higher after 100 mumole/kg than after 300 mumole/kg.7. The loss following 300 mumole/kg was increased if that dose were preceded by a dose of 100 mumole/kg 40 min earlier; this suggests some additional disposal mechanism(s) had been activated by the first dose.8. Three minutes after the injection of choline 300 mumole/kg, about 60% was present as free choline in the tissues studied. The order of the concentration increases was kidneys > liver > muscle > lung > small intestine (caecal end) > heart > intestine > small intestine (duodenal end) > brain.9. Forty minutes after the injection of choline 300 mumole/kg, only 11% could be accounted for as free choline. Only the levels in the kidney, liver, muscle and lung were significantly above normal at this time.10. Infusion of choline 0.8 mumole/kg. min or greater produced rises in plasma choline that corresponded to a clearance of 32 ml. plasma/kg. min.11. After the infusion of 300 mumole/kg over a period of 1 hr, raised levels of choline were detected in all tissues assayed, but the amount found accounted for only 14% of the choline administered. The concentrations in the kidney, liver and lung were similar to those found 40 min after the injection of 300 mumole/kg.12. There was no change in the concentration of choline in the erythrocytes after the injection of choline 100 or 300 mumole/kg, nor during the infusion of choline at the rate of 5 mumole/kg. min for 1 hr.13. The plasma volume appeared to be affected by the injection of the large doses of choline; after choline 300 mumole/kg the plasma volume was reduced. No effect on the plasma volume was observed during the infusion of the same dose.

Animals↗
PubMed1994

Phosphatidylcholine biosynthesis via the CDP-choline pathway in Saccharomyces cerevisiae. Multiple mechanisms of regulation.

Multiple mechanisms of regulation in the CDP-choline pathway for phosphatidylcholine (PC) synthesis were revealed by exploring the effects of choline and inositol on this pathway in Saccharomyces cerevisiae. At exogenous choline concentrations below 100 microM, phosphocholine cytidylyltransferase was rate-limiting; at higher choline concentrations the conversion of choline to phosphocholine by choline kinase became rate-limiting. Choline and inositol were found to regulate choline uptake; this established another regulatory mechanism by which PC synthesis is regulated in yeast. Inositol addition did not immediately affect labeled choline uptake or its incorporation into PC in actively dividing cells; however, preculturing the cells in the presence of choline decreased the rate of choline uptake, and this effect was amplified by the concomitant addition of inositol and choline. Additionally, a growth phase dependent effect of inositol supplementation was observed. Inositol addition to stationary phase cells resulted in an increase in choline uptake and subsequent PC production in these cells. This increase was shown to be due to an increase in the rate of choline transport into the cell. In the presence of inositol, choline transport is the main regulatory mechanism controlling flux through the CDP-choline pathway in S. cerevisiae. Inositol supplementation resulted in changes in the levels of enzyme activity detected in vitro. However, the effects observed in vivo correlated exclusively with changes in choline uptake. Choline transporter assays were consistent with these results. Since both the CPT1 and EPT1 gene products catalyze the cholinephosphotransferase reaction in vitro (Hjelmstad, R. H., and Bell, R. M. (1991) J. Biol. Chem. 266, 4357-4365), the effect of inositol on these two separate routes for PC biosynthesis was investigated. The data revealed that only cells harboring a functional CPT1 gene synthesized PC in vivo. These cells (ept1-delta 1::URA3) also displayed an identical mode of regulation in response to inositol as did cells containing an intact EPT1 gene (wild type) indicating there is no requirement for an alternate functional CDP-amino-alcohol pathway for inositol to regulate PC synthesis via the CDP-choline pathway.

Base Sequence↗
PubMed2005

Choline status in newborns, infants, children, breast-feeding women, breast-fed infants and human breast milk.

This study assessed the choline status in newborns, infants, children, breast-feeding women, breast milk, infant formula, breast-fed and formula-fed infants. The serum free choline level was 35.1+/-1.1 micromol/L at birth and decreased to 24.2+/-1.6, 18.1+/-0.8, 16.3+/-0.9, 14.3+/-0.8, 12.9+/-0.6 or 10.9+/-0.6 micromol/L at 22-28, 151-180, 331-365, 571-730, 731-1095 or 4016-4380 days after birth, respectively. The serum phospholipid-bound choline level was 1997+/-75 micromol/L at birth and increased gradually to 2315+/-190 or 2572 +/-100 micromol/L at 571-730 or 4016-4380 days after birth, respectively. In breast-feeding women, serum free and phospholipid-bound choline levels were doubled at 12-28 days after birth, they decreased toward the control values with time. Free choline, phosphocholine and glycerophosphocholine were major choline compounds in breast milk. Their concentrations in mature milk were much greater than in colostrum and serum. Choline contents of breast milk varied greatly between mothers, and milk free choline levels were correlated with serum free choline (r=.541; P<.001), phospholipid-bound choline (r=.527; P<.001) and glycerophosphocholine (r=.299; P<.01) concentrations and lactating days (r=.520; P<.001). In breast-fed infants, serum free choline concentrations were correlated with free choline (r=.47; P<.001), phosphocholine (r=.345; P<.002), glycerophosphocholine (r=.311; P<.01) and total choline (r=.306; P<.01) contents of breast milk. Serum free choline concentration in formula-fed infants was lower than breast-fed infants. These data show that (a) circulating choline status is elevated during infancy and lactation, (b) choline contents of breast milk vary between mothers and milk free choline contents are influenced by maternal circulating choline status, and (c) the choline contents of breast milk can influence infants' circulating choline status.

Adult↗
PubMed1994

Immunologically and enzymatically distinct rat choline kinase isozymes.

The purification and characterization of choline kinase R1 expressed in Escherichia coli, using expression plasmid pKCK(H) constructed with rat liver choline kinase cDNA [Uchida, T. and Yamashita, S. (1992) J. Biol. Chem. 267, 10156-10162], were carried out. Choline kinase R1 was chromatographically, enzymatically, and immunologically distinct from rat brain choline kinase [Uchida, T. and Yamashita, S. (1990) Biochim. Biophys. Acta 1043, 281-288]. This report, for the first time, definitively demonstrates the presence of two types of choline kinase isozymes. Rat choline kinase was immunologically classified into choline kinases P and R. Choline kinase P was purified from rat brain as described previously. Choline kinase R was designated as the choline kinase generated from the choline kinase R gene for choline kinase R1. Choline kinases from cytosol were chromatographically separated into two fractions, that is, one passed through a Blue-Toyopearl column and the other was retained on it. The choline kinase retained on the Blue-Toyopearl column was highly purified from rat liver cytosol and characterized in this study, being found to comprise isoforms of choline kinase R, and a choline kinase which was immunologically similar to choline kinase P. Choline kinases P and R are co-distributed in rat tissues. The carcinogen, 3-methylcholanthrene, and hepatotoxic carbon tetrachloride increased the enzyme activity, and the transcript and protein levels of choline kinase R in rat liver. The relation of choline kinase isozymes to previously purified enzymes was discussed.

Animals↗
PubMed1996

Tight connection between choline transport and phosphatidylcholine synthesis in MDCK cells.

In MDCK cells, choline uptake, the first step in the CDP-choline pathway for the biosynthesis of choline-containing phospholipids and osmolytes, occurs via both a transport system highly specific for choline and a non-specific pathway. The specific choline carrier is present at the apical domain of cells grown on dishes and is sodium-independent. Growing the cells on a permeant support results in the preferential localization of the specific choline carrier at the basolateral domain. To characterize the relationships between the choline uptake sites and the synthesis of phosphatidylcholine, MDCK cells were incubated with [Me-3H]choline and/or [Me-14C]choline for various times (up to 36 h) and the incorporation of label into phospholipids and water-soluble molecules was determined. For cells grown on dishes, addition of [Me-3H]choline at the apical side was followed by rapid incorporation of the label into the successive intermediates of the CDP-choline pathway. A comparable situation was found when growing the cells on a permeant support and adding the labelled choline at the basolateral side of the culture. On the other hand, radioactive choline added to the apical bath entered the CDP pathway to only a very low extent. Efflux experiments on cells loaded with choline from either the apical or the basolateral side demonstrate the existence of intracellular pools of choline. Addition of hemicholinium-3, an inhibitor of the specific choline carrier, markedly reduced the metabolism of choline taken up by the cells on the basolateral side but had no effect on that transported at the apical side. These results strongly suggest the existence of a tight connection between the entry of choline through the specific choline carrier and phosphatidylcholine synthesis in MDCK cells.

Animals↗
PubMed1997

Methyl-group donors cannot prevent apoptotic death of rat hepatocytes induced by choline-deficiency.

Choline-deficiency causes liver cells to die by apoptosis, and it has not been clear whether the effects of choline-deficiency are mediated by methyl-deficiency or by lack of choline moieties. SV40 immortalized CWSV-1 hepatocytes were cultivated in media that were choline-sufficient, choline-deficient, choline-deficient with methyl-donors (betaine or methionine), or choline-deficient with extra folate/vitamin B12. Choline-deficient CWSV-1 hepatocytes were not methyl-deficient as they had increased intracellular S-adenosylmethionine concentrations (132% of control; P < 0.01). Despite increased phosphatidylcholine synthesis via sequential methylation of phosphatidylethanol-amine, choline-deficient hepatocytes had significantly decreased (P < 0.01) intracellular concentrations of choline (20% of control), phosphocholine (6% of control), glycerophosphocholine (15% of control), and phosphatidylcholine (55% of control). Methyl-supplementation in choline-deficiency enhanced intracellular methyl-group availability, but did not correct choline-deficiency induced abnormalities in either choline metabolite or phospholipid content in hepatocytes. Methyl-supplemented, choline-deficient cells died by apoptosis. In a rat study, 2 weeks of a choline deficient diet supplemented with betaine did not prevent the occurrence of fatty liver and the increased DNA strand breakage induced by choline-deficiency. Though dietary supplementation with betaine restored hepatic betaine concentration and increased hepatic S-adenosylmethionine/S-adenosylhomocysteine ratio, it did not correct depleted choline (15% of control), phosphocholine (6% control), or phosphatidylcholine (48% of control) concentrations in deficient livers. These data show that decreased intracellular choline and/or choline metabolite concentrations, and not methyl deficiency, are associated with apoptotic death of hepatocytes.

Animals↗
PubMed1994

The responses to choline ions induced by transition metal ions in single water fibers of the frog glossopharyngeal nerve.

In single water-sensitive fibers (water fibers) of the frog glossopharyngeal nerve, application of a solution of 500 mM choline C1 to the tongue elicited responses of varying magnitude. Some water fibers (plain choline-insensitive water fibers) barely responded to the solution, while some water fibers (plain choline-sensitive water fibers) exhibited a considerable response to this solution. NiCl2, which is barely effective in producing neural response at concentrations below 5 mM, induced the response of plain choline-insensitive water fibers to choline+ ions. It was confirmed, in a collision test, that the Ni(2+)-induced responses to choline+ ions were derived from water fibers. However, NiCl2 did not affect the magnitude of the response generated by choline+ ions in plain choline-sensitive water fibers. The concentration-response curve for choline C1 in the presence of 1 mM NiCl2 for plain choline-insensitive water fibers was similar to the curves obtained in the absence of NiCl2 for plain choline-sensitive water fibers. Other organic salts, such as tris(hydroxymethyl)aminomethane-HCl, triethanolamine-HCl and tetraethylammonium C1, elicited no response or only a very small response from water fibers, and NiCl2 did not affect these responses. It is suggested that there exists a choline receptor for the response to choline+ ions in the apical membrane of frog taste cells and that Ni2+ ions expose the sites of such choline receptors, which are deeply embedded in the receptor membrane, to the outside medium. The effect of Ni2+ ions results in an increase in the number of the choline receptor sites available for binding of choline+ ions. The rank order of effectiveness of transition metal ions in eliciting the appearance or enhancement of the response to choline C1 was Ni2+ > Co2+ > Mn2+. Mg2+ ions had no effect on the response to choline+ ions. A similar rank order was previously obtained in enhancement of the responses to Ca2+, Mg2+ and Na+ ions (Kitada, 1994a). It seems likely that the mechanism for enhancement or elicitation of the response to choline+ ions by the transition metal ions has features in common with that for enhancement of the responses to Ca2+, Mg2+ and Na+ ions.

Animals↗
PubMed1997

A new method for determining the availability of choline in soybean meal.

Studies were conducted to evaluate the availability of choline in soybean meal, using a new approach: The basal diet contained soybean meal with a lowered choline content (partially extracted with methanol). Choline was added to the basal diet 1) by substituting intact soybean meal for the methanol washed soybean meal; or 2) from crystalline choline Cl. Four experiments were conducted with day-old male broilers housed in battery brooders and fed the experimental diets from 4 to 18 d posthatching. The basal diet contained corn, soybean meal, and isolated soy protein in Experiments 1 to 3, and also glucose in Experiment 4. In Experiment 1, chicks fed the basal diet (1,140 mg/kg choline, 0.61% sulfur amino acids) had a growth response to 1,000 mg/kg choline or 0.10% methionine (P < 0.05), but not to 0.10% cysteine (P > 0.05) supplementation. Supplementation of choline (0, 50, 100, 200, 400, 800, and 1,200 mg/kg) to the basal diet (1,230 mg/kg choline) in Experiment 2 resulted in a linear increase in growth up to 122 +/- 22 mg/kg supplemental choline (1,352 +/- 22 mg/kg total choline), reaching a plateau after that. In the slope ratio assays to determine choline availability, the basal diets in Experiments 3 (1,098 mg/kg choline) and 4 (920 mg/kg choline) were supplemented with either 50 or 100 mg/ kg choline (from choline Cl) or had approximately 50 or 100 mg/kg choline added with intact soybean meal (at the expense of washed soybean meal). There were significant linear responses of weight gain vs choline intake from either source (P < 0.001). The availability of choline in soybean meal was calculated to be 97 and 105% in Experiments 3 and 4, respectively. These results indicate that choline availability in soybean meal is close to 100%.

Animals↗
PubMed1975

Distribution and metabolism of intravenously administered choline[methyl- 3-H] and synthesis in vivo of acetylcholine in various tissues of guinea pigs.

The biosynthesis of acetylcholine and the fate of intravenously administered choline [methyl- 3-H] were studied in guinea pigs anesthetized with pentobarbital. Choline and acetylcholine were isolated by paper electrophoresis and estimated by use of a specific enzymatic (choline kinase) - radioisotopic assay. The concentration of acetylcholine ranged from 25.5 to 1.1 nmol/g in the following tissues (in order of decreasing concentration): duodenum, corpus striatum, stomach, cerebral cortex, spinal cord, abdominal fat, submaxillary gland, kidney, adrenal gland, spleen, liver, lung, heart and diaphragm. Choline [methyl- 3-H] was converted in the tissues to acetylcholine within 3 minutes after intravenous administration of the precursor. Virtually all the radioactivity in plasma at that time was present as free choline, suggesting that free choline from plasma is the immediate precursor for acetylcholine synthesized in the tissues cited. The concentration of free choline in tissues ranged from 344 nmol/g in adrenals to 40 nmol/g in heart, while that in plasma was 15 nmol/g. The initial half-life of choline in plasma, estimated from the rate of disappearance of choline after intravenous administration of either a tracer dose of choline [methyl- 3-H] (0.031 mumol/kg) or a high dose of choline chloride (200 mumol/kg), was less than 1 minute. This rapid removal of choline from plasma resulted from uptake (or binding) by tissues, with kidney and liver removing about 50% of the administered dose of choline [methyl- 3-H] within 3 minutes after its administration. Uptake of choline occurred in all tissues cited above, but there was a 20-fold difference in the uptake by the most active tissues (kidney and adrenals), as compared to that of the least active (central nervous system). Within 60 minutes after administration of choline [methyl- 3-H], most of the radioactive choline taken up by tissues had been converted to organic-soluble metabolites and to water-soluble metabolites that behaved like either phosphorylcholine or betaine during paper electrophoresis and chromatography. Betaine was the principal metabolite of choline in plasma. Radioactivity was excreted slowly into urine, which contained primarily free choline, betaine and a large amount of an unidentified metabolite. These findings indicate that the principal mechanism for the rapid removal of choline from plasma is uptake into tissues followed by metabolism.

Acetylcholine↗
PubMed1995

Decreased brain choline uptake in older adults. An in vivo proton magnetic resonance spectroscopy study.

OBJECTIVE: To test the hypothesis that uptake of circulating choline into the brain decreases with age, because alterations in metabolism of choline may be a factor contributing to age-related degenerative changes in the brain. DESIGN: Cohort comparison in younger and older adults. PARTICIPANTS: Subjects were chosen consecutively from lists of healthy volunteers screened by medical and psychiatric interviews and laboratory tests. Younger adults (n = 12) were between the ages of 20 and 40 years (mean age, 32 years), and older adults (n = 16) were between the ages of 60 and 85 years (mean age, 73 years). INTERVENTIONS: After fasting overnight, subjects received choline, as the bitartrate, to yield free choline equal to 50 mg/kg of body weight. Blood was drawn for determination of plasma choline concentration by high-performance liquid chromatography, and proton magnetic resonance spectroscopy (1H-MRS) was performed to determine the relative concentration of cytosolic choline-containing compounds in the brain at baseline and after ingestion of choline. MAIN OUTCOME MEASURES: Plasma choline and cytosolic choline-containing compounds in the brain, estimated as the ratio of the choline resonance to the creatine resonance on 1H-MRS scans of the basal ganglia, were compared following blinded analyses of data from subject cohorts studied at baseline and 3 hours after choline ingestion. RESULTS: Levels of plasma choline and cytosolic choline-containing compounds in brain were similar at baseline in younger and older subjects. Following ingestion of choline, plasma choline concentration increased by similar proportions (76% and 80%) in both younger and older subjects. Brain cytosolic choline--containing compounds increased substantially in younger subjects (mean increase, 60%; P < .001 vs baseline). Older subjects showed a much smaller increase in brain choline-containing compounds (mean, 16%; P < .001 vs the increase in younger subjects). CONCLUSION: Uptake of circulating choline into the brain decreases with age. Given the key role of choline in neuronal structure and function, this change may be a contributing factor in onset in late life of neurodegenerative, particularly dementing, illnesses in which cholinergic neurons show particular susceptibility to loss.

Adult↗
PubMed1983

C Tracer Evidence for Synthesis of Choline and Betaine via Phosphoryl Base Intermediates in Salinized Sugarbeet Leaves.

Like other chenopods, sugarbeets (Beta vulgaris L. cv Great Western D-2) accumulate glycine betaine when salinized; this may be an adaptive response to stress. The pathway of betaine synthesis in leaves of salinized (150-200 millimolar NaCl) sugarbeet plants was investigated by supplying [(14)C]formate, phosphoryl[(14)C]monomethylethanolamine ([(14)C][unk] MME) or phosphoryl[(14)C]choline ([(14)C][unk] choline) to leaf discs and following (14)C incorporation into prospective intermediates. The (14)C kinetic data were used to develop a computer model of the betaine pathway.When [(14)C]formate was fed, [unk] MME, phosphoryldimethylethanolamine ([unk] DME) and [unk] choline were the most prominent methylated products at short labeling times, after which (14)C appeared in free choline and in betaine. Phosphatidylcholine labeled more slowly than [unk] choline, choline, and betaine, and behaved as a minor end product. Very little (14)C entered the free methylethanolamines. When [(14)C][unk] MME was supplied, a small amount was hydrolyzed to the free base but the major fate was conversion to [unk] DME, [unk] choline, free choline, and betaine; label also accumulated slowly in phosphatidylcholine. Label from supplied [(14)C][unk] choline entered choline and betaine rapidly, while phosphatidylcholine labeled only slowly and to a small extent.These results are consistent with the pathway [unk] MME -->[unk] DME --> [unk] choline --> choline --> --> betaine, with a minor side branch leading from [unk] choline into phosphatidylcholine. This contrasts markedly (a) with the pathway of stress-induced choline and betaine synthesis in barley, in which phosphatidylcholine apparently acts as an intermediate (Hitz, Rhodes, Hanson 1981, Plant Physiol 68: 814-822); (b) with choline biogenesis in mammalian liver and microorganisms. Computer modeling of the experimental data pointed strongly to regulation at the [unk] choline --> choline step, and also indicated that the rate of [unk] choline synthesis is subject to feedback inhibition by [unk] choline.

Journal Article↗
PubMed1998

Expression, purification, and characterization of choline kinase, product of the CKI gene from Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) is the product of the CKI gene. Choline kinase catalyzes the committed step in the synthesis of phosphatidylcholine by the CDP-choline pathway. The yeast enzyme was overexpressed 106-fold in Sf-9 insect cells and purified 71.2-fold to homogeneity from the cytosolic fraction by chromatography with concanavalin A, Affi-Gel Blue, and Mono Q. The N-terminal amino acid sequence of purified choline kinase matched perfectly with the deduced sequence of the CKI gene. The minimum subunit molecular mass (73 kDa) of purified choline kinase was in good agreement with the predicted size (66.3 kDa) of the CKI gene product. Native choline kinase existed in oligomeric structures of dimers, tetramers, and octomers. The amounts of the tetrameric and octomeric forms increased in the presence of the substrate ATP. Antibodies were raised against the purified enzyme and were used to identify choline kinase in insect cells and in S. cerevisiae. Maximum choline kinase activity was dependent on Mg2+ ions (10 mM) at pH 9.5 and at 30 degrees C. The equilibrium constant (0.2) for the reaction indicated that the reverse reaction was favored in vitro. The activation energy for the reaction was 6.26 kcal/mol, and the enzyme was labile above 30 degrees C. Choline kinase exhibited saturation kinetics with respect to choline and positive cooperative kinetics with respect to ATP (n = 1.4-2.3). Results of the kinetic experiments indicated that the enzyme catalyzes a sequential Bi Bi reaction. The Vmax for the reaction was 138.7 micromol/min/mg, and the Km values for choline and ATP were 0.27 mM and 90 microM, respectively. The turnover number per choline kinase subunit was 153 s-1. Ethanolamine was a poor substrate for the purified choline kinase, and it was also poor inhibitor of choline kinase activity. ADP inhibited choline kinase activity (IC50 = 0.32 mM) in a positive cooperative manner (n = 1.5), and the mechanism of inhibition with respect to ATP and choline was complex. The regulation of choline kinase activity by ATP and ADP may be physiologically relevant.

Adenosine Diphosphate↗
PubMed1979

Choline and phospholipid metabolism and the synthesis of acetylcholine in rat brain.

The metabolism of choline by rat brain, plasma, and liver was investigated using combined gas chromatography mass spectrometry following microwave irradiation and treatment with deuterium-labeled choline. Methods were established to measure simultaneously the concentrations of six choline-containing compounds and the incorporation of labeled choline into each of them. Intravenous injection of [2H4]-choline led to initial labeling of choline, acetylcholine, and phosphocholine in rat brain, with all of the label eventually entering the phosphocholine pool. When labeled choline was administered in the diet its rate of incorporation into choline, phosphatidylcholine, and combined choline plasmalogen and lysophosphatidylcholine in the plasma and liver and into choline, acetylcholine, phosphocholine, glycerophosphorylcholine, phosphatidylcholine, and combined choline plasmalogen and lysophosphatidylcholine in the brain were determined. Choline, phosphatidylcholine, and combined choline plasmalogen and lysophosphatidylcholine in the plasma had similar specific activities. In the cortex and the striatum, choline and combined choline plasmalogen and lysophosphatidylcholine fraction generally had the highest specific activities. The time course of the post-mortem release of choline by the brain was measured, and the sources of this choline were, sequentially, acetylcholine, glycerophosphoryl-choline, and phospholipids.

Acetylcholine↗
PubMed1994

Choline transport in human placental brush-border membrane vesicles.

Pathways for transport of choline by human placental epithelia were investigated using brush border membrane vesicles isolated by divalent cation precipitation. The presence of choline transport mechanisms mediating Na(+)-choline cotransport, choline/H+ exchange and facilitated diffusion were assessed from [3H]choline tracer flux measurements. The rate and magnitude of intravesicular choline accumulation was unaffected by the imposition of an inwardly directed Na+ gradient suggesting an absence of a mechanism mediating brush border membrane Na(+)-choline cotransport. The imposition of inside-acid or inside-alkaline pH gradients was observed to have no significant effect on choline uptake suggesting choline is not a substrate for placental epithelial organic cation/H+ exchange. Conditions favoring the development of an inside-negative K+ diffusion potential was observed to induce a concentrative accumulation of choline to levels exceeding equilibrium suggesting the presence of a conductive uptake pathway for choline in placental brush border membrane. Evidence to suggest conductive choline uptake resulted from a mediated transport process includes a demonstration of the counterflow phenomena, the concentration-dependent inhibition by hemicholinium-3 (IC50 approximately equal to 100 microM) and the saturable rate of conductive choline uptake (Km approximately equal to 300 microM, Vmax approximately equal to 30 nmol/mg per min). Substrate specificity studies of the mechanism mediating conductive choline uptake suggest the interaction of choline with the transport protein occurs at a minimum of two sites: a site of negativity with the positively charged nitrogen group and a site of hydrogen bonding to the primary alcohol. Several commonly prescribed pharmaceuticals known to cross the placental barrier including imipramine, verapamil, propranolol, quinine, flurazepam, amiloride and ritodrin were observed to inhibit conductive choline uptake suggesting an interaction with the mechanism mediating conductive choline transport. Conductive choline uptake was unaffected by the presence of the basic amino acids lysine, arginine and histidine; the neurotransmitters serotonin, dopamine and histamine and the vitamins thiamine and carnitine which suggests the mechanism mediating conductive choline transport is not a pathway for placental uptake of these compounds.

Carrier Proteins↗
PubMed1996

Development of an experimental diet for determining bioavailable choline concentration and its application in studies with soybean lecithin.

Attempts to determine choline bioavailability have encountered criticism of experimental diets and protocol. Our objectives were to develop a choline-deficient soy isolate diet to quantify bioavailable choline concentration of soybean lecithin and to compare results to those obtained with a purified diet. In Assay 1, weight gain of chicks fed a choline-free crystalline amino acid diet responded linearly (P < .01) to graded doses of choline chloride, fluid lecithin (FL), or deoiled lecithin (DL). Multiple linear regression analysis indicated a bioavailable choline content of 2.3 and 3.7% for FL and DL, respectively. In Assay 2, a choline-deficient soy isolate diet was supplemented with 2-amino-2-methyl-1-propanol (AMP) to inhibit choline biosynthesis. Weight gain, feed intake, and feed efficiency increased (P < .05) markedly with the addition of choline chloride, but supplemental methionine or betaine had no effect (P > .10). Addition of 10% soybean meal to the diet severely deficient in choline per se produced a growth response (P < .05), whereas the same addition to the diet made adequate in choline did not elicit a growth response. In Assay 3, addition of graded levels of choline chloride, FL, or DL to the choline-deficient soy isolate diet containing AMP resulted in a linear (P < .01) increase in weight gain and feed intake. Multiple linear regression analysis indicated a bioavailable choline content of 2.0 and 3.5% for FL and DL, respectively. The AMP-containing soy isolate diet seems well suited for determination of bioavailable choline content in products containing secondary nutrients. Bioavailable choline content of FL and DL was similar to estimates of total choline content, suggesting that the choline in these products was fully available.

Animals↗
PubMed1984

The choline-depleted type II pneumonocyte. A model for investigating the synthesis of surfactant lipids.

When type II pneumonocytes from adult rats were maintained in a medium that lacked choline, the incorporation of [14C]glycerol into phosphatidylcholine was not greatly diminished during the period that the cells displayed characteristics of type II pneumonocytes. Cells that were maintained in choline-free medium that contained choline oxidase and catalase, however, became depleted of choline and subsequent synthesis of phosphatidylcholine by these cells was responsive to choline in the extracellular medium. Incorporation of [14C]glycerol into phosphatidylcholine by choline-depleted cells was stimulated maximally (approx. 6-fold) by extracellular choline at a concentration (0.05 mM) that also supported the greatest incorporation into phosphatidylglycerol. The incorporation of [14C]glycerol into other glycerophospholipids by choline-depleted cells was not increased by extracellular choline. When cells were incubated in the presence of [3H]cytidine, the choline-dependent stimulation of the synthesis of phosphatidylcholine and phosphatidylglycerol was accompanied by an increased recovery of [3H]CMP. This increased recovery of [3H]CMP reflected an increase in the intracellular amount of CMP from 48 +/- 9 to 76 +/- 16 pmol/10(6) cells. Choline-depleted cells that were exposed to [3H]choline contained [3H]CDP-choline as the principal water-soluble choline derivative. As the extracellular concentration of choline was increase, however, the amount of 3H in phosphocholine greatly exceeded that in all other water-soluble derivatives. Choline-depletion of cells resulted in an increase in the specific activity of CTP:phosphocholine cytidylyltransferase in cell homogenates (from 0.40 +/- 0.15 to 1.31 +/- 0.20 nmol X min-1 X mg of protein-1). These data are indicative that the biosynthesis of phosphatidylcholine is integrated with that of phosphatidylglycerol and are consistent with the proposed involvement of CMP in this integration. The choline-depleted type II pneumonocyte provides a new model for investigating the regulation of CTP:phosphocholine cytidylyltransferase activity.

Animals↗
PubMed1970

Choline permeability in cardiac muscle cells of the cat.

Permeability of the cardiac cell membrane to choline ions was estimated by measuring radioactive choline influx and efflux in cat ventricular muscle. Maximum values for choline influx in 3.5 and 137 mM choline were respectively 0.56 and 9 pmoles/cm(2).sec. In 3.5 mM choline the intracellular choline concentration was raised more than five times above the extracellular concentration after 2 hr of incubation. In 137 mM choline, choline influx corresponded to the combined loss of intracellular Na and K ions. Paper chromatography of muscle extracts indicated that choline was not metabolized to any important degree. The accumulation of intracellular choline rules out the existence of an efficient active pumping mechanism. By measuring simultaneously choline and sucrose exchange, choline efflux was analyzed in an extracellular phase, followed by two intracellular phases: a rapid and a slow one. Efflux corresponding to the rapid phase was estimated at 16-45 pmoles/cm(2).sec in 137 mM choline and at 1.3-3.5 pmoles/cm(2).sec in 3.5 mM choline; efflux in 3.5 mM choline was proportional to the intracellular choline concentration. The absolute figures for unidirectional efflux were much larger than the net influx values. The data are compared to Na and Li exchange in heart cells. Possible mechanisms for explaining the choline behavior in heart muscle are discussed.

Animals↗